GlyH-101 and CaCCinh-A01 Inhibited HT-29 Proliferation by Activating the Mitochondrial Apoptosis Pathway and Arresting the Cell Cycle

Background/Aim: GlyH-101 and CaCCinh-A01 are effective blockers of cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated chloride channels (CaCCs), respectively. Available evidence suggests that GlyH-101 and CaCCinh-A01 can suppress cell proliferation, block invasion and metastasis, and cause several cancer cell types to undergo apoptosis, demonstrating their anti-tumor properties. The aim of this study was to investigate the effect of GlyH-101 and CaCCinh-A01 on HT 29 cell activity and to suggest the possible molecular mechanisms by which inhibitors of CFTR and CaCCs inhibit HT-29 cell activity. Materials and Methods: Human colon HT-29 cancer cells were treated with GlyH-101 or CaCCinh- A01 or GlyH-101 plus CaCCinh-A01 complex. Cell viability was determined by MTT assay, the apoptosis and cell cycle were determined by flow cytometry, and reactive oxygen species (ROS) leves were determined by 2',7'Dichlorodihydrofluorescein diacetate staining. The expression of proteins related to apoptosis and cell cycle regulation was measured by western blotting. Results: The proliferative ability of HT-29 cells was dose-and time-dependently reduced by GlyH-101 and CaCCinh-A01. Treatment with GlyH-101 and CaCCinh-A01 resulted in cell necrosis and apoptosis, up-regulated ROS levels, activated the mitochondrial apoptosis pathway, prompted arrest of the cell cycle in S phase, and increased the levels of proteins related to the cell cycle. Additionally, the combination of these two inhibitors had a stronger regulatory effect on HT-29 cell proliferation than either GlyH-101 or CaCCinh-A01 treated alone. Conclusion: GlyH-101 and CaCCinh-A01 inhibited cell proliferation through cell cycle arrest and mitochondrialrelated pathways in vitro. The combination of these inhibitors could further enhance their anti-proliferative effects. Our findings propose new lead compounds with anti-colon cancer activity, and also provide new evidence for the effectiveness of chloride channels-targeted therapy in anticancer therapy.