敲低前列腺癌相关基因PC-1的表达抑制前列腺癌细胞C4-2雄激素非依赖性生长

目的:研究敲低癌基因D52家族成员PC-1的表达对前列腺癌细胞雄激素非依赖性生长的影响。方法:利用RNA干扰技术构建PC-1稳定低表达的C4-2细胞株;利用四唑盐(MTT)比色实验检测敲低PC-1基因表达对C4-2细胞雄激素非依赖生长的影响。结果:敲低PC-1表达抑制前列腺癌C4-2细胞的生长,并降低了C4-2细胞雄激素非依赖性生长的能力。结论:PC-1基因参与了前列腺癌向雄激素非依赖阶段发展和维持的过程,为进一步研究PC-1在促进前列腺癌细胞发生发展过程中的作用奠定了基础。     

前列腺癌雄激素受体和PI3K/Akt信号通路相互作用研究进展

前列腺癌的发生、进展依赖于雄激素,因此去势手术成为治疗晚期前列腺癌的标准疗法。但是去势后大多前列腺癌最终将转化为雄激素非依赖性前列腺癌,甚至进展为激素难治性前列腺癌,使得肿瘤的进展不受低水平雄激素的影响。即使如此,大多数激素非依赖性前列腺癌,依然阳性表达雄激素受体。因而雄激素受体在前列腺癌发生发展中起着重要作用。而PI3K/Akt信号通路能够通过维持细胞生存、抑制细胞凋亡、促进细胞代谢及血管生成等促进前列腺癌进展。本综述旨在总结前人研究,阐述雄激素受体和PI3K/Akt信号通路之间相互作用关系。研究表明Akt信号通路能够正性或者负性调控AR蛋白表达、蛋白的稳定性及其转录活性,从而维持细胞的生存、代谢。而AR即可以通过基因转录途径抑制Akt活化又能通过非转录基因途径激活Akt及其下游蛋白。因此,AR和Akt信号通路相互协同促进前列腺癌的发生及其向雄激素非依赖性前列腺癌进展。     

前列腺癌雄激素受体和PI3K/Akt 信号通路相互作用研究进展

前列腺癌的发生、进展依赖于雄激素,因此去势手术成为治疗晚期前列腺癌的标准疗法。但是去势后大多前列腺癌最终将转化为雄激素非依赖性前列腺癌,甚至进展为激素难治性前列腺癌,使得肿瘤的进展不受低水平雄激素的影响。即使如此,大多数激素非依赖性前列腺癌,依然阳性表达雄激素受体。因而雄激素受体在前列腺癌发生发展中起着重要作用。而PI3K/Akt信号通路能够通过维持细胞生存、抑制细胞凋亡、促进细胞代谢及血管生成等促进前列腺癌进展。本综述旨在总结前人研究,阐述雄激素受体和PI3K/Akt信号通路之间相互作用关系。研究表明Akt信号通路能够正性或者负性调控AR蛋白表达、蛋白的稳定性及其转录活性,从而维持细胞的生存、代谢。而AR即可以通过基因转录途径抑制Akt活化又能通过非转录基因途径激活Akt及其下游蛋白。因此,AR和Akt信号通路相互协同促进前列腺癌的发生及其向雄激素非依赖性前列腺癌进展。     

齐墩果酸诱导人非小细胞肺癌A549细胞凋亡的作用机制研究

齐墩果酸(oleanolic acid,OA)是一种广泛存在于自然界中的五环三萜类天然化合物,已有研究表明OA具有抗肺癌活性,但是具体机制尚未完全阐释清楚。本研究旨在证实OA是否能够诱导非小细胞肺癌A549细胞凋亡,并探索该效应是否与线粒体凋亡通路相关。我们将不同浓度的OA作用于细胞后,使用MTT法检测A549细胞的生长情况。采用AV/PI法进行染色后,经流式细胞仪检测其凋亡率,同时使用Hoechst 33342染色并使用荧光显微镜观察并拍照;Westen-blotting检测OA对A549细胞的线粒体凋亡通路相关蛋白相对表达水平的影响。MTT结果显示OA能够抑制A549细胞的生长,并呈时间剂量依赖性。AV/PI法染色结果显示OA能够诱导A549细胞凋亡,亦呈时间剂量依赖性。Hoechst 33342法染色结果相似。Westen-blotting结果表明OA能够上调A549细胞线粒体凋亡通路相关蛋白Bax,t Bid,Cleaved caspase 3的相对表达水平,同时下调该通路Bcl-xl,Bcl-2的相对表达水平。以上所有的结果表明OA能够显著抑制肺癌A549细胞的生长并通过调节线粒体凋亡通路相关蛋白的表达水平而诱导其凋亡。     

