薄荷醇激活瞬时受体电位M8对雄激素非依赖性前列腺癌DU145细胞生物学行为影响的研究

发布时间：2018-05-02 14:23

本文选题：TRPM8 + TRPA1　；参考：《武汉大学》2014年博士论文

【摘要】：前列腺癌是泌尿外科最常见的恶性肿瘤之一,对男性健康威胁巨大。全球范围内,前列腺癌的发病率占据男性所有恶性肿瘤发病率的第一位,高达28%,是导致男性癌症死亡的第二大诱因(11%)。在不同的地区和种族中前列腺癌的发病率有所不同,例如在美国,前列腺癌已经超过了肺癌,跃居肿瘤威胁男性健康的首位。在我国,最初前列腺癌发病率较低,但是随着人民生活水平的不断改善及因此导致的饮食结构的变化、社会发展所致人口老龄化的加剧以及诊疗技术的不断进步,前列腺癌的检出率逐年增加,呈现出了惊人的升高趋势。在前列腺癌早期即雄激素依赖阶段,癌细胞的生存及增值依赖雄激素,所以抗雄激素治疗可以显著的抑制肿瘤的生长及发展,但是抗雄激素治疗并不能阻止前列腺癌最终进展到雄激素非依赖阶段。肿瘤的复发及广泛转移将最终导致前列腺癌患者的死亡,而复发和转移同时也是治疗的难点。在全球范围内还没有有效的治疗雄激素非依赖性前列腺癌的方法。 Ca2+作为细胞内第二信使参与了细胞增殖和凋亡这两个相互矛盾的过程,Ca2+平衡失调在肿瘤的发生、发展中起至关重要的作用,因而Ca2+通道具有非常高的潜能成为肿瘤治疗的新靶点。瞬时受体电位(TRP)家族中具有Ca2+通透性的成员也因此受到了大家的广泛关注,其中一个重要的成员就是trpm8最初被称为trp-p8。trpm8的转录产物是阳离子通道蛋白TRPM8,该通道对Ca2+具有透过性的,可以被低温和薄荷醇激活,参与细胞内ca2+浓度的调控[5-7]。在正常组织中,只有感觉神经和前列腺中可以检测到大量的trpm8表达,因此它被认为是具有前列腺特异性的基因。trpm8在前列腺癌中的表达较正常前列腺相比显著增高,并且其表达水平与前列腺癌分级密切相关,因此有研究者认为trpm8基因具有作为一种诊断指标和瘤标的潜力。这种TRPM8的表达变化也许与细胞Ca2+平衡失调以及肿瘤的发生、发展有关,但TRPM8的确切生理功能还不清楚。Henshall等人的研究显示TRPM8的表达可能受雄激素的调控,因为抗雄激素治疗能显著的降低其在前列腺癌中的表达。而小片段干扰RNA(siRNA)沉默前列腺上皮细胞内TRPM8表达的实验则表明它的沉默能诱导细胞凋亡,这充分证明了TRPM8对维持前列腺上皮细胞内Ca2+稳态具有至关重要的作用,而且与siRNA效果相似,TRPM8通道阻断剂也能诱导前列腺癌LNCaP细胞的凋亡,这些结果说明了TRPM8通道是前列腺上皮细胞存活所必需的。薄荷醇(mentho1)是自然界的一种化合物,提取自植物薄荷。因为可以给人带来凉爽的感觉,薄荷醇被广泛的应用于化妆品、调味品及制药业。薄荷醇具有止痒、止痛、刺激和抗炎等作用,可用于治疗头痛、神经痛、声音嘶哑等,在非处方用药中薄荷醇的使用浓度高达16%[15]。研究证实薄荷醇是TRPM8通道蛋白特异性的激动剂,TRPM8通道蛋白在感觉神经中大量表达,可被低温激活,激活后给人体带来凉爽的感觉,至此薄荷醇诱导凉爽感觉的机制才得以揭示[16,17]。另外有报道称薄荷醇可以诱导前列腺癌雄激素依赖性LNCaP细胞发生持续性的Ca2+内流,从而导致细胞凋亡。近年来随着研究的深入,有研究发现薄荷醇对于TRPA1通道具有双峰作用,即在低浓度时激活该通道,而在高浓度时抑制该通道[18,19]。TRPA1是TRP家族中另一位冷感觉感受器,与TRPM8的同源性较低,对于该通道的研究主要集中于温度感受,较少涉及肿瘤。本课题拟在DU145细胞中讨论TRPM8的功能；具体的方法是使用薄荷醇激动TRPM8通道,以此作为干预因素观察DU145细胞的生长增殖能力、运动能力和凋亡的变化,从而初步探索TRPM8通道在雄激素非依赖性前列腺癌中的功能以及作为治疗靶点的可能性,为开发雄激素非依赖性前列腺癌治疗药物以及基因治疗前列腺癌提供理论基础。