| Colorectal cancer included colon and rectum cancer, what is one of the world’s third largest malignant tumors. In American and European countries, the incidence of colorectal cancer reached up to35-50/10million and ranked third in cancer-related mortality. Which colorectal cancer compared with colon cancer incidence, about1.5:1, and displayed gradually close. In China, with the change of people’s living standards improve and diet, the incidence of colorectal cancer had been rising. Colorectal cancer incidence and mortality in malignant tumors were10.56%and7.8%for respectively, ranking first three and fifth. Colorectal cancer and colon ratio were close, and a larger proportion of males. Colorectal cancer detection, diagnosis and treatment were becoming more and more attention. Surgical treatment was still the main treatment for colorectal cancer. Total mesorectal excision (TME) had become the standard surgical treatment of colorectal cancer. Complete mesocolic resection (CME) was also effective in reducing the rate of local recurrence, but patients often due to surgical trauma was too large, the quality of life impacted. In order to improved surgical resection rate and sphincter preservation rate, preoperative adjuvant radiation therapy had been widely carried out; without surgery or surgery can not be fully resected patients were to give comprehensive approach, in terms of postoperative radiation therapy, anti-cancer chemotherapy and biological therapy, to the possible tumor debulking, improve the local control rate and prolong survival. Therefore, radiation therapy played an important role in the treatment of colorectal cancer patients in the whole, how to choose effective radiosensitizing drugs had become the focus of current research.Methods1MTT method was used to observe the inhibiting proliferation of human colorectal adenocarcinoma HT-29cell lines rate by cetuximab combined with celecoxib.2The influence on radiosensitivity of human colorectal adenocarcinoma HT-29cell lines by using cetuximab combined with celecoxib were observed by plate clone formation assay.3The onfluence on cell cycle and apoptosis was detected by flow cytometry to survey the cetuximab and celecoxib combined with radiation’s infulence on human colorectal adenocarcinoma HT-29cell lines.4Western blot method was used to detect the expression of caspase-3of colorectal adenocarcinoma HT-29cell lines before and affter treated by cetuximab and celecoxib combined with radiation.Results1MTT detected the result shows that:gave25μg/ml,50μg/ml,100μg/ml,200μg/ml,400μg/ml,800μg/ml,1600μg/ml and blank control group of cetuximab to HT-29cells respectively after24h, the cell proliferation inhibition rate were:3.57%,20.11%,32.99%,47.36%,53.75%,68.79%,83.92%, the IC50alue concentration of cetuximab is:281μg/ml. Gave100μg/ml,200μg/ml,400μg/ml,800μg/ml,1600μg/ml,3200μg/ml,6400μg/ml and blank control group of celecoxib to HT-29cells respectively after24h, the IC50alue concentration of celecoxib is:2720μg/ml. Blend cetuximab and celecoxib with the ratio of1:2,1:1and2:1, role in HT-29cells for24h, measured combined drug group IC50alue concentration are:632μg/ml,351μg/ml, 193μg/ml. Cell growth inhibition rate of combined cetuximab and celecoxib group was obviously higher than that of single drug group, and the difference was statistically significant (P<0.05).2Plate clone formation assay result shows that:drugs combined with radiation group of25μg/ml,50μg/ml’s Do value (mean lethal dose), Dq value (prospective domain doses), N value (radiation demage repair ability), SF2(survival fraction at2Gy) were:2.505,2.804,3.064,0.826and2.184,2.370,2.960,0.772. All of them were less than the radiation group:3.368,3.993,3.273,0.922. And index decreases with the increase of drug concentration. That combined with cetuximab and celecoxib enhanced HT-29cell lines’radiosensitivity,SER (Do) were:1.345,1.542; SER (Dq) were:1.424,1.685. Cetuximab combined with celecoxib can effectively weaken the HT-29cell lines sublethal damage repair(SLDR) and radioresistance, enhance the radiosensitizing effect.3The flow cytometry results showed that:simple drug group induced HT-29cell apoptosis percentage were7.21%,11.74%, and radiation alone group (2Gy,4Gy,6Gy,8Gy,) induced the apoptosis rate were:10.93%,16.45%,29.84%,39.57%for respectively;25μg/ml drug combined irradiated group’s apoptosis rate were:17.89%,25.02%,40.32%,48.33%for respectively; and50μg/ml drug combined irradiated group of HT-29cell apoptosis rate were:22.06%,34.39%,47.57%,54.03%for respectively. GO/G1phase cell cycle arrest phenomenon can be found in drugs alone group and in radiation group of low irradiation dose, and the degree of GO/G1phase cycle arrest is positively related to durg concentration, and the difference was statistically significant (P<0.05). Drug combined irradiated group has the obvious S phase cell cycle arrest phenomenon in high irradiation dose (6Gy,8Gy), the difference was statistically significant (P<0.05). The lowest degree of S phase cell cycle arrest was11.78%, when50μg/ml drug combined irradiated group accepted8Gy dose. G2/M phase cell cycle arrest phenomenon can be found in radiation group(4Gy,6Gy,8Gy), the difference was statistically significant (P<0.05). The lowest degree of G2/M phase cell cycle arrest was27.63%, when50μg/ml drug combined irradiated group accepted8Gy dose.4Western blot results showed that:protein expression of caspase-3in radiation alone group, drugs combined irradiated group were lower than the blank control group (P<0.05), and a statistically significant difference can be found between the radiation alone group with the drug combined irradiated group (P<0.05). There were significant difference between the high and low concentrations of drugs in the same group (P<0.05). The protein expression of caspase-3was positively related to the drug concentration,.Conclusion1In this experimental range, cetuximab combined with celecoxib could effectively inhibit the proliferation of colorectal adenocarcinoma HT-29cell lines, which presents concentration-dependently.2The clone formation rate of colorectal adenocarcinoma HT-29cell lines was negatively related to radiation dose, in the same dose, The clone formation rate in cetuximab and celecoxib combined with radiation group was obviously lower than that of irradiated group. The surviving fraction of HT-29was negtively related to grug concentration. Cetuximab combined with celecoxib can effectively enhance the radiosensitizing effect.3GO/G1phase cell cycle arrest phenomenon can be found in low concentration group of cetuximab combined celecoxib. High concentration group of cetuximab combined celecoxib can effectively decreased the number of HT-29cells in S phase, and increased the number of HT-29cells in G2/M phase which was sensitve with radiation. The enhancement of radiosensitizing effect was related to G2/M phase cell cycle arrest phenomenon.4In the same dose, the protein expression of caspase-3of colorectal adenocarcinoma HT-29cell lines treated by cetuximab and celecoxib combined with radiation was obviously higher than that of only irradiated group. Cetuximab combined celecoxib can enhance the radiosensitivity, it may be associated with the synergy enhancement of protein caspase-3expression. |
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