树胶脂毒素对人膀胱癌RT4细胞抑制作用的体外研究
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摘要
摘要：膀胱癌是泌尿外科最常见的恶性肿瘤之一,组织学类型主要为膀胱尿路上皮癌(urothelial cell carcinoma, UCC),又称移形细胞癌(transitional cell carcinoma, TCC)。大多数(70%-80%)膀胱癌病例为为非肌层浸润性膀胱癌(Non-muscle-invasive bladder cancer,NMIBC),手术治疗是目前NMEBC的首选的治疗方法,所有非肌层浸润性膀胱癌患者行TURBT术后均建议行膀胱腔内灌注化疗药物或免疫制剂,降低肿瘤复发率。但由于膀胱癌具有多发性原位癌不易通过膀胱镜检查发现等特点,TURBT术后行化疗药物灌注后,1年内的复发率仍高达15%-61%,而5年内更有31%-78%患者出现肿瘤复发。
目前常用的灌注化疗药物包括：吡柔比星、丝裂霉素C、表柔比星、阿霉素、羟喜树碱。但这些常见药物均会产生化学性膀胱炎等副作用,并在TURBT术后即刻灌注化疗后更明显。卡介苗(BCG)是已知预防复发和进展最有效的灌注药物,但行BCG灌注治疗后,肿瘤复发率仍有30%-45%,大量患者出现严重膀胱刺激征,并有部分患者出现反应性前列腺炎、附睾炎、肝炎等严重副反应,无法耐受治疗,致其临床应用受到很大限制。为了寻找疗效更好、副作用更小的膀胱癌化疗药物,国内外学者都在进行不懈的努力。
近年来,香草酸受体1(vanilloid receptor1,VR1)作为新的抗肿瘤药物的靶点进入了人们的视线。VR1是瞬时感受器电位亚家族成员之一,是一种非选择性的阳离子通道,广泛表达在初级传入感觉神经元及粘膜组织中,在多种恶性肿瘤组织中也有表达。使用香草酸类药物如树胶脂毒素(resiniferatoxin)、辣椒素(capsaisin)等,可以激活VR1通道,引起强烈的钙离子内流,引起细胞内钙超载导致明显的细胞毒作用,研究证明了这种钙超载可以激活线粒体凋亡途径诱导的肿瘤细胞凋亡。
树胶脂毒素(resiniferatoxin, RTX)是一种从大戟类树胶植物的乳汁中分离出的刺激性香草酸类分子,对VR1的激动活性大大强于辣椒素,因此又被称为辣椒素类似物、超强辣素,是已知最强的VR1激动剂,可以诱发更强、持续时间更长的钙离子内流。已经在临床研究中应用于膀胱灌注治疗我科间质性膀胱炎及神经源性膀胱等疾病,无明显不良反应。在抗肿瘤领域,国外学者研究了树胶脂毒素对皮肤鳞状细胞癌细胞及胰腺癌细胞的体外抑制作用,并对作用机制进行了研究。而对膀胱癌细胞树胶脂毒素是否具有抗肿瘤活性,是否可以作为新的膀胱灌注化疗药物是我们感兴趣的地方。
本研究应用了MTT、流式细胞术、倒置显微镜研究了树胶脂毒素对人膀胱癌RT4细胞的增殖的抑制作用、对细胞周期的阻滞作用、对细胞凋亡的诱导作用以及对RT4细胞形态学的影响。又应用荧光钙离子成像技术、Western-blot技术初步探讨了树胶脂毒素对RT4细胞抑制作用的分子生物学机制。
实验目的：研究RTX干预对膀胱癌RT4细胞增殖、细胞周期、细胞凋亡的影响。
实验方法：
1.采用MTT法以0nmol/L RTX作为阴性对照组,观察不同浓度(50,100,200,500,1000,1500nmol/L)RTX作用不同时间(24h,48h,72h)对RT4细胞增殖的抑制作用。
2.采用倒置相差显微镜观察不同浓度(0,50,100,200,500,1000nmol/L) RTX作用不同时间(8h,24h,48h)后RT4细胞形态学变化
3.采用流式细胞术(PI染色)检测不同浓度(0,500,1000nmol/L)RTX作用48h后RT4细胞周期分布。
4.采用流式细胞术(Annexin V/PI双染)分析不同浓度(0,500,1000nmol/L) RTX作用48h后RT4细胞凋亡情况.
