Roles Of CFTR And Ca²⁺-activated Chloride Channel In The Proliferation And Migration Of HT-29 Cell

Cancer severely threatens human's health and lives.It has been estimated that there are more than 14 million new cases each year.Recurrence and metastasis are the main causes of cancer death.Tumor cell proliferation and migration,complex processes involved in various factors and regulators,are critical for recurrence and metastasis.In recent years,reports indicated that expression and/or function of cystic fibrosis transmembrane conductance regulator?CFTR?and Ca²⁺-activated chloride channel?CaCC?were closely related to metastasis in different kinds of cancers,while precise mechanisms remains unclear.CFTR is a cAMP-dependent chloride channel that plays key roles in transepithelial salt and fluid transport.CaCC is ubiquitously expressed in various cells including epithelial cells,vascular endothelial cell and smooth muscle cells,and has many physiological functions.In the present study,CFTR and CaCC selective inhibitors(GlyH101 and CaCC_inh-A01)were used to systematically study roles of CFTR and CaCC in proliferation and migration of the colonic adenocarcinoma HT-29 cells.The results showed that :?1?Both GlyH101 and CaCC_inh-A01 time-and dosedependently inhibited the proliferation of HT-29 cells as shown in the MTT assay,with IC50 values of 9.11 ± 0.13?M ? 32.88 ± 0.08?M,respectively.Further study showed that the two compounds had additive inhibitory effect in HT-29 proliferation.?2?PI staining analysis showed that GlyH101 and CaCC_inh-A01 could induce S phase arrest in HT-29 cell;following western blotting study indicated that these compounds upregulated the expression of cell cycle related proteins of CDK4,CDK6,CyclinD1 and CyclinE.?3?Hochest 33342 staining results indicated that GlyH101 and CaCC_inh-A01 could cause significant nuclear condensation,DNA fragmentation and apoptotic body formation in HT-29 cells;Annexin V-FITC/PI staining study showed that these compounds could induce apoptosis cell death in HT-29 cells;Western blotting results showed that the compounds downregulated the expression of Bcl-2,upregulated the expression of Bax,caspase-8,cleaved-caspase-3 and PARP.?4?Cell wound healing study showed GlyH101 and CaCC_inh-A01 had little effect on HT-29 cell migration.In conclusion,the present study showed that CFTR and CaCC selective inhibitors GlyH101 and CaCC_inh-A01 could inhibit proliferation in HT-29 cells via apoptosis and cell cycle arrest pathways.Induction of apoptosis in HT-29 cells may involve in mitochondriarelated apoptotic pathway.Our finding with the others reports confirmed that chloridechannels are closely related to the proliferation of tumor cells,and thus providing new molecule targets for the therapy of cancer.