Influence Of Curcumin Combined With Epirubicin On PI3K/AKT Signal Pathway And Expression Of FOXP3and TIPE2in HepG2

Hepatocellular carcinoma (HCC) is one of the most common malignanttumors in the world. And China is the high incidence area of liver cancer.Because liver cancer has the characteristics of onset occult and rapiddevelopment. So many patients were in the late stage when they werediagnosed. At that time, patients lost the opportunity of operation. AndClinical curative effect of radiofrequency ablation, hepatic arteryinterventional chemotherapy and embolization, percutaneous ethanol injectiontherapy is not ideal. Hepatocellular carcinoma tissue and cell are not sensitiveto the commonly used chemotherapy drug such as epirubicin, mitomycin C,5-FU, cisplatin. Traditional Chinese medicine can inhibit the growth andmetastasis of tumor cells, and it enhanced the sensitivity of tumor cells tochemotherapeutic drugs. Curcumin is the active component extracted fromturmeric.It has the role of antioxidant, antiviral, anti-inflammatory, preventionand inhibition of tumor growth. Previous studies showed that curcumin alsohas certain immune regulating effect on tumor patients. But the effect ofcurcumin combined with common chemotherapy drugs in inhibiting thegrowth of hepatoma cells and regulating immune function is currently unclear.Tumor necrosis factor-alpha-induced2protein-8like-2(TNFAIP8L2,TIPE2) is an important immune molecule recently discovered. It cannegatively regulate both acquired immunity and innate immunity and beregarded as an important regulator of cellular immunity. TIPE2cancommunicate with inflammation and tumor and plays an important role on thedevelopment of tumor. FOXP3(forkhead box P3，FOXP3) is a specific markerof CD4~+CD25~+Regulatory T cells and could limit immune cell activation andexpansion, control the development and function of CD4~+CD25~+Regulatory Tcells. As an immune molecules, FOXP3mainly expressed on T cells, and also can be expressed on tumor cells to prevent tumor cells from immunesurveillance. But the expression and effects of FOXP3on tumor cells were notfully understood.Epirubicin, as a conventional anti-turmor drug, not only can promote theapoptosis of tumor cells, but also can influence the anti-turmor immunefunction through regulating a variety of immune cells and molecules. However,the inhibition effect of epirubicin on hepatoma cells is still not satisfactory, solooking for effective drugs and methods to inhibit human hepatoma cellsgrowth has become the important work of scientific researchers. Curcumin isthe active component extracted from turmeric and has the role of antioxidation,antivirus, anti-inflammatory, prevention and inhibition of tumor growth.Meanwhile, curcumin has also some immune regulating effect on tumorpatients. But, the effect of curcumin combined with commonly chemotherapydrugs to inhibit the growth of hepatoma cells and regulate immune function iscurrently unclear.A wide variety of human tumors such as gastric cancer, colorectal cancer,breast cancer, liver cancer, renal cell carcinoma is closely related tophosphatidylinositol3-kinase (phosphatidylinositol-3-kinases, PI3K)/protein serine threonine kinase (protein-serine-threonine kinase, AKT)signaling pathway. This pathway can regulate apoptosis of tumor cell andexpression of inflammatory factor in tumor microenvironment. Previousstudies showed that PI3K/AKT signal pathway can also regulated thedevelopment of tumor through regulating immune molecular and immunecells. Therefore, we respectively intervened HepG2cells by epirubicin andepirubicin combined with curcumin in vitro to observe the percentage ofviable cells of HepG2cells, the expression of FOXP3and TIPE2and theinfluence of PI3K/AKT signaling pathway, discussing whether epirubicin orcurcumin combined with epirubicin can influence the expression of FOXP3,TIPE2and PI3K/AKT signaling and the underlying molecular mechanism.Objective: To investigate the influence of epirubicin and epirubicincombined with curcumin on HepG2cells and observe