实验结果：
1.MTT结果示,不同浓度(50,100,200,500,1000,1500nmol/L) RTX作用人膀胱癌RT4细胞不同时间(24h,48h,72h)后,抑制率为(3.48%-29.73%),(8.49%-50.23%),(13.54%-72.76%),呈剂量及时间依赖性；各浓度组抑制率与阴性对照组(Onmol/L)比较,均有显著性差异(P<0.01).
2.倒置相差显微镜观察结果示：RTX干预后,显微镜下可见RTX干预后RT4细胞数量逐渐减少,并出现典型凋亡细胞特征。效应呈时间及剂量依赖性。
3.流式细胞术(PI染色)结果示：不同浓度(500,1000nmol/L) RTX干预可诱导人膀胱癌RT4细胞G0/G1期细胞周期阻滞,RTX干预48小时后,各组均出现G0/G1期细胞比例增加,依次为：42.37%,48.79%。与阴性对照组(35.02%)比较,差异均具有统计学意义(P<0.05)
4.流式细胞术(Annexin V/PI双染)结果示：RTX干预48小时后,早期凋亡细胞的比例升高,分别：3.21%,5.42%,与阴性对照组(0.65%)比较差异具有统计学意义(P<0.05),晚期凋亡细胞的比例升高,分别：10.23%,19.87%,与阴性对照组(2.34%)比较差异具有统计学意义(P<0.05)。总凋亡细胞细胞的比例也升高,分别：13.93%,25.29%±6.21%,与阴性对照组(2.99%)比较差异具有统计学意义(P<0.05),提示RTX干预诱导RT4细胞凋亡,并呈剂量依赖性。
实验结论：
1.RTX体外干预可抑制RT4细胞的体外增殖,其抑制作用呈剂量及时间依赖性
2.RTX体外干预可诱导RT4细胞周期G0/G1期阻滞,呈剂量依赖性。
3.RTX体外干预可诱导RT4细胞凋亡,呈剂量依赖性。
实验目的：初步研究RTX对RT4细胞抑制作用的分子生物学机制。
实验方法：
1.采用荧光钙离子成像技术检测不同浓度(0,500,1000nmol/L) RTX作用不同时间(Oh,8h,24h)后RT4细胞内钙离子浓度变化。
2.采用Western-blot技术检测RTX(1000nmol/L)干预4个时间点(Oh,8h,24h,48h)细胞周期调控蛋白P53、P21、细胞周期依赖性激酶(CDK)2表达变化。
3.采用Western-blot技术检测RTX(1000nmol/L)干预4个时间点(Oh,8h,24h,48h)细胞凋亡相关蛋白caspase-8, caspase-3、以及胞浆内细胞色素C的表达变化。
实验结果：
1.荧光钙离子成像结果证明细胞内钙离子水平在RTX干预后明显提高,效果呈浓度依赖性及时间依赖性。
2.P53蛋白在RTX干预早期(8h)出现表达高峰,随后逐渐下降。P21在8h后表达升高并维持。CDK2结果显示随时间推移,表达逐渐降低。
3. caspase-8在RTX干预后随时间的推移,表达逐渐升高,caspase-3结果显示随时间推移表达逐渐增高,细胞色素C在干预后随时间推移表达升高。
实验结论：
1.RTX干预可造成膀胱癌RT4细胞内钙超载并呈时间依赖性及浓度依赖性。
2.RTX干预可通过激活P53-P21-CDK2通路造成膀胱癌RT4细胞G0/G1期细胞周期阻滞。
3.RTX干预可通过激活caspase-8-caspase-3通路并激活细胞凋亡的线粒体途径诱发膀胱癌RT4细胞凋亡。
Abstract:Bladder cancer (BC) is the most common malignant tumor of urinary system,about90%of which is comprised of urothelial cell carcinoma (UCC)(also known as transitional cell carcinoma, TCC).About70%-80%bladder cancer cases are Non-muscle-invasive bladder cancer (NMIBC).At present,all the patients with NMIBC are suggested to have surgical resection.But the recurrence rate is still above60%in3-5years after the surgery. So it is important to have intravesical chemotherapy drugs or immunomodulating agents,which can kill the epibiotic tumor cells and reduce the risk of relapses of the tumor.Because of NMIBC always are multiple malignant tumor and the tumor in Tis stage can hardly be diagnosed, about15%-61%of the patients presented to us with recurrent tumors after received intravesical therapy in the first year after operation and31%-78%in five years.
