Reversal Effect Of Sorafenib On Multidrug Resistance In Hepatoma Cells

BackgroundPrimary liver cancer is one of the most common malignant tumors in the world, and about half of the patients with liver cancer focused in China,where about 110,000 people die from liver cancer each year,ranking the second in malignant tumor mortality.Presently,there still existed some problems in clinical diagnosis and treatment of liver cancer,such as a difficult early diagnosis to make,high rate of recurrence and metastasis,and lack of relevance for treatment,original innovative drugs and various treatment means.Chemotherapy is one of the important ways in the comprehensive treatment for liver cancer,while the tumor multidrug resistance (MDR) is one of the main reasons for significant effects on chemotherapy and patients'survival.The tumor MDR has been widespread in clinical tumor therapy, becoming one of the main obstacles in chemotherapy.MDR refers to the drug resistance of tumor cells to a kind of antitumor drug, simultaneously,cross resistance also occurs to other antitumor drugs with different structure and mechanism.At present,the production mechanism of MDR remains unclear,the main MDR mechanisms of molecular biology in tumor chemotherapy are considered as:â‘ transmembrane proteins,reduce the antitumor drug accumulation in cells by pumping chemotherapy drugs from tumor cells,mainly including MDR1 encoded P-glycoprotein(P-gp),MDR-related protein(MRP) and lung resistance related protein(LRP);â‘¡enzyme systems which relate to oxidation reduction and detoxification in cells change,such as reduced glutathione(GSH) and glutathione S-transferase(GST);â‘¢target molecule of drug change,like protein kinase C(PKC) and DNA topoisomeraseâ…¡(TOPOâ…¡);â‘£genes and proteins which control apoptosis change,such as Ras,Bcl-2,P53,c-fos and c-jun.With people go increased deep into the tumor biological behavior,molecular target therapy--another powerful mean for tumor treatment after the three major traditional methods(surgery,chemotherapy and radiotherapy)--has attracted wide attention.Molecular target therapy for tumor aim at the links which may lead to cell carcinogenesis,such as cell signaling pathway,proto-oncogene,tumor suppressor gene,cytokine,receptor,anti-tumor angiogenesis and suicide gene.It is a new biotherapy mode for inhibiting,even dissipating the tumor cell by reversing the malignant biological behavior from molecular level.Because the treatment is specific for the target molecule and signal transduction pathway which play a key role in the development of tumor,it not only possess specificity and selectivity of anti-tumor-treatment, but also avoid side effects and drug resistance caused by the no-choice conventional chemotherapy.Molecular targeted drug is a new research field on tumor therapy.For the high selectivity and small side-effect of this kind of drugs,it has become a hot research and a development trend on anticancer drugs in recent years,as well as a research direction to enhance the curative effect of liver cancer.Sorafenib(BAY 43-9006) is a new type of oral multi-target antitumor drug.It is a type of oral multikinase inhibitor of dual-aryl ure with dual anti-tumor effect.One hand,it inhibits tumor growth directly by inhibiting the RAF/MEK/ERK signal transduction pathway;the other hand,by inhibiting the activity of several tyrosine kinase receptors related to tumor angiogenesis and development,including vascular endothelial growth factor -2 (VEGFR-2),vascular endothelial growth factor -3(VEGFR-3),platelet-derived growth factor-β(PDGFR-β) and c-kit proto-oncogene,it blocks tumor angiogenesis, indirectly inhibiting tumor cell growth,thus playing the effect on anti-tumor.Intracellular and intercellular signal transduction is the basis of various biological behaviors of organisms,and the abnormal signal pathway is closely related to tumor.The physiological activities of various cells involved in the development of MDR depend on the intracellular complex signal transduction system and the activation of various kinase.Mitogen-activated protein kinase(MAPK) signal transduction pathway is the important signal transduction system to mediate extracellular stimulation to intracellular response,regulating