卡培他滨与放射线照射联合治疗子宫颈腺癌的效果与机制探讨
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摘要
前言
卡培他滨(capecitabine,CAP)是一种口服型5-氟尿嘧啶(5-fluorouracil,5-FU)前体类抗肿瘤新药。CAP的药物设计,使其本身无毒无活性,在胃肠道可以原药形式快速吸收,在肝脏和肿瘤组织中三种酶的作用下转化形成5-FU而发挥抗肿瘤活性。最终5-FU由二氢嘧啶脱氢酶(dihydropyrimidine dehydrogenase,DPD)代谢失活。在其转化过程中,胸腺嘧啶核苷磷酸化酶(thymidine phosphorylase,TP)是其活化最后一环的一个限制酶,TP的特点在多数实体肿瘤组织中浓度较高,明显高于正常组织。这使得CAP在正常组织中生成5-FU量较少,而在肿瘤组织局部可有高浓度5-FU生成,大大增强了抗肿瘤活性,降低了5-FU对正常组织的毒副作用。因此,CAP全身毒性反应轻且选择性抗肿瘤,是目前口服制剂中唯一能超过静脉给药的氟尿嘧啶类药物,弥补了5FU毒性大用量受限的缺撼。临床上已被用于紫杉醇、阿霉素等耐药的复发转移性乳腺癌及晚期结肠癌等患者的治疗,取得了很好的效果。关于CAP疗效方面的研究,由于TP是CAP选择性作用于肿瘤的关键酶,而成为研究的焦点。人们致力于增加TP的表达来提高CAP的疗效。研究显示,放射可使多种肿瘤组织TP表达明显增高,CAP和放射联合应用无论用于辅助治疗、术前放化疗、还是姑息治疗,均能得到较好的效果,是一种有前途的肿瘤治疗方法。另外研究还发现,DPD在CAP治疗中也发挥着重要作用,TP/DPD的比值更直接的关系到CAP的疗效。提示在化疗之前测定肿瘤相关酶的水平,能选择到更适合接受CAP治疗的人群。
子宫颈癌有着较高的发病率,其治疗主要还是以手术和放疗为主,可使部分患者得到根治或缓解,但有部分患者单纯放疗仍然预后不佳。尤其是宫颈腺癌,不如宫颈鳞癌对放射线敏感,临床期别较晚、淋巴结有转移的患者,初始治疗后很快又复发、转移。且其发病还有逐渐增加趋势。因此为改善预后,临床迫切需要探讨对宫颈腺癌更为有效的疗法。近年来新辅助化疗即术前或放疗前进行化疗,确实一定程度上提高了手术或/和放疗的效果。故而寻找对宫颈腺癌更有效的新辅助化疗方案的有关研究出现许多。CAP也已被用于多种人宫颈癌细胞株动物移植模型中进行研究,显示出了一定的抑癌效果,在人宫颈腺癌HeLa细胞株动物移植模型中CAP的抑瘤率可达34.6%。但CAP与放射联合在宫颈腺癌中的作用如何尚未见报道。介于CAP高效低毒的药物特点和5-FU所具有的放射增敏作用,将CAP与放射联合用于宫颈腺癌,效果值得期待。因此本研究采用人子宫颈腺癌细胞株异种移植于裸鼠建立动物模型,观察了CAP和放射线联合治疗在宫颈腺癌中的抗肿瘤效果。并通过放射线照射对宫颈腺癌细胞TP、DPD酶影响的研究,及观察联合治疗后宫颈腺癌细胞周期的变化,对其联合治疗效果的机制进行了探讨。
实验材料和方法
一、实验材料
1、实验动物:雌性BALB/c nu/nu裸鼠(5或6周龄,体重约20克)约220只。
2、细胞株:人宫颈腺癌CAC-1细胞株从日本札幌医科大学获得,人宫颈腺癌HeLa细胞株由中国医科大学盛京医院感染科实验室提供。
3、主要试剂及药品:CAP由F-Hofmann-Roche(Besle Switzerland)提供。5-FU购买于Kyowa Hakko Co(Tokyo Japan)和上海旭东海普药业公司。培养基(海克隆生物化学制品北京有限公司);0.1mg/ml考马斯亮蓝溶液(进口分装,上海化学试剂采购供应站);牛血清白蛋白(Sigma公司);ELISA法测定TP酶的试剂盒(德国罗氏公司);总RNA提取液Trizol(东胜创新实验技术有限公司);cDNA第一链合成逆转录试剂盒、PCR试剂盒、Taq DNA、聚合酶DNA Marker(大连宝生物工程有限公司);TP、DPD引物(上海Invitrogen英俊生物技术有限公司合成);四甲基偶氮唑蓝(MTT)(Sigma公司),DMSO试剂二甲基亚砜(北京夏斯生物技术有限公司),碘化丙啶(PI)(sigma公司)。
4、主要实验仪器和设备:
