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

Sicheng Guo; Hanyu Yan; Liqin Wang; Yue Liu; B O Yu

doi:10.21873/anticanres.16523

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

Anticancer Res. 2023 Aug;43(8):3471-3477. doi: 10.21873/anticanres.16523.

Authors

Sicheng Guo¹, Hanyu Yan¹, Liqin Wang¹, Yue Liu¹, B O Yu²

Affiliations

¹ Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, School of Life Sciences, Liaoning Normal University, Dalian, P.R. China.
² Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, School of Life Sciences, Liaoning Normal University, Dalian, P.R. China yubo821208@163.com.

PMID: 37500166
DOI: 10.21873/anticanres.16523

Abstract

Background/aim: GlyH-101 and CaCC_inh-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 CaCC_inh-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 CaCC_inh-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 CaCC_inh-A01 or GlyH-101 plus CaCC_inh-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 CaCC_inh-A01. Treatment with GlyH-101 and CaCC_inh-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 CaCC_inh-A01 treated alone.

Conclusion: GlyH-101 and CaCC_inh-A01 inhibited cell proliferation through cell cycle arrest and mitochondrial-related 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.

Keywords: CaCCinh-A01; GlyH-101; HT-29; apoptosis; cell cycle.

MeSH terms

Apoptosis
Cell Cycle
Cell Cycle Checkpoints
Cell Proliferation
Chloride Channels* / metabolism
Cystic Fibrosis Transmembrane Conductance Regulator* / pharmacology
HT29 Cells
Humans
Reactive Oxygen Species / metabolism

Substances

Chloride Channels
Cystic Fibrosis Transmembrane Conductance Regulator
6-t-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahydrobenzo(b)thiophene-3-carboxylic acid
N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide
Reactive Oxygen Species