【作者】 黎勤；

【导师】 王行环；

【作者基本信息】 南方医科大学 ， 泌尿外科， 2010， 博士

【摘要】 膀胱癌是泌尿系统常见恶性病变,居男性恶性肿瘤发病率第四位,死亡率第八位。尽管大部分新发患者可以通过经尿道膀胱肿瘤电切(TUR-Bt)治疗,但术后肿瘤复发率高,因此,如何降低患者术后肿瘤复发率是目前膀胱癌相关研究的焦点。目前研究已显示术后膀胱灌注化疗和免疫治疗可以降低患者术后复发率,但现有药物对术后肿瘤复发率缓解作用有限,且部分副作用难以耐受,需要进一步寻找安全高效的药物来降低膀胱癌患者TUR-Bt术后复发的难题。薄荷醇是从唇形科植物薄荷中提取的薄荷挥发油中主要成分,是一种已被应用了几千年的植物药,并被美国食品药品管理局(FDA)视为安全无毒药物,在医药食品等多领域广泛应用。研究表明薄荷醇对前列腺癌、黑色素瘤、白血病、胃癌等癌细胞生长有抑制作用,但同时也有试验提示薄荷醇可以促进神经内分泌瘤细胞、神经胶质母细胞瘤细胞的分泌功能及迁徙能力,促进肿瘤的生长。而就其机制而言,研究已显示薄荷醇不但能通过其特异性受体一瞬时受体电位通道M8(TRPM8,或称为CMR1或Trpp8)抑制肿瘤生长,尚可通过非TRPM8通道抑制肿瘤细胞的生长。因此,薄荷醇与恶性肿瘤之间的研究才拉开序幕。本课题拟分三部分来探讨薄荷醇对膀胱癌增殖的影响及机制,以期为膀胱癌的临床治疗和薄荷醇的新药开发奠定理论基础。本课题的创新点：1)首次观察了人膀胱癌细胞系T24细胞中TRPM8通道的表达和功能情况；2)首次观察了薄荷醇对T24细胞增殖的影响；3)深入探讨了薄荷醇调节肿瘤细胞生长的可能机制。第一章人膀胱癌T24细胞中TRPM8的表达及活性的研究目的：探讨人膀胱癌T24细胞中是否存在TRPM8通道的表达及其亚细胞结构分布和功能方法：体外常规培养人膀胱癌T24细胞、HEK293细胞及HEK293-TRPM8细胞,使用Trizol法和RIPA法分别提取细胞总RNA和总蛋白,进一步使用聚合酶链式反应(PCR)和western蛋白印迹试验(western blotting)分别检测TRPM8基因的mRNA和蛋白表达情况。并使用免疫荧光杂交检测在共聚焦显微镜下观察人膀胱癌T24细胞TRPM8蛋白的表达及分布。利用钙成像技术检测T24细胞中TRPM8通道在低温刺激下对钙离子(Ca2+)的调控功能。结果：1)人膀胱癌细胞系T24细胞表达TRPM8 mRNA及蛋白PCR产物通过凝胶电泳分离后,T24细胞和HEK-293-TRPM8细胞(阳性对照),在200bp和300-400bp之间可以清晰看到两条亮带,而HEK-293细胞(阴性对照),与试验设计PCR产物吻合,表明T24细胞表达TRPM8 mRNA。进一步western-blotting检测显示T24细胞和HEK-293-TRPM8细胞在35KD附近及130KD附近出现两条特异性条带,与TRPM8和GAPDH蛋白分子量基本吻合,表明T24细胞表达TRPM8蛋白。试验初步表明人膀胱癌细胞系T24细胞表达TRPM8 mRNA及蛋白。2)TRPM8在T24细胞的细胞核外表达为进一步证实上述结果,试验中应用免疫荧光杂交反应观察了TRPM8蛋白在T24细胞的表达和分布。结果显示,Cy3红色荧光在T24细胞的细胞核外充分结合表达,表明细胞核外存在与TRPM8蛋白结合的特异性anti-TRPM8抗体,从而进一步证实T24细胞存在TRPM8蛋白表达,且可能分布于细胞核外,细胞膜和／或胞浆细胞器。3)人膀胱癌T24细胞细胞表达具有生物活性的TRPM8通道试验结果显示,在37℃状态下平衡20s后,当温度调节至20℃时,T24细胞内Fura3-AM荧光增强,表明细胞内Ca2+浓度([Ca2+]i)升高,当温度降至7℃时,荧光进一步增强,表明[Ca2+]i进一步升高。虽然冷温刺激下,含Ca2+和无Ca2+的HBSS溶液均可导致[Ca2+]i升高,但无Ca2+状态下,[Ca2+]i升高低于含Ca2+状态,尤以7℃时明显。这表明TRPM8通道在T24细胞中具有生物活性,可以介导外界冷刺激,导致细胞内Ca2+增高；另外,该试验也表明TRPM8在T24细胞的胞膜和胞浆均有分布。结论：人膀胱癌细胞系T24细胞表达具有活性功能的TRPM8通道,可以介导Ca2+通透,且TRPM8蛋白在T24细胞的细胞核外细胞器表达,分布于胞膜和胞浆。第二章薄荷醇抑制人膀胱癌细胞株T24细胞增殖的研究目的：探讨TRPM8通道及薄荷醇对人膀胱癌T24细胞增殖的影响方法：利用基因调控技术构建pcDNA3-TRPM8表达型质粒和siRNA-TRPM8干扰单体,调控T24细胞中TRPM8的表达,并使用CCK-8检测观察,在TRPM8蛋白表达变化时,T24细胞的增殖情况。同时使用TRPM8通道的激活剂-薄荷醇干预,观察T24细胞的增殖情况。进一步使用薄荷醇和siRNA-TRPM8单体共同干预T24细胞,CCK-8检测观察T24细胞的增殖情况,以了解TRPM8通道的增殖活性。统计学数据均以x±s表示,统计学采用单因素方差分析(one-way ANOVA),且以p<0.05具有统计学意义。结果：1)TRPM8表达变化不影响T24细胞的增殖为观察TRPM8表达对人膀胱癌细胞系T24细胞的增殖的影响,试验中使用CCK-8试剂检测了TRPM8表达变化时细胞的增殖情况,结果显示：对照组、siRNA-1、siRNA-2、siRNA-3、pcDNA3和pcDNA3-TRPM8各干预组(n=9)的光密度(OD值)依次为0.580±0.041、0.535±0.043、0.545±0.079、0.526±0.06、0.532±0.07和0.538±0.04；统计学方差分析显示：总体样本F值=1.037,p值=0.407,样本间无差异。表明TRPM8表达的变化并不影响T24细胞的生长。2)薄荷醇呈剂量依赖性抑制人膀胱癌细胞系T24细胞生长为进一步了解TRPM8通道在T24细胞中的作用,试验中使用TRPM8通道的激动剂-薄荷醇(menthol)对细胞进行干预并观察其生长状况。结果显示,OuM、10uM、100uM、500uM、1000uM和2000uM menthol干预组(n=9)的OD值依次为0.820±0.064、0.701±0.072、0.611±0.059、0.548±0.072、0.454±0.054和0.355±0.040；统计学方差分析显示：总体样本F值=67.340,p值=0.000,存在样本差异；组间比较显示,均p<0.05,存在统计学差异。表明薄荷醇呈剂量依赖性抑制T24细胞的生长。3)薄荷醇依赖TRPM8通道抑制人膀胱癌细胞系T24细胞的生长试验中进一步使用siRNA预先沉默T24细胞中TRPM8表达,再观察薄荷醇对T24细胞的抑制作用。结果显示对照组(menthol=OuM)、siRNA+menthol(1000uM)组及menthol(1000uM)组的OD值依次为0.782±0.042、0.738±0.043、0.452±0.065；统计学方差分析显示：整体样本F值=111.145,p值=0.000,存在样本间差异。组间对比发现,与对照组相比,siRNA+menthol组的p=0.075,而menthol组p=0.000；而与siRNA+menthol比较时,仅menthol组p=0.000,有显著性差异。