超净工作台(苏州净化设备厂);瓦里安直线加速器2300C/D-SN293(美国);超声粉碎机UP200H(德国);DV600分光光度计(美国);振荡机RS-232(美国);酶标仪GIS-1000 Tanon(上海天能科技有限公司);紫外分光光度仪Wellscan MK3(德国);电泳仪BIO-RADpower/PAC 300(美国);PCR仪PTC-100(美国);凝胶成像分析系统(基因有限公司);流式细胞仪(FACS-440型);Mitsubishi电子射线装置(机型EXL-15DP,4MeV,剂量率5Gy/分)。
二、实验方法
1、人宫颈腺癌CAC-1细胞株动物移植模型建立及治疗效果评价
在每只裸鼠左下肢外上方皮下,接种含2×10~6个细胞的CAC-1单细胞悬液0.2ml。在局部有肿瘤块生长并达一定程度时开始进行肿瘤治疗。治疗前将携瘤鼠分成对照组、CAP组、5-FU组、放射组、CAP+放射组和5-FU+放射组。并设肿瘤大小不同的两大组,在大、小瘤组又分别分出对照组、CAP组、放射组、CAP+放射组。CAP、5-FU每天被分别投给它们最大耐受剂量(MTD)的1/2或2/3。每周投药6天,连续投药两周。对需行放射的携瘤鼠分别于投药第4天进行一次性4、5、6Gy局部照射,以及投药周的第1、2、4、5天进行日一次2Gy的局部照射,两周共照射8天。药物、照射等的疗效评价通过观察肿瘤体积的变化来进行。计算肿瘤增殖百分率,将肿瘤增殖百分率与所经过的时间绘制成肿瘤增殖曲线图,以此评价肿瘤被抑制程度。T2.5表示肿瘤体积从治疗第1天的体积到生长达其2.5倍时所需的时间,以此推算肿瘤生长延迟天数。
2、人宫颈腺癌HeLa细胞TP,DPD酶表达的测定
将宫颈腺癌HeLa细胞按X线照射的不同剂量分成对照组(0Gy)及2Gy、6Gy放射组。分别于照射前1天(第0天),照射后第1天,第3天,第5天,第7天,第9天收集细胞,制成测定样本冻存。ELISA测定HeLa细胞照射前后TP酶蛋白含量。RT-PCR检测HeLa细胞放射前后TP,DPD酶mRNA表达。
3、人宫颈腺癌HeLa细胞周期的测定
先以MTT法行5-FU处理48h半数抑制浓度(IC50)测定,为125μg/ml。再将HeLa细胞分成对照组(不加药,不照射)、5-FU组、放射组、5-FU+放射组(药物和放射同时投给)。各组根据不同时间经过又分成12、24、48h三组。药物组是以48h IC50浓度的5-FU处理相应各组细胞,12、24、48h后将细胞收集。需照射的各组细胞在室温下进行单次6Gy的X射线照射,照射后继续培养12、24、48h再收集细胞。行冰乙醇固定备测。细胞周期测定前先行PI染色,后以流式细胞仪检测各组细胞周期变化,测定G_0/G_1、S、G_2/M期细胞。
实验结果
1、不同剂量的单纯药物及单纯放射的治疗效果
2/3MTD药物(CAP、5-FU)和4、5、6、2×8Gy放射分别施与后的效果,与对照组相比,单纯CAP治疗显示了一定的抑制效果,并且效果要好于5-FU,与单纯6Gy放射相比两者效果近似。而不同剂量的单纯放射治疗组与对照组相比,2Gy×8回放射在各治疗条件中显示出的抑制效果最好,持续抑制的时间也长,其次是6Gy放射。总的来看子宫颈腺癌CAC-1对单纯CAP及单纯放射都有一定的效果,但不明显。1/2MTD CAP、6Gy放射施与后的结果,CAP的抑制效果也近似于单纯6Gy放射的效果,二者联合的效果虽有,但也不是很明显。
2、药物与不同剂量放射及不同方法放射联合施与后的治疗效果
与对照组相比2/3MTD CAP分别和各个不同剂量放射联合施与的抑制效果,均较单纯药物或单纯放射显示的效果明显。2/3MTD 5-FU和6Gy放射联合施与的抑制效果,明显不如2/3MTD CAP和6Gy放射联合的效果,只见到了延缓生长作用,没有消瘤作用。2/3MTD CAP分别和5、6、2×8Gy剂量放射联合施与后的抑制效果明显,在治疗后的一周开始明显显示,不仅使肿瘤生长延迟而且有消瘤作用,抑制效果并能持续一周左右;特别在与2Gy/日×8回的小剂量多次放射方法联合施与后,其显示出的抑制效果,较CAP分别和5、6Gy单次放射方法联合的效果更为明显,并且显效快抑瘤时间长;而CAP与4Gy放射联合施与后的效果不佳。另外2/3MTD CAP和4Gy放射联合的效果与2/3MTD 5-FU和6Gy放射联合的效果相近,提示放射与CAP联合治疗,同样效果需要的射线剂量小,CAP对放射的增强作用比5-FU强。
