薄荷醇通过TRPM8通道诱导人膀胱癌T24细胞株死亡的试验研究
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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。
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/ERK1/2
When 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.
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。
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/ERK1/2
When 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.
引文
[1]Jemal A, Thun MJ, Ries LA, et al.Annual report to the nation on the status of cancer,1975-2005,featuring trends in lung cancer, tobacco use, and tobacco control.J Natl Cancer Inst.2008 Dec 3;100(23):1672-94.
[2]Voutsinas GE, Stravopodis DJ. Molecular targeting and gene delivery in bladder cancer therapy. J Buon.2009 Sep;14 Suppl 1:S69-78.
[3]Barocas DA, Clark PE. Bladder cancer. Current opinion in oncology.2008 May;20(3):307-14.
[4]Bochner BH. Gene therapy in bladder cancer. Current opinion in urology.2008 Sep;18(5):519-23.
[5]Cassileth B.Complementary therapies, herbs, and other OTC agents. Oncology (Williston Park, NY.2009 Sep;23(10):904.
[6]Bhatia SP, McGinty D, Letizia CS, Api AM. Fragrance material review on menthol. Food Chem Toxicol.2008 Nov;46 Suppl 11:S209-14.
[7]Vriens J, Nilius B,Vennekens R. Herbal Compounds and Toxins Modulating TRP Channels. Current neuropharmacology.2008 Mar;6(1):79-96.
[8]Fleetwood-Walker SM, Proudfoot CW, Garry EM, et al. Cold comfort pharm. Trends in pharmacological sciences.2007 Dec;28(12):621-8.
[9]Patel T, Ishiuji Y, Yosipovitch G. Menthol:a refreshing look at this ancient compound. Journal of the American Academy of Dermatology.2007 Nov;57 (5):873-8.
[1.0]Werley MS,Coggins CR, Lee PN. Possible effects on smokers of cigarette mentholation:a review of the evidence relating to key research questions. Regul Toxicol Pharmacol.2007 Mar;47(2):189-203.
[11]Heck JD.A review and assessment of menthol employed as a cigarette flavoring ingredient. Food Chem Toxicol. Jan;48 Suppl 2:S1-38.
[12]McKemy DD, Neuhausser WM, Julius D.Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature.2002 Mar 7;416(6876):52-8.
[13]Peier AM,Moqrich A, Hergarden AC, et al. A TRP channel that senses cold stimuli and menthol.Cell.2002 Mar 8;108(5):705-15.
[14]Thebault S,Flourakis M, Vanoverberghe K, et al.Differential role of transient receptor potential channels in Ca2+ entry and proliferation of prostate cancer epithelial cells. Cancer Res.2006 Feb 15;66(4):2038-47.
[15]Zhang L, Barritt GJ. Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival of prostate cancer cells. Cancer Res.2004 Nov 15;64(22):8365-73.
[16]Yamamura H, Ugawa S,Ueda T, et al. TRPM8 activation suppresses cellular viability in human melanoma. Am J Physiol Cell Physiol.2008 Aug;295 (2):C296-301.
[17]Bernhardt G, Biersack B, Bollwein S, et al.Terpene conjugates of diaminedichloridoplatinum(II) complexes:antiproliferative effects in HL-60 leukemia,518A2 melanoma, and HT-29 colon cancer cells.Chem Biodivers. 2008 Aug;5(8):1645-59.
[18]Lin JP, Lu HF, Lee JH, et al. (-)-Menthol inhibits DNA topoisomerases Ⅰ, Ⅱ alpha and beta and promotes NF-kappaB expression in human gastric cancer SNU-5 cells. Anticancer Res.2005 May-Jun;25(3B):2069-74.
[19]Mergler S,Strowski MZ, Kaiser S,et al. Transient receptor potential channel TRPM8 agonists stimulate calcium influx and neurotensin secretion in neuroendocrine tumor cells. Neuroendocrinology.2007;85(2):81-92.
[20]Wondergem R, Ecay TW, Mahieu F, et al. HGF/SF and menthol increase human glioblastoma cell calcium and migration. Biochem Biophys Res Commun.2008 Jul 18;372(1):210-5.
[21]Kim SH, Nam JH, Park EJ, et al. Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells.Biochim Biophys Acta.2009 Jan;1792 (1):33-8.
[22]Patapoutian A, Peier AM, Story GM, Viswanath V.ThermoTRP channels and beyond:mechanisms of temperature sensation. Nat Rev Neurosci.2003 Jul;4(7):529-39.
[23]Ma S,G G, Ak VE, et al. Menthol derivative WS-12 selectively activates transient receptor potential melastatin-8 (TRPM8) ion channels.Pak J Pharm Sci. 2008 Oct;21(4):370-8.
[24]Macpherson LJ, Hwang SW, Miyamoto T, et al.More than cool:promiscuous relationships of menthol and other sensory compounds.Molecular and cellular neurosciences.2006 Aug;32(4):335-43.
[25]Rohacs T, Lopes CM, Michailidis I, Logothetis DE. PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain. Nature neuroscience.2005 May;8(5):626-34.
[26]Roderick HL, Cook SJ. Ca2+ signalling checkpoints in cancer:remodelling Ca2+for cancer cell proliferation and survival. Nat Rev Cancer.2008 May; 8(5):361-75.
[27]Whitfield JF. Calcium signals and cancer. Crit Rev Oncog.1992;3(1-2):55-90.
[28]Cook SJ, Lockyer PJ. Recent advances in Ca(2+)-dependent Ras regulation and cell proliferation. Cell calcium.2006 Feb;39(2):101-12.
[29]Berridge MJ, Bootman MD, Roderick HL. Calcium signalling:dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol.2003 Jul;4(7):517-29.
[30]Rizzuto R, Pinton P, Ferrari D, et al.Calcium and apoptosis:facts and hypotheses. Oncogene.2003 Nov 24;22(53):8619-27.
[31]Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer:targeting Ca2+ transport. Nat Rev Cancer.2007 Jul;7(7):519-30.
[32]Takuwa N, Zhou W, Kumada M, Takuwa Y.Ca(2+)-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts. The Journal of biological chemistry.1993 Jan 5;268(1):138-45.
[33]Szabadkai G,Bianchi K, Varnai P, et al.Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. The Journal of cell biology.2006 Dec 18;175(6):901-11.
[1]Patapoutian A, Peier AM, Story GM, Viswanath V.ThermoTRP channels and beyond:mechanisms of temperature sensation. Nat Rev Neurosci.2003 Jul;4(7):529-39.
[2]Tsavaler L, Shapero MH, Morkowski S,Laus R. Trp-p8,a novel prostate-specific gene,is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer research.2001 May 1;61(9):3760-9.
[3]Yang XR, Lin MJ, McIntosh LS, Sham JS.Functional expression of transient receptor potential melastatin-and vanilloid-related channels in pulmonary arterial and aortic smooth muscle. American journal of physiology.2006 Jun;290(6):L1267-76.
[4]Johnson CD, Melanaphy D, Purse A, et al.Transient receptor potential melastatin 8 channel involvement in the regulation of vascular tone. Am J Physiol Heart Circ Physiol.2009 Jun;296(6):H1868-77.
[5]Lashinger ES,Steiginga MS,Hieble JP, et al. AMTB, a TRPM8 channel blocker: evidence in rats for activity in overactive bladder and painful bladder syndrome. Am J Physiol Renal Physiol.2008 Sep;295(3):F803-10.
[6]Fuessel S,Sickert D, Meye A, et al. Multiple tumor marker analyses (PSA, hK2, PSCA, trp-p8) in primary prostate cancers using quantitative RT-PCR. International journal of oncology.2003 Jul;23(1):221-8.
[7]Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer:targeting Ca2+ transport. Nature reviews.2007 Jul;7(7):519-30.
[8]Du S,Araki I, Kobayashi H, et al.Differential expression profile of cold (TRPA1)and cool (TRPM8) receptors in human urogenital organs. Urology. 2008 Aug;72(2):450-5.
