A recombinant adenovirus expressing wild-type Bax induces apoptosis in prostate cancer cells independently of their Bcl-2 status and androgen sensitivity

Using a binary co-transfection strategy of Ad/GT Bax and Ad/PGK-GV16, we have succeeded in inducing overexpression of Bax protein in three prostate cell lines (androgen-insensitive DU145 and PC3, and androgen-sensitive LNCaP). The expression of Bax protein by this system was sufficient to induce all three prostate lines to undergo apoptosis. The fact that DU145 cells which have a p53 mutation and are deficient in Bax, responded to this treatment, suggests that this effect is independent of these pathways. Initiation of the cleavage of Caspase-3 (CPP32/Yama/apopain) and PARP (poly (ADP-ribose) polymerase) by the introduction of Bax were confirmed by western blot analysis. Bcl-2 expression is relevant in the progression of prostate cancer and contributes to an androgen, apoptotic-resistant phenotype in the advanced stages. We examined stable Bcl-2 overexpressing DU145, PC3 and LNCaP cell lines as models of advanced prostate cancer. The adenoviral co-transfection system induced Bax protein expression and apoptosis even in these Bcl-2 transfected cell lines. Taken together, our results suggest that this Bax expression system might represent a useful gene therapy strategy when applied to the treatment of prostate cancer and its efficacy would be independent of the Bcl-2 status and androgen sensitivity of these cancers.     

miRNA-135在调控前列腺癌细胞增殖中的应用及机制

目的 观察miRNA-135在调控激素非依赖性前列腺癌细胞增殖能力中的作用及其机制.方法 利用微小核糖核酸(miRNA)基因芯片检测激素非依赖性前列腺癌和激素依赖性前列腺癌组织中miRNA-135的表达差异.经过体外转染miRNA-135后对DU145和PC3细胞进行MTT检测,了解细胞的增殖情况.利用miRNA-135靶基因预测软件miRwalk和miRanda预测,初筛miRNA-135调控的基因和相关通路.结果 miRNA基因芯片结果显示,激素依赖性前列腺癌组织中miRNA-135的表达量高于激素非依赖性前列腺癌组织(P﹤0.05);miR-NA-135可以抑制DU145和PC3细胞的生长,尤其在第48 h和第72 h以后(P﹤0.05);转染miRNA-135后,前列腺癌细胞株DU145和PC3中STAT6的表达水平较转染NC的细胞株明显下调.结论 miRNA-135能够抑制激素非依赖性前列腺细胞DU145和PC3的增殖能力,有望成为前列腺癌治疗的新靶标.     

Expression and function of the complement membrane attack complex inhibitor protectin (CD59) in human prostate cancer

Protectin (CD59) inhibits homologous complement-mediated cytolysis by preventing formation of the membrane attack complex at the point of insertion and polymerization of C9 into cell membranes. The present study investigated the expression and function of CD59 on human prostatic tumor cells in situ and on 5 human prostate cell lines in vitro originating from either metastatic tumors or benign prostate hypertrophy epithelial cells. Immunohistochemical staining of prostate carcinoma tissue with monoclonal antibody (MAb) MEM43 revealed weak to moderately strong expression of CD59 by prostate glandular epithelial cells. Flow cytometry with MEM43 demonstrated that the 5 prostate cell lines expressed different relative quantities of CD59. Indirect immunofluorescence analysis revealed uniform membrane staining of DU145 and PC3 cell lines with no membranous granularity in the staining pattern. Western immunoblots with MAb BRIC 229 showed that PC3 and DU145 cells express CD59 with a m.w. of 18-25 kDa. Treatment of DU145 and PC3 cells with phosphatidylinositol-specific phospholipase C caused a significant decrease of CD59 expression indicating that the CD59 expressed by prostate cancer cells is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage. PC3 and DU145 cells were completely resistant to human complement-mediated cytolysis but became sensitive to killing in the presence of the CD59-neutralizing MAb YTH53.1. We conclude that malignant and benign human prostate cells express CD59 that is GPI-linked to the cell surface and that CD59 may regulate the immunological response to cancerous prostate cells by protecting the cells from the cytolytic activity of complement. Int. J. Cancer 71: 1049-1055, 1997.© 1997 Wiley-Liss Inc.     

