Curcumin Enhances The Sensitivity Of Hepatocellular Carcinoma Cells To Sorafenib By Inhibiting The PI3K/AKT Pathway

Background:Hepatocellular carcinoma(HCC)is one of the few malignant tumors with persistent high morbidity and mortality.The early symptoms of HCC patients are not obvious,and most of them are in the late stage when they are discovered and diagnosed,so the choice of treatment methods is greatly limited.Sorafenib is the only drug approved for the treatment of advanced HCC.It can significantly improve the overall survival rate of patients,and the patient's tolerance is good.However,the problem of susceptibility decline and even drug resistance is the main reason for its treatment failure.Therefore,it is necessary to clarify the mechanism of Sorafenib susceptibility decline in HCC cells and to find a reasonable way to improve the HCC cells?sensitivity to Sorafenib.Breast cancer resistance protein(BCRP)is a member of the ABC transporter superfamily,which can use the energy provided by ATP hydrolysis to expel drugs out of the cell,which is an important cause of drug resistance in many cancer cells.It has been reported that Sorafenib is the substrate of BCRP,which may be the main reason for the decreased sensitivity or resistance of HCC cells to Sorafenib.In addition,Curcumin can be used as a selective inhibitor of BCRP in vivo.So,if Curcumin can enhance Sorafenib's anti-liver cancer effect when combined with Sorafenib? What is its mechanism?Objective:1.BCRP was used as a starting point to explore the mechanism of decreased sensitivity of Hepatocellular carcinoma cells to Sorafenib.2.Finding ways to improve the sensitivity of Hepatocellular carcinoma cells to Sorafenib and elucidate the molecular mechanisms.Methods:1.The effects of Sorafenib and Curcumin on the proliferation of Hepatocellular carcinoma cells(Hep G2,Huh-7,SMMC-7721,SNU-368 and SNU-739 cell lines)were measured by MTT assay.2.The effects of Sorafenib,Curcumin,Sorafenib combined with Curcumin on apoptosis of HCC cells were detected by flow cytometry.Then the expression levels of apoptotic-related proteins(Bax,Bcl-2,PARP,Caspase-3)were measured using western blot experiments.3.The expression levels of BCRP protein in Hep G2 or Huh-7 cell were knocked down or overexpressed through transient transfection method.Then MTT assay and Western blot assay were used to detect the changes of HCC cells?sensitivity to Sorafenib.4.Western blot assay,high-context cell imaging assay,high performance liquid chromatography,biotin labeling and immunofluorescence were used to clarify the mechanism for Curcumin enhancing the sensitivity of Hepatocellular carcinoma cells to Sorafenib.5.Hepatocellular carcinoma models were established based on BALB/c mice.Then the effects of Sorafenib combined with Curcumin on the proliferation of solid tumors were evaluated.Furthermore,the expression levels of BCRP,p-AKT and apoptotic-related proteins in tumors were detected by immunohistochemical experiments.Results:1.The results of MTT experiments showed that both Sorafenib and Curcumin could inhibit the proliferation of HCC cells and showed a dose-dependent effect;when Sorafenib combined with Curcumin,the antitumor effect was enhanced,the specific manifestation was that the cell viability was obviously weakened and the IC50 value was decreased.2.Compu Syn software was used to analyzed MTT data.When Sorafenib and Curcumin were combined at concentrations ratio of 1:1 or 1:2,the CI value was <1.These results indicated that Sorafenib had synergistic effect with Curcumin on the proliferation HCC cells.3.The results of flow cytometry assay and western blot assay showed that the apoptosis caused by the combination of Sorafenib and Curcumin is more obvious than that by Sorafenib alone,which indicated the synergistic antitumor effect of Sorafenib and Curcumin.4.Knockdown of BCRP enhanced the sensitivity of HCC cells to Sorafenib;Overexpression of BCRP weakened the sensitivity of HCC cells to Sorafenib;The the sensitivity of HCC cells to Sorafenib was enhanced after Sorafenib combination with Curcumin.These results indicated that BCRP played an important role in the sensitivity of HCC cells to Sorafenib,and Curcumin may enhance the sensitivity of HCC cells to Sorafenib by regulating BCRP.5.Western blot assay results showed that Curcumin inhibited the protein expression of BCRP.Through further exploration,it was found that curcumin regulated the expression of BCRP by inhibiting the PI3K/AKT pathway.6.The results of high connotation cell imaging experiment,high performance liquid chromatography(HPLC),biotin-labeled membrane protein experiment and immunofluorescence labeling experiment showed that Curcumin could also inhibit the efflux function of BCRP through inhibiting the activation of the PI3K/AKT pathway.7.Animal experiments showed that the tumor growth of BALB/c mice was significantly inhibited when Curcumin was administered in combination with Sorafenib.The results of western blot assay and immunohistochemistry assay showed that the p-AKT,BCRP and Bcl-2 was down-regulated and cleaved Caspase-3 was up-regulated when Sorafenib was combined with Curcumin.Conclusion:Curcumin could inhibit the protein expression and the efflux transport function of BCRP though inhibiting the activation of PI3K/AKT signaling pathway.Then the accumulation of Sorafenib in cells was increased,which in turn enhanced the sensitivity of HCC cells to Sorafenib..