第一部分雄激素非依赖性前列腺癌DU145细胞中TRPM8和TRPAl的表达及活性的研究目的：检测TRPM8和TRIA1在雄激素非依赖性前列腺癌DU145细胞中是否表达,以及薄荷醇是否通过作用于TRPM8通道来影响DU145细胞的生物学行为。方法：通过RT-PCR实验、Western blot实验和免疫组织化学实验从不同水平例如mRNA水平、蛋白水平以及直观观察层面检测TRPM8和TRPA1在DU145细胞中的表达；通过Ca2+成像实验观察薄荷醇能否诱导DU145细胞内Ca2+浓度的变化,以此判断DU145细胞中的TRPM8通道是否具有生物活性。结果：RT-PCR结果显示TRPM8在雄激素非依赖性前列腺癌DU145细胞中有表达,而且发现可被薄荷醇激活的TRPA1在DU145细胞中表达缺失。PCR产物纯化后进行DNA序列测定,将测序所得结果运用BLAST与GenBank数据库中发布序列进行序列分析,结果显示高度相同。为进一步验证PCR结果,我们采用Western blot和免疫组织化学法检测了DU145细胞中TRPM8和TRPA1的蛋白表达,结果证实TRPM8在DU145细胞中有大量表达,而TRPA1也未检测到表达。为了验证DU145细胞中表达的TRPM8通道是否具有活性,我们采用荧光染色Ca2+成像法检测薄荷醇对TRPM8通道的激活能力。结果显示,在未添加薄荷醇干预时,DU145细胞中有极低量的荧光,随着薄荷醇的滴入,细胞中荧光亮度急剧升高,具有典型的TRPM8通道激活曲线,由此可证明DU145细胞中确有TRPM8通道蛋白的表达,且具有生物活性；薄荷醇可激活TRPM8通道引起Ca2+内流。结论：雄激素非依赖性前列腺癌DU145细胞中有大量的具有生物活性的TRPM8通道蛋白表达,而可同被薄荷醇激活的TRPA1却未被检测到表达。薄荷醇可激活DU145细胞中的TRPM8通道诱导Ca2+内流。第二部分薄荷醇激活TRPM8通道对雄激素非依赖性前列腺癌DU145细胞生物学行为影响的研究目的：以薄荷醇作为干预因素,观察TRPM8通道的激活对DU145细胞的增殖、迁移/侵袭和凋亡的产生何种影响,从而初步探索TRPM8通道在雄激素非依赖性前列腺癌中的功能以及作为治疗靶点的可能性,期望以TRPM8为突破口找寻治疗雄激素非依赖性前列腺癌的新途径。方法：采用MTT法检测梯度浓度的薄荷醇对DU145细胞增殖能力的影响,筛选出合适的薄荷醇浓度以备后用。而对于薄荷醇对细胞周期以及迁移/侵袭的影响则分别采用流式细胞周期实验、划痕实验和transwell实验来检测。Hochest33258染色及流式细胞凋亡实验检测薄荷醇对DU145细胞凋亡的影响。Western blot检测周期相关蛋白Cdk2、Cdk4、Cdk6及迁移相关蛋白磷酸化FAK(FAK-pY-397). 结果：MTT实验结果显示,与未经薄荷醇处理的细胞相比,不同浓度(25,50,75,100μM)薄荷醇处理组的细胞数量明显下降。将未处理的细胞设定为100%,则处理组与其相比分别为100%±2.68%VS90.66%±6.60%,82.78%±7.24%,70.12%±9.96%,53.41%±6.45%,25μM组p0.05其余组p0.01。细胞经TRPM8通道阻断剂BCTC预处理20分钟后,薄荷醇所致的细胞增殖抑制被部分恢复(p0.01)。流式细胞周期检测结果显示,与空白组相比,细胞经100μM薄荷醇处理24h(p0.05)、48h和72h(p0.01)后,其处于Go/G1期的细胞量显著增加(49.12%±1.92%VS61.71%±2.70%,77.65%±1.63%,71.81%±2.46%),然而流式细胞凋亡检测及Hochest33258染色实验提示,100μM薄荷醇处理并未引起细胞凋亡。