本试验提示薄荷醇需依赖T24细胞中TRPM8通道的活性进一步抑制T24细胞的生长。结论：对于人膀胱癌T24细胞,TRPM8蛋白的表达变化不影响其生长增殖,但TRPM8通道的激活剂-薄荷醇,可以呈剂量依赖性抑制T24细胞的生长,但该种效应需要TRPM8通道参与。第三章薄荷醇抑制人膀胱癌T24细胞生长的机制的研究目的：探讨薄荷醇抑制人膀胱癌T24细胞生长的机制方法：利用流式细胞学细胞筛选法,分别使用PI单染和Annexin V-FITC/PI双染观察不同浓度薄荷醇干预下,T24细胞的各细胞周期细胞比例及凋亡和死亡细胞比例。进一步使用钙成像技术,检测不同浓度薄荷醇对T24细胞内[Ca2+]i的影响。并使用JC-1荧光探针法和DCFH-DA荧光探针法在共聚焦显微镜下分别观察不同浓度薄荷醇对T24细胞中线粒体膜电位和细胞内活性氧(ROS)的影响。再进一步使用N-乙酰半胱氨酸(NAC)和薄荷醇共同干预,CCK-8检测观察其对T24细胞增殖的影响。最后使用western蛋白印迹法检测ROS生长相关通路信号蛋白的表达变化。统计学数据均以x±s表示,统计学采用单因素方差分析(one-way ANOVA),且以p<0.05具有统计学意义。结果：1)薄荷醇不能诱导人膀胱癌T24细胞细胞周期阻滞流式细胞学细胞周期筛选结果显示0、10、100、500、1000、2000uM各浓度作用下,S期细胞比例分别为：39.463±3.042%、38.867±3.056%、37.383±3.049%、37.643±2.338%、37.81±2.511%和37.617±2.447%；方差分析提示：整体样本F=0.274,p=0.918,样本间没有统计学差异。G0／G1期分别为47.733±3.477%、47.737±5.877%、46.667±1.475%、48.267±5.524%、48.977±1.66%及47.96±3.144%；同样方差分析提示：整体样本F值为0.112,p值为0.987,样本间无差异。G2／M期为：12.803±1.819%、13.397±2.913%、15.95±4.518%、14.09±3.683%、13.213±2.368%和14.423±3.888%；方差分析提示：整体样本F=0.347,p=0.875,表明样本间没有统计学差异。试验结果提示薄荷醇不能诱导T24细胞的细胞周期阻滞。2)薄荷醇诱导人膀胱癌T24细胞死亡Annexin V-FITC/PI双染结果显示,薄荷醇各干预组细胞凋亡率依次为OuM(3.663±0.479%)、10uM(3.353±1.577%)、100uM(3.273±0.355%)、500uM(3.81±0.655%)、1000uM(3.4±0.9%)、2000uM(3.54±0.694%);统计学分析显示,整体样本F值为0.163,p值为0.971,无样本差异。而死亡细胞明显增加,相对于对照组(薄荷醇=0uM,细胞死亡率0.99±0.24%),10、100、500、1000、2000uM各浓度作用下,细胞死亡率依次为13.973±319%、26.2±3.573%、35.11±2.983%、48.463±4.149%及70.467±5.118%,呈明显的剂量依赖性；统计学分析显示,整体样本F=145.566,p=0.000,样本见存在显著性差异；组间比较显示,各组间均p<0.05,有显著性差异。结果表明薄荷醇可以诱导T24细胞死亡,而非凋亡。3)薄荷醇诱导人膀胱癌细胞系T24细胞[Ca2+]i增加钙成像技术检测显示,T24细胞在负载Fluo-3AM后,相比对照组(薄荷醇=OuM),100uM、1000uM、2000uM薄荷醇均可诱发T24细胞瞬间荧光增强,由于钙成像技术中,荧光强度与[Ca2+]i威正比,因此本实验提示薄荷醇可以诱导T24细胞[Ca2+]i增加,并且随薄荷醇浓度增加而增高,显示剂量依赖趋势。4)薄荷醇诱导T24细胞线粒体膜的去极化JC-1荧光染探针检测显示,当薄荷醇浓度达到100uM时,其明显降低T24细胞所激发的红／绿色荧光比值(红／绿色荧光比值与线粒体膜电位呈正比,本试验中以两者比值代表线粒体膜电位),即降低线粒体膜电位。各组红／绿色荧光比值依次为,OuM(0.886±0.119),10uM(0.801±0.027).100uM(0.729±0.049)、500uM(0.588±0.025)、1000uM(0.512±0.026)、2000uM(0.307±0.0309);统计学分析显示,整体样本F值为122.866,p值为0.000,表明存在样本间差异；组间比较显示,除对照组与10uM细胞组之间比较时p=0.508,无统计学差异,而其他各组间均p<0.05,有统计学差异。表明当薄荷醇达到100uM时,可呈剂量依赖性降低T24细胞线粒体膜电位。5)薄荷醇增加T24细胞内ROS产生DCFH-DA荧光探针检测显示,各组T24细胞红色荧光值依次为0uM(344.222±32.081)、10uM(447.111±55.784)、100uM(514.444±72.224),500uM (618.889±95.995)、1000uM(757.667±74.746)、2000uM(866.556±76.069);方差分析显示,整体样本F值为69.226,p值为0.000,存在样本间差异；组间比较显示,各组均p<0.05，有统计学差异。表明薄荷醇呈剂量依赖性诱导T24细胞内ROS的产生。6)NAC(ROS清除剂)可以逆转薄荷醇对T24细胞增殖的影响NAC与薄荷醇共同干预下,CCK-8试剂检测显示,各组细胞OD值分别为：对照组0.705±0.06,NAC组0.726±0.079,menthol组0.418±0.102,menthol+NAC组为0.693±0.559。统计学分析显示,整体样本F值为32.792,p值为0.000,存在样本间差异；组间比较显示,与对照组相比,NAC组p=0.982,menthol组p=0.000,且menthol+NAC组p=0.998；而与NAC组比较时,menthol+NAC组p=0.872,而menthol组p=0.000；且与]menthol+NAC组比较时,仅menthol组p=0.000。表明仅1000uM薄荷醇可以诱导T24细胞生长抑制,但NAC可以逆转其生长抑制作用。7)薄荷醇通过ROS/Ras/ERK1/2调控通路诱导T24细胞生长抑制为进一步探讨薄荷醇诱导T24细胞死亡的ROS下游可能通路,试验中进一步检测了ROS生长相关信号通路的经典通路蛋白。结果显示10mMNAC可以阻断1000uM薄荷醇诱导的T24细胞内ROS产生。且蛋白印迹检测进一步提示薄荷醇可以降低Ras蛋白表达,并全面抑制Raf-MEK1/2-ERK1/2通路蛋白的磷酸化状态,但NAC可以逆转这种变化；但薄荷醇对于T24细胞中的p38和JNK蛋白,及它们的磷酸化蛋白没有影响。结果表明,ROS/Ras/ERK1/2通路可能参与了薄荷醇诱导T24细胞死亡的过程。结论：薄荷醇通过增加人膀胱癌T24细胞内[Ca2+]i,导致线粒体去极化或非线粒体通路激活,而诱发细胞内ROS的产生,并进一步抑制Raf-MEK1/2-ERK1/2信号通路,最终导致T24细胞死亡而出现生长抑制。更多还原

