| Objective:1.To detect the m RNA and protein expressions of ULK1 in human renal carcinoma tissues and corresponding adjacent non-tumor tissues,and to investigate the relationship between the expression of ULK1 and the occurrence and development of renal cell carcinoma.2.To explore the effect of ULK1 inhibitor SBI-0206965 on the proliferation and apoptosis of human renal cell carcinoma cell line A498 and to explore the potential value of SBI-0206965 in renal cancer targeted therapy.Method:1.The expression level of ULK1 m RNA was measured in 20 pairs of cc RCC and matched adjacent tissues by q RT-PCR.2.The expression level of ULK1 protein was detected in 54 pairs of cc RCC and matched adjacent tissues by Western Blotting.3.The expression of ULK1 protein in A498,Caki-1,HEK293 T and HK-C cell lines were detected by Western Blotting.4.The effects of different doses of SBI-0206965(0,2.5,5,10,20,40μM)on the proliferation of A498 were measured by MTS assay under different nutritional conditions(0.5% FBS,EBSS).5.The expression of PARP and Caspase8 in A498 treated with different doses of SBI-0206965(0,5,10,20μM)were detected by Western Blotting under different nutritional conditions(0.5% FBS,EBSS).6.The apoptotic levels of A498 cells treated with different doses of SBI-0206965(0,5,10,20μM)were detected by Cell Death Detection ELISA kit under different nutritional conditions(0.5% FBS,EBSS).Results:1.Our q RT-PCR results showed that among 20 patients with RCC,70%(14/20)of which had significantly higher expression levels of ULK1 m RNA than their corresponding normal adjacent tissues.On the contrary,the expression level of ULK1 m RNA in 5 cases normal adjacent tissues were higher than their matched cc RCC tissues.There was no significant difference between the two cases,which accounting for 5%(1/20).The difference of ULK1 m RNA expression between cc RCC tissues and adjacent tissues was statistically significant(P = 0.0107).2.As seen in Western blotting results :the levels of ULK1 protein in 34 cases cc RCC tissues were significantly higher than their adjacent non-tumor tissues(62.96%,34/54);9 cases demonstrated the protein expression of ULK1 in RCC tissue were lower than their matched adjacent tissues.However,the expression of ULK1 protein was similar in 11 cases.The difference of ULK1 relative gray value between cc RCC tissues and corresponding adjacent tissues was statistically significant.3.Western blotting was used to detect the expression of ULK1 protein in HEK293 T,HK-C,A498 and Caki-1 cells,which indicated that ULK1 protein was expressed in A498,Caki-1,HEK293 T and HK-C cells.The expression level of ULK1 protein was the highest in A498 cells.4.The results of MTS showed that the inhibitory rate of A498 cells increased with the increase of doses of SBI-0206965 under low serum nutritional conditions(0.5%FBS).There was no significant difference in the inhibitory rate of proliferation at2.5μM compared with the control group(0μM)(P> 0.05).The difference between the other drug treatment groups(5,10,20,40 μM)and the control group was statistically significant(P<0.01).In addition,the difference in proliferation inhibition rate between the each doses groups(2.5 μM vs 5 μM,5 μM vs 10 μM,10μM vs 20 μM,20 μM vs 40 μM)were also statistically significant(P <0.01).Similar to the results of low serum nutritional conditions,the inhibition rate of A498 cell proliferation was increased with the increase of SBI-0206965 concentration under starvation condition(EBSS).There was significant difference between different drug doses groups(P<0.01).The results showed thatSBI-0206965 can inhibit the proliferation of renal cell carcinoma cell A498 in a dose-dependent manner.5.Western blotting was used to detect the expression of apoptotic proteins PARP and Caspase8 in A498 cell lines treated with different dose of SBI-0206965(5μM,10μM,20μM)under low serum nutritional conditions and starvation conditions,the results show that different doses of SBI-0206965 could degrade the apoptosis-related protein PARP and Caspase8 in A498 cells compared with the control group(0μM),and the degradation degree of which was enhanced with the increase of drug dose.6.Cell Death Detection ELISA kit was used to detect the apoptotic levels of A498 cells treated with different doses of SBI-0206965(0,5,10,20 μM)under low serum nutritional conditions and starvation conditions,the results show that with the increase of dose of SBI-0206965,the apoptosis level of A498 cells increased.Conclusion:1.The expression of ULK1 m RNA and protein in cc RCC tissues were significantly higher than corresponding adjacent tissues.2.ULK1 inhibitor SBI-0206965 can induce apoptosis and inhibits proliferation of A498 in vitro.3.ULK1 may become a new target for the treatment of renal cell carcinoma,ULK1 inhibitor SBI-0206965 is a potential anti-renal cancer targeted drug. |
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