划痕实验提示,将空白组的迁移率设定为100%,则薄荷醇处理24h和48h组与之相比分别为100%VS58.62%±11.55%和48.21%±11.11%。细胞经TRPM8通道阻断剂BCTC预处理20分钟后,薄荷醇诱导的细胞迁移抑制被部分恢复(p0.05)。Transwell实验显示,薄荷醇抑制了细胞的侵袭能力,且Western blot检测发现周期相关蛋白Cdk2、Cdk4、Cdk6及迁移相关蛋白磷酸化FAK表达量显著下调。结论：薄荷醇通过激活TRPM8通道抑制了DU145细胞的增殖和迁移、侵袭能力,但未诱导细胞凋亡。实验结果证实了通过激活TRPM8通道治疗雄激素非依赖性前列腺癌的可能性,为TRPM8靶向治疗激素抵抗型前列腺癌提供了理论依据。
[Abstract]:Prostate cancer is one of the most common malignant tumors in the Department of Urology and has a great threat to male health. The incidence of prostate cancer is the first of all the male malignant tumors worldwide, up to 28%, which is the second major cause of male cancer death (11%). The incidence of prostate cancer in different regions and races has been found. Different, for example, in the United States, prostate cancer has exceeded lung cancer and is leaping cancer to the top of the male health. In China, the incidence of prostate cancer is low in the country, but with the improvement of the living standard of the people and the change of the diet structure, the aging of the population caused by the social development and the continuous progress of diagnosis and treatment technology. In the early stage of prostate cancer, the survival and increment of cancer cells depend on androgens, so anti androgen therapy can significantly inhibit the growth and development of cancer, but anti androgen therapy does not prevent the final progression of prostate cancer. The recurrence and extensive metastasis of the tumor will eventually lead to the death of the prostate cancer patients, and the recurrence and metastasis are also the difficulty of the treatment. There is no effective treatment for androgen independent prostate cancer worldwide.