3、不同大小肿瘤对CAP与放射联合治疗效果的影响,
1/2MTD CAP和6Gy放射联合施与,大瘤组肿瘤对联合治疗反应敏感,有明显抑制。而小瘤组肿瘤对联合治疗反应不敏感,效果仅同于单纯药物或单纯放射的治疗效果。2/3MTD CAP和6Gy放射联合施与,加大CAP剂量后,虽然大、小瘤组肿瘤均对联合治疗有效,但大瘤组肿瘤受抑明显,优于小瘤组肿瘤。
4、从肿瘤生长延迟天数来看,CAP分别和6、5、2×8Gy放射联合施与的三个治疗组,肿瘤生长分别被延迟了23.9、22、48.4天。各联合治疗组的肿瘤生长延迟天数,都超过了它们单纯药物组及相应的单纯放射组延迟天数的总和,延迟天数并非为单纯药物组及相应的单纯放射组延迟天数的简单相加。
5、放射后HeLa细胞TP酶蛋白含量的ELISA测定
与对照组相比,HeLa细胞照射后,两个放射组的TP酶蛋白水平均有明显提高,放射后第3天都升高达高峰,以后逐渐下降,持续高水平约有一周左右时间。2Gy放射组TP酶蛋白水平升高程度,较6Gy放射组升高有更加明显的趋势,尤其放射后第3天两者的差别显著。2Gy放射组TP酶处于高水平的持续时间更长,超过9天。
6、放射后HeLa细胞TP、DPD酶mRNA表达RT-PCR检测
放射后HeLa细胞TP酶的mRNA表达,在照射后第3天迅速升高达到高峰,明显高于未经照射的对照组,以后出现逐渐下降的趋势。其升高时间与ELISA测定的TP酶蛋白水平的变化情况基本相吻合。而HeLa细胞DPD酶mRNA的表达,在放射组与对照组之间未见明显差异。
7、HeLa细胞周期构成比的变化
与对照组比较,5-FU处理组HeLa细胞S期增加明显,G_2/M期及G_0/G_1期比例减少,出现S期明显阻滞,药物处理24小时后阻滞最显著;X线照射组G_0/G_1期细胞有所增加,而S期细胞减少,照射24小时后变化最明显;5-FU与放射联合组对HeLa细胞的影响,主要是S期增多5-FU的作用为主,G_0/G_1期细胞明显减少。G_2/M期也有减少,但联合作用组G_2/M期细胞减少没有5-FU组那么明显。5-FU与放射联合作用时,HeLa细胞G_2/M、S及G_0/G_1各期细胞均受影响,但其对5-FU的反应比对放射线更敏感。联合作用引起的细胞周期变化也是处理后24小时最显著;照射前、后的HeLa细胞,均是5-FU处理组引起了S期阻滞,而单纯照射前、后HeLa细胞S期比例变化不大。
结论
1、对于宫颈腺癌CAC-1应用CAP与放射联合治疗能产生相互协同增效作用,有明显的肿瘤抑制效果,是一种有效的治疗子宫颈腺癌的方法。其效果优于单纯CAP及单纯放射的治疗,也优于5-FU直接和放射联合的效果。CAP对放射的增强作用比5-FU强。
2、CAP与放射联合治疗的效果与CAP药物浓度、放射剂量、照射方法及肿瘤大小有关。2/3MTD CAP分别和5、6Gy及2Gy×8放射联合治疗时,肿瘤抑制的效果较好。CAP与小剂量多次放射方法的联合治疗,较CAP与单次放射方法联合,更显示出了明显的抑瘤效果。体积较大的肿瘤,对CAP与放射联合治疗的反应敏感,比体积较小的肿瘤治疗效果好,可能CAP与放射联合治疗对较晚期子宫颈腺癌更为有效。
3、放射线照射,能提高子宫颈腺癌HeLa细胞TP酶的mRNA表达和TP酶蛋白水平,而DPD酶mRNA表达不受影响,因此放射后HeLa细胞TP/DPD比值升高,能增加其对CAP的敏感性。
4、5-FU与放射联合,两者对HeLa细胞周期均有影响,其细胞G_2/M、S及G_0/G_1各期都有变化,但主要还是以S期细胞阻滞即5-FU的影响为主。其联合作用引起的细胞周期变化,在处理后的24小时最为显著。HeLa细胞表现出对5-FU的作用比对放射更敏感,可以认为在两者的联合作用中,5-FU通过改变HeLa细胞周期而对放射效果的影响较大,相反放射对5-FU影响较小。
Introduction
Capecitabine(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 oneⅣ-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 Methods
Materials
1、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 University