the cell proliferation, differentiation,apoptosis and interaction.MAPK is a class of serine / threonine protein kinase widely distributed in mammalian cells.At present,4 parallel MAPK signal transduction pathway have been found,namely,extracellular signal-regulated kinase(ERK) pathway,c-Jun N-terminal kinase(JNK) pathway,p38 pathway and ERK5/BMK1 pathway.It is showed that MAPK pathway played an important role in chemotherapy MDR,antitulnor drug could induce the activation of MAPK signal transduction system and the emergence of MDR.It is considered in most current studies that the excessive activation of ERK and chemotherapy MDR of many tumors showed significant positive difference,its possible mechanism might be the regulation of drug resistance gene and protein expression.It may become a new method to reverse MDR by regulating the expression of MARK kinase system in tumor cells.With the development of molecular biological technique and the further understanding of the pathogenesis on cells and molecular level,the targeted tumor therapy goes into a new era.The relevant research of the effect of molecular targeted drug on tumor resistance-associated protein has not been found in China,and only most in vitro studies in other countries.It has been reported that monoclonal antibody - cetuximab could reverse colorectal cancer resistance to chemotherapeutic drugs, properties,but no impact of multi-targeted drugs on drug-resistant protein has been reported.Therefore,a further study of the molecular mechanism of Sorafenib reverse liver cancer resistance can help to the treatment by the designed combination of chemotherapy drugs and Sorafenib,obtaining the best curative effect,maximizing the improvement for patient survival quality,and providing a theoretical foundation for the comprehensive treatment and efficacy enhancement for liver cancer.ObjectiveThe multi-target drug Sorafenib blocks tumor angiogenesis and tumor cell proliferation on both the level of growth factor combining with its membrane receptor and intracellular signal transduction.It can effectively inhibit hepotoma cell growth and angiogenesis.This study was to determine the reversal effect of Sorafenib on liver cancer cell MDR,look for the effect of Sorafenib on MDR protein,and explore the possible reversal mechanism of Sorafenib to chemotherapy drug MDR from molecular level and cellular signal transduction.Methods1.BEL-7402 cell of hepatoma cell line and BEL-7402/5-FU cell of hepatoma cell line resistant were selected as experimental cell.Their growth curve,cell doubling time and cell mitotic index were measured,and expression rate of P-gp protein in cell surface was checked by flow cytometry.2.By MTT colorimetric assay,the dose-response curve of(1,2,4,8,16)μmol/L Sorafenib on BEL-7402/5-FU cell was drawn,and with the flow cytometry to detect the effect of Sorafenib with above-mentioned dose on Rho123 concentration in BEL-7402/5-FU cell,then,the appropriate dose of Sorafenib was selected according to the above experimental results. 3.It was divided into three groups:BEL-7402 cell,BEL-7402/5-FU cell,and BEL-7402/5-FU cell + sorafenib(4μmol/L) group.3.1 The effect of the four kind of chemotherapy drugs,including adriamycin (ADM),gemcitabine(GEM),cisplatin(DDP),fluorouracil(5-FU),was taken on the above three groups of cells.Changes in cell survival rate were detected by MTT colorimetric assay,and the growth inhibition effect of these drugs on the three groups of cells was evaluated,coming to the half cell inhibition rate(IC50),thus,the sensitivity of chemotherapeutic drugs was determined and the reversal multiple of Sorafenib to hepatoma drug resistance cells.3.2 P-gp protein expression rate in cell surface,Rho123 accumulation and cell cycle distribution of the cells in three groups were determined by flow cytometry;3.3 Immunocytochemistry method was applied to detect P-gp and MRP protein expression;3.4 Semi-quantitative PCR and fluorescent real-time quantitative PCR were adopted to detect the mRNA level of MDR1 and MRP gene;3.5 With the western-blot method,protein level of P-gp,MRP,ERK,pERK and pJNK was determined and the relevance was analyzed.4.Statistical MethodsAll data were analyzed with software SPSS 13.0,the independent sample t was adopted to check the two-sample compare.Multi-group compare was analyzed with One-way ANOVA to detect the differences,LSD analysis to detect homogeneity of variance;while Pseudo F test(Welch) and Tamhane analysis used if uneven variance. The results were expressed as mean±standard deviation((?)