Therapeutic Effect And Mechanism Of Combination Of Capecitabine And Radiation On Cervical Adenocarcinoma

IntroductionCapecitabine(CAP)is a new orally available prodrug for the treatment of carcinoma.CAP,being 5-fluorouracil(5-FU),is developed as nontoxic and inactive form which is absorbed as original one in GI tract.It is then converted to active drug 5-FU by three kinds of enzymes which are found in the liver and tumor tissue.Finally, 5-FU is catalyzed by DPD(dihydropyrimidine dehydrogenase) to inactive metabolite. During the process,TP(thymidine phosphorylase) is a restriction enzyme in the ultimate stage of activation.Advantage of TP is that its concentration in most tumor tissues is much higher than that in normal tissues which results in high-concentration 5-FU in tumor tissues so that greatly increases activation for tumor therapy and decreases toxicity to normal tissue.CAP with benefits of less toxicity,selective tumor therapy and compensation to 5-FU limitation of less cumulative dose and great toxicity is only oneâ…£-injection fluorouracil medicine among those taken by mouth.Currently, CAP has been used and made positive results for the treatment of metastatic and recurrent breast cancer and colon carcinoma in patients who are resistant to Doxorubicin Hydrochloride and Paclitaxil.TP has become research focus now because it is pivotal enzyme that makes CAP play selective tumor therapy.It is the goal for researchers to increase TP to enhance CAP curative effect.Experiments show that combination of CAP and radiation applications is a tumor treatment with bright future for assistant therapy,preoperative chemotherapy and appeasement therapy due to radiation which may increase TP in multiple tumor tissues,on the other hand,DPD also plays important role in therapy with CAP as the ratio of TP/DPD has close relations with CAP curative effect which indicates that test of related enzymes can help to select patients who are optimal for therapy with CAP.Operation and radiotherapy are the major traditional therapeutic methods for patients with cervical cancer which has relatively high morbidity.By the two methods, some patients recover,some,however,the majority of them become worse,especially for cervical adenocarcinoma which is less sensitive to radiation compared with cervical SC(Squanous carcinoma),the same as patients who suffer adenocarcinoma in relatively terminal stage,lymphatic metastasis or recurrence after first time treatment.In order to improve prognosis,it is urgent to find out a better way to cure cervical adenocarcinoma. Recently,new chemotherapy plan before operation or radiotherapy indeed improves therapeutic effect of operation or/and radiotherapy so a lot of new assistant chemotherapy plans have appeared.CAP has also been used as chemotherapy in many researches on animal models where many kinds of human cells of cervical adenocarcinoma has been transplanted.The plan shows some restraint on tumor which is about 34.6%.The plan of CAP combined with radiation in the treatment of cervical adenocarcinoma has not been reported until now but maybe has a good result as expected due to CAP of high efficiency & low toxicity and 5-FU with characteristics that radiation may increase its sensitivity.Nude mice models are established in the experiments that cells of human cervical adenocarcinoma are transplanted to mice.The researches show therapeutic effect in the treatment of tumors with new plan of combination of CAP and radiation,at the same time,explore the therapeutic mechanism of the plan through studies on influence of radiation on TP and DPD of cervical adenocarcinoma cells and change of cell cycle. Materials and MethodsMaterials1,Animals:200 female BALB/c nude mice(5 or 6 years old,weight 20g)2,Cell Strain:CAC-1 cell strain of human cervical adenocarcinoma from Japan Sapporo Medical UniversityHeLa cell strain of human cervical adenocarcinoma from laboratory of infection department in Shengjing Hospital affiliated to China Medical University3,Major Reagents and Medicine:CAP is provided by F-Hofmann-Roche(Besle Switzerland),5-FU is from Kyowa Hakko Co(Tokyo Japan) and Shanghai Xudonghaipu Pharmaceutical company Culture medium(Beijing),0.1mg/ml coomassie brilliant blue(Shanghai),Bovine serum albumin(Sigma company),Test box for TP by ELISA(Germany),RNA extract Trizol(Dongsheng chuangxin),cDNA test box for first chain synthesis reverse transcription,PCR test box,Taq DNA polymerase DNA Marker(TakaRa Biotechnology Dalian Co.,Ltd),TP,DPD primer (Shanghai Invitrogen),MTT(Sigma company),DMSO(Beijing),PI(sigma company)4,Major Apparatus:Superclean bench(Suzhou),Varian