| Hepatocellular carcinoma (HCC) is one of the most common malignant diseases with600,000new cases reported each year worldwide, and more than50%of the new HCC casesand deaths have occurred in China. Although aggressive surgery offers significant rates ofcure, only15%of patients are eligible for optimal resection at diagnosis. The efficacy ofchemotherapy and radiotherapy for HCC remain disappointing. Radiotherapy represents amajor therapeutic option for HCC patients, but the efficacy of this therapy is limited byintrinsic radioresistance of the tumor cells. Ionizing radiation (IR) can result in lethal celldamage, which is correlated with DNA damage induction and repair. The activity of the DNAdamage repair pathway is the major factor leading to radioresistance in tumors, includinghepatoma. DNA-repair systems play an important role in protecting the genomic stabilizationand integrity. However, an elevated DNA repair capacity in tumor cells is associated withdrug or radiation resistance.The human apurinic/apyrimidinic endonuclease (here-after, APE1) is a key enzyme inthe DNA base excision repair (BER) pathway, which plays a critical role in repairing DNAdamaged by irradiation. In addition to its DNA repair function, APE1maintains a number oftranscriptional factors including p53by both redox-dependent and-independent mechanismsin their reduced and active state, thereby regulating their DNA-binding activity, influencinggene expression and maintaining genomic stability. As known that, the p53gene is mutated inapproximately50%of hepatoma cells. In our previous study, we found APE1high expressionwas associated with mutp53status. The inhibition rate of Ad5/F35-APE1siRNA+Ad5-wtp53+IR group was higher than that of Ad5-wtp53+IR group. These suggest that APE1actswith mtp53to regulate the radioresistance of HCC. APE1can enhance the DNA-bindingactivity of wtp53by redox-dependent and independent mechanisms. Moreover, theredox–independent activation of wtp53is due to a regulatory interaction of APE1with the p53C-terminal regulatory domain (CRD), which is intact in most of the frequentlyencountered mutp53proteins, containing the most common R249S mutation in HCC. In thisstudy, we detect the impact of APE1regulation on mtp53and the possible mechanism toelucidate the mechanisms of HCC radioresistance and provide new evidences for HCCtherapyObjective1. To investigate the “gain of function” oncogenic effect of mtp53and explore itscorrelation with radiosensitivity of HCC in vitro;2. To investigate the inhibitory effects of Ad5/F35-APE1siRNA combined withradiotherapy on human HCC cells in vitro.3. To investigate the mechanism of regulatory role of APE1on mtp53;4. To investigate the inhibitory impacts of Ad5/F35-APE1siRNA combined withradiotherapy on HCC xenografts with p53mutation.Materials and Methods1. The oncogenic effect of mtp53and its correlation with radiosensitivity of HCC: Theradiosensitivity of human HCC cell lines with different p53status (HepG2, wtp53; Hep3B,p53-/-; MHCC97L,mtp53) was detected by MTT assay, colony formation assay and flowcytometry analysis; Hep3B was infected with pCMV-wtp53or pCMV-mtp53R249S, then theradiosensitivity was also detected.2. The regulation effects of APE1on mtp53R249S: The human HCC HepG2, Hep3Band MHCC97L cells were infected with Ad5/F35-APE1siRNA or Ad5/F35-EGFP, then theradiosensitivity was detected by MTT assay, colony formation assay and flow cytometryanalysis; Meanwhile, Western Blot and qRT-PCR were used to detect APE1、P53and P21protein and mRNA expression.3. The mechanism of regulatory role of APE1on mtp53: The p53response element ofthe p21Waf1-promoter (p53-RE) was prepared either in its linear form or in a stem loopconformation. EMSA was used to detect the DNA binding activity of latent and reducedmtp53R249Son P53RE-Iine and p53RE-structure; Chromatin immunoprecipitation (ChIP)assays were performed to analyze the DNA binding affinity of p53to the p21promoter regionof its downstream genes. 