[9]Stein RJ, Santos S, Nagatomi J, et al. Cool (TRPM8) and hot (TRPV1)receptors in the bladder and male genital tract. The Journal of urology.2004 Sep;172(3): 1175-8.
[10]Yang ZH, Wang XH, Wang HP, Hu LQ. Effects of TRPM8 on the proliferation and motility of prostate cancer PC-3 cells. Asian J Androl.2009 Mar; 11(2): 157-65.
[11]Zhang L, Barritt GJ. Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival of prostate cancer cells.Cancer Res.2004 Nov 15;64(22):8365-73.
[12]Cosens DJ, Manning A. Abnormal electroretinogram from a Drosophila mutant. Nature.1969 Oct 18;224(5216):285-7.
[13]Montell C, Rubin GM.Molecular characterization of the Drosophila trp locus:a putative integral membrane protein required for phototransduction. Neuron. 1989 Apr;2(4):1313-23.
[14]Montell C, Birnbaumer L, Flockerzi V.The TRP channels, a remarkably functional family.Cell.2002 Mar 8;108(5):595-8.
[15]Montell C, Birnbaumer L, Wissenbach U, et al. Modulation of the contractions of the smooth muscles of the rat vas deferens by TRPM8 channel activator menthol. Fiziol Zh.2009;55(6):30-40.
[16]Bodding M. TRP proteins and cancer. Cell Signal.2007 Mar; 19(3):617-24.
[17]Voets T, Droogmans G, Wissenbach U, et al. The principle of temperature-dependent gating in cold-and heat-sensitive TRP channels. Nature.2004 Aug 12;430(7001):748-54.
[18]Rohacs T, Lopes CM, Michailidis I, Logothetis DE. PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain. Nat Neurosci.2005 May;8(5):626-34.
[19]Bidaux G, Flourakis M, Thebault S,et al.Prostate cell differentiation status determines transient receptor potential melastatin member 8 channel subcellular localization and function. The Journal of clinical investigation.2007 Jun;117(6): 1647-57.
[20]Sabnis AS,Shadid M, Yost GS,Reilly CA. Human lung epithelial cells express a functional cold-sensing TRPM8 variant. Am J Respir Cell Mol Biol.2008 Oct;39(4):466-74.
[21]Birder LA. TRPs in bladder diseases. Biochim Biophys Acta.2007 Aug; 1772(8):879-84.
[1]Tsavaler L, Shapero MH, Morkowski S,Laus R. Trp-p8,a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer research.2001 May 1;61(9):3760-9.
[2]Fuessel S,Sickert D, Meye A, et al.Multiple tumor marker analyses (PSA, hK2, PSCA, trp-p8) in primary prostate cancers using quantitative RT-PCR. International journal of oncology.2003 Jul;23(1):221-8.
[3]Yamamura H, Ugawa S,Ueda T, et al.TRPM8 activation suppresses cellular viability in human melanoma. Am J Physiol Cell Physiol.2008 Aug;295 (2):C296-301.
[4]Bernhardt G, Biersack B, Bollwein S, et al.Terpene conjugates of diaminedichloridoplatinum(II) complexes:antiproliferative effects in HL-60 leukemia,518A2 melanoma, and HT-29 colon cancer cells. Chem Biodivers. 2008 Aug;5(8):1645-59.
[5]Lin JP, Lu HF, Lee JH, et al. (-)-Menthol inhibits DNA topoisomerases I, II alpha and beta and promotes NF-kappaB expression in human gastric cancer SNU-5 cells.Anticancer Res.2005 May-Jun;25(3B):2069-74.
[6]Bhatia SP, McGinty D, Letizia CS,Api AM.Fragrance material review on menthol.Food Chem Toxicol.2008 Nov;46 Suppl 11:S209-14.
[7]Patel T, Ishiuji Y, Yosipovitch G. Menthol:a refreshing look at this ancient compound. J Am Acad Dermatol.2007 Nov;57(5):873-8.
[8]Zhang L, Barritt GJ. Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival of prostate cancer cells. Cancer research.2004 Nov 15;64(22):8365-73.
[9]Bidaux G, Roudbaraki M, Merle C, et al. Evidence for specific TRPM8 expression in human prostate secretory epithelial cells:functional androgen receptor requirement. Endocrine-related cancer.2005 Jun;12(2):367-82.
[10]Zhang L, Barritt GJ. TRPM8 in prostate cancer cells:a potential diagnostic and prognostic marker with a secretory function? Endocrine-related cancer.2006 Mar;13(1):27-38.
[11]Prevarskaya N, Skryma R, Bidaux G, et al.Ion channels in death and differentiation of prostate cancer cells. Cell death and differentiation.2007 Jul;14(7):1295-304.
[12]Henshall SM, Afar DE, Hiller J, et al. Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse. Cancer research.2003 Jul 15;63(14):4196-203.
[13]Yang ZH, Wang XH, Wang HP, Hu LQ. Effects of TRPM8 on the proliferation and motility of prostate cancer PC-3 cells. Asian J Androl.2009 Mar;11 (2):157-65.
[14]Thebault S,Flourakis M, Vanoverberghe K, et al.Differential role of transient receptor potential channels in Ca2+ entry and proliferation of prostate cancer epithelial cells. Cancer Res.2006 Feb 15;66(4):2038-47.
[15]Macpherson LJ, Hwang SW, Miyamoto T, et al. More than cool:promiscuous relationships of menthol and other sensory compounds. Molecular and cellular neurosciences.2006 Aug;32(4):335-43.
[16]Ma S,G G, Ak VE, et al. Menthol derivative WS-12 selectively activates transient receptor potential melastatin-8 (TRPM8) ion channels. Pak J Pharm Sci. 2008 Oct;21(4):370-8.
[17]Kim SH, Nam JH, Park EJ, et al.Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells. Biochim Biophys Acta.2009 Jan;1792(1):33-8.
[1]Yamamura H, Ugawa S,Ueda T, et al.TRPM8 activation suppresses cellular viability in human melanoma. Am J Physiol Cell Physiol.2008 Aug;295 (2):C296-301.
[2]Bernhardt G, Biersack B, Bollwein S,et al. Terpene conjugates of diaminedichloridoplatinum(II) complexes:antiproliferative effects in HL-60 leukemia,518A2 melanoma, and HT-29 colon cancer cells. Chem Biodivers. 2008 Aug;5(8):1645-59.
[3]Whitfield JF. Calcium signals and cancer. Crit Rev Oncog.1992;3(1-2):55-90.
[4]Cook SJ, Lockyer PJ. Recent advances in Ca(2+)-dependent Ras regulation and cell proliferation. Cell calcium.2006 Feb;39(2):101-12.
[5]Takuwa N, Zhou W, Kumada M, Takuwa Y. Ca(2+)-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts. The Journal of biological chemistry.1993 Jan 5;268(1):138-45.
[6]Szabadkai G, Bianchi K, Varnai P, et al.Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+channels. The Journal of cell biology.2006 Dec 18;175(6):901-11.
[7]Wondergem R, Bartley JW. Menthol increases human glioblastoma intracellular Ca2+, BK channel activity and cell migration. Journal of biomedical science. 2009;16:90.
[8]Kim SH, Nam JH, Park EJ, et al.Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells. Biochim Biophys Acta.2009 Jan;1792(1):33-8.
[9]Fulda S,Gorman AM, Hori O, Samali A. Cellular stress responses:cell survival and cell death. International journal of cell biology.2010:214074.
[10]Platini F, Perez-Tomas R, Ambrosio S, Tessitore L. Understanding autophagy in cell death control. Current pharmaceutical design.16(1):101-13.
[11]Grimm S,Noteborn M. Anticancer genes:inducers of tumour-specific cell death signalling. Trends in molecular medicine. Feb;16(2):88-96.