【Abstract】 Bladder cancer is the fourth most common cancer in males and has the eighth highest cancer-related mortality rate in adult men. Whereas most of new cases can be treated by transurethral resection (TUR-Bt), the tumor recurrence rate following transurethral resection is high. So the central field in bladder cancer are focused on the issue, how to decrease the postoperative tumor recurrence rate. It shows that postoperative intravesical chemotherapy and immunotherapy can control the recurrence rate in patients with a high risk of recurrence. However, the present medicines just play a little role in the relief of trouble, and some of which has serious side-effects. Thereafter, it is a challenge for urologists and patients to find out new effective medicine but no side-effects to control the postoperative recurrence.Menthol is a naturally occurring cyclic terpene alcohol of Mentha hap localyx Briq.It has been used for medicinal purposes for more than 2000 years.Menthol is identified as generally recognized as safe (GRAS) by US Food and Drug Administration and can be obtained over the counter (OTC), and has been in a wide application. The present reports have showed menthol could inhibit the growth of human prostate cancer, but also human melanoma cells, human promyelocytic leukemia cancer cells, human colon cancer cells and gastric cancer cells. Whereas, few reports showed menthol also could enhance the secretion and the migration of human neuroendocrine tumor cells and human glioblastoma cells resulting in the growth. To the mechanism of bioactivity by menthol, the present investigation has shown that menthol can inhibit the growth of malignant tumor in TRPM8 (transient receptor potential cation channel subfamily M member 8,formerly called as CMR1 or Trpp8))-dependant way, also in a non-TRPM8-dependent manner. Thus, the complicated net between menthol and malignant cancer is just uncovered.Basing on the above mentioned, we designed the present study with three parts to observe the potential effect and mechanism of menthol on the proliferation of bladder cancer cells in vitro, which lay the theoretical foundation to treat bladder cancer and parmaceutical development of menthol.The innovation of this present study highlight three points:1)It is the first time to study the expression and function of TRPM8 channel in human bladder cancer cell line T24 cells;2) It is the newest report about the role of menthol on T24 cell; 3)It has a deeper investigation on the potential mechanism of menthol to regulate the growth of malignant cells.Chapter 1 The expression and function of TRPM8 channel in human bladder cancer T24 cells.Objective:To investigate whether TRPM8 channel is expressed in T24 cells, also its sub cellular location and its function.Methods:T24 cells、HEK293 cells and HEK293-TRPM8 cells were cultured in vitro, then total RNA and protein were respectively obtained in the way of Trizol and RIPA. After that, TRPM8 mRNA and protein were detected by PCR and western blotting respectively. Immnocytochemistry under confocal microscope