The Study Of Reversal Of Multidrug Resistance In Human Renal Cell Carcinoma By Inhibition Agents Of Protein Kinase C Alpha

ObjectiveProtein Kinase C is one of the earliest established Protein Kinase, made up of serine/threonine protien kinase family, whose construction close to each other, on which calcfium and phosphatide rely, activated by diacylglycerol(DG). PKC adjust different function of various kinds of cells, including tumor cell, though serine/threonine phosphorylation. PKC has important effect on cellular growth, differerntiate and such of signal conductions. For the past few years, it is known that participation of PKC in tumorous development and growth is key point factor of tumour cell adhesion, motion,invasion and transfusion.Study on cDNA of isozymes of PKC indicate that the polypeptide chain N-terminal sequence is regulatory domain, and the carboxyl terminal is catalytic domain is exposed. There is a hinge domain which can be hydrolyzed by protease between them. When enzyme were activated, the hinge domain is degradated so thar PKC active site. The difference between pseudosubstrate site in regulatory domain and true substrate is that alanina in phosphorylation site displace serine/threonine residue, and close enzyme activation center instead of phosphorylation.PKC usually reside with inactivestyle in endochylema under quiescent condition. With surrounding stimulation, PKC is activated because of translation to cell membrane. Temporal PKC activation seems important effect in short-term events,(such as ion inflow, secrete, and so on), which induced in signal conduction access. Persistent PKC activation seems consequence effect in cell multiplication, differentiate, tumorigenesis and so on. PKC is tumor promoter phorbol 12-myristate-13-acetate intracellular receptor. Its over-expression induces cell growth disorder and triggers tumorous form.Efferth et al. investigate PKC express in 18 kinds of human renal cell carcinoma system with immmuocytochemistry. There are intimate correlations between PKC high express and drug resistances of cancer cell for Adriamycin and P glucoprotein. PKC participate renal cell carcinoma endogenous drug resistances though PKC phosphorylation.PKC generally exist in animal, microbeorganism, and present in overall tissue and organ. There are four categories and thirteen subtypes in PKC based on different grades in isozyme construction, enzymic character, activating agent. Classical PKC(cPKC):α,βⅠ,βⅡ,γ,which are activated by calcium, phosphatide, diacyglycerol(DAG) or phorbol ester. Novel PKC(nPKC):ι/η,δ,ε,θ, which are activated by phosphatide, diacyglycerol(DAG) or phorbol ester instead of calcium. Atypical PKC(aPKC):Ï„/λ,ξ, which are only activated by phospholipin. Recently a kind of PKC is found, which is activated by phosphatidylinositol-4,5-diphosphate instead of calcium and DAG and is similar to the others in construction. So this kind of PKC is called the fourth kind of PKC. Various kinds of isozyme have different specificity of distribution and effects.We use research in vitro to discuss mechanism of MDR in renal cell carcinoma at the PKC subtype level, to provide the experiments about treating renal cell carcinoma.The experiment divides three parts:One: To investigated the expression of PKCαin membrane and plasma of 30 cases renal cell carcinoma and normal adjacent tissue by Western blotting technology.Two: LR\BR recombination reactions were used to generate mammalian expression vectors of protein kinase C alpha cDNA with C-terminal fused GFP, and vectors were transfected into human RCC cell with Lipofectimine 2000. RT-PCR,Western Blot and Inverted Fluorescent Microscope were used to determine the expression of PKCαgene in RCC cells transferred by PKCαcDNA. RT-PCR were used to determine the expression of MDR related gene MDR1,MRP1,LRP in RCC cells transferred by PKCαcDNA.Three: To identify the alteration of Multidrug resistance-related genes of PKCα/786-0 cells with and without PMA or Calphostin C treatment, MDR1 was detected using RT-PCR. To elucidate a putative synergistic effect of the PKCαexcitomotor or inhibitor and ADM used for RCC therapy, PKCα/786-0 cells were cultured in the presence of ADM together with different concentrations of Calphostin C or PMA, Cell viability analysis and the cytotoxity of ADM by MTT reduction assay.MethodsExpression and significance of PKCαin renal cell carcinoma and normal adjacent tissueTo investigated the expression of PKCαin membrane and plasma of 30 cases renal cell carcinoma and normal adjacent tissue by Western blot technology.Expression of PKCαin membrane and plasma of renal cell carcinoma or normal adjacent tissue.Compared to that of adjacent normal tissue, the expression of PKCαin plasma is decreased significantly, while its expression in membrane is increased significantly and the ratio of membrane/plasma increases significantly(P<0.01). The difference has statistical significance. In membrane of renal cell carcinoma tissue, the expression of PKCαis increased with the elevation of pathological stages and cell grading of tumor(P<0.01); while in plasma of tumor, the expression of PKCαis decreased with the elevation of pathological stages and cell grading of tumor(P<0.01), and the ratio of membrane/plasma increases significantly.Cells culture786-0, was maintained in RPMI-1640 medium, supplemented with 10% fetal bovine serum(FBS), 2mM glutamine, 25mM HEPES