Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage

Bcl-2 is an anti-apoptotic oncoprotein and its protein levels are inversely correlated with prognosis in many cancers.However, the role of Bcl-2 in the progression of prostate cancer is not clear. Here we report that Bcl-2 is required for theprogression of LNCaP prostate cancer cells from an androgen-dependent to an androgen-independent growth stage. ThemRNA and protein levels of Bcl-2 are significantly increased in androgen-independent prostate cancer cells. shRNA-medi-ated gene silencing of Bcl-2 in androgen-independent prostate cancer cells promotes UV-induced apoptosis and suppressesthe growth of prostate tumors in vivo. Growing androgen-dependent cells under androgen-deprivation conditions resultsin formation of androgen-independent colonies; and the transition from androgen-dependent to androgen-independentgrowth is blocked by ectopic expression of the Bcl-2 antagonist Bax or Bcl-2 shRNA. Thus, our results demonstratethat Bcl-2 is not only critical for the survival of androgen-independent prostate cancer cells, but is also required for theprogression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage.     

Knockdown of SRPX2 inhibits the proliferation,migration, and invasion of prostate cancer cells through the PI3K/Akt/mTOR signaling pathway

Sushi repeat‐containing protein X‐linked 2 (SRPX2), a novel chondroitin sulfate proteoglycan, is reported to play a critical role in tumorigenesis. However, the expression and functional role of SRPX2 in prostate cancer have not been defined. Thus, the aim of this study was to investigate the expression and functional role of SRPX2 in human prostate cancer. Our results showed that the expression of SRPX2 was obviously increased in human prostate cancer tissues and cell lines. In addition, knockdown of SRPX2 inhibited the proliferation, migration, and invasion of prostate cancer cells, as well as prevented the epithelial‐mesenchymal transition process in prostate cancer cells. Mechanically, knockdown of SRPX2 efficiently inhibited the activation of PI3K/Akt/mTOR pathway in prostate cancer cells. Taken together, these data demonstrated that knockdown of SRPX2 inhibits the proliferation and metastasis in human prostate cancer cells, partly through the PI3K/Akt/mTOR signaling pathway. Thus, SRPX2 may be a novel therapeutic target for the treatment of prostate cancer.     

Sensitization of prostate carcinoma cells to Apo2L/TRAIL by a Bcl-2 family protein inhibitor

Overexpression of anti-apoptotic Bcl-2 family proteins may play an important role in the aggressive behavior of prostate cancer cells and their resistance to therapy. The Bcl-2 homology 3 domain (BH3) is a uniquely important functional element within the pro-apoptotic class of the Bcl-2-related proteins, mediating their ability to dimerize with other Bcl-2-related proteins and promote apoptosis. The BH3 inhibitors (BH3Is) function by disrupting the interactions mediated by the BH3 domain between pro- and anti-apoptotic members of the Bcl-2 family and liberating more Bax/Bak to induce mitochondrial membrane permeabilization. LNCaP-derived C4-2 human prostate cancer cells are quite resistant to non-tagged, human recombinant soluble Apo2 ligand [Apo2L, also Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL], a tumor specific drug that is now in clinical trials. However, when Apo2L/TRAIL was combined with the Bcl-xL inhibitor, BH3I-2′, it induced apoptosis synergistically through activation of Caspase-8 and the proapoptotic Bcl-2 family member Bid, resulting in the activation of effector Caspase-3 and proteolytic cleavage of Poly(ADP-ribose) polymerase, events that were blocked by the pan-caspase inhibitor zVAD-fmk. Our data indicate that, in combination with the BH3 mimetic, BH3I-2′, Apo2L/TRAIL synergistically induces apoptosis in C4-2 human prostate cancer cells through both the extrinsic and intrinsic apoptotic pathways.     

The significance of galectin-3 as a new basal cell marker in prostate cancer

Prostate cancer may originate from distinct cell types, resulting in the heterogeneity of this disease. Galectin-3 (Gal-3) and androgen receptor (AR) have been reported to play important roles in the progression of prostate cancer, and their heterogeneous expressions might be associated with different cancer subtypes. Our study found that in various prostate cancer cell lines Gal-3 expression was always opposite to AR expression and other luminal cell markers but consistent with basal cell markers including glutathione S-transferase-π and Bcl-2. This expression pattern was confirmed in human prostate cancer tissues. Our results also showed that prostate cancer cells positive with basal cell markers were more aggressive. Downregulation of Gal-3 expression resulted in increased apoptotic potential and decreased metastasis potential of prostate cancer cells. Our findings demonstrate for the first time that Gal-3 may serve as a new marker for basal characteristics of prostate cancer epithelium. This study helps us to better understand the heterogeneity of prostate cancer. The clinical significance of this study lies in the application of Gal-3 to distinguish prostate cancer subtypes and improve treatment efficacy with designed personalized therapy.     