Now the most common chemotherapy drugs are pirarubicin, mitomycin, pharmorubicin, doxorubicine, hydroxycamptothecine.All those drugs lead to side effect such as chemical cystitis, which is obvious at the time of the immediate postoperative intravesical. Bacillus Calmette-Guerin (BCG) is considered to be the most effective intravesical drug.But tumor recurrence in30%-45%of the patients received intravesical BCG therapy.Some of the patients have severe irritation symptoms of bladder, prostatitis, epididymitis, hepatitis. So its usage is greatly limited. Doctors among the world are making unremitting efforts to search new drug or new drug targets.
In recent years,VR1(vanilloid receptor1) is rediscovered as a antineoplastic drug target. Vanilloid receptor1, a non-selective cation channel belonging to the transient receptor potential family of ion channels, is expressed in the spinal cord, brain and a wide-range of non-neuronal cells such as urothelium and some tumor tissue.Vanilloids (capsaicin and resiniferatoxin),as the VR1agonists can strongly activates VR1producing a large influx of calcium, resulting in calcium-induced inactivation and cytotoxicity lead to mitochondrial damage.
Resiniferatoxin, a diterpine derived from the latex of the plant Euphorbia resinifera used by the ancient physicians of Roman times, was found to share structural similarity to capsaicin by containing a common vanilloid moiety essential for activity. In the anti-tumor field Hail investigate the inhibitory effect of resiniferatoxin on SCC cell line.Hartel investigate the inhibitory effect of resiniferatoxin on human pancreatic cancer line. What we interested in is if RTX has antineoplastic activity against the UCC cell line.
Object:To investigate the inhibitory effect of resiniferatoxin on growth inhibition cell cycle and induction on apoptosis of human bladder carcinoma RT4cell.
Methods:
1.The inhibitory effect of RTX on human bladder carcinoma RT4cells was determined with varying concentration of RTX (50,100,200,500,1000,1500nmol/L) treatment for24,48and72hours by MTT assay taking0nmol/L RTX as a negtive control.
2.Morphologic changes of RT4cells were studied with inverted phase contrast microscope with different concentration (0,50,100,200,500,1000nmol/L) of RTX at different time (Oh,8h,24h,48h)
3.The cell cycle of RT4cells treated by RTX (0,500,1000nmol/L) for48hours were detected by flow cytometiy (FCM) with PI staining method.
4.The extent of apoptosis of RT4cells treated by RTX (0,500,1000nmol/L) for48hours were detected by (FCM) with annexin V and PI staining method.
Results:
1. Using the human bladder carcinoma RT4cell line, we found that RTX treatment resulted in dose-dependent (50,100,200,500,1000,1500nmol/L) inhibition of cellular proliferation and cell viability. The inhibition rate of cellular proliferation with RTX treatment at concentrations of (50,100,200,500,1000,1500nmol/L) treatment after24hours ranged from3.48%to29.73%respectively. The inhibition rate of cellular proliferation with RTX treatment was significantly different compared with negative control group(P<0.01). The inhibition of cellular proliferation with RTX treatment at concentrations of (50,100,200,500,1000,1500nmol/L) after48and72hours ranged from8.49%to50.23%,13.54%to72.76%respectively in a time-dependent manner.
2. In lower concentrations (50,100nmol/L) treatment of RTX for8hours, it shows a moderate apoptotic effect, while in higher concentrations1000nmol/L it showed more apoptotic cells observed with inverted phase contrast microscope.After24hours in higher concentrations(1000nmol/L), more typical apoptosis body was observed,while after48hours a large number of apoptotic cells was observed.