miR-221对于前列腺癌细胞的增殖及侵袭能力作用的研究

目的:研究miR-221不同时期前列腺癌细胞系中表达差异,探讨miR-221在雄激素非依赖前列腺癌的生长及侵袭能力中发挥的作用.方法:Northern检测雄激素依赖性与雄激素非依赖性前列腺癌细胞系中差异表达的miRNA.在不同前列腺癌细胞系中,通过CCK-8及流式细胞学检测基因转染前后miR-221对于前列腺癌细胞系的生长影响,以及通过侵袭实验检测细胞侵袭能力的变化.结果:①Northern显示LNCaP-AI细胞中miR-221水平明显高于LNCaP细胞;②miR-221促进LNCaP及LNCaP-AI细胞的增殖能力③下调miR-221会减弱LNCaP-AI细胞的侵袭能力.结论:miR-221促进不同时期前列腺癌细胞系的增殖,并增强LNCaP-AI的侵袭能力.     

澳洲茄碱诱导胆管癌QBC939细胞凋亡及作用机制

研究澳洲茄碱对人胆管癌上皮细胞系QBC939凋亡的诱导与作用机制。采用MTT法检测不同浓度澳洲茄碱对QBC939细胞的增殖抑制作用;流式细胞术检测澳洲茄碱对QBC939细胞的凋亡诱导情况;Western blot检测澳洲茄碱对QBC939细胞中凋亡相关蛋白(Bax、Bcl-2、caspase3、caspase7、PARP、cleaved PARP)的表达影响。结果发现澳洲茄碱以浓度依赖方式能够抑制QBC939细胞的增殖;澳洲茄碱能够显著诱导QBC939细胞的凋亡;澳洲茄碱可以上调Bax、caspase3、caspase7和cleaved PARP蛋白表达,下调Bcl-2和PARP蛋白表达。表明澳洲茄碱能够通过改变凋亡相关蛋白表达,诱导胆管癌QBC939细胞的凋亡,这对于研发和治疗胆管癌相关药物具有一定的潜在价值。     

雄激素受体共调节因子与雄激素非依赖性前列腺癌

雄激素介导的雄激素受体（AR）信号途径对雄性胚胎的发育及雄激素依赖性靶组织的分化发育是必需的。异常的AR活性与前列腺癌由雄激素依赖转变为雄激素非依赖性密切相关。已证实AR共调节因子参与前列腺癌的发生和发展,并在雄激素非依赖性前列腺癌细胞的增殖中扮演着重要角色。它们的表达失衡,可导致AR转录活性的改变,促进晚期前列腺癌的进展。简要综述了AR共调节因子的类型和功能,及其与雄激素非依赖性前列腺癌的关系。     

硫利达嗪通过线粒体应激信号通路诱导肺腺癌细胞发生免疫原性死亡

硫利达嗪作为吩噻嗪类抗精神病类药物,具有诱导肿瘤发生免疫原性死亡(ICD)、激活特异性免疫反应的潜力。本研究利用A549和H1299细胞探讨了硫利达嗪诱导肺腺癌细胞免疫原性死亡及其分子机制。将不同浓度的硫利达嗪与A549和H1299细胞共孵育24 h后,利用四甲基偶氮唑蓝(MTT)法检测细胞的存活及生长情况,利用流式细胞术分析细胞凋亡率,利用ATP检测试剂盒检测细胞上清中的ATP含量,利用免疫荧光法检测细胞表面钙网蛋白(CRT)的表达,利用蛋白质印迹(Western blot)法检测凋亡相关蛋白裂解的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase 3)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt C)的表达水平。结果表明,硫利达嗪能够显著抑制A549和H1299细胞的增殖,促进细胞凋亡,细胞增殖抑制及凋亡呈浓度依赖性。ATP分泌量增加及细胞表面CRT的表达水平上调,表明上述细胞发生了免疫原性死亡。而Bcl-2表达水平下降,Bax和Cyt C以及Cleaved caspase 3表达水平上调,进一步证明了硫利达嗪可诱导肿瘤细胞凋亡。上述结果表明,硫利达嗪可通过线粒体应激信号通路诱导肺腺癌细胞发生免疫原性死亡,从而抑制肿瘤细胞的增殖。     