[12]Lu HF, Hsueh SC, Yu FS,et al. The role of Ca2+in(-)-menthol-induced human promyelocytic leukemia HL-60 cell death. In Vivo.2006 Jan-Feb;20(1):69-75.
[13]Ma S,G G, Ak VE, et al.Menthol derivative WS-12 selectively activates transient receptor potential melastatin-8 (TRPM8) ion channels.Pak J Pharm Sci. 2008 Oct;21(4):370-8.
[14]Macpherson LJ, Hwang SW, Miyamoto T, et al.More than cool:promiscuous relationships of menthol and other sensory compounds. Molecular and cellular neurosciences.2006 Aug;32(4):335-43.
[15]Du S,Araki I, Kobayashi H, et al.Differential expression profile of cold (TRPA1)and cool (TRPM8) receptors in human urogenital organs. Urology. 2008 Aug;72(2):450-5.
[16]Capiod T, Shuba Y, Skryma R, Prevarskaya N. Calcium signalling and cancer cell growth. Sub-cellular biochemistry.2007;45:405-27.
[17]Monteith GR, McAndrew D,Faddy HM,Roberts-Thomson SJ. Calcium and cancer:targeting Ca2+ transport. Nat Rev Cancer.2007 Jul;7(7):519-30.
[18]Ott M, Gogvadze V, Orrenius S,Zhivotovsky B. Mitochondria, oxidative stress and cell death. Apoptosis.2007 May;12(5):913-22.
[19]Sattler UG, Mueller-Klieser W. The anti-oxidant capacity of tumour glycolysis. International journal of radiation biology.2009 Nov;85(11):963-71.
[20]Trachootham D, Alexandre J, Huang P.Targeting cancer cells by ROS-mediated mechanisms:a radical therapeutic approach? Nature reviews.2009 Jul;8 (7):579-91.
[21]Pan JS,Hong MZ, Ren JL. Reactive oxygen species:a double-edged sword in oncogenesis. World J Gastroenterol.2009 Apr 14;15(14):1702-7.
[1]Gupta GP, Massague J. Cancer metastasis:building a framework. Cell.2006 Nov 17;127(4):679-95.
[2]Roderick HL, Cook SJ. Ca2+ signalling checkpoints in cancer:remodelling Ca2+ for cancer cell proliferation and survival. Nature reviews.2008 May;8(5):361-75.
[3]Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer:targeting Ca2+ transport. Nature reviews.2007 Jul;7(7):519-30.
[4]Rizzuto R, Pinton P, Ferrari D, et al. Calcium and apoptosis:facts and hypotheses.Oncogene.2003 Nov 24;22(53):8619-27.
[5]Cook SJ, Lockyer PJ. Recent advances in Ca(2+)-dependent Ras regulation and cell proliferation. Cell calcium.2006 Feb;39(2):101-12.
[6]Nilius B,Owsianik G, Voets T, Peters JA. Transient receptor potential cation channels in disease. Physiological reviews.2007 Jan;87(1):165-217.
[7][7] Bidaux G, Flourakis M, Thebault S, et al.Prostate cell differentiation status determines transient receptor potential melastatin member 8 channel subcellular localization and function. The Journal of clinical investigation.2007 Jun;117(6):1647-57.
[8]Thebault S, Lemonnier L,Bidaux G, et al. Novel role of cold/menthol-sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store-operated channels in LNCaP human prostate cancer epithelial cells. The Journal of biological chemistry.2005 Nov 25;280(47):39423-35.
[9]Xu XZ, Moebius F, Gill DL, Montell C.Regulation of melastatin, a TRP-related protein, through interaction with a cytoplasmic isoform. Proceedings of the National Academy of Sciences of the United States of America.2001 Sep 11;98(19):10692-7.
[10]Wisnoskey BJ, Sinkins WG, Schilling WP. Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry. The Biochemical journal.2003 Jun 1;372(Pt 2):517-28.
[11]Giampa C, DeMarch Z, Patassini S,et al.Immunohistochemical localization of TRPC6 in the rat substantia nigra. Neurosci Lett.2007 Sep 13;424(3):170-4.
[12]Montell C, Birnbaumer L, Flockerzi V. The TRP channels, a remarkably functional family. Cell.2002 Mar 8;108(5):595-8.
[13]Benarroch EE. TRP channels:functions and involvement in neurologic disease. Neurology.2008 Feb 19;70(8):648-52.
[14]Clark K, Middelbeek J, van Leeuwen FN. Interplay between TRP channels and the cytoskeleton in health and disease. European journal of cell biology.2008 Sep;87(8-9):631-40.
[15]Jordt SE, Ehrlich BE. TRP channels in disease. Sub-cellular biochemistry. 2007;45:253-71.
[16]Cosens DJ, Manning A. Abnormal electroretinogram from a Drosophila mutant. Nature.1969 Oct 18;224(5216):285-7.
[17]Montell C, Rubin GM. Molecular characterization of the Drosophila trp locus:a putative integral membrane protein required for phototransduction. Neuron. 1989 Apr;2(4):1313-23.
[18]Hardie RC, Minke B.The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors. Neuron.1992 Apr;8(4):643-51.
[19]Petersen CC, Berridge MJ, Borgese MF, Bennett DL. Putative capacitative calcium entry channels:expression of Drosophila trp and evidence for the existence of vertebrate homologues.The Biochemical journal.1995 Oct 1;311 (Pt 1):41-4.
[20]Thebault S, Flourakis M, Vanoverberghe K, et al.Differential role of transient receptor potential channels in Ca2+ entry and proliferation of prostate cancer epithelial cells. Cancer research.2006 Feb 15;66(4):2038-47.
[21]Wissenbach U, Niemeyer BA, Fixemer T, et al.Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer. The Journal of biological chemistry.2001 Jun 1;276(22):19461-8.
[22]Fixemer T, Wissenbach U, Flockerzi V, Bonkhoff H. Expression of the Ca2+-selective cation channel TRPV6 in human prostate cancer:a novel prognostic marker for tumor progression. Oncogene.2003 Oct 30;22(49): 7858-61.
[23]Duncan LM, Deeds J, Hunter J, et al. Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis.Cancer research. 1998 Apr 1;58(7):1515-20.
[24]Pienta KJ, Bradley D. Mechanisms underlying the development of androgen-independent prostate cancer. Clin Cancer Res.2006 Mar 15;12(6):1665-71.
[25]Sorensen KD,Orntoft TF. Discovery of prostate cancer biomarkers by microarray gene expression profiling. Expert review of molecular diagnostics. Jan;10(1):49-64.
[26]Zhang L, Barritt GJ. Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival of prostate cancer cells. Cancer Res.2004 Nov 15;64(22):8365-73.
[27]Bidaux G, Roudbaraki M, Merle C, et al.Evidence for specific TRPM8 expression in human prostate secretory epithelial cells:functional androgen receptor requirement. Endocrine-related cancer.2005 Jun;12(2):367-82.
[28]Fuessel S,Sickert D, Meye A, et al. Multiple tumor marker analyses (PSA, hK2, PSCA, trp-p8) in primary prostate cancers using quantitative RT-PCR. Int J Oncol.2003 Jul;23(1):221-8.
[29]Kiessling A, Fussel S,Schmitz M, et al.Identification of an HLA-A *0201-restricted T-cell epitope derived from the prostate cancer-associated protein trp-p8.Prostate.2003 Sep 1;56(4):270-9.
[30]Tsavaler L, Shapero MH, Morkowski S,Laus R. Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins.Cancer Res.2001 May 1;61(9):3760-9.
[31]Henshall SM,Afar DE,Hiller J, et al.Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse. Cancer research.2003 Jul 15;63(14):4196-203.
[32]Wilkens MR,Kunert-Keil C, Brinkmeier H, Schroder B.Expression of calcium channel TRPV6 in ovine epithelial tissue. Vet J.2009 Nov;182(2):294-300.
