Chimeric Adenoviral Vector Ad5/F35 Delivery Of APE1 SiRNA Enhances Sensitivity Of Human Colorectal Cancer Cells To Chemotherapy And Radiotherapy

Colorectal cancer(CRC) is one of the most common malignancies, and the incidence tends to increase in China. The frequency of lymph node and liver metastases is high in CRC patients. The 3 main types of treatment for colorectal cancer are surgery, radiation therapy and chemotherapy. However,the clinical efficacy of chemoradiotherapy is poor. Moreover, the CRC patients with similar tumor location, tumor size, and histologic subtype display different therapeutic response to chemoradiotherapy. It suggests that intrinsic sensitivity of CRC to chemoradiotherapy is significant different. DNA-repair systems, as the molecular basis of defending against environmental damage to cell DNA, play an important role in protecting the genomic stabilization and integrity. However, an elevated DNA repair capacity in tumor cells leads to drug or radiation resistance and severely limits the efficacy of these agents. Interference with DNA repair has emerged as an important approach in combination therapy against cancer.The human apurinic/apyrimidinic endonuclease(APE1), is abundant in human cells and accounts for nearly all of the abasic site cleavage activity observed in cellular extracts. In addition to its DNA repair functions, APE1 is also a multifunctional protein that participates in other crucial cellular processes, including the response to oxidative stress, regulation of transcription factors, such as nuclear factorκB (NF-κB). The transcription factors are associated with chemoresistance and radioresistance. Therefore, targeting inhibition of APE1 display an emerging cancer therapeutic opportunity. In this study, we first constructed chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA and examined its infectivity to human colorectal cancer cells in vitro and in vivo. Then, we investigated its effect on the expression of APE1 and the sensitivity to chemotherapy and radiotherapy in human colorectal cancer cells.Objective 1. To investigate the role of APE1 in the carcinogenesis and progression of CRC, and determinate that APE1 is an effective therapeutic target gene of CRC.2. To investigate the perspective of clinical application of gene therapy targeting APE1 gene enhancing the sensitivity of CRC cell to chemotherapy and radiotherapy.3. To investigate the perspective of clinical application of chimeric adenoviral vector Ad5/35 of gene transfer for CRC.Materials and Methods1. Expression feature of APE1 and its significance in CRC: Expression of APE1 was examined by SP immunohistochemical technique in 40 cases of normal colorectal mucosa, 60 cases of adjacent colorectal mucosa with CRC, 72 cases of colorectal adenoma and 125 cases of colorectal carcinoma.2. Construction and identification of chimeric adenoviral vector Ad5/35 carrying human APE1 siRNA: First,the foreign APE1 siRNA cDNA was inserted into a small shuttle plasmid called pDC316-U6-EGFP. Then, the obtained shutle plasmids pDC316-U6-EGFP-APE1 siRNA were cotransfected with rescue plasmid pBHG35 into 293 cells to acquire recombinant adenovirus Ad5/F35-APE1 siRNA. The recombinant adenoviruses'titers were measured after amplification by means of TCID50 assay. The infection efficiency of Ad5/35-EGFP, Ad5/F35-APE1siRNA and Ad5-EGFP to human CRC LOVO cell line was measured by flow cytometry and observed by fluorescence microscopy. The expression of human APE1 protein was detected after Ad5/F35-APE1 siRNA or Ad5/F35-EGFP was transfered into LOVO cells by western blot analysis and immunohistochemical technique in vitro and in vivo, respectively. [γ-32P]ATP-labeled oligonucleotide containing AP site was used for AP endonuclease assay.3. Study of chimeric Ad5/F35-APE1 siRNA enhancing sensitivity of human colorectal cells to radiotherapy: Classic clone formation assay and tumor xenograft model were used to detect radiobiological reaction in vitro and in vivo, respectively. Alkaline comet assay was used to detect DNA damage. Cell apoptosis was determined by TUNEL. The expression of human APE1 protein was detected by western blot analysis, indirect immunofluorescence and immunohistochemical technique. We also assayed for NF-κB DNA binding by EMSA.4. Study of chimeric Ad5/F35-APE1 siRNA enhancing sensitivity of human colorectal cells to 5-FU: Cells were treated with 5-FU at various concentrations 48 hours after Ad5/F35-APE1 siRNA or Ad5/F35-EGFP was transfered into LOVO cells, and the cellular proliferation capacity was observed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was determined by TUNEL. The expression of human APE1 protein was detected by western blot analysis and indirect immunofluorescence. NF-κB DNA binding was detected by EMSA.Results1. Expression feature of APE1 and its significance in CRC: In normal colorectal mucosa, APE1 was detected in nucleus of epithelial