miR-125b在前列腺癌1E8细胞运动转移中的作用及其分子机制的初步探讨

目的 研究 miR-125b在前列腺癌高低转移潜能细胞中的表达差异及其对高转移细胞株1E8细胞的运动转移中的作用和可能的分子机制.方法 realtime PCR法检测前列腺癌高低转移潜能配对细胞系中 miR-125b的表达差异.通过划痕实验及transwell实验观察1E8细胞及转染 miR-125b 抑制剂及其阴性对照后该细胞运动转移能力的变化.结果 realtime PCR结果显示高转移潜能1E8细胞中miR-125b表达水明显高于低转移潜能2B4细胞;下调miR-125b会减弱1E8细胞的运动转移能力.结论 miR-125b可促进前列腺癌细胞的运动转移能力.     

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.     

Bcl-2和COX-2在宫颈鳞癌中的表达及其临床意义

目的：观察Bcl-2和COX-2在正常宫颈和宫颈鳞癌中的表达情况,并探讨其与宫颈鳞癌发生发展的关系。方法：应用免疫组织化学s-P法检测40例宫颈鳞癌组织、10例正常宫颈组织中Bcl-2和COX-2的表达情况。结果：（1）Bcl-2在正常宫颈组织和宫颈鳞癌组织中的阳性表达率分别为30．0％、72．5％（P〈0．05）,而COX-2在正常宫颈组织和宫颈鳞癌组织中的阳性表达率分别为0．0％、60．0％（P〈0．05）。（2）在宫颈鳞癌中,Bcl-2的表达与宫颈鳞癌的病理分级、临床分期以及淋巴结转移无关（P〉0．05）,而COX-2的表达与宫颈鳞癌的病理分级及淋巴转移有关（P〈0．05）,与临床分期无关（P〉0．05）。（3）Spearman等级相关性分析显示宫颈鳞癌组织中Bcl-2和COX-2的表达呈正相关（r=0．517,P〈0．01）。结论：Bcl-2和COX-2在宫颈鳞癌中的表达升高并呈显著正相关,且COX-2的表达与宫颈鳞癌的淋巴转移有关,二者在宫颈癌的发生发展中可能起重要作用,有可能作为评估宫颈鳞癌淋巴结转移的参考指标。     

Docetaxel/zoledronic acid combination triggers apoptosis synergistically through downregulating antiapoptotic Bcl-2 protein level in hormone-refractory prostate cancer cells

Docetaxel, a semi-synthetic taxane analogue, is used effectively in the treatment of metastatic prostate cancer. Zoledronic acid, the most potent member of bisphosphonates, has shown pleiotropic anti-tumoral effects on prostate cancer cells. We have explored the possible additive/synergistic effects and the apoptotic pathways induced by combination treatment of docetaxel and zoledronic acid in hormone and drug refractory, PC-3 and DU-145 prostate cancer cells. Combination of docetaxel and zoledronic acid synergistically inhibits cell growth in PC-3 and DU-145 cells. Moreover, this effect was due to downregulation of antiapoptotic protein Bcl-2 in PC-3 and DU-145 cells. In conclusion, docetaxel/zoledronic acid combination is potentially a novel and effective approach for the treatment of prostate cancer.     

Knockout of Akt1/2 suppresses the metastasis of human prostate cancer cells CWR22rv1 in vitro and in vivo

Although primary androgen deprivation therapy resulted in tumour regression, unfortunately, majority of prostate cancer progress to a lethal castration-resistant prostate cancer, finally die to metastasis. The mutual feedback between AKT and AR pathways plays a vital role in the progression and metastasis of prostate cancer. Therefore, the treatment of a single factor will eventually inevitably lead to failure. Therefore, better understanding of the molecular mechanisms underlying metastasis is critical to the development of new and more effective therapeutic agents. In this study, we created prostate cancer CWR22rv1 cells with the double knockout of Akt1 and Akt2 genes through CRISPR/Cas9 method to investigate the effect of Akt in metastasis of prostate cancer. It was found that knockout of Akt1/2 resulted in markedly reduced metastasis in vitro and in vivo, and appeared to interfere AR nuclear translocation through regulating downstream regulatory factor, FOXO proteins. It suggests that some downstream regulatory factors in the AKT and AR interaction network play a vital role in prostate cancer metastasis and are potential targeting molecules for prostate cancer metastasis treatment.     