Ca2+, as the second messenger within the cell, participates in the two contradictory processes of cell proliferation and apoptosis, and the imbalance of Ca2+ plays a vital role in the development of the tumor. Therefore, the Ca2+ channel has a very high potential to be a new target for cancer treatment. The members of the transient receptor potential (TRP) family with Ca2+ permeability are also the cause of the disease. It has attracted wide attention. One of the important members is that TRPM8, which was originally called trp-p8.trpm8, is a cationic channel protein TRPM8, which is transmissive to Ca2+, can be activated by low temperature and menthol, and participates in the regulation of the intracellular ca2+ concentration of [5-7]. in normal tissues, only sensory nerves and prostaglandin A large number of TRPM8 expressions can be detected in the glands, so it is believed that the expression of the prostate specific gene.Trpm8 is significantly higher in the prostate cancer than in the normal prostate, and the expression level is closely related to the classification of the prostate cancer. Therefore, some researchers believe that the TRPM8 gene is a diagnostic marker and a tumor marker. Potential. Changes in the expression of this TRPM8 may be related to the imbalance of Ca2+ balance and the development of tumor, but the exact physiological function of TRPM8 is not clear. The study of.Henshall et al. Shows that the expression of TRPM8 may be regulated by androgens, because anti androgen therapy can significantly reduce the expression of TRPM8 in prostate cancer. The effect of RNA (siRNA) silence on the expression of TRPM8 in the epithelial cells of the prostate showed that its silence could induce apoptosis, which fully demonstrated that TRPM8 played a crucial role in maintaining Ca2+ homeostasis in the prostate epithelial cells, and was similar to the effect of siRNA. The TRPM8 channel inhibitor could also induce the apoptosis of LNCaP cells in prostate cancer. These results indicate that TRPM8 channels are necessary for the survival of prostatic epithelial cells.
Menthol (mentho1) is a natural compound extracted from plant menthol. Because it can bring a cool feeling to people, menthol is widely used in cosmetics, condiments and pharmaceuticals. Menthol has antipruritic, analgesic, irritation and anti-inflammatory effects, which can be used to treat headache, neuralgia, hoarseness and so on. It is thinner in non prescription drugs. The use of the concentration of alcohols up to 16%[15]. confirmed that menthol is a specific excitant of TRPM8 channel protein. The TRPM8 channel protein is expressed in a large number of sensory nerves, which can be activated at low temperature and activate to bring cool feeling to the human body. At this point, the mechanism of inducing cool sensation by menthol can reveal [16,17]. in addition to menthol. The prostatic cancer androgen dependent LNCaP cells can induce persistent Ca2+ inflow and lead to cell apoptosis. In recent years, some studies have shown that menthol has the effect on the TRPA1 channel in Shuangfeng, that is to activate the channel at low concentration, and the inhibition of the channel [18,19].TRPA1 at the high concentration is the other one in the TRP family. The location of cold sensation receptors is lower than that of TRPM8. The study of this channel mainly focuses on temperature perception and less on tumors.
The purpose of this study is to discuss the function of TRPM8 in DU145 cells. The specific method is to use menthol to stimulate the TRPM8 channel as an intervention factor to observe the growth and proliferation of DU145 cells, exercise ability and apoptosis, so as to explore the function of TRPM8 channel in androgen independent prostate cancer and to be a therapeutic target. It provides a theoretical basis for the development of androgen independent prostate cancer drugs and gene therapy for prostate cancer.
Part one: expression and activity of TRPM8 and TRPAl in androgen independent prostate cancer DU145 cells
Objective: to detect the expression of TRPM8 and TRIA1 in androgen independent prostate cancer DU145 cells and whether menthol affects the biological behavior of DU145 cells by acting on the TRPM8 channel.
Methods: the expression of TRPM8 and TRPA1 in DU145 cells was detected at different levels, such as mRNA level, protein level and visual observation at different levels, such as mRNA level, protein level and visual observation at different levels, such as the level of mRNA, protein level and visual observation, and the changes in the concentration of Ca2+ in DU145 cells were observed by the Ca2+ imaging experiment to judge the DU145 cells by the RT-PCR experiment and the immunohistochemical experiment. Whether the TRPM8 channel has biological activity.