at 37℃in a humidified, 5% CO₂ incubator.Plasmid constructThe PKCαcDNA was subcloned into pDONR^TM 201 donor vector by PCR with Pyrobest DNA Polymerase using BP Clonase Reaction system. Entry clone was identified by DNA sequencing analysis. pcDNA-PKCα-GFP recombinant plasmid expressing PKCαwith C-terminal fused Green Fluorescent Protein(GFP) was constructed with entry clone and GFP expression vector pcDNA-DEST47 using Gateway LR recombination reaction system. primers were designed as follows: 5'-ATG GCT GAC GTT TTC CCG GGC AA-3'; 5'-CAT ACT GCA CTC TGT AAG ATG GGG-3'.Cell transfectionThe cells were transfected with pcDNA-PKCα-GFP vector by Lipofectimine2000 reagent. Positive clones were identified by GFP fluorescence microscopy.Selection of stable expressionThe stable expression clones(786-0/PKCα-GFP) were established by the selection with G418 at a concentration of 400μg/ml for four weeks. clone Isolated clones were analyzed for PKCα-GFP expression by western blot, RT-PCR analysis and fluorescence microscopy.Multidrug resistance correlated genes assaysTo identify the alteration of Multidrug resistance correlated genes, MDR1, MRP1, LRP gene were assayed using RT-PCR, primers were designed as follows: MDR1: 5'-ACACCCGACTTACAGATGATGTCTC-3', 5'-CGAGATGGGTAACTGAAGTGAACAT-3', length of fragment:623bp; MRP1: 5'-AGTGACCTCTGGTCCTTAAACAAGG-3', 5'-GAGGTAGAGAGCAAGGATGACTTGC-3', length of fragment:657bp; LRP: 5'-GAGCAGTTCACAGTGTTGTCC-3', 5'-GCGTGACGACAGAAACCGAAA-3'; length of fragment:342bp.MDR1 were detected using RT-PCR To identify the alteration of Multidrug resistance-related genes of PKCα/786-0 cells with and without PMA or Calphostin C treatment, MDR1 was detected using RT-PCR.Cell viability analysis and the cytotoxity of ADM by MTT reduction assayTo elucidate a putative synergistic effect of the PKCαexcitomotor or inhibitor and ADM used for RCC therapy, PKCα/786-0 cells were cultured in the presence of ADM together with different concentrations of Calphostin C or PMA, Cell viability analysis and the cytotoxity of ADM by MTT reduction assay.ResultsPKCαcDNA was identified by DNA sequencing analysis.To investigate the effect of PKCαon the drug resistance of renal cell carcinoma cells, we established stable cell clones expressing PKCα-GFP using 786-0 drug-sensitive RCC cell line. Three highest expression clones were identified by western blot analysis. In order to further identify the character of expressed PKCα-GFP, fluorescence microscopy was conducted to examine the distribution of PKCα-GFP. As the result, these data demonstrated that stably expressed PKCα-GFP has the same character as endogenous PKCα.Effect of PKCαon the drug resistance of 786-0MDR1,MRP1, LRP genes were detected in PKCα/786-0 cells, and,for comparison, 786-0 cells. While 786-0 cells expressed MDR1,MRP1 and LRP were detected by RT-PCR, MDR1 expression in transfected 786-0 cells was significantly higher than that in homologous cells. Nevertheless for MRP1 and LRP, the diversification of expression were indistinguishable between two parallelized cells.MDR1 were detected using RT-PCRWe detected MDR1 expression in PKCα/786-0 cells with and without PMA or Calphostin C treatment, the results shows that MDR1 expression in PKCα/786-0 cells with PMA treatment was significantly higher than that in homologous cells. Nevertheless, MDR1 expression in PKCα/786-0 cells with Calphostin C treatment was significantly lower than that in homologous cells.Cell viability analysis and the cytotoxity of ADM by MTT reduction assayCell viability analysis showed that, at a concentration of 200 nM, Calphostin C suppressed the number of viable cells with 59.6%, at a concentration of 50 nM, Calphostin C suppressed the number of viable cells with 13.9%, which indicates that a PKCαisoform has a positive effect on Rcc cell growth., whereas PMA displayed a substantially higher resistance to ADM. MTT assay was conducted on 786-0 and PKCα/786-0 cells with and without PMA or Calphostin C treatment, following the incubation with ADM at various concentrations. The results clearly showed that, in the normal growth condition, PKCα/786-0 cells showed a stronger growth ability than parental 786-0 cell under the addition of ADM(P<0.05). After PMA induction for 2h, resistance potential of PKCα/786-0 cells with PMA was 1.62-fold higher than those without treatment(P<0.05). On the contrary, Calphostin C treated PKCα/786-0 cells revealed a remarkably decreased growth ability compared with PKCα/786-0 cells without treatment(17.93 fold, P<0.01).Conclusions1. PKCαmay be play an important regulatory role in the occurrence and development process of renal cell carcinoma; Membrane transposition of PKCαis present in renal cell carcinoma tissue; The expression of PKCa increases in membrane of tumor and decreases in plasma, and concerns with the grade and stage of renal cell carcinoma closely; PKCαis one of the ideal indexes to judge the differentiation and prognosis of renal cell carcinoma.2. Protein kinase C alpha cDNA can transfect successfully into human renal cell carcinoma 786-0 cells and express stably.3. The expression of MDR1mRNA in786—0 cell line can be up—regulated by PKCαcDNA, which suggests PKCαcDNA can induces the increase of multidrug resistance in renal cell carcinoma.4. This study raises the potential to use PKCαisoforms as targets to both partially suppress proliferation and to attenuate the multidrug resistance of RCC cells.