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1
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Zhou AS, Tucker JB, Scribano CM, Lynch AR, Carlsen CL, Pop-Vicas ST, Pattaswamy SM, Burkard ME, Weaver BA. Diverse microtubule-targeted anticancer agents kill cells by inducing chromosome missegregation on multipolar spindles. PLoS Biol 2023; 21:e3002339. [PMID: 37883329 PMCID: PMC10602348 DOI: 10.1371/journal.pbio.3002339] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Accepted: 09/18/2023] [Indexed: 10/28/2023] Open
Abstract
Microtubule-targeted agents are commonly used for cancer treatment, though many patients do not benefit. Microtubule-targeted drugs were assumed to elicit anticancer activity via mitotic arrest because they cause cell death following mitotic arrest in cell culture. However, we recently demonstrated that intratumoral paclitaxel concentrations are insufficient to induce mitotic arrest and rather induce chromosomal instability (CIN) via multipolar mitotic spindles. Here, we show in metastatic breast cancer and relevant human cellular models that this mechanism is conserved among clinically useful microtubule poisons. While multipolar divisions typically produce inviable progeny, multipolar spindles can be focused into near-normal bipolar spindles at any stage of mitosis. Using a novel method to quantify the rate of CIN, we demonstrate that cell death positively correlates with net loss of DNA. Spindle focusing decreases CIN and causes resistance to diverse microtubule poisons, which can be counteracted by addition of a drug that increases CIN without affecting spindle polarity. These results demonstrate conserved mechanisms of action and resistance for diverse microtubule-targeted agents. Trial registration: clinicaltrials.gov, NCT03393741.
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Affiliation(s)
- Amber S. Zhou
- Molecular and Cellular Pharmacology Graduate Training Program, University of Wisconsin, Madison, Wisconsin, United States of America
| | - John B. Tucker
- Cancer Biology Graduate Training Program, University of Wisconsin, Madison, Wisconsin, United States of America
| | - Christina M. Scribano
- Molecular and Cellular Pharmacology Graduate Training Program, University of Wisconsin, Madison, Wisconsin, United States of America
| | - Andrew R. Lynch
- Cellular and Molecular Pathology Graduate Training Program, University of Wisconsin, Madison, Wisconsin, United States of America
| | - Caleb L. Carlsen
- Cellular and Molecular Biology Graduate Training Program, University of Wisconsin, Madison, Wisconsin, United States of America
| | - Sophia T. Pop-Vicas
- Department of Cell and Regenerative Biology, University of Wisconsin, Madison, Wisconsin, United States of America
| | - Srishrika M. Pattaswamy
- Department of Cell and Regenerative Biology, University of Wisconsin, Madison, Wisconsin, United States of America
| | - Mark E. Burkard
- Department of Medicine, University of Wisconsin, Madison, Wisconsin, United States of America
- Department of Oncology/McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin, United States of America
- Carbone Cancer Center, University of Wisconsin, Madison, Wisconsin, United States of America
| | - Beth A. Weaver
- Department of Cell and Regenerative Biology, University of Wisconsin, Madison, Wisconsin, United States of America
- Department of Oncology/McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin, United States of America
- Carbone Cancer Center, University of Wisconsin, Madison, Wisconsin, United States of America
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2
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Mahserejian SM, Scripture JP, Mauro AJ, Lawrence EJ, Jonasson EM, Murray KS, Li J, Gardner M, Alber M, Zanic M, Goodson HV. Quantification of Microtubule Stutters: Dynamic Instability Behaviors that are Strongly Associated with Catastrophe. Mol Biol Cell 2022; 33:ar22. [PMID: 35108073 PMCID: PMC9250389 DOI: 10.1091/mbc.e20-06-0348] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting DI mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA) that identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term “stutters.” During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our in vitro experiments and dimer-scale MT simulations, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anticatastrophe factor CLASP2γ works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared with previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.
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Affiliation(s)
- Shant M Mahserejian
- Department of Applied and Computational Mathematics and Statistics, University of Notre Dame, IN 46556.,Pacific Northwest National Laboratory, Richland, WA 99352
| | - Jared P Scripture
- Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556
| | - Ava J Mauro
- Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556.,Department of Mathematics and Statistics, University of Massachusetts Amherst, Amherst MA, 01003
| | - Elizabeth J Lawrence
- Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37240
| | - Erin M Jonasson
- Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556.,Department of Natural Sciences, Saint Martin's University, Lacey, WA 98503
| | - Kristopher S Murray
- Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556
| | - Jun Li
- Department of Applied and Computational Mathematics and Statistics, University of Notre Dame, IN 46556
| | - Melissa Gardner
- Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
| | - Mark Alber
- Department of Applied and Computational Mathematics and Statistics, University of Notre Dame, IN 46556.,Department of Mathematics, University of California Riverside, Riverside, CA 92521
| | - Marija Zanic
- Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37240.,Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN 37235.,Department of Biochemistry, Vanderbilt University, Nashville, TN 37205
| | - Holly V Goodson
- Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556
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3
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Tu Y, Wang X. Tracking cell migration by cellular force footprint recorded with a mechano-optical biosensor. Biosens Bioelectron 2021; 193:113533. [PMID: 34343934 DOI: 10.1016/j.bios.2021.113533] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 06/21/2021] [Accepted: 07/23/2021] [Indexed: 12/31/2022]
Abstract
Conventional cell migration assays require time-lapse imaging of live cells to trace cell migration paths, consequently demanding cumbersome hardware setup and suffering from low data throughput. In this work, we developed an assay named Tracking Cells by Footprint (TCF) based on a mechano-optical biosensor that irreversibly becomes fluorescent when sensing local cell adhesive force. Cell migration paths are visualized and recorded as fluorescent footprints on glass or elastic substrates coated with such biosensor. From the footprints, cell migration ranges, speeds and persistence are analyzed and quantified without the need of time-lapse imaging. The feasibility of TCF assays was demonstrated with three types of cells with different migratory capabilities. TCF was then applied to evaluating cell motility affected by biochemical or biomechanical cues. The results show that fibroblast motility is reduced by blebbistatin and vinblastine but promoted by bFGF (basic fibroblast growth factor), and the motility correlates with the substrate rigidity. TCF is also compatible with 96-well plates which, combined with static imaging and large-area scanning, provides high data throughput with minimal additional effort.
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Affiliation(s)
- Ying Tu
- Department of Physics and Astronomy, Iowa State University, Ames, IA, 50011, USA
| | - Xuefeng Wang
- Department of Physics and Astronomy, Iowa State University, Ames, IA, 50011, USA; Molecular, Cellular, and Development Biology Interdepartmental Program, Iowa State University, Ames, IA, 50011, USA.
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4
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Jepps TA. Kv7 channel trafficking by the microtubule network in vascular smooth muscle. Acta Physiol (Oxf) 2021; 232:e13692. [PMID: 34021973 PMCID: PMC8365713 DOI: 10.1111/apha.13692] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 05/17/2021] [Accepted: 05/19/2021] [Indexed: 12/17/2022]
Abstract
In arterial smooth muscle cells, changes in availability of integral membrane proteins influence the regulation of blood flow and blood pressure, which is critical for human health. However, the mechanisms that coordinate the trafficking and membrane expression of specific receptors and ion channels in vascular smooth muscle are poorly understood. In the vasculature, very little is known about microtubules, which form a road network upon which proteins can be transported to and from the cell membrane. This review article summarizes the impact of the microtubule network on arterial contractility, highlighting the importance of the network, with an emphasis on our recent findings regarding the trafficking of the voltage‐dependent Kv7 channels.
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Affiliation(s)
- Thomas A Jepps
- Vascular Biology Group Department of Biomedical Sciences University of Copenhagen Blegdamsvej 3 2200 Copenhagen N Denmark
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5
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Quader S, Liu X, Toh K, Su YL, Maity AR, Tao A, Paraiso WKD, Mochida Y, Kinoh H, Cabral H, Kataoka K. Supramolecularly enabled pH- triggered drug action at tumor microenvironment potentiates nanomedicine efficacy against glioblastoma. Biomaterials 2020; 267:120463. [PMID: 33130321 DOI: 10.1016/j.biomaterials.2020.120463] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Revised: 09/26/2020] [Accepted: 10/18/2020] [Indexed: 02/06/2023]
Abstract
The crucial balance of stability in blood-circulation and tumor-specific delivery has been suggested as one of the challenges for effective bench-to-bedside translation of nanomedicines (NMs). Herein, we developed a supramolecularly enabled tumor-extracellular (Tex) pH-triggered NM that can maintain the micellar structure with the entrapped-drug during systemic circulation and progressively release drug in the tumor by rightly sensing heterogeneous tumor-pH. Desacetylvinblastine hydrazide (DAVBNH), a derivative of potent anticancer drug vinblastine, was conjugated to an aliphatic ketone-functionalized poly(ethylene glycol)-b-poly(amino acid) copolymer and the hydrolytic stability of the derived hydrazone bond was efficiently tailored by exploiting the compartmentalized structure of polymer micelle. We confirmed an effective and safe therapeutic application of Tex pH-sensitive DAVBNH-loaded micelle (Tex-micelle) in orthotopic glioblastoma (GBM) models, extending median survival to 1.4 times in GBM xenograft and 2.6 times in GBM syngeneic model, compared to that of the free DAVBNH. The work presented here offers novel chemical insights into the molecular design of smart NMs correctly sensing Tex-pH via programmed functionalities. The practical engineering strategy based on a clinically relevant NM platform, and the encouraging therapeutic application of Tex-micelle in GBM, one of the most lethal human cancers, thus suggests the potential clinical translation of this system against other types of common cancers, including GBM.