linear accelerator 2300C/D-SN293(USA),Ultrasonic disintegrator UP200H(Germany),DV600 spectrophotometer(USA),Oscillator RS-232(USA),Absorbance Microplate Readers GIS-1000 Tanon(Shanghai),Ultraviolet spectrophotometer Wellscan MK3 (Germany),Electorphoresis BIO-RADpower/PAC 300(USA),PCR:PTC-100 (USA),Analysis system for gel imaging(Gene limited Co.,Ltd),flow cytometry (FACS-440),Mitsubishi electron ray apparatus(EXL-15DP,4MeV,dose 5Gy/m).Methods1,model establishment and therapeutic evaluation of CAC-1 cells of human cervical adenocarcinoma transplanted to animals:Inoculate 0.2ml monoplast suspension of CAC-1 of 2×10⁶ subcutaneously in the upper part of left legs of nude mice.Treatment begins when tumors grow up to some extent.Before treatment,all mice with tumors are divided into comparison group,CAP group,5-FU group,radiation group,group with CAP and radiation,group with 5-FU and radiation.According to tumor size,mice are divided into large-size group and small-size group in which group mice are divided into comparison group,CAP group, radiation group,group with CAP and radiation.Mice are fed with CAP and 5-FU of 1/2 or 2/3 MTD daily for two weeks,6 days/week.For radiation group,local radiation with doses of 4,5,6Gy is given on the 4^th day after medicine and radiation with dose of 2Gy daily in 1^st,2^nd,4^th and 5^th day respectively in the week when medicine is applied which last for two weeks.Make Therapeutic evaluation of medicine or radiation depends on the change of tumor size.Calculate tumor growth percentage and make curve chart between tumor growth percentage and time needed so as to evaluate extent of tumor restraint.T2.5 represents the time that tumor grows to 2.5 times of its original size since the 1^st day of treatment.Based on T2.5,we calculate the delay time of tumor growth.2,test of TP,DPD in HeLa cells of human cervical adenocarcinoma:According to different X-ray doses,we divide the HeLa cells of cervical adenocarcinoma into three groups:comparison group(0Gy),groupl(2Gy),group2(6Gy): collect cells and store samples in refrigerator respectively at the day before radiation and 1^st,3^rd,5^th,7^th and 9^th day after radiation.TP enzyme protein level of HeLa cells before and after radiation is tested by ELISA.DPD mRNA of TP in HeLa cells is tested by RT-PCR.3,test of HeLa cell cycle of human cervical adenocarcinoma:Cells are prepared with 5-FU(MTT) with IC50 125 ug/ml for 48h.Divide HeLa cells into comparison group(no medicine and radiation),5-FU group,radiation group and group of combination of 5-FU and radiation.For above-mentioned each group, cells are divided to three subgroups according to different time of 12,24 and 48h and treated by 5-FU of IC50 for 48h.Cells in radiation group receive 6Gy X-ray radiation for single time,then cultivate for 12,24 and 48h,finally,fixed by ice alcohol.Test of cell cycle:first,PI dying,then,test cell cycle change of different groups with flow cytometry,finally,test cells in G₀/G₁,S,G₂/M phases.Results1,therapeutic effect of separate medicine and separate radiation of different dose:Compared with comparison group among the different kinds of combinations of 2/3MTD(CAP,5-FU) and radiotherapy of 4,5,6,2×8Gy,therapy with CAP shows some restraint effect on tumor and better than 5-FU,similar to the effect of therapy with only 6Gy radiotherapy.Between the comparison group and radiotherapy groups with different dose,the radiotherapy of 2Gyx8 has the best and longest restraint effect, the next one is 6 Gy radiation.In conclusion,to CAC-1 of cervical adenocarcinoma, separate use of CAP or radiation has some extent of effect but not obvious. Combination of 1/2 MTD CAP and 6Gy has the similar result.2,therapeutic effect of combination of medicine and radiotherapy of different dose and different methods:Compared with comparison group,therapeutic effects on tumors of combination of 2/3MTD CAP with different-dose radiotherapy are obviously better than those using medicine or radiation separately.Restraint effect of the combination of 2/3MTD CAP and 6Gy radiotherapy is better than that of combination of 2/3MTD 5-FU and 6Gy radiotherapy which only delays tumor growth instead of eliminating tumors. Combination of 2/3MTD CAP and radiotherapy with doses of 5,6,2×8Gy separately has evident effect of eliminating and restraining tumor which begins one week after combining therapy and lasts for about one week,especially when CAP combines with multiple small-dose radiotherapy of 2Gy/d×8 times,its restraint effect on tumors is more obvious and lasting than combination of CAP and single radiotherapy with doses of 5,6Gy;the effect