3.As shown by FCM with PI staining method, RTX treatment (500,1000nmol/L) of the RT4cells resulted in significant G0/G1-phase cell arrest ranging from42.37to48.79%, and each concentration of RTX group was significantly different compared with negative control group35.02%(P<0.05).
4. As shown by FCM with PI and Annexin V staining method, we found that RTX caused a dose-dependent increase in RT4cell apoptosis. It was observed that treatment of RT4cells with varying concentration of RTX (0,500,1000nmol/L) for48hours increased the number of early apoptotic cells from3.21%to5.42%, and total apoptotic cells from13.93%to25.29%in a dose-dependent manner, and significantly different compared with negative control group0.65%and2.99%respectively (P<0.05).
Conclusion:
1.Resiniferatoxin (RTX) can inhibit the proliferation of human bladder carcinoma RT4cells effectively in a dose and time-dependent manner in vitro.
2. RTX can arrest the RT4cells in G0/G1phase at a dose-dependent manner.
3. RTX induces human bladder carcinoma RT4cells apoptosis at a dose-dependent manner.
Object:to investigate the mechnism of the inhibitory effect of resiniferatoxin on human bladder carcinoma RT4cell.
Methods:
1. After treated by RTX (0,500,1000nmol/L) for different time (Oh,8h,24h) the concentration of Ca2+in RT4cells is determined with fluoreacent labeling method.
2.After treated by RTX (1000nmol/L) for different time (Oh,8h,24h,48h),detect three proteins of apoptosis related proteins caspase-8, caspase-3and evaluate cytochrome c release with the approsch of western-blot analysis.
3.After treated by RTX (1000nmol/L) for different time (Oh,8h,24h,48h),detect three cell cycle regulatory protein p53, p21, CDK2with the approsch of western-blot analysis.
Results:
1. After a48hour treatment with RTX, the expressions of P53and P21were up-regulated in contrary to the expression of CDK2.
3.RTX induced caspase-8activation at8h that increased at24-48h. Caspase-3activation occurred at12h and persisted until24h after treatment.8hours after treatment, a band of12kDa corresponding to cytochrome c was observed and declined at later time points
Conclusion:
1. RTX can arrest the RT4cells in G0/G1phase by modulating the expression of P53, P21and CDK2.
2.Treatment of cells with RTX induced apoptosis accompanied by activation of caspase-8,caspase-3.and increased mitochondrial cytochrome c release.
目前常用的灌注化疗药物包括：吡柔比星、丝裂霉素C、表柔比星、阿霉素、羟喜树碱。但这些常见药物均会产生化学性膀胱炎等副作用,并在TURBT术后即刻灌注化疗后更明显。卡介苗(BCG)是已知预防复发和进展最有效的灌注药物,但行BCG灌注治疗后,肿瘤复发率仍有30%-45%,大量患者出现严重膀胱刺激征,并有部分患者出现反应性前列腺炎、附睾炎、肝炎等严重副反应,无法耐受治疗,致其临床应用受到很大限制。为了寻找疗效更好、副作用更小的膀胱癌化疗药物,国内外学者都在进行不懈的努力。