猪丁型冠状病毒诱导的细胞线粒体凋亡

猪丁型冠状病毒(Porcine deltacoronavirus,PDCoV)是一种新型的猪肠道致病性冠状病毒,可引起猪群剧烈腹泻及呕吐,但致病机制尚不清楚。本研究检测了PDCoV感染诱导的细胞凋亡。Caspase酶活性检测显示,在PDCoV感染的细胞中,caspase 3、caspase 8和caspase 9的活性随病毒感染量的增多而显著提高,类似的现象未能在紫外灭活病毒感染的细胞中观察到,表明PDCoV感染可同时激活内源性与外源性细胞凋亡通路,并暗示细胞凋亡的诱导依赖于病毒复制。为深入探究PDCoV诱导的内源性细胞凋亡,分别检测胞浆和线粒体中细胞色素C与凋亡诱导因子。结果显示,与正常细胞相比,PDCoV感染细胞从线粒体释放到胞浆的细胞色素C显著增多,且其释放量随着感染时间的延长而增多,而凋亡诱导因子始终定位于线粒体,提示PDCoV感染通过促使线粒体膜间隙的细胞色素C进入胞浆而启动caspase依赖的线粒体凋亡通路。本研究初步揭示了PDCoV诱导细胞凋亡的机制。     

Nutlins类似物NL-86诱导HeLa细胞凋亡的研究

目的 观察新型Nutlins类似物NL-86在体外诱导宫颈癌HeLa细胞凋亡的作用,并初步探讨其作用的分子机制.方法 采用MTT法检测NL-86化合物对HeLa细胞增殖的影响;用 FITC-Annexin V及碘化丙锭（PI）双染法,通过流式细胞仪（FCM）检测NL-86诱导HeLa细胞凋亡情况;用Western 印迹检测PARP[poly（ADP-ribose）polymerase]、pro-caspase 3、8、9的变化,并利用活力检测试剂盒检测caspase 3、8、9的活力,初步确定其诱导凋亡的通路.结果 不同浓度的NL-86 对于HeLa细胞的存活率均具有一定影响,并以浓度依赖的方式诱导HeLa细胞凋亡;随着NL-86浓度的增加,PARP被切割、HeLa 细胞pro-casepase 3、8的含量下降,但 pro-caspase 9无变化.活力测定结果显示caspase 3、8被激活,caspase 9无变化.结论 NL-86化合物能够有效地在体外诱导HeLa 细胞凋亡,且可能依赖于caspase 3、8相关的死亡受体凋亡途径,为进一步研发治疗宫颈癌等肿瘤疾病的药物奠定了实验基础.     

Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3

Prostate carcinoma is one of the most common malignant tumors and has become a more common cancer in men. Previous studies demonstrated that evodiamine (EVO) exhibited anti-tumor activities on several cancers, but its effects on androgen-independent prostate cancer are unclear. In the present study, the action mechanisms of EVO on the growth of androgen-independent prostate cancer cells (DU145 and PC3 cells) were explored. EVO dramatically inhibited the growth and elevated cytotoxicity of DU145 and PC3 cells. The flow cytometric analysis of EVO-treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest was accompanied by elevated Cdc2 kinase activity, an increase in expression of cyclin B1 and phosphorylated Cdc2 (Thr 161), and a decrease in expression of phosphorylated Cdc2 (Tyr 15), Myt-1, and interphase Cdc25C. TUNEL examination showed that EVO-induced apoptosis was observed at 72 h. EVO elevated the activities of caspase 3, 8, and 9 in DU145 cells, while in PC3 cells only the activities of caspase 3 and 9 were elevated. EVO also triggered the processing of caspase 3 and 9 in both DU145 and PC3 cells. We demonstrate that roscovitine treatment result in the reversion of G2/M arrest in response to EVO in both DU145 and PC3. However, inhibitory effect of roscovitine on EVO-induced apoptosis could only be observed in DU145 rather than PC3. In DU145, G2/M arrest might be a signal for initiation of EVO-triggered apoptosis. Whereas EVO-triggered PC3 apoptosis might be independent of G2/M arrest. These results suggested that EVO inhibited the growth of prostate cancer cell lines, DU145 and PC3, through an accumulation at G2/M phase and an induction of apoptosis.     