Brefeldin A enhances docetaxel-induced growth inhibition and apoptosis in prostate cancer cells in monolayer and 3D cultures

Docetaxel is a commonly used chemotherapeutic drug for patients with late stage prostate cancer. However, serious side effect and drug resistance limit its clinical success. Brefeldin A is a 16-membered macrolide antibiotic from mangrove-derived Fungus Aspergillus sp. (9Hu), which exhibited potent cytotoxicity against human cancer cells. In the present study, we determined the effect of brefeldin A on docetaxel-induced growth inhibition and apoptosis in human prostate cancer PC-3 cells. Brefeldin A in combination with docetaxel inhibited the growth of PC-3 cells in monolayer and in three dimensional cultures. The combination also potently stimulated apoptosis in PC-3 cells as determined by propidium iodide staining and morphological assessment. Mechanistic studies showed that growth inhibition and apoptosis in PC-3 cells treated with brefeldin A and docetaxel were associated with decrease in the level of Bcl-2. The present study indicates that combined brefeldin A with docetaxel may represent a novel approach for improving the efficacy of docetaxel, and Bcl-2 may serve as a target for brefeldin A to enhance the effects of docetaxel chemotherapy.     

A natural BH3 mimetic induces autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2–Beclin1 interaction at endoplasmic reticulum

A natural BH3-mimetic, small-molecule inhibitor of Bcl-2, (−)-gossypol, shows promise in ongoing phase II and III clinical trials for human prostate cancer. In this study we show that (−)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent (AD) cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy through blocking Bcl-2–Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1, and activating the autophagic pathway. The (−)-gossypol-induced autophagy is dependent on Beclin1 and Atg5. Our results show for the first time that (−)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (−)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which will facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.     

A survey of the anti-apoptotic Bcl-2 subfamily expression in cancer types provides a platform to predict the efficacy of Bcl-2 antagonists in cancer therapy

We investigated the mRNA expression levels of all six antiapoptotic Bcl-2 subfamily members in 68 human cancer cell lines using qPCR techniques and measured the ability of known Bcl-2 inhibitors to induce cell death in 36 of the studied tumor cell lines. Our study reveals that Mcl-1 represents the anti-apoptotic Bcl-2 subfamily member with the highest mRNA levels in the lung, prostate, breast, ovarian, renal, and glioma cancer cell lines. In leukemia/lymphoma and melanoma cancer cell lines, Bcl-2 and Bfl-1 had the highest levels of mRNA, respectively. The observed correlation between the cell killing properties of known Bcl-2 inhibitors and the relative mRNA expression levels of anti-apoptotic Bcl-2 proteins provide critical insights into apoptosis-based anticancer strategies that target Bcl-2 proteins. Our data may explain current challenges of selective Bcl-2 inhibitors in the clinic, given that severe expression of Bcl-2 seems to be limited to leukemia cell lines. Furthermore, our data suggest that in most cancer types a strategy targeted to Mcl-1 inhibition, or combination of Bfl-1 and Mcl-1 inhibition for melanoma, may prove to be more successful than therapies targeting only Bcl-2.     

非小细胞肺癌中细胞凋亡与P53、Bcl-2蛋白的表达及对预后的影响

目的研究细胞凋亡及凋亡相关蛋白P53和Bcl-2在非小细胞肺癌组织中的表达水平,及其对预后的影响。方法应用DNA缺口末端标记技术和免疫组化方法检测111例肺癌患者组织中细胞凋亡、突变型P53和Bcl-2蛋白表达水平。结果111例肺癌中,细胞凋亡高表达53例(47.7%),突变型P53蛋白阳性45例(40.5%),Bcl-2蛋白阳性59例(53.2%)。COX模型多因素分析显示,淋巴结的转移和Bcl-2蛋白的阳性表达是本组肺癌患者的预后不良因素(P<0.05)。结论凋亡及凋亡相关蛋白P53和Bcl-2影响肺癌患者的生物学行为及预后。     

Mechanisms for 2-methoxyestradiol-induced apoptosis of prostate cancer cells

Prostate and breast carcinomas are sex hormone-related carcinomas, which are known to be associated with an over-expression of the proto-oncogene Bcl-2. Here, we report that 2-methoxyestradiol (2-ME), an endogenous metabolite of estrogen that does not bind to nuclear estrogen receptors, effectively induces apoptosis in Bcl-2-expressing human prostate and breast carcinoma cells in vitro and in a rat prostate tumor model in vivo. In several cell lines derived from prostate, breast, liver and colorectal carcinomas, 2-ME treatment led to an activation of c-Jun N-terminal kinase (JNK) and phosphorylation of Bcl-2, which preceded the induction of apoptosis. In summary, our data suggest that 2-ME induces apoptosis in epithelial carcinomas by causing phosphorylation of JNK, which appears to be correlated with phosphorylation of Bcl-2.     