the changes of immune molecules TIPE2and FOXP3and PI3K/AKT signaling pathway, discussingthe influence of TIPE2on FOXP3through the PI3K/AKT.Methods: HepG2cells were cultured in vitro. The cells in logarithmicgrowth phase were seeded in a96-well plate at a density of1×104cells well,cultured for48h and treated with different concentrations of curcumin(5μmol/L,10μmol/L,20μmol/L,40μmol/L,50μmol/L,60μmol/L,80μmol/L)and epirubicin (1μg/mL、2μg/mL、4μg/mL、8μg/mL、16μg/mL) to observesurvival rate of HepG2cell. After incubation for24and48h, we determine thelowest effective concentration of curcumin (20μmol/L) and epirubicin(2μg/mL). And epirubicin (2μg/mL) and epirubicin (2μg/mL) combined withcurcumin (20μmol/L) intervented HepG2in the same way. The survival rate ofHepG2was detected respectively at24and48hours by MTT assay. The cellsin the logarithmic growth phase divided into three groups (control group,epirubicin group, curcumin combined with epirubicin group). Each group wasrespectively treated with drug concentration as MTT assay. The total cellularRNA and protein were extracted for inspection in24and48hours respectively.The expression of TIPE2, PTEN, AKT, NF-KB and FOXP3in HepG2wasexamined by RT-PCR assay. The expression of PTEN, AKT was examined bywestern-blot.Results:1Effect of Curcumin, epirubicin and epirubicin combined with curcuminon growth and survival of HepG2cells:With the increase of the concentration and time of curcumin, the survival rateof HepG2cells decreased gradually. The survival rate of HepG2cells did notdecreased significantly in5μmol/L and10μmol/L. But the cell survival ratebegan to decline rapidly in20μmol/L. it is indicated that the lowest effectiveconcentration of curcumin was in20μmol/L. The survival rate in in5μmol/Land the survival rate in10umol/L are not significantly different (P>0.05). Thesurvival rate in40μmol/L and the survival rate in50μmol/L are notsignificantly different (P>0.05). Survival rate in other groups are different(P<0.05). Epirubicin also can lead to decrease HepG2cell survival rate. With the increase of the concentration and time of Epirubicin, cell survival rate alsodecreased gradually. At the same time the lowest effective concentration ofepirubicin was in2μg/ml. The survival rates in all the groups are different(P<0.05). And epirubicin (2μg/ml) combined curcumin (20μmol/L) coulddecreased cell survival rate more effectively than that in epirubicin groupalone. The difference was statistically significant (P<0.05).2Effect of epirubicinon and epirubicinon combined with curcumin on theexpression of TIPE2, PTEN, AKT, NF-KB and FOXP3mRNA and PTEN,AKT protein in HepG2cells:RT-PCR test show: Compared with control group, epirubicin coulddown-regulate AKT, NF-KB and FOXP3mRNA expression and up-regulatePTEN and TIPE2mRNA expression (P<0.05). The difference was statisticallysignificant. Compared with epirubicin group, curcumin combined withepirubicin decreased AKT, NF-κB and FOXP3mRNA expression andincreased PTEN and TIPE2mRNA expression (P<0.05). The difference wasstatistically significant.Western-blot test show: Compared with control group, epirubicin coulddown-regulate AKT protein expression and up-regulate PTEN proteinexpression (P<0.05). The difference was statistically significant. Comparedwith epirubicin group, curcumin combined with epirubicin decreased AKTprotein expression and increased PTEN protein expression (P<0.05). Thedifference was statistically significant.Conclusion:1Curcumin and epirubicin in the concentration and time-dependent waycauses cell survival rate of HepG2decreased. And the lowest effectiveconcentrations of curcumin and epirubicin were respectively20umol/L and2ug/ml by MTT assay.2Curcumin (20μmol/L) combined with epirubicin (2μmol/L) increased theeffect of epirubicin in inhiting the cell survival rate.3Epirubicin decreases the function of FOXP3through increasing expressionof TIPE2and then inhibiting PI3K/AKT signal pathway. Curcumin combined with epirubicin strengthen this effect of epirubicin alone, improving theanti-tumor immune function.