Connexin32 activates necroptosis through Src-mediated inhibition of caspase 8 in hepatocellular carcinoma.

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.

Gap junctions enhance the antiproliferative effect by transfer of microRNAs in glioma cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the permeability of gap junction composed of connexin 43 (Cx43) for microRNAs (miRNAs) and the impact of gap junction-mediated transfer of miRNAs in glioma U87 cells. METHODS Co-culture assay demonstrated the transmission of miRNAs through gap junction channel into adjacent cells. U87 cells were labeled with green fluorescein protein (GFP) as receivers and cells were transfected miRNAs as donors. Receiver cells and donor cells were mixed together in a ratio of 1:1. After 12 h co-culture, cells were separated using a BD influx flow cytometer based on the GFP labeled. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied detect to the expressions of miRNAs and Cx43 mRNA. Western blotting was performed to detect the protein expressions of Cx43 and GFP in U87 cells. CCK-8 assay is used to detect cell growth. RESULTS Co-culture assays demonstrated miR-34a could transfer between U87 cells. The role of the contact independent could also transfer of miR-34a. Gap junctions inhibitor (CBX and 18-α-GA) showed lower miR-34a expression than co-culture group, whereas gap junctions enhancer (RA and Galanglin) enhanced miR-34a expression. Knockdown of Cx43 could significantly decrease the transferring of miR-34a between U87 cells. Different length of miRNAs (miR-1827, miR-144, miR-203a and miR-1183) were similar to the expression of miR-34a between U87 cells. Additionally, we demonstrated that gap junctions mediate the effect of antiproliferation mediated by miR- 34a in U87 cells. The functional inhibition of gap junctions using either siRNA or inhibition eliminated the miR- 34a mediated antiproliferation, whereas the enhancement of gap junctions treatment augmented this miR34a-mediated antiproliferation. CONCLUSION Our study demonstrates that gap junction composed of Cx43-mediated transfer miRNAs in different length of nucleotides and gap junction-mediated transfer of miR-34a enhance the antiproliferative effect in glioma U87 cells.