Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 28, 2017; 23(24): 4341-4353
Published online Jun 28, 2017. doi: 10.3748/wjg.v23.i24.4341
Indole phytoalexin derivatives induce mitochondrial-mediated apoptosis in human colorectal carcinoma cells
Viera Tischlerova, Martin Kello, Mariana Budovska, Jan Mojzis
Viera Tischlerova, Martin Kello, Jan Mojzis, Department of Pharmacology, Faculty of Medicine, Pavol Jozef Safarik University, 04011 Kosice, Slovak Republic
Mariana Budovska, Department of Organic Chemistry, Institute of Chemistry, Faculty of Science, Pavol Jozef Safarik University, 04154 Kosice, Slovak Republic
Author contributions: Tischlerova V designed the procedure; conducted in vitro experiments; wrote the manuscript; Kello M designed the procedure, conducted in vitro experiments; Budovska M synthesized the studied compounds; Mojzis J revised the paper and gave final approval.
Supported by the Slovak Grant Agency for Science VEGA, No. 1/0322/14 and No. 1/0753/17.
Institutional review board statement: No human or animal subjects were involved in our experiments.
Institutional animal care and use committee statement: No animal subjects were involved in our experiments.
Conflict-of-interest statement: All authors declared that there was no conflict of interest related to this study.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Jan Mojzis, Professor, DVM, DSc, Department of Pharmacology, Faculty of Medicine, Pavol Jozef Safarik University, 04011 Kosice, Slovak Republic.
Telephone: +421-55-2343485 Fax: +421-55-2343485
Received: January 26, 2017
Peer-review started: February 2, 2017
First decision: February 15, 2017
Revised: March 27, 2017
Accepted: May 19, 2017
Article in press: May 19, 2017
Published online: June 28, 2017

To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines.


Changes in cell proliferation and the cytotoxic effect of the tested compounds on human colorectal cancer cell lines and human fibroblasts were evaluated using MTS and BrdU assay, allowing us to choose the most potent substance. Cell cycle alterations were analyzed using flow cytometric analysis. The apoptosis-inducing effect of compound K-453 on the HCT116 cell line was examined with annexin V/PI double staining using flow cytometry, as well as acridine orange/propidium iodide (AO/PI) staining. The flow cytometry method also allowed us to measure changes in levels or activation states of other factors associated with apoptosis, such as poly (ADP-ribose) polymerase (PARP), caspase-3 and -9, cytochrome c, Bcl-2 family proteins, and also the integrity of the mitochondrial membrane. To evaluate activity of the transcription factors and proteins involved in signaling pathways we used Western blot analysis together with flow cytometry.


Among the ten tested compounds, compound K-453 {(±)-trans-1,2-dimethoxy-2’-(3,5-bis-trifluoromethylphenylamino)spiro{indoline-3,5’[4’,5’]dihydrothiazol} exhibited the most potent activity with IC50 = 32.22 ± 1.14 μmol/L in human colorectal HCT116 cells and was thus selected for further studies. Flow cytometric analysis revealed a K-453-induced increase in the population of cells with sub-G1 DNA content, which is considered as a marker of apoptotic cell death. The apoptosis-inducing effect of compound K453 was also confirmed by annexin V/PI double staining and AO/PI staining. The apoptosis was associated with the loss of mitochondrial membrane potential, PARP cleavage, caspase-3 and caspase-9 activation, release of cytochrome c, as well as changes in the levels of Bcl-2 family members. Moreover, flow cytometry showed that compound K-453 stimulates phosphorylation of p38 MAPK but decreases phosphorylation of Akt and Erk 1/2. Activation of p38 MAPK was also confirmed using Western blot analysis. This analysis also revealed down-regulation of NF-κB1 (p50) and RelA (p65) proteins and the loss of their anti-apoptotic activity.


In our study compound K-453 exhibited an antiproliferative effect by induction of intrinsic apoptosis as well as modulation of several signaling pathways.

Keywords: Colorectal carcinoma, Indole phytoalexins, Antiproliferative, Apoptosis, Intrinsic pathway

Core tip: In this study we inform about our first results obtained from cell proliferation assays, flow cytometry analysis and Western blot analysis of HCT116 cells after treatment with compound K-453, which belongs to a group of synthetic derivatives of indole phytoalexins originally isolated from cruciferous vegetables. This study demonstrates that our compound induces the mitochondrial pathway of apoptosis and that the antiproliferative effect of our substance may be linked to inhibition of several signaling pathways. Our results provide a rationale for further in vivo anticancer efficacy studies.