| PART ONEEFFECT OF GAMBOGIC ACID ON PROLIFERATION OF RENALCARCINOMA CELL LINES RC-2AND RC-2CELL LINES-DERIVED EXOSOMESObjective: To observe the effect of gambogic acid on proliferation ofrenal carcinoma cells RC-2and its-derived exosomes.Methods: MTT assay was used to detect the changes of RC-2cellline proliferation before or after the treatment of Garcinia acid. After renalcarcinoma RC-2cell lines treated by Garcinia acid, supernatant of RC-2cell lines was selected, exosomes were extracted by sucrose gradientcentrifugation, its morphology was observed by transmission electron, itssecretion quantity was compared, BCA method was used to compare itsprotein concentration and then calculate the total protein content, WesternBlot method was used to compare exosomes surface antigen moleculeICAM-1, HSP-70, and G250content differences, Survivin protein expression was compared in cells(treated or untreated by Garciniaacid), wild type p53mRNA expression changes were compared before orafter cells were treated by Garcinia acid by RT-PCR.Results: MTT assay showed that Garcinia acid could inhibit the renalcancer cell lines RC-2proliferation in a concentration and timedependent manner. After Garcinia acid treatment, transmission electronmicroscopy showed there was no obvious morphological changes forexosomes, while its secretion quantity and protein content significantlyincreased than untreatment group(P <0.05), there was no obvious changefor protein content of its surface molecules ICAM-1, HSP-70and andG250antigen than untreatment group (P>0.05), its protein expression ofSurvivin was significantly reduced (P<0.05), wild type p53mRNAexpression in treated cells was significantly increased than untreatedcells(P <0.05).Conclusion: Garcinia acid could inhibit the renal cancer celllines RC-2proliferation in a concentration and time dependent manner.After Garcinia acid treatment, there was a significant increase for theamount of renal cancer cell lines-derived exosomes and protein quality inexosomes, but not the surface molecules, and apoptosisinhibitory protein(Survivin) contained in exosomes was significantlyreduced.These changes may be associated with Garcinia acid-induced upregulation of the expression of wild type p53mRNA in kidney cancer cell lines. PART TWOINVESTIGATE THE EFFECT OF RENAL CARCINOMA RC-2CELL LINES(TREATED BY GARCINIA ACID)-DERIVEDEXOSOMES ON THE PROLIFERATION AND APOPTOSIS OFRENAL CANCER RC-2CELL LINESObjective: To observe the effect of exosomes secreted from renalcarcinoma cell lines (treated by Garcinia acid) on the proliferation andapoptosis of renal cancer cell lines RC-2(stable expression of hepaCAMgene).Methods: To construct RC-2cell lines which hasstable gene expression of hepaCAM after adenoviral transfection, thenWestern blot analysis and RT-PCR was used to detect hepaCAM mRNAand protein expression, RC-2cell lines(treated by Garcinia acid)derived-exosomes and RC-2cell lines were cocultured as the experimentalgroup, the same amount of RC-2cell lines(Garcinia acid-untreated)derived-exosomes and RC-2cell lines were co-cultured as the control group, their proliferation was compared by MTT assay, AnnexinV-FITC/PI double staining flow cytometry was used to detect their changesin apoptosis, Western blot analysis was used to detect hepaCAM, AKTand p-AKT, p-ERKl/2, ERKl/2protein expression after48h cellscultured. After MK-2206was added at every time spots in the controlgroup, expression changes of hepaCAM, AKT and p-AKT protein wereobserved. The mRNA expression of hepaCAM, PI3K and AKT in58casesof renal carcinoma and their adjacent normal tissues were detected byreverse transcription-polymerase chain reaction (RT-PCR), the proteinexpression of hepaCAM, p-AKT in the same tissues were detected byimmunohistochemical method, then the correlation between them wasanalyzed.Results: After adenoviral transfection, expression ofhepaCAM gene and protein was detected in RC-2cell lines (P <0.05).MTT assay showed that exosomes promote proliferation in a timedependent manner on RC-2cell lines. However, proliferation force in theexperimental group at48h,72h was significantly decreased than the controlgroup(P<0.05). It showed that exosomes inhibited apoptosis of RC-2celllines in a time dependent mode, while the apoptosis rate of48h,72h inexperimental group was significantly increased compared with controlgroup (P<0.05). Western