DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation

The majority of human prostate cancer cell lines, including the two "classical" cell lines DU-145 and PC-3, are reported to be androgen receptor (AR)-negative. However, other studies have provided evidence that the DU-145 and PC-3 cell lines express AR mRNA. These contradictory observations prompted us to investigate whether DU-145 and PC-3 cell lines express the androgen receptor. Using antipeptide antibodies directed against three distinct regions of the human AR protein and an improved method to detect AR protein in immunoblotting, we report that DU-145 and PC-3 cell lines express AR protein. We found that the relative levels of the AR mRNA and protein that were detected in DU-145 and PC-3 cell lines were lower than the LNCaP, an AR-positive cell line. Moreover, the antibody directed against the non-variant region (amino acids 299-315), but not the variant N- or C-terminal region (amino acids 1-20 and 900-919, respectively) of the human AR protein, detected the expression of AR in all prostate cancer cell lines. Notably, treatment of these cell lines with dihydrotestosterone (DHT) resulted in measurable increases in the AR protein levels and considerable nuclear accumulation. Although, treatment of DU-145 and PC-3 cells with DHT did not result in stimulation of the activity of an AR-responsive reporter, knockdown of AR expression in PC-3 cells resulted in decreases in p21(CIP1) protein levels, and a measurable decrease in the activity of the p21-luc-reporter. Our observations demonstrate the expression of AR protein in DU-145 and PC-3 prostate cancer cell lines.     

Prostate tumor CXC-chemokine profile correlates with cell adhesion to endothelium and extracellular matrix

Though chemokines of the CXC family are thought to play key roles in neoplastic transformation and tumor invasion, information about CXC chemokines in prostate cancer is sparse. To evaluate the involvement of CXC chemokines in prostate cancer, we analyzed the CXC coding mRNA of both chemokine ligands (CXCL) and chemokine receptors (CXCR), using the prostate carcinoma cell lines PC-3, DU-145 and LNCaP. CXCR proteins were further evaluated by Western blot, CXCR surface expression by flow cytometry and confocal microscopy. The expression pattern was correlated to adherence of the tumor cells to an endothelial cell monolayer or to extracellular matrix components. Based on growth and adhesion capacity, PC-3 and DU-145 were identified to be highly aggressive tumor cells (PC-3>DU-145), whereas LNCaP belonged to the low aggressive phenotype. CXCL1, CXCL3, CXCL5 and CXCL6 mRNA, chemokines with pro-angiogenic activity, were strongly expressed in DU-145 and PC-3, but not in LNCaP. CXCR3 and CXCR4 surface level differed in the following order: LNCaP>DU-145>PC-3. The differentiation factor, fatty acid valproic acid, induced intracellular CXCR accumulation. Therefore, prostate tumor malignancy might be accompanied by enhanced synthesis of angiogenesis stimulating CXC chemokines. Further, shifting CXCR3 and CXCR4 from the cell surface to the cytoplasm might activate pro-tumoral signalling events and indicate progression from a low to a highly aggressive phenotype.     

Over-expression of Zip-13 mRNA in kidney and lung during dietary zinc deficiency in Wistar rats

Zinc is an essential nutrient for all organisms, which is involved in the function of numerous key enzymes in metabolism. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while Zip transporters increase intracellular zinc. Previous studies in our laboratory have shown that Zip-1, ZnT-1, Zip-2 and LIV-1 mRNA are associated with zinc level in established human breast cancer in nude mice model. In this study, six zinc transporters: ZnT-1, ZnT-2, ZnT-4, Zip-1, Zip-8 and Zip-13 were chosen. We aim to determine the relation between zinc transporters and zinc level in kidney and lung of Wistar rats. Eighteen Wistar rats were randomly divided into three groups: normal group, zinc-deficiency group and pair-fed group. After 22 days, the rats were killed and organs samples were taken, then zinc transporters mRNA were detected by RT-PCR. Compared with the normal group, Zip-13 shows an up-regulation (P < 0.05) in zinc-deficiency group both in kidney and lung, and Zip-8 was significantly lower (P < 0.05) in zinc-deficiency group in kidney.     

