Sutherlandia Frutescens提取物抗前列腺肿瘤的研究

发布时间：2018-06-04 04:05

本文选题：前列腺肿瘤 + Gli/Hedgehog信号通路　；参考：《西北农林科技大学》2014年博士论文

【摘要】：前列腺癌是男性泌尿生殖系统的恶性肿瘤之一。在西方国家，它仅次于肺癌，是导致男性因癌症死亡的第二大疾病。治疗前列腺肿瘤的主要方法为激素疗法和化学疗法，但是这两种治疗方法在治愈后经常复发高致病力肿瘤。因此，新的治疗方法，如通过调节前列腺肿瘤中异常表达的信号通路来达到抑制前列腺肿瘤的生长具有重要的临床应用前景。 Gli/Hedgehog(Hh)信号通路是癌细胞增殖和肿瘤生长所必须的，同时它在调节前列腺上皮生长方面也发挥着重要的作用。此信号通路靶向基因的表达量升高会驱使前列腺前体细胞向癌细胞转化，甚至导致癌细胞扩散。已有研究表明利用Gli/Hh信号通路的抑制剂环巴胺可以在体内或体外显著地抑制前列腺癌细胞的生长。但是，由于环巴胺本身存在多种缺点，如：半衰期短，非特异性毒性和它在高剂量时的非有效靶向作用都导致其不是最佳的抑制剂。因此，筛选Gli/Hh信号通路的高效抑制剂可能是预防或者治疗前列腺肿瘤的一种有效方法。 Sutherlandia Frutescens（S.Frutescens）是一种传统药用植物，它常用来治疗发热、咳嗽、感染、胃病、糖尿病和癌症等多种人类疾病。近来，它又在改善艾滋病患者的身体机能方面发挥了重要的作用。已有研究证明，S.Frutescens提取物可以抑制人前列腺癌细胞的增殖，但是它的作用机理还不是十分清楚。一些来源于植物的活性成分可以通过对Gli/Hh信号通路的调控来抑制前列腺癌细胞的增殖。因此，本研究在体外检测了S. Frutescens甲醇提取物（SLE）对前列腺癌细胞中Gli/Hh信号通路的影响；利用稳定转染Gli报告基因的小鼠胚胎成纤维细胞Shh Light II筛选SLE中抑制Gli/Hh信号通路的主要化学活性成分。在体内检测了S. Frutescens对TRAMP小鼠肿瘤的影响。得到了以下研究结果： 1.利用不同浓度的SLE处理人前列腺癌细胞PC3、LNCaP和小鼠前列腺癌细胞TRAMP-C224h、48h和72h后，发现SLE对三种癌细胞的生长抑制与剂量和时间呈正相关，即浓度越高或作用时间越长，SLE对细胞的抑制力也越强。而且在72h时，它对三种癌细胞的增殖抑制达到一半。它对三种前列腺肿瘤细胞的半数抑制浓度分别为：PC3167μg/ml、LNCaP200μg/ml和TRAMP-C2100μg/ml。但是，SLE对人正常前列腺上皮细胞RWPE-1无影响。 2.利用稳定转染Gli报告基因的小鼠胚胎成纤维细胞Shh Light II和小鼠前列腺癌细胞TRAMP-C2QGli检测SLE对Gli/Hh信号通路的影响。荧光素酶实验检测发现，无论是否用Gli/Hh信号通路的激活剂CM或者SAG激活，SLE均抑制Gli报告基因的表达，并随着剂量增加对Gli报告基因的抑制作用增强。但是，环巴胺（5μM）对Gli报告基因在未被激活的Shh Light II细胞中的表达没有影响。以上实验证明S. frutescens提取物不仅可以抑制处于活化状态的Gli/Hh信号通路，而且对非活化状态细胞Gli基础水平的表达也有抑制作用。 3.利用实时定量PCR检测SLE对人和小鼠前列腺癌细胞中Gli/Hh信号通路的影响。结果显示，当小鼠前列腺癌细胞株TRAMP-C2用含有Gli/Hh信号通路配体（ShhN+）的CM激活时，SLE和环巴胺都显著地抑制Gli1和Ptch1的表达；反之，，若没有CM激活，SLE和环巴胺的抑制作用消失。在人前列腺癌细胞PC3上，即使用CM激活PC细胞，环巴胺对Gli1和Ptch1的表达没有明显影响，SLE却仍然能显著抑制Gli1和Ptch1的表达。利用免疫印迹试验得出SLE抑制蛋白GLI1在人骨髓横纹肌肉癌细胞RMS13细胞中的表达。 4.利用高效液相色谱-蒸发光散射检测法（HPLC-ELSD）从SLE中分离鉴定环木菠萝烷Sutherlandiosides（A-D）和黄酮类Sutherlandins（A-D）的化合物或者同系化合物的混合物。荧光素酶实验筛选比较发现Sutherlandioside D对Gli报告基因的抑制力最强，Sutherlandioside D对Gli报告基因的半数抑制浓度（IC50）为1.8μg/ml，而SLE的IC50则是30μg/ml，分析发现Sutherlandioside D在SLE中的含量约为0.3%，这些数据提示Sutherlandioside D很可能是SLE中调节Gli/Hh信号通路的主要化学活性成分。 