Correlation Of Protein Kinase C Activity, Expression And Subcellular Distribution And Multidrug Resistance In KBV200 Cells

OBJECTIVE: Multidrug resistance (MDR) to chemotherapy is a majorobstacle to successful cancer treatment. The tumor often becomes resistant not only to the drug used in the therapy, but also to a broad spectrum of unrelated cytotoxic drugs as well. The mechanisms of MDR are complicated in tumors. It has been demonstrated that PKC may play an important role in MDR, especially the involvement of PKC a . But the mechanism of PKC regulating MDRremains unclear. In this study, the author investigated PKC activity, expression and subcellular distribution in drug-sensitive KB and multidrug-resistant KBV200 cells. The aim of the study was to elucidate the importance andmechanism of PKC in MDR, and to provide new ideas and clues to seek new effective methods for preventing and reversing the MDR in tumor as well.METHODS: The cell inhibitory rate was evaluated by MTT. Concentrationof protein was measured by Bradford method. PKC activity was assayed by the measurement of the incorporation of 32P from [ Y -32P]ATP into peptide substrates. Western blot was used for the assessment of PKC isoform expression. Fluorescence intensity(FI) of PKC isoforms was determined by flow cytometry(FCM). PKC activitor PMA and PKC inhibitor SP were used to preincubate KBV200 cells to evaluate the importance of PKC in MDR. And SPSS 8.0 was used to analyze data and make charts.RESULTS: The values of IC50 of vincristine(VCR) and adriamycin(ADR) in KB cells were 21.16 nmol/L and 5.82 nmol/L , and those in KBV200 cells were 1375.97 nmol/L and 115.26 nmol/L respectively^ < 0.01, vs KB cells). The resistance indexes of VCR and ADR were 65.03 and 19.8 respectively. PKC activity of membrane and cytosol fraction was higher in KBV200 cells than in KB cells. Total PKC activity in KBV200 cells was1.12-fold higher than that in KB cells. And the percentage of PKC activity of membrane fraction was higher in KBV200 cells than that in KB cells(40.41 % vs 30.34%). It was found that the level of PKC a expressed on membrane andcytosol fraction in KBV200 cells was higher than that in KB cells. The PKC (3 and e expression in KBV200 cells had no significant different from those in KB cells. No PKC Y and C expression was found in KB and KBV200cells. The FI of PKC a, which was higher than those of PKC P and e, was2.12-fold higher in KBV200 cells than in KB cells.In order to elucidate the role of PKC a in MDR, the KBV200 cells weredivided into three groups: Control group (no activitor or inhibitor), PMA group( 200 nmol/L PMA preincubation ) and SP group ( 100 nmol/L SPpreincubation). The PKC activity of total and membrane fraction were higherin PMA group than those in control group, while PKC activity of cytosolfraction was lower. PKC a expression was upregulated in membrane fraction and downregulated in cytosol fraction in KBV200 cells after PMA preincubation. PKC P expression was higher slightly in cytosol fraction but no alteration in membrane fraction in PMA group compared with control group. PKC e expression was not changed by PMA preincubation. The FI of PKC a in PMA group was as 6.91-fold high as that control group. Thepercentage of PKC a positive cells was increased after PMA preincubation. The values of IC50 of VCR and ADR in PMA group were increased to 2275.5 nmol/L and 233.25 nmol/L respectively(P < 0.01, vs control).The PKC activity of membrane and cytosol fraction was lower in SP group than that in control group(P < 0.01). PKC a expression was downregulatedespecially in membrane fraction after SP preincubation. PKC P expression was increased slightly in cytosol fraction but disappeared in membrane fraction and PKC ?expression was not altered in SP group. After SP preincubatin, thepercentage of PKC a positive cells was lowered and the FI of PKC a wasdecreased to 19.88% of that in control group. The percentage of positive cellsand FI of PKCP and e were not altered by SP preincubation. The values of IC50 of VCR and ADR in SP group were decreased to 251.8 nmol/L and 58.69 nmol/L re...