HeLa cell strain of human cervical adenocarcinoma from laboratory of infection department in Shengjing Hospital affiliated to China Medical University
3、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).
Methods
1、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 2×10~6 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 G_0/G_1、S、G_2/M phases.
Results
1、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 G_0/G_1 phase increase and cells in S phase decrease;for group using 5-FU and radiotherapy,cells in G_0/G_1 decrease sharply but cells in S phase increase which enhance the effect of 5-FU.Cells in G_2/M phase decrease in combination group less than those in 5-FU group.HeLa cells in G_2/M、S and G_0/G_1 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.
Conclusions
1、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 G_2/M、S and G_0/G_1 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.
卡培他滨(capecitabine,CAP)是一种口服型5-氟尿嘧啶(5-fluorouracil,5-FU)前体类抗肿瘤新药。CAP的药物设计,使其本身无毒无活性,在胃肠道可以原药形式快速吸收,在肝脏和肿瘤组织中三种酶的作用下转化形成5-FU而发挥抗肿瘤活性。最终5-FU由二氢嘧啶脱氢酶(dihydropyrimidine dehydrogenase,DPD)代谢失活。在其转化过程中,胸腺嘧啶核苷磷酸化酶(thymidine phosphorylase,TP)是其活化最后一环的一个限制酶,TP的特点在多数实体肿瘤组织中浓度较高,明显高于正常组织。这使得CAP在正常组织中生成5-FU量较少,而在肿瘤组织局部可有高浓度5-FU生成,大大增强了抗肿瘤活性,降低了5-FU对正常组织的毒副作用。因此,CAP全身毒性反应轻且选择性抗肿瘤,是目前口服制剂中唯一能超过静脉给药的氟尿嘧啶类药物,弥补了5FU毒性大用量受限的缺撼。临床上已被用于紫杉醇、阿霉素等耐药的复发转移性乳腺癌及晚期结肠癌等患者的治疗,取得了很好的效果。关于CAP疗效方面的研究,由于TP是CAP选择性作用于肿瘤的关键酶,而成为研究的焦点。人们致力于增加TP的表达来提高CAP的疗效。研究显示,放射可使多种肿瘤组织TP表达明显增高,CAP和放射联合应用无论用于辅助治疗、术前放化疗、还是姑息治疗,均能得到较好的效果,是一种有前途的肿瘤治疗方法。另外研究还发现,DPD在CAP治疗中也发挥着重要作用,TP/DPD的比值更直接的关系到CAP的疗效。提示在化疗之前测定肿瘤相关酶的水平,能选择到更适合接受CAP治疗的人群。