Imaging agents for in vivo molecular profiling of disseminated prostate cancer--targeting EGFR receptors in prostate cancer: comparison of cellular processing of [111In]-labeled affibody molecule Z(EGFR:2377) and cetuximab

Expression of receptor tyrosine-kinase (RTK) EGFR is low in normal prostate, but increases in prostate cancer. This receptor is significantly up-regulated as tumors progress into higher grade, androgen-insensitive and metastatic lesions. The up-regulated receptors could serve as targets for novel selective anti-cancer drugs, e.g. antibodies and tyrosine kinase inhibitors. Radionuclide imaging of RTK can facilitate patient stratification and monitoring of anti-RTK therapy of prostate cancer. The goal of the study was to evaluate binding and cellar processing of radiolabeled EGFR-targeting conjugates by prostate cancer cell lines. Receptor expression of EGFR was studied in three prostate cancer cell lines: DU145 (brain metastasis of PC, hormone insensitive), PC3 (bone metastasis of PC) and LNCaP (lymph node metastasis of PC, androgen and estrogen receptor positive). Uptake and internalization of anti-EGFR mAbs (cetuximab) and affibody molecule (Z2377) labeled with indium-111 was investigated. EGFR expression on prostate cancer cell lines was clearly demonstrated. Both labelled conjugates 111In-Z2377 and 111In-cetuximab bound to prostate cancer cells in the receptor mediated model. Expression levels were modest but correlate with degree of hormone independence. Internalization of Affibody molecules was relatively slow in all cell lines. Internalization of mAbs was more rapid. The level of EGFR expression in these cell lines is sufficient for in vivo molecular imaging. Slow internalization indicates possibility of the use of non-residualizing labels for affibody molecules.     

Apoptotic signaling in bufalin‐ and cinobufagin‐treated androgen‐dependent and ‐independent human prostate cancer cells

Prostate cancer has its highest incidence in the USA and is becoming a major concern in Asian countries. Bufadienolides are extracts of toxic glands from toads and are used as anticancer agents, mainly on leukemia cells. In the present study, the antiproliferative and apoptotic mechanisms of bufalin and cinobufagin on prostate cancer cells were investigated. Proliferation of LNCaP, DU145, and PC3 cells was measured by 3‐(4,5‐dimethylthiazol‐2‐yle)‐2,5‐diphenyltetrazolium bromide assay and the doubling time (tD) was calculated. Bufalin and cinobufagin caused changes in the tD of three prostate cancer cell lines, which were more significant than that of human mesangial cells. In addition, bufadienolides induced prostate cancer cell apoptosis more significantly than that in breast epithelial cell lines. After treatment, the caspase‐3 activity and protein expression of caspase‐3, ‐8, and ‐9 were elevated. The expression of other apoptotic modulators, including mitochondrial Bax and cytosolic cytochrome c, were also increased. However, expression of p53 was only enhanced in LNCaP cells. Downregulation of p53 by antisense TP53 restored the cell viability suppressed by bufalienolides. Furthermore, the increased expression of Fas was more significant in DU145 and PC3 cells with mutant p53 than in LNCaP cells. Transfection of Fas small interfering RNA restored cell viability in the bufadienolide‐treated cells. These results suggest that bufalin and cinobufagin suppress cell proliferation and cause apoptosis in prostate cancer cells via a sequence of apoptotic modulators, including Bax, cytochrome c, and caspases. The upstream mediators might be p53 and Fas in androgen‐dependent LNCaP cells and Fas in androgen‐independent DU145 and PC3 cells. (Cancer Sci 2008; 99: 2467–2476)     

Prostate cancer mediates osteoclastogenesis through two different pathways

The present study was undertaken to test the effects of prostate cancer cell lines (LNCaP, DU145, PC3, and MDA PCa 2b) on osteoclastogenesis. Crude conditioned medium (CM) from all four prostate cancer cell lines enhanced expression of the mRNA for receptor activator of NF-kappaB ligand (RANKL) in a mouse osteoblast cell line, MC3T3-E1; however, CM had no effect on expression of osteoprotegerin (OPG) mRNA. Coculture of MC3T3-E1 with prostate cancer cells yielded similar results. The number of mature osteoclasts induced by soluble RANKL increased significantly when osteoclast precursor cells were cultured with CM from LNCaP and DU145 cells. CM from LNCaP and DU145 cells also induced maturation from precