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Illangeswaran RSS, Jebanesan DZP, Sivakumar KK, Vidhyadharan RT, Rajamani BM, Janet NB, David E, Velayudhan SR, Mathews V, Balasubramanian P. Chemotherapeutic drugs elicit stemness and metabolic alteration to mediate acquired drug-resistant phenotype in acute myeloid leukemia cell lines. Leuk Res 2023; 128:107054. [PMID: 36906941 DOI: 10.1016/j.leukres.2023.107054] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2022] [Revised: 02/25/2023] [Accepted: 03/01/2023] [Indexed: 03/06/2023]
Abstract
Chemotherapy resistance leading to disease relapse is a significant barrier in treating acute myeloid leukemia (AML). Metabolic adaptations have been shown to contribute to therapy resistance. However, little is known about whether specific therapies cause specific metabolic changes. We established cytarabine-resistant (AraC-R) and Arsenic trioxide-resistant (ATO-R) AML cell lines, displaying distinct cell surface expression and cytogenetic abnormalities. Transcriptomic analysis revealed a significant difference in the expression profiles of ATO-R and AraC-R cells. Geneset enrichment analysis showed AraC-R cells rely on OXPHOS, while ATO-R cells on glycolysis. ATO-R cells were also enriched for stemness gene signatures, whereas AraC-R cells were not. The mito stress and glycolytic stress tests confirmed these findings. The distinct metabolic adaptation of AraC-R cells increased sensitivity to the OXPHOS inhibitor venetoclax. Cytarabine resistance was circumvented in AraC-R cells by combining Ven and AraC. In vivo, ATO-R cells showed increased repopulating potential, leading to aggressive leukemia compared to the parental and AraC-R. Overall, our study shows that different therapies can cause different metabolic changes and that these metabolic dependencies can be used to target chemotherapy-resistant AML.
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Affiliation(s)
| | | | | | | | | | - Nancy Beryl Janet
- Department of Haematology, Christian Medical College, Vellore, India
| | - Ernest David
- Department of Biotechnology, Thiruvalluvar University, Vellore, India
| | - Shaji Ramachandran Velayudhan
- Department of Haematology, Christian Medical College, Vellore, India; Center for Stem Cell Research (A Unit of InStem, Bengaluru, India), Christian Medical College, Vellore, India
| | - Vikram Mathews
- Department of Haematology, Christian Medical College, Vellore, India
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The Potential of Lonidamine in Combination with Chemotherapy and Physical Therapy in Cancer Treatment. Cancers (Basel) 2020; 12:cancers12113332. [PMID: 33187214 PMCID: PMC7696079 DOI: 10.3390/cancers12113332] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Accepted: 11/09/2020] [Indexed: 02/07/2023] Open
Abstract
Simple Summary The unique characteristics of tumor energy metabolism (highly dependent on aerobic glycolysis, namely, the Warburg effect) make it an interesting and attractive target for drug discovery. Radio- and chemoresistance are closely associated with the Warburg effect. Lonidamine (LND), as a glycolytic inhibitor, although having low anticancer activity when used alone, exhibits selectivity to various tumors, and its adverse effects do not overlap when combined with other chemotherapeutic drugs. Therefore, LND may be very promising as a sensitizer of tumors to chemotherapeutic agents and physical therapies. This review summarizes the advance of LND in combination with chemotherapy and physical therapy over the past several decades, as well as the promising LND derivative adjudin (ADD). The underlying sensitizing mechanisms were also analyzed and discussed, which may contribute to an improved therapeutic effect in future clinical cancer treatment. Abstract Lonidamine (LND) has the ability to resist spermatogenesis and was first used as an anti-spermatogenic agent. Later, it was found that LND has a degree of anticancer activity. Currently, LND is known to target energy metabolism, mainly involving the inhibition of monocarboxylate transporter (MCT), mitochondrial pyruvate carrier (MPC), respiratory chain complex I/II, mitochondrial permeability transition (PT) pore, and hexokinase II (HK-II). However, phase II clinical studies showed that LND alone had a weak therapeutic effect, and the effect was short and reversible. Interestingly, LND does not have the common side effects of traditional chemotherapeutic drugs, such as alopecia and myelosuppression. In addition, LND has selective activity toward various tumors, and its toxic and side effects do not overlap when combined with other chemotherapeutic drugs. Therefore, LND is commonly used as a chemosensitizer to enhance the antitumor effects of chemotherapeutic drugs based on its disruption of energy metabolism relating to chemo- or radioresistance. In this review, we summarized the combination treatments of LND with several typical chemotherapeutic drugs and several common physical therapies, such as radiotherapy (RT), hyperthermia (HT), and photodynamic therapy (PDT), and discussed the underlying mechanisms of action. Meanwhile, the development of novel formulations of LND in recent years and the research progress of LND derivative adjudin (ADD) as an anticancer drug were also discussed.
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Alves A, Mamede A, Alves M, Oliveira P, Rocha S, Botelho M, Maia C. Glycolysis Inhibition as a Strategy for Hepatocellular Carcinoma Treatment? Curr Cancer Drug Targets 2018; 19:26-40. [DOI: 10.2174/1568009618666180430144441] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2017] [Revised: 03/05/2018] [Accepted: 03/10/2018] [Indexed: 12/19/2022]
Abstract
Hepatocellular carcinoma (HCC) is the most frequently detected primary malignant liver tumor, representing a worldwide public health problem due to its high morbidity and mortality rates. The HCC is commonly detected in advanced stage, precluding the use of treatments with curative intent. For this reason, it is crucial to find effective therapies for HCC. Cancer cells have a high dependence of glycolysis for ATP production, especially under hypoxic environment. Such dependence provides a reliable possible strategy to specifically target cancer cells based on the inhibition of glycolysis. HCC, such as other cancer types, presents a clinically well-known upregulation of several glycolytic key enzymes and proteins, including glucose transporters particularly glucose transporter 1 (GLUT1). Such enzymes and proteins constitute potential targets for therapy. Indeed, for some of these targets, several inhibitors were already reported, such as 2-Deoxyglucose, Imatinib or Flavonoids. Although the inhibition of glycolysis presents a great potential for an anticancer therapy, the development of glycolytic inhibitors as a new class of anticancer agents needs to be more explored. Herein, we propose to summarize, discuss and present an overview on the different approaches to inhibit the glycolytic metabolism in cancer cells, which may be very effective in the treatment of HCC.
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Affiliation(s)
- A.P. Alves
- Centro de Investigacao em Ciencias da Saude (CICS-UBI), Universidade da Beira Interior, Covilha, Portugal
| | - A.C. Mamede
- Centro de Investigacao em Ciencias da Saude (CICS-UBI), Universidade da Beira Interior, Covilha, Portugal
| | - M.G. Alves
- Centro de Investigacao em Ciencias da Saude (CICS-UBI), Universidade da Beira Interior, Covilha, Portugal
| | - P.F. Oliveira
- Department of Microscopy, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS) and Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto, Porto, Portugal
| | - S.M. Rocha
- Centro de Investigacao em Ciencias da Saude (CICS-UBI), Universidade da Beira Interior, Covilha, Portugal
| | - M.F. Botelho
- Biophysics Unit, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
| | - C.J. Maia
- Centro de Investigacao em Ciencias da Saude (CICS-UBI), Universidade da Beira Interior, Covilha, Portugal
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Liu Y, Zhang X, Zhou M, Nan X, Chen X, Zhang X. Mitochondrial-Targeting Lonidamine-Doxorubicin Nanoparticles for Synergistic Chemotherapy to Conquer Drug Resistance. ACS APPLIED MATERIALS & INTERFACES 2017; 9:43498-43507. [PMID: 29171954 DOI: 10.1021/acsami.7b14577] [Citation(s) in RCA: 58] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Lonidamine (LND) can act on mitochondria and inhibit energy metabolism in cancer cells and therefore has been used together with chemotherapy drugs for synergistically enhanced therapeutic efficacy. However, its use is hindered by the poor solubility and slow diffusion in the cytoplasm. To address these problems, we designed and prepared aqueous dispersible nanoparticles (NPs) containing integrated components including triphenylphosphine (TPP) to target the mitochondria of cells and LND and doxorubicin (DOX) for synergistic cancer treatment and conquering drug resistance. This design allows the NPs to concentrate in the mitochondria of cells, solve the low solubility of LND, and contain very high load of LND and DOX in comparison with previously reported drug-delivery systems based on various carrier nanomaterials. Detailed mechanism studies reveal that TPP-LND-DOX NPs could induce significant reactive oxygen species production, mitochondrial membrane potential decrease, and mitochondrial apoptosis pathway, thereby leading to great cytotoxicity in cancer cells. In vivo anticancer activities indicate that TPP-LND-DOX NPs exhibit the highest efficacy in tumor inhibition among all tested groups and show high effectiveness in drug-resistant model. This work demonstrates the potential use of our TPP-LND-DOX NPs to jointly promote the mitochondria apoptosis pathway and contribute to conquer drug resistance in cancer therapy.
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Affiliation(s)
- Yanqiu Liu
- Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Joint International Research Laboratory of Carbon-Based Functional Materials and Devices, Soochow University , Suzhou, Jiangsu 215123, P.R. China
| | - Xiujuan Zhang
- Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Joint International Research Laboratory of Carbon-Based Functional Materials and Devices, Soochow University , Suzhou, Jiangsu 215123, P.R. China
| | - Mengjiao Zhou
- Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Joint International Research Laboratory of Carbon-Based Functional Materials and Devices, Soochow University , Suzhou, Jiangsu 215123, P.R. China
| | - Xueyan Nan
- Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Joint International Research Laboratory of Carbon-Based Functional Materials and Devices, Soochow University , Suzhou, Jiangsu 215123, P.R. China
| | - Xianfeng Chen
- School of Engineering, Institute for Bioengineering, University of Edinburgh , Edinburgh EH9 3JL, United Kingdom
| | - Xiaohong Zhang
- Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Joint International Research Laboratory of Carbon-Based Functional Materials and Devices, Soochow University , Suzhou, Jiangsu 215123, P.R. China
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Kharroubi W, Nury T, Ahmed SH, Andreoletti P, Sakly R, Hammami M, Lizard G. Induction by arsenate of cell-type-specific cytotoxic effects in nerve and hepatoma cells. Hum Exp Toxicol 2017; 36:1256-1269. [PMID: 28071239 DOI: 10.1177/0960327116687893] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The aim of the study was to compare the effect of sodium arsenate (AsV) on two different cell types: 158N murine oligodendrocytes and HepG2 human hepatoma cells. Exposure of 158N cells to AsV (0.1-400 µM; 48 h) induced a biphasic cytoxic effect defined as hormesis. Thus, low concentrations of AsV stimulate cell proliferation, as shown by phase-contrast microscopy, cell counting with trypan blue, and crystal violet assay, whereas high concentrations induce cell death associated with a loss of cell adhesion. These side effects were confirmed by staining with propidium iodide and cell cycle analysis, characterized by the presence of a subG1 peak, a criterion of apoptosis. The effects of AsV on mitochondrial function, as determined by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, the measurement of mitochondrial transmembrane potential with 3,3'-dihexyloxacarbocyanine iodide, and the rate of mitochondrial adenosine triphosphate confirm the impact of AsV on the mitochondria. In contrast to 158N cells, HepG2 cells were susceptible to all AsV concentrations as shown by microscopic observations, by counting with trypan blue. However, no alteration is noted in the cell membrane integrity, which indicated an apoptotic mode of cell death, and this side effect is confirmed by the cycle analysis, which revealed a subG1 peak. Of note, there was a loss of MTT, suggesting that AsV induces mitochondrial complex II dysfunction. Altogether, our data show that the cytotoxic characteristics of AsV depend on the cell type considered.
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Affiliation(s)
- Wafa Kharroubi
- 1 Laboratory Bio-PeroxIL, Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism EA7270/INSERM, Faculty of Sciences Gabriel, University of Bourgogne Franche Comté, Dijon, France.,2 Laboratory of Nutrition-Functional Foods and Vascular Diseases, Faculty of Medicine, University of Monastir, Monastir, Tunisia
| | - Thomas Nury
- 1 Laboratory Bio-PeroxIL, Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism EA7270/INSERM, Faculty of Sciences Gabriel, University of Bourgogne Franche Comté, Dijon, France
| | - Samia Haj Ahmed
- 1 Laboratory Bio-PeroxIL, Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism EA7270/INSERM, Faculty of Sciences Gabriel, University of Bourgogne Franche Comté, Dijon, France.,2 Laboratory of Nutrition-Functional Foods and Vascular Diseases, Faculty of Medicine, University of Monastir, Monastir, Tunisia
| | - Pierre Andreoletti
- 1 Laboratory Bio-PeroxIL, Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism EA7270/INSERM, Faculty of Sciences Gabriel, University of Bourgogne Franche Comté, Dijon, France
| | - Rachid Sakly
- 2 Laboratory of Nutrition-Functional Foods and Vascular Diseases, Faculty of Medicine, University of Monastir, Monastir, Tunisia
| | - Mohamed Hammami
- 2 Laboratory of Nutrition-Functional Foods and Vascular Diseases, Faculty of Medicine, University of Monastir, Monastir, Tunisia
| | - Gérard Lizard
- 1 Laboratory Bio-PeroxIL, Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism EA7270/INSERM, Faculty of Sciences Gabriel, University of Bourgogne Franche Comté, Dijon, France
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Ganta KK, Mandal A, Chaubey B. Depolarization of mitochondrial membrane potential is the initial event in non-nucleoside reverse transcriptase inhibitor efavirenz induced cytotoxicity. Cell Biol Toxicol 2016; 33:69-82. [PMID: 27639578 DOI: 10.1007/s10565-016-9362-9] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2016] [Accepted: 09/05/2016] [Indexed: 12/23/2022]
Abstract
Efavirenz is a non-nucleoside reverse transcriptase inhibitor (NNRTI) and an active constituent of the highly active antiretroviral therapy regime. It has significantly contributed in control and management of human immunodeficiency virus propagation. However, EFV administration has also led to severe adverse effects, several reports highlighted the role of EFV in mitochondrial dysfunction and toxicity but the molecular mechanism has been poorly understood. In present study, human hepatoma cells Huh 7.5 were treated with clinically relevant concentrations of EFV and parameters like cytotoxicity, mitochondrial transmembrane potential, mitochondrial morphology, cytochrome c release, mitochondria-mediated apoptosis, mtDNA and mtRNA levels and EFV distribution into mitochondrial compartment were evaluated to understand sequence of events leading to cell death in EFV-treated cells. EFV at its clinically relevant concentration was significantly toxic after 48 and 72 h of treatments. EFV-mediated toxicity is initiated with the permeabilization of mitochondrial outer membrane and change in mitochondrial membrane potential (Δψm) which triggers a series of events like cytochrome c release, alteration in mitochondrial morphology and mitochondria-mediated apoptosis. Total mitochondrial content is reduced after 48 h of EFV treatment at IC50 concentration which is also reflected in reduced mitochondrial DNA and RNA levels. After detecting EFV in mitochondrial compartment after 12 h of incubation with EFV, we hypothesize that EFV being a lipophilic molecule is internalized into the mitochondrial compartment causing depolarization of Δψm which subsequently leads to a cascade of events causing cell death.
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Affiliation(s)
- Krishna Kumar Ganta
- Functional Genomics Lab., Centre for Advanced Study, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, India
| | - Anirban Mandal
- Functional Genomics Lab., Centre for Advanced Study, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, India
| | - Binay Chaubey
- Functional Genomics Lab., Centre for Advanced Study, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, India. .,Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
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Hauge M, Bruserud Ø, Hatfield KJ. Targeting of cell metabolism in human acute myeloid leukemia - more than targeting of isocitrate dehydrogenase mutations and PI3K/AKT/mTOR signaling? Eur J Haematol 2015; 96:211-21. [DOI: 10.1111/ejh.12690] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/06/2015] [Indexed: 12/20/2022]
Affiliation(s)
- Michelle Hauge
- Department of Medicine; Haukeland University Hospital; Bergen Norway
| | - Øystein Bruserud
- Department of Medicine; Haukeland University Hospital; Bergen Norway
- Department of Clinical Science; University of Bergen; Bergen Norway
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Chen ZP, Li M, Zhang LJ, He JY, Wu L, Xiao YY, Duan JA, Cai T, Li WD. Mitochondria-targeted drug delivery system for cancer treatment. J Drug Target 2015; 24:492-502. [DOI: 10.3109/1061186x.2015.1108325] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Affiliation(s)
- Zhi-Peng Chen
- Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, P.R. China and
| | - Man Li
- Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, P.R. China and
| | - Liu-Jie Zhang
- Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, P.R. China and
| | - Jia-Yu He
- Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, P.R. China and
| | - Li Wu
- Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, P.R. China and
| | - Yan-Yu Xiao
- Department of Pharmacy, China Pharmaceutical University, Nanjing, P.R. China
| | - Jin-Ao Duan
- Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, P.R. China and
| | - Ting Cai
- Department of Pharmacy, China Pharmaceutical University, Nanjing, P.R. China
| | - Wei-Dong Li
- Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, P.R. China and
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Cervantes-Madrid D, Romero Y, Dueñas-González A. Reviving Lonidamine and 6-Diazo-5-oxo-L-norleucine to Be Used in Combination for Metabolic Cancer Therapy. BIOMED RESEARCH INTERNATIONAL 2015; 2015:690492. [PMID: 26425550 PMCID: PMC4575731 DOI: 10.1155/2015/690492] [Citation(s) in RCA: 83] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/27/2015] [Revised: 06/23/2015] [Accepted: 08/16/2015] [Indexed: 01/20/2023]
Abstract
Abnormal metabolism is another cancer hallmark. The two most characterized altered metabolic pathways are high rates of glycolysis and glutaminolysis, which are natural targets for cancer therapy. Currently, a number of newer compounds to block glycolysis and glutaminolysis are being developed; nevertheless, lonidamine and 6-diazo-5-oxo-L-norleucine (DON) are two old drugs well characterized as inhibitors of glycolysis and glutaminolysis, respectively, whose clinical development was abandoned years ago when the importance of cancer metabolism was not fully appreciated and clinical trial methodology was less developed. In this review, a PubMed search using the words lonidamine and 6-diazo-5-oxo-L-norleucine (DON) was undertaken to analyse existing information on the preclinical and clinical studies of these drugs for cancer treatment. Data show that they exhibit antitumor effects; besides there is also the suggestion that they are synergistic. We conclude that lonidamine and DON are safe and potentially effective drugs that need to be reevaluated in combination as metabolic therapy of cancer.
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Affiliation(s)
| | - Yair Romero
- Facultad de Ciencias, Universidad Nacional Autónoma de México, 04510 Mexico City, DF, Mexico
| | - Alfonso Dueñas-González
- Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México/Instituto Nacional de Cancerología, 14080 Mexico City, DF, Mexico
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Abstract
There are many approaches used to control breast cancer, although the most efficient strategy is the reactivation of apoptosis. Since mitochondria play an important role in cellular metabolism and homeostasis, as well as in the regulation of cell death pathways, we focus here on metabolic remodeling and mitochondrial alterations present in breast tumor cells. We review strategies including classes of compounds and delivery systems that target metabolic and specific mitochondrial alterations to kill tumor cells without affecting their normal counterparts. We present here the arguments for the improvement of already existent molecules and the design of novel promising anticancer drug candidates that target breast cancer mitochondria.
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Ghani S, Khan N, Koriyama C, Akiba S, Yamamoto M. N‑acetyl‑L‑cysteine reduces arsenite‑induced cytotoxicity through chelation in U937 monocytes and macrophages. Mol Med Rep 2014; 10:2961-6. [PMID: 25310083 DOI: 10.3892/mmr.2014.2612] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2013] [Accepted: 05/14/2014] [Indexed: 11/06/2022] Open
Abstract
In the present study, in order to clarify the preventive mechanism of N‑acetyl‑L‑cysteine (NAC) on arsenite‑induced apoptosis in U937 cells, which lack functional p53, the cytotoxicity among U937 cells [monocytes and 12‑O‑tetradecanoylphorbol‑13‑acetate (TPA)‑treated macrophages] receiving NAC treatment at different times post arsenite treatment was examined. TPA‑treated macrophages were more resistant to arsenite‑induced apoptosis than monocytes, which may be associated with the induction of Bcl‑2 expression. Pretreatment with 20 mM NAC prior to arsenite exposure suppressed apoptosis up to 75% in the monocytes and 100% in the macrophages. However, 6‑h NAC pretreatment and subsequent washing out of NAC from the culture medium prior to arsenite treatment did not inhibit the arsenite‑induced apoptosis. Post‑treatment by NAC up to 1 h following arsenite exposure almost completely inhibited the cytotoxic effects of arsenite in U937 monocytes and macrophages. The results of the current study indicate that the preventive mechanism of NAC on arsenite‑induced apoptosis in U937 monocytes and macrophages mainly involves chelation of arsenite in culture medium.
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Affiliation(s)
- Sidra Ghani
- Department of Epidemiology and Preventive Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima 890‑8544, Japan
| | - Noureen Khan
- Department of Epidemiology and Preventive Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima 890‑8544, Japan
| | - Chihaya Koriyama
- Department of Epidemiology and Preventive Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima 890‑8544, Japan
| | - Suminori Akiba
- Department of Epidemiology and Preventive Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima 890‑8544, Japan
| | - Megumi Yamamoto
- Integrated Physiology Section, Department of Basic Medical Science, National Institute for Minamata Disease, Minamata, Kumamoto 867‑0008, Japan
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Xie QR, Liu Y, Shao J, Yang J, Liu T, Zhang T, Wang B, Mruk DD, Silvestrini B, Cheng CY, Xia W. Male contraceptive Adjudin is a potential anti-cancer drug. Biochem Pharmacol 2012. [PMID: 23178657 DOI: 10.1016/j.bcp.2012.11.008] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Adjudin, also known as AF-2364 and an analog of lonidamine (LND), is a male contraceptive acting through the induction of premature sperm depletion from the seminiferous epithelium when orally administered to adult rats, rabbits or dogs. It is also known that LND can target mitochondria and block energy metabolism in tumor cells. However, whether Adjudin exhibits any anti-cancer activity remains to be elucidated. Herein we described the anti-proliferative activity of Adjudin on cancer cells in vitro and on lung and prostate tumors inoculated in nude mice. We found that Adjudin induced apoptosis in cancer cells through a Caspase-3-dependent pathway. Further experiments revealed that Adjudin could trigger mitochondrial dysfunction in cancer cells, apparently affecting the mitochondrial mass, inducing the loss of mitochondrial membrane potential and reducing cellular ATP levels. Intraperitoneal administration of Adjudin to tumor-bearing athymic nude mice also significantly suppressed the lung and prostate tumor growth. When used in combination with cisplatin, Adjudin enhances the sensitivity to cisplatin-induced cancer cell cytotoxicity. Taken together, these findings have demonstrated that Adjudin may be a potential drug for cancer therapy.
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Affiliation(s)
- Qian Reuben Xie
- School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, People's Republic of China
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Naranmandura H, Chen X, Tanaka M, Wang WW, Rehman K, Xu S, Chen Z, Chen SQ, Suzuki N. Release of Apoptotic Cytochrome c From Mitochondria by Dimethylarsinous Acid Occurs Through Interaction With Voltage-Dependent Anion Channel In Vitro. Toxicol Sci 2012; 128:137-46. [DOI: 10.1093/toxsci/kfs154] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
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Zhang Y, Zhang G, Hendrix LR, Tesh VL, Samuel JE. Coxiella burnetii induces apoptosis during early stage infection via a caspase-independent pathway in human monocytic THP-1 cells. PLoS One 2012; 7:e30841. [PMID: 22303462 PMCID: PMC3267756 DOI: 10.1371/journal.pone.0030841] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2011] [Accepted: 12/27/2011] [Indexed: 11/25/2022] Open
Abstract
The ability of Coxiella burnetii to modulate host cell death may be a critical factor in disease development. In this study, human monocytic THP-1 cells were used to examine the ability of C. burnetii Nine Mile phase II (NMII) to modulate apoptotic signaling. Typical apoptotic cell morphological changes and DNA fragmentation were detected in NMII infected cells at an early stage of infection. FACS analysis using Annexin-V-PI double staining showed the induction of a significant number of apoptotic cells at an early stage of NMII infection. Double staining of apoptotic cell DNA and intracellular C. burnetii indicates that NMII infected cells undergoing apoptosis. Interestingly, caspase-3 was not cleaved in NMII infected cells and the caspase-inhibitor Z-VAD-fmk did not prevent NMII induced apoptosis. Surprisingly, the caspase-3 downstream substrate PARP was cleaved in NMII infected cells. These results suggest that NMII induces apoptosis during an early stage of infection through a caspase-independent pathway in THP-1 cells. In addition, NMII-infected monocytes were unable to prevent exogenous staurosporine-induced apoptotic death. Western blot analysis indicated that NMII infection induced the translocation of AIF from mitochondria into the nucleus. Cytochrome c release and cytosol-to-mitochondrial translocation of the pore-forming protein Bax in NMII infected cells occurred at 24 h post infection. These data suggest that NMII infection induced caspase-independent apoptosis through a mechanism involving cytochrome c release, cytosol-to-mitochondrial translocation of Bax and nuclear translocation of AIF in THP-1 monocytes. Furthermore, NMII infection increased TNF-α production and neutralization of TNF-α in NMII infected cells partially blocked PARP cleavage, suggesting TNF-α may play a role in the upstream signaling involved in NMII induced apoptosis. Antibiotic inhibition of C. burnetii RNA synthesis blocked NMII infection-induced PARP activation. These results suggest that both intracellular C. burnetii replication and secreted TNF-α contribute to NMII infection-triggered apoptosis during an early stage of infection.
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Affiliation(s)
- Yan Zhang
- Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A & M Health Science Center, Bryan, Texas, United States of America
- Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri, United States of America
| | - Guoquan Zhang
- Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri, United States of America
- * E-mail: (JS); (GZ)
| | - Laura R. Hendrix
- Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A & M Health Science Center, Bryan, Texas, United States of America
| | - Vernon L. Tesh
- Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A & M Health Science Center, Bryan, Texas, United States of America
| | - James E. Samuel
- Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A & M Health Science Center, Bryan, Texas, United States of America
- * E-mail: (JS); (GZ)
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15
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Calviño E, Estañ MC, Simón GP, Sancho P, Boyano-Adánez MDC, de Blas E, Bréard J, Aller P. Increased apoptotic efficacy of lonidamine plus arsenic trioxide combination in human leukemia cells. Reactive oxygen species generation and defensive protein kinase (MEK/ERK, Akt/mTOR) modulation. Biochem Pharmacol 2011; 82:1619-29. [DOI: 10.1016/j.bcp.2011.08.017] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2011] [Revised: 08/19/2011] [Accepted: 08/22/2011] [Indexed: 01/13/2023]
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16
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Toxoplasma gondii infection inhibits the mitochondrial apoptosis through induction of Bcl-2 and HSP70. Parasitol Res 2010; 107:1313-21. [PMID: 20680337 DOI: 10.1007/s00436-010-1999-3] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2010] [Accepted: 07/16/2010] [Indexed: 10/19/2022]
Abstract
Heat-shock protein 70 (HSP70) is highly expressed in Toxoplasma gondii-infected cells. However, the role of this protein is not well understood, especially during apoptosis. This study addresses the mechanism behind the antiapoptotic chaperone activity of HSP70 in Toxoplasma-infected host cells using a human macrophage cell line, THP-1 by Western blot, DNA fragmentation assay, immunoprecipitation, and a caspase-3/7 activity assay based on cleavage of the colorimetric substrate DEVD-pNA. Apoptosis induced by arsenic trioxide (As(2)O(3)) was inhibited in T. gondii-infected THP-1 cells, but not in uninfected cells. Without As(2)O(3) induction of apoptosis, T. gondii infection caused increased expression of Bcl-2 and HSP70, but not caspase-3. However, active form caspase-3 levels were lower in As(2)O(3)-treated infected cells as compared with As(2)O(3)-treated uninfected cells. Bcl-2 expression in As(2)O(3)-treated infected cells was similar to that in cells infected with T. gondii. Translocation of apoptosis-inducing factor (AIF) and release of cytochrome c from mitochondria were inhibited in As(2)O(3)-treated infected cells as compared with As(2)O(3)-treated uninfected cells. Increased parasite loads in Toxoplasma-infected macrophages caused higher HSP70 and Bcl-2 expression in whole-cell extracts and fractionated components, respectively. However, expression of AIF and cytochrome c was unaffected. Toxoplasma dose-dependently inhibited caspase-3 activation, thus revealing an anti-apoptotic parasite activity on cytochrome c-mediated caspase activation in subcellular components. In addition, immunoprecipitation analysis suggested that HSP70 is capable of binding to the pro-apoptotic factors AIF and Apaf-1, but not to cytochrome c or procaspase-9. Taken together, these data demonstrate that T. gondii infection inhibits mitochondrial apoptosis through overproduction of anti-apoptotic Bcl-2 as well as HSP70, which are increased parasite loads dependently.
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17
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Arsenic trioxide as an anti-tumour agent: mechanisms of action and strategies of sensitization. J Appl Biomed 2010. [DOI: 10.2478/v10136-009-0027-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
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18
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Singh S, Okamura T, Ali-Osman F. Serine phosphorylation of glutathione S-transferase P1 (GSTP1) by PKCα enhances GSTP1-dependent cisplatin metabolism and resistance in human glioma cells. Biochem Pharmacol 2010; 80:1343-55. [PMID: 20654585 DOI: 10.1016/j.bcp.2010.07.019] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2010] [Revised: 07/09/2010] [Accepted: 07/12/2010] [Indexed: 01/20/2023]
Abstract
Recently, we reported that the human GSTP1 is phosphorylated and functionally activated by the PKC class of serine/threonine kinases. In this study, we investigated the contribution of this post-translational modification of GSTP1 to tumor cisplatin resistance. Using two malignant glioma cell lines, MGR1 and MGR3, the ability of PKCα-phosphorylated GSTP1 to catalyze the conjugation of cisplatin to glutathione was assessed and correlated with cisplatin sensitivity and cisplatin-induced DNA interstrand cross-links and apoptosis of the cells. The results showed PKCα activation and associated phosphorylation of GSTP1 to correlate significantly with increased glutathionylplatinum formation, decreased DNA interstrand cross-link formation and increased cisplatin resistance. Following PKC activation, the IC(50) of cisplatin increased from 13.63μM to 36.49μM in MGR1 and from 20.75μM to 38.45μM in MGR3. In both cell lines, siRNA-mediated GSTP1 or PKCα transcriptional suppression similarly decreased cisplatin IC(50) and was associated with decreased intracellular levels of glutathionylplatinum metabolite. Combined inhibition/transcriptional suppression of both PKCα and GSTP1 was synergistic in enhancing cisplatin sensitivity. Although, cisplatin-induced apoptosis was associated with the translocation of Bax to mitochondria, release of cytochrome c and caspase-3/7 activation, the levels of relocalized Bax and cytochrome c were significantly greater following GSTP1 knockdown. These results support a mechanism of cisplatin resistance mediated by the PKCα-dependent serine phosphorylation of GSTP1 and its associated increased cisplatin metabolism, and suggest the potential of simultaneous targeting of GSTP1 and PKCα to improve the efficacy of cisplatin therapy.
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Affiliation(s)
- Simendra Singh
- Department of Surgery, The Preston Robert Tisch Brain Tumor Center, Durham, NC 27710, USA.
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19
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Burchiel SW, Mitchell LA, Lauer FT, Sun X, McDonald JD, Hudson LG, Liu KJ. Immunotoxicity and biodistribution analysis of arsenic trioxide in C57Bl/6 mice following a 2-week inhalation exposure. Toxicol Appl Pharmacol 2009; 241:253-9. [PMID: 19800901 PMCID: PMC2843624 DOI: 10.1016/j.taap.2009.09.019] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2009] [Revised: 09/22/2009] [Accepted: 09/25/2009] [Indexed: 02/06/2023]
Abstract
In these studies the immunotoxicity of arsenic trioxide (ATO, As(2)O(3)) was evaluated in mice following 14 days of inhalation exposures (nose only, 3 h per day) at concentrations of 50 microg/m(3) and 1 mg/m(3). A biodistribution analysis performed immediately after inhalation exposures revealed highest levels of arsenic in the kidneys, bladder, liver, and lung. Spleen cell levels were comparable to those found in the blood, with the highest concentration of arsenic detected in the spleen being 150 microg/g tissue following the 1 mg/m(3) exposures. No spleen cell cytotoxicity was observed at either of the two exposure levels. There were no changes in spleen cell surface marker expression for B cells, T cells, macrophages, and natural killer (NK) cells. There were also no changes detected in the B cell (LPS-stimulated) and T cell (Con A-stimulated) proliferative responses of spleen cells, and no changes were found in the NK-mediated lysis of Yac-1 target cells. The primary T-dependent antibody response was, however, found to be highly susceptible to ATO suppression. Both the 50 microg/m(3) and 1 mg/m(3) exposures produced greater than 70% suppression of the humoral immune response to sheep red blood cells. Thus, the primary finding of this study is that the T-dependent humoral immune response is extremely sensitive to suppression by ATO and assessment of humoral immune responses should be considered in evaluating the health effects of arsenic containing agents.
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Affiliation(s)
- Scott W Burchiel
- Toxicology and Pharmaceutical Sciences Program, The University of New Mexico College of Pharmacy, Albuquerque, NM 87131, USA.
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20
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Carney DA. Arsenic trioxide mechanisms of action--looking beyond acute promyelocytic leukemia. Leuk Lymphoma 2008; 49:1846-51. [PMID: 18949607 DOI: 10.1080/10428190802464745] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Affiliation(s)
- Dennis A Carney
- Department of Hematology and Medical Oncology, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia.
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21
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Effect of differentiating agents (all-trans retinoic acid and phorbol 12-myristate 13-acetate) on drug sensitivity of HL60 and NB4 cells in vitro. Folia Histochem Cytobiol 2008; 46:323-30. [DOI: 10.2478/v10042-008-0080-x] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
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22
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Chow JM, Liu CR, Lin CP, Lee CN, Cheng YC, Lin S, Liu HE. Downregulation of c-Myc determines sensitivity to 2-methoxyestradiol–induced apoptosis in human acute myeloid leukemia. Exp Hematol 2008; 36:140-8. [DOI: 10.1016/j.exphem.2007.10.004] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2007] [Revised: 10/15/2007] [Accepted: 10/25/2007] [Indexed: 12/15/2022]
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23
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Vejux A, Lizard G, Tourneur Y, Riedinger JM, Frouin F, Kahn E. Effects of caspase inhibitors (z-VAD-fmk, z-VDVAD-fmk) on Nile Red fluorescence pattern in 7-ketocholesterol-treated cells: investigation by flow cytometry and spectral imaging microscopy. Cytometry A 2007; 71:550-62. [PMID: 17458884 DOI: 10.1002/cyto.a.20410] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND The 7-ketocholesterol (7KC)-induced cell death has some characteristics of apoptosis and is associated with polar lipid accumulation. So, we investigated the effects of the broad-spectrum caspase inhibitor z-VAD-fmk and of the caspase-2 inhibitor z-VDVAD-fmk on lipid profile evaluated by staining with Nile Red (NR). METHODS The 7KC-treated human monocytic U937 cells were cultured in the absence or in the presence of the caspase inhibitors z-VAD-fmk or z-VDVAD-fmk. When staining with NR is performed, neutral and polar lipids have yellow and orange/red emission, respectively, and fluorescence was then analyzed by flow cytometry (FCM) and by confocal laser scanning microscopy (CLSM) combined with subsequent image processing. The 3D-image sequences were obtained by means of CLSM using spectral analysis, and were analyzed by the factor analysis of medical image sequences algorithm to differentiate spectra inside mixed fluorescence emission and get corresponding specific images. RESULTS By FCM, comparatively to untreated cells, higher percentages of red fluorescent cells were identified in 7KC-treated cells. Factor curves and images reveal orange and red fluorescence emissions in 7KC-treated cells and show yellow, orange, and red fluorescence emissions in 7KC-treated cells cultured in the presence of z-VAD-fmk or z-VDVAD-fmk. CONCLUSIONS Our data support that investigation by FCM and by spectral analysis in CLSM associated with subsequent image processing provides useful tools to determine the effect of caspase inhibitors on lipid content evaluated with NR. They also favor the hypothesis of relationships between caspase activity and polar lipid accumulation.
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Affiliation(s)
- Anne Vejux
- INSERM UMR 866 (Lipides, Nutrition et Cancer; équipe Biochimie Métabolique et Nutritionnelle), IFR Santé STIC, Université de Bourgogne-Faculté des Sciences Gabriel, 21000 Dijon, France
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24
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Droin N, Cathelin S, Jacquel A, Guéry L, Garrido C, Fontenay M, Hermine O, Solary E. A role for caspases in the differentiation of erythroid cells and macrophages. Biochimie 2007; 90:416-22. [PMID: 17905508 DOI: 10.1016/j.biochi.2007.08.007] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2007] [Accepted: 08/24/2007] [Indexed: 10/22/2022]
Abstract
Several cysteine proteases of the caspase family play a central role in many forms of cell death by apoptosis. Other enzymes of the family are involved in cytokine maturation along inflammatory response. In recent years, several caspases involved in cell death were shown to play a role in other cellular processes such as proliferation and differentiation. In the present review, we summarize the current knowledge of the role of caspases in the differentiation of erythroid cells and macrophages. Based on these two examples, we show that the nature of involved enzymes, the pathways leading to their activation in response to specific growth factors, and the specificity of the target proteins that are cleaved by the activated enzymes strongly differ from one cell type to another. Deregulation of these pathways is thought to play a role in the pathophysiology of low-grade myelodysplastic syndromes, characterized by excessive activation of caspases and erythroid precursor apoptosis, and that of chronic myelomonocytic leukemia, characterized by a defective activation of caspases in monocytes exposed to M-CSF, which blocks their differentiation.
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25
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Yin W, Cheng W, Shen W, Shu L, Zhao J, Zhang J, Hua ZC. Impairment of Na(+),K(+)-ATPase in CD95(APO-1)-induced human T-cell leukemia cell apoptosis mediated by glutathione depletion and generation of hydrogen peroxide. Leukemia 2007; 21:1669-78. [PMID: 17554377 DOI: 10.1038/sj.leu.2404791] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Human T-cell leukemia is a malignant disease that needs various regimens of cytotoxic chemotherapy to overcome drug resistance. Recently, Na(+),K(+)-ATPase has emerged as a potential target for cancer therapy. However, its exact signaling pathway in human T-cell leukemia cell death has not been well defined. In the current study, we found CD95(APO-1) was able to trigger the internalization of plasma membrane Na(+),K(+)-ATPase in Jurkat cells or primary T cells as a mechanism to suppress its activity. This internalization was closely relevant to intracellular glutathione (GSH) depletion in Jurkat cells downstream of Fas-associated death domain protein (FADD) and caspase 8. GSH depletion in Fas L-treated Jurkat cells induced the generation of hydrogen peroxide (H(2)O(2)), which subsequently increased the serine phosphorylation of Na(+),K(+)-ATPase alpha1 subunit. Exogenous H(2)O(2) even mimicked the effect of Fas L to upregulate the serine phosphorylation of Na(+),K(+)-ATPase alpha1 subunit and suppress Na(+),K(+)-ATPase activity. Overall, our results indicate that CD95(APO-1) induces the FADD- and caspase 8-dependent internalization of Na(+),K(+)-ATPase through intracellular GSH loss, and the subsequent generation of H(2)O(2)-mediated serine phosphorylation of Na(+),K(+)-ATPase alpha1 subunit. Taken together, this study presents a novel regulatory mechanism of Na(+),K(+)-ATPase in CD95(APO-1)-mediated human T-leukemia cell apoptosis.
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Affiliation(s)
- W Yin
- The State Key Lab of Pharmaceutical Biotechnology, College of Life Science, Nanjing University, Nanjing, People's Republic of China
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26
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Ramos AM, Fernandez C, Amrán D, Esteban D, de Blas E, Palacios MA, Aller P. Pharmacologic inhibitors of extracellular signal-regulated kinase (ERKs) and c-Jun NH(2)-terminal kinase (JNK) decrease glutathione content and sensitize human promonocytic leukemia cells to arsenic trioxide-induced apoptosis. J Cell Physiol 2007; 209:1006-15. [PMID: 16972261 DOI: 10.1002/jcp.20806] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Treatment with 1-4 microM As(2)O(3) slightly induced apoptosis in U-937 human promonocitic leukemia cells. This effect was potentiated by co-treatment with MEK/ERK (PD98059, U0126) and JNK (SP600125, AS601245) inhibitors, but not with p38 (SB203580, SB220025) inhibitors. However, no potentiation was obtained using lonidamine, doxorubicin, or cisplatin instead of As(2)O(3). Apoptosis potentiation by mitogen-activated protein kinase (MAPK) inhibitors involved both the intrinsic and extrinsic executionary pathways, as demonstrated by Bax activation and cytochrome c release from mitochondria, and by caspase-8 activation and Bid cleavage, respectively; and the activation of both pathways was prevented by Bcl-2 over-expression. Treatment with MEK/ERK and JNK inhibitors, but not with p38 inhibitors, caused intracellular glutathione (GSH) depletion, which was differentially regulated. Thus, while it was prevented by N-acetyl-L-cysteine (NAC) in the case of U0126, it behaved as a NAC-insensitive process, regulated at the level of DL-buthionine-(S,R)-sulfoximine (BSO)-sensitive enzyme activity, in the case of SP600125. The MEK/ERK inhibitor also potentiated apoptosis and decreased GSH content in As(2)O(3)-treated NB4 human acute promyelocytic leukemia (APL) cells, but none of these effects were produced by the JNK inhibitor. MEK/ERK and JNK inhibitors did not apparently affect As(2)O(3) transport activity, as measured by intracellular arsenic accumulation. SP600126 greatly induced reactive oxygen species (ROS) accumulation, while BSO and U0126 had little or null effects. These results, which indicate that glutathione is a target of MAP kinases in myeloid leukemia cells, might be exploited to improve the antitumor properties of As(2)O(3), and provide a rationale for the use of kinase inhibitors as therapeutic agents.
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Affiliation(s)
- Adrian M Ramos
- Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040-Madrid, Spain
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27
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Martin G, Cagnon N, Sabido O, Sion B, Grizard G, Durand P, Levy R. Kinetics of occurrence of some features of apoptosis during the cryopreservation process of bovine spermatozoa. Hum Reprod 2006; 22:380-8. [PMID: 17092986 DOI: 10.1093/humrep/del399] [Citation(s) in RCA: 96] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
BACKGROUND Cryopreservation/thawing of bovine spermatozoa induces a reduction in cell viability and is possibly associated with a form of programmed cell death that we previously named 'apoptosis-like phenomenon'. METHODS In this study, we specified, by flow cytometry, the moment of appearance of some characteristics of apoptosis during the cryopreservation process. We also studied the presence and/or activation in bovine sperm cells of specific proteins involved in somatic cell apoptosis by western blot and fluorimetry. RESULTS A decrease of the mitochondrial membrane potential (DeltaPsim) was detectable 5 min after sperm dilution in the cryopreservation medium, caspase activation after 3 h of equilibration and an increase in plasma membrane permeability after the complete process of cryopreservation/thawing. The presence of the pro-apoptotic factor Bax, a protein that facilitates the formation of mitochondrial pores, was observed in bovine spermatozoa, but the anti-apoptotic factor Bcl-2 was not detectable. Moreover, it was observed that bovine spermatozoa contain cytochrome c and apoptosis-inducing factor (AIF), two proteins usually released from the mitochondria during the apoptotic process. Activated caspase-9, involved in the mitochondrial pathway, was detected in bovine spermatozoa but not caspase-3 and -8. CONCLUSIONS The early features of apoptosis appear as ordered events during the cryopreservation/thawing process of bovine sperm cells. Bovine spermatozoa contain the machinery necessary to proceed to apoptosis involving especially the mitochondrial pathway.
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Affiliation(s)
- G Martin
- Laboratoire de Biologie de la Reproduction-GIMAP, Hôpital Nord, Saint-Etienne, France
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28
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Fontenay M, Cathelin S, Amiot M, Gyan E, Solary E. Mitochondria in hematopoiesis and hematological diseases. Oncogene 2006; 25:4757-67. [PMID: 16892088 DOI: 10.1038/sj.onc.1209606] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Mitochondria are involved in hematopoietic cell homeostasis through multiple ways such as oxidative phosphorylation, various metabolic processes and the release of cytochrome c in the cytosol to trigger caspase activation and cell death. In erythroid cells, the mitochondrial steps in heme synthesis, iron (Fe) metabolism and Fe-sulfur (Fe-S) cluster biogenesis are of particular importance. Mutations in the specific delta-aminolevulinic acid synthase (ALAS) 2 isoform that catalyses the first and rate-limiting step in heme synthesis pathway in the mitochondrial matrix, lead to ineffective erythropoiesis that characterizes X-linked sideroblastic anemia (XLSA), the most common inherited sideroblastic anemia. Mutations in the adenosine triphosphate-binding cassette protein ABCB7, identified in XLSA with ataxia (XLSA-A), disrupt the maturation of cytosolic (Fe-S) clusters, leading to mitochondrial Fe accumulation. In addition, large deletions in mitochondrial DNA, whose integrity depends on a specific DNA polymerase, are the hallmark of Pearson's syndrome, a rare congenital disorder with sideroblastic anemia. In acquired myelodysplastic syndromes at early stage, exacerbation of physiological pathways involving caspases and the mitochondria in erythroid differentiation leads to abnormal activation of a mitochondria-mediated apoptotic cell death pathway. In contrast, oncogenesis-associated changes at the mitochondrial level can alter the apoptotic response of transformed hematopoietic cells to chemotherapeutic agents. Recent findings in mitochondria metabolism and functions open new perspectives in treating hematopoietic cell diseases, for example various compounds currently developed to trigger tumor cell death by directly targeting the mitochondria could prove efficient as either cytotoxic drugs or chemosensitizing agents in treating hematological malignancies.
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Affiliation(s)
- M Fontenay
- Inserm U567, Institut Cochin, Department of Hematology, Paris, Cedex, France
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29
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Filomenko R, Prévotat L, Rébé C, Cortier M, Jeannin JF, Solary E, Bettaieb A. Caspase-10 involvement in cytotoxic drug-induced apoptosis of tumor cells. Oncogene 2006; 25:7635-45. [PMID: 16767158 DOI: 10.1038/sj.onc.1209733] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Anticancer drugs can induce tumor cell death by caspase-dependent apoptosis. The observation that procaspase-10 expression decreased in leukemic cells from acute myeloblastic leukemia patients at first relapse led us to explore the role of caspase-10 in cytotoxic drug-induced apoptosis. We show that caspase-10 is activated in etoposide-treated cells in a dose- and time-dependent manner. A caspase-10 peptide inhibitor, a caspase-10 dominant-negative mutant or a small interfering RNA (siRNA)-mediated downregulation of the enzyme negatively interfere with drug-induced cell death and caspase-2, -3, -8 and -9 activation. The extrinsic pathway to apoptosis is not involved in drug-induced caspase-10 activation that occurs downstream of Bax redistribution to mitochondria and cytochrome c release from this organelle. siRNA-mediated downregulation of Apaf-1 prevents etoposide-mediated activation of caspase-10. In a cell-free assay, cytochrome c and dATP treatment of cell extracts after immunodepletion of either caspase-3 or caspase-9 indicates that caspase-10 is activated downstream of caspase-9. Then, caspase-10 is involved in a feedback amplification loop that amplifies caspase-9 and -3 activities. Altogether, these data indicate an active role for caspase-10 in cytotoxic drug-induced tumor cell death, downstream of the mitochondria.
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Affiliation(s)
- R Filomenko
- Inserm U 517, Ecole Pratique des Hautes Etudes, IFR100, Faculty of Medicine, Dijon cedex, France
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Chang PY, Miyamoto S. Nuclear factor-kappaB dimer exchange promotes a p21(waf1/cip1) superinduction response in human T leukemic cells. Mol Cancer Res 2006; 4:101-12. [PMID: 16513841 DOI: 10.1158/1541-7786.mcr-05-0259] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The nuclear factor-kappaB (NF-kappaB)/Rel transcription factors are recognized as critical apoptosis regulators. We reported previously that NF-kappaB contributes to chemoresistance of CEM human T leukemic cells in part through its ability to induce p21(waf1/cip1). Here, we provide evidence that sequential NF-kappaB-activating signals induce heightened NF-kappaB DNA binding and p21(waf1/cip1) induction in CEM and additional T leukemic cell lines. This response arises from exceedingly low basal expression of the p105/p50 NF-kappaB subunit encoded by the NFKB1 gene in these cell lines. An initial NF-kappaB activation event enhances the recruitment of p65 and ELF1 to the NFKB1 promoter, leading to p65- and ELF1-dependent synthesis of p105/p50, which promotes an exchange of NF-kappaB complexes to p50-containing complexes with an increased DNA-binding activity to certain NF-kappaB target elements. Subsequent stimulation of these cells with an anticancer agent, etoposide, results in augmented NF-kappaB-dependent p21(waf1/cip1) induction and increased chemoresistance of the leukemia cells. Thus, we propose that low basal NFKB1 expression coupled with sequential NF-kappaB activation events can promote increased chemoresistance in certain T leukemic cells.
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Affiliation(s)
- Pei-Yun Chang
- Program in Molecular and Cellular Pharmacology, Department of Pharmacology, University of Wisconsin-Madison, 301 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706, USA
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Bouchier-Hayes L, Lartigue L, Newmeyer DD. Mitochondria: pharmacological manipulation of cell death. J Clin Invest 2005; 115:2640-7. [PMID: 16200197 PMCID: PMC1236694 DOI: 10.1172/jci26274] [Citation(s) in RCA: 149] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Cell death by apoptosis or necrosis is often important in the etiology and treatment of disease. Since mitochondria play important roles in cell death pathways, these organelles are potentially prime targets for therapeutic intervention. Here we discuss the mechanisms through which mitochondria participate in the cell death process and also survey some of the pharmacological approaches that target mitochondria in various ways.
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Affiliation(s)
- Lisa Bouchier-Hayes
- La Jolla Institute for Allergy and Immunology, Department of Cellular Immunology, San Diego, California 92121, USA
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32
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Tian X, Ma X, Qiao D, Ma A, Yan F, Huang X. mCICR is required for As2O3-induced permeability transition pore opening and cytochrome c release from mitochondria. Mol Cell Biochem 2005; 277:33-42. [PMID: 16132712 DOI: 10.1007/s11010-005-4818-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2005] [Accepted: 04/01/2005] [Indexed: 01/21/2023]
Abstract
The permeability transition pore (PTP) is central for apoptosis by acting as a good candidate pathway for the release of Cyt. c and apoptosis induction factors (AIF). Arsenite induces apoptosis via a direct effect on PTP. To characterize the exact mechanism for arsenite induces PTP opening, the effect of Ca2+ on As2O3-induced PTP opening, the relationship between As2O3-induced PTP opening and Cyt. c release from mitochondria and calcium-induced calcium release from mitochondria (mCICR), and the effects of As2O3 on Ca2+-induced PTP opening were studied. The results showed As2O3 induces Cyt. c release by triggering PTP opening. Ca2+ is necessary for As2O3-induced PTP opening. As2O3-induced PTP opening and Cyt. c release depends on mCICR. As2O3 promotes PTP opening by lowering Ca2+-threshold. These results indicated As2O3 induce Cyt. c release from mitochondria by lowering Ca2+-threshold for PTP and triggering mCICR-dependent PTP opening. Suggesting that it is possible to control apoptosis by altering Ca2+ threshold and mCICR to modulate PTP opening and Cyt. c release.
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Affiliation(s)
- Xuemei Tian
- Deparment of Histology and Embryology, Southern Medical University, Guangzhou, Guangdong, 510515, China
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Launay S, Hermine O, Fontenay M, Kroemer G, Solary E, Garrido C. Vital functions for lethal caspases. Oncogene 2005; 24:5137-48. [PMID: 16079910 DOI: 10.1038/sj.onc.1208524] [Citation(s) in RCA: 153] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Caspases are a family of cysteine proteases expressed as inactive zymogens in virtually all animal cells. These enzymes play a central role in most cell death pathways leading to apoptosis but growing evidences implicate caspases also in nonapoptotic functions. Several of these enzymes, activated in molecular platforms referred to as inflammasomes, play a role in innate immune response by processing some of the cytokines involved in inflammatory response. Caspases are requested for terminal differentiation of specific cell types, whether this differentiation process leads to enucleation or not. These enzymes play also a role in T and B lymphocyte proliferation and, in some circumstances, appear to be cytoprotective rather than cytotoxic. These pleiotropic functions implicate caspases in the control of life and death but the fine regulation of their dual effect remains poorly understood. The nonapoptotic functions of caspases implicate that cells can restrict the proteolytic activity of these enzymes to selected substrates. Deregulation of the pathways in which caspases exert these nonapoptotic functions is suspected to play a role in the pathophysiology of several human diseases.
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Affiliation(s)
- Sophie Launay
- INSERM U-517, IFR100, Faculty of Medicine, 7 Boulevard Jeanne d'Arc, 21033 Dijon, France
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Navakauskiene R, Treigyte G, Savickiene J, Gineitis A, Magnusson KE. Alterations in protein expression in HL-60 cells during etoposide-induced apoptosis modulated by the caspase inhibitor ZVAD.fmk. Ann N Y Acad Sci 2005; 1030:393-402. [PMID: 15659822 DOI: 10.1196/annals.1329.0049] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
DNA topoisomerase inhibitors induce a specific signaling cascade that promotes an active apoptotic caspase-dependent cell death process. However, little is known about the initial signals elicited by these agents. In the present study, we compared apoptosis in HL-60 cells treated either with the chemotherapeutic drug etoposide (VP16) alone or combined with the broad caspase inhibitor ZVAD.fmk. Apoptosis was assessed by changes in cell morphology and agarose gel electrophoresis of extracted cell DNA. We found that ZVAD.fmk prevents VP16-induced DNA fragmentation and the appearance of an increased number of apoptotic cells in the culture. We also compared the effects of etoposide alone or together with the pan-caspase inhibitor ZVAD.fmk on proliferating cell nuclear antigen, Bcl-2, and actin expression in human promyelocytic leukemia HL-60 cells. In addition, we screened for proteins that were initially upregulated in a caspase-dependent manner. Indeed, some proteins were induced in the cytoplasm and subsequently accumulated in the nuclei after etoposide treatment. This process was slightly inhibited by the caspase inhibitor ZVAD.fmk. We suggest that these proteins are associated with the induction of specific signaling cascades that characterize the apoptotic cell death process.
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Affiliation(s)
- Ruta Navakauskiene
- Department of Developmental Biology, Institute of Biochemistry, LT-08662 Vilnius, Lithuania.
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35
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Shao QS, Ye ZY, Ling ZQ, Ke JJ. Cell cycle arrest and apoptotic cell death in cultured human gastric carcinoma cells mediated by arsenic trioxide. World J Gastroenterol 2005; 11:3451-6. [PMID: 15948253 PMCID: PMC4316002 DOI: 10.3748/wjg.v11.i22.3451] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effect of arsenic trioxide on human gastric cancer cell line MKN45 with respect to both cytotoxicity and induction of apoptosis in vitro.
METHODS: MKN45 cells were treated with arsenic trioxide (As2O3) at the concentration of 1, 5, and 10 µmol/L, respectively, for three successive days. Cell growth and proliferation were observed by cell counting and trypan blue exclusion. Cytotoxicity of As2O3 was determined by MTT assay. Morphologic changes were studied with light microscopy. Flow cytometry was used to assay cell DNA distribution and apoptotic cells were confirmed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and DNA electrophoresis.
RESULTS: The growth of MKN45 cells was significantly inhibited by As2O3 which was confirmed by colony-forming assay. After 7 d of culture with various concentrations of As2O3, colony-forming capacity of MKN45 cells decreased with As2O3 increment in comparison with that of control group. The inhibitory rate of colony-formation was 38.5%, 99.1%, and 99.5% when the concentration of As2O3 was 1, 5, and 10 μmol/L in culture medium, respectively. The cell number of a single colony in drug treatment groups was less than that of control group. The cell-killing rate of As2O3 to MKN45 cells was both dose- and time-dependent with an IC50 of (11.05±0.25) µmol/L. After incubation in 10 μmol/L As2O3 for 24 h, the cell-killing rate was 27.1%, and it was close to 50% after 48 h. The results showed that As2O3 induced time- and dose-dependent apoptosis in MKN45 cells, blocked at G2/M phase. The apoptotic peak (sub-G1 phase) appeared and cell apoptotic rate in MKN45 cells was 18.3-32.5% after treatment by 10 μmol/L As2O3 for 48 h. The percentage of G2/M cell of the experimental groups was 2.0-5.0 times than that of the control group. Gel electrophoresis of DNA from cells treated with each concentration of As2O3 for 48 h revealed a “ladder” pattern, indicating preferential DNA degradation at the internucleosomal, linker DNA sections. TUNEL also demonstrated strand breaks in DNA of MKN45 cells treated with As2O3, while control cells showed negative labeling.
CONCLUSION: As2O3 can induce apoptosis of human gastric carcinoma cells MKN45, which is the basis of its effectiveness. It shows great potential in the treatment of gastric carcinoma.
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Affiliation(s)
- Qin-Shu Shao
- Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang Province, China
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36
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Ramos AM, Fernández C, Amrán D, Sancho P, de Blas E, Aller P. Pharmacologic inhibitors of PI3K/Akt potentiate the apoptotic action of the antileukemic drug arsenic trioxide via glutathione depletion and increased peroxide accumulation in myeloid leukemia cells. Blood 2005; 105:4013-20. [PMID: 15665116 DOI: 10.1182/blood-2004-07-2802] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Abstract
Treatment for 14 to 24 hours with low concentrations of arsenic trioxide (As2O3, 1-4 μM) caused apoptosis in U-937 promonocytes and other human myeloid leukemia cell lines (HL-60, NB4). This effect was potentiated by cotreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin, and the Akt inhibitor Akti5. However, the inhibitors did not increase the toxicity of the mitochondria-targeting drug lonidamine, and the DNA-specific drugs camptothecin and cisplatin, when used under similar experimental conditions as As2O3. The potentiation of As2O3-provoked apoptosis involved the increased disruption of mitochondrial transmembrane potential, increased caspase-3 activation and cytochrome c release from mitochondria, increased Bax and Bid activation, and attenuation of 27-kDa heat shock protein (HSP27) expression; the potentiation was prevented by Bcl-2 overexpression. The PI3K/Akt inhibitors decreased the intracellular glutathione content, and caused intracellular oxidation, as measured by peroxide accumulation. Cotreatment with subcytotoxic concentrations of hydrogen peroxide increased apoptosis induction by As2O3. On the other hand, the treatments did not significantly affect glutathione S-transferase π expression and activity. These results, which indicate that glutathione is a target of PI3K/Akt in myeloid leukemia cells, may partially explain the selective increase of As2O3 toxicity by PI3K/Akt inhibitors, and may provide a rationale to improve the efficacy of these inhibitors as therapeutic agents.
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Affiliation(s)
- Adrián M Ramos
- Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040-Madrid, Spain
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37
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Del Bufalo D, Trisciuoglio D, Scarsella M, D'Amati G, Candiloro A, Iervolino A, Leonetti C, Zupi G. Lonidamine causes inhibition of angiogenesis-related endothelial cell functions. Neoplasia 2005; 6:513-22. [PMID: 15548359 PMCID: PMC1531654 DOI: 10.1593/neo.04133] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
The aim of this study was to assess whether lonidamine (LND) interferes with some steps in angiogenesis progression. We report here, for the first time, that LND inhibited angiogenic-related endothelial cell functions in a dose-dependent manner (1-50 microg/ml). In particular, LND decreased proliferation, migration, invasion, and morphogenesis on matrigel of different endothelial cell lines. Zymographic and Western blot analysis assays showed that LND treatment produced a reduction in the secretion of matrix metalloproteinase-2 and metalloproteinase-9 by endothelial cells. Vessel formation in a matrigel plug was also reduced by LND. The viability, migration, invasion, and matrix metalloproteinase production of different tumor cell lines were not affected by low doses of LND (1-10 microg/ml), whereas 50 microg/ml LND, which corresponds to the dose used in clinical management of tumors, triggered apoptosis both in endothelial and tumor cells. Together, these data demonstrate that LND is a compound that interferes with endothelial cell functions, both at low and high doses. Thus, the effect of LND on endothelial cell functions, previously undescribed, may be a significant contributor to the antitumor effect of LND observed for clinical management of solid tumors.
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Affiliation(s)
- Donatella Del Bufalo
- Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Rome, Italy.
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38
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Abstract
Cancer cells are defined by their unlimited replicative potential and resistance to cell death stimuli. It is generally considered that a point of no return in apoptotic cell death is the permeabilisation of the mitochondrial membranes. For this reason, agents that permeabilise cancer cell mitochondria have the potential to circumvent their resistance to apoptotic cell death. Fortunately, the proliferative and bioenergetic differences between normal and cancerous cells provide an opportunity to selectively target cancer cell mitochondria.
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Affiliation(s)
- Anthony S Don
- Centre for Vascular Research, University of New South Wales, Department of Haematology, Prince of Wales Hospital, Sydney NSW 2052, Australia
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39
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Karasavvas N, Cárcamo JM, Stratis G, Golde DW. Vitamin C protects HL60 and U266 cells from arsenic toxicity. Blood 2005; 105:4004-12. [PMID: 15677571 PMCID: PMC1895087 DOI: 10.1182/blood-2003-03-0772] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Although there is no compelling evidence that vitamin C has antitumor activity in humans, clinical trials are testing the hypothesis that ascorbic acid (AA) will enhance the efficacy of arsenic trioxide (As2O3) in myeloma. In vitro, AA cytotoxicity depends on its interaction with free transition metal ions in culture media leading to the generation of H2O2 and other reactive oxygen species (ROSs). Therefore, to circumvent the extracellular in vitro pro-oxidant effects of AA, we loaded HL60, U266, and RPMI-8226 cells with vitamin C by incubation with dehydroascorbic acid (DHA). Loading cells in this manner resulted in prominent, dose-dependent protection of As2O3-treated cells as measured by viability, colony formation, and apoptosis assays. Glutathione depletion enhanced cell sensitivity to the cytotoxic effects of As2O3 and vitamin C loading provided protection. AA was found to generate cytotoxic concentrations of H2O2 in culture medium without cells and copper/iron chelators inhibited this reaction. However, AA did not generate H2O2 in simple buffer or human plasma. Direct incubation with AA resulted in increased intracellular ROSs, whereas DHA incubation decreased it. These results clarify an apparent paradox and indicate that vitamin C loading in HL60, U266, and RPMI-8226 cells ameliorates As2O3 cytotoxicity.
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Affiliation(s)
- Nicos Karasavvas
- Memorial Sloan-Kettering Cancer Center, Box 451, 1275 York Ave, New York, NY 10021, USA
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40
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Pelicano H, Carney D, Huang P. ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 2004; 7:97-110. [PMID: 15158766 DOI: 10.1016/j.drup.2004.01.004] [Citation(s) in RCA: 1649] [Impact Index Per Article: 78.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2004] [Accepted: 01/29/2004] [Indexed: 11/29/2022]
Abstract
Reactive oxygen species (ROS) are constantly generated and eliminated in the biological system, and play important roles in a variety of normal biochemical functions and abnormal pathological processes. Growing evidence suggests that cancer cells exhibit increased intrinsic ROS stress, due in part to oncogenic stimulation, increased metabolic activity, and mitochondrial malfunction. Since the mitochondrial respiratory chain (electron transport complexes) is a major source of ROS generation in the cells, the vulnerability of the mitochondrial DNA to ROS-mediated damage appears to be a mechanism to amplify ROS stress in cancer cells. The escalated ROS generation in cancer cells serves as an endogenous source of DNA-damaging agents that promote genetic instability and development of drug resistance. Malfunction of mitochondria also alters cellular apoptotic response to anticancer agents. Despite the negative impacts of increased ROS in cancer cells, it is possible to exploit this biochemical feature and develop novel therapeutic strategies to preferentially kill cancer cells through ROS-mediated mechanisms. This article reviews ROS stress in cancer cells, its underlying mechanisms and relationship with mitochondrial malfunction and alteration in drug sensitivity, and suggests new therapeutic strategies that take advantage of increased ROS in cancer cells to enhance therapeutic activity and selectivity.
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Affiliation(s)
- Helene Pelicano
- Department of Molecular Pathology, Box 89, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA
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41
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Han J, Goldstein LA, Gastman BR, Rabinovitz A, Wang GQ, Fang B, Rabinowich H. Differential involvement of Bax and Bak in TRAIL-mediated apoptosis of leukemic T cells. Leukemia 2004; 18:1671-80. [PMID: 15356645 DOI: 10.1038/sj.leu.2403496] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
TRAIL-induced apoptosis has been considered a promising therapeutic approach for tumors that are resistant to chemotherapy, which is usually mediated via mitochondrial apoptotic cascades. Recent studies have shown that in certain cancer cells, TRAIL-mediated apoptosis is also dependent on mitochondrial involvement, suggesting that similar mechanisms of resistance to chemotherapy might be implicated in the resistance of tumor cells to TRAIL. We have used TRAIL-resistant leukemic cells that are deficient in both Bax and Bak to determine the roles of these Bcl-2 members in TRAIL-mediated apoptosis. Exposure of these cells to TRAIL did not have an impact on cell viability, although it induced the processing of caspase-3 to its active p20 subunit. The activity of the p20 caspase-3 appeared to be inhibited as no autoprocessing of this p20 subunit or cleavage of known caspase-3 substrates were detected. Also, in the absence of Bax and Bak, no release of mitochondrial apoptogenic proteins was observed following TRAIL treatment. Adenoviral transduction of the Bax, but not the Bak gene, to the Bax/Bak-deficient leukemic cells rendered them TRAIL-sensitive as assessed by enhanced apoptotic death and caspase-3 processing. These findings demonstrate preferential utilization of Bax over Bak in leukemic cell response to specific apoptotic stimulation.
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Affiliation(s)
- J Han
- Department of Pathology, The University of Pittsburgh School of Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
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42
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Berthoux L, Sebastian S, Sokolskaja E, Luban J. Lv1 inhibition of human immunodeficiency virus type 1 is counteracted by factors that stimulate synthesis or nuclear translocation of viral cDNA. J Virol 2004; 78:11739-50. [PMID: 15479815 PMCID: PMC523245 DOI: 10.1128/jvi.78.21.11739-11750.2004] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2004] [Accepted: 07/16/2004] [Indexed: 12/19/2022] Open
Abstract
Human immunodeficiency virus type 1 (HIV-1) cDNA synthesis is inhibited in cells from some nonhuman primates by an activity called Lv1. Sensitivity to restriction by Lv1 maps to a region of the HIV-1 CA required for interaction with the cellular protein cyclophilin A. A similar antiviral activity in mammalian cells, Ref1, inhibits reverse transcription of murine leukemia virus (MLV), but only with viral strains bearing N-tropic CA. Disruption of the HIV-1 CA-cyclophilin A interaction inhibits Lv1 restriction in some cells and, paradoxically, seems to render HIV-1 sensitive to Ref1. Lv1 and Ref1 activities are overcome by high-titer infection and are saturable with nonreplicating, virus-like particles encoded by susceptible viruses. Two compounds that disrupt mitochondrial membrane potential, As(2)O(3) and m-Cl-CCP, reduce Ref1 activity. Here we show that these drugs, as well as a third compound with similar effects on mitochondria, PK11195, attenuate Lv1 activity in rhesus macaque and African green monkey cells. Effects of PK11195 and virus-like particles on HIV-1 infectivity in these cells were largely redundant, each associated with increased HIV-1 cDNA. Comparison of acutely infected macaque and human cells suggested that, in addition to effects on cDNA synthesis, Lv1 inhibits the accumulation of nuclear forms of HIV-1 cDNA. Disruption of the HIV-1 CA-cyclophilin A interaction caused a minimal increase in total viral cDNA but increased the proportion of viral cDNA in the nucleus. Consistent with a model in which Lv1 inhibits both synthesis and nuclear translocation of HIV-1 cDNA, complete suppression of macaque or African green monkey Lv1 was achieved by the additive effect of factors that stimulate both processes.
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Affiliation(s)
- Lionel Berthoux
- Department of Microbiology Department, Columbia University, 701 W. 168th St., New York, NY 10032, USA
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43
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Plenchette S, Cathelin S, Rébé C, Launay S, Ladoire S, Sordet O, Ponnelle T, Debili N, Phan TH, Padua RA, Dubrez-Daloz L, Solary E. Translocation of the inhibitor of apoptosis protein c-IAP1 from the nucleus to the Golgi in hematopoietic cells undergoing differentiation: a nuclear export signal-mediated event. Blood 2004; 104:2035-43. [PMID: 15187025 DOI: 10.1182/blood-2004-01-0065] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractThe caspase inhibitor and RING finger-containing protein cellular inhibitor of apoptosis protein 1 (c-IAP1) has been shown to be involved in both apoptosis inhibition and signaling by members of the tumor necrosis factor (TNF) receptor family. The protein is regulated transcriptionally (eg, is a target for nuclear factor-κB [NF-κB]) and can be inhibited by mitochondrial proteins released in the cytoplasm upon apoptotic stimuli. The present study indicates that an additional level of regulation of c-IAP1 may be cell compartmentalization. The protein is present in the nucleus of undifferentiated U937 and THP1 monocytic cell lines. When these cells undergo differentiation under phorbol ester exposure, c-IAP1 translocates to the cytoplasmic side of the Golgi apparatus. This redistribution involves a nuclear export signal (NES)-mediated, leptomycin B-sensitive mechanism. Using site-directed mutagenesis, we localized the functional NES motif in the caspase recruitment domain (CARD) of c-IAP1. A nucleocytoplasmic redistribution of the protein was also observed in human monocytes as well as in tumor cells from epithelial origin when undergoing differentiation. c-IAP1 does not translocate from the nucleus of cells whose differentiation is blocked (ie, in cell lines and monocytes from transgenic mice overexpressing B-cell lymphoma 2 [Bcl-2] and in monocytes from patients with chronic myelomonocytic leukemia). Altogether, these observations associate c-IAP1 cellular location with cell differentiation, which opens new perspectives on the functions of the protein. (Blood. 2004;104:2035-2043)
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Affiliation(s)
- Stéphanie Plenchette
- Institut National de la Sante et de la Recherche Médicale U517, INSERM EPI 106, IFR100, Dijon, France
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44
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Evens AM, Tallman MS, Gartenhaus RB. The potential of arsenic trioxide in the treatment of malignant disease: past, present, and future. Leuk Res 2004; 28:891-900. [PMID: 15234563 DOI: 10.1016/j.leukres.2004.01.011] [Citation(s) in RCA: 132] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2004] [Revised: 01/22/2004] [Accepted: 01/26/2004] [Indexed: 11/18/2022]
Abstract
Arsenic trioxide (As2O3) is an effective therapy for acute promyelocytic leukemia (APL), and there has been promising activity noted in other hematologic and solid tumors. The mechanism of action of As2O3 such as differentiation and apoptosis has prompted study into combination therapy. Furthermore, the connection of the sensitivity of diseases such as APL and multiple myeloma to oxidative damage has allowed the investigation of pharmacologic modulation of the cellular redox state for potentiation of As2O3. Continued study of As2O3 as a single-agent and in combination therapy will allow identification of the safest and most effective treatment regimens for malignant disease.