[33]Schwarz EC, Wissenbach U, Niemeyer BA, et al. TRPV6 potentiates calcium-dependent cell proliferation. Cell calcium.2006 Feb;39(2):163-73.
[34]Soejima M, Tachida H, Koda Y. Sequence analysis of human TRPV6 suggests positive selection outside Africa. Biochemical genetics.2009 Feb;47(1-2): 147-53.
[35]Kessler T, Wissenbach U, Grobholz R, Flockerzi V.TRPV6 alleles do not influence prostate cancer progression. BMC cancer.2009;9:380.
[36]Bolanz KA, Kovacs GG, Landowski CP, Hediger MA. Tamoxifen inhibits TRPV6 activity via estrogen receptor-independent pathways in TRPV6-expressing MCF-7 breast cancer cells. Mol Cancer Res.2009 Dec;7(12): 2000-10.
[37]Irnaten M, Blanchard-Gutton N, Harvey BJ. Rapid effects of 17beta-estradiol on epithelial TRPV6 Ca2+channel in human T84 colonic cells. Cell calcium.2008 Nov;44(5):441-52.
[38]Gallin WJ, Greenberg ME. Calcium regulation of gene expression in neurons: the mode of entry matters. Current opinion in neurobiology.1995 Jun;5 (3):367-74.
[39]Hoenderop JG, Nilius B,Bindels RJ. Calcium absorption across epithelia. Physiological reviews.2005 Jan;85(1):373-422.
[40]Bogenrieder T, Herlyn M. Axis of evil:molecular mechanisms of cancer metastasis. Oncogene.2003 Sep 29;22(42):6524-36.
[41]Baffy G, Yang L, Michalopoulos GK, Williamson JR. Hepatocyte growth factor induces calcium mobilization and inositol phosphate production in rat hepatocytes.Journal of cellular physiology.1992 Nov;153(2):332-9.
[42]Comoglio PM, Trusolino L. Invasive growth:from development to metastasis. The Journal of clinical investigation.2002 Apr;109(7):857-62.
[43]Zhang YW, Vande Woude GF. HGF/SF-met signaling in the control of branching morphogenesis and invasion. Journal of cellular biochemistry.2003 Feb 1;88(2):408-17.
[44]Vriens J, Janssens A, Prenen J, et al.TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells. Cell calcium.2004 Jul;36(1):19-28.
[45]Wondergem R, Ecay TW, Mahieu F, et al.HGF/SF and menthol increase human glioblastoma cell calcium and migration. Biochemical and biophysical research communications.2008 Jul 18;372(1):210-5.
[46]Waning J, Vriens J, Owsianik G, et al. A novel function of capsaicin-sensitive TRPV1 channels:involvement in cell migration. Cell calcium.2007 Jul;42(1):17-25.
[47]El Boustany C, Bidaux G, Enfissi A, et al.Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation. Hepatology (Baltimore, Md.2008 Jun;47(6):2068-77.
[48]LeRoith D, Werner H, Beitner-Johnson D, Roberts CT, Jr. Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocrine reviews. 1995 Apr;16(2):143-63.
[49]Kojima I, Mogami H, Shibata H, Ogata E. Role of calcium entry and protein kinase C in the progression activity of insulin-like growth factor-I in Balb/c 3T3 cells. The Journal of biological chemistry.1993 May 15;268(14):10003-6.
[50]Hopkins A, Crowe PJ, Yang JL. Effect of type 1 insulin-like growth factor receptor targeted therapy on chemotherapy in human cancer and the mechanisms involved. Journal of cancer research and clinical oncology. May; 136(5): 639-50.
[51]Kanzaki M, Zhang YQ, Mashima H, et al. Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I. Nature cell biology.1999 Jul;1(3):165-70.
[52]Bruchim I, Attias Z, Werner H. Targeting the IGF1 axis in cancer proliferation. Expert opinion on therapeutic targets.2009 Oct; 13(10):1179-92.
[53]Chen RR, Silva EA, Yuen WW, Mooney DJ. Spatio-temporal VEGF and PDGF delivery patterns blood vessel formation and maturation. Pharmaceutical research.2007 Feb;24(2):258-64.
[54]Smith GC, Wear H. The perinatal implications of angiogenic factors.Current opinion in obstetrics & gynecology.2009 Apr;21(2):111-6.
[55]Munaron L, Fiorio Pla A. Endothelial calcium machinery and angiogenesis: understanding physiology to interfere with pathology. Current medicinal chemistry.2009;16(35):4691-703.
[56]Hellberg C, Ostman A, Heldin CH. PDGF and vessel maturation. Recent results in cancer research Fortschritte der Krebsforschung.180:103-14.
[57]Ogawa A, Firth AL, Yao W, et al. Prednisolone inhibits PDGF-induced nuclear translocation of NF-kappaB in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol.2008 Oct;295(4):L648-57.
[58]Guilbert A, Dhennin-Duthille I, Hiani YE, et al.Expression of TRPC6 channels in human epithelial breast cancer cells. BMC cancer.2008;8:125.
[59]Stamm S, Ben-Ari S, Rafalska I, et al.Function of alternative splicing. Gene. 2005 Jan 3;344:1-20.
[60]Prevarskaya N, Zhang L, Barritt G.TRP channels in cancer. Biochim Biophys Acta.2007 Aug;1772(8):937-46.
[61]Nagasawa M, Nakagawa Y, Tanaka S,Kojima I. Chemotactic peptide fMetLeuPhe induces translocation of the TRPV2 channel in macrophages. Journal of cellular physiology.2007 Mar;210(3):692-702.
[62]Wang C, Hu HZ, Colton CK, et al.An alternative splicing product of the murine trpvl gene dominant negatively modulates the activity of TRPV1 channels.The Journal of biological chemistry.2004 Sep 3;279(36):37423-30.
[63]Lazzeri M, Vannucchi MG, Spinelli M, et al. Transient receptor potential vanilloid type 1 (TRPV1)expression changes from normal urothelium to transitional cell carcinoma of human bladder. European urology.2005 Oct;48(4):691-8.
[64]Oancea E, Vriens J, Brauchi S,et al.TRPM1 forms ion channels associated with melanin content in melanocytes.Science signaling.2009;2(70):ra21.
[65]Miller AJ, Du J, Rowan S, et al.Transcriptional regulation of the melanoma prognostic marker melastatin (TRPM1)by MITF in melanocytes and melanoma. Cancer research.2004 Jan 15;64(2):509-16.
[66]Asai Y, Holt JR, Geleoc GS.A quantitative analysis of the spatiotemporal pattern of transient receptor potential gene expression in the developing mouse cochlea. J Assoc Res Otolaryngol. Mar;11(1):27-37.
[67]Voets T, Owsianik G, Nilius B.Trpm8.Handbook of experimental pharmacology. 2007(179):329-44.
[68]Mahieu F, Owsianik G, Verbert L, et al. TRPM8-independent menthol-induced Ca2+ release from endoplasmic reticulum and Golgi.The Journal of biological chemistry.2007 Feb 2;282(5):3325-36.
[69]Bai VU, Murthy S,Chinnakannu K, et al. Androgen regulated TRPM8 expression:a potential mRNA marker for metastatic prostate cancer detection in body fluids.International journal of oncology. Feb;36(2):443-50.
[70]Wissenbach U, Niemeyer B, Himmerkus N, et al. TRPV6 and prostate cancer: cancer growth beyond the prostate correlates with increased TRPV6 Ca2+ channel expression. Biochemical and biophysical research communications. 2004 Oct1;322(4):1359-63.
[71]Fang D, Setaluri V.Expression and Up-regulation of alternatively spliced transcripts of melastatin, a melanoma metastasis-related gene, in human melanoma cells. Biochemical and biophysical research communications.2000 Dec 9;279(1):53-61.
[72]Kiselyov K, Soyombo A, Muallem S.TRPpathies.The Journal of physiology. 2007 Feb 1;578(Pt 3):641-53.