Results: RT-PCR results showed that TRPM8 was expressed in androgen independent prostate cancer DU145 cells, and it was found that the deletion of.PCR products expressed by menthol activated TRPA1 in DU145 cells was purified to carry out DNA sequencing. The sequencing results were analyzed with the sequence of BLAST and GenBank data base. In order to further verify the PCR results, we detected the protein expression of TRPM8 and TRPA1 in DU145 cells by using Western blot and immunohistochemistry. The results confirmed that TRPM8 was expressed in DU145 cells, and TRPA1 was not detected. In order to verify whether TRPM8 channels expressed in DU145 cells were active, we picked up the activity of TRPM8 in the DU145 cells. The activation ability of menthol to TRPM8 channel was detected by fluorescein Ca2+ imaging. The results showed that there was a very low fluorescence in DU145 cells without menthol intervention. With the drop of menthol, the luminosity of the cells increased sharply, with a typical TRPM8 channel activation curve, which proved that there was a TRPM8 channel in DU145 cells. Protein expression and bioactivity; menthol can activate TRPM8 channel to cause Ca2+ influx.
Conclusion: a large number of bioactive TRPM8 channel proteins are expressed in androgen independent prostate cancer DU145 cells, but the expression of TRPA1, which is activated by menthol, is not detected. Menthol activates the TRPM8 channel in DU145 cells to induce the Ca2+ inflow. The second part of the TRPM8 channel activates the TRPM8 channel and is not dependent on the androgen. Study on the biological behavior of prostate cancer DU145 cells
Objective: To observe the effect of TRPM8 channel activation on the proliferation, migration / invasion and apoptosis of DU145 cells by using menthol as an intervention factor, so as to explore the function of TRPM8 channel in androgen independent prostate cancer and the possibility of as a target for treatment. It is expected to search for the treatment of androgen by TRPM8 as a breakthrough. A new way to rely on prostate cancer.
Methods: the effect of the gradient concentration of menthol on the proliferation of DU145 cells was detected by MTT method, and a suitable concentration of menthol was screened for subsequent use. The effects of menthol on cell cycle and migration / invasion were measured by flow cytometry, scratch test and Transwell test to detect.Hochest33258 staining and flow. The effect of menthol on apoptosis of DU145 cells by apoptosis assay,.Western blot detection of cycle related protein Cdk2, Cdk4, Cdk6 and phosphorylation of migration related proteins FAK (FAK-pY-397).
Results: the results of MTT test showed that the number of cells in the menthol treatment group with different concentrations (25,50,75100 mu M) decreased significantly compared with those without menthol treatment. The untreated cells were set to 100%, and the treatment group was 100% + 2.68%VS90.66% + 6.60%, 82.78% + 7.24%, 70.12% + 9.96%, 53.41% + 6.45%, 25 mu M P0.05, respectively. The cell proliferation inhibition induced by menthol was partially restored after 20 minutes of preconditioning with TRPM8 channel blocker BCTC for 20 minutes (P0.01). Flow cytometry showed that cell volume in Go/G1 phase increased significantly after 24h (P0.05), 48h and 72h (P0.01) treated with 100 mu M menthol (49.12% + 1.92%VS). 61.71% + 2.70%, 77.65% + 1.63%, 71.81% + 2.46%), however, flow cytometry and Hochest33258 staining showed that 100 - M menthol treatment did not cause cell apoptosis. The scratch test suggested that the mobility of the blank group was set to 100%, then the menthol treatment 24h and 48h group were 100%VS58.62% + 11.55% and 48.21% + 11.11%., respectively. After the cells were pretreated with TRPM8 channel blocker BCTC for 20 minutes, the cell migration inhibition induced by menthol was partially restored (P0.05).Transwell experiment showed that menthol inhibited the cell invasiveness, and the Western blot detection found that the cycle related protein Cdk2, Cdk4, Cdk6 and ex migration related protein phosphorylation FAK expression decreased significantly.
Conclusion: Menthol inhibits the proliferation, migration and invasion of DU145 cells by activating the TRPM8 channel, but does not induce apoptosis. The experimental results confirm the possibility of the treatment of androgen independent prostate cancer by activating the TRPM8 channel, which provides a theoretical basis for the targeted treatment of hormone resistant prostate cancer by TRPM8.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

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