5.用含有三种浓度的S. frutescens（0.05%、0.25%和1%）日粮饲喂转基因前列腺癌小鼠（TRAMP：Transgenic adenocarcinoma of the mouse prostate），与对照组相比，0.05%的S. frutescens能明显降低TRAMP小鼠前列腺低度分化肿瘤（gross-PDC）的发生率，而且，0.05%的S. frutescens实验组的小鼠具有最小的前列腺重量。但是，在TRAMP小鼠皮下注射环巴胺既不能明显降低前列腺肿瘤的发生率，也不能推迟前列腺肿瘤的发生，甚至对前列腺肿瘤的大小也没有明显的作用。 6.免疫组化检测GLI1蛋白在0.05%S. frutescens实验组TRAMP小鼠不同分化类型肿瘤中的表达，结果表明GLI1蛋白的表达不会随着TRAMP小鼠前列腺肿瘤的癌性损伤而改变。研究表明，S. frutescens提取物在体外可以通过对Gli/Hh信号通路主要元件Gli1和Ptch1的调控来抑制人和小鼠前列腺癌细胞的生长；在体内，用含有0.05%S. frutescens日粮饲喂TRAMP小鼠可以显著地抑制小鼠前列腺低度分化肿瘤的发生率。
[Abstract]:Prostate cancer is one of the malignant tumors in the male genitourinary system. In western countries, it is second only to lung cancer, which is the second major disease that causes death for men. The main method for treating prostate cancer is hormone therapy and chemotherapy, but these two treatments often relapse high pathogenic tumor after cure. Therefore, new treatment is a new treatment. Therapies such as regulating abnormal signaling pathways in prostate tumors to inhibit the growth of prostate cancer have important clinical application prospects.
Gli/Hedgehog (Hh) signal pathway is necessary for cancer cell proliferation and tumor growth, and it also plays an important role in regulating the growth of prostate epithelium. The increase of the target gene expression in this signal pathway will drive the prostatic cells to transform to the cancer cells and even lead to the proliferation of cancer cells. Research has shown that the use of Gli/Hh The signal pathway inhibitor, cyclic amine, can significantly inhibit the growth of prostate cancer cells in vivo or in vitro. However, due to the existence of a variety of shortcomings, such as short half-life, non specific toxicity, and its non effective targeting at high doses, it is not the best inhibitor. Therefore, the Gli/Hh signal pathway is screened. Highly effective inhibitors may be an effective way to prevent or treat prostate cancer.