近年来,香草酸受体1(vanilloid receptor1,VR1)作为新的抗肿瘤药物的靶点进入了人们的视线。VR1是瞬时感受器电位亚家族成员之一,是一种非选择性的阳离子通道,广泛表达在初级传入感觉神经元及粘膜组织中,在多种恶性肿瘤组织中也有表达。使用香草酸类药物如树胶脂毒素(resiniferatoxin)、辣椒素(capsaisin)等,可以激活VR1通道,引起强烈的钙离子内流,引起细胞内钙超载导致明显的细胞毒作用,研究证明了这种钙超载可以激活线粒体凋亡途径诱导的肿瘤细胞凋亡。
树胶脂毒素(resiniferatoxin, RTX)是一种从大戟类树胶植物的乳汁中分离出的刺激性香草酸类分子,对VR1的激动活性大大强于辣椒素,因此又被称为辣椒素类似物、超强辣素,是已知最强的VR1激动剂,可以诱发更强、持续时间更长的钙离子内流。已经在临床研究中应用于膀胱灌注治疗我科间质性膀胱炎及神经源性膀胱等疾病,无明显不良反应。在抗肿瘤领域,国外学者研究了树胶脂毒素对皮肤鳞状细胞癌细胞及胰腺癌细胞的体外抑制作用,并对作用机制进行了研究。而对膀胱癌细胞树胶脂毒素是否具有抗肿瘤活性,是否可以作为新的膀胱灌注化疗药物是我们感兴趣的地方。
本研究应用了MTT、流式细胞术、倒置显微镜研究了树胶脂毒素对人膀胱癌RT4细胞的增殖的抑制作用、对细胞周期的阻滞作用、对细胞凋亡的诱导作用以及对RT4细胞形态学的影响。又应用荧光钙离子成像技术、Western-blot技术初步探讨了树胶脂毒素对RT4细胞抑制作用的分子生物学机制。
实验目的：研究RTX干预对膀胱癌RT4细胞增殖、细胞周期、细胞凋亡的影响。
实验方法：
1.采用MTT法以0nmol/L RTX作为阴性对照组,观察不同浓度(50,100,200,500,1000,1500nmol/L)RTX作用不同时间(24h,48h,72h)对RT4细胞增殖的抑制作用。
2.采用倒置相差显微镜观察不同浓度(0,50,100,200,500,1000nmol/L) RTX作用不同时间(8h,24h,48h)后RT4细胞形态学变化
3.采用流式细胞术(PI染色)检测不同浓度(0,500,1000nmol/L)RTX作用48h后RT4细胞周期分布。
4.采用流式细胞术(Annexin V/PI双染)分析不同浓度(0,500,1000nmol/L) RTX作用48h后RT4细胞凋亡情况.
实验结果：
1.MTT结果示,不同浓度(50,100,200,500,1000,1500nmol/L) RTX作用人膀胱癌RT4细胞不同时间(24h,48h,72h)后,抑制率为(3.48%-29.73%),(8.49%-50.23%),(13.54%-72.76%),呈剂量及时间依赖性；各浓度组抑制率与阴性对照组(Onmol/L)比较,均有显著性差异(P<0.01).
2.倒置相差显微镜观察结果示：RTX干预后,显微镜下可见RTX干预后RT4细胞数量逐渐减少,并出现典型凋亡细胞特征。效应呈时间及剂量依赖性。
3.流式细胞术(PI染色)结果示：不同浓度(500,1000nmol/L) RTX干预可诱导人膀胱癌RT4细胞G0/G1期细胞周期阻滞,RTX干预48小时后,各组均出现G0/G1期细胞比例增加,依次为：42.37%,48.79%。与阴性对照组(35.02%)比较,差异均具有统计学意义(P<0.05)
4.流式细胞术(Annexin V/PI双染)结果示：RTX干预48小时后,早期凋亡细胞的比例升高,分别：3.21%,5.42%,与阴性对照组(0.65%)比较差异具有统计学意义(P<0.05),晚期凋亡细胞的比例升高,分别：10.23%,19.87%,与阴性对照组(2.34%)比较差异具有统计学意义(P<0.05)。总凋亡细胞细胞的比例也升高,分别：13.93%,25.29%±6.21%,与阴性对照组(2.99%)比较差异具有统计学意义(P<0.05),提示RTX干预诱导RT4细胞凋亡,并呈剂量依赖性。
实验结论：
1.RTX体外干预可抑制RT4细胞的体外增殖,其抑制作用呈剂量及时间依赖性
2.RTX体外干预可诱导RT4细胞周期G0/G1期阻滞,呈剂量依赖性。
3.RTX体外干预可诱导RT4细胞凋亡,呈剂量依赖性。
实验目的：初步研究RTX对RT4细胞抑制作用的分子生物学机制。
实验方法：
1.采用荧光钙离子成像技术检测不同浓度(0,500,1000nmol/L) RTX作用不同时间(Oh,8h,24h)后RT4细胞内钙离子浓度变化。
2.采用Western-blot技术检测RTX(1000nmol/L)干预4个时间点(Oh,8h,24h,48h)细胞周期调控蛋白P53、P21、细胞周期依赖性激酶(CDK)2表达变化。
3.采用Western-blot技术检测RTX(1000nmol/L)干预4个时间点(Oh,8h,24h,48h)细胞凋亡相关蛋白caspase-8, caspase-3、以及胞浆内细胞色素C的表达变化。
实验结果：
1.荧光钙离子成像结果证明细胞内钙离子水平在RTX干预后明显提高,效果呈浓度依赖性及时间依赖性。
2.P53蛋白在RTX干预早期(8h)出现表达高峰,随后逐渐下降。P21在8h后表达升高并维持。CDK2结果显示随时间推移,表达逐渐降低。
3. caspase-8在RTX干预后随时间的推移,表达逐渐升高,caspase-3结果显示随时间推移表达逐渐增高,细胞色素C在干预后随时间推移表达升高。
实验结论：
1.RTX干预可造成膀胱癌RT4细胞内钙超载并呈时间依赖性及浓度依赖性。
2.RTX干预可通过激活P53-P21-CDK2通路造成膀胱癌RT4细胞G0/G1期细胞周期阻滞。
3.RTX干预可通过激活caspase-8-caspase-3通路并激活细胞凋亡的线粒体途径诱发膀胱癌RT4细胞凋亡。