±SD),P＜0.05 was treated as statistical significance. Results1.BEL-7402 cell and BEL-7402/5-FU cell showed epithelioid single-layer arrange,with passage once every 3 to 4 days,the survival rates of the two kind of cells in logarithmic growth phase were＞95%.The multinucleated giant cells in BEL-7402/5-FU cell increased significantly compared with which in BEL-7402 cell (P=0.000).The doubling time of BEL-7402 cell was 23 h,and BEL-7402/5-FU cell 32 h,significantly higher than that of BEL-7402 cell(P=0.007).2.The expression rate of P-gp protein in BEL-7402 cell surface was 11.64%±1.03%,while which in BEL-7402/5-FU cell was 68.94%±1.45%,significantly higher than which of the parental cell(P=0.000).The mitotic index of BEL-7402 cell gradually increased after passage,reached the peak in the 4th day,accounting for 69.33‰±0.96‰.After 5 d cultivation,the mitotic index began to decline,was the lowest in the 8th day.The mitotic index of BEL-7402/5-FU cell was also on the rise after passage,reached the peak in the 5th day,accounting for 69.83‰±1.82‰.After 6 d cultivation,the mitotic index began to decline,and was the lowest in the 8th day.3.Sorafenib showed no obvious toxicity to BEL-7402/5-FU cell under the dose of 4μmol/L,and its toxicity showed dose effect once over this concentration,IC₅₀ was(30.69±0.93)μmol/L.At(1～8)μmol/L concentration,intracellular Rho123 fluorescence intensity increased with the Sorafenib concentration;While over the concentration of 8μmol/L,the intracellular Rho 123 fluorescence intensity would no longer increase,indicating higher reverse efficiency and less side-effects of Sorafenib at the concentration of 4μmol/L.4.To all these four chemotherapy drugs - ADM,GEM,DDP and 5-FU, BEL-7402/5-FU cell showed resistance,and their IC₅₀ were(16.74±1.12)μmol/L, (253.56±2.41)μmol/L,(11.48±5.45)μmol/L and(893.02±2.55)μmol/L, respectively.While BEL-7402 cell are relatively sensitive,the IC₅₀ were respectively (3.56±0.03)μmol/L,(17.82±0.13)μmol/L,(3.35±0.05)μmol/L and(38.31±0.09)μmol/L.Compared with parental cells,the resistance multiple of drug-resistant cells to the four chemotherapy drugs were 4.70,14.23,3.30 and 23.36,respectively.Under the action of 4μmol/L Sorafenib,the sensitivity of the drug-resistant cell BEL-7402/5-FU enhanced to all the anticancer drugs,the IC50 decreased significantly(P(?)0.001),were(5.57±0.57)μmol/L,(35.63±1.30)μmol/L,(5.45±0.88)μmol/L and(88.31±1.60)μmol/L,respectively.The reversal multiple were 2.98,7.16,1.99 and 10.08.5.After the 24 h effect of 4μmol/L Sorafenib on BEL-7402/5-FU cell,P-gp expression rate was 36.34%±1.12%,P-gp expression rate of BEL-7402/5-FU cell was 67.44%±1.69%,and the expression rate of BEL-7402 cell was 30.66%±1.17%. P-gp expression rate of hepatoma cell drug-resistant cell BEL-7402/5-FU significantly decreased after Sorafenib effect(P=0.002).6.In Rho123 accumulation experiment,the Rho123 fluorescence was very strong in BEL-7402 cell,and the expression rate was 76.01%±3.47%;while which in BEL-7402/5-FU cell was weak,the expression rate was only 19.83%±1.44%. After 4μmol/L Sorafenib was added,the peak shifted to right,the intracellular fluorescence markedly increased,and the expression rate was 49.80%±1.35%.It indicated that Sorafenib could markedly increase the accumulation of drug-resistant cells to Rho123(P=0.000).7.The cell cycle detected with flow cytometry showed that Sorafenib could block cell cycle in G0/G1 phase.After 24 h effect of 4μmol/L Sorafenib,the cell ratio of BEL-7402 cell,BEL-7402/5-FU cell and BEL-7402/5-FU + sorafenib group in G0/G1 phase,were respectively:76.48%±1.23%,38.29%±1.40%and 56.45%±2.08%.While which in S phase were 15.69%±1.13%,48.15%±1.37%and 34.70%±2.79%.The cell ratio of BEL-7402/5-FU cell in G0/G1 phase after adding with Sorafenib was significantly higher than that before treatment(P=0.000),the cell ratio of S phase was significantly lower than that before treatment(P=0.000).8.With