Sutherlandia Frutescens (S.Frutescens) is a traditional medicinal plant. It is often used to treat a variety of human diseases such as fever, cough, infection, stomach disease, diabetes and cancer. Recently, it has played an important role in improving the physical function of AIDS patients. Research has shown that the S.Frutescens extract can inhibit the human prostate. Cancer cell proliferation, but its mechanism is not very clear. Some of the active components of the plant can inhibit the proliferation of prostate cancer cells by regulating the Gli/Hh signaling pathway. Therefore, the effect of S. Frutescens methanol extract (SLE) on the Gli/Hh signaling pathway in prostate cancer cells is detected in this study. The main chemical active components of the Gli/Hh signaling pathway in SLE were screened using Shh Light II, a mouse embryonic fibroblast stable transfected with Gli reporter gene. The effects of S. Frutescens on the TRAMP mouse tumor were detected in the body. The following results were obtained:
1. using different concentrations of SLE to treat human prostate cancer cells PC3, LNCaP and mouse prostate cancer cells TRAMP-C224h, 48h and 72h, it was found that the growth inhibition of SLE on three kinds of cancer cells was positively correlated with the dose and time, that is, the higher the concentration or the longer the action time, the stronger the inhibition of SLE to the cells. And at 72h, it is to three kinds of cancer cells. The inhibitory concentration of proliferation to three kinds of prostate tumor cells was PC3167 mu g/ml, LNCaP200 mu g/ml and TRAMP-C2100 micron g/ml., but SLE had no effect on human normal prostate epithelial cells RWPE-1.
2. the effect of SLE on Gli/Hh signaling pathway was detected by Shh Light II in mouse embryonic fibroblasts and TRAMP-C2QGli of mouse prostate cancer cells with stable transfection of Gli reporter gene. The luciferase test found that SLE inhibited the expression of the Gli reporter gene, whether or not it was activated by the activator of Gli/Hh signaling pathway, and with the agent. The inhibition of the Gli reporter gene was enhanced. However, the expression of the Gli reporter gene was not affected by the expression of the Gli reporter gene in the non activated Shh Light II cells. The above experiments showed that the S. frutescens extract could not only inhibit the Gli/Hh signaling pathway in the activated state, but also the table of the non activated state cell Gli base level. It also has an inhibitory effect.
3. the effect of SLE on the Gli/Hh signaling pathway in human and mouse prostate cancer cells was detected by real-time quantitative PCR. The results showed that both SLE and cyclic amine significantly inhibited the expression of Gli1 and Ptch1 when the mouse prostate cancer cell line TRAMP-C2 was activated by CM containing the Gli/Hh signaling ligand (ShhN+); conversely, if no CM activation was performed, SLE and cyclic amines were not activated. The inhibitory effect disappeared. On human prostate cancer cell PC3, the use of CM to activate PC cells, the expression of Gli1 and Ptch1 was not significantly affected by cyclic amine, but SLE still significantly inhibited the expression of Gli1 and Ptch1. The expression of SLE suppressor protein GLI1 in the RMS13 cells of human bone marrow rhabdomystriated muscle cancer cells was obtained by immunoblot test.
4. high performance liquid chromatography - evaporative light scattering detection (HPLC-ELSD) was used to separate and identify compounds or homologous compounds of pineapple pineapple Sutherlandiosides (A-D) and flavonoids Sutherlandins (A-D) from SLE. The luciferase test showed that Sutherlandioside D had the strongest inhibitory effect on the Gli reporter gene, Sutherlandio. The median inhibitory concentration (IC50) of side D to the Gli reporter gene was 1.8 mu g/ml, while the IC50 of SLE was 30 mu g/ml. The analysis found that Sutherlandioside D was about 0.3% in SLE.
5. the transgenic prostate cancer mice (TRAMP:Transgenic adenocarcinoma of the mouse prostate) were fed with three concentrations of S. frutescens (0.05%, 0.25% and 1%). Compared with the control group, 0.05% S. frutescens could significantly reduce the incidence of low-grade prostate cancer (gross-PDC) in TRAMP mice, and 0.05% The mice in the experimental group had the smallest weight of the prostate. However, the subcutaneous injection of cyclic amine in TRAMP mice could not significantly reduce the incidence of prostate tumors, nor delayed the occurrence of prostate tumors, and even had no significant effect on the size of the prostate tumor.
6. immunohistochemistry was used to detect the expression of GLI1 protein in 0.05%S. frutescens experimental group TRAMP mice with different differentiated types of tumor. The results showed that the expression of GLI1 protein did not change with the carcinomatous damage of the prostate tumor of TRAMP mice.
The study shows that S. frutescens extract can inhibit the growth of human and mouse prostate cancer cells by regulating the main components of Gli/Hh signaling pathway Gli1 and Ptch1 in vitro. In vivo, the rate of low differentiation tumor of mouse anterior gland can be inhibited significantly by feeding TRAMP mice with 0.05%S. frutescens diet.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

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