ClC-3 Promote Tumor Drug Resistance By Raising P-gp

Objective:To explore the role of Cl C-3(voltage-gated chloride channel 3) in drug-resistant tumor; to set a new direction for researches on Cl C-3 and provide theory basis for reverse of drugresistant tumor treatment, through determination on the fact that the regμlation of Pglycoprotein(P-gp) mediates drug-resistant tumor.Methods:(1) Western blot was used to detect Cl C-3 and P-gp in human lung adenocarcinoma cell line A549, paclitaxel-resistant cell line A549/T, human breast cancer cell line MCF-7 and adriamycin-resistant cell line MCF-7/ADR.(2) A549 and MCF-7/ADR cells were transfected with Cl C-3 small interfering RNA(Si-Cl C-3), which inhibited Cl C-3 expression; A549 and MCF-7 cells were transfected with Cl C-3 expression plasmid(pc DNA3.1/Cl C-3), which enhanced Cl C-3 expression; MTT assay was used to detect the effects of paclitaxel on proliferation of cells intervened by Cl C-3.(3) A549/T and MCF-7/ADR cells were transfected with Si-Cl C-3 while A549 and MCF-7 were transfected with pc DNA3.1/Cl C-3. Immunofluorescence was combined with laser confocal technology and Western blot to detect Cl C-3 and P-gp.(4) A549/T and MCF-7/ADR cells were transfected with multi-drug resistance gene 1(MDR1) small interfering RNA(Si-MDR1) to inhibit P-gp expression. A549 and MCF-7 cells were transfected with MDR1 expression plasmid(pc DNA3.1/ MDR1) to enhance P-gp expression. Immunofluorescence and Western blot were to detect Cl C-3 and P-gp.(5) MMTV-Py MT single transgenic mice were raised. MMTVPy MT/CLCN3 double transgenic mice with spontaneous breast tumor and overexpression of Cl C-3 were established and bred. The mice were given chemotherapy intervention with paclitaxel and adriamycin. According to paclitaxel chemotherapy regimens, the mice were divided into 4 groups, including M-N1 group(single transgenic mice given saline), M-PTX group(single transgenic mice given paclitaxel), MC-N1 group(double transgenic mice given saline) and MC-PTX group(double transgenic mice given paclitaxel); while according to adriamycin chemotherapy regimens, the mice were divided into 4 groups, including M-N2 group(single transgenic mice given saline), M-ADM group(single transgenic mice given adriamycin), MC-N2 group(double transgenic mice given saline) and MC-ADM(double transgenic mice given adriamycin); the mice were measured for body weight, tumor weight, tumor size and survival period.(6) Real-time PCR was used to detect m RNA expression of MDR1 and Cl C-3 in tumor tissues; Western blot and immunohistochemistry were used to detect protein expression of P-gp and Cl C-3.Resμlts:(1) With regarded to comparison between MCF-7/ADR and MCF-7 as well as between A549/T and A549, expression of Cl C-3 and P-gp were obviously higher in drug-resistant cell line than non-resistant cell line.(2) If down-regμlateed the expression of Cl C-3 in A549/T and MCF-7/ADR cells, the inhibition role of paclitaxel on cell proliferation was obviously increased; while if up-regμlateed the expression of Cl C-3 in A549 and MCF-7 cells, the inhibition role of paclitaxel on cell proliferation was obviously decreased.(3) Inhibition on the expression of Cl C-3 in A549/T and MCF-7/ADR cells leaded to downregμlation of P-gp expression, while enhancement on expression of Cl C-3 in A549 and MCF-7 cells leaded to up-regμlation of P-gp expression.(4) Inhibition on MDR1 expression in A549/T and MCF-7/ADR cells or enhancement on MDR1 expression in A549 and MCF-7 cells had no obvious effects on P-gp expression.(5) Tumor size and weight of MC-N1 group and MC-N2 group were significantly higher than those of M-N1 group and M-N2 group. For comparison between MC-PTX group and M-PTX group, inhibition rate of paclitaxel on tumor growth was decreased, while for comparison between MC-ADM group and M-ADM group, inhibition rate of adriamycin on tumor growth was also reduced. Paclitaxel treatment improved the survival rate of MMTV-Py MT single transgenic mice, but had no effect on MMTV-Py MT/ CLCN3 double transgenic mice.(6) Compared to MMTV-Py MT single transgenic mice, MDR1 m RNA and P-gp expressions were both increased in MMTV-Py MT/ CLCN3 double transgenic mice.Conclusion:There is a positive correlation between Cl C-3 and P-gp expression in tumor cells. Cl C-3 plays an important role in drug resistance of tumor cells. Its medication on drug-resistant tumor cells is related to up-regμlation on the expression of P-gp.