optical microscopy,the positive response of P-gp and MRP protein showed brown-yellow,mainly located in cytoplasm,showing uniform fine granular distribution.The expression of drug-resistant cell BEL-7402/5-FU group increased, the color deepened,widely distributed in the nucleus and cytoplasm;After the application of 4μmol/L Sorafenib,P-gp and MRP protein expression of drug-resistant cells decreased significantly,the color became shallow,the brown-yellow granules in cytoplasm reduced significantly.9.Semi-quantitative PCR results showed that:after 24 h effect of 4μmol/L Sorafenib on BEL-7402/5-FU cell,the MDR1/β-actin ratio of BEL-7402, BEL-7402/5-FU and BEL-7402/5-FU + sorafenib group were respectively 0.484±0.065,0.776±0.069 and 0.503±0.082.After Sorafenib treatment,MDR1 gene expression of BEL-7402/5-FU cell decreased 27.3%compared with that before the treatment(P=0.004).The MRP/β-actin ratio were:0.350±0.026,0.698±0.123 and 0.493±0.024,respectively.After the effect of Sorafenib on BEL-7402/5-FU cell, MRP gene expression decreased 20.5%compare with that before the treatment(P= 0.000).10.Real-time quantitative PCR results showed that:the CT value of MDR1 gene in BEL-7402 group was 23.65±0.21,and the CT value ofβ-actin was 20.87±0.16; The CT value of MDR1 gene in BEL-7402/5-FU group was 23.85±0.23,and the CT value ofβ-actin was 19.32±0.13;The CT value of MDR1 gene in BEL-7402/5-FU + sorafenib group was 23.33±0.22,and the CT value ofβ-actin was 19.43±0.15.2^{-△△CT} was calculated as 2^-0.63 according to formula,so the content of MDR1 gene in BEL-7402/5-FU + sorafenib group was about 64.62%of which in BEL-7402/5-FU group,the reversal rate was about 35.38%.The CT value of MRP gene in BEL-7402 group was 26.83±0.21,and the CT value ofβ-actin was 20.87±0.16;The CT value of MRP gene in BEL-7402/5-FU group was 26.87±0.23,and the CT value ofβ-actin was 19.32±0.13;The CT value of MRP gene in BEL-7402/5-FU + sorafenib group was 26.51±0.22,and the CT value ofβ-actin was 19.43±0.15.2^{-△△CT} was calculated as 2^-0.47 according to formula,so the content of MRP gene in BEL-7402/5-FU + sorafenib group was about 72.20%of which in BEL-7402/5-FU group,the reversal rate was about 27.80%.11.Western-blot results showed that after the application of 4μmol/L Sorafenib, P-gp protein(0.257±0.014) expression in BEL-7402/5-FU cell was decreased significantly compared with that before the treatment(0.400±0.014)(P=0.000); MRP protein(0.253±0.024) expression decreased significantly compared with that of before treatment(0.396±0.007)(P=0.000);ERK protein expression (0.913±0.006) showed no significant change compared with that before treatment (0.917±0.018)(P=0.683);pERK protein(0.663±0.009) expression decreased significantly compared with that before treatment(0.934±0.006)(P=0.000); pJNK protein(0.536±0.007) expression significantly increased compared with that before treatment(0.305±0.009)(P=0.000).Conclusion1.Sorafenib could markedly enhance the sensitivity of drug-resistant hepatocellular carcinoma cells to chemotherapeutic drug in vitro,and block cell cycle in G0/G1 phase,reduce the cell ratio of S phase,thus blocking the tumor cell proliferation.2.The results of immunocytochemistry,semi-quantitative and real-time fluorescence quantitative PCR and western blot showed that the possible mechanisms of Sorafenib reverse hepatoma MDR in vitro were the down-regulated MDR1 and MRP gene expression inhibited the function of the transmembrane transporter protein, decreased the chemotherapy drugs pump out of the cells,increased concentration of chemotherapeutic drugs in cells,thus enhancing the killing effect to drug-resistant cells.3.Sorafenib could also inhibit the ERK signal transduction pathway;activate the JNK signal transduction pathway,thus blocking the tumor cell proliferation.It indicated that the mechanism of Sorafenib reverse hepatoma MDR might also relate to the molecule which impacted the MAPK signal transduction pathway.4.It was proved in this experiment that Sorafenib showed strong effect of reversing hepatoma cell MDR in vitro,reversal of multidrug resistance of hepatocellular carcinoma cell function,providing an important clue for the further experimental and clinical MDR reversing in vivo of animal.