was used to confer the expression and sub cellular location of TRPM8 in T24 cells. Moreover, the function of TRPM8 channel in T24 cells, being stimulated by cold, was observed by Ca2+ imaging. Results:1)T24 cells expressed functional TRPM8 at level of mRNA and protein.After the PCR product was separated by gel electrophoresis, two light bands were observed respectively at 200bp and 300-400bp in T24 cells and HEK-293-TRPM8 cells (positive control), which coincided with the design. It showed T24 cells had TRPM8 mRNA. In T24 cells and HEK-293-TRPM8 cells,two specific bands were recorded respectively around 35KD and 130KD,when detected by western-blotting, which coincide with the molecular weight of TRPM8 and GAPDH protein.Thus, TRPM8 mRNA and protein were expressed in human bladder cancer T24 cells.2) TRPM8 protein was located outside of the uncle in T24 cells.Immunocytochemistry showed the red fluorescence of Cy3 was distributed outside of uncle of T24 cells, which suggested specific anti-TRPM8,binding of TRPM8 protein, located outside of uncle of T24 cells. It indicated that TRPM8 protein was distributed at membrane and/or cytoplasmic organelles of T24 cells.3)TRPM8 channel in human bladder cancer T24 cells was functional.After balanced at 37℃for 20 seconds, the fluorescence intensity of Fura3-AM in T24 cells was enhanced when the temperature modulated to 20℃.Which indicated [Ca2+]I was increased. When the temperature down to 7℃, the fluorescence intensity further increased, which suggested T24 cells have a more further rise of [Ca2+]i. Whereas[Ca2+]i was increased in the solution of HBSS with Ca2+ or not when T24 cells were activated by cold (<28℃),the level of [Ca2+]i in the HBSS without Ca2+ was much lower than in the HBSS with Ca2+, especially at 7℃.Thus, the above indicated that TRPM8 channel in T24 cells was functional, and result in the increase of [Ca2+]i when activated by cold. Moreover, it conferred that TRPM8 protein was distributed both at membrane and cytoplasm of T24 cells. Conclusions:TRPM8 channel was functional in human bladder cancer T24 cells, what could mediate the permeability of Ca2+, and located both at membrane and cytoplasm.Chapter 2.Menthol inhibited the growth of human bladder cancer T24 cells.Objective:To investigate the effect of TRPM8 channel and menthol on the growth of human bladder cancer T24 cells.Methods:To construct the plasmid of pcDNA3-TRPM8 and siRNA-TRPM8 oligos by gene control to regulate the expression of TRPM8 in T24 cells.Then we observed the proliferation of T24 cells with CCK-8 assay when TRPM8 protein was altered. Other hand, the growth was investigated when T24 cells was treated with menthol (agonist to TRPM8 channel).Finally, we detected the proliferation with CCK-8 assay when T24 cells were intervened by menthol and siRNA-TRPM8. Statistics Data were expressed as x±s, and one-way ANOVA was used in statistical analysis, also p<0.05 was statistically significant.Results:1)The alteration of TRPM8 level had no effect on the proliferation of human bladder cancer T24 cells.With