PKI-587 Enhances Chemosensitivity Of Oxaliplatin In Hepatocellular Carcinoma

BACKGROUND&AIMS:Hepatocellular carcinoma(HCC)is the second leading cause of cancer-related deaths worldwide and is the most common primary liver cancer.Because the early symptoms of liver cancer patients are not significant and the early diagnosis technology is not complete,it is easy to make the disease progress to advanced stages.For early patients,surgical resection is a better treatment,but for patients with poor body load and in the advanced stage,systemic chemotherapy is a very important treatment.Oxaliplatin,as a first-line treatment for advanced hepatocellular carcinoma,is widely used in the treatment of patients with advanced tumors,but resistance has limited its effectiveness in the treatment of HCC.In recent years,many scholars have done a lot of work on exploring the mechanism of tumor resistance,and found that the abnormal activation of signal pathways after the application of chemotherapy drugs plays an important role in it.Among them,abnormal activation of the PI3K/AKT/mTOR pathway has been associated with decreased survival of HCC patients,anti-apoptosis after chemotherapeutic drug-induced DNA damage,and chemoresistance.In this study,we evaluated the effect of the PI3K/mTOR dual inhibitor PKI-587 on the chemosensitivity of oxaliplatin in HCC.METHODS:MTT assay and clone formation assay were applied to evaluate the proliferation abilities of HepG2 and SK-Hep1 cells;Cell scratch assay and Transwell assay were used to evaluate the migration abilities of HepG2 and SK-Hep1 cells;indirect immunofluorescence assay was used to detect the formation of DNA breakpoints;flow cytometry assay was applied to evaluate the cell cycle of HepG2 and SK-Hepl cells;AnnexinV-FITC/PI/Hoechst33258 staining was used to evaluate the apoptosis of HepG2 and SK-Hep1 cells by fluorescence microscopy;JC-1 staining was used to evaluate the cell membrane potential of HepG2 and SK-Hep1 cells by fluorescence microscopy;a HepG2 tumor-bearing model was used to assess the in vivo effects of the combination of the two compounds by measuring tumor volume changes after administration of drugs;western blotting assay was used to analyze the expression levels of related protein molecules which regulation of cell DNA damage response,proliferation,clonogenic survival,cycle and apoptosis in HepG2 and SK-Hep1 cells lines and tumor tissues,respectively.RESULTS:In HCC cells,oxaliplatin stably activates the PI3K/AKT/mTOR signaling pathway and DNA damage repair pathways(non-homologous end joining(NHEJ)and homologous recombination(HR)),which were attenuated by PKI-587.In vitro,compared with the drug alone,oxaliplatin combined with PKI-587 can more inhibit the proliferation of HCC cells and down-regulate the expression of eIF4EBP1 and S6K1 that regulate cells proliferation,and more inhibit HCC cells migration and down-regulate the expression of MMP2 and MMP9 that regulate cells migration,and more suppress the repair of HCC cells DSB,increase the generation of DSB breakpoints,and could down-regulate the expression of p-DNAPKcs(Ser2056),p-ATM(Ser1981)and p-ATR(Ser428),and more reduce mitochondrial membrane potential and up-regulate the expression of Bad,Bax,Puma,Cytochrome C,Apaf-1,and increase activities of Caspase3,Caspase9 and PARP.In addition,oxaliplatin alone can increase the percentage of S phase cells,while PKI-587 combined with oxaliplatin can increase the percentage of G0/G1 phase cells,and can down-regulate the expression of p-Rb and cyclin D1,which are regulating cell transitioned from G0/G1 phase to S phase.In vivo,compared with oxaliplatin alone,the combined treatment of PKI-587 and oxaliplatin can inhibit the tumor growth.Anti-tumor effects were associated with stimulation of Cytochrome C,generation of y-H2AX foci and induction of Caspase3 and PARP by inhibiting the activation of the PI3K/AKT/mTOR signaling pathway.Conclusion:PKI-587 enhances chemosensitivity of oxaliplatin in hepatocellular carcinoma through suppressing DNA damage repair pathway(NHEJ and HR)and PI3K/AKT/mTOR pathway.The combination of PKI-587 and oxaliplatin appears to be a promising regimen for the treatment of HCC.Figure[20]table[0]reference[43]...