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Affiliation(s)
- Sabina Quader
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 212-0821, Japan.
| | - Xueying Liu
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 212-0821, Japan
| | - Kazuko Toh
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 212-0821, Japan
| | - Yu-Lin Su
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 212-0821, Japan
| | - Amit Ranjan Maity
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 212-0821, Japan
| | - Anqi Tao
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
| | - West Kristian D Paraiso
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 212-0821, Japan
| | - Yuki Mochida
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 212-0821, Japan
| | - Hiroaki Kinoh
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 212-0821, Japan
| | - Horacio Cabral
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
| | - Kazunori Kataoka
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 212-0821, Japan; Institute for Future Initiatives, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
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6
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Jost M, Chen Y, Gilbert LA, Horlbeck MA, Krenning L, Menchon G, Rai A, Cho MY, Stern JJ, Prota AE, Kampmann M, Akhmanova A, Steinmetz MO, Tanenbaum ME, Weissman JS. Pharmaceutical-Grade Rigosertib Is a Microtubule-Destabilizing Agent. Mol Cell 2020; 79:191-198.e3. [PMID: 32619469 PMCID: PMC7332992 DOI: 10.1016/j.molcel.2020.06.008] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2019] [Revised: 05/13/2020] [Accepted: 06/01/2020] [Indexed: 11/17/2022]
Abstract
We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.
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Affiliation(s)
- Marco Jost
- Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Yuwen Chen
- Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Luke A Gilbert
- Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Helen Diller Family Comprehensive Cancer Center, Department of Urology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Max A Horlbeck
- Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Lenno Krenning
- Hubrecht Institute - KNAW and University Medical Center Utrecht, 3584CT Utrecht, the Netherlands
| | - Grégory Menchon
- Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland
| | - Ankit Rai
- Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3548CH Utrecht, the Netherlands
| | - Min Y Cho
- Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Jacob J Stern
- Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Andrea E Prota
- Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland
| | - Martin Kampmann
- Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Institute for Neurodegenerative Diseases and Department of Biochemistry & Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Chan-Zuckerberg Biohub, San Francisco, CA 94158, USA
| | - Anna Akhmanova
- Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3548CH Utrecht, the Netherlands
| | - Michel O Steinmetz
- Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland; Biozentrum, University of Basel, 4056 Basel, Switzerland
| | - Marvin E Tanenbaum
- Hubrecht Institute - KNAW and University Medical Center Utrecht, 3584CT Utrecht, the Netherlands.
| | - Jonathan S Weissman
- Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
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7
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Shiri F, Bakhshayesh S, Ghasemi JB. Computer-aided molecular design of (E)-N-Aryl-2-ethene-sulfonamide analogues as microtubule targeted agents in prostate cancer. ARAB J CHEM 2019. [DOI: 10.1016/j.arabjc.2014.11.063] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
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8
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Thirupathi A, Shanmugavadivelu CM, Natarajan S. Fastidious Anatomization of Biota Procured Compounds on Cancer Drug Discovery. Curr Pharm Biotechnol 2019; 21:354-363. [PMID: 31778106 DOI: 10.2174/1389201020666191128145015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2019] [Revised: 11/20/2019] [Accepted: 11/20/2019] [Indexed: 11/22/2022]
Abstract
BACKGROUND Natural products are the rootstock for identifying new drugs since ancient times. In comparison with synthetic drugs, they have abounding beneficial effects in bestowing protection against many diseases, including cancer. Cancer has been observed as a major threat in recent decades, and its prevalence is expected to increase over the next decades. Also, current treatment methods in cancer therapy such as radiation therapy and chemotherapy cause severe adverse side effects among the cancer population. Therefore, it is exigent to find a remedy without any side effects. METHODS In recent years, research has focused on obtaining naturally derived products to encounter this complication. The current pace of investigations, such as gene identification and advancement in combinatorial chemistry, leads to the aberrant access to a wide range of new synthetic drugs. In fact, natural products act as templates in structure predictions and synthesis of new compounds with enhanced biological activities. RESULTS Recent developments in genomics have established the importance of polymorphism, which implies that patients require different drugs for their treatment. This demands the discovery of a large number of drugs, but limited sources restrict the pharmaceutical industry to overcome these major obstacles. The use of natural products and their semisynthetic and synthetic analogues could alleviate these problems. However, the lack of standardization in terms of developing methods for evaluating the chemical composition, efficacy, isolation and international approval is still a major limitation in this field. In the past few years, several drug-approval authorities, including the FDA and WHO have allowed using these naturally derived compounds in humans. CONCLUSION In this review, we described the use of some natural products from plant and marine sources in cancer treatment and shed some light on semi-synthetic and synthetic compounds derived from natural sources used in cancer therapy.
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Affiliation(s)
- Anand Thirupathi
- Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei Normal University, Shijiazhuang 050024, Hebei Province, China
| | | | - Sampathkumar Natarajan
- Department of Chemistry, SSM Institute of Engineering and Technology, Dindigul, Tamil Nadu, India
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9
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Zhang Z, Lu C, Wang P, Li A, Zhang H, Xu S. Structural Basis and Mechanism for Vindoline Dimers Interacting with α,β-Tubulin. ACS OMEGA 2019; 4:11938-11948. [PMID: 31460305 PMCID: PMC6682054 DOI: 10.1021/acsomega.9b00947] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/03/2019] [Accepted: 06/26/2019] [Indexed: 06/10/2023]
Abstract
Vinblastine and its derivatives used in clinics as antitumor drugs often cause drug resistance and some serious side effects; thus, it is necessary to study new vinblastine analogues with strong anticancer cytotoxicity and low toxicity. We designed a dimer molecule using two vindoline-bonded dimer vindoline (DVB) and studied its interaction with α,β-tubulin through the double-sided adhesive mechanism to explore its anticancer cytotoxicity. In our work, DVB was docked into the interface between α-tubulin and β-tubulin to construct a complex protein structure, and then it was simulated for 100 ns using the molecular dynamics technology to become a stable and refined complex protein structure. Based on such a refined structure, the quantum chemistry at the level of the MP2/6-31G(d,p) method was used to calculate the binding energies for DVB interacting with respective residues. By the obtained binding energies, the active site residues for interaction with DVB were found. Up to 20 active sites of residues within α,β-tubulin interacting with DVB are labeled in β-Asp179, β-Glu207, β-Tyr210, β-Asp211, β-Phe214, β-Pro222, β-Tyr224, and β-Leu227 and α-Asn249, α-Arg308, α-Lys326, α-Asn329, α-Ala333, α-Thr334, α-Lys336, α-Lys338, α-Arg339, α-Ser340, α-Thr349, and α-Phe351. The total binding energy between DVB and α,β-tubulin is about -251.0 kJ·mol-1. The sampling average force potential (PMF) method was further used to study the dissociation free energy (ΔG) along the separation trajectory of α,β-tubulin under the presence of DVB based on the refined structure of DVB with α,β-tubulin. Because of the presence of DVB within the interface between α- and β-tubulin, ΔG is 252.3 kJ·mol-1. In contrast to the absence of DVB, the separation of pure β-tubulin needs a free energy of 196.9 kJ·mol-1. The data show that the presence of DVB adds more 55.4 kJ·mol-1 of ΔG to hinder the normal separation of α,β-tubulin. Compared to vinblastine existing, the free energy required for the separation of α,β-tubulin is 220.5 kJ·mol-1. Vinblastine and DVB can both be considered through the same double-sided adhesive mechanism to give anticancer cytotoxicity. Because of the presence of DVB, a larger free energy is needed for the separation of α,β-tubulin, which suggests that DVB should have stronger anticancer cytotoxicity than vinblastine and shows that DVB has a broad application prospect.
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Affiliation(s)
- Zhengqiong Zhang
- College
of Chemical Science and Technology and Pharmacy, Key Laboratory of
Education Ministry for Medicinal Chemistry of Natural Resource, Yunnan University, Kunming 650091, China
| | - Chengqi Lu
- College
of Chemical Science and Technology and Pharmacy, Key Laboratory of
Education Ministry for Medicinal Chemistry of Natural Resource, Yunnan University, Kunming 650091, China
| | - Pei Wang
- College
of Chemical Science and Technology and Pharmacy, Key Laboratory of
Education Ministry for Medicinal Chemistry of Natural Resource, Yunnan University, Kunming 650091, China
| | - Aijing Li
- College
of Chemical Science and Technology and Pharmacy, Key Laboratory of
Education Ministry for Medicinal Chemistry of Natural Resource, Yunnan University, Kunming 650091, China
| | - Hongbo Zhang
- College
of Mathematics, Yunnan Normal University, Kunming 650500, China
| | - Sichuan Xu
- College
of Chemical Science and Technology and Pharmacy, Key Laboratory of
Education Ministry for Medicinal Chemistry of Natural Resource, Yunnan University, Kunming 650091, China
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10
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Guo C, Williams JC, Polenova T. Conformational Flexibility of p150 Glued(1-191) Subunit of Dynactin Assembled with Microtubules. Biophys J 2019; 117:938-949. [PMID: 31445682 DOI: 10.1016/j.bpj.2019.07.036] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2019] [Revised: 07/12/2019] [Accepted: 07/25/2019] [Indexed: 02/05/2023] Open
Abstract
Microtubule (MT)-associated proteins perform diverse functions in cells. These functions are dependent on their interactions with MTs. Dynactin, a cofactor of dynein motor, assists the binding of dynein to various organelles and is crucial to the long-distance processivity of dynein-based complexes. The largest subunit of dynactin, the p150Glued, contains an N-terminus segment that is responsible for the MT-binding interactions and long-range processivity of dynactin. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150Glued N-terminal region, both free and in complex with polymerized MTs. This 191-residue region encompasses the cytoskeleton-associated protein glycine-rich domain, the basic domain, and serine/proline-rich (SP-rich) domain. We demonstrate that the basic and SP-rich domains are intrinsically disordered in solution and significantly enhance the binding affinity to MTs as these regions contain the second MT-binding site on the p150Glued subunit. The majority of the basic and SP-rich domains are predicted to be random coil, whereas the segments S111-I116, A124-R132, and K144-T146 in the basic domain contain short α-helical or β-sheet structures. These three segments possibly encompass the MT-binding site. Surprisingly, the protein retains a high degree of flexibility upon binding to MTs except for the regions that are directly involved in the binding interactions with MTs. This conformational flexibility may be essential for the biological functions of the p150Glued subunit.
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Affiliation(s)
- Changmiao Guo
- Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware
| | - John C Williams
- Department of Molecular Medicine, Beckman Research Institute of City of Hope, Duarte, California
| | - Tatyana Polenova
- Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware.
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11
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Ramos SV, Hughes MC, Perry CGR. Altered skeletal muscle microtubule-mitochondrial VDAC2 binding is related to bioenergetic impairments after paclitaxel but not vinblastine chemotherapies. Am J Physiol Cell Physiol 2019; 316:C449-C455. [PMID: 30624982 DOI: 10.1152/ajpcell.00384.2018] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Microtubule-targeting chemotherapies are linked to impaired cellular metabolism, which may contribute to skeletal muscle dysfunction. However, the mechanisms by which metabolic homeostasis is perturbed remains unknown. Tubulin, the fundamental unit of microtubules, has been implicated in the regulation of mitochondrial-cytosolic ADP/ATP exchange through its interaction with the outer membrane voltage-dependent anion channel (VDAC). Based on this model, we predicted that disrupting microtubule architecture with the stabilizer paclitaxel and destabilizer vinblastine would impair skeletal muscle mitochondrial bioenergetics. Here, we provide in vitro evidence of a direct interaction between both α-tubulin and βII-tubulin with VDAC2 in untreated single extensor digitorum longus (EDL) fibers. Paclitaxel increased both α- and βII-tubulin-VDAC2 interactions, whereas vinblastine had no effect. Utilizing a permeabilized muscle fiber bundle preparation that retains the cytoskeleton, paclitaxel treatment impaired the ability of ADP to attenuate H2O2 emission, resulting in greater H2O2 emission kinetics. Despite no effect on tubulin-VDAC2 binding, vinblastine still altered mitochondrial bioenergetics through a surprising increase in ADP-stimulated respiration while also impairing ADP suppression of H2O2 and increasing mitochondrial susceptibility to calcium-induced formation of the proapoptotic permeability transition pore. Collectively, these results demonstrate that altering microtubule architecture with chemotherapeutics disrupts mitochondrial bioenergetics in EDL skeletal muscle. Specifically, microtubule stabilization increases H2O2 emission by impairing ADP sensitivity in association with greater tubulin-VDAC binding. In contrast, decreasing microtubule abundance triggers a broad impairment of ADP's governance of respiration and H2O2 emission as well as calcium retention capacity, albeit through an unknown mechanism.
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Affiliation(s)
- Sofhia V Ramos
- School of Kinesiology and Health Science, Muscle Health Research Centre, York University , Toronto, Ontario , Canada
| | - Meghan C Hughes
- School of Kinesiology and Health Science, Muscle Health Research Centre, York University , Toronto, Ontario , Canada
| | - Christopher G R Perry
- School of Kinesiology and Health Science, Muscle Health Research Centre, York University , Toronto, Ontario , Canada
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12
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Advani S, Maresca TJ, Ross JL. Creation and testing of a new, local microtubule-disruption tool based on the microtubule-severing enzyme, katanin p60. Cytoskeleton (Hoboken) 2018; 75:531-544. [PMID: 30176123 DOI: 10.1002/cm.21482] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2018] [Revised: 06/25/2018] [Accepted: 07/13/2018] [Indexed: 12/18/2022]
Abstract
Current methods to disrupt the microtubule cytoskeleton do not easily provide rapid, local control with standard cell manipulation reagents. Here, we develop a new microtubule-disruption tool based on katanin p60 severing activity and demonstrate proof-of-principle by targeting it to kinetochores in Drosophila melanogaster S2 cells. Specifically, we show that human katanin p60 can remove microtubule polymer mass in S2 cells and an increase in misaligned chromosomes when globally overexpressed. When katanin p60 was targeted to the kinetochores via Mis12, we were able to recapitulate the misalignment only when using a phosphorylation-resistant mutant katanin p60. Our results demonstrate that targeting an active version of katanin p60 to the kinetochore can reduce the fidelity of achieving full chromosome alignment in metaphase and could serve as a microtubule disruption tool for the future.
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Affiliation(s)
- Siddheshwari Advani
- Molecular and Cellular Biology Graduate Program, University of Massachusetts Amherst, Amherst, Massachusetts
| | - Thomas J Maresca
- Molecular and Cellular Biology Graduate Program, University of Massachusetts Amherst, Amherst, Massachusetts.,Department of Biology, University of Massachusetts Amherst, Amherst, Massachusetts
| | - Jennifer L Ross
- Molecular and Cellular Biology Graduate Program, University of Massachusetts Amherst, Amherst, Massachusetts.,Department of Physics, University of Massachusetts Amherst, Amherst, Massachusetts
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13
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Small-Molecule Modulation of Lipid-Dependent Cellular Processes against Cancer: Fats on the Gunpoint. BIOMED RESEARCH INTERNATIONAL 2018; 2018:6437371. [PMID: 30186863 PMCID: PMC6114229 DOI: 10.1155/2018/6437371] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/18/2018] [Accepted: 07/22/2018] [Indexed: 12/27/2022]
Abstract
Lipid cell membrane composed of various distinct lipids and proteins act as a platform to assemble various signaling complexes regulating innumerous cellular processes which are strongly downregulated or altered in cancer cells emphasizing the still-underestimated critical function of lipid biomolecules in cancer initiation and progression. In this review, we outline the current understanding of how membrane lipids act as signaling hot spots by generating distinct membrane microdomains called rafts to initiate various cellular processes and their modulation in cancer phenotypes. We elucidate tangible drug targets and pathways all amenable to small-molecule perturbation. Ranging from targeting membrane rafts organization/reorganization to rewiring lipid metabolism and lipid sorting in cancer, the work summarized here represents critical intervention points being attempted for lipid-based anticancer therapy and future directions.
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14
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Ternary complex of Kif2A-bound tandem tubulin heterodimers represents a kinesin-13-mediated microtubule depolymerization reaction intermediate. Nat Commun 2018; 9:2628. [PMID: 29980677 PMCID: PMC6035175 DOI: 10.1038/s41467-018-05025-7] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2018] [Accepted: 06/11/2018] [Indexed: 12/22/2022] Open
Abstract
Kinesin-13 proteins are major microtubule (MT) regulatory factors that catalyze removal of tubulin subunits from MT ends. The class-specific “neck” and loop 2 regions of these motors are required for MT depolymerization, but their contributing roles are still unresolved because their interactions with MT ends have not been observed directly. Here we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers in a head-to-tail array, providing a view of these interactions. The neck of Kif2A binds to one tubulin dimer and the motor core to the other, guiding insertion of the KVD motif of loop 2 in between them. AMPPNP-bound Kif2A can form stable complexes with tubulin in solution and trigger MT depolymerization. We also demonstrate the importance of the neck in modulating ATP turnover and catalytic depolymerization of MTs. These results provide mechanistic insights into the catalytic cycles of kinesin-13. The kinesin-13 family of microtubule (MT) depolymerases are major regulators of MT dynamics. Here the authors provide insights into the MT depolymerization mechanism by solving the crystal structure of a kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers.
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15
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Bioactive carbon dots lights up microtubules and destabilises cell cytoskeletal framework – A robust imaging agent with therapeutic activity. Colloids Surf B Biointerfaces 2017; 159:662-672. [DOI: 10.1016/j.colsurfb.2017.07.054] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2017] [Revised: 07/11/2017] [Accepted: 07/24/2017] [Indexed: 02/05/2023]
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16
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Singireesu SSNR, Misra S, Mondal SK, Yerramsetty S, Sahu N, K SB. Costunolide induces micronuclei formation, chromosomal aberrations, cytostasis, and mitochondrial-mediated apoptosis in Chinese hamster ovary cells. Cell Biol Toxicol 2017; 34:125-142. [PMID: 28914393 DOI: 10.1007/s10565-017-9411-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2017] [Accepted: 08/25/2017] [Indexed: 12/18/2022]
Abstract
Costunolide (CE) is a sesquiterpene lactone well-known for its antihepatotoxic, antiulcer, and anticancer activities. The present study focused on the evaluation of the cytogenetic toxicity and cellular death-inducing potential of CE in CHO cells, an epithelial cell line derived from normal ovary cells of Chinese hamster. The cytotoxic effect denoting MTT assay has shown an IC50 value of 7.56 μM CE, where 50% proliferation inhibition occurs. The oxidative stress caused by CE was confirmed based on GSH depletion induced cell death, conspicuously absent in N-acetylcysteine (GSH precursor) pretreated cells. The evaluation of genotoxic effects of CE using cytokinesis block micronucleus assay and chromosomal aberration test has shown prominent induction of binucleated micronucleated cells and aberrant metaphases bearing chromatid and chromosomal breaks, indicating CE's clastogenic and aneugenic potential. The apoptotic death in CE treated cells was confirmed by an increase in the number of cells in subG1 phase, exhibiting chromatin condensation and membranous phosphatidylserine translocation. The apoptosis induction follows mitochondrial mediation, evident from an increase in the BAX/Bcl-2 ratio, caspase-3/7 activity, and mitochondrial membrane permeability. CE also induces cytostasis in addition to apoptosis, substantiated by the reduced cytokinetic (replicative indices) and mitotic (mitotic indices and histone H3 Ser-10 phosphorylation) activities. Overall, the cellular GSH depletion and potential genotoxic effects by CE led the CHO cells to commit apoptosis and lowered cell division. The observed sensitivity of CHO cells doubts unintended adverse effects of CE on normal healthy cells, suggesting higher essentiality of further studies in order to establish its safety efficacy in therapeutic explorations.
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Affiliation(s)
| | - Sunil Misra
- Genetic Toxicology Laboratory, Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500007, India
| | - Sujan Kumar Mondal
- Biomaterials Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500007, India
| | - Suresh Yerramsetty
- Chemical Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500007, India
| | - Nivedita Sahu
- Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500007, India
| | - Suresh Babu K
- Natural Products Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500007, India
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17
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De Vos KJ, Hafezparast M. Neurobiology of axonal transport defects in motor neuron diseases: Opportunities for translational research? Neurobiol Dis 2017; 105:283-299. [PMID: 28235672 PMCID: PMC5536153 DOI: 10.1016/j.nbd.2017.02.004] [Citation(s) in RCA: 147] [Impact Index Per Article: 18.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2016] [Revised: 01/26/2017] [Accepted: 02/20/2017] [Indexed: 12/12/2022] Open
Abstract
Intracellular trafficking of cargoes is an essential process to maintain the structure and function of all mammalian cell types, but especially of neurons because of their extreme axon/dendrite polarisation. Axonal transport mediates the movement of cargoes such as proteins, mRNA, lipids, membrane-bound vesicles and organelles that are mostly synthesised in the cell body and in doing so is responsible for their correct spatiotemporal distribution in the axon, for example at specialised sites such as nodes of Ranvier and synaptic terminals. In addition, axonal transport maintains the essential long-distance communication between the cell body and synaptic terminals that allows neurons to react to their surroundings via trafficking of for example signalling endosomes. Axonal transport defects are a common observation in a variety of neurodegenerative diseases, and mutations in components of the axonal transport machinery have unequivocally shown that impaired axonal transport can cause neurodegeneration (reviewed in El-Kadi et al., 2007, De Vos et al., 2008; Millecamps and Julien, 2013). Here we review our current understanding of axonal transport defects and the role they play in motor neuron diseases (MNDs) with a specific focus on the most common form of MND, amyotrophic lateral sclerosis (ALS).
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Affiliation(s)
- Kurt J De Vos
- Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield S10 2HQ, UK.
| | - Majid Hafezparast
- Neuroscience, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
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18
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Ramkumar A, Murthy D, Raja DA, Singh A, Krishnan A, Khanna S, Vats A, Thukral L, Sharma P, Sivasubbu S, Rani R, Natarajan VT, Gokhale RS. Classical autophagy proteins LC3B and ATG4B facilitate melanosome movement on cytoskeletal tracks. Autophagy 2017; 13:1331-1347. [PMID: 28598240 DOI: 10.1080/15548627.2017.1327509] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Macroautophagy/autophagy is a dynamic and inducible catabolic process that responds to a variety of hormonal and environmental cues. Recent studies highlight the interplay of this central pathway in a variety of pathophysiological diseases. Although defective autophagy is implicated in melanocyte proliferation and pigmentary disorders, the mechanistic relationship between the 2 pathways has not been elucidated. In this study, we show that autophagic proteins LC3B and ATG4B mediate melanosome trafficking on cytoskeletal tracks. While studying melanogenesis, we observed spatial segregation of LC3B-labeled melanosomes with preferential absence at the dendritic ends of melanocytes. This LC3B labeling of melanosomes did not impact the steady-state levels of these organelles but instead facilitated their intracellular positioning. Melanosomes primarily traverse on microtubule and actin cytoskeletal tracks and our studies reveal that LC3B enables the assembly of microtubule translocon complex. At the microtubule-actin crossover junction, ATG4B detaches LC3B from melanosomal membranes by enzymatic delipidation. Further, by live-imaging we show that melanosomes transferred to keratinocytes lack melanocyte-specific LC3B. Our study thus elucidates a new role for autophagy proteins in directing melanosome movement and reveal the unconventional use of these proteins in cellular trafficking pathways. Such crosstalk between the central cellular function and housekeeping pathway may be a crucial mechanism to balance melanocyte bioenergetics and homeostasis.
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Affiliation(s)
- Amrita Ramkumar
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India.,b Academy of Scientific and Innovative Research , Rafi Marg, New Delhi , India
| | - Divya Murthy
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India
| | - Desingu Ayyappa Raja
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India.,b Academy of Scientific and Innovative Research , Rafi Marg, New Delhi , India
| | - Archana Singh
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India
| | - Anusha Krishnan
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India.,b Academy of Scientific and Innovative Research , Rafi Marg, New Delhi , India
| | - Sangeeta Khanna
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India
| | - Archana Vats
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India
| | - Lipi Thukral
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India
| | - Pushkar Sharma
- c National Institute of Immunology , Aruna Asaf Ali Marg, New Delhi , India
| | - Sridhar Sivasubbu
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India
| | - Rajni Rani
- c National Institute of Immunology , Aruna Asaf Ali Marg, New Delhi , India
| | - Vivek T Natarajan
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India.,b Academy of Scientific and Innovative Research , Rafi Marg, New Delhi , India
| | - Rajesh S Gokhale
- a CSIR- Institute of Genomics and Integrative Biology , Mathura Road, New Delhi , India.,b Academy of Scientific and Innovative Research , Rafi Marg, New Delhi , India.,c National Institute of Immunology , Aruna Asaf Ali Marg, New Delhi , India.,d Jawaharlal Nehru Center for Advanced Scientific Research , Jakkur, Bangalore , India
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19
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Gosselin P, Rando G, Fleury-Olela F, Schibler U. Unbiased identification of signal-activated transcription factors by barcoded synthetic tandem repeat promoter screening (BC-STAR-PROM). Genes Dev 2017; 30:1895-907. [PMID: 27601530 PMCID: PMC5024686 DOI: 10.1101/gad.284828.116] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2016] [Accepted: 08/02/2016] [Indexed: 12/23/2022]
Abstract
Gosselin et al. designed a widely applicable method, dubbed BC-STAR-PROM, to identify signal-activated TFs without any prior knowledge of their properties. To establish proof of concept for BC-STAR-PROM, they applied it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. The discovery of transcription factors (TFs) controlling pathways in health and disease is of paramount interest. We designed a widely applicable method, dubbed barcorded synthetic tandem repeat promoter screening (BC-STAR-PROM), to identify signal-activated TFs without any a priori knowledge about their properties. The BC-STAR-PROM library consists of ∼3000 luciferase expression vectors, each harboring a promoter (composed of six tandem repeats of synthetic random DNA) and an associated barcode of 20 base pairs (bp) within the 3′ untranslated mRNA region. Together, the promoter sequences encompass >400,000 bp of random DNA, a sequence complexity sufficient to capture most TFs. Cells transfected with the library are exposed to a signal, and the mRNAs that it encodes are counted by next-generation sequencing of the barcodes. This allows the simultaneous activity tracking of each of the ∼3000 synthetic promoters in a single experiment. Here we establish proof of concept for BC-STAR-PROM by applying it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. BC-STAR-PROM revealed that serum response factor (SRF) is the only immediate early TF induced by both actin polymerization and microtubule depolymerization. Such changes in cytoskeleton dynamics are known to occur during the cell division cycle, and real-time bioluminescence microscopy indeed revealed cell-autonomous SRF–myocardin-related TF (MRTF) activity bouts in proliferating cells.
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Affiliation(s)
- Pauline Gosselin
- Department of Molecular Biology, University of Geneva, CH-1211 Geneva, Switzerland
| | - Gianpaolo Rando
- Department of Molecular Biology, University of Geneva, CH-1211 Geneva, Switzerland
| | | | - Ueli Schibler
- Department of Molecular Biology, University of Geneva, CH-1211 Geneva, Switzerland
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20
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Understanding the Effectiveness of Natural Compound Mixtures in Cancer through Their Molecular Mode of Action. Int J Mol Sci 2017; 18:ijms18030656. [PMID: 28304343 PMCID: PMC5372668 DOI: 10.3390/ijms18030656] [Citation(s) in RCA: 205] [Impact Index Per Article: 25.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2017] [Revised: 03/13/2017] [Accepted: 03/15/2017] [Indexed: 12/19/2022] Open
Abstract
Many approaches to cancer management are often ineffective due to adverse reactions, drug resistance, or inadequate target specificity of single anti-cancer agents. In contrast, a combinatorial approach with the application of two or more anti-cancer agents at their respective effective dosages can achieve a synergistic effect that boosts cytotoxicity to cancer cells. In cancer, aberrant apoptotic pathways allow cells that should be killed to survive with genetic abnormalities, leading to cancer progression. Mutations in apoptotic mechanism arising during the treatment of cancer through cancer progression can consequently lead to chemoresistance. Natural compound mixtures that are believed to have multiple specific targets with minimal acceptable side-effects are now of interest to many researchers due to their cytotoxic and chemosensitizing activities. Synergistic interactions within a drug mixture enhance the search for potential molecular targets in cancer cells. Nonetheless, biased/flawed scientific evidence from natural products can suggest false positive therapeutic benefits during drug screening. In this review, we have taken these factors into consideration when discussing the evidence for these compounds and their synergistic therapeutic benefits in cancer. While there is limited evidence for clinical efficacy for these mixtures, in vitro data suggest that these preparations merit further investigation, both in vitro and in vivo.
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21
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Castle BT, McCubbin S, Prahl LS, Bernens JN, Sept D, Odde DJ. Mechanisms of kinetic stabilization by the drugs paclitaxel and vinblastine. Mol Biol Cell 2017; 28:1238-1257. [PMID: 28298489 PMCID: PMC5415019 DOI: 10.1091/mbc.e16-08-0567] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2016] [Revised: 02/21/2017] [Accepted: 02/28/2017] [Indexed: 12/20/2022] Open
Abstract
Chemotherapeutic agents that target microtubule dynamics promote a universal phenotype of kinetic stabilization. Integrated computational modeling and fluorescence microscopy identify the fundamental kinetic and thermodynamic mechanisms that result in kinetic stabilization, specifically by the drugs paclitaxel and vinblastine. Microtubule-targeting agents (MTAs), widely used as biological probes and chemotherapeutic drugs, bind directly to tubulin subunits and “kinetically stabilize” microtubules, suppressing the characteristic self-assembly process of dynamic instability. However, the molecular-level mechanisms of kinetic stabilization are unclear, and the fundamental thermodynamic and kinetic requirements for dynamic instability and its elimination by MTAs have yet to be defined. Here we integrate a computational model for microtubule assembly with nanometer-scale fluorescence microscopy measurements to identify the kinetic and thermodynamic basis of kinetic stabilization by the MTAs paclitaxel, an assembly promoter, and vinblastine, a disassembly promoter. We identify two distinct modes of kinetic stabilization in live cells, one that truly suppresses on-off kinetics, characteristic of vinblastine, and the other a “pseudo” kinetic stabilization, characteristic of paclitaxel, that nearly eliminates the energy difference between the GTP- and GDP-tubulin thermodynamic states. By either mechanism, the main effect of both MTAs is to effectively stabilize the microtubule against disassembly in the absence of a robust GTP cap.