of CAP and 4Gy radiotherapy is not as good as previous plan. Besides,similar effect occurs between the combination of 2/3 MTD CAP & 4Gy and combination of 2/3 MTD 5-FU and 6Gy which indicates radiotherapy combined with CAP achieve the same effect with less radiation dose and CAP has stronger therapeutic enhancement to radiotherapy than 5-FU.3,therapeutic effect of combination of CAP and radiation on tumors of different size:Combination therapy of 1/2MTD CAP and 6Gy radiotherapy indicates that large-size tumors are sensitive to the restraint effect,otherwise,small-size tumors are not sensitive to the therapy and has the same therapeutic result as single usage of medicine or radiotherapy.Large-size tumors have better response than small-size ones to increasing CAP dose in combination of 2/3 MTD CAP and 6Gy.4,Delay time of tumor growth are 23.9,22 and 48.4 days among the three groups of combined therapy of CAP and radiotherapy with doses of 6,5,2×8Gy,longer than the delay time by separate use of medicine or radiation.The delay time of combining therapy is not equal to the simple cumulation of that of separate therapy.5,ELISA test of TP enzyme protein in HeLa cells after radiotherapy:Compared with comparison group,after radiation to HeLa cells,TP enzyme protein level of two groups with radiotherapy rises sharply,reaches peak at the third day,then decreases gradually,lasts for one week at the high level.The level of TP enzyme protein in group with radiotherapy of 2Gy is higher and remain at high level for more than 9 days compared with that in group with radiotherapy of 6Gy.6,RT-PCR test for TP in HeLa cells and DPD mRNA after radiotherapy:TP mRNA rises sharply and reaches peak at the third day after radiation which is obviously higher than that of comparison group without radiation,then decrease gradually.The change is the same as the variation of TP enzyme protein tested with ELISA.Regarding DPD mRNA in HeLa cells,there is no apparent difference between radiotherapy group and comparison group.7,ratio change of components in HeLa cell cycle: Compared with comparison group,in group using 5-FU,HeLa cells in S phase increase greatly,decrease in G2/M and G0/G1 phase,especially prominent 24 hours after use of medicine;for group with X-ray radiation,cells in G₀/G₁ phase increase and cells in S phase decrease;for group using 5-FU and radiotherapy,cells in G₀/G₁ decrease sharply but cells in S phase increase which enhance the effect of 5-FU.Cells in G₂/M phase decrease in combination group less than those in 5-FU group.HeLa cells in G₂/M,S and G₀/G₁ phases are more sentive to 5-FU than to x-ray radiation.Major change of cell cycle also occurs at 24 hours after using 5-FU and radiotherapy.In the group treated by 5-FU,cells are restrained in S phase after radiation;In the other group with only radiation,no component change happened after x-ray.Conclusions1,Combination of CAP and radiation in the treatment of adenocarcinoma CAC-1 of cervix is an effective way with obvious restraint effect to tumor and promotion to each other.The method is better than separate CAP or radiation therapy,at the same time,it is also better than direct combination of 5-FU and radiation.CAP has stronger enhancement than radiation does.2,Treatment effect of combination of CAP and radiation is affected by a number of factors including CAP dose and concentration,radiation dose,radiation method and tumor size.2/3MTD CAP combining with 5,6Gy and 2Gy×8 radiation separately is a better way for tumor restraint.Compared with combination of CAP with single radiation,CAP combined with multiple small-dose radiotherapy shows more prominent tumor restraint effect.Tumors with large size are more sensitive to combination of CAP and radiation than smaller ones.There is possibility that this combination has better curative effect on cervical carcinoma in terminal stage.3,TP/DPD increasement of Hela cell after radiation makes Hela cells more sensitive to CAP because radiation increases level of TP enzyme protein and amount of active mRNA in TP enzyme of Hela cells in cervical adenocarcinoma,at the same time, no influence on mRNA of DPD.4,Cells change in G₂/M,S and G₀/G₁ under the influence of 5-FU and radiation proves that two elements exert effect on cells but 5-FU is more important which lead major restraint on cells in S phase.The largest influence on cell cycle occurs 24 hours after use of 5-FU and radiation.Hela cells are more sensitive to 5-FU than to radiation so it is conclusion that 5-FU has greater influence on radiation through changing Hela cell cycle,on the contrary,radiation plays a little impact on 5-FU.