Cell fate regulation by reticulon-4 in human prostate cancers

Reticulon-4 (RTN4), a reticulon family protein localized in the endoplasmic reticulum, is reported to be involved in multiple physiological processes like neuroendocrine secretion and membrane trafficking in neuroendocrine cells. Previous studies have presented a great potential of RTN4 for the treatment of autoimmune-mediated demyelinating diseases and spinal cord injury regeneration. While interaction with Bcl-2 and Bcl-2-like family in apoptosis modulation implicated its possible role in various human cancers. However, the investigation of this gene in prostate cancer is mainly ignored. Here in our current study, we focused on its role in prostate cancer and found that RTN4 DNA copy numbers were higher in prostate cancer than normal prostate gland while its RNA and protein expressions were relatively lower. Chromosomal neighbor gene EML6 had similar expression patterns with RTN4 in prostate cancer tissues and cell lines, and further research found that they could be both targeted by miR-148a-3p. Lentivirus-mediated RTN4 overexpression potently inhibited DU145 and LNCaP cells proliferation. Cell cycle was blocked in G2/M phase and significant cell senescence was observed in RTN4 overexpressed prostate cancer cells. Finally, interaction networks in the normal prostate gland and cancer tissues further revealed that RTN4 maybe phosphorylated by MAPKAPK2 and FYN at tyrosine 591 and serine 107, respectively. All these results implied that RTN4 might somehow participate in prostate tumor progression, and this elicits possibility to develop or identify selective agents targeting RTN4 for prostate cancer therapy.     

黄精凝集素PCL-2诱导人前列腺癌LNCap细胞凋亡及机制研究

研究黄精凝集素PCL-2对人前列腺癌LNCap细胞生物活性和可能的抗肿瘤机制。从黄精药材中提取分离得到黄精凝集素PCL-2,体外培养LNCap细胞,采用WST-1和集落形成实验评价PCL-2对LNCap细胞的增殖作用;2,7-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针法检测LNCap细胞内ROS含量;qRT-PCR和Western blot法检测PCL-2对LNCap细胞中相关基因和蛋白表达的影响。研究结果显示PCL-2能显著抑制LNCap细胞的增殖,促进细胞中ROS生成;PCL-2通过上调LNCap细胞中Bax、Caspase-3及Caspase-9基因mRNA和蛋白表达并下调Bcl-2基因mRNA和蛋白表达水平诱导LNCap细胞凋亡。本研究结果表明PCL-2诱导LNCap细胞凋亡活性作用显著,可作为抑制前列腺癌的潜在药物。     

Annexin II/annexin II receptor axis regulates adhesion, migration, homing, and growth of prostate cancer

One of the most life-threatening complications of prostate cancer is skeletal metastasis. In order to develop treatment for metastasis, it is important to understand its molecular mechanisms. Our work in this field has drawn parallels between hematopoietic stem cell and prostate cancer homing to the marrow. Our recent work demonstrated that annexin II expressed by osteoblasts and endothelial cells plays a critical role in niche selection. In this study, we demonstrate that annexin II and its receptor play a crucial role in establishing metastasis of prostate cancer. Prostate cancer cell lines migrate toward annexin II and the adhesion of prostate cancer to osteoblasts and endothelial cells was inhibited by annexin II. By blocking annexin II or its receptor in animal models, short-term and long-term localization of prostate cancers are limited. Annexin II may also facilitate the growth of prostate cancer in vitro and in vivo by the MAPK pathway. These data strongly suggest that annexin II and its receptor axis plays a central role in prostate cancer metastasis, and that prostate cancer utilize the hematopoietic stem cell homing mechanisms to gain access to the niche.     

Targeting of distinct signaling cascades and cancer-associated fibroblasts define the efficacy of Sorafenib against prostate cancer cells

Sorafenib, a multi-tyrosine kinase inhibitor, kills more effectively the non-metastatic prostate cancer cell line 22Rv1 than the highly metastatic prostate cancer cell line PC3. In 22Rv1 cells, constitutively active STAT3 and ERK are targeted by sorafenib, contrasting with PC3 cells, in which these kinases are not active. Notably, overexpression of a constitutively active MEK construct in 22Rv1 cells stimulates the sustained phosphorylation of Bad and protects from sorafenib-induced cell death. In PC3 cells, Src and AKT are constitutively activated and targeted by sorafenib, leading to an increase in Bim protein levels. Overexpression of constitutively active AKT or knockdown of Bim protects PC3 cells from sorafenib-induced killing. In both PC3 and 22Rv1 cells, Mcl-1 depletion is required for the induction of cell death by sorafenib as transient overexpression of Mcl-1 is protective. Interestingly, co-culturing of primary cancer-associated fibroblasts (CAFs) with 22Rv1 or PC3 cells protected the cancer cells from sorafenib-induced cell death, and this protection was largely overcome by co-administration of the Bcl-2 antagonist, ABT737. In summary, the differential tyrosine kinase profile of prostate cancer cells defines the cytotoxic efficacy of sorafenib and this profile is modulated by CAFs to promote resistance. The combination of sorafenib with Bcl-2 antagonists, such as ABT737, may constitute a promising therapeutic strategy against prostate cancer.