[73]Zhang L, Barritt GJ. TRPM8 in prostate cancer cells:a potential diagnostic and prognostic marker with a secretory function? Endocrine-related cancer.2006 Mar;13(1):27-38.
[2]Voutsinas GE, Stravopodis DJ. Molecular targeting and gene delivery in bladder cancer therapy. J Buon.2009 Sep;14 Suppl 1:S69-78.
[3]Barocas DA, Clark PE. Bladder cancer. Current opinion in oncology.2008 May;20(3):307-14.
[4]Bochner BH. Gene therapy in bladder cancer. Current opinion in urology.2008 Sep;18(5):519-23.
[5]Cassileth B.Complementary therapies, herbs, and other OTC agents. Oncology (Williston Park, NY.2009 Sep;23(10):904.
[6]Bhatia SP, McGinty D, Letizia CS, Api AM. Fragrance material review on menthol. Food Chem Toxicol.2008 Nov;46 Suppl 11:S209-14.
[7]Vriens J, Nilius B,Vennekens R. Herbal Compounds and Toxins Modulating TRP Channels. Current neuropharmacology.2008 Mar;6(1):79-96.
[8]Fleetwood-Walker SM, Proudfoot CW, Garry EM, et al. Cold comfort pharm. Trends in pharmacological sciences.2007 Dec;28(12):621-8.
[9]Patel T, Ishiuji Y, Yosipovitch G. Menthol:a refreshing look at this ancient compound. Journal of the American Academy of Dermatology.2007 Nov;57 (5):873-8.
[1.0]Werley MS,Coggins CR, Lee PN. Possible effects on smokers of cigarette mentholation:a review of the evidence relating to key research questions. Regul Toxicol Pharmacol.2007 Mar;47(2):189-203.
[11]Heck JD.A review and assessment of menthol employed as a cigarette flavoring ingredient. Food Chem Toxicol. Jan;48 Suppl 2:S1-38.
[12]McKemy DD, Neuhausser WM, Julius D.Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature.2002 Mar 7;416(6876):52-8.
[13]Peier AM,Moqrich A, Hergarden AC, et al. A TRP channel that senses cold stimuli and menthol.Cell.2002 Mar 8;108(5):705-15.
[14]Thebault S,Flourakis M, Vanoverberghe K, et al.Differential role of transient receptor potential channels in Ca2+ entry and proliferation of prostate cancer epithelial cells. Cancer Res.2006 Feb 15;66(4):2038-47.
[15]Zhang L, Barritt GJ. Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival of prostate cancer cells. Cancer Res.2004 Nov 15;64(22):8365-73.
[16]Yamamura H, Ugawa S,Ueda T, et al. TRPM8 activation suppresses cellular viability in human melanoma. Am J Physiol Cell Physiol.2008 Aug;295 (2):C296-301.
[17]Bernhardt G, Biersack B, Bollwein S, et al.Terpene conjugates of diaminedichloridoplatinum(II) complexes:antiproliferative effects in HL-60 leukemia,518A2 melanoma, and HT-29 colon cancer cells.Chem Biodivers. 2008 Aug;5(8):1645-59.
[18]Lin JP, Lu HF, Lee JH, et al. (-)-Menthol inhibits DNA topoisomerases Ⅰ, Ⅱ alpha and beta and promotes NF-kappaB expression in human gastric cancer SNU-5 cells. Anticancer Res.2005 May-Jun;25(3B):2069-74.
[19]Mergler S,Strowski MZ, Kaiser S,et al. Transient receptor potential channel TRPM8 agonists stimulate calcium influx and neurotensin secretion in neuroendocrine tumor cells. Neuroendocrinology.2007;85(2):81-92.
[20]Wondergem R, Ecay TW, Mahieu F, et al. HGF/SF and menthol increase human glioblastoma cell calcium and migration. Biochem Biophys Res Commun.2008 Jul 18;372(1):210-5.
[21]Kim SH, Nam JH, Park EJ, et al. Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells.Biochim Biophys Acta.2009 Jan;1792 (1):33-8.
[22]Patapoutian A, Peier AM, Story GM, Viswanath V.ThermoTRP channels and beyond:mechanisms of temperature sensation. Nat Rev Neurosci.2003 Jul;4(7):529-39.
[23]Ma S,G G, Ak VE, et al. Menthol derivative WS-12 selectively activates transient receptor potential melastatin-8 (TRPM8) ion channels.Pak J Pharm Sci. 2008 Oct;21(4):370-8.
[24]Macpherson LJ, Hwang SW, Miyamoto T, et al.More than cool:promiscuous relationships of menthol and other sensory compounds.Molecular and cellular neurosciences.2006 Aug;32(4):335-43.
[25]Rohacs T, Lopes CM, Michailidis I, Logothetis DE. PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain. Nature neuroscience.2005 May;8(5):626-34.
[26]Roderick HL, Cook SJ. Ca2+ signalling checkpoints in cancer:remodelling Ca2+for cancer cell proliferation and survival. Nat Rev Cancer.2008 May; 8(5):361-75.
[27]Whitfield JF. Calcium signals and cancer. Crit Rev Oncog.1992;3(1-2):55-90.
[28]Cook SJ, Lockyer PJ. Recent advances in Ca(2+)-dependent Ras regulation and cell proliferation. Cell calcium.2006 Feb;39(2):101-12.
[29]Berridge MJ, Bootman MD, Roderick HL. Calcium signalling:dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol.2003 Jul;4(7):517-29.
[30]Rizzuto R, Pinton P, Ferrari D, et al.Calcium and apoptosis:facts and hypotheses. Oncogene.2003 Nov 24;22(53):8619-27.
[31]Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer:targeting Ca2+ transport. Nat Rev Cancer.2007 Jul;7(7):519-30.
[32]Takuwa N, Zhou W, Kumada M, Takuwa Y.Ca(2+)-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts. The Journal of biological chemistry.1993 Jan 5;268(1):138-45.
[33]Szabadkai G,Bianchi K, Varnai P, et al.Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. The Journal of cell biology.2006 Dec 18;175(6):901-11.
[1]Patapoutian A, Peier AM, Story GM, Viswanath V.ThermoTRP channels and beyond:mechanisms of temperature sensation. Nat Rev Neurosci.2003 Jul;4(7):529-39.
[2]Tsavaler L, Shapero MH, Morkowski S,Laus R. Trp-p8,a novel prostate-specific gene,is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer research.2001 May 1;61(9):3760-9.
[3]Yang XR, Lin MJ, McIntosh LS, Sham JS.Functional expression of transient receptor potential melastatin-and vanilloid-related channels in pulmonary arterial and aortic smooth muscle. American journal of physiology.2006 Jun;290(6):L1267-76.
[4]Johnson CD, Melanaphy D, Purse A, et al.Transient receptor potential melastatin 8 channel involvement in the regulation of vascular tone. Am J Physiol Heart Circ Physiol.2009 Jun;296(6):H1868-77.
[5]Lashinger ES,Steiginga MS,Hieble JP, et al. AMTB, a TRPM8 channel blocker: evidence in rats for activity in overactive bladder and painful bladder syndrome. Am J Physiol Renal Physiol.2008 Sep;295(3):F803-10.
[6]Fuessel S,Sickert D, Meye A, et al. Multiple tumor marker analyses (PSA, hK2, PSCA, trp-p8) in primary prostate cancers using quantitative RT-PCR. International journal of oncology.2003 Jul;23(1):221-8.
[7]Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer:targeting Ca2+ transport. Nature reviews.2007 Jul;7(7):519-30.
[8]Du S,Araki I, Kobayashi H, et al.Differential expression profile of cold (TRPA1)and cool (TRPM8) receptors in human urogenital organs. Urology. 2008 Aug;72(2):450-5.
[9]Stein RJ, Santos S, Nagatomi J, et al. Cool (TRPM8) and hot (TRPV1)receptors in the bladder and male genital tract. The Journal of urology.2004 Sep;172(3): 1175-8.