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Affiliation(s)
- Brian T Castle
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455
| | - Seth McCubbin
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109
| | - Louis S Prahl
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455
| | - Jordan N Bernens
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455
| | - David Sept
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109
| | - David J Odde
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455
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22
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Sueth-Santiago V, Decote-Ricardo D, Morrot A, Freire-de-Lima CG, Lima MEF. Challenges in the chemotherapy of Chagas disease: Looking for possibilities related to the differences and similarities between the parasite and host. World J Biol Chem 2017; 8:57-80. [PMID: 28289519 PMCID: PMC5329715 DOI: 10.4331/wjbc.v8.i1.57] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2016] [Revised: 12/30/2016] [Accepted: 01/14/2017] [Indexed: 02/05/2023] Open
Abstract
Almost 110 years after the first studies by Dr. Carlos Chagas describing an infectious disease that was named for him, Chagas disease remains a neglected illness and a death sentence for infected people in poor countries. This short review highlights the enormous need for new studies aimed at the development of novel and more specific drugs to treat chagasic patients. The primary tool for facing this challenge is deep knowledge about the similarities and differences between the parasite and its human host.
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23
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Jana B, Sarkar J, Mondal P, Barman S, Mohapatra S, Bhunia D, Pradhan K, Saha A, Adak A, Ghosh S, Ghosh S. A short GC rich DNA derived from microbial origin targets tubulin/microtubules and induces apoptotic death of cancer cells. Chem Commun (Camb) 2016; 51:12024-7. [PMID: 26121245 DOI: 10.1039/c5cc03432a] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
A short GC rich DNA derived from microbial origin interacts with tubulin/microtubules activates p53 over expression and induces apoptotic death of human breast cancer (MCF-7) cells.
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Affiliation(s)
- Batakrishna Jana
- Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India.
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24
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Marchetti F, Massarotti A, Yauk CL, Pacchierotti F, Russo A. The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2016; 57:87-113. [PMID: 26581746 DOI: 10.1002/em.21986] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/27/2015] [Revised: 10/13/2015] [Accepted: 10/13/2015] [Indexed: 06/05/2023]
Abstract
The Organisation for Economic Co-operation and Development (OECD) has launched the Adverse Outcome Pathway (AOP) Programme to advance knowledge of pathways of toxicity and improve the use of mechanistic information in risk assessment. An AOP links a molecular initiating event (MIE) to an adverse outcome (AO) through intermediate key events (KE). Here, we present the scientific evidence in support of an AOP whereby chemicals that bind to tubulin cause microtubule depolymerization resulting in spindle disorganization followed by altered chromosome alignment and segregation and the generation of aneuploidy in female germ cells, ultimately leading to aneuploidy in the offspring. Aneuploidy, an abnormal number of chromosomes that is not an exact multiple of the haploid number, is a well-known cause of human disease and represents a major cause of infertility, pregnancy failure, and serious genetic disorders in the offspring. Among chemicals that induce aneuploidy in female germ cells, a large majority impairs microtubule dynamics and spindle function. Colchicine, a prototypical chemical that binds to tubulin and causes microtubule depolymerization, is used here to illustrate the AOP. This AOP is specific to female germ cells exposed during the periovulation period. Although the majority of the data come from rodent studies, the available evidence suggests that the MIE and KEs are conserved across species and would occur in human oocytes. The development of AOPs related to mutagenicity in germ cells is expected to aid the identification of potential hazards to germ cell genomic integrity and support regulatory efforts to protect population health.
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Affiliation(s)
- Francesco Marchetti
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Canada
| | - Alberto Massarotti
- Dipartimento Di Scienze Del Farmaco, Università Degli Studi Del Piemonte Orientale "A. Avogadro", Novara, Italy
| | - Carole L Yauk
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Canada
| | - Francesca Pacchierotti
- Division of Health Protection Technologies, Laboratory of Biosafety and Risk Assessment, ENEA CR Casaccia, Rome, Italy
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25
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Zulkipli IN, David SR, Rajabalaya R, Idris A. Medicinal Plants: A Potential Source of Compounds for Targeting Cell Division. Drug Target Insights 2015; 9:9-19. [PMID: 26106261 PMCID: PMC4468949 DOI: 10.4137/dti.s24946] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2015] [Revised: 04/07/2015] [Accepted: 04/20/2015] [Indexed: 01/28/2023] Open
Abstract
Modern medicinal plant drug discovery has provided pharmacologically active compounds targeted against a multitude of conditions and diseases, such as infection, inflammation, and cancer. To date, natural products from medicinal plants remain a solid niche as a source from which cancer therapies can be derived. Among other properties, one favorable characteristic of an anticancer drug is its ability to block the uncontrollable process of cell division, as cancer cells are notorious for their abnormal cell division. There are numerous other documented works on the potential anticancer activity of drugs derived from medicinal plants, and their effects on cell division are an attractive and growing therapeutic target. Despite this, there remains a vast number of unidentified natural products that are potentially promising sources for medical applications. This mini review aims to revise the current knowledge of the effects of natural plant products on cell division.
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Affiliation(s)
- Ihsan N Zulkipli
- PAP Rashidah Sa'adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Gadong, Brunei Darussalam
| | - Sheba R David
- PAP Rashidah Sa'adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Gadong, Brunei Darussalam
| | - Rajan Rajabalaya
- PAP Rashidah Sa'adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Gadong, Brunei Darussalam
| | - Adi Idris
- PAP Rashidah Sa'adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Gadong, Brunei Darussalam
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Azarenko O, Jordan MA, Wilson L. Erucin, the major isothiocyanate in arugula (Eruca sativa), inhibits proliferation of MCF7 tumor cells by suppressing microtubule dynamics. PLoS One 2014; 9:e100599. [PMID: 24950293 PMCID: PMC4065051 DOI: 10.1371/journal.pone.0100599] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2014] [Accepted: 05/28/2014] [Indexed: 01/25/2023] Open
Abstract
Consumption of cruciferous vegetables is associated with reduced risk of various types of cancer. Isothiocyanates including sulforaphane and erucin are believed to be responsible for this activity. Erucin [1-isothiocyanato-4-(methylthio)butane], which is metabolically and structurally related to sulforaphane, is present in large quantities in arugula (Eruca sativa, Mill.), kohlrabi and Chinese cabbage. However, its cancer preventive mechanisms remain poorly understood. We found that erucin inhibits proliferation of MCF7 breast cancer cells (IC50 = 28 µM) in parallel with cell cycle arrest at mitosis (IC50 = 13 µM) and apoptosis, by a mechanism consistent with impairment of microtubule dynamics. Concentrations of 5-15 µM erucin suppressed the dynamic instability of microtubules during interphase in the cells. Most dynamic instability parameters were inhibited, including the rates and extents of growing and shortening, the switching frequencies between growing and shortening, and the overall dynamicity. Much higher erucin concentrations were required to reduce the microtubule polymer mass. In addition, erucin suppressed dynamic instability of microtubules reassembled from purified tubulin in similar fashion. The effects of erucin on microtubule dynamics, like those of sulforaphane, are similar qualitatively to those of much more powerful clinically-used microtubule-targeting anticancer drugs, including taxanes and the vinca alkaloids. The results suggest that suppression of microtubule dynamics by erucin and the resulting impairment of critically important microtubule-dependent cell functions such as mitosis, cell migration and microtubule-based transport may be important in its cancer preventive activities.
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Affiliation(s)
- Olga Azarenko
- Department of Molecular, Cellular, and Developmental Biology, and the Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, California, United States of America
| | - Mary Ann Jordan
- Department of Molecular, Cellular, and Developmental Biology, and the Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, California, United States of America
| | - Leslie Wilson
- Department of Molecular, Cellular, and Developmental Biology, and the Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, California, United States of America
- * E-mail:
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Zaytseva N, Lynn JG, Wu Q, Mudaliar DJ, Sun H, Kuang PQ, Fang Y. Resonant waveguide grating biosensor-enabled label-free and fluorescence detection of cell adhesion. SENSORS AND ACTUATORS. B, CHEMICAL 2013; 188:10.1016/j.snb.2013.08.012. [PMID: 24319319 PMCID: PMC3852437 DOI: 10.1016/j.snb.2013.08.012] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/03/2023]
Abstract
Cell adhesion to extracellular matrix (ECM) is fundamental to many distinct aspects of cell biology, and has been an active topic for label-free biosensors. However, little attention has been paid to study the impact of receptor signaling on the cell adhesion process. We here report the development of resonant waveguide grating biosensor-enabled label-free and fluorescent approaches, and their use for investigating the adhesion of an engineered HEK-293 cell line stably expressing green fluorescent protein (GFP) tagged β2-adrenergic receptor (β2-AR) onto distinct surfaces under both ambient and physiological conditions. Results showed that cell adhesion is sensitive to both temperature and ECM coating, and distinct mechanisms govern the cell adhesion process under different conditions. The β2-AR agonists, but not its antagonists or partial agonists, were found to be capable of triggering signaling during the adhesion process, leading to an increase in the adhesion of the engineered cells onto fibronectin-coated biosensor surfaces. These results suggest that the dual approach presented is useful to investigate the mechanism of cell adhesion, and to identify drug molecules and receptor signaling that interfere with cell adhesion.
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Affiliation(s)
- Natalya Zaytseva
- Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, New York 14831, USA
| | - Jeffery G. Lynn
- Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, New York 14831, USA
| | - Qi Wu
- Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, New York 14831, USA
| | | | | | - Patty Q. Kuang
- Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, New York 14831, USA
| | - Ye Fang
- Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, New York 14831, USA
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Salmela AL, Kallio MJ. Mitosis as an anti-cancer drug target. Chromosoma 2013; 122:431-49. [PMID: 23775312 DOI: 10.1007/s00412-013-0419-8] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2013] [Revised: 05/23/2013] [Accepted: 05/27/2013] [Indexed: 12/15/2022]
Abstract
Suppression of cell proliferation by targeting mitosis is one potential cancer intervention. A number of existing chemotherapy drugs disrupt mitosis by targeting microtubule dynamics. While efficacious, these drugs have limitations, i.e. neuropathy, unpredictability and development of resistance. In order to overcome these issues, a great deal of effort has been spent exploring novel mitotic targets including Polo-like kinase 1, Aurora kinases, Mps1, Cenp-E and KSP/Eg5. Here we summarize the latest developments in the discovery and clinical evaluation of new mitotic drug targets.