子宫颈癌有着较高的发病率,其治疗主要还是以手术和放疗为主,可使部分患者得到根治或缓解,但有部分患者单纯放疗仍然预后不佳。尤其是宫颈腺癌,不如宫颈鳞癌对放射线敏感,临床期别较晚、淋巴结有转移的患者,初始治疗后很快又复发、转移。且其发病还有逐渐增加趋势。因此为改善预后,临床迫切需要探讨对宫颈腺癌更为有效的疗法。近年来新辅助化疗即术前或放疗前进行化疗,确实一定程度上提高了手术或/和放疗的效果。故而寻找对宫颈腺癌更有效的新辅助化疗方案的有关研究出现许多。CAP也已被用于多种人宫颈癌细胞株动物移植模型中进行研究,显示出了一定的抑癌效果,在人宫颈腺癌HeLa细胞株动物移植模型中CAP的抑瘤率可达34.6%。但CAP与放射联合在宫颈腺癌中的作用如何尚未见报道。介于CAP高效低毒的药物特点和5-FU所具有的放射增敏作用,将CAP与放射联合用于宫颈腺癌,效果值得期待。因此本研究采用人子宫颈腺癌细胞株异种移植于裸鼠建立动物模型,观察了CAP和放射线联合治疗在宫颈腺癌中的抗肿瘤效果。并通过放射线照射对宫颈腺癌细胞TP、DPD酶影响的研究,及观察联合治疗后宫颈腺癌细胞周期的变化,对其联合治疗效果的机制进行了探讨。
实验材料和方法
一、实验材料
1、实验动物:雌性BALB/c nu/nu裸鼠(5或6周龄,体重约20克)约220只。
2、细胞株:人宫颈腺癌CAC-1细胞株从日本札幌医科大学获得,人宫颈腺癌HeLa细胞株由中国医科大学盛京医院感染科实验室提供。
3、主要试剂及药品:CAP由F-Hofmann-Roche(Besle Switzerland)提供。5-FU购买于Kyowa Hakko Co(Tokyo Japan)和上海旭东海普药业公司。培养基(海克隆生物化学制品北京有限公司);0.1mg/ml考马斯亮蓝溶液(进口分装,上海化学试剂采购供应站);牛血清白蛋白(Sigma公司);ELISA法测定TP酶的试剂盒(德国罗氏公司);总RNA提取液Trizol(东胜创新实验技术有限公司);cDNA第一链合成逆转录试剂盒、PCR试剂盒、Taq DNA、聚合酶DNA Marker(大连宝生物工程有限公司);TP、DPD引物(上海Invitrogen英俊生物技术有限公司合成);四甲基偶氮唑蓝(MTT)(Sigma公司),DMSO试剂二甲基亚砜(北京夏斯生物技术有限公司),碘化丙啶(PI)(sigma公司)。
4、主要实验仪器和设备:
超净工作台(苏州净化设备厂);瓦里安直线加速器2300C/D-SN293(美国);超声粉碎机UP200H(德国);DV600分光光度计(美国);振荡机RS-232(美国);酶标仪GIS-1000 Tanon(上海天能科技有限公司);紫外分光光度仪Wellscan MK3(德国);电泳仪BIO-RADpower/PAC 300(美国);PCR仪PTC-100(美国);凝胶成像分析系统(基因有限公司);流式细胞仪(FACS-440型);Mitsubishi电子射线装置(机型EXL-15DP,4MeV,剂量率5Gy/分)。
二、实验方法
1、人宫颈腺癌CAC-1细胞株动物移植模型建立及治疗效果评价
在每只裸鼠左下肢外上方皮下,接种含2×10~6个细胞的CAC-1单细胞悬液0.2ml。在局部有肿瘤块生长并达一定程度时开始进行肿瘤治疗。治疗前将携瘤鼠分成对照组、CAP组、5-FU组、放射组、CAP+放射组和5-FU+放射组。并设肿瘤大小不同的两大组,在大、小瘤组又分别分出对照组、CAP组、放射组、CAP+放射组。CAP、5-FU每天被分别投给它们最大耐受剂量(MTD)的1/2或2/3。每周投药6天,连续投药两周。对需行放射的携瘤鼠分别于投药第4天进行一次性4、5、6Gy局部照射,以及投药周的第1、2、4、5天进行日一次2Gy的局部照射,两周共照射8天。药物、照射等的疗效评价通过观察肿瘤体积的变化来进行。计算肿瘤增殖百分率,将肿瘤增殖百分率与所经过的时间绘制成肿瘤增殖曲线图,以此评价肿瘤被抑制程度。T2.5表示肿瘤体积从治疗第1天的体积到生长达其2.5倍时所需的时间,以此推算肿瘤生长延迟天数。
2、人宫颈腺癌HeLa细胞TP,DPD酶表达的测定
将宫颈腺癌HeLa细胞按X线照射的不同剂量分成对照组(0Gy)及2Gy、6Gy放射组。分别于照射前1天(第0天),照射后第1天,第3天,第5天,第7天,第9天收集细胞,制成测定样本冻存。ELISA测定HeLa细胞照射前后TP酶蛋白含量。RT-PCR检测HeLa细胞放射前后TP,DPD酶mRNA表达。
3、人宫颈腺癌HeLa细胞周期的测定