Role of protein kinase C-iota in transformed non-malignant RWPE-1 cells and androgen-independent prostate carcinoma DU-145 cells

Prostate cancer is one of the leading causes of death among men in the USA.
Objective:  In this study, we investigated the role of atypical protein kinase C-iota (PKC-ι) in androgen-independent prostate DU-145 carcinoma cells compared to transformed non-malignant prostate RWPE-1 cells.
Materials and methods:  Western blotting and immunoprecipitations demonstrated that PKC-ι is associated with cyclin-dependent kinase activating kinase (CAK/Cdk7) in RWPE-1 cells, but not in DU-145 cells.
Results:  Treatment of prostate RWPE-1 cells with PKC-ι silencing RNA (siRNA) decreased cell viability, cell-cycle accumulation at G₂/M phase, and phosphorylation of Cdk7 and Cdk2. In addition, PKC-ι siRNA treatment caused less phosphorylation of Bad at ser-155, ser-136, and greater Bad/Bcl-x_L heterodimerization, leading to apoptosis. In DU-145 cells, PKC-ι was anti-apoptotic and was required for cell survival. Treatment with PKC-ι siRNA blocked increase in cell number, and inhibited G₁/S transition by accumulation of cells in G₀/G₁ phase. In addition to cell-cycle arrest, both RWPE-1 and DU-145 cells underwent apoptosis due to mitochondrial dysfunction and apoptosis cascades, such as release of cytochrome c, activation of caspase-7, and poly (ADP-ribose) polymerase (PARP) cleavage.
Conclusion:  Our results suggest that PKC-ι is required for cell survival in both transformed non-malignant prostate RWPE-1 cells and androgen-independent malignant prostate DU-145 cells, whereas suppressing PKC-ι lead to apoptosis in DU-145 prostate cells.     

Inhibition of cell survival signal protein kinase B/Akt by curcumin in human prostate cancer cells

Although curcumin has been shown to inhibit prostate tumor growth in animal models, its mechanism of action is not clear. To better understand the anti-cancer effects of curcumin, we investigated the effects of curcumin on cell survival factor Akt in human prostate cancer cell lines, LNCaP, PC-3, and DU-145. Our results demonstrated differential activation of Akt. Akt was constitutively activated in LNCaP and PC-3 cells. Curcumin inhibited completely Akt activation in both LNCaP and PC-3 cells. The presence of 10% serum decreased the inhibitory effect of curcumin in PC-3 cells whereas complete inhibition was observed in 0.5% serum. Very little or no activation of Akt was observed in serum starved DU-145 cells (0.5% serum). The presence of 10% serum activated Akt in DU-145 cells and was not inhibited by curcumin. Results suggest that one of the mechanisms of curcumin inhibition of prostate cancer may be via inhibition of Akt. To our knowledge this is the first report on the curcumin inhibition of Akt activation in LNCaP and PC-3 but not in DU-145 cells.     

Olfactory ensheathing glia and Schwann cells: two of a kind?

