Impaired gap junctions in human hepatocellular carcinoma limit intrinsic oxaliplatin chemosensitivity: A key role of connexin 26

Abstract

Hepatocellular carcinoma (HCC) is generally believed to have low sensitivity to chemotherapeutic agents including oxaliplatin (OXA). Studies have demonstrated that gap junctions (GJs) composed of connexin (Cx) proteins have the potential to modulate drug chemosensitivity in multiple tumor cells. In the present study, we investigated the characteristics of Cx and GJs in HCC at both histologic and cytologic levels, and the effects of GJ and its effective components on OXA cytotoxicity in HCC cells in vitro. Immunohistochemistry was performed in 76 HCCs and 20 normal liver tissues to detect and locate the expression of Cx26, Cx32 and Cx43. At cytologic levels, the expression and localization of Cxs were evaluated by RT-PCR, western blot and immunofluorescence assay, respectively. The GJ function between adjacent cells was detected using dye transfer assay. The role of GJs in the modulation of OXA toxicity in HCC cells was explored using pharmacologic and molecular biologic methods. We found that Cx expression in HCC tissues was significantly lower than in normal liver tissues, and the 'internalization' from cell membrane to cytoplasm was remarkable. In vitro experiments revealed the presence of functional GJs in the SMMC-7721 HCC cells due to a small amount of Cx protein along the plasma membrane at cell-cell contacts. Regulation of this part of GJs positively influenced OXA cytotoxicity. Using RNA interference, only specific inhibition of Cx26 but not Cx32 or Cx43 reduced OXA cytotoxicity. Conversely, Cx26 overexpression by transfection of Cx26 plasmid DNA enhanced OXA cytotoxicity. This study demonstrated that during hepatocarcinogenesis, the reduced expression and internalization of Cx proteins impaired the GJ function, which further attenuated OXA cytotoxicity. Impaired GJ function may contribute to low intrinsic chemosensitivity of HCC cells to OXA, mediated by Cx26.

Downregulated connexin32 promotes EMT through the Wnt/β-catenin pathway by targeting Snail expression in hepatocellular carcinoma

“…Our previous study found that compared with normal liver tissue, the expression of Cx32 in HCC tissue is downregulated, and the positively expressed protein shows significant ectopic expression from the cell membrane to the cytoplasm (18). We further analyzed its relationship to clinicopathological characteristics and found that the expression of Cx32 was not correlated with age, sex, tumor size, TNM stage, background of liver disease, or vascular embolus (all P>0.05), but negatively correlated with histological grade and lymph node metastasis (all P<0.05) (Table III).…”

“…Archival normal liver (20 cases) and HCC (76 cases) paraffin blocks were collected as described in an earlier study (18) to determine the relationship of Cx32 expression to clinicopathological parameters. Another set of 34 cases of archival paraffin-embedded formalin-fixed HCC tissues were also collected at our institution from January 2014 to June 2015 to define the correlation between Cx32 and other indicators.…”

“…H&E and IHC staining was performed as previously described (18). The Cx32 immunoreactivity was performed using a combined scoring system based on the fraction of positive tumor cells and the predominant staining intensity in the tumors as described by Regidor et al (19).…”

“…The experimental procedure was performed according to our previous report (18). The concentrations of primary antibodies were used all at 1:200 (diluted with 2% BSA).…”

Cx32 mediates cisplatin resistance in human ovarian cancer cells by affecting drug efflux transporter expression and activating the EGFR‑Akt pathway

“…Recently, numerous studies have indicated that Cx contributes to chemoresistance in several types of tumour. Upregulated Cx26 contributed to gefitinib resistance in non-small cell lung cancer cells (10), while increased Cx26 expression was able to reverse oxaliplatin resistance in hepatocellular carcinoma (11). Upregulation of Cx43 led to the insensitivity…”