[10]Yang ZH, Wang XH, Wang HP, Hu LQ. Effects of TRPM8 on the proliferation and motility of prostate cancer PC-3 cells. Asian J Androl.2009 Mar; 11(2): 157-65.
[11]Zhang L, Barritt GJ. Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival of prostate cancer cells.Cancer Res.2004 Nov 15;64(22):8365-73.
[12]Cosens DJ, Manning A. Abnormal electroretinogram from a Drosophila mutant. Nature.1969 Oct 18;224(5216):285-7.
[13]Montell C, Rubin GM.Molecular characterization of the Drosophila trp locus:a putative integral membrane protein required for phototransduction. Neuron. 1989 Apr;2(4):1313-23.
[14]Montell C, Birnbaumer L, Flockerzi V.The TRP channels, a remarkably functional family.Cell.2002 Mar 8;108(5):595-8.
[15]Montell C, Birnbaumer L, Wissenbach U, et al. Modulation of the contractions of the smooth muscles of the rat vas deferens by TRPM8 channel activator menthol. Fiziol Zh.2009;55(6):30-40.
[16]Bodding M. TRP proteins and cancer. Cell Signal.2007 Mar; 19(3):617-24.
[17]Voets T, Droogmans G, Wissenbach U, et al. The principle of temperature-dependent gating in cold-and heat-sensitive TRP channels. Nature.2004 Aug 12;430(7001):748-54.
[18]Rohacs T, Lopes CM, Michailidis I, Logothetis DE. PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain. Nat Neurosci.2005 May;8(5):626-34.
[19]Bidaux G, Flourakis M, Thebault S,et al.Prostate cell differentiation status determines transient receptor potential melastatin member 8 channel subcellular localization and function. The Journal of clinical investigation.2007 Jun;117(6): 1647-57.
[20]Sabnis AS,Shadid M, Yost GS,Reilly CA. Human lung epithelial cells express a functional cold-sensing TRPM8 variant. Am J Respir Cell Mol Biol.2008 Oct;39(4):466-74.
[21]Birder LA. TRPs in bladder diseases. Biochim Biophys Acta.2007 Aug; 1772(8):879-84.
[1]Tsavaler L, Shapero MH, Morkowski S,Laus R. Trp-p8,a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer research.2001 May 1;61(9):3760-9.
[2]Fuessel S,Sickert D, Meye A, et al.Multiple tumor marker analyses (PSA, hK2, PSCA, trp-p8) in primary prostate cancers using quantitative RT-PCR. International journal of oncology.2003 Jul;23(1):221-8.
[3]Yamamura H, Ugawa S,Ueda T, et al.TRPM8 activation suppresses cellular viability in human melanoma. Am J Physiol Cell Physiol.2008 Aug;295 (2):C296-301.
[4]Bernhardt G, Biersack B, Bollwein S, et al.Terpene conjugates of diaminedichloridoplatinum(II) complexes:antiproliferative effects in HL-60 leukemia,518A2 melanoma, and HT-29 colon cancer cells. Chem Biodivers. 2008 Aug;5(8):1645-59.
[5]Lin JP, Lu HF, Lee JH, et al. (-)-Menthol inhibits DNA topoisomerases I, II alpha and beta and promotes NF-kappaB expression in human gastric cancer SNU-5 cells.Anticancer Res.2005 May-Jun;25(3B):2069-74.
[6]Bhatia SP, McGinty D, Letizia CS,Api AM.Fragrance material review on menthol.Food Chem Toxicol.2008 Nov;46 Suppl 11:S209-14.
[7]Patel T, Ishiuji Y, Yosipovitch G. Menthol:a refreshing look at this ancient compound. J Am Acad Dermatol.2007 Nov;57(5):873-8.
[8]Zhang L, Barritt GJ. Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival of prostate cancer cells. Cancer research.2004 Nov 15;64(22):8365-73.
[9]Bidaux G, Roudbaraki M, Merle C, et al. Evidence for specific TRPM8 expression in human prostate secretory epithelial cells:functional androgen receptor requirement. Endocrine-related cancer.2005 Jun;12(2):367-82.
[10]Zhang L, Barritt GJ. TRPM8 in prostate cancer cells:a potential diagnostic and prognostic marker with a secretory function? Endocrine-related cancer.2006 Mar;13(1):27-38.
[11]Prevarskaya N, Skryma R, Bidaux G, et al.Ion channels in death and differentiation of prostate cancer cells. Cell death and differentiation.2007 Jul;14(7):1295-304.
[12]Henshall SM, Afar DE, Hiller J, et al. Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse. Cancer research.2003 Jul 15;63(14):4196-203.
[13]Yang ZH, Wang XH, Wang HP, Hu LQ. Effects of TRPM8 on the proliferation and motility of prostate cancer PC-3 cells. Asian J Androl.2009 Mar;11 (2):157-65.
[14]Thebault S,Flourakis M, Vanoverberghe K, et al.Differential role of transient receptor potential channels in Ca2+ entry and proliferation of prostate cancer epithelial cells. Cancer Res.2006 Feb 15;66(4):2038-47.
[15]Macpherson LJ, Hwang SW, Miyamoto T, et al. More than cool:promiscuous relationships of menthol and other sensory compounds. Molecular and cellular neurosciences.2006 Aug;32(4):335-43.
[16]Ma S,G G, Ak VE, et al. Menthol derivative WS-12 selectively activates transient receptor potential melastatin-8 (TRPM8) ion channels. Pak J Pharm Sci. 2008 Oct;21(4):370-8.
[17]Kim SH, Nam JH, Park EJ, et al.Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells. Biochim Biophys Acta.2009 Jan;1792(1):33-8.
[1]Yamamura H, Ugawa S,Ueda T, et al.TRPM8 activation suppresses cellular viability in human melanoma. Am J Physiol Cell Physiol.2008 Aug;295 (2):C296-301.
[2]Bernhardt G, Biersack B, Bollwein S,et al. Terpene conjugates of diaminedichloridoplatinum(II) complexes:antiproliferative effects in HL-60 leukemia,518A2 melanoma, and HT-29 colon cancer cells. Chem Biodivers. 2008 Aug;5(8):1645-59.
[3]Whitfield JF. Calcium signals and cancer. Crit Rev Oncog.1992;3(1-2):55-90.
[4]Cook SJ, Lockyer PJ. Recent advances in Ca(2+)-dependent Ras regulation and cell proliferation. Cell calcium.2006 Feb;39(2):101-12.
[5]Takuwa N, Zhou W, Kumada M, Takuwa Y. Ca(2+)-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts. The Journal of biological chemistry.1993 Jan 5;268(1):138-45.
[6]Szabadkai G, Bianchi K, Varnai P, et al.Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+channels. The Journal of cell biology.2006 Dec 18;175(6):901-11.
[7]Wondergem R, Bartley JW. Menthol increases human glioblastoma intracellular Ca2+, BK channel activity and cell migration. Journal of biomedical science. 2009;16:90.
[8]Kim SH, Nam JH, Park EJ, et al.Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells. Biochim Biophys Acta.2009 Jan;1792(1):33-8.
[9]Fulda S,Gorman AM, Hori O, Samali A. Cellular stress responses:cell survival and cell death. International journal of cell biology.2010:214074.
[10]Platini F, Perez-Tomas R, Ambrosio S, Tessitore L. Understanding autophagy in cell death control. Current pharmaceutical design.16(1):101-13.
[11]Grimm S,Noteborn M. Anticancer genes:inducers of tumour-specific cell death signalling. Trends in molecular medicine. Feb;16(2):88-96.
[12]Lu HF, Hsueh SC, Yu FS,et al. The role of Ca2+in(-)-menthol-induced human promyelocytic leukemia HL-60 cell death. In Vivo.2006 Jan-Feb;20(1):69-75.