CCK-8 assay, we studied the proliferation of T24 cells when the expression of TRPM8 protein was regulated. It showed the OD value of the control、siRNA-1 group、siRNA-2 group、siRNA-3 group、pcDNA3 group and pcDNA3-TRPM8 group(n=9)was respectively 0.580±0.041、0.535±0.043、0.545±0.079.0.526±0.06.0.532±0.07和0.538±0.04. Statistical analysis showed total F value was 1.037, and p=0.407.There was no statistical difference in samples. It indicated the regulation of TRPM8 level could not affect the growth of T24 cells. 2) Menthol could inhibit the proliferation of human bladder cancer T24 cells in a dose way.To have a deep knowledge about the role of TRPM8 channel in T24 cells, menthol (the agonist to TRPM8 channel) was used to interfere the growth of T24 cells. The results indicated that the OD value was 0.820±0.064、0.701±0.072、0.611±0.059、0.548±0.072、0.454±0.054 and 0.355±0.040, respectively in the menthol group (OuM、10uM、100uM、500uM、1000uM and 2000uM, n=9).Statistical analysis suggested total F=67.340 and p=0.000, which mean there was significant difference among the samples. When compared each other, it showed p<0.05 among all groups, and there was significant difference.Thus, menthol could induce T24 cells growth arrest in a dose manner.3) Menthol could inhibit the growth of human bladder cancer T24 cells through TRPM8 channel.Pre-silencing TRPM8 expression of T24 cells with siRNA-TRPM8 oligo, the proliferation of T24 cells was assessed by CCK-8 assay when menthol was added. It showed the OD value was 0.782±0.042、0.738±0.043、0.452±0.065 respectively in the control (menthol=OuM)、siRNA+menthol(1000uM) group and menthol(1000uM) group. Statistical analysis suggested total F=111.145,p=0.000, which mean there was significant difference among the samples. When compared with the control, siRNA+menthol group was p=0.075, while menthol group wasp=0.000. It suggested there was significant difference between the control and the menthol group, but not between the control and the siRNA+menthol group. Moreover, when compared with the siRNA+menthol group, the menthol group was also p=0.000.It was significantly different. Thus, menthol induced T24 cells growth arrest in a TRPM8-dependent manner.Conclusions:In the section, we concluded that the alteration of TRPM8 protein level could not affect the growth of human bladder cancer T24 cells, however, menthol could induce T24 cells proliferation arrest in a TRPM8-dependent way.Chapter 3.The study of the potential mechanism that menthol inhibited human bladder cancer T24 cells growth.Objective:To investigate the potential mechanism that menthol induced human bladder cancer T24 cells growth arrest.Methods:Firstly, cell cycle and apoptosis, death were respectively detected with PI staining and Annexin V-FITC/PI staining by flow cytometry, when T24 cells were treated with different concentration of menthol.Then [Ca2+]i was detected with Ca2+ imaging under confocal microscope when activated by different dose of menthol. Next, the mitochondrial membrane potential and reactive osygen species (ROS) was