Pretreatment of docetaxel enhances TRAIL-mediated apoptosis in prostate cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In this study, we examined whether treatment of docetaxel (DTX) can enhance apoptotic cell death by TRAIL against androgen-independent prostate cancer (AIPC). The cell death effect of combinations of TRAIL and docetaxel on prostate cancer cell lines (androgen-dependent LNCaP and its derived androgen-independent, metastatic C4-2B) was evaluated by synergisms of apoptosis. Western blot assay and DNA fragmentation assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL induced apoptosis in the presence of docetaxel. In addition, we investigated the in vitro anti-tumor effects of combined docetaxel and TRAIL using MAP kinase inhibitors. Docetaxel itself could not induce apoptotic cell death in 24 h even in high concentration. Apoptotic cell death, however, was drastically enhanced by pretreatment of docetaxel 20 h before TRAIL treatment. Docetaxel enhanced the PARP-1 cleavage and caspases activation by TRAIL especially in androgen-independent, metastatic C4-2B cell line, mainly by phosphorylation of Bcl-2 by JNK activation. It appears that apoptotic cell death was protected by the JNK inhibitor SP600125. The results of our study show that pretreatment of docetaxel is able to enhance the apoptosis produced by TRAIL in prostate cancer cells, especially in hormone-refractory prostate cancer (HRPC).     

Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB

Background

Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC) with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.

Methodology/Principal Findings

We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1), with IC₅₀ in the range of 1–2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.

Conclusions/Significance

Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be developed as a new therapeutic agent for hormone-refractory prostate cancer.     

Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer

The tumor necrosis factor (TNF) receptor super family comprises of members that induce two distinct signaling cascades, leading to either cell survival or apoptosis. However, in prostate cancer (PCa), TNF-mediated prosurvival signaling is the predominant pathway that leads to cell survival and resistance to therapy. Although inhibition of TNF signaling by pharmacological agents or monoclonal antibodies has gained importance in the field of cancer therapy, toxicity to normal cells has impaired their extensive use for cancer treatment. We previously identified a natural, nontoxic compound psoralidin that inhibited viability and induced apoptosis in androgen independent prostate cancer (AIPC) cells. Thus, the goal of our study is to investigate whether psoralidin inhibits TNF-mediated prosurvival signaling in AIPC cells. Our results suggest that psoralidin inhibits constitutive and TNF-induced expression of TNF-α and its downstream prosurvival signaling molecules such as NF-κB and Bcl-2 in AIPC cells. On the other hand, psoralidin simultaneously induces the death receptor (DR)-mediated apoptotic signaling eventually causing the activation of caspase cascade and resultant induction of apoptosis. Oral administration of psoralidin inhibits expression of TNF-α and NF-κB/p65 in tumor sections, resulting in tumor regression in PC-3 xenografts. Our results suggest that psoralidin inhibits TNF-mediated survival signaling in AIPC and thus is a potent therapeutic agent for prostate cancer.     

2-Methoxyestradiol induces G2/M arrest and apoptosis in prostate cancer

Few therapeutic treatment options are available for patients suffering from metastatic androgen-independent prostate cancer. We investigated the ability of the estrogen metabolite 2-methoxyestradiol to inhibit the proliferation of a variety of human prostate cancer cell lines in vitro and to inhibit the growth of androgen-independent prostate cancer in a transgenic mouse model in vivo. Our results showed that 2-methoxyestradiol is a powerful growth inhibitor of LNCaP, DU 145, PC-3, and ALVA-31 prostate cancer cells. Cell flow cytometry of 2-methoxyestradiol-treated DU 145 cells showed a marked accumulation of cells in the G2/M phase of the cell cycle and an increase in the sub-G1 fraction (apoptotic). In addition, staining for annexin V, changes in nuclear morphology, and inhibition of caspase activity support a role for apoptosis. More importantly, we showed that 2-methoxyestradiol inhibits prostate tumor progression in the Ggamma/T-15 transgenic mouse model of androgen-independent prostate cancer without toxic side effects. These results in cell culture and an animal model support investigations into the clinical use of 2-methoxyestradiol in patients with androgen-independent prostate cancer.     

Docetaxel and thalidomide as a treatment option for androgen- independent, nonmetastatic prostate cancer

Prostate cancer usually presents with early-stage disease, yet a significant proportion of patients present or will progress to androgen-independent, nonmetastatic prostate cancer (AIPC). Chemotherapy has demonstrated statistically significant improvements in palliation of AIPC. Docetaxel in particular has demonstrated high response rates as a single agent. Thalidomide is effective in treating many malignancies, including prostate cancer. Thalidomide may act synergistically with docetaxel through their antiangiogenic effects. We performed a phase II trial of docetaxel with or without thalidomide in patients with AIPC and demonstrated encouraging response rates with combination therapy. We advocate further investigation of this promising combination regimen.     

