The Study On The Role Of BCCIP In The Radiation Sensitivity Of Celecoxib In The Colorectal Cancer

Part I The proliferation inhibition and radiosensitizing effects ofCelecoxib on the tumor cell lines with different levels of Cox-2Objective: To observe the proliferation inhibition and radiosensitizing effects of COX-2 selective inhibitor Celecoxib on the tumor cell lines with different levels of COX-2, and on the expression of BCCIP gene in vitro.Methods: Human lung carcinoma cell line A549, cervical carcinoma cell line Hela and colon carcinoma cell line HCT116 were used in vitro. CCK-8 was used to examine the viability of cells which were incubated with different concentrations of Celecoxib after different incubation duration. The effects of radiosensitivity on cells were observed by colony formation assay. And the expression of proteins were investigated by Western blot. All the measurement data were presented as means ± standard deviation(means+SD). The differences between the two groups are compared by paired samples T test. Cell survival curves were generated by Graph Pad software. Linear-quadratic formula and "Multitarget-single hitting" model were used to fit those survival curves. The parameters of the radiosensitivity, such as D0, Dq, N and K, were calculated directly via Graph Pad software.Results: The expressions of COX-2 were positive in both Human lung carcinoma cell line A549 cells and cervical carcinoma cell line Hela cells, however, it was negative in colon carcinoma cell lines HCT116. The proliferations of three cell lines were significantly inhibited by the different concentrations and incubation durations of Celecoxib. High proliferation-inhibition ratios resulted from high concentrations and long incubation duration, which suggested in a concentration and time dependent manner. The radiosensitizing roles of 30μmol/L Celecoxib were observed in those three cell lines, with the SERD0 values: 1.254 for A549, 1.213 for Hela and 1.224 for HCT116. The BCCIP proteins were normally expressed in the three cell lines. The up regulation of BCCIP protein was observed in HCT116 cells with negative COX-2 expression after incubating with 30μmol/L Celecoxib for 6 hours.Conclusion: The radiotherapy sensitization effect of Celecoxib in the tumor cells is not related to the COX-2 expression level. When treated with 30μmol/L Celecoxib for 6 hours, the BCCIP expression was up-regulated significantly in HCT116 cells, whose COX-2 expression is negative.Part II Construction of colon cancer cell line with stable knock-down ofBCCIP geneObjective: To construct a colon cancer cell line with stable knock-down of BCCIP and observe the different effects of Celecoxib on the radiation sensitivity of the colon cells with or without BCCIP expression.Method: A BCCIP knocked down cell model C12 was constructed by transfecting HCT116 cell with BCCIP-sh RNA plasmid, and the expression levels of both BCCIP transcript and BCCIP protein in transfected HCT116 cells was detected by RT-PCR and Western blot. Influences of Celecoxib on radiotherapy sensitization effects of homologous HCT116 cell lines with different BCCIP expression levels were detected by colony formation assays.Results: The HCT116 cells, which were transfected by the plasmid combinations of p PUR/U6-311+p Silencer2.1Hyg-633 or p PUR/U6-311+p Silencer2.1Hyg-730, showed a significant decrease of BCCIP transcript expression level, which was more than 95%(P<0.05). The cellular total proteins of stable transfected monoclonal HCT116 cells after puromycin selection(named C12) and the control cells were isolated, and BCCIP protein expression were examined by Western blot. In comparison with non-transfected cells, HCT116 cells with p PUR/U6-311+p Silencer2.1Hyg-730 transfection had significant reduction of BCCIP at protein expression level(P<0.05). 30μmol/L Celecoxib obviously increased the radiosensitivity of HCT116 cells, sensitization enhancement ratio SERD0=1.224, however radiotherapy sensitization effects of Celecoxib were not found in C12 cells with stabe BCCIP knock down, SERD0=1.038.Conclusion: C12 cell line,which was HCT116 cells with stable BCCIP gene knock down, could be successfully constructed and passaged. 30μmol/L Celecoxib had little effect on its radiotherapy sensitization compared with HCT116 with normal BCCIP expression.Part III The mechanisms of BCCIP gene acting on the radiosensitivityenhencement of HCT116 cells caused by CelecoxibObjective: To investigate the impact of BCCIP gene on the radiosensitivity enhancement of HCT116 cells caused by Celecoxib, and study the possible mechanisms.Method: Western blot was carried out to observe the protein expression of two kinds of cells with different BCCIP expression levels, which were treated by 30μmol/L Celecoxib, irradiation(6Gy X-rays) or 30μmol/L Celecoxib+irradiation(6Gy). The expression of γ-H2 AX, phosphorylated ATM and Chk2 proteins were examined in these two cells at different time points after irradiation, and the expressions of cell cycle and apoptosis related