Abstract:Bladder cancer (BC) is the most common malignant tumor of urinary system,about90%of which is comprised of urothelial cell carcinoma (UCC)(also known as transitional cell carcinoma, TCC).About70%-80%bladder cancer cases are Non-muscle-invasive bladder cancer (NMIBC).At present,all the patients with NMIBC are suggested to have surgical resection.But the recurrence rate is still above60%in3-5years after the surgery. So it is important to have intravesical chemotherapy drugs or immunomodulating agents,which can kill the epibiotic tumor cells and reduce the risk of relapses of the tumor.Because of NMIBC always are multiple malignant tumor and the tumor in Tis stage can hardly be diagnosed, about15%-61%of the patients presented to us with recurrent tumors after received intravesical therapy in the first year after operation and31%-78%in five years.
Now the most common chemotherapy drugs are pirarubicin, mitomycin, pharmorubicin, doxorubicine, hydroxycamptothecine.All those drugs lead to side effect such as chemical cystitis, which is obvious at the time of the immediate postoperative intravesical. Bacillus Calmette-Guerin (BCG) is considered to be the most effective intravesical drug.But tumor recurrence in30%-45%of the patients received intravesical BCG therapy.Some of the patients have severe irritation symptoms of bladder, prostatitis, epididymitis, hepatitis. So its usage is greatly limited. Doctors among the world are making unremitting efforts to search new drug or new drug targets.
In recent years,VR1(vanilloid receptor1) is rediscovered as a antineoplastic drug target. Vanilloid receptor1, a non-selective cation channel belonging to the transient receptor potential family of ion channels, is expressed in the spinal cord, brain and a wide-range of non-neuronal cells such as urothelium and some tumor tissue.Vanilloids (capsaicin and resiniferatoxin),as the VR1agonists can strongly activates VR1producing a large influx of calcium, resulting in calcium-induced inactivation and cytotoxicity lead to mitochondrial damage.
Resiniferatoxin, a diterpine derived from the latex of the plant Euphorbia resinifera used by the ancient physicians of Roman times, was found to share structural similarity to capsaicin by containing a common vanilloid moiety essential for activity. In the anti-tumor field Hail investigate the inhibitory effect of resiniferatoxin on SCC cell line.Hartel investigate the inhibitory effect of resiniferatoxin on human pancreatic cancer line. What we interested in is if RTX has antineoplastic activity against the UCC cell line.
Object:To investigate the inhibitory effect of resiniferatoxin on growth inhibition cell cycle and induction on apoptosis of human bladder carcinoma RT4cell.
Methods:
1.The inhibitory effect of RTX on human bladder carcinoma RT4cells was determined with varying concentration of RTX (50,100,200,500,1000,1500nmol/L) treatment for24,48and72hours by MTT assay taking0nmol/L RTX as a negtive control.