Prostatic carcinoma affects 1 in 11 men and targets bone with sclerotic metastases. The study of prostate carcinoma growth in bone has been hampered by the lack of suitable animal models. We have developed an in vivo model of prostate carcinoma growth in bone by inoculating three human prostate carcinoma cell lines (PC-3, DU-145, and LNCaP) into the tibia of congenitally athymic mice. Developing tumors were analyzed by radiographic, histologic, immunohistochemical, and in situ hybridization examination. Seven of the nine PC-3 inoculated mice and all (9/9) of the DU-145 inoculated mice developed tumors in the injected limb. In contrast, inoculation with LNCaP cells failed to produce tumors (0/9). Radiologically, the tumors had a mixed sclerotic/lytic appearance with extracortical extension. All the PC-3 tumors invaded the bone marrow cavity, cortical bone, and surrounding soft tissue. The DU-145 tumors were confined to the bone marrow cavity in 7/9 animals. CK18 and Ki67 localization identified the human tumor cells and their proliferative activity, respectively. The PC-3- and DU-145-induced tibial tumors expressed alpha(1)I procollagen and osteopontin mRNA, to varying degrees. All the tumors demonstrated an up-regulation of osteoclasts at the bone/tumor interface compared with the control limbs. Thus, this is a reliable and reproducible in vivo model of prostate carcinoma growth in bone enabling the study of the interactions that occur between prostate cancer cells and bone at an important part of the metastatic cascade, namely, growth and invasion at a distant site.     

Prostate-specific kallikreins-2 and -4 enhance the proliferation of DU-145 prostate cancer cells through protease-activated receptors-1 and -2

A major characteristic of prostate cancer is the elevation of serum levels of prostate-specific antigen (hK3) and hK2, which are tumor markers that correlate with advancing stages of disease. Including hK4, these three kallikrein serine proteases are almost exclusively produced by the prostate. Prostate cancer cells have been recently shown to overexpress protease-activated receptors (PAR), which can be potentially activated by kallikreins and can regulate tumor growth. Here, we show that recombinant hK2 and hK4 activate ERK1/2 signaling of DU-145, PC-3, and LNCaP prostate cancer cells, which express both PAR1 and PAR2. These kallikreins also stimulate the proliferation of DU-145 cells. Pretreatment of hK2 and hK4 with the serine protease inhibitor, aprotinin, blocks the responses in DU-145 cells, and small interfering RNA against PAR1 and PAR2 also inhibits ERK1/2 signaling. To determine which PAR is activated by hK2 and hK4, a cell line that expresses a single PAR, a PAR1 knockout mouse lung fibroblast cell line transfected with PAR1 (KOLF-PAR1) or PAR2 (KOLF-PAR2) was used. hK4 activates both PAR1 and PAR2, whereas hK2 activates PAR2. hK4 generates more phosphorylated ERK1/2 than hK2. These data indicate that prostatic kallikreins (hK2 and hK4) directly stimulate prostate cancer cell proliferation through PAR1 and/or PAR2 and may be potentially important targets for future drug therapy for prostate cancer.     

Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months after treatment with a median overall survival time of 1-2 years after relapse. LNCaP is a commonly used cell line established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We previously established two relapsed androgen receptor (AR)-rich androgen-independent LNCaP sublines 104-R1 (androgen depleted for 12 months) and 104-R2 cells (androgen depleted for 24 months) from AR-positive androgen-dependent LNCaP 104-S cells. LNCaP 104-R1 and 104-R2 mimics the AR-positive hormone-refractory relapsed tumors in patients receiving androgen ablation therapy. Androgen treatment stimulates proliferation of 104-S cells, but causes growth inhibition and G1 cell cycle arrest in 104-R1 and 104-R2 cells. We investigated the protein expression profile difference between LNCaP 104-S vs. LNCaP 104-R1, 104-R2, PC-3, and DU-145 cells as well as examined the sensitivity of these prostate cancer cells to different chemotherapy drugs and small molecule inhibitors. Compared to 104-S cells, 104-R1 and 104-R2 cells express higher protein levels of AR, PSA, c-Myc, Skp2, BCL-2, P53, p-MDM2 S166, Rb, and p-Rb S807/811. The 104-R1 and 104-R2 cells express higher ratio of p-Akt S473/Akt, p-EGFR/EGFR, and p-Src/Src, but lower ratio of p-ERK/ERK than 104-S cells. PC-3 and DU-145 cells express higher c-Myc, Skp2, Akt, Akt1, and phospho-EGFR but less phospho-Akt and phospho-ERK. Overexpression of Skp2 increased resistance of LNCaP cells to chemotherapy drugs. Paclitaxel, androgen, and inhibitors for PI3K/Akt, EGFR, Src, or Bcl-2 seem to be potential choices for treatment of advanced prostate cancers. Our study provides rationale for targeting Akt, EGFR, Src, Bcl-2, and AR signaling as a treatment for AR-positive relapsed prostate tumors after hormone therapy.     