respectively investigated with JC-1 fluorescent probe and DCFH-DA fluorescent probe by confocal microscope. In the end, co-interference with menthol and NAC (inhibitor of ROS production), the cell viability was detected by CCK-8 assay, and ROS production was tested with DCFH-DA fluorescent probe, also some cell growth-related signaling protein was detected with western-blot. Statistics Data were expressed as x±s, and one-way ANOVA was used in statistical analysis, also p <0.05 was statistically significant.Results:1)Menthol could not induce human bladder cancer T24 cells cell cycle arrest.The results of cell cycle examination with flow cytometry showed the percentage of S phase cells was 39.463±3.042%、38.867±3.056%、37.383±3.049%、37.643±2.338%、37.81±2.511% and 37.617±2.447%, respectively in the concentration of 0、10、100、500、1000、2000uM. Statistical analysis suggested total F=0.274,p=0.918,which mean there was no significant difference among the samples. It also showed the percentage of G0/G1 phase cells was 47.733±3.477%、47.737±5.877%、46.667±1.475%、48.267±5.524%、48.977±1.66% and 47.96±3.144%, respectively in the concentration of 0.10、100±500±1000±2000uM menthol. Statistical analysis suggested total F=0.112,p=0.987, which mean there was no significant difference among the samples.Moreover, the percentage of G2/M phase cells was respectively 12.803±1.819%、13.397±2.913%、15.95±4.518%、14.09±3.683%±13.213±2.368% and 14.423±3.888% in different dose of menthol. Statistical analysis suggested total F=0.347, p=0.875.It was no difference among samples. It indicated menthol could not induce T24 cells cell cycle arrest.2) Menthol induced human bladder cancer T24 cells death.The apoptosis percentage, assessed by Annexin V-FITC/PI double staining with flow cytometry, was respectively OuM (3.663±0.479%)、10uM (3.353±1.577%)、100uM(3.273±0.355%)、500uM(3.81±0.655%)、1000uM(3.4±0.9%)and 2000uM (3.54±0.694%).Statistical analysis suggested total F=0.163,p=0.971,which mean there was no significant difference among the samples.However, the dead cell was increased. The death percentage of T24 cells was 0.99±0.24%、13.973±3.3199%、26.2±3.573%、35.11±2.983%、48.463±4.149% and 70.467±5.118%, respectively at above dose. Statistical analysis suggested total F=145.566 and p=0.000, which suggested there was significant difference among the samples. When compared each other, it showed p<0.05 among all groups, and there was significant difference.Thus, menthol could induce T24 cells death arrest in a dose manner, but not apoptosis.3)Menthol increased [Ca2+]i of human bladder cancer T24 cells.After loaded with Fura3-AM, the fluorescence intensity in T24 cells, compared with the control, was enhanced when 100uM、1000uM、2000uM menthol was added. For fluorescence intensity is directly proportional to [Ca2+]i with Ca2+ imaging, which indicated menthol could induce T24 cells a rise of [Ca2+]i,And it showed a trend in a dose-dependent manner.4) Menthol induced T24 cells mitochondrial membrane depolarization.The results suggested 100uM menthol significantly decreased the ratio