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Affiliation(s)
- Andrew M Evens
- Department of Medicine, Divison of Hematology/Oncology, Northwestern University, Feinberg School of Medicine, Robert H. Lurie Comprehensive Cancer Center, 676 N. St. Clair, Suite 850, Chicago, IL 60611, USA
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45
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Martin G, Sabido O, Durand P, Levy R. Cryopreservation Induces an Apoptosis-Like Mechanism in Bull Sperm. Biol Reprod 2004; 71:28-37. [PMID: 14973261 DOI: 10.1095/biolreprod.103.024281] [Citation(s) in RCA: 206] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
Cryopreservation induces many changes in sperm cells, including membrane disorders and cell death. We tested the hypothesis that apoptosis, a form of programmed cell death, can contribute to the fatal effect of cryopreservation on sperm cells. A multiparametric study of apoptosis on bovine sperm is proposed, using flow cytometry, including mitochondrial membrane potential (DeltaPsi(m)), caspase activation, membrane permeability, nucleus condensation, DNA fragmentation, and phosphatidylserine (PS) externalization. The relevance of each test was first validated on a human somatic cell line, U937. Cryopreservation and/or thawing induced significant changes in all apoptotic markers in living bull sperm cells except those concerning the nucleus. After cryopreservation, 44.9% +/- 17% (vs. 11.3% +/- 10.6% before cryopreservation) of sperm cells showed low DeltaPsi(m), 12% +/- 6.3% (vs. 2.2% +/- 1.0% before) contained active caspases, and 10.8% +/- 5.8% (vs. 1.4% +/- 1.1% before) exhibited high membrane permeability. However, cryopreservation had no effect on DNA fragmentation (9.1% +/- 7.7% before vs. 11.1% +/- 5.7% after cryopreservation) or on nucleus condensation (46% +/- 12.7% before vs. 43.8% +/- 13.1% after). Cryopreservation acts as an apoptotic mechanism inducer in bovine sperm cells, where the earliest but not the latest features of cells undergoing apoptosis occur. We have named this abortive process an apoptosis-like phenomenon.
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Affiliation(s)
- Guillaume Martin
- Laboratoire de Biologie de la Reproduction-GIMAP, Hopital Nord, 42055 Saint-Etienne, France
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46
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Sordet O, Liao Z, Liu H, Antony S, Stevens EV, Kohlhagen G, Fu H, Pommier Y. Topoisomerase I-DNA complexes contribute to arsenic trioxide-induced apoptosis. J Biol Chem 2004; 279:33968-75. [PMID: 15178684 DOI: 10.1074/jbc.m404620200] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Topoisomerase I is an essential enzyme that relaxes DNA supercoiling by forming covalent DNA cleavage complexes, which are normally transient. Topoisomerase I-DNA complexes can be trapped by anticancer drugs (camptothecins) as well as by endogenous and exogenous DNA lesions. We show here that arsenic trioxide (a potent inducer of apoptosis that induces the intracellular accumulation of reactive oxygen species and targets mitochondria) induces cellular topoisomerase I cleavage complexes. Bcl-2 overexpression and quenching of reactive oxygen species, which prevent arsenic trioxide-induced apoptosis, also prevent the formation of topoisomerase I-DNA complexes, whereas enhancement of reactive oxygen species accumulation promotes these complexes. The caspase inhibitor, benzyloxycarbonyl-VAD partially prevents arsenic trioxide-induced topoisomerase I-DNA complexes and apoptosis, suggesting that activated caspases further maintain intracellular levels of reactive oxygen species that induce the formation of topoisomerase I-DNA complexes. Down-regulation of topoisomerase I expression decreases arsenic trioxide-induced apoptotic DNA fragmentation. Thus, we propose that arsenic trioxide induces topoisomerase I-DNA complexes that participate in chromatin fragmentation and programmed cell death during apoptosis.
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Affiliation(s)
- Olivier Sordet
- Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA
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47
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Pommier Y, Sordet O, Antony S, Hayward RL, Kohn KW. Apoptosis defects and chemotherapy resistance: molecular interaction maps and networks. Oncogene 2004; 23:2934-49. [PMID: 15077155 DOI: 10.1038/sj.onc.1207515] [Citation(s) in RCA: 407] [Impact Index Per Article: 19.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Intrinsic (innate) and acquired (adaptive) resistance to chemotherapy critically limits the outcome of cancer treatments. For many years, it was assumed that the interaction of a drug with its molecular target would yield a lethal lesion, and that determinants of intrinsic drug resistance should therefore be sought either at the target level (quantitative changes or/and mutations) or upstream of this interaction, in drug metabolism or drug transport mechanisms. It is now apparent that independent of the factors above, cellular responses to a molecular lesion can determine the outcome of therapy. This review will focus on programmed cell death (apoptosis) and on survival pathways (Bcl-2, Apaf-1, AKT, NF-kappaB) involved in multidrug resistance. We will present our molecular interaction mapping conventions to summarize the AKT and IkappaB/NF-kappaB networks. They complement the p53, Chk2 and c-Abl maps published recently. We will also introduce the 'permissive apoptosis-resistance' model for the selection of multidrug-resistant cells.
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Affiliation(s)
- Yves Pommier
- Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, NIH, DHHS, Bethesda, MD 20892, USA.
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48
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Fernández C, Ramos AM, Sancho P, Amrán D, de Blas E, Aller P. 12-O-Tetradecanoylphorbol-13-acetate May Both Potentiate and Decrease the Generation of Apoptosis by the Antileukemic Agent Arsenic Trioxide in Human Promonocytic Cells. J Biol Chem 2004; 279:3877-84. [PMID: 14610070 DOI: 10.1074/jbc.m310665200] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Arsenic trioxide (As(2)O(3)) caused apoptosis in U-937 human promonocytic cells. This effect was potentiated by the simultaneous addition of the glutathione (GSH) synthesis inhibitor DL-buthionine-(R,S)-sulfoximine or the protein kinase C activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin 1. In addition TPA decreased the intracellular GSH content, caused ERK activation, and potentiated the As(2)O(3)-provoked activation of p38 and JNK. The addition of N-acetyl-L-cysteine, the PKC inhibitor GF109203X, and the MEK/ERK inhibitors PD98059 and U0126 attenuated both apoptosis induction and GSH decrease, whereas the p38 inhibitor SB203580 and the JNK inhibitor SP600125 were ineffective. TPA also potentiated ERK activation and GSH depletion when added simultaneously to cadmium chloride (CdCl(2)) and doxorubicin. However, TPA only enhanced apoptosis in the case of CdCl(2), which is a GSH-sensitive agent, whereas it reduced the toxicity of doxorubicin and other DNA-specific drugs. Finally, preincubation for 14-24 h with TPA did not potentiate but, instead, attenuated the As(2)O(3)- and CdCl(2)-provoked apoptosis. The same result was obtained by preincubation with bryostatin 1 and other differentiation inducers. It is concluded that TPA increases the apoptotic action of As(2)O(3), an effect mediated by ERK activation and GSH depletion. However, the increase in apoptosis is only effective in non-differentiated cells.
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Affiliation(s)
- Carlos Fernández
- Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain
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Yang HH, Ma MH, Vescio RA, Berenson JR. Overcoming drug resistance in multiple myeloma: the emergence of therapeutic approaches to induce apoptosis. J Clin Oncol 2003; 21:4239-47. [PMID: 14615454 DOI: 10.1200/jco.2003.06.001] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Drug resistance remains a major clinical challenge for cancer treatment. Early studies suggested that overexpression of P-glycoprotein was a major contributor to the chemotherapy resistance of myeloma cells and other tumor cells. Attempts in several clinical studies to reverse multidrug resistance protein (MDR) by using MDR modulators have not yet generated promising results. Recently, the emerging knowledge about the importance of overcoming antiapoptosis and drug resistance in treating a variety of malignancies, including multiple myeloma (MM), raises new hope of improving the treatment outcome for patients with cancer. The therapeutic value of targeting therapies that aim to reverse the antiapoptotic process in MM cells has been explored in a number of experimental systems, and the results have been promising. The proteasome inhibitor PS-341 is a new specifically targeted proapoptotic therapy that has been tested in clinical studies. The results indicate that PS-341 alone is an effective therapy for patients with MM who experience disease relapse. Recent in vitro data also demonstrate that PS-341 can markedly sensitize chemotherapy-resistant MM cells to various chemotherapeutic agents. On the basis of these encouraging results, clinical studies are underway to test the efficacy of PS-341 and chemotherapeutic agents as combination therapy in treating patients with refractory and relapsed MM.
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Affiliation(s)
- Hank H Yang
- Institute for Myeloma and Bone Cancer Research, Los Angeles, CA, USA.
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Papaldo P, Lopez M, Cortesi E, Cammilluzzi E, Antimi M, Terzoli E, Lepidini G, Vici P, Barone C, Ferretti G, Di Cosimo S, Nistico C, Carlini P, Conti F, Di Lauro L, Botti C, Vitucci C, Fabi A, Giannarelli D, Marolla P. Addition of either lonidamine or granulocyte colony-stimulating factor does not improve survival in early breast cancer patients treated with high-dose epirubicin and cyclophosphamide. J Clin Oncol 2003; 21:3462-8. [PMID: 12972521 DOI: 10.1200/jco.2003.03.034] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
PURPOSE Lonidamine (LND) can enhance the activity of anthracyclines in patients with metastatic breast cancer. A multicenter, prospective, randomized trial was designed to determine whether the association of LND with high-dose epirubicin plus cyclophosphamide (EC) could improve disease-free survival (DFS) in patients with early breast cancer (BC) compared with EC alone. Granulocyte colony-stimulating factor (G-CSF) was added to maintain the EC dose-intensity. PATIENTS AND METHODS From October 1991 to April 1994, 506 patients with stage I/II BC were randomly assigned to four groups: (A) epirubicin 120 mg/m2 and cyclophosphamide 600 mg/m2 administered intravenously on day 1 every 21 days for four cycles (124 patients); (B) EC plus LND 450 mg/d administered orally (125 patients); (C) EC plus G-CSF administered subcutaneously (129 patients); (D) EC plus LND plus G-CSF (128 patients). RESULTS Median follow-up was 55 months. Five-year DFS rate was similar for LND (B+D groups; 69.6%) versus non-LND arms (A+C groups; 70.3%) and G-CSF (C+D groups; 67.2%) versus non-G-CSF arms (A+B groups; 72.9%). Five-year overall survival (OS) was comparable in LND (79.1%) versus non-LND arms (81.3%) and in G-CSF (80.6%) versus non-G-CSF arms (79.6%). DFS and OS distributions in LND and G-CSF arms did not change according to tumor size, node, receptor, and menopausal status. G-CSF dramatically reduced hematologic toxicity without having a significant impact on dose-intensity (98.1% v 95.5% for C+D and A+B groups, respectively). CONCLUSION EC is active and well tolerated in patients with early breast cancer. The addition of LND or G-CSF does not improve DFS or OS.
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Affiliation(s)
- Paola Papaldo
- Division of Medical Oncology A, Regina Elena Cancer Institute, Via Elio Chianesi 53, 00144 Rome, Italy.
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