cells, the shifts of APE1 from nucleus to cytoplasm was observed in 6 of 60 (10%) adjacent colorectal mucosa tissues. The shifts of APE1 from nucleus to cytoplasm was observed in 92 of 125 (73.6%) CRC tissues and 60 of 72 (83.3%) colorectal adenoma, which was significantly higher than that of normal colorectal mucosa and adjacent colorectal mucosa tissues with CRC(P<0.01). Statistic analysis showed that the shifts of APE1 from nucleus to cytoplasm was associated with Dukes stage and lymph node metastasis in CRC(P<0.01, P<0.05).2. Construction and identification of chimeric adenoviral vector Ad5/35 carrying human APE1 siRNA: The recombinant chimeric adenoviral vector Ad5/35 carrying human APE1 siRNA(Ad5/F35-APE1 siRNA) was constructed successfully.The titer was 1.6×1010 IU/mL after amplification. The infectivity of Ad5/35-EGFP or Ad5/F35-APE1 siRNA was greater than that of Ad5-EGFP. The results showed that the infection efficiency of Ad5/35 and Ad5 vector at 20 multiplicity of infection (MOI) to LOVO cells was 99% and 15%, respectively. In vivo experiment, a number of fluorescence positive cells were observed in LOVO xenografts 3 days after 5×108 IU Ad5/35-EGFP or Ad5/F35-APE1 siRNA was injected intratumorally, but few fluorescence positive cells were observed in Ad5-EGFP groups. Infection of LOVO cells with Ad5/F35-APE1 siRNA resulted in a dose-dependent suppression of APE1 protein and AP endonuclease activity in vitro. Ad5/F35-APE1 siRNA also suppressed significantly the expression of APE1 protein in vivo.3. Study of Ad5/F35-APE1 siRNA enhancing sensitivity of human colorectal cells to radiotherapy: Clone formation assay showed that Ad5/F35-APE1 siRNA enhanced sensitivity of LOVO cells to radiotherapy, and SER (sensitive enhancement ratio) of D0 and Dq is 1.23, and 5.75, respectively. Ad5/F35-APE1 siRNA also increased cell apoptosis induction by irradiation. Alkaline comet assay showed that radiation induced increased DNA damage in LOVO cells pretreatmented with Ad5/F35-APE1 siRNA comparing with Ad5/35-EGFP. Ad5/F35-APE1 siRNA also enhanced sensitivity of LOVO cells to radiotherapy in vivo. The tumor-inhibition rate of Ad5/F35-APE1 siRNA alone, combination of Ad5/35-EGFP and radiotherapy, combination of Ad5/F35-APE1 siRNA and radiotherapy treatment group was 15.06%, 41.41% and 70.90%, respectively. We found that the protein expression of APE1 in LOVO cells was induced by irradiation in a dose-dependent manner, accompanied with the enhancement of the DNA binding activity of NF-κB, and Ad5/F35-APE1 siRNA can effectively suppress the APE1 expression and NF-κB activation induced by irradiation.4. Study of Ad5/F35-APE1 siRNA enhancing sensitivity of human colorectal cells to 5-FU: The result of MTT showed that Ad5/F35-APE1 siRNA enhanced sensitivity of LOVO cells to 5-FU. The 50% inhibitory concentration (IC50) value for LOVO cells pretreatmented with Ad5/F35-APE1 siRNA and Ad5/35-EGFP at 72 h after 5-FU treatment was 1.69μM and 7.04μM, respectively. Ad5/F35-APE1 siRNA also increased cell apoptosis induction by 5-FU. The protein expression of APE1 in LOVO cells was induced by 5-FU in a dose-dependent manner, accompanied with the enhancement of the DNA binding activity of NF-κB, and Ad5/F35-APE1 siRNA can effectively suppress the APE1 expression and NF-κB activation induced by 5-FU.Conclusion1. The shifts of APE1 from nucleus to cytoplasm might play a pivotal role in the carcinogenesis, progression and metastasis of CRC. It suggests that APE1 is a potential molecular therapeutic target of CRC.2. Chimeric adenoviral vector Ad5/35 is more suitable for CRC gene therapy because of its significantly higher infection efficiency to CRC cells compared with Ad5 adenoviral vector.3. The protein expression of APE1 in CRC cells is induced by irradiation and 5-FU in a dose-dependent manner, accompanied with increase of the DNA binding activity of NF-κB. Ad5/F35-APE1 siRNA can effectively suppress the APE1 expression and NF-κB activation induced by irradiation and 5-FU. Therefore, APE1 may play an important role in chemoresistance and radioresistance of cancer. 4. Ad5/F35-APE1 siRNA can specifically knock down the APE1 protein expression in CRC cells, and the inhibition rate of expression of APE1 protein reached in 95%.5. Targeting inhibition of APE1 enhances sensitivity of CRC cells to irradiation in vitro and in vivo, and two main molecular mechanisms involved: one is that suppression of the cellular repair capacity responding to sub-lethal lesion caused by ionizing radiation, the other is that suppression of the transcription activity of radioresistance-related transcription factors such as NF-κB.6. Targeting inhibition of APE1 enhances sensitivity of CRC cells to 5-FU in vitro, and the main molecular mechanisms involved may be downregulation of base excision repair capacity and transcription activity of 5-FU-resistant-related transcription factors such as NF-κB in CRC cells.