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The roles of gap junctions (GJs) and its components, connexins, in the autophagy of cervical cancer cells have been rarely investigated. Our previous study demonstrated that connexin 32 (Cx32) exerted an anti‑apoptotic effect on cervical cancer. However, as an important regulator of apoptosis, whether the autophagy is involved in the function of Cx32 on cervical cancer cells is not well defined. The present study aimed to investigate the role of Cx32 on autophagy and apoptosis inhibition in cervical cancer cells. The expression levels of Cx32 and the autophagy‑associated protein LC3‑Ⅱ in paracancerous cervical tissues (n=30) and cervical cancer (n=50) tissues were determined via western blotting. In total, 45 cervical cancer specimens were used to evaluate the clinical relevance of Cx32 and LC3‑Ⅱ. It was found that both Cx32 and LC3‑Ⅱ were upregulated in cervical cancer tissues compared with those in paracancerous cervical tissues. The effect of Cx32 on autophagy was examined by detecting the change of LC3‑Ⅱ using western blotting, transfection with enhanced green fluorescent protein‑LC3 plasmid and transmission electron microscopy analysis. Overexpression of Cx32 significantly enhanced autophagy in HeLa‑Cx32 cells, whereas knockdown of Cx32 suppressed autophagy in C‑33A cells. The flow cytometry results demonstrated that Cx32 inhibited the apoptosis of cervical cancer cells by promoting autophagy. Moreover, Cx32 triggered autophagy via the activation of the AMP‑activated protein kinase (AMPK) signalling, regardless of the presence or absence of GJs. Collectively, it was identified that Cx32 exerted its anti‑apoptotic effect by activating autophagy via the AMPK pathway in cervical cancer, which demonstrates a novel mechanism for Cx32 in human cervical cancer progression.

Necroptosis is an alternative form of programmed cell death that generally occurs under apoptosis-deficient conditions. Our previous work showed that connexin32 (Cx32) promotes the malignant progress of hepatocellular carcinoma (HCC) by enhancing the ability of resisting apoptosis in vivo and in vitro. Whether triggering necroptosis is a promising strategy to eliminate the apoptosis-resistant HCC cells with high Cx32 expression remains unknown. In this study, we found that Cx32 expression was positively correlated with the expression of necroptosis protein biomarkers in human HCC specimens, cell lines, and a xenograft model. Treatment with shikonin, a well-used necroptosis inducer, markedly caused necroptosis in HCC cells. Interestingly, overexpressed Cx32 exacerbated shikonin-induced necroptosis, but downregulation of Cx32 alleviated necroptosis in vitro and in vivo. Mechanistically, Cx32 was found to bind to Src and promote Src-mediated caspase 8 phosphorylation and inactivation, which ultimately reduced the activated caspase 8-mediated proteolysis of receptor-interacting serine-threonine protein kinase 1/3, the key molecule for necroptosis activation. In conclusion, we showed that Cx32 contributed to the activation of necroptosis in HCC cells through binding to Src and then mediating the inactivation of caspase 8. The present study suggested that necroptosis inducers could be more favorable than apoptosis inducers to eliminate HCC cells with high expression of Cx32., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Our previous study demonstrated that connexin 32 (Cx32) was upregulated and redistributed to the cytoplasm in A2780 human ovarian cancer cells with acquired resistance to cisplatin; this increased Cx32 feedback promoted cisplatin resistance. To further investigate the mechanism underlying Cx32‑mediated cisplatin resistance, alterations in drug transporters, the DNA repair system and the anti‑apoptotic signalling pathway were investigated by overexpressing or knocking down Cx32 in parental cells (A2780); cisplatin‑resistant human ovarian cancer cells (A2780/CDDP) were also acquired. Upregulation of efflux transporters [multi‑drug resistance protein 2 (MRP‑2), ATPase copper transporting α (ATP7A) and ATPase copper transporting β] and downregulation of the influx transporter copper uptake protein 1 mediated cisplatin resistance in A2780/CDDP cells. A2780/CDDP cells also exhibited increased expression of the DNA repair enzyme excision repair cross‑complementation group 1 (ERCC1) and activation of the epidermal growth factor receptor (EGFR) signalling pathway. Small interfering RNA‑mediated knockdown of Cx32 in A2780/CDDP cells decreased the expression of efflux transporters (MRP‑2 and ATP7A). Knockdown of Cx32 in A2780/CDDP cells also decreased the expression of ERCC1, inhibited the activation of the EGFR signalling pathway and enhanced the cytotoxicity of cisplatin. When Cx32 was overexpressed in A2780 cells, an opposite effect on the expression of efflux transporters (MRP‑2 and ATP7A) and the activation of the EGFR signalling pathway was observed, which resulted in insensitivity to cisplatin‑induced apoptosis. Thus, Cx32 expression may induce cisplatin resistance by modulating drug efflux transporter expression and activating the EGFR‑protein kinase B signalling pathway in ovarian cancer cells.