Indole phytoalexin derivatives induce apoptosis in human colorectal carcinoma cells

[Speaker] Jan Mojzis:1
[Co-author] Viera Tischlerova:1, Martin Kello:1, Gabriela Mojzisova:2, Mariana Budovska:3, Ladislav Mirossay:1
1:Department of Pharmacology, University of P. J. Safarik, Faculty of Medicine, Slovakia, 2:Department of Experimental Medicine, Faculty of Medicine, Pavol Jozef Safarik University, Kosice, Slovakia, 3:Institute of Chemistry, Faculty of Science, Pavol Jozef Safarik University, Kosice, Slovakia

Background: The aim of the study was to investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives in human colorectal HCT116 cell line.
Methods: Changes in cell proliferation and the cytotoxic effect of the tested compounds were evaluated using MTS and BrdU assay. The apoptosis-inducing effect of compound K-453 was examined with annexin V/PI and acridine orange/propidium iodide (AO/PI) stainings. The flow cytometry method was used to measure cell cycle alterations and changes in factors associated with apoptosis, such as poly (ADP-ribose) polymerase (PARP), caspase-3 and -9, cytochrome c, Bcl-2 family proteins, and the integrity of the mitochondrial membrane. Activity of the transcription factors and signaling pathways proteins was evaluated by Western blot analysis.
Results: Among the ten tested compounds, K-453 exhibited the most potent activity with IC50 = 32.22 microM and was thus selected for further studies. Flow cytometric analysis revealed a K-453-induced increase in cell population with sub-G1 DNA content, which is considered as a marker of cell apoptosis. It was confirmed by annexin V/PI double staining and AO/PI staining. Apoptosis was associated with the loss of mitochondrial membrane potential, PARP cleavage, caspase-3 and -9 activation, cytochrome c release, and the levels of Bcl-2 family members changes. Moreover, K-453 stimulated phosphorylation of p38 MAPK (confirmed using Western blot analysis) but decreased phosphorylation of Akt and Erk 1/2. Down-regulation of NF-kappaB (p50) and RelA (p65) proteins and the loss of their anti-apoptotic activity was also observed.
Conclusion: K-453 exhibited an antiproliferative effect by modulation of several signaling pathways and induction of intrinsic apoptosis.
Acknowledgments: This study was supported by the Slovak Research and Development Agency under the contract no. APVV-16-0446 and the Grant Agency of Ministry of Education, Science, Research, and Sport of the Slovak Republic (VEGA 1/0018/16 and VEGA 1/0753/17).

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