| PartⅠ: The expression of PPARα in renal cell carcinomaObject: To investigate the expression characteristics of PPARα(Peroxisome proliferator-activated receptor)in clear cell renal cell carcinoma(cc RCC),compared with the adjacent normal renal tissues.Methods: 40 cases of cc RCC tissues and the adjacent tissues(2cm away from the tumor region)were collected,from patients who were initially diagnosed,did not take any medicine and undewent radical nephrectomy.786-O cell line was applied,and HK-2 human renal tubular epithelial cell line were applied as controlled groups.Immunohistochemistry was applied to explore the difference of PPARα expression.And quantitative real-time polymerase chain reaction(q RT-PCR)and Western blot was also applied.Results: Compared with normal renal tissues,PPARα was poorly expressed in cc RCC tissues(P<0.05).Compared with HK-2 cell line,PPARα was also poorly expressed in both of the m RNA and protein level in 786-O cell line(P<0.05).Conclusion: PPARα expression was significantly downregulated in cc RCC tissues.PartⅡ The role of PPARα antagonist GW6471 on autophagy and growth status in 786-O cellsObject: To investigate the effect of PPARα antagonist GW6471 on the autophagy and growth status in 786-O cells.Methods: 786-O cells were treated by GW6471.HK-2 cells were worked as controlled groups.Different concentration gradients of GW6471 were estabolished,0,12.5,25,50,75μM GW6471,respectively.After the optimal concentraion(25μM)was determined,the optimal timer(6,12,24,or 48 hours)was explored.The dye of Acridine Orange(AO)was utilized for staining lysosomes in 786-O cells treated by GW6471 alone or hydroxychloroquine together.The selected markers of autophagy,LC3-Ⅰ and LC3-Ⅱ,and p62 was detected by Western blot.CCK-8 kit was applied to measure the cell viability.Flow cytometry was used for calculating the ratio of apoptosis.The markers of apoptosis,cleaved-PARP and cleaved-caspase3,were detected by Western blot.Results: The cell viability of 786-O cells was decreased by GW6471 in the manner of dose-dependent(P<0.05).No time dose effect on the 786-O cells treated by GW6471 was observed(P>0.05).No drug-dose effect or time-dose effect on the HK-2 cells treated with GW6471 were observed.The fluorescence signal results of AO showed that 786-O cells treated by GW6471 had much brighter signal than that in the untreated cells(P<0.05).After786-O cells were co-treated by GW6471 and hydroxychloroquine,the signal of lysosomes was brighter than that in GW6471 treated group(P<0.05).Western blotting bands suggested that,the ratio of LC3-Ⅱ/LC3-Ⅰwas increased in the GW6471 group compared with the untreated group,but p62 was significantly down-regulated in 786-O cells treated by GW6471 compared with the untreated groups(P<0.05).After 786-O cells were co-treated by GW6471 and hydroxychloroquine,the ratio of LC3-Ⅱ/LC3-Ⅰ was the highest compared with the other treatment groups,and p62 accumulation was observed.The CCK-8detection results also suggested that the cell viability was significantly downregulated in the GW6471 and hydroxychloroquine cotreatment groups,compared with GW6471 alone treatment group(P<0.05).Flow cytometry results showed that the ratio of apoptotic cells in the GW6471 and hydroxychloroquine cotreatment groups was higher than that in GW6471 alone treatment group(P<0.05).And Western blotting results showed that,cleaved-PARP and cleaved-caspase3,were significantly highly expressed in 786-O cells co-treated by GW6471 and hydroxychloroquine than that in GW6471 alone treatment groups(P<0.05).Conclusion: GW6471 could induce the level of autophagy in 786-O cells,and this autophagy flux could be blocked by hydroxychloroquine.Co-treatment with GW6471 and hydroxychloroquine would increase the level of apoptosis in 786-O cells,but no effect on cell viability in HK-2 cells.Combined with GW6471 and hydroxychloroquine may be the potential treatment way on cc RCC.PartⅢ: The effect of co-treatment with GW6471 and hydroxychloroquine on lipid metabolism in 786-O cellsObject: To investigate the effect of GW6471 and hydroxychloroquine on lipid metabolism in 786-O cellsMethods: The dye of Oil O was applied for staining lipid droplets in 786-O cell.The expression of lipid droplets,Perilipin2 was detected by western blot.The content of ATP was measured by the creatine kinase-phosphomolybdic acid.The content of lactic acid and pyruvic acid was measured by commercial kits.Results: After 786-O cells were treated by GW6471 alone,the number of lipid droplets and the expression level of perilipin2 were all not significantly varied compared with those in untreated groups.However,after 786-O cells were co-treated by GW6471 and hydroxychloroquine,the number of lipid droplets and the expression level of perilipin2 was all significantly down-regulated than those in cells treated by GW6471 alone(P<0.05).The level of ATP was decreased in 786-O cells treated by GW6471 alone,compared with untreated group(P<0.05);And the level of ATP was much lower than that in GW6471 alone,compared with GW6471 and hydroxychloqoquine cotreatment groups(P<0.05).The content of lactic acid and pyruvic acid was higher in GW6471 alone treated cells than that in the untreated cells,and co-treatment of GW6471 with hydroxycholroquine(P<0.05).Conclusion: Co-treatment with GW6471 and hydroxychloroquine can inhibit the synthesis of lipid droplets in 786-O cells,maybe associating with the low level of ATP production and the lack of energy originated from the metabolite of glycolysis. |