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Affiliation(s)
- Anna-Leena Salmela
- VTT Biotechnology for Health and Wellbeing, VTT Technical Research Centre of Finland, Itäinen Pitkäkatu 4C, Pharmacity Bldg, 4th Floor, P.O. Box 106, 20521, Turku, Finland
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29
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Reddy MVR, Mallireddigari MR, Pallela VR, Cosenza SC, Billa VK, Akula B, Subbaiah DRCV, Bharathi EV, Padgaonkar A, Lv H, Gallo JM, Reddy EP. Design, synthesis, and biological evaluation of (E)-N-aryl-2-arylethenesulfonamide analogues as potent and orally bioavailable microtubule-targeted anticancer agents. J Med Chem 2013; 56:5562-86. [PMID: 23750455 DOI: 10.1021/jm400575x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
A series of novel (E)-N-aryl-2-arylethenesulfonamides (6) were synthesized and evaluated for their anticancer activity. Some of the compounds in this series showed potent cytotoxicity against a wide spectrum of cancer cell-lines (IC50 values ranging from 5 to 10 nM) including all drug resistant cell-lines. Nude mice xenograft assays with compound (E)-N-(3-amino-4-methoxyphenyl)-2-(2',4',6'-trimethoxyphenyl)ethenesulfonamide (6t) showed dramatic reduction in tumor size, indicating their in vivo potential as anticancer agents. A preliminary drug development study with compound 6t is predicted to have increased blood-brain barrier permeability relative to many clinically used antimitotic agents. Mechanistic studies indicate that 6t and some other analogues disrupted microtubule formation, formation of mitotic spindles, and arrest of cells in mitotic phase. Compound 6t inhibited purified tubulin polymerization in vitro and in vivo and circumvented drug resistance mediated by P-glycoprotein. Compound 6t specifically competed with colchicine binding to tubulin and with similar avidity as podophylltoxin, indicating its binding site on tubulin.
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Affiliation(s)
- M V Ramana Reddy
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai , 1425 Madison Avenue, New York, New York 10029-6514, United States
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30
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End-binding proteins sensitize microtubules to the action of microtubule-targeting agents. Proc Natl Acad Sci U S A 2013; 110:8900-5. [PMID: 23674690 DOI: 10.1073/pnas.1300395110] [Citation(s) in RCA: 80] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Microtubule-targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and a detailed understanding of the mechanism of their action is important for the development of improved microtubule-directed therapies. Although there is a large body of data on the interactions of different MTAs with purified tubulin and microtubules, much less is known about how the effects of MTAs are modulated by microtubule-associated proteins. Among the regulatory factors with a potential to have a strong impact on MTA activity are the microtubule plus end-tracking proteins, which control multiple aspects of microtubule dynamic instability. Here, we reconstituted microtubule dynamics in vitro to investigate the influence of end-binding proteins (EBs), the core components of the microtubule plus end-tracking protein machinery, on the effects that MTAs exert on microtubule plus-end growth. We found that EBs promote microtubule catastrophe induction in the presence of all MTAs tested. Analysis of microtubule growth times supported the view that catastrophes are microtubule age dependent. This analysis indicated that MTAs affect microtubule aging in multiple ways: destabilizing MTAs, such as colchicine and vinblastine, accelerate aging in an EB-dependent manner, whereas stabilizing MTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process.
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31
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Cotruvo JA, Stubbe J. Class I ribonucleotide reductases: metallocofactor assembly and repair in vitro and in vivo. Annu Rev Biochem 2011; 80:733-67. [PMID: 21456967 DOI: 10.1146/annurev-biochem-061408-095817] [Citation(s) in RCA: 165] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Incorporation of metallocofactors essential for the activity of many enyzmes is a major mechanism of posttranslational modification. The cellular machinery required for these processes in the case of mono- and dinuclear nonheme iron and manganese cofactors has remained largely elusive. In addition, many metallocofactors can be converted to inactive forms, and pathways for their repair have recently come to light. The class I ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides and require dinuclear metal clusters for activity: an Fe(III)Fe(III)-tyrosyl radical (Y•) cofactor (class Ia), a Mn(III)Mn(III)-Y• cofactor (class Ib), and a Mn(IV)Fe(III) cofactor (class Ic). The class Ia, Ib, and Ic RNRs are structurally homologous and contain almost identical metal coordination sites. Recent progress in our understanding of the mechanisms by which the cofactor of each of these RNRs is generated in vitro and in vivo and by which the damaged cofactors are repaired is providing insight into how nature prevents mismetallation and orchestrates active cluster formation in high yields.
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Affiliation(s)
- Joseph A Cotruvo
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
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KAPOOR SONIA, RANJITH P, PANDA DULAL. ENGINEERING AND THERAPEUTIC APPLICATIONS OF MICROTUBULES. INTERNATIONAL JOURNAL OF NANOSCIENCE 2011. [DOI: 10.1142/s0219581x11009325] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Living organisms are fascinating systems. The macromolecules that make up a living cell possess equally astounding structural and functional characteristics. By taking simple cues from how these biopolymers organize and work inside the cell, one can draw inspiration to utilize them outside their natural environment for several purposes. Microtubules are example of biopolymers that demonstrate extraordinary properties of hierarchical self-organization, dynamic remodeling and mechanical rigidity. Mimicking the principles and properties of microtubules and improving them have opened novel engineering avenues. In addition, due to the functions that microtubules perform during cell division, they are excellent therapeutic drug targets for anticancer agents. In this work, we describe the biological properties and functions of microtubules, and discuss their engineering and therapeutic applications.
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Affiliation(s)
- SONIA KAPOOR
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, 400076, India
| | - P. RANJITH
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, 400076, India
| | - DULAL PANDA
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, 400076, India
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Kelly EB, Tuszynski JA, Klobukowski M. QM and QM/MD simulations of the Vinca alkaloids docked to tubulin. J Mol Graph Model 2011; 30:54-66. [PMID: 21798777 DOI: 10.1016/j.jmgm.2011.06.005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2011] [Revised: 06/03/2011] [Accepted: 06/08/2011] [Indexed: 10/18/2022]
Abstract
The Vinca alkaloids are a class of pharmaceutically relevant binary indole-indoline alkaloids based on and including natural extracts of the periwinkle plant, Catharanthus rosea. Two natural products, vinblastine and vincristine, have been in clinical use as important chemotherapy agents for over four decades. Two semi-synthetic Vinca alkaloids, vindesine and vinorelbine, are currently in investigational chemotherapy programs, and a third semi-synthetic, vinflunine, is in advanced clinical trials. In addition to these five compounds studied in the present work, there are hundreds of other natural and semi-synthetic Vinca alkaloids known, although most are not clinically advantageous. The Vinca alkaloids are anti-mitotic agents that affect the cellular protein tubulin and bind to a specific site known as the Vinca domain located on β-tubulin. While the Vinca domain is well established, the specific binding mode of each drug is not. However, there is much insight into the binding mode and this has provided a strong base of information to begin simulations and to make comparisons against. Complicating the issue, however, is the large size of the Vinca alkaloids and their complex molecular structure, including a rotatable single bond joining the indole and indoline portions of each compound. The differential geometric and tubulin-binding properties of the drugs are not fully known. In the present work, the projection of the potential energy surface on the major torsional angle was calculated at the semi-empirical AM1 level, through in vacuo geometry optimizations. QM/MD simulations were performed, with the drugs at the AM1 level, of each Vinca alkaloid free in TIP3P water, and also bound to β-tubulin. A single equilibrium structure, resembling a known crystallographic vinblastine structure, for the free drugs was found. Further, the 1Z2B crystal structure of vinblastine bound to tubulin appears to be a valid starting point for simulations of all five Vinca alkaloids studied here.
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Affiliation(s)
- Evan B Kelly
- Department of Chemistry, University of Alberta, Edmonton, Alberta T6G2G2, Canada
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34
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Lopus M, Oroudjev E, Wilson L, Wilhelm S, Widdison W, Chari R, Jordan MA. Maytansine and cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule dynamics by binding to microtubules. Mol Cancer Ther 2011; 9:2689-99. [PMID: 20937594 DOI: 10.1158/1535-7163.mct-10-0644] [Citation(s) in RCA: 161] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. Recently, antibody-conjugated maytansine derivatives have been developed to overcome these drawbacks. Several conjugates show promising early clinical results. We evaluated the effects on microtubule polymerization and dynamic instability of maytansine and two cellular metabolites (S-methyl-DM1 and S-methyl-DM4) of antibody-maytansinoid conjugates that are potent in cells at picomolar levels and that are active in tumor-bearing mice. Although S-methyl-DM1 and S-methyl-DM4 inhibited polymerization more weakly than maytansine, at 100 nmol/L they suppressed dynamic instability more strongly than maytansine (by 84% and 73%, respectively, compared with 45% for maytansine). However, unlike maytansine, S-methyl-DM1 and S-methyl-DM4 induced tubulin aggregates detectable by electron microscopy at concentrations ≥2 μmol/L, with S-methyl-DM4 showing more extensive aggregate formation than S-methyl-DM1. Both maytansine and S-methyl-DM1 bound to tubulin with similar K(D) values (0.86 ± 0.2 and 0.93 ± 0.2 μmol/L, respectively). Tritiated S-methyl-DM1 bound to 37 high-affinity sites per microtubule (K(D), 0.1 ± 0.05 μmol/L). Thus, S-methyl-DM1 binds to high-affinity sites on microtubules 20-fold more strongly than vinblastine. The high-affinity binding is likely at microtubule ends and is responsible for suppression of microtubule dynamic instability. Also, at higher concentrations, S-methyl-DM1 showed low-affinity binding either to a larger number of sites on microtubules or to sedimentable tubulin aggregates. Overall, the maytansine derivatives that result from cellular metabolism of the antibody conjugates are themselves potent microtubule poisons, interacting with microtubules as effectively as or more effectively than the parent molecule.