先以MTT法行5-FU处理48h半数抑制浓度(IC50)测定,为125μg/ml。再将HeLa细胞分成对照组(不加药,不照射)、5-FU组、放射组、5-FU+放射组(药物和放射同时投给)。各组根据不同时间经过又分成12、24、48h三组。药物组是以48h IC50浓度的5-FU处理相应各组细胞,12、24、48h后将细胞收集。需照射的各组细胞在室温下进行单次6Gy的X射线照射,照射后继续培养12、24、48h再收集细胞。行冰乙醇固定备测。细胞周期测定前先行PI染色,后以流式细胞仪检测各组细胞周期变化,测定G_0/G_1、S、G_2/M期细胞。
实验结果
1、不同剂量的单纯药物及单纯放射的治疗效果
2/3MTD药物(CAP、5-FU)和4、5、6、2×8Gy放射分别施与后的效果,与对照组相比,单纯CAP治疗显示了一定的抑制效果,并且效果要好于5-FU,与单纯6Gy放射相比两者效果近似。而不同剂量的单纯放射治疗组与对照组相比,2Gy×8回放射在各治疗条件中显示出的抑制效果最好,持续抑制的时间也长,其次是6Gy放射。总的来看子宫颈腺癌CAC-1对单纯CAP及单纯放射都有一定的效果,但不明显。1/2MTD CAP、6Gy放射施与后的结果,CAP的抑制效果也近似于单纯6Gy放射的效果,二者联合的效果虽有,但也不是很明显。
2、药物与不同剂量放射及不同方法放射联合施与后的治疗效果
与对照组相比2/3MTD CAP分别和各个不同剂量放射联合施与的抑制效果,均较单纯药物或单纯放射显示的效果明显。2/3MTD 5-FU和6Gy放射联合施与的抑制效果,明显不如2/3MTD CAP和6Gy放射联合的效果,只见到了延缓生长作用,没有消瘤作用。2/3MTD CAP分别和5、6、2×8Gy剂量放射联合施与后的抑制效果明显,在治疗后的一周开始明显显示,不仅使肿瘤生长延迟而且有消瘤作用,抑制效果并能持续一周左右;特别在与2Gy/日×8回的小剂量多次放射方法联合施与后,其显示出的抑制效果,较CAP分别和5、6Gy单次放射方法联合的效果更为明显,并且显效快抑瘤时间长;而CAP与4Gy放射联合施与后的效果不佳。另外2/3MTD CAP和4Gy放射联合的效果与2/3MTD 5-FU和6Gy放射联合的效果相近,提示放射与CAP联合治疗,同样效果需要的射线剂量小,CAP对放射的增强作用比5-FU强。
3、不同大小肿瘤对CAP与放射联合治疗效果的影响,
1/2MTD CAP和6Gy放射联合施与,大瘤组肿瘤对联合治疗反应敏感,有明显抑制。而小瘤组肿瘤对联合治疗反应不敏感,效果仅同于单纯药物或单纯放射的治疗效果。2/3MTD CAP和6Gy放射联合施与,加大CAP剂量后,虽然大、小瘤组肿瘤均对联合治疗有效,但大瘤组肿瘤受抑明显,优于小瘤组肿瘤。
4、从肿瘤生长延迟天数来看,CAP分别和6、5、2×8Gy放射联合施与的三个治疗组,肿瘤生长分别被延迟了23.9、22、48.4天。各联合治疗组的肿瘤生长延迟天数,都超过了它们单纯药物组及相应的单纯放射组延迟天数的总和,延迟天数并非为单纯药物组及相应的单纯放射组延迟天数的简单相加。
5、放射后HeLa细胞TP酶蛋白含量的ELISA测定
与对照组相比,HeLa细胞照射后,两个放射组的TP酶蛋白水平均有明显提高,放射后第3天都升高达高峰,以后逐渐下降,持续高水平约有一周左右时间。2Gy放射组TP酶蛋白水平升高程度,较6Gy放射组升高有更加明显的趋势,尤其放射后第3天两者的差别显著。2Gy放射组TP酶处于高水平的持续时间更长,超过9天。
6、放射后HeLa细胞TP、DPD酶mRNA表达RT-PCR检测
放射后HeLa细胞TP酶的mRNA表达,在照射后第3天迅速升高达到高峰,明显高于未经照射的对照组,以后出现逐渐下降的趋势。其升高时间与ELISA测定的TP酶蛋白水平的变化情况基本相吻合。而HeLa细胞DPD酶mRNA的表达,在放射组与对照组之间未见明显差异。
7、HeLa细胞周期构成比的变化
与对照组比较,5-FU处理组HeLa细胞S期增加明显,G_2/M期及G_0/G_1期比例减少,出现S期明显阻滞,药物处理24小时后阻滞最显著;X线照射组G_0/G_1期细胞有所增加,而S期细胞减少,照射24小时后变化最明显;5-FU与放射联合组对HeLa细胞的影响,主要是S期增多5-FU的作用为主,G_0/G_1期细胞明显减少。G_2/M期也有减少,但联合作用组G_2/M期细胞减少没有5-FU组那么明显。5-FU与放射联合作用时,HeLa细胞G_2/M、S及G_0/G_1各期细胞均受影响,但其对5-FU的反应比对放射线更敏感。联合作用引起的细胞周期变化也是处理后24小时最显著;照射前、后的HeLa细胞,均是5-FU处理组引起了S期阻滞,而单纯照射前、后HeLa细胞S期比例变化不大。
