Abstract
Purpose: To determine the apoptotic effect of trichlorophenyl-benzoxime (TCPB) on colorectal cancer (CRC) cells, and to elucidate the mechanism of action.
 Methods: Colon carcinoma cell lines (DLD-1 and HT-29) were used in this study. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin at 37 ˚C in an atmosphere of 5 % CO2 and 95 % air. When the cells attained 60 - 70 % confluency, they were treated with serum-free medium and graded concentrations of TCPB (1.0 – 6.0 μM) for 24 h. Cell viability and apoptosis were assessed using 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) and flow cytometric assays, respectively. Western blotting and 2', 7' dichlorofluorescein diacetate (DCFH DA) assays were used for the determination of expression levels of apoptotic proteins, and levels of reactive oxygen species (ROS), respectively.
 Results: Treatment of DLD-1 and HT-29 cells with TCPB led to significant and dose-dependent reductions in their viability, as well as significant and dose-dependent increases in the number of apoptotic cells (p < 0.05). Treatment of HT-29 cells with TCPB led to significant increases in the population of cells in the G0/G1 phase, but significant reduction of cell proportion in S and G2/M phases (p < 0.05). It also significantly and dose-dependently upregulated the expressions of caspase-3 and bax, down-regulation of the expression of bcl-2 (p < 0.05). TCPB treatment upregulated the expressions of p53, cytochrome c (cyt c), procaspase-3, and procaspase-9, but down-regulated the expression of pAkt dose-dependently (p < 0.05). The expression of Akt in HT-29 cells was not significantly affected by TCPB (p > 0.05). However, TCPB significantly enhanced the cleavage of PARP1, and significantly and dose-dependently increased the levels of ROS in HT-29 cells (p < 0.05).
 Conclusion: These results suggest that TCPB exerts apoptotic effect on CRC cells via activation of mitochondria-dependent pathway, and thus can be suitably developed for the management of colon cancer.
Highlights
EXPERIMENTALColorectal cancer (CRC) is the third most common cancer of the gastrointestinal tract (GIT), and one of the most frequently diagnosed malignancies of the intestines [1,2]
The present study investigated the apoptotic effect of TCPB on CRC cells, and the underlying mechanism
The results of MTT assay showed that treatment of DLD-1 and HT-29 cells with TCPB significantly and dose-dependently reduced their viability
Summary
Colorectal cancer (CRC) is the third most common cancer of the gastrointestinal tract (GIT), and one of the most frequently diagnosed malignancies of the intestines [1,2]. Death receptors on surfaces of cells are involved in the induction of apoptosis via the extrinsic pathway [9]. These surface receptors elicit their effects by activating and upregulating the expression of caspase-8 [9]. The intrinsic pathway of apoptosis involves several stress stimuli which change the level of bcl-2 protein resulting in the release of cytochrome c from mitochondria. TCPB (1.0 – 6.0 μM) was added to the cells and incubated for 72 h. The cells treated with TCPB (1.0 – 6.0 μM) were washed with PBS after an initial incubation for 72 h. Values of p < 0.05 were considered statistically significant
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