4. Study of inhibitory impacts of Ad5/F35-APE1siRNA combined with radiotherapy onHCC xenografts with p53mutation: Nude mice hepatocarcinoma xenograft model wasestablished by using human HCC HepG2and MHCC97L cell lines. There are four groups:Ad5/F35-EGFP control group, Ad5/F35-APE1siRNA group, Ad5/F35-EGFP+IR group andAd5/F35-APE1siRNA+IR group. We measured the tumor volumes and plotted the tumorgrowth curves. The tumor growth inhibition ratio was observed by Ki-67immunohisto-chemistry. Apoptosis index was detected by using TUNEL.Results1. The oncogenic effect of mtp53and its correlation with radiosensitivity of HCC:HepG2、Hep3B and MHCC97L cells were irradiated with different doses of X ray, and thenthe radiosensitivity was detected. After irradiation, the cell survival and colony formation ratewas decreased, and tumor cells apoptosis was increased in a dose-dependent manner; at samedose of X ray, the cell survival and colony formation rate of MHCC97L was higher than thatof HepG2and Hep3B cells, and the apoptosis rate of MHCC97L was lower than two celllines；the cell survival and colony formation rate of Hep3B was higher than that of HepG2atthe same dose of radiation.Hep3B were infected with pCMV-wtp53and pCMV-mtp53, and then irradiated with Xray. The cell survival and colony formation rate was decreased, and tumor cells apoptosis wasincreased in a dose-dependent manner; at the same dose of X ray, the cell survival and colonyformation rate was pCMV-mtp53group>non-infection group>pCMV-wtp53group, andapoptosis rate was pCMV-wtp53group> non-infection group> pCMV-mtp53group.2. The regulation effects of APE1on mtp53R249S:HepG2、 Hep3B and MHCC97L were infected with Ad5/F35-APE1siRNA orAd5/F35-EGFP; and then irradiated with X ray. The cell survival and colony formation ratedecreased, apoptosis rate increased in a dose-dependent manner after irradiation. At the samedose of irradiation, the cell survival and colony formation rate in Ad5/F35-APE1siRNAgroup was much higher, and the apoptosis rate was lower, compared with Ad5/F35-EGFPgroup.After irradiation, the expression of APE1protein increased in a dose-dependent manner,and reached the highest level at6Gee dose. HepG2and MHCC97L cells were infected with Ad5/F35-APE1siRNA or Ad5/F35-EGFP, and irradiated with6Gy X-ray. Western Blot wasperformed to detect the protein expression of APE1、P53and P21. Ad5/F35-APE1siRNAcould inhibit irradiation-induced APE1expression in both HepG2and MHCC97L cells.Ad5/F35-APE1siRNA also suppressed irradiation-induced WTP53and P21proteinexpressions in HepG2cells. Silencing of APE1inhibited irradiation-induced MTP53proteinexpression in MHCC97L cells, but no P21protein was found.3. The mechanism of regulatory role of APE1on mtp53:Reduced wtp53bound effectively to p53RE-line and p53RE-structure, but reducedmtp53bound to nonlinear DNA, not to linear DNA. APE1stimulated wtp53DNA binding tolinear and nonlinear DNA, and promoted mutant p53DNA binding to nonlinear DNA.WTAPE1enhanced the p21DNA activity, p21mRNA and protein expression in HepG2cells, whereas Ad5/F35-APE1siRNA inhibited them. WTAPE1could enhance p21DNAactivity in MHCC97L cells, but not p21mRNA and protein expression; Knowdown ofAPE1suppressed the p21DNA binding activity, but not p21mRNA and protein expression.4. Study of inhibitory impacts of Ad5/F35-APE1siRNA combined with radiotherapy onHCC xenografts with p53mutation: The tumor growth inhibition ratio and apoptosis index inAd5/F35-APE1siRNA+IR group were significantly higher, compared with the Ad5/F35-EGFP control group, Ad5/F35-APE1siRNA group and Ad5/F35-EGFP+IR group. There wasno significant difference of the tumor growth inhibition ratio and apoptosis index in Ad5/F35-APE1siRNA group and Ad5/F35-EGFP+IR group, but it was higher than in control group.Conclusion1. The radiosensitivity of human HCC cell lines was HepG2>Hep3B>MHCC97L; Afterinfection with pCMV-wtp53and pCMV-mtp53in Hep3B cells, the radiosensitivity waspCMV-wtp53> non-infection group> pCMV-mtp53group. These results indicated thatmtp53was related to radioresistance of HCC.2. Ad5/F35-APE1siRNA can enhance the radiosensitivity of HCC cell lines; APE1may act with Mtp53protein, activate the p53down scream gene, but not p21gene, exert thecooperative effect on radioresistance of HCC.3. Reduced Mtp53bound strongly and specifically to nonlinear DNA; APE1stimulatedmutant p53DNA binding to nonlinear DNA. 4. The combination of Ad5/F35-APE1siRNA and radiotherapy can significantly inhibitthe growth of HCC cells, and promote apoptosis of tumor cells. Therefore, combine genetherapy targeting APE1with radiotherapy may be a promising new approach for treatment ofunresectable HCC in the future. |
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