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Affiliation(s)
- Manu Lopus
- Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, California 93106-9610, USA
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Chakrabarty S, Das A, Bhattacharya A, Chakrabarti G. Theaflavins depolymerize microtubule network through tubulin binding and cause apoptosis of cervical carcinoma HeLa cells. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2011; 59:2040-2048. [PMID: 21323312 DOI: 10.1021/jf104231b] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/30/2023]
Abstract
Here we studied the antiproliferative activity of theaflavins in cervical carcinoma HeLa cells by investigating their effects on cellular microtubules and purified goat brain tubulin. Theaflavins inhibited proliferation of HeLa cells with IC(50) value of 110 ± 2.1 μg/mL (p = < 0.01), caused cell cycle arrest at G(2)/M phase and induced apoptosis with alteration of expression of pro- and antiapoptotic proteins. Along with these antiproliferative activities, theaflavins act as microtubule depolymerizers. Theaflavins disrupted the microtubule network accompanied by alteration of cellular morphology and also decreased the polymeric tubulin mass of the cells. The polymerization of cold treated depolymerized microtubules in HeLa cells was prevented in the presence of theaflavins. In vitro polymerization of purified tubulin into microtubules was also inhibited by theaflavins with an IC(50) value of 78 ± 2.43 μg/mL (P < 0.01). Thus, disruption of cellular microtubule network of HeLa cells through microtubule depolymerization may be one of the possible mechanisms of antiproliferative activity of theaflavins.
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Affiliation(s)
- Subhendu Chakrabarty
- Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700019, India
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36
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Stanton RA, Gernert KM, Nettles JH, Aneja R. Drugs that target dynamic microtubules: a new molecular perspective. Med Res Rev 2011; 31:443-81. [PMID: 21381049 DOI: 10.1002/med.20242] [Citation(s) in RCA: 394] [Impact Index Per Article: 28.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Microtubules have long been considered an ideal target for anticancer drugs because of the essential role they play in mitosis, forming the dynamic spindle apparatus. As such, there is a wide variety of compounds currently in clinical use and in development that act as antimitotic agents by altering microtubule dynamics. Although these diverse molecules are known to affect microtubule dynamics upon binding to one of the three established drug domains (taxane, vinca alkaloid, or colchicine site), the exact mechanism by which each drug works is still an area of intense speculation and research. In this study, we review the effects of microtubule-binding chemotherapeutic agents from a new perspective, considering how their mode of binding induces conformational changes and alters biological function relative to the molecular vectors of microtubule assembly or disassembly. These "biological vectors" can thus be used as a spatiotemporal context to describe molecular mechanisms by which microtubule-targeting drugs work.
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37
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Kiris E, Ventimiglia D, Sargin ME, Gaylord MR, Altinok A, Rose K, Manjunath BS, Jordan MA, Wilson L, Feinstein SC. Combinatorial Tau pseudophosphorylation: markedly different regulatory effects on microtubule assembly and dynamic instability than the sum of the individual parts. J Biol Chem 2011; 286:14257-70. [PMID: 21288907 DOI: 10.1074/jbc.m111.219311] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias. A huge literature has led to the widely held notion that aberrant Tau hyperphosphorylation is central to these disorders. Unfortunately, our mechanistic understanding of the functional effects of combinatorial Tau phosphorylation remains minimal. Here, we generated four singly pseudophosphorylated Tau proteins (at Thr(231), Ser(262), Ser(396), and Ser(404)) and four doubly pseudophosphorylated Tau proteins using the same sites. Each Tau preparation was assayed for its abilities to promote microtubule assembly and to regulate microtubule dynamic instability in vitro. All four singly pseudophosphorylated Tau proteins exhibited loss-of-function effects. In marked contrast to the expectation that doubly pseudophosphorylated Tau would be less functional than either of its corresponding singly pseudophosphorylated forms, all of the doubly pseudophosphorylated Tau proteins possessed enhanced microtubule assembly activity and were more potent at regulating dynamic instability than their compromised singly pseudophosphorylated counterparts. Thus, the effects of multiple pseudophosphorylations were not simply the sum of the effects of the constituent single pseudophosphorylations; rather, they were generally opposite to the effects of singly pseudophosphorylated Tau. Further, despite being pseudophosphorylated at different sites, the four singly pseduophosphorylated Tau proteins often functioned similarly, as did the four doubly pseudophosphorylated proteins. These data lead us to reassess the conventional view of combinatorial phosphorylation in normal and pathological Tau action. They may also be relevant to the issue of combinatorial phosphorylation as a general regulatory mechanism.
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Affiliation(s)
- Erkan Kiris
- Neuroscience Research Institute and Department of Molecular and Cellular and Developmental Biology, University of California, Santa Barbara, California 93106, USA
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38
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Synthetic conjugates of genistein affecting proliferation and mitosis of cancer cells. Bioorg Med Chem 2011; 19:295-305. [DOI: 10.1016/j.bmc.2010.11.024] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2010] [Revised: 11/04/2010] [Accepted: 11/08/2010] [Indexed: 12/30/2022]
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Nguyen V, Tang J, Oroudjev E, Lee CJ, Marasigan C, Wilson L, Ayoub G. Cytotoxic effects of bilberry extract on MCF7-GFP-tubulin breast cancer cells. J Med Food 2010; 13:278-85. [PMID: 20132040 DOI: 10.1089/jmf.2009.0053] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Bilberry (European blueberry) has been reported to have many biological effects, including anticancer activity. In this study, we investigated the antiproliferative effects of bilberry extract in relation to its ability to induce apoptosis and affect microtubule assembly and organization in MCF7 human breast cancer cells. We observed that bilberry extract inhibited cell proliferation in a concentration-dependent fashion with a 50% inhibitory concentration of 0.3-0.4 mg/mL, in concert with induction of apoptotic cell death. At these concentrations there was no selective inhibition of mitosis or any other cell cycle stage, nor was there any apparent effect on the microtubule or actin cytoskeletons. However, somewhat higher extract concentrations (0.5-0.9 mg/mL) did cause an increase in the fraction of cells at the G(2)/M phase of the cell cycle, together with destruction of microtubules and formation of punctate tubulin aggregates in the cells. Bilberry extract at 0.3-0.4 mg/mL did not appreciably inhibit microtubule polymerization in vitro, but significant inhibition of polymerization (approximately 30%) did occur at higher extract concentrations (0.5-1 mg/mL). We conclude that bilberry extract as ingested by humans, not just the purified anthocyanins it contains, inhibits proliferation of and induces apoptosis in breast cancer cells at its lowest effective concentrations via a mechanism that does not involve action on microtubules or on mitosis. We further conclude that at somewhat higher concentrations the extract modifies microtubule organization in cells and causes accumulation of cells at mitosis by a direct action on microtubules.
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Affiliation(s)
- Vy Nguyen
- Department of Molecular, Cellular & Developmental Biology, University of California Santa Barbara, Santa Barbara, California 93106, USA
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40
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Quantitative Analysis of MAP-Mediated Regulation of Microtubule Dynamic Instability In Vitro. Methods Cell Biol 2010; 95:481-503. [DOI: 10.1016/s0091-679x(10)95024-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
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Yenjerla M, Lopus M, Wilson L. Analysis of dynamic instability of steady-state microtubules in vitro by video-enhanced differential interference contrast microscopy with an appendix by Emin Oroudjev. Methods Cell Biol 2010; 95:189-206. [PMID: 20466136 DOI: 10.1016/s0091-679x(10)95011-5] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Microtubules are major constituents of the cytoskeleton which display dynamic properties. They exhibit dynamic instability which is defined as the stochastic switching between growing and shortening at microtubule ends. Dynamic instability plays an important role in diverse cellular functions including cell migration and mitosis. Many successful antimitotic drugs and microtubule-associated proteins (MAPs) are known to modulate microtubule dynamics, and it is important to analyze the in vitro dynamic instability of microtubules to study the mechanism of action of microtubule-targeted therapeutics and MAPs. In this chapter, we describe a method to analyze the in vitro dynamic instability of microtubules at steady state using video-enhanced differential contrast (VE-DIC) microscopy in detail. In this method, microtubules are assembled to steady state at 30 degrees C with MAP-free tubulin in a slide chamber in the presence of GTP, using sea urchin axonemes as nucleating seeds. Images of microtubules are enhanced and recorded in real time by a video camera and an image processor connected to a DIC microscope which is maintained at 30 degrees C. We use two software programs to track and analyze the growing and shortening of plus or minus ends of microtubules in the real-time images recorded using VE-DIC. In this chapter, we describe the instructions to use the tracking software Real Time Measurement II (RTM II) program. The instructions to use the analysis software Microtubule Life History Analysis Procedures (MT-LHAP) in Igor Pro software have been described in detail in an appendix (Oroudjev, 2010) following this chapter.
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Affiliation(s)
- Mythili Yenjerla
- Department of Molecular, Cellular, and Developmental Biology, The Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA
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42
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Zhu ZC, Gupta KK, Slabbekoorn AR, Paulson BA, Folker ES, Goodson HV. Interactions between EB1 and microtubules: dramatic effect of affinity tags and evidence for cooperative behavior. J Biol Chem 2009; 284:32651-61. [PMID: 19778897 DOI: 10.1074/jbc.m109.013466] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.
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Affiliation(s)
- Zhiqing C Zhu
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
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Lee CJ, Wilson L, Jordan MA, Nguyen V, Tang J, Smiyun G. Hesperidin suppressed proliferations of both Human breast cancer and androgen-dependent prostate cancer cells. Phytother Res 2009; 24 Suppl 1:S15-9. [DOI: 10.1002/ptr.2856] [Citation(s) in RCA: 65] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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Azarenko O, Okouneva T, Singletary KW, Jordan MA, Wilson L. Suppression of microtubule dynamic instability and turnover in MCF7 breast cancer cells by sulforaphane. Carcinogenesis 2008; 29:2360-8. [PMID: 18952594 DOI: 10.1093/carcin/bgn241] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Sulforaphane (SFN), a prominent isothiocyanate present in cruciferous vegetables, is believed to be responsible along with other isothiocyanates for the cancer preventive activity of such vegetables. SFN arrests mitosis, possibly by affecting spindle microtubule function. A critical property of microtubules is their rapid and time-sensitive growth and shortening dynamics (dynamic instability), and suppression of dynamics by antimitotic anticancer drugs (e.g. taxanes and the vinca alkaloids) is central to the anticancer mechanisms of such drugs. We found that at concentrations that inhibited proliferation and mitosis of MCF7-green fluorescent protein-alpha-tubulin breast tumor cells by approximately 50% (~15 microM), SFN significantly modified microtubule organization in arrested spindles without modulating the spindle microtubule mass, in a manner similar to that of much more powerful antimitotic drugs. By using quantitative fluorescence video microscopy, we determined that at its mitotic concentration required to inhibit mitosis by 50%, SFN suppressed the dynamic instability of the interphase microtubules in these cells, strongly reducing the rate and extent of growth and shortening and decreasing microtubule turnover, without affecting the polymer mass. SFN suppressed the dynamics of purified microtubules in a similar fashion at concentrations well below those required to depolymerize microtubules, indicating that the suppression of dynamic instability by SFN in cells is due to a direct effect on the microtubules. The results indicate that SFN arrests proliferation and mitosis by stabilizing microtubules in a manner weaker than but similar to more powerful clinically used antimitotic anticancer drugs and strongly support the hypothesis that inhibition of mitosis by microtubule stabilization is important for SFN's chemopreventive activity.
