| Objective:To investigate the alterations of expression of connexin26(Cx26) and its gap junction(GJ) function in the process of hepatocellular carcinoma(HCC) development.Based on the above findings, the effect of modulation of Cx26 and its GJ function on antineoplastic activity of oxalipatin(OXA) in vitro was further explored. The study will give new insight into the mechanism of poor chemotherapy response of HCC.Methods:The expression and localization of Cx26 in 20 cases of normal liver tissue and 76 cases of HCC were detected by immunohistochemistry. A human normal liver cell line(LO2) and an Asian ethnic-origin HCC cell line(SMMC-7721) were further used to verify the histological results. RT-PCR and western blot analysis were conducted to detect the m RNA and protein expression of Cx26, immunofluorescence was performed to determine the localization of the protein, and dye transfer assay(also named “parachute assay”) was used to measure the GJ function between adjacent cells.Based on the findings of Cx26 and its composed GJ function alterations, MTT method was used to observe OXA antineoplastic activity in SMMC-7721 cells after modulation of GJ function by high and low cell density seeding, pharmacological manipulation and specific regulation of the expression of Cx26.Results:1. The alterations of Cx26 and its GJ function in the process of HCC development 1.1 Cx26 expression in normal liver and HCC tissues Immunohistochemistry analysis showed that all normal liver samples were positive for Cx26 expression, while the positive rate was markedly decreased in HCC tissues(χ2 =18.045,P=0.000). Cx26 staining in normal liver samples located mainly on the membranes of hepatocytes at intercellular contacts, however, positive staining for Cx26 in HCC tissues was observed mainly intracytoplasmically where no chance to form GJ channels between adjacent cells occurring. The translocation of protein from cell membrane to the cytoplasm called "internalization". The internalization rate of Cx26 in normal liver tissue and HCC group was 15% and 100%, respectively, and the differences between the two groups were significant(χ2 =43.938, P =0.000).1.2 Cx26 expression and its GJ function in normal liver cell line(LO2)and HCC cell line(SMMC-7721)RT-PCR and western blotting assay concurrently showed that the expression of Cx26 in LO2 cells was relatively higher than that in SMMC-7721 cells.Immunofluorescence assay demonstrated Cx26 located mainly on the membranes of LO2 cell at intercellular contacts, however, Cx26 in SMMC-7721 cells was observed mainly in the cytoplasm. Note is that a small amount of Cx26 protein along the plasma membrane at cell-cell contacts could also be detected in SMMC-7721 cells.For GJ function level, the intercellular dye coupling between adjacent cells was rich in LO2 cells and impaired in SMMC-7721 cells, as evidenced by the results of parachute assay.2. Effect of GJ function modulation on OXA antineoplastic activity in HCC cells2.1 OXA antineoplastic activity in SMMC-7721 cells MTT assay was used to observe cell survival of SMMC-7721 cells treated with oxaliplatin(0, 1, 2, 4, 8, 16, 32, 64, 128 μg/ml) for 24 h, 48 h, 72 h, respectively, result showed that survival rate decreased as the OXA concentration increased or treatment time prolonged. The anti-proliferative effect of OXA was obvious and moderate in 32, 64, 128 μg/ml, thus these three doses were used in the subsequent experiments.2.2 Influence of GJ formation on OXA antineoplastic activity By seeding cells at high or low density, confluent cells(tight contact with each other, with GJ formation) and nonconfluent cells(can not contact with each other,with no GJ formation) were obtained respectively. Results showed that the antineoplastic effect of OXA treatment for 24 h in SMMC-7721 cells at high density group was substantially greater than that at low density group(P<0.05).2.3 Influence of GJ modulation by pharmacological manipulation on OXA antineoplastic activity2.3.1 Effect of pharmacological tool drugs on GJ function in SMMC-7721 cells Parachute assay showed that the intercellular dye coupling through GJs in SMMC-7721 cells increased when the cells treated with 10 μM all-transretinoic acid(ATRA) for 24 h, with the enhancement rate of 72%. And the intercellular dye coupling through GJs decreased when the cells treated with 5 μM 18-α-glycyrrhetinic acid(18-α-GA) for 1 h, with the inhibition rate of 63%.2.3.2 Effect of GJ modulation by pharmacological tool drugs on OXA antineoplastic activity The results showed that, GJ potentiator ATRA itself at the concentration of 10μM for 24 h exerted no cytotoxic effect on the cell survival, and its pretreatment for24 h before OXA(64 μg/ml) application, added a substantial portion of the toxicity of OXA(P<0.05). Meanwhile, GJ inhibitor 18-α-GA itself at the concentration of 5 μM for 1 h exerted no cytotoxic effect on the cell survival, and its pretreatment for 24 h caused an increased cell survival compared to that of OXA treatment alone(P<0.05).2.4 Influence of specific regulation of Cx26 expression on OXA antineoplastic activity The results of using si RNA interference technology to inhibit the expression of Cx26 transiently in SMMC-7721 cells showed that, cell survival by OXA treatment was increased after Cx26 expression inhibition(P<0.05). To the contrary, the results of using c DNA transfection technology to enhance the expression of Cx26 transiently in SMMC-7721 cells showed that, cell survival by OXA treatment was decreased after Cx26 overexpression(P<0.05).Conclusion:1. Cx26 expression downregulation and abnormal localization, leading to impairment of GJ, are associated with HCC development.2. Enhancement of GJ function can significantly improve OXA antineoplastic effect in SMMC-7721 cells, while its inhibition can reduce OXA antineoplastic effect in SMMC-7721 cells. |
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