Proteomic Profiling of Androgen-independent Prostate Cancer Cell Lines Reveals a Role for Protein S during the Development of High Grade and Castration-resistant Prostate Cancer

Androgen deprivation constitutes the principal therapy for advanced and metastatic prostate cancers. However, this therapeutic intervention usually results in the transition to a more aggressive androgen-independent prostate cancer. The elucidation of molecular alterations during the progression to androgen independence is an integral step toward discovering more effective targeted therapies. With respect to identifying crucial mediators of this transition, we compared the proteomes of androgen-independent (PC3, DU145, PPC1, LNCaP-SF, and 22Rv1) and androgen-dependent (LNCaP and VCaP) and/or normal prostate epithelial (RWPE) cell lines using mass spectrometry. We identified more than 100 proteins that were differentially secreted in the androgen-independent cell lines. Of these, Protein S (PROS1) was elevated in the secretomes of all of the androgen-independent prostate cancer cell lines, with no detectable secretion in normal and androgen-dependent cell lines. Using quantitative PCR, we observed significantly higher (p < 0.05) tissue expression levels of PROS1 in prostate cancer samples, further indicating its importance in prostate cancer progression. Similarly, immunohistochemistry analysis revealed elevation of PROS1 in high grade prostate cancer (Gleason grade ≥8), and further elevation in castration-resistant metastatic prostate cancer lesions. We also observed its significant (p < 0.05) elevation in high grade prostate cancer seminal plasma samples. Taken together, these results show that PROS1 is elevated in high grade and castration-resistant prostate cancer and could serve as a potential biomarker of aggressive disease.     

Valproic acid inhibits the invasion of PC3 prostate cancer cells by upregulating the metastasis suppressor protein NDRG1

Valproic acid (VPA) is a clinically available histone deacetylase inhibitor with promising anticancer attributes. Recent studies have demonstrated the anticancer effects of VPA on prostate cancer cells. However, little is known about the differential effects of VPA between metastatic and non-metastatic prostate cancer cells and the relationship between the expression of metastasis suppressor proteins and VPA. In the present study, we demonstrate that inhibition of cell viability and invasion by VPA was more effective in the metastatic prostate cancer cell line PC3 than in the tumorigenic but non-metastatic prostate cell line, RWPE2. Further, we identified that the metastasis suppressor NDRG1 is upregulated in PC3 by VPA treatment. In contrast, NDRG1 was not increased in RWPE2 cells. Also, the suppressed invasion of PC3 cells by VPA treatment was relieved by NDRG1 knockdown. Taken together, we suggest that the anticancer effect of VPA on prostate cancer cells is, in part, mediated through upregulation of NDRG1. We also conclude that VPA has differential effects on the metastasis suppressor gene and invasion ability between non-metastatic and metastatic prostate cancer cells.     

Combined effect of sodium selenite and docetaxel on PC3 metastatic prostate cancer cell line

Docetaxel and sodium selenite are well known for their anticancer properties. While resistance to docetaxel remains an obstacle in prostate cancer chemotherapy, sodium selenite, has been exploited as a new therapeutic approach. Currently, development of therapies affecting a multitude of cell targets, have been proposed as a strategy to overcome drug resistance. This association may reduce systemic toxicity counteracting a wide range of side effects.Here we report the effect of docetaxel and sodium selenite combination on the PC3 prostate cancer cell line, derived from bone metastasis. Therefore we evaluate cell growth, cell cycle progression, viability, mitochondria membrane potential, cytochrome C, Bax/Bcl2 ratio, caspase-3 expression and reactive oxygen species production.Our results suggest that sodium selenite and docetaxel combination have a synergistic effect on cell growth inhibition (67%) compared with docetaxel (22%) and sodium selenite (24%) alone. This combination also significantly induced cell death, mainly by late apoptosis vs necrosis, which is correlated with mitochondria membrane potential depletion. On the other hand, cytochrome C, Bax/Bcl2 ratio and caspase-3, known as proapoptotic factors, significantly increased in the presence of sodium selenite alone, but not in the presence of docetaxel in monotherapy or in combination with sodium selenite.These findings suggest that docetaxel and sodium selenite combination may be more effective on prostate cancer treatment than docetaxel alone warranting further evaluation of this combination in prostate cancer therapeutic approach.