【作者】 时平；

【导师】 张建业；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 前列腺癌不仅是美国男性最普遍发生的恶性肿瘤,而且在亚洲男性中的发病率也呈逐渐升高的趋势。发生转移的前列腺癌病人虽然受益于最初的雄激素去势疗法,但大部分病人死于后期复发的激素顽固性前列腺癌(HPRC)。流行病学研究认为亚洲地区大肠癌低发病率的一部分原因是由于印度和东南亚国家食物中添加了植物成分姜黄素(Curcumin,Cur)。对于Cur在动物模型中的分子生物学机制的研究显示出了它具有抑制多种肿瘤细胞生长和增殖的特性。Hung等发现Cur在鼠的纤维细胞中可以抑制肿瘤激活剂佛波酯(TPA)诱导肿瘤发生的转录因子C-Jun/AP-1,从而抑制C-Jun/AP-1介导的基因表达。Kawamori等人研究Cur还可以通过抑制PKC抑制佛波酯诱导的进展期的大肠癌的发展。Cur通过抑制特异的蛋白激酶来抑制许多原癌基因如c-fos,c-jun和c-myc等等。同时Cur可以诱导人T细胞和HL-60细胞的凋亡。这些研究都显示了Cur本身具有肿瘤化学防御的特点。Cur由于它的几乎可以被忽略的系统毒性已经被列为安全类药,所以它是一种开发前景很好的抗肿瘤药物。 1994年,Zou等人通过减数杂交和差异显示发现Maspin(mammary serpin)表达于正常的乳腺上皮细胞中。Maspin所编码的蛋白产物是丝氨酸蛋白酶抑制剂serpin(serine protease inhibitor)家族的成员,因此命名为maspin(mammary serine更多还原

【Abstract】 Prostate cancer is the most common US cancer in men and is the second leading cause of male cancer deaths in the western world. Current therapies, such as surgery, chemotherapy, and radiation therapy, are of limited efficacy, especially in advanced disease, and metastasis disease remains inCurable. Androgen reduction therapy is commonly used to control hormone-sensitive tumor cells; however, hormone refractory clones often emerge after hormonal therapy. Therefore, novel therapies, including biotherapy, are needed to treat adenocarcinoma of prostate while it is still confined to the gland and potentially Curable. In contrast to the high incidence of prostate cancer in North America, the incidence of this disease in Japan and China is very low. The low incidence of prostate cancer in Asian men has been attributed to the dietary consumption of large amounts of plant-based foods rich in phytochemicals. Because of these observations, nutritional supplements such as soybean, garlic, green tea, and so on, which are rich in polyphenolic compounds, have been used to augment the anticancer therapies. One such unique dietary factor in Asian cuisine, apart from the widespread use of soy-based products, is Curcumin (turmeric), extracted from the rhizome of the plant Curcuma longa L, which has received a great deal of attention recently as chemoprotective agent for the prevention of colon and breast cancer and may have clinical application in the prevention of prostate cancer.The molecular mechanisms of Curcumin action include effects on a number of signaling pathways and transcription factors. Curcumin has been shown to decrease the expression and activities of proto-oncogenes such as c-fos, c-jun and c-myc, possibly through the inhibition of specific protein kinases.21. It also inhibits a number of kinase and enzyme activities, including protein kinase C, EGF receptor (EGF-R), and erbB2. Curcumin affects the expression of a number of cell cycle proteins, including thymidine kinase, ras, cyclin E, PCNA, and p21. It has also been found to alter the expression of apoptosis related proteins such as Bax, Bcl-2, and p53.In spite of these studies, large gaps exist in our knowledge of how Curcumin functions as an anti-tumorigenic agent. For example, little information is available regarding the effects of Curcumin on tumor suppressor genes. One such gene, maspin (mammary serine protease inhibitor), was identified by subtractive hybridization and differential display analysis to compare normal mammary epithelium to invasive carcinoma cells. In human breast cancer specimens, maspin expression inversely correlated with tumor progression and malignancy grade. Recently, it was found the maspin works as an effective inhibitor of angiogenesis. Maspin has been classified as a class II tumor suppressor gene because no genetic mutations, deletions, or rearrangements have been found in maspin in association with its loss of expression. The following research showed that maspin is a tumor suppressor gene to inhibit tumor growth, cell motility and metastasis. The development of the prostate depends on androgenic hormones and therefore, its tumorigenesis may result from similar molecular events as that in breast cancer. In human primary PC s (prostate cancer), maspin expression consistently appears to be down-regulated at the critical transition from noninvasive, low-grade disease to highly invasive, high-grade PC. Maspin is regulated at transcriptional level differently in normal and carcinoma prostate cancer cells. By promoter analysis, two cis elements— the Ets and hormonal responsive element (HRE) sites have been identified as involved in transcriptional activation and repression. In prostate tumor cells, the Ets element is inactive in up-regulation of maspin expression while the negative element HRE remains active which isrecognized by the androgen receptor. With the predominance of negative regulation by HRE, the expression of maspin protein is lost during tumor progression. From the therapeutic point of view, re-expression of maspin in the prostate tumors offers great hope for reversing the tumor phenotypes. In our experiment, curcumin down-regulates the AR gene not only in protein level but also in its transcriptional activity in androgen-depended prostate cancer cell-LNCaP. The suppression of AR transaction may affect the AR-regulated genes such as maspin. In the Current study, we evaluated the effects on the growth inhibition of LNCaP and the regulation of maspin both in promoter and expression levels. Functional AR maybe the a factor that works between Curcumin and maspin. The findings of this study uncover a novel function of Curcumin in altering a cancer suppressive gene*.Objective: the aim of our experiment is to detect the effect of Curcumin on the apoptosis of LNCaP and the effect of Curcumin on the regulation and expression of maspin.Methods and results: Different doses of Curcumin were added to the culture of LNCaP. After different periods, the viability of the cells was assayed through MTT. Curcumin has the maximum inhibition rate of viability at the nontoxic dose (40uM) after 48h. Then we added the maximum nontoxic dose of Cur to the culture of LNCaP. After different period, we collected the general cell DNA. The electrophoresis of DNA demonstrated the DNA ladder and the apoptosis rate increased with the time extension. The following we did was that we treated LNCaP with different doses from lOuJVI to 40uM and total cell RNA and cytoplasm protein were extracted. Then we detect the effect of Curcumin on both RNA and protein level of maspin through RT-PCR and Western-blotting. The outcome showed that Curcumin increased the expression of maspin with dose and time dependence in both levels. Based on above outcome, pGLi-maspin luciferase expression vector containing 847bp (-764—h83 ) DNA of maspin gene 5; promoter region was transfected into LNCaP cell with lipofectamin? 2000. pRL-TK was cotransfected as an intern control. Through detecting the activity of luciferase, the effect of Curcumin on the promoter of maspin was studied. Our experiment showed that after 24h of 40uM Curcumin addition, theluciterase activity reached its optimal increase. The increase activity is both dose and time dependence. To further prove whether the effect of Cur on the expresson of maspin is through inhibiting the expression of AR gene, we use the RT-PCR and Western-blotting to detect the change of AR after Cur addition. Conclusion:1) Cur inhibits the viability of LNCaP cell. The inhibition rate is 40% under 40uM dose of Cur afer 48h.2) Cur indices apoptosis of LNCaP cell. The effect is time dependence and reaches the maximum effect with 40uM treatment.3) Cur increases the expression of maspin protein through maspin promoter. Cur can inhibit the combination of AR with ARE (the negtive responsive element.in the promoter of maspin) by supressing the expression of AR protein.更多还原