Abstract
Purpose: To investigate the effect of N-(3-(1H-tetrazol-1-yl)phenyl) isonicotinamide derivative (TPIN) on prostate cancer cells, and the mechanism involved.Methods: The cytotoxicity of TPIN in DU145 and PC3 cells was determined using Cell Counting Kit-8, while apoptosis induction was assayed by flow cytometry using Annexin V-fluorescein isothiocyanate dye. Changes in expressions of F-actin, RAC-α and paxillin were determined by western blot assay.Results: Cell proliferation was effectively inhibited by TPIN in the concentration range of 0.75-15 μM. The values of half-minimum inhibitory concentration (IC50) of TPIN for DU145 and PC3 cells at 48 h were 5.6 and 10.2 μM, respectively (p < 0.05). Treatment with 5.6 μM TPIN increased apoptosis to 59.64 % in DU145 cells, and 54.21% in PC3 cells. Cleaved caspase-3 and caspase-9 levels were increased by TPIN treatment in both cell lines (p < 0.05). Moreover, the levels of F-actin and paxillin were significantly downregulated by TPIN treatment in DU145 and PC3 cells (p < 0.05). In TPIN-treated DU145 and PC3 cells, cofilin-1expression was up-regulated, relative to control cells.Conclusion: TPIN exhibits cytotoxic effect on prostate cancer cells via activation of apoptosis. It elevates cofilin-1 and the expressions of targets F-actin and paxillin in prostate cancer cells. Thus, TPIN is a potential chemotherapeutic agent for prostate cancer. However, further investigations, including clinical trials are required to authenticate these findings.
 Keywords: Prostate cancer, F-actin, Paxillin, Apoptosis, Caspases
Highlights
Prostate cancer, one of the highly malignant tumors, is the fourth highest cause of cancerrelated mortality in developed countries [1]
The proliferations of DU145 and PC3 cells were inhibited by TPIN in the concentration range of 0.75-15 μM
The proliferation of DU145 cells was decreased to 43 % on treatment with 15 μM TPIN at 24 h, while treatment with 15 μM TPIN suppressed PC3 cell proliferation to 51 % after 24 h
Summary
One of the highly malignant tumors, is the fourth highest cause of cancerrelated mortality in developed countries [1]. The cells were plated at 2 x105 cells/well and maintained for 24 h, followed by incubation with TPIN at doses of 0.75, 1.5, 3.0, 6.0, 12 and 15 μM at 37 ̊C for 48 h. The cells were seeded in well-plates at a density of 2 x105 cells per well, and treated with 5.6 μM TPIN, followed by incubation at 37 ̊C for 48 h, and washing in cold PBS. The cells were plated at a density of 2 x105 cells per well, and treated with 5.6 μM TPIN at 37 ̊C for 48 h They were incubated with reagents for caspase-3 and caspase-9 (100 μL) for 3 h at room temperature. The cells were lysed by treatment with RIPA buffer, and the insoluble protein lysate was centrifuged at 11,000 g for 35 min at 4 ̊C. Treatment at doses of 5.6 μM for 48 h enhanced PC3 cell apoptotic count to 54.21 %, when compared to 2.97 % in control
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