[13]Ma S,G G, Ak VE, et al.Menthol derivative WS-12 selectively activates transient receptor potential melastatin-8 (TRPM8) ion channels.Pak J Pharm Sci. 2008 Oct;21(4):370-8.
[14]Macpherson LJ, Hwang SW, Miyamoto T, et al.More than cool:promiscuous relationships of menthol and other sensory compounds. Molecular and cellular neurosciences.2006 Aug;32(4):335-43.
[15]Du S,Araki I, Kobayashi H, et al.Differential expression profile of cold (TRPA1)and cool (TRPM8) receptors in human urogenital organs. Urology. 2008 Aug;72(2):450-5.
[16]Capiod T, Shuba Y, Skryma R, Prevarskaya N. Calcium signalling and cancer cell growth. Sub-cellular biochemistry.2007;45:405-27.
[17]Monteith GR, McAndrew D,Faddy HM,Roberts-Thomson SJ. Calcium and cancer:targeting Ca2+ transport. Nat Rev Cancer.2007 Jul;7(7):519-30.
[18]Ott M, Gogvadze V, Orrenius S,Zhivotovsky B. Mitochondria, oxidative stress and cell death. Apoptosis.2007 May;12(5):913-22.
[19]Sattler UG, Mueller-Klieser W. The anti-oxidant capacity of tumour glycolysis. International journal of radiation biology.2009 Nov;85(11):963-71.
[20]Trachootham D, Alexandre J, Huang P.Targeting cancer cells by ROS-mediated mechanisms:a radical therapeutic approach? Nature reviews.2009 Jul;8 (7):579-91.
[21]Pan JS,Hong MZ, Ren JL. Reactive oxygen species:a double-edged sword in oncogenesis. World J Gastroenterol.2009 Apr 14;15(14):1702-7.
[1]Gupta GP, Massague J. Cancer metastasis:building a framework. Cell.2006 Nov 17;127(4):679-95.
[2]Roderick HL, Cook SJ. Ca2+ signalling checkpoints in cancer:remodelling Ca2+ for cancer cell proliferation and survival. Nature reviews.2008 May;8(5):361-75.
[3]Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer:targeting Ca2+ transport. Nature reviews.2007 Jul;7(7):519-30.
[4]Rizzuto R, Pinton P, Ferrari D, et al. Calcium and apoptosis:facts and hypotheses.Oncogene.2003 Nov 24;22(53):8619-27.
[5]Cook SJ, Lockyer PJ. Recent advances in Ca(2+)-dependent Ras regulation and cell proliferation. Cell calcium.2006 Feb;39(2):101-12.
[6]Nilius B,Owsianik G, Voets T, Peters JA. Transient receptor potential cation channels in disease. Physiological reviews.2007 Jan;87(1):165-217.
[7][7] Bidaux G, Flourakis M, Thebault S, et al.Prostate cell differentiation status determines transient receptor potential melastatin member 8 channel subcellular localization and function. The Journal of clinical investigation.2007 Jun;117(6):1647-57.
[8]Thebault S, Lemonnier L,Bidaux G, et al. Novel role of cold/menthol-sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store-operated channels in LNCaP human prostate cancer epithelial cells. The Journal of biological chemistry.2005 Nov 25;280(47):39423-35.
[9]Xu XZ, Moebius F, Gill DL, Montell C.Regulation of melastatin, a TRP-related protein, through interaction with a cytoplasmic isoform. Proceedings of the National Academy of Sciences of the United States of America.2001 Sep 11;98(19):10692-7.
[10]Wisnoskey BJ, Sinkins WG, Schilling WP. Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry. The Biochemical journal.2003 Jun 1;372(Pt 2):517-28.
[11]Giampa C, DeMarch Z, Patassini S,et al.Immunohistochemical localization of TRPC6 in the rat substantia nigra. Neurosci Lett.2007 Sep 13;424(3):170-4.
[12]Montell C, Birnbaumer L, Flockerzi V. The TRP channels, a remarkably functional family. Cell.2002 Mar 8;108(5):595-8.
[13]Benarroch EE. TRP channels:functions and involvement in neurologic disease. Neurology.2008 Feb 19;70(8):648-52.
[14]Clark K, Middelbeek J, van Leeuwen FN. Interplay between TRP channels and the cytoskeleton in health and disease. European journal of cell biology.2008 Sep;87(8-9):631-40.
[15]Jordt SE, Ehrlich BE. TRP channels in disease. Sub-cellular biochemistry. 2007;45:253-71.
[16]Cosens DJ, Manning A. Abnormal electroretinogram from a Drosophila mutant. Nature.1969 Oct 18;224(5216):285-7.
[17]Montell C, Rubin GM. Molecular characterization of the Drosophila trp locus:a putative integral membrane protein required for phototransduction. Neuron. 1989 Apr;2(4):1313-23.
[18]Hardie RC, Minke B.The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors. Neuron.1992 Apr;8(4):643-51.
[19]Petersen CC, Berridge MJ, Borgese MF, Bennett DL. Putative capacitative calcium entry channels:expression of Drosophila trp and evidence for the existence of vertebrate homologues.The Biochemical journal.1995 Oct 1;311 (Pt 1):41-4.
[20]Thebault S, Flourakis M, Vanoverberghe K, et al.Differential role of transient receptor potential channels in Ca2+ entry and proliferation of prostate cancer epithelial cells. Cancer research.2006 Feb 15;66(4):2038-47.
[21]Wissenbach U, Niemeyer BA, Fixemer T, et al.Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer. The Journal of biological chemistry.2001 Jun 1;276(22):19461-8.
[22]Fixemer T, Wissenbach U, Flockerzi V, Bonkhoff H. Expression of the Ca2+-selective cation channel TRPV6 in human prostate cancer:a novel prognostic marker for tumor progression. Oncogene.2003 Oct 30;22(49): 7858-61.
[23]Duncan LM, Deeds J, Hunter J, et al. Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis.Cancer research. 1998 Apr 1;58(7):1515-20.
[24]Pienta KJ, Bradley D. Mechanisms underlying the development of androgen-independent prostate cancer. Clin Cancer Res.2006 Mar 15;12(6):1665-71.
[25]Sorensen KD,Orntoft TF. Discovery of prostate cancer biomarkers by microarray gene expression profiling. Expert review of molecular diagnostics. Jan;10(1):49-64.
[26]Zhang L, Barritt GJ. Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival of prostate cancer cells. Cancer Res.2004 Nov 15;64(22):8365-73.
[27]Bidaux G, Roudbaraki M, Merle C, et al.Evidence for specific TRPM8 expression in human prostate secretory epithelial cells:functional androgen receptor requirement. Endocrine-related cancer.2005 Jun;12(2):367-82.
[28]Fuessel S,Sickert D, Meye A, et al. Multiple tumor marker analyses (PSA, hK2, PSCA, trp-p8) in primary prostate cancers using quantitative RT-PCR. Int J Oncol.2003 Jul;23(1):221-8.
[29]Kiessling A, Fussel S,Schmitz M, et al.Identification of an HLA-A *0201-restricted T-cell epitope derived from the prostate cancer-associated protein trp-p8.Prostate.2003 Sep 1;56(4):270-9.
[30]Tsavaler L, Shapero MH, Morkowski S,Laus R. Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins.Cancer Res.2001 May 1;61(9):3760-9.
[31]Henshall SM,Afar DE,Hiller J, et al.Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse. Cancer research.2003 Jul 15;63(14):4196-203.
[32]Wilkens MR,Kunert-Keil C, Brinkmeier H, Schroder B.Expression of calcium channel TRPV6 in ovine epithelial tissue. Vet J.2009 Nov;182(2):294-300.
[33]Schwarz EC, Wissenbach U, Niemeyer BA, et al. TRPV6 potentiates calcium-dependent cell proliferation. Cell calcium.2006 Feb;39(2):163-73.