2.Morphologic changes of RT4cells were studied with inverted phase contrast microscope with different concentration (0,50,100,200,500,1000nmol/L) of RTX at different time (Oh,8h,24h,48h)
3.The cell cycle of RT4cells treated by RTX (0,500,1000nmol/L) for48hours were detected by flow cytometiy (FCM) with PI staining method.
4.The extent of apoptosis of RT4cells treated by RTX (0,500,1000nmol/L) for48hours were detected by (FCM) with annexin V and PI staining method.
Results:
1. Using the human bladder carcinoma RT4cell line, we found that RTX treatment resulted in dose-dependent (50,100,200,500,1000,1500nmol/L) inhibition of cellular proliferation and cell viability. The inhibition rate of cellular proliferation with RTX treatment at concentrations of (50,100,200,500,1000,1500nmol/L) treatment after24hours ranged from3.48%to29.73%respectively. The inhibition rate of cellular proliferation with RTX treatment was significantly different compared with negative control group(P<0.01). The inhibition of cellular proliferation with RTX treatment at concentrations of (50,100,200,500,1000,1500nmol/L) after48and72hours ranged from8.49%to50.23%,13.54%to72.76%respectively in a time-dependent manner.
2. In lower concentrations (50,100nmol/L) treatment of RTX for8hours, it shows a moderate apoptotic effect, while in higher concentrations1000nmol/L it showed more apoptotic cells observed with inverted phase contrast microscope.After24hours in higher concentrations(1000nmol/L), more typical apoptosis body was observed,while after48hours a large number of apoptotic cells was observed.
3.As shown by FCM with PI staining method, RTX treatment (500,1000nmol/L) of the RT4cells resulted in significant G0/G1-phase cell arrest ranging from42.37to48.79%, and each concentration of RTX group was significantly different compared with negative control group35.02%(P<0.05).
4. As shown by FCM with PI and Annexin V staining method, we found that RTX caused a dose-dependent increase in RT4cell apoptosis. It was observed that treatment of RT4cells with varying concentration of RTX (0,500,1000nmol/L) for48hours increased the number of early apoptotic cells from3.21%to5.42%, and total apoptotic cells from13.93%to25.29%in a dose-dependent manner, and significantly different compared with negative control group0.65%and2.99%respectively (P<0.05).
Conclusion:
1.Resiniferatoxin (RTX) can inhibit the proliferation of human bladder carcinoma RT4cells effectively in a dose and time-dependent manner in vitro.
2. RTX can arrest the RT4cells in G0/G1phase at a dose-dependent manner.
3. RTX induces human bladder carcinoma RT4cells apoptosis at a dose-dependent manner.
Object:to investigate the mechnism of the inhibitory effect of resiniferatoxin on human bladder carcinoma RT4cell.
Methods:
1. After treated by RTX (0,500,1000nmol/L) for different time (Oh,8h,24h) the concentration of Ca2+in RT4cells is determined with fluoreacent labeling method.
2.After treated by RTX (1000nmol/L) for different time (Oh,8h,24h,48h),detect three proteins of apoptosis related proteins caspase-8, caspase-3and evaluate cytochrome c release with the approsch of western-blot analysis.
3.After treated by RTX (1000nmol/L) for different time (Oh,8h,24h,48h),detect three cell cycle regulatory protein p53, p21, CDK2with the approsch of western-blot analysis.
Results:
1. After a48hour treatment with RTX, the expressions of P53and P21were up-regulated in contrary to the expression of CDK2.
3.RTX induced caspase-8activation at8h that increased at24-48h. Caspase-3activation occurred at12h and persisted until24h after treatment.8hours after treatment, a band of12kDa corresponding to cytochrome c was observed and declined at later time points
Conclusion:
1. RTX can arrest the RT4cells in G0/G1phase by modulating the expression of P53, P21and CDK2.
2.Treatment of cells with RTX induced apoptosis accompanied by activation of caspase-8,caspase-3.and increased mitochondrial cytochrome c release.
引文
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