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Affiliation(s)
- Olga Azarenko
- Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA
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Galmarini CM, Treilleux I, Cardoso F, Bernard-Marty C, Durbecq V, Gancberg D, Bissery MC, Paesmans M, Larsimont D, Piccart MJ, Di Leo A, Dumontet C. Class III beta-tubulin isotype predicts response in advanced breast cancer patients randomly treated either with single-agent doxorubicin or docetaxel. Clin Cancer Res 2008; 14:4511-6. [PMID: 18628466 DOI: 10.1158/1078-0432.ccr-07-4741] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
PURPOSE To evaluate the role of microtubule-associated variables as potential predictors of response and clinical outcome in patients with advanced breast cancer receiving single-agent docetaxel or doxorubicin chemotherapy. EXPERIMENTAL DESIGN The analysis was done on 173 tumor samples from patients with locally advanced or metastatic breast cancer who have participated in the TAX-303 phase III trial in which patients were randomly assigned to receive docetaxel or doxorubicin. Expression of total alpha- and beta-tubulin, classes II to IV beta-tubulin isotypes, and tau protein was evaluated by immunohistochemistry on formalin-fixed, paraffin-embedded tumors from the primary breast cancer. RESULTS We observed that patients with "high" expression of class III beta-tubulin isotype had a higher probability of response to docetaxel than to doxorubicin treatment (odds ratio, 1.9; 95% confidence interval, 1.01-3.7; P = 0.05). No difference was observed in terms of time to progression or in terms of overall survival. CONCLUSIONS This study suggests that the superiority of docetaxel over doxorubicin seems to be confined to the subgroup of patients with "high" expression of class III beta-tubulin isotype.
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Drake DM, Pack DW. Biochemical investigation of active intracellular transport of polymeric gene-delivery vectors. J Pharm Sci 2008; 97:1399-413. [PMID: 17712850 DOI: 10.1002/jps.21106] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
To design safe, efficient synthetic gene therapy vectors, it is desirable to understand the intracellular mechanisms that facilitate their delivery from the cell surface to the nucleus. Elements of the cytoskeleton and molecular motor proteins are known to play a pivotal role in most intracellular active transport processes. The actin depolymerizer cytochalasin D and microtubule effectors colchicine and paclitaxel were used to evaluate the function of these components of the cytoskeleton in the trafficking of polyethylenimine (PEI)-DNA complexes. In addition, ATPase inhibitors erythro-9[3-(2-hydroxynonyl)] adenine (EHNA), vanadate, adenylylimidodiphosphate (AMP-PNP), and rose bengal lactone (RBL), which have inhibitory activity against dynein and kinesin, were used to examine to the effects of these molecular motors on PEI-DNA delivery. Disruption of microfilaments decreased the delivery efficiency of PEI polyplexes 60-80%, though cytochalasin D did not significantly inhibit uptake. Depolymerization of microtubules by colchicine decreased transfection efficiency by 75%. Microtubule stabilization with paclitaxel, however, facilitated a 20-fold increase in gene expression. Treatment with EHNA and vanadate caused 50% and 80% decreases in transfection efficiency, respectively. Transfection efficiency was also decreased by RBL (80%) and AMP-PNP (98%). Our findings confirm the importance of microfilament- and microtubule-based active transport of PEI-DNA complexes. Further, the strong decrease in transfection efficiency caused by ATPase inhibitors that possess inhibitory activity against kinesin implies an unexpected role for these motors in gene delivery.
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Affiliation(s)
- David M Drake
- Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana, Illinois 61801, USA
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Manna T, Honnappa S, Steinmetz MO, Wilson L. Suppression of microtubule dynamic instability by the +TIP protein EB1 and its modulation by the CAP-Gly domain of p150glued. Biochemistry 2007; 47:779-86. [PMID: 18081319 DOI: 10.1021/bi701912g] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The EB1+TIP protein family and its binding partners track growing plus ends of microtubules in cells and are thought to regulate their dynamics. Here we determined the effects of EB1 and the N-terminal CAP-Gly domain (p150n) of one of its major binding partners, p150Glued, both separately and together, on the dynamic instability parameters at plus ends of purified steady-state microtubules. With EB1 alone, the shortening rate, the extent of shortening, and the catastrophe frequency were suppressed in the absence of significant effects on the growth rate or rescue frequency. The effects of EB1 on dynamics were significantly different when p150n was added together with EB1. The rate and extent of shortening and the catastrophe frequency were suppressed 3-4 times more strongly than with EB1 alone. In addition, the EB1-p150n complex increased the rescue frequency and the mean length the microtubules grew, parameters that were not significantly affected by EB1 alone. Similarly, deletion of EB1's C-terminal tail, which is a crucial binding region for p150n, significantly increased the ability of EB1 to suppress shortening dynamics. EB1 by itself bound along the length of the microtubules with 1 mol of EB1 dimer bound per approximately 12 mol of tubulin dimer. Approximately twice the amount of EB1 was recruited to the microtubules in the presence of p150n. Our results indicate that inactivation of EB1's flexible C-terminal tail significantly changes EB1's ability to modulate microtubule dynamics. They further suggest that p150Glued may activate and thereby facilitate the recruitment of EB1 to the tips of microtubules to regulate their dynamics.
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Affiliation(s)
- Tapas Manna
- Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, California 93106, USA
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Ray A, Okouneva T, Manna T, Miller HP, Schmid S, Arthaud L, Luduena R, Jordan MA, Wilson L. Mechanism of action of the microtubule-targeted antimitotic depsipeptide tasidotin (formerly ILX651) and its major metabolite tasidotin C-carboxylate. Cancer Res 2007; 67:3767-76. [PMID: 17440090 DOI: 10.1158/0008-5472.can-06-3065] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Tasidotin (ILX-651), an orally active synthetic microtubule-targeted derivative of the marine depsipeptide dolastatin-15, is currently undergoing clinical evaluation for cancer treatment. Tasidotin inhibited proliferation of MCF7/GFP breast cancer cells with an IC(50) of 63 nmol/L and inhibited mitosis with an IC(50) of 72 nmol/L in the absence of detectable effects on spindle microtubule polymer mass. Tasidotin inhibited the polymerization of purified tubulin into microtubules weakly (IC(50) approximately 30 micromol/L). However, it strongly suppressed the dynamic instability behavior of the microtubules at their plus ends at concentrations approximately 5 to 10 times below those required to inhibit polymerization. Its major actions were to reduce the shortening rate, the switching frequency from growth to shortening (catastrophe frequency), and the fraction of time the microtubules grew. In contrast with all other microtubule-targeted drugs thus far examined that can inhibit polymerization, tasidotin did not inhibit the growth rate. In contrast to stabilizing plus ends, tasidotin enhanced microtubule dynamic instability at minus ends, increasing the shortening length, the fraction of time the microtubules shortened, and the catastrophe frequency and reducing the rescue frequency. Tasidotin C-carboxylate, the major intracellular metabolite of tasidotin, altered dynamic instability of purified microtubules in a qualitatively similar manner to tasidotin but was 10 to 30 times more potent. The results suggest that the principal mechanism by which tasidotin inhibits cell proliferation is by suppressing spindle microtubule dynamics. Tasidotin may be a relatively weak prodrug for the functionally active tasidotin C-carboxylate.
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Affiliation(s)
- Anasuya Ray
- Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute, University of California-Santa Barbara, Santa Barbara, CA 93106, USA
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Manna T, Grenningloh G, Miller HP, Wilson L. Stathmin family protein SCG10 differentially regulates the plus and minus end dynamics of microtubules at steady state in vitro: implications for its role in neurite outgrowth. Biochemistry 2007; 46:3543-52. [PMID: 17311410 DOI: 10.1021/bi061819d] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
SCG10 (superior cervical ganglia neural-specific 10 protein) is a neuron specific member of the stathmin family of microtubule regulatory proteins that like stathmin can bind to soluble tubulin and depolymerize microtubules. The direct actions of SCG10 on microtubules themselves and on their dynamics have not been investigated previously. Here, we analyzed the effects of SCG10 on the dynamic instability behavior of microtubules in vitro, both at steady state and early during microtubule polymerization. In contrast to stathmin, whose major action on dynamics is to destabilize microtubules by increasing the switching frequency from growth to shortening (the catastrophe frequency) at microtubule ends, SCG10 stabilized the plus ends both at steady state and early during polymerization by increasing the rate and extent of growth. For example, early during polymerization at high initial tubulin concentrations (20 microM), a low molar ratio of SCG10 to tubulin of 1:30 increased the growth rate by approximately 50%. In contrast to its effects at plus ends, SCG10 destabilized minus ends by increasing the shortening rate, the length shortened during shortening events, and the catastrophe frequency. Consistent with its ability to modulate microtubule dynamics at steady state, SCG10 bound to purified microtubules along their lengths. The dual activity of SCG10 at opposite microtubule ends may be important for its role in regulating growth cone microtubule dynamics. SCG10's ability to promote plus end growth may facilitate microtubule extension into filopodia, and its ability to destabilize minus ends could provide soluble tubulin for net plus end elongation.
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Affiliation(s)
- Tapas Manna
- Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA
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