proteins in HCT116 and C12 were also detected. The γ-H2 AX foci formation were observed in the cells with the forementioned treatment at different time points by immunofluorescent assay. Cell cycle distribution and cell apoptosis of cells in untreated group, 30μmol/L Celecoxib group, different radiation dose groups and different radiation doses+30μmol/L Celecoxib groups were detected by flow cytometry assay.Result: 1h and 3h after 30μmol/L Celecoxib+6Gy irradiation treatment, HCT116 cells expressed much more γ-H2 AX than irradiation group and other groups, however this phenomenon was not observed in C12 cells; the same results were found in immunofluorescent detection. Combined use of 30μmol/L Celecoxib six hours in advance could greatly increase G2-M arrest induced by irradiation, and the differences is statistically meaningful(P<0.01), nevertheless this difference was not observed in C12 cells(P>0.05). Apoptosis could be induced by irradiation in both HCT116 cells and C12 cells, and it was increased when the HCT116 cells were treated with 30μmol/L Celecoxib prior to irradiation significantly(P<0.05), while the apoptosis increasement of C12 cells was limited when given the same treatment(P>0.05). With the treatment of 30μmol/L Celecoxib for 6h, the BCCIP, p53 and p21 proteins were up-regulated, Cyclin B1 proteins down-regulated, while expressions of all those proteins in C12 cells were not changed.Conclusion: BCCIP gene may be a radiosensitivity-related gene. Celecoxib could affect the functions of p53 and inhibit the recovery from the irradiated-induced injury by regulating BCCIP, then regulate expressions of genes such as p21 and Cyclin B1 to enhance radiosensitivity of HCT116 cells.Part IV The predictive value of the expression level of BCCIP for thetreatment of Celecoxib with chemoradiotherapy in rectal cancerObjective: To investigate the relationship between the expressions of BCCIP, COX-2 and P53 protein of biopsy tissue samples from rectal cancer of patients before treatment and the treatment efficacy of Celecoxib given with neo-adjuvant chemoradiotherapy. And the predictive value of the expressions of BCCIP in rectal cancer tissue for the radiosensitivity enhancement of Celecoxib were also explored.Methods: Those patients were enrolled, including age between 18 and 70, performance status from 0 to 2, rectal adenocarcinoma before treating by pathologic biopsy, never receiving colorectal surgery or radiochemotherapy. The conventional histopathologic sections and tissue microarrays from biopsy tumor tissue samples were used to detect the protein expressions immunohistochemically. Every patient had one pelvic magnetic resonance imaging(MRI) before surgery and chemotradioherapy, those who couldn’t receive MRI for various reasons would be scanned by contrast-enhanced pelvic CT. The professional radiologists had the interpretations of MRI or CT and compared the tumor regressions before and after neo-adjuvant treatment. Those tissue microarrays were stained by immunohistochemistry, and the expressions of BCCIP, COX-2 and p53 proteins were detected. Patients were given Celecoxib with chemoradiotherapy before surgery. The evaluation of radiosensitivity of surgical specimens was determined by TRG score. According to the tumor regressions before and after chemoradiotherapy, the clinical efficacy was evaluated by Response Evaluation Criteria in Solid Tumors(RECIST) from the images of MRI or CT scan. The correlations between TRG and RECIST scores were analyzed by the Spearman rank correlation test. The correlations of differences among therapeutic effects and clinical data or expressions of BCCIP, COX-2 and p53 in cancer tissue were analyzed by Fisher’s exact test.Results: There were 31 patients with colorectal cancer who met the diagnostic criteria were administered Celecoxib with chemoradiotherapy before surgery in the Department of Radiation Oncology, the First Affiliated Hospital of Soochow University from Jan 2012 to Dec 2014. All 31 patients completed the treatments and well-tolerated. 3 patients displayed obvious tumor regression and refused surgery due to old age or surgical contraindication. Among 31 patients, 7 were BCCIP expression negative, 2 were COX-2 expression negative, and 4 were p53 expression positive. Univariate analysis indicated that BCCIP expression was strongly related to the objective validity of tumor TRG and RECIST after therapy, but less related to p CR(p=0.290), non related to COX-2 expression. The expression of p53 had little relationship with p CR and TRG as well. However patients with wild type p53 showed more obvious tumor regression in images(P=0.028).Conclusion: The application of regimen of Celecoxib with chemoradiotherapy in local advanced rectal cancer was safe and effective. BCCIP could be a predictive factor for curative effect of neo-adjuvant chemoradiotherapy in local advanced rectal cancer and it was hopeful as an index in primary screening for local advanced rectal cancer patients who would accept Celecoxib with radiotherapy.