Effects Of Curcumin On Human Renal Cell Carcinoma 786-O Cells In Vitro And Its Mechanisms

Objective: To study the effects of curcumin on human renal carcinoma cell line 786-O in vitro and its mechanisms.Meanwhile, to investigate the effects of curcumin combined with sorafenib on human renal carcinoma cell line786-O in vitro.Methods: 786-O cells were incubated in culture medium in vitro. The 786-O cells were divided to 4 groups as control group,sorafenib group,curcumin group and sorafenib- curcumin group. Sorafenib and curcumin were used in single agent or in combination against 786-O in vitro. The inhibitory effects of sorafenib and/or curcumin on proliferation of 786-O were determined by MTT assay at 24h,48h,72h after the addition of the drugs to 786-O culture and the OD values were computed. Apoptosis and distribution of cell cycle were examined with flow cytometry after treated with curcumin. The morphological changes of 786-O cells were observed by light microscopy and electron microscopy after treated with curcumin.In addition, the expression of Survivin,NF-KB and Caspase-3 in 786-O cells was analysed by SP immunohistochemistry after treated with curcumin. Results: 1.MTT colorimetric method showed that curcumin(6.25,12.5,25,50μmol/L)and /or sorafenib(1.5,3,6,12μmol/L)in single agent could inhibit the proliferation of 786-O cells. After treated with curcumin and/or sorafenib for 24h to 72h, compared with control group, the OD values of curcumin-treated and/or sorafenib-treated groups decreased, and there was statistically significant difference between control group and every treatment group(P<0.05). Among every treatment group, there was also statistically significant difference (P<0.05). Furthermore, with the increasing concentration of curcumin and prolonging of treatment time, the OD values decreased gradually, that was, curcumin and/or sorafenib inhibited the proliferation of 786-O cells significantly in a dose-and time-dependent manner. Treatment with curcumin for 24h,48h,72h, the IC50 was 38.7μmol/L,29.2μmol/L and 21.8μmol/L separately, Treatment with sorafenib for 24h,48h,72h, the IC50 was 7.7μmol/L,6.5μmol/L and 5.1μmol/L separately. 2.MTT colorimetric method was performed to evaluate the potential cytostatic effect of combining curcumin with anti-cancer agents sorafenib in 786-O cells. combining curcumin at 12.5μmol/L, 25μmol/L with sorafenib at 1.5μmol/L,3μmol/L,6μmol/L,12μmol/L respectively for 24h,48h,72h increased the growth inhibition rate of 786-O cells significantly (P<0.05). These results suggested additive actions of the two drugs. The IC50 of sorafenib in 786-O cells alone was 7.7-5.1μmol/L.When combined with 12.5μmol/L curcumin,the IC50 decreased to 5.02-2.36μmol/L;when combined with 25μmol/L curcumin,the IC50 decreased to 2.53-1.38μmol/L;Thus, we have a conclusion,in the range of concentration, curcumin had the enhancing synergistic cytostatic effect in 786-O cells combined with sorafenib with the increasing concentration. 3.When treated with curcumin, 786-O cells had significant morphological changes:The cells shrinked and became into cambiform.With the increasing concentration of curcumin and prolonging of treatment time, the cells rounded gradually and more and more detached from the wall of culture flask.There were punctiform-lamellar cell pieces around the cells.Vacuolus could be observed in the kytoplasm of some cells. The cell ultramicrostructure changes were observed by transmission electron microscope: nucelus overshoot in acute angle outwards, chromatin concentratede highly, we also observed karyopycnosis. 4.When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the results were: After treatment with 12.5μmol/L,25μmol/L,50μmol/L curcumin for 48h, the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase and G2/M phase decreased grudually. That was, curcumin could induce an arrest of cell cycle in G1 phase by a dose-dependent manner. In addition, after treated with 12.5μmol/L,25μmol/L,50μmol/L curcumin for 48h, the typical apoptotic peak which enhanced gradually with the increasing concentration of curcumin was observed and the apoptotic percentage was 12.19%,22.12%,33.40% seperatelyand there was statistically significant difference between control group and every treatment group (P<0.05). 5.The results of SP immunohistochemistry:The expression of Survivin and NF-kB was decreased while the expression of Caspase-3 was increased in 786-O cells treated with curcumin at 25μmol/L for 48h. and there was statistically significant difference between control group and every treatment group(P<0.05).Conclusions:1.The reseach showed that curcumin had the effect of anti-renal cancer cell line 786-O, which provided a new theoretical basement for reasonable clinical treatment of renal cancer. 2.We confirmed that curcumin and/or sorafenib in single agent had the capability of inhibiting the proliferation of 786-O cells in vitro by a dose-and time-dependent manner significantly.3.Through MTT colorimetric method, we confirmed that curcumin combined with sorafenib had a synergistic effect on inhibiting the proliferation of 786-O cells. 4. We confirmed that curcumin induced an arrest of cell cycle in G1 phase as well as apoptosis in 786-O cells. 5.Curcumin's effects of inhibiting proliferation and inducing apoptosis in 786-O cells seem to due to up-regulation of the expression of Caspase-3 and down-regulation of the expression of Survivin,NF-kB. 6.This research proved that curcumin had the effect of anti-renal cancer, and in riched the treatment of renal cancer through combining sorafenib with curcumin.