of red/green fluorescence, representing the mitochondrial membrane potential, which indicated the decrease of mitochondrial membrane potential of T24 cells. The ratio of red/green fluorescence was respectively 0.886±0.119 (OuM)、0.801±0.027(10uM)、0.729±0.049(100uM)、0.588±0.025(500uM)、0.512±0.026(1000uM)、0.307±0.0309 (2000uM).Statistical analysis suggested total F=122.866 and p=0.000, which suggested there was significant difference among the samples.When compared each other, it showed p<0.05 among most of groups.While between the control and 10uM menthol group, it was p=0.508, and there was no significant difference. Thus, menthol could decrease the mitochondrial membrane potential of T24 cells in a dose-dependent way when the concentration of menthol was up to 100uM.5) Menthol increased the ROS production in T24 cells.The assay of DCFH-DA fluorescent probe showed the fluorescence intensity in T24 cells was respectively 344.222±32.081 (OuM)、447.111±55.784(10uM)、514.444±72.224(100uM)、618.889±95.995(500uM)、757.667±74.746(1000uM)and 866.556±76.069 (2000uM).Statistical analysis suggested total F=69.226, and p=0.000, which suggested there was significant difference among the samples. When compared each other, it showed p<0.05 among all groups. It was significantly different. Thus, menthol could induce T24 cells ROS production in a concentration way.6) NAC (inhibitor of ROS production) could deteriorate the role of menthol on the growth of human bladder cancer T24 cells.When co-interfered with NAC and menthol, the OD value, detected by CCK-8 assay, was respectively 0.705±0.06 (the control, no interference)、0.726±0.079 (10mM NAC group)、0.418±0.102(1mM menthol group)、0.693±0.559 (1mM menthol+lOmM NAC group).Statistical analysis suggested total F=32.792 and p=0.000, which suggested there was significant difference among the samples. When compared with the control, NAC group was p=0.982, and menthol+NAC group was p=0.998, while menthol group wasp=0.000. However, when compared with NAC group, menthol+NAC group was p=0.872, and menthol group was p=0.000. Moreover,when compared with menthol+NAC group,menthol group was also p=0.000. There was significant difference. Thus, NAC could deteriorate growth arrest of T24 cells induced by menthol.7) Menthol inhibited the growth of human bladder cancer T24 cells in the pathway of ROS/Ras/ERK1/2When detected with western blotting, 1000uM menthol could significantly decrease the band gray of Ras protein, and fully inhibited Raf-MEK1/2-ERK1/2 pathway protein phosphorylation state, which could be deteriorated by 10mM NAC. However, menthol had no effect on p38 and JNK, also their phosphorylation state. It proved ROS/Ras/ERK1/2 pathway played a role in the growth arrest induced by menthol.Conclusions:Menthol increased[Ca+]i of human bladder cancer T24 cells, resulting in T24 cells mitochondrial membrane depolarization or other pathway activated, to increase ROS production, which activated Raf-MEK1/2-ERK1/2 signaling way, eventually induced T24 cells death resulting in the proliferation inhibited.更多还原