blot analysis showed expression of hepaCAM inthe control group was significantly reduced (P <0.05) after48h treatment of exosomes, compared with protein expression in the experimental group,while p-AKT protein expression was significantly increased in the controlgroup (P<0.05) compared with the expression of that in the experimentalgroup, while the p-ERKl/2, ERKl/2, AKT expression changes in the twogroups was not statistically difference (P>0.05). Whenp-AKT inhibitor MK-2206was added in the control group at different timepot, hepaCAM expression inhibition induced by exosomes wassignificantly weakened compared with MK-2206untreated group (P<0.05).RT-PCR showed the mRNA expression levels of PI3K and AKT in renalcarcinoma tissues were higher than those of the adjacent normal tissues(P<0.05)，while mRNA expression level of hepaCAM was lower thanthose of the adjacent normal tissues (P<0.05). HepaCAM mRNAexpression was negative correlated with PI3K mRNA and AKT mRNAexpression (P<0.05) respectively. The protein expression level of p-AKT inrenal carcinoma tissues was higher than those of the adjacent normaltissues (P<0.05), while the protein expression level of hepaCAM in renalcarcinoma tissues was lower than those of the adjacent normal tissue(P<0.05). HepaCAM protein expression was negative correlated withp-AKT protein expression (P<0.05) by immuno-histochemistry.Conclusion: Kidney cancer cell lines RC-2-derived exosomes couldpromote the proliferation and inhibit apoptosis of RC-2cell lines(stableexpression of hepaCAM), while the promotion was significantly inhibited for Garcinia acid-treated RC-2celllines-derived exosomes. Their proliferative capacity was likely ina p-AKT pathway dependent manner to inhibit hepaCAM protein in RC-2cells. HepaCAM and PI3K/AKT expression in renal cell carcinoma hasa negative correlation. PART THREEA EXPERIMENTAL STUDY OF GARCINIA ACID COMBINEDWITH SUNITINIB INHIBITED RENAL CARCINOMA CELLS INVIVO AND IN VITROObjective: To observe the effect of Garcinia acid combinedwith sunitinib inhibited kidney cancer786-0cells proliferation, invasion,and so on.Methods: Garcinia acid alone or in combination with sunitinib, wereused to treat786-0cell lines, then MTT assay and flow cytometry wereused to detect changes in cell viability and cycle, cell migrationand invasion assay were used to detect changes in cell migration andmetastasis, Enzyme-linked immunosorbent assay (ELISA) was used todetect change of VEGF secreted from786-0cells, Western Blot was used to detect cell cycle and metastasis-related protein expressionchanges. Xenograft model of786-0cells was constructed, after treatedby Garcinia acid alone or in combination with sunitinib, tumorgrowth and blood vessel growth in young mice were observed andmeasured by immunohistochemistry assay.Results: The combination use of Garcinia acid and sunitinib showedmore pronounced[Proliferation rate(63.2±5.7)%] thanmonotherapy treatment in inhibiting cell proliferation(P<0.05), andthere were more cells[(27.43±3.11)%] gathered in sub-G1phase of cellcycle in combined treatment group than in monotherapy treatmentgroup(P<0.05), the combination treatment group couldsignificantly inhibit cell migration and invasion (P<0.05), significantlyinhibit the cells to secrete more VEGF[Concentration (141.72±3.98pg/mL/105cells)] than untreatment group (P<0.05), WesternBlot showed that the cell cycle regulatory protein Bcl-2expression wassignificantly reduced in combined treatment group than inthe monotherapy group, P21expression was significantly increased incombined treatment group than in the monotherapy group, and there was asignificant decrease in VEGF protein and MMP-2in combinedtreatment group than in the monotherapy group, while there was nosignificant change about the expression of CyclinB1and MMP-9proteinthan in the monotherapy group.Animal experiments had shown: Xenograft and vascular formation was significantly inhibited in thecombined treatment group than a monotherapy group(P<0.05).Conclusion: The combination use of Garcinia acid and sunitinib canbe more significant than the individual drugs to inhibit kidney tumor786-0cells proliferation and invasion in vitro. The combination treatmentof Garcinia acid and sunitinib can be more significant than theindividual drugs to inhibit tumor growth and angiogenesis in vivo. |
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