[34]Soejima M, Tachida H, Koda Y. Sequence analysis of human TRPV6 suggests positive selection outside Africa. Biochemical genetics.2009 Feb;47(1-2): 147-53.
[35]Kessler T, Wissenbach U, Grobholz R, Flockerzi V.TRPV6 alleles do not influence prostate cancer progression. BMC cancer.2009;9:380.
[36]Bolanz KA, Kovacs GG, Landowski CP, Hediger MA. Tamoxifen inhibits TRPV6 activity via estrogen receptor-independent pathways in TRPV6-expressing MCF-7 breast cancer cells. Mol Cancer Res.2009 Dec;7(12): 2000-10.
[37]Irnaten M, Blanchard-Gutton N, Harvey BJ. Rapid effects of 17beta-estradiol on epithelial TRPV6 Ca2+channel in human T84 colonic cells. Cell calcium.2008 Nov;44(5):441-52.
[38]Gallin WJ, Greenberg ME. Calcium regulation of gene expression in neurons: the mode of entry matters. Current opinion in neurobiology.1995 Jun;5 (3):367-74.
[39]Hoenderop JG, Nilius B,Bindels RJ. Calcium absorption across epithelia. Physiological reviews.2005 Jan;85(1):373-422.
[40]Bogenrieder T, Herlyn M. Axis of evil:molecular mechanisms of cancer metastasis. Oncogene.2003 Sep 29;22(42):6524-36.
[41]Baffy G, Yang L, Michalopoulos GK, Williamson JR. Hepatocyte growth factor induces calcium mobilization and inositol phosphate production in rat hepatocytes.Journal of cellular physiology.1992 Nov;153(2):332-9.
[42]Comoglio PM, Trusolino L. Invasive growth:from development to metastasis. The Journal of clinical investigation.2002 Apr;109(7):857-62.
[43]Zhang YW, Vande Woude GF. HGF/SF-met signaling in the control of branching morphogenesis and invasion. Journal of cellular biochemistry.2003 Feb 1;88(2):408-17.
[44]Vriens J, Janssens A, Prenen J, et al.TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells. Cell calcium.2004 Jul;36(1):19-28.
[45]Wondergem R, Ecay TW, Mahieu F, et al.HGF/SF and menthol increase human glioblastoma cell calcium and migration. Biochemical and biophysical research communications.2008 Jul 18;372(1):210-5.
[46]Waning J, Vriens J, Owsianik G, et al. A novel function of capsaicin-sensitive TRPV1 channels:involvement in cell migration. Cell calcium.2007 Jul;42(1):17-25.
[47]El Boustany C, Bidaux G, Enfissi A, et al.Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation. Hepatology (Baltimore, Md.2008 Jun;47(6):2068-77.
[48]LeRoith D, Werner H, Beitner-Johnson D, Roberts CT, Jr. Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocrine reviews. 1995 Apr;16(2):143-63.
[49]Kojima I, Mogami H, Shibata H, Ogata E. Role of calcium entry and protein kinase C in the progression activity of insulin-like growth factor-I in Balb/c 3T3 cells. The Journal of biological chemistry.1993 May 15;268(14):10003-6.
[50]Hopkins A, Crowe PJ, Yang JL. Effect of type 1 insulin-like growth factor receptor targeted therapy on chemotherapy in human cancer and the mechanisms involved. Journal of cancer research and clinical oncology. May; 136(5): 639-50.
[51]Kanzaki M, Zhang YQ, Mashima H, et al. Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I. Nature cell biology.1999 Jul;1(3):165-70.
[52]Bruchim I, Attias Z, Werner H. Targeting the IGF1 axis in cancer proliferation. Expert opinion on therapeutic targets.2009 Oct; 13(10):1179-92.
[53]Chen RR, Silva EA, Yuen WW, Mooney DJ. Spatio-temporal VEGF and PDGF delivery patterns blood vessel formation and maturation. Pharmaceutical research.2007 Feb;24(2):258-64.
[54]Smith GC, Wear H. The perinatal implications of angiogenic factors.Current opinion in obstetrics & gynecology.2009 Apr;21(2):111-6.
[55]Munaron L, Fiorio Pla A. Endothelial calcium machinery and angiogenesis: understanding physiology to interfere with pathology. Current medicinal chemistry.2009;16(35):4691-703.
[56]Hellberg C, Ostman A, Heldin CH. PDGF and vessel maturation. Recent results in cancer research Fortschritte der Krebsforschung.180:103-14.
[57]Ogawa A, Firth AL, Yao W, et al. Prednisolone inhibits PDGF-induced nuclear translocation of NF-kappaB in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol.2008 Oct;295(4):L648-57.
[58]Guilbert A, Dhennin-Duthille I, Hiani YE, et al.Expression of TRPC6 channels in human epithelial breast cancer cells. BMC cancer.2008;8:125.
[59]Stamm S, Ben-Ari S, Rafalska I, et al.Function of alternative splicing. Gene. 2005 Jan 3;344:1-20.
[60]Prevarskaya N, Zhang L, Barritt G.TRP channels in cancer. Biochim Biophys Acta.2007 Aug;1772(8):937-46.
[61]Nagasawa M, Nakagawa Y, Tanaka S,Kojima I. Chemotactic peptide fMetLeuPhe induces translocation of the TRPV2 channel in macrophages. Journal of cellular physiology.2007 Mar;210(3):692-702.
[62]Wang C, Hu HZ, Colton CK, et al.An alternative splicing product of the murine trpvl gene dominant negatively modulates the activity of TRPV1 channels.The Journal of biological chemistry.2004 Sep 3;279(36):37423-30.
[63]Lazzeri M, Vannucchi MG, Spinelli M, et al. Transient receptor potential vanilloid type 1 (TRPV1)expression changes from normal urothelium to transitional cell carcinoma of human bladder. European urology.2005 Oct;48(4):691-8.
[64]Oancea E, Vriens J, Brauchi S,et al.TRPM1 forms ion channels associated with melanin content in melanocytes.Science signaling.2009;2(70):ra21.
[65]Miller AJ, Du J, Rowan S, et al.Transcriptional regulation of the melanoma prognostic marker melastatin (TRPM1)by MITF in melanocytes and melanoma. Cancer research.2004 Jan 15;64(2):509-16.
[66]Asai Y, Holt JR, Geleoc GS.A quantitative analysis of the spatiotemporal pattern of transient receptor potential gene expression in the developing mouse cochlea. J Assoc Res Otolaryngol. Mar;11(1):27-37.
[67]Voets T, Owsianik G, Nilius B.Trpm8.Handbook of experimental pharmacology. 2007(179):329-44.
[68]Mahieu F, Owsianik G, Verbert L, et al. TRPM8-independent menthol-induced Ca2+ release from endoplasmic reticulum and Golgi.The Journal of biological chemistry.2007 Feb 2;282(5):3325-36.
[69]Bai VU, Murthy S,Chinnakannu K, et al. Androgen regulated TRPM8 expression:a potential mRNA marker for metastatic prostate cancer detection in body fluids.International journal of oncology. Feb;36(2):443-50.
[70]Wissenbach U, Niemeyer B, Himmerkus N, et al. TRPV6 and prostate cancer: cancer growth beyond the prostate correlates with increased TRPV6 Ca2+ channel expression. Biochemical and biophysical research communications. 2004 Oct1;322(4):1359-63.
[71]Fang D, Setaluri V.Expression and Up-regulation of alternatively spliced transcripts of melastatin, a melanoma metastasis-related gene, in human melanoma cells. Biochemical and biophysical research communications.2000 Dec 9;279(1):53-61.
[72]Kiselyov K, Soyombo A, Muallem S.TRPpathies.The Journal of physiology. 2007 Feb 1;578(Pt 3):641-53.
[73]Zhang L, Barritt GJ. TRPM8 in prostate cancer cells:a potential diagnostic and prognostic marker with a secretory function? Endocrine-related cancer.2006 Mar;13(1):27-38.