miR-182在前列腺癌组织中的表达及其对前列腺癌细胞增殖和凋亡的影响

目的:观察前列腺癌组织及不同前列腺癌细胞系中miR-182的表达,并探讨下调其表达对前列腺癌细胞增殖和凋亡的影响及机制。方法:采用实时荧光定量PCR(q RT-PCR)检测30例前列腺癌组织和30例相应的癌旁组织以及前列腺正常上皮RWPE-1细胞、前列腺癌PC-3、LNCa P和DU145细胞中miR-182的表达,进一步采用Lipfectamine 2000脂质体转染miRNA-182 inhibitor和阴性对照miRNA于PC-3细胞后,通过噻唑蓝(MTT)比色法检测细胞增殖情况,流式细胞术检测细胞凋亡率,免疫印迹(Western blot)法检测转录因子FOXO1、血管内皮生长因子(VEGF)和抑癌基因p53蛋白的表达。结果:miR-182在前列腺癌组织中的表达明显高于癌旁组织(P0.05);miR-182在前列腺癌细胞系PC-3、LNCa P和DU145中的表达均高于前列腺正常上皮细胞RWPE-1(P0.05),其中PC-3细胞中miR-182表达水平最高。转染miRNA-182 inhibitor至PC-3细胞成功下调miR-182表达后,细胞的增殖能力明显受到抑制,细胞凋亡能力明显增强,FOXO1表达水平显著升高,VEGF和p53的表达明显降低,差异均具有统计学意义(P0.05)。结论:miR-182在前列腺癌组织及细胞中呈高表达,下调miR-182的表达可能通过增加FOXO1的表达并减少VEGF和p53的表达,抑制前列腺癌细胞增殖并诱导细胞凋亡。     

Downregulation of PTEN/MMAC/TEP1 expression in human prostate cancer cell line DU145 by growth stimuli

Genetic alterations and/or deletion of the tumor suppressor gene PTEN/MMAC/TEP1 occur in many types of human cancer including prostate cancer. We describe the production of monoclonal antibody against recombinant human PTEN and the study of PTEN gene and protein expression in three commercially available human prostate cancer cell lines, PC-3, LNCaP, and DU 145. Northern blotting analyses showed that LNCaP and DU145 but not PC-3 cells expressed PTEN mRNA. However, Western blotting analyses using a monoclonal antibody against PTEN demonstrated the expression of PTEN protein in DU145 but not LNCaP cells. In DU145 cells, PTEN expression at both the mRNA and protein levels inversely correlated with serum concentrations and levels of PKB/Akt phosphorylation. In addition, the basal activity of PKB/Akt as indicated by level of phosphorylation was higher in prostate cancer cells which do not express PTEN than that in the cells expressing wild type PTEN. Thus, PTEN may play a critical role in regulating cellular signaling in prostate cancer cells.     

Thalidomide analogues demonstrate dual inhibition of both angiogenesis and prostate cancer

The identification of agents with antiproliferative activity against endothelial cells has significant value for the treatment of many angiogenesis-dependent pathologies. Herein, we describe the discovery of a series of thalidomide analogues possessing inhibitory effects against both endothelial and prostate cancer cells. More specifically, several analogues exhibited low micromolar to mid-nanomolar potency in the inhibition of human microvascular endothelial cell (HMEC) proliferation, both in the presence and absence of vascular endothelial growth factor (VEGF), with the tetrafluorophthalimido class of compounds demonstrating the greatest potency. Additionally, all the compounds were screened against two different androgen independent prostate cancer cell lines (PC-3 and DU-145). Again, the tetrafluorophthalimido analogues exhibited the greatest effect with GI(50) values in the low micromolar range. Thalidomide was found to demonstrate selective inhibition of androgen receptor positive LNCaP prostate cancer cells. Furthermore, we showed that, as an example, tetrafluorophthalimido analogue 19 was able to completely inhibit the prostate specific antigen (PSA) secretion by the LNCaP cell line, while thalidomide demonstrated a 70% inhibition. We have also demonstrated that a correlation exists between HMEC and prostate cancer cell proliferation for this structural class. Altogether, our study suggests that these analogues may serve as promising leads for the development of agents that target both androgen dependent and independent prostate cancer and blood vessel growth.     

Selenoprotein expression is regulated at multiple levels in prostate cells

Selenium supplementation in a population with low basal blood selenium levels has been reported to decrease the incidence of several cancers including prostate cancer. Based on the clinical findings, it is likely that the antioxidant function of one or more selenoproteins is responsible for the chemopreventive effect, although low molecular weight seleno-compounds have also been posited to selectively induce apoptosis in transformed cells. To address the effects of selenium supplementation on selenoprotein expression in prostate cells, we have undertaken an analysis of antioxidant selenoprotein expression as well as selenium toxicity in non-tumorigenic prostate epithelial cells （RWPE- 1 ） and prostate cancer cells （LNCaP and PC-3）. Our results show that two of the glutathione peroxidase family members （GPX1 and GPX4） are highly induced by supplemental selenium in prostate cancer cells but only slightly induced in RWPE-1 cells. In addition, GPX 1 levels are dramatically lower in PC-3 cells as compared to RWPE- 1 or LNCaP cells. GPX2 protein and mRNA, however, are only detectable in RWPE-1 cells. Of the three selenium compounds tested （sodium selenite, sodium selenate and selenomethionine）, only sodium selenite shows toxicity in a physiological range of selenium concentrations. Notably and in contrast to previous studies, RWPE-1 cells were significantly more sensitive to selenite than either of the prostate cancer cell lines. These results demonstrate that selenoproteins and selenium metabolism are regulated at multiple levels in prostate cells.     

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.     

MicroRNA-Dependent Regulation of <Emphasis Type="Italic">IGF1R</Emphasis> Gene Expression in Hormone-Sensitive and Hormone-Resistant Prostate Cancer Cells

Using multiple parallel sequencing on Illumina platform, we identified eight microRNAs that showed significant opposite changes of gene expression in cells of the hormone-sensitive LNCaP prostate cancer cell line and in cells of the hormone-resistant DU-145 cell line, in comparison to the microRNA expression in the normal prostate tissue cells. We found that the insulin-like growth factor 1 receptor (IGF1R) gene is a target of five microRNAs whose expression is increased in LNCaP cells and reduced in DU-145 cells.     

Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins

Cancer of the prostate commonly metastasizes to bony sites where cells acquire an aggressive, rapidly proliferating, androgen-independent phenotype. The interaction between bone and prostate, thus, becomes a key factor in disease progression. Fluctuations in intracellular ionized Ca2+ [Ca2+]i are rapid, regulated signal transduction events often associated with cell proliferation. Hence, Ca2+ signals provide a convenient measure of early events in cancer cell growth. This study developed single cell fluorescent imaging techniques to visualize Ca2+ signals in Fura-2 loaded prostatic cancer cell lines of various metastatic phenotypes. Solubilized bone fractions containing extracellular matrix and associated proteins were tested for the ability to trigger Ca2+ signals in prostate cancer cell lines. Fractions representing the complete repertoire of non-collagenous proteins present in mineralized bone were tested. Results demonstrated that two bone fractions termed D3b- and D4a-triggered Ca2+ signals in prostate cancer cells derived from bone (PC-3), but not brain (DU-145) metastases of prostate cancer. Lymph-node derived LNCaP cells also did not produce a Ca2+ signal in response to addition of soluble bone matrix. No other bone fractions produced a Ca2+ signal in PC-3 cells. It is of interest that bone fractions D3b and D4a contain a number of non-collagenous matrix proteins including osteonectin (SPARC) and osteopontin (OPN), as well as prothrombin. Moreover, antibody LM609 that recognizes the alpha v beta 3 integrin, blocks the ability of OPN to trigger a Ca2+ transient in PC-3 cells. These studies support a conclusion that bone-matrix proteins play a role in the growth and progression of metastatic prostate cancer, and that prior growth in bone may be associated with development of a bone-matrix-responsive phenotype.     

Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc.

A cDNA encoding a zinc transporter (ZnT-1) was isolated from a rat kidney cDNA expression library by complementation of a mutated, zinc-sensitive BHK cell line. This cDNA was used to isolate the homologous mouse ZnT-1 gene. The proteins predicted for these transporters contain six membrane-spanning domains, a large intracellular loop and a C-terminal tail. ZnT-1 is homologous to zinc and cobalt resistance genes of yeast. Immunocytochemistry with an antibody to a myc epitope added to the C-terminus of ZnT-1 revealed localization to the plasma membrane. Transformation of normal cells with a mutant ZnT-1 lacking the first membrane-spanning domain conferred zinc sensitivity on wild-type cells, suggesting that ZnT-1 functions as a multimer. Deletion of the first two membrane-spanning domains resulted in a non-functional molecule, whereas deletion of the C-terminal tail produced a toxic phenotype. Mutant cells have a slightly higher steady-state level of intracellular zinc and high basal expression of a zinc-dependent reporter gene compared with normal cells. Mutant cells have a lower turnover of 65Zn compared with normal cells or mutant cells transformed with ZnT-1. We propose that ZnT-1 transports zinc out of cells and that its absence accounts for the increased sensitivity of mutant cells to zinc toxicity.     

miRNA-191对前列腺癌的增殖、迁移侵袭能力的影响及其机制*

目的:观察miRNA-191对前列腺癌的增殖、迁移和侵袭能力的影响,并探讨其机制。方法:分别检测4种人前列癌细胞系(PC-3、DU-145、LNCa P、22RU1)及人正常前列腺细胞RWPE-2中miRNA-191的表达水平,并选择前列腺癌细胞系PC-3作为实验对象。将PC-3细胞分为3组:空白对照组(不转染)、miRNA-191 NC组(Inhibitor NC转染PC-3细胞)、miRNA-191 Inhibitor组(miRNA-191 Inhibitor转染PC-3细胞),每组设置3个复孔。采用RT-qPCR法检测PC-3细胞miRNA-191和PLCD1的mRNA表达水平;采用CCK8法检测PC-3细胞增殖水平;采用划痕实验和侵袭实验分别检测PC-3细胞迁移能力和侵袭能力;通过Targetscan靶基因预测网站,筛选PLCD1作为miRNA-191的靶向蛋白,并用双荧光素酶靶标实验验证;采用Western blot法检测PC-3细胞PLCD1的蛋白表达。结果:与RWPE-2细胞相比,人前列癌细胞中miNRA-191的表达水平显著升高(P＜0.05),且miRNA-191的表达水平在PC-3中较其他3种细胞系显著上调(P＜0.05)。抑制miRNA-191的表达水平后,PLCD1表达水平显著升高,PC-3细胞增殖能力受到抑制,迁移和侵袭能力较空白对照组和miRNA-191 NC组显著降低(P＜0.05)。双荧光素酶报告基因实验结果显示,PLCD1基因是miRNA-191的靶基因。结论:miRNA-191通过靶向PLCD1促进前列腺癌PC-3细胞的增殖、迁移和侵袭能力。