结论
1、对于宫颈腺癌CAC-1应用CAP与放射联合治疗能产生相互协同增效作用,有明显的肿瘤抑制效果,是一种有效的治疗子宫颈腺癌的方法。其效果优于单纯CAP及单纯放射的治疗,也优于5-FU直接和放射联合的效果。CAP对放射的增强作用比5-FU强。
2、CAP与放射联合治疗的效果与CAP药物浓度、放射剂量、照射方法及肿瘤大小有关。2/3MTD CAP分别和5、6Gy及2Gy×8放射联合治疗时,肿瘤抑制的效果较好。CAP与小剂量多次放射方法的联合治疗,较CAP与单次放射方法联合,更显示出了明显的抑瘤效果。体积较大的肿瘤,对CAP与放射联合治疗的反应敏感,比体积较小的肿瘤治疗效果好,可能CAP与放射联合治疗对较晚期子宫颈腺癌更为有效。
3、放射线照射,能提高子宫颈腺癌HeLa细胞TP酶的mRNA表达和TP酶蛋白水平,而DPD酶mRNA表达不受影响,因此放射后HeLa细胞TP/DPD比值升高,能增加其对CAP的敏感性。
4、5-FU与放射联合,两者对HeLa细胞周期均有影响,其细胞G_2/M、S及G_0/G_1各期都有变化,但主要还是以S期细胞阻滞即5-FU的影响为主。其联合作用引起的细胞周期变化,在处理后的24小时最为显著。HeLa细胞表现出对5-FU的作用比对放射更敏感,可以认为在两者的联合作用中,5-FU通过改变HeLa细胞周期而对放射效果的影响较大,相反放射对5-FU影响较小。
Introduction
Capecitabine(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 oneⅣ-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 Methods
Materials
1、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 University
HeLa cell strain of human cervical adenocarcinoma from laboratory of infection department in Shengjing Hospital affiliated to China Medical University
3、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).
Methods
1、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 2×10~6 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 G_0/G_1、S、G_2/M phases.
Results
1、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 G_0/G_1 phase increase and cells in S phase decrease;for group using 5-FU and radiotherapy,cells in G_0/G_1 decrease sharply but cells in S phase increase which enhance the effect of 5-FU.Cells in G_2/M phase decrease in combination group less than those in 5-FU group.HeLa cells in G_2/M、S and G_0/G_1 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.
Conclusions
1、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 G_2/M、S and G_0/G_1 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.
引文
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