非核苷类抗HIV药物TMC120的抗肿瘤作用及其机制研究

发布时间：2018-04-13 17:13

  本文选题：TMC120 + 细胞凋亡　； 参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景： 尽管高效抗逆转录病毒治疗(HAART)使人类免疫缺陷病毒(HIV)感染者的死亡率有所下降,艾滋病相关并发症的发生率及死亡率也大幅降低,且艾滋病患者的平均寿命也显著增加。但是恶性肿瘤始终是HIV感染者死亡的第二大常见原因。 最常见于艾滋病患者的肿瘤并发症前三位分别是卡波西肉瘤,非霍奇金淋巴瘤,侵袭性宫颈癌,他们也称为艾滋病特异性肿瘤。 与不断下降的艾滋病相关并发症的发生率与死亡率相比,艾滋病特异性肿瘤以及艾滋病非特异性肿瘤,如肺癌、肛管直肠癌等,却是逐年增长的。 虽然对于艾滋病并发恶性肿瘤的患者,联合应用抗肿瘤化疗和HAART已经显示出可行性和有效性,但是,很多抗肿瘤药物与HAART合用可能导致药物的蓄积和毒性增加、或者药物的排泄增加和效力降低。不幸的是,揭示药物相互作用,并可用于安全指导HAART和抗肿瘤化疗联合应用的研究数据非常有限。对于抗肿瘤药物与抗逆转录病毒药物的联合应用,缺乏基于临床试验的普遍可行的详细指南。所以癌症,始终是艾滋病患者生存的重大隐患。 因此,我们选取常用的各类抗HIV药物作用于肿瘤细胞,筛查抗HIV药物是否存在抗肿瘤细胞毒性,结果发现,TMC120单独应用时对多种肿瘤细胞有显著的细胞毒性。 TMC120(也叫达匹韦林),非核苷类逆转录酶抑制剂(NNRTI),目前主要研究作为阴道的杀微生物剂,以阴道环的形式在体外实验及间接体内疗法中均能有效地阻止HIV-1的感染。TMC120在抗HIV的临床前研究中,其活性得以充分肯定,如果其兼具抗肿瘤活性,无疑给肿瘤患者,特别是艾滋病并发肿瘤的患者带来福音。最重要的是,TMC120的应用避免了抗HIV药物与抗肿瘤药物联合应用带来的潜在的相互作用。 因此,TMC120有望用于预防及治疗艾滋病并发肿瘤的患者,基于其潜在的抗肿瘤及抗逆转录病毒活性。 目的： 研究非核苷类抗HIV药物TMC120的抗肿瘤活性,探讨TMC120的抗肿瘤机制。并且,通过建立裸鼠宫颈癌移植瘤模型,进一步研究TMC120体内的抗肿瘤活性。 方法： 1.MTT法检测细胞活力 多种抗HIV药物及顺铂(20μmol/L)处理结肠癌HT-29细胞,多种抗HIV药物及吉非替尼(40μmol/L)处理肺癌A549细胞,多种抗HIV药物及紫杉醇(10μmol/L)处理宫颈癌HeLa细胞,48h后停止培养,加入用培养基现配的MTT溶液,处理3h后,弃上清,加入DMSO振荡混匀,于酶标仪中测定吸光度值(OD570)。 多类肿瘤细胞,如肺癌细胞(A549、95D),肝癌细胞(HepG2),结肠癌细胞(colon26、 HT-29),食管癌细胞(EC109),乳腺癌细胞(MCF-7),子宫内膜癌细胞(HEC-1),宫颈癌细胞(HeLa、 SiHa)与TMC120(0、1.56、3.13、6.25、12.5、25、50μmol/L)共同孵育48h后停止培养,加入用培养基现配的MTT溶液,处理3h后,弃上清,加入DMSO振荡混匀,于酶标仪中测定吸光度值(OD570)。2.流式细胞仪检测细胞凋亡情况 EC109细胞、HeLa细胞与0、6.25、12.5μmol/L的TMC120, SiHa细胞与0、、12.5、25μmol/L的TMC120共同孵育48h后停止培养,用不含EDTA的胰蛋白酶消化细胞,离心后细胞用PBS洗2次,再用FITC Annexin V Apoptosis Detection Kit中的1×缓冲液100μL重悬细胞,加入FITC和PI各5μL,避光,室温放置15min。每管细胞中再分别加入400μL的1×缓冲液,充分混匀后于1h内上样,用流式细胞仪检测细胞凋亡情况。3.流式细胞仪测细胞周期分布 EC109, HeLa和SiHa细胞分别与0、6.25.12.5、25μmol/L的TMC120共同孵育24h后收集细胞,细胞用PBS洗2次,再用预冷的体积分数为75%乙醇混悬细胞,置于4℃冰箱密封保存过夜。离心收集细胞,PBS洗1次,每管细胞加入500μL含PI(50μg/mL)及RNA酶(10μg/mL)的PBS溶液,细胞置于37℃避光孵育30rmin,后于1h内上样,用流式细胞仪检测细胞周期的分布情况。 4. Western blotting测G2/M检测点相关蛋白及M期标志蛋 TMC120(12.5μmol/L)分别与HeLa和SiHa细胞共同孵育0、1、3、6、12、24、48h,紫杉醇(100nmol/L)与HeLa细胞共同孵育0、1、3、6、12、24、48h。裂解细胞,蛋白定量,Western Blotting检测G2/M检查点相关蛋白p21waf1/cip1、cyclin B1、 phospho-cdc2(Tyrl5),以及M期标志蛋白phospho-Histone H3(Ser10)、phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198)的表达情况。 5.免疫荧光共聚焦检测细胞微管形态 HeLa和SiHa细胞分别用TMC120(12.5μmol/L)和紫杉醇(100nmol/L或1μmol/L)处理12h后,用4%多聚甲醛固定15min,再用100%甲醇通透,置于4℃孵育10min,吸弃甲醇,细胞用10%山羊血清室温封闭1h,后用含有抗a/β-tubulin一抗的山羊血清于4℃孵育过夜。细胞用PBS洗2次,再用含有CY3荧光标记的二抗室温孵育2h。细胞用PBS洗2次,最后加入DAIP标记细胞核,于共聚焦荧光显微镜下观察细胞微管形态,并拍照。 6.微管蛋白聚合实验 微管蛋白聚合检测试剂盒测在TMC120或紫杉醇作用下,体外纯微管蛋白的聚合情况。即酶标仪温度恒定37℃,每分钟一次动态检测OD340的值,以吸光度值表示微管蛋白聚合程度。 7.裸鼠移植瘤模型研究TMC120的体内抗肿瘤活性 4到6周的雌性BALB/c nu/nu裸鼠用于实验研究。将SiHa细胞悬液皮下接种到裸鼠的左腋下,每只裸鼠接种约3×106个细胞。然后3次／周用游标卡尺测量肿瘤的大小。肿瘤的大小按照以下公式计算：V(体积)=L×W2/2,其中L是肿瘤的长度,W是肿瘤的宽度。接种后10天,所有老鼠都长出明显肿瘤。将裸鼠随机分为5组,按以下方案给药：(1)对照组；(2) TMC12012.5mg/kg，，每周给药3次；(3)TMC12025mg/kg,每周给药3次；(4) TMC12050mg/kg,每周给药3次；(5)紫杉醇15mg/kg,每周给药2次。5组裸鼠均采用腹腔注射给药,给药18天后处死裸鼠并取出肿瘤组织。 当对照组肿瘤体积超过1000mm3后结束给药,处死裸鼠取出肿瘤组织。肿瘤组织用4%的多聚甲醛固定,脱水后石蜡包埋；自动切片机将包埋组织切成5μm厚度的石蜡切片。石蜡切片脱蜡再水化组织,按TUNEL试剂盒的指示,将TUNEL反应混合液加入样品上,湿盒、暗室、37℃中加盖孵育60min；加入转化-POD于样品上,湿盒、暗室、37℃中加盖孵育30min；然后加入DAB底物,于室温孵育10min；镜检前用苏木素复染,中性树脂封片保存。正置显微镜下拍照并计数凋亡细胞(即棕褐色的TUNEL阳性细胞)所占比例。 8.统计学分析 数据分析和作图均是由GraphPad Prism5.0(数据分析和作图软件)提供,数据结果以x±s表示；多组间均数比较采用One way-ANOVA方法,各组分别与对照组比较采用Dunnett检验以及各组间比较采用Tukey检验。 结果： 1、MTT结果显示ritonavir和TMC120单独用药时对结肠癌、肺癌、宫颈癌细胞都具有明显的细胞毒性作用。 2、MTT结果显示TMC120对肿瘤细胞具有浓度依耐性的抑制作用,且其对于各类肿瘤细胞的半数抑制率IC50值基本上在10μmol/L以内。这表明TMC120对多种肿瘤细胞具有较强的细胞毒性作用。 3、流式测细胞凋亡的结果显示,与对照组相比较,经TMC120处理的肿瘤细胞,其FITC Annexin V阳性细胞比率显著增加。结果表明,与抗肿瘤药物顺铂类似,TMC120也能诱导肿瘤细胞的凋亡,通过介导凋亡而抑制肿瘤细胞的增殖。 4、通过流式细胞仪对细胞周期分布的检测表明,经TMC120处理的细胞,其G2/M期所占比率呈浓度依耐性的增多。即TMC120能够促使肿瘤细胞阻滞在G2/M期,发挥类似于紫杉醇一样的作用。 5、Western Blotting结果表明TMC120发挥类似紫杉醇的作用特征,即能够削弱G2/M检测点功能,使宫颈癌细胞阻滞在有丝分裂期。 6、Confocal的结果显示,类似于紫杉醇,TMC120处理的宫颈癌细胞的微管伸展受限、短缩在核周围,且有丝分裂期细胞出现异常的、单极的纺锤体结构。结果表明,TMC120能够引起微管的形态变化,破坏正常的有丝分裂期纺锤体结构,从而使肿瘤细胞难以维持正常的有丝分裂。 7、体外微管聚合实验显示,TMC120呈现出剂量依耐性的促进微管聚合的作用,表现出堪比于紫杉醇的微管稳定效应。结果证实,TMC120能够靶向微管,发挥稳定微管的作用,从而破坏微管的动态平衡,干扰细胞的有丝分裂。 8、裸鼠移植瘤实验显示,TMC120能够明显延迟肿瘤的生长,具有抗肿瘤体内活性,且在各剂量的药物处理中均没有出现明显的毒性反应；并且,TMC120能够介导体内肿瘤组织的细胞凋亡,即通过介导肿瘤细胞的凋亡而抑制肿瘤组织的增殖。 结论： TMC120对多种肿瘤细胞均有较强的毒性作用,能够介导肿瘤细胞的凋亡。类似于紫杉醇的作用特征,TMC120能够削弱G2/M检测点功能,使宫颈癌细胞阻滞在有丝分裂期；能够促进微管蛋白聚合,稳定微管,改变微管的正常形态,从而使肿瘤细胞难以进行分裂增殖。裸鼠体内实验证明,TMC120能够延缓肿瘤的增长,并且介导裸鼠肿瘤细胞的凋亡。
[Abstract]:Background of Study :

Although high - efficiency antiretroviral therapy ( HAART ) has decreased the mortality rate of HIV - infected persons , the incidence and mortality of AIDS - related complications have also significantly decreased , and the average life expectancy of AIDS patients is also significantly increased . However , malignancies have always been the second most common cause of the death of HIV - infected persons .

The first three of the most common tumor complications in AIDS patients were Kaposi ' s sarcoma , non - Hodgkin ' s lymphoma , invasive cervical cancer , also known as AIDS - specific tumors .

Compared with the declining incidence and mortality of AIDS - related complications , AIDS - specific tumors and non - specific HIV / AIDS tumors , such as lung cancer , anal and rectal cancer , are growing year by year .

Although the combination of anti - tumor chemotherapy and HAART has shown the feasibility and effectiveness for patients with AIDS complicated with malignant tumors , the combination of many anti - tumor drugs with HAART may lead to increased accumulation and toxicity of drugs , or increased excretion and efficacy of drugs . Unfortunately , there is a very limited study data available for safety - directed combination of HAART and anti - retroviral drugs .

Therefore , we select the common anti - HIV drugs to act on tumor cells and screen anti - HIV drugs for anti - tumor cytotoxicity . As a result , it is found that the TMC120 has significant cytotoxicity to multiple tumor cells when used alone .

TMC120 , a non - nucleoside reverse transcriptase inhibitor ( NNRTI ) , is currently being used as a vaginal microbicide to effectively prevent the infection of HIV - 1 in both in vitro and indirect in vivo therapy . TMC120 , in preclinical studies against HIV , is sufficiently active to provide the gospel to patients with tumors , especially AIDS patients , in preclinical studies against HIV . Most importantly , the use of TMC120 avoids potential interactions between anti - HIV drugs and anti - tumor drugs .

Therefore , TMC120 is expected to be used in the prevention and treatment of AIDS patients with tumors , based on their potential anti - tumor and anti - retroviral activity .

Purpose :

To study the anti - tumor activity of non - nucleoside anti - HIV drug TMC120 and to investigate the anti - tumor mechanism of TMC120 .

Method :

1 . MTT assay to detect cell viability

A variety of anti - HIV drugs and cisplatin ( 20 渭mol / L ) were used to treat HT - 29 cells of colon cancer , multiple anti - HIV drugs and gemcitabine ( 40 渭mol / L ) for the treatment of human lung cancer A549 cells , multiple anti - HIV drugs and paclitaxel ( 10 渭mol / L ) for cervical cancer HeLa cells . After 48 hours culture was stopped , the MTT solution prepared with the culture medium was added , the supernatant was discarded , the DMSO oscillation was added , and the absorbance value was measured in the microplate reader ( OD570 ) .

Multiple tumor cells , such as lung cancer cells ( A549 , 95D ) , liver cancer cells ( HepG2 ) , colon cancer cells ( A549 , HT - 29 ) , esophageal cancer cells ( EC109 ) , breast cancer cells ( MCF - 7 ) , endometrial cancer cells ( HEC - 1 ) , cervical cancer cells ( HeLa , SiHa ) and TMC120 ( 0 , 1 . 56 , 3 . 13 , 6.25 , 12.5 , 25 , 50 渭mol / L ) were incubated for 3 h .

EC109 cells , HeLa cells and 0 , 6.25 , 12.5 渭mol / L TMC120 , SiHa cells and 0 , 12.5 , 25 渭mol / L TMC120 were incubated for 48 hours . Cells were digested with PBS without EDTA . After centrifugation , the cells were washed twice with PBS and incubated at room temperature for 15 min .

EC109 , HeLa and SiHa cells were incubated with 0 , 6.25 . 12.5 , 25 渭mol / L TMC120 for 24 h . The cells were collected by PBS . The cells were incubated overnight at 4.degree . C.

4 . Western blotting for G2 / M detection point - related protein and M - phase marker eggs

TMC120 ( 12.5 渭mol / L ) was incubated with HeLa and SiHa cells for 0 , 1 , 3 , 6 , 12 , 24 , 48 h , and paclitaxel ( 100 nmol / L ) was incubated with HeLa cells for 0 , 1 , 3 , 6 , 12 , 24 , 48 h . The expression of p21 waf1 / cip1 , cyclin B1 , bcl - cdc2 ( Tyrol 5 ) , and M - phase marker protein kinase - Histone H3 ( Ser10 ) , Ser - Aurora A ( Th28288 ) / Aurora B ( Thr 232 ) / Aurora C ( Thr 198 ) were detected by Western Blotting .

5 . Immunofluorescence confocal detection of cell microtubules morphology

HeLa and SiHa cells were treated with TMC120 ( 12.5 渭mol / L ) and paclitaxel ( 100 nmol / L or 1 渭mol / L ) for 15 min . After 12 h incubation with 100 % methanol , the cells were incubated overnight at 4.degree . C.

6 . Microtube protein polymerization experiment

In the presence of TMC120 or paclitaxel , the micro - tube protein polymerization detection kit was used to measure the polymerization of pure microtube protein in vitro , i.e . , the temperature of the microplate reader was constant 37 鈩

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