Effect Of Apatinib On The Invasion,metastasis And Multidrug Resistance Of Human Liver Cancer Cells And Its Mechanism

Part 1 Apatinib inhibits the invasion and metastasis of liver cancer cells by downregulating MMP-related proteins via regulation of the NF-κB signaling pathway[Purpose] We aimed to investigate whether apatinib has an inhibitory effect on the invasion and metastasis of liver cancer in vitro.[Methods](1)The anti-invasion and anti-metastasis effects of apatinib in Hep G2,Hep3 B,Huh7and SMMC-7721 liver cancer cell lines were tested by the wound-healing and transwell invasion assays.(2)Real-time PCR and Western blot were used to detect the influence of apatinib on the gene expression of MMPs,TIMPs and constituents of the NF-κB signaling pathway in the Hep G2 and Hep3 B liver cell lines.[Results](1)Apatinib has a significant inhibitory effect on the metastasis and invasion of liver cancer cells.(2)The expression levels of MMP-1,-2,-3,-7,-9,-10,-11 and-16 were downregulated,while the expression levels of TIMP-3 and-4 were upregulated by apatinib treatment at both the m RNA and protein level.(3)The phosphorylation of IκBα and NF-κB p65 was significantly reduced compared with that in the control group.[Conclusions](1)Apatinib inhibits the invasion and metastasis of human liver cancer cells by down-regulating the expression of MMP-1,-2,-3,-7,-9,-10,-11 and-16 in MMP family genes;(2)Down-regulation of MMP-related gene expression is related to up-regulation of its endogenous tissue inhibitors TIMP-1 and TIMP-2;(3)The above effects of apatinib may be achieved by inhibiting the activation of the NF-κB signaling pathway.Part 2 Apatinib inhibits the multidrug resistance of liver cancer cells by downregulating MDR-related proteins via regulation of the NF-κB signaling pathway[Purpose] We aimed to investigate whether apatinib has an inhibitory effect on the multidrug resistance of liver cancer in vitro and in vivo.[Methods](1)The Hep3B/5-Fu resistant cell line was established by increasing the concentration of 5-Fu in the culture medium.(2)The CCK8 test and flow cytometry assay confirmed the drug resistance of Hep3B/5-Fu cells and the reversal effect of apatinib on multidrug resistance.(3)NF-κB si RNA was transfected into Hep3B/5-Fu multidrug-resistant cell lines to detect the expression changes of MDR and NF-κB pathway-related genes after apatinib treatment.(4)Nude mice with subcutaneous xenograft tumors were established by implanting Hep3B/5-Fu cells and randomly divided into eight groups: control group,Apatinib monotherapy group(50 mg / kg / day),5-Fu monotherapy group(20 mg / kg,administered twice a week),Oxaliplatin monotherapy group(6 mg / kg,administered twice a week),Apatinib + 5-Fu group,Apatinib + Oxaliplatin group,5-Fu + Oxaliplatin group and Apatinib + 5-Fu + Oxaliplatin group.(5)Nude mice were sacrificed and tumors were removed 24 hours after the last administration.The expression levels of the multidrug resistance genes in tumor tissues were detected by real-time PCR,immunohistochemistry and Western blot analysis.[Results](1)Hep3B/5-Fu human liver cancer cell multidrug resistance line was successfully established.Hep3B/5-Fu cells showed high resistance to 5-Fu with a drug resistance index of 46.14±10.26.The resistance indexes of epirubicin and oxaliplatin were 4.31±0.90 and6.61±0.78,respectively.(2)Apatinib reversed the drug resistance of Hep3B/5-Fu cells and promoted its apoptosis.The reversal index was 2.19–3.86 and increased with increasing apatinib concentration.(3)After transfection of NF-κB si RNA,the expression of resistance-related proteins P-gp and LRP in Hep3B/5-Fu cells were significantly down-regulated.(4)Testing in Hep3B/5-Fu multidrug-resistant cells transfected with NF-κB p65 si RNA,it was found that compared with the control group,the MDR-related proteins(P-gp,LRP,GST-pi)and NF-κB signaling pathway related proteins(p-IκBα,p-p65)expression levels were significantly reduced in the apatinib treatment groups.(5)The results of animal experiments showed that the tumor growth rate of the combined treatment(apatinib plus chemotherapy)groups was significantly lower than that of the chemotherapy group.The difference was statistically significant(P < 0.05).Among the combination treatments,the three-drug combination group(Apatinib + 5-Fu +Oxaliplatin)showed the slowest growth and the smallest tumor volume.(6)Extraction of proteins and m RNA from stripped xenografts,real-time quantitative PCR,immunohistochemistry and western blot analysis showed that apatinib can inhibit the expression of multidrug resistance-related genes MDR1,LRP,MRP2 and GST-pi at the protein level.[Conclusions](1)Apatinib can promote the apoptosis of Hep3B/5-Fu cell line and reverse its multidrug resistance;(2)Apatinib inhibits the multidrug resistance of human liver cancer cells to chemotherapeutic drugs by down-regulating the expression of multidrug resistance-related genes MDR1,LRP,MRP2 and GST-pi;(3)This may be achieved through inhibiting the activation of the NF-κB signaling pathway.