| Objective:Hepatocellular carcinoma is one of the most commonly malignant neoplasms. Multidrug resistance (MDR) of hepatocellular carcinoma is the dominant factor against to chemotherapy. P-glycoprotein was one of mechanisms of MDR and its activastion is always depends on protein kinase C (PKC). There was a relationship between PKC a and the expression of P-glycoprotein (P-gp). The present study was to investigate the induction of Ca2+on PKCa and the activation of P-gp in Human drug resistant hepatocellular carcinoma cells BEL-7402/ADM.Methods:1.Groups dividing:1.1For the experiments of laser scanning confocal microscopy, the Human hepatocellular carcinoma cell BEL-7402and the drug resistant hepatocellular carcinoma cell BEL-7402/ADM were divided into six groups, BEL-7402, BEL-7402+TMP, BEL-7402+VRP, BEL-7402+IP3, BEL-7402/ADM, BEL-7402/ADM+TMP, BEL-7402/ADM+VRP and BEL-7402/ADM+IP3. For other experiments, BEL-7402and BEL-7402/ADM were divided into ten groups:BEL-7402, BEL-7402+TMP, BEL-7402+VRP, BEL-7402+TMP+IP3, BEL-7402+VRP+IP3, BEL-7402/ADM, BEL-7402/ADM+TMP, BEL-7402/ADM+VRP, BEL-7402/ADM+TMP+IP3and BEL-7402/ADM+VRP+IP3.2. Methods:2.1To investigate the changes of intracellular calcium in BEL-7402/ADM. Treated with/without Inositol-triphosphate (IP3), Verapamil (VRP) or Tetramethypyrazine (TMP), the average intensity of the fluorescence of intracellular Ca2+was detected by laser scanning confocal microscope (LSCM) in both BEL-7402and BEL-7402/ADM.2.2To investigate the induction of calcium on protein kinase Ca in BEL-7402/ADM, MTT assay was used to detect the IC50value of the ten groups; Immunofluorescence microscope was used to detect the Immunofluorescence staining of PKCa of the ten groups demonstrated above. Fluorescence microscope, flow cytometry (FCM) assay and high performance liquid chromatography (HPLC) were used to detect the accumulation of Doxorubicin in the ten groups, respectively.Results:(1) The average intensity of the fluorescence of intracellular Ca2+of the ten groups was collected. The index for BEL-7402was574.08±3.30and the index for BEL-7402/ADM was665.16±2.89there is a significant difference between the two group (P<0.05), while the index of BEL-7402, BEL-7402+IP3, BEL-7402+TMP, BEL-7402+VRP, BEL-7402/ADM, BEL-7402/ADM+IP3, BEL-7402/ADM+TMP and BEL-7402/ADM+VRP were777.78±6.35,479.17±5.83,478.26±4.08,800.00±8.45,541.67±8.15and522.73±7.23respectively. There was a significant difference between parental and resistant groups, and cells with drugs and control groups (P<0.05)(2) The IC50value for BEL-7402, BEL-7402+TMP, BEL-7402+VRP, BEL-7402+TMP+IP3, BEL-7402+VRP+IP3, BEL-7402/ADM, BEL-7402/ADM+TMP, BEL-7402/ADM+VRP, BEL-7402/ADM+TMP+IP3, BEL-7402/ADM+VRP+IP3cells were56.68±0.21,28.58±0.43,37.95±0.29,29.52±0.37,41.35±0.52,74.03±0.30,48.38±0.18,51.62±0.34,48.04±0.13,56.06±0.12, respectively. There was a significant difference between parental and resistant groups, and cells with/without drugs (P<0.05).(3) Compared with the BEL-7402, the intensity of green fluorescence of PKCa in the BEL-7402+TMP and BEL-7402+VRP was obviously decreased. Compared with BEL-7402+TMP and BEL-7402+VRP respectively, the intensity of the BEL-7402+TMP+IP3and BEL-7402+VRP+IP3was obviously increased. The intensity of green fluorescence of PKCa of BEL-7402/ADM, BEL-7402/ADM+TMP, BEL-7402/ADM+VRP, BEL-7402/ADM+TMP+IP3and BEL-7402/ADM+TMP+IP3was consistented with the tendency of five groups of the same treatment of BEL-7402. Compared with used the same treatment of the BEL-7402, the intensity of green fluorescence of BEL-7402/ADM group was obviously stronger than former.(4) Compared with the BEL-7402, the intensity of red fluorescence of Doxorubicin in the BEL-7402+TMP and BEL-7402+VRP was obviously increased. Compared with BEL-7402+TMP and BEL-7402+VRP, respectively, the intensity of the BEL-7402+TMP+IP3and BEL-7402+VRP+IP3was obviously decreased. The tendency of the intensity of the fluorescence of ADM in BEL-7402/ADM, BEL-7402/ADM+TMP, BEL-7402/ADM+VRP, BEL-7402/ADM+TMP+IP3and BEL-7402/ADM+TMP+IP3were consistented with the tendency of five groups of the same treatment of BEL-7402. Compared with the same treatment of the BEL-7402, the intensity of red fluorescence of BEL-7402/ADM group was obviously weaker than the former.(5) The percentage of the cells in which Doxorubicin accumulated by FCM analysis. Compared with total cells number, the percentage of BEL-7402cells group was64.75+3.13, the percentage of BEL-7402+TMP group was71.75±1.69, the percentage of BEL-7402cellls treated with VRP group ws77.13±2.97, the percentage of BEL-7402+TMP+IP3was66.25±3.25, the percentage of BEL-7402+VRP+IP3was67.14±1.89, the percentage of BEL-7402/ADM cells was43.15±4.97, the percentage of BEL-7402/ADM+TMP was49.55±1.96, the percentage BEL-7402/ADM+VRP was59.05±2.39, the percentage of BEL-7402/ADM+TMP+IP3was47.55±4.69and the percentage BEL-7402/ADM fore-treated with VRP than treated with IP3were48.25±1.48. There was a significant difference between parental and resistant groups, and cells with/without drugs (P<0.05).(6) HPLC analysis the concentration of the ten groups cells accumulation of hydroxyldaunorubicin were:the concentration of BEL-7402, BEL-7402+TMP, BEL-7402+VRP, BEL-7402+TMP+IP3, BEL-7402+VRP+IP3was0.73±0.18μg/mL,1.75±0.41μg/mL,1.89±0.44μg/mL,1.29±0.30μg/mL, and0.96±0.23μg/mL, respectively. The concentration of BEL-7402/ADM, BEL-7402/ADM+TMP, BEL-7402/ADM+VRP, BEL-7402/ADM+TMP+IP3and BEL-7402/ADM+VRP+IP3was0.65±0.16μg/mL,1.43±0.34μg/mL,1.71±0.40μg/mL,0.83±0.20μg/mL and1.09±0.26μg/mL, respectively. There was a significant difference between parental and resistant groups, and cells with/without drugs (P<0.05).Conclusion:Ca2+can induce PKCa in Human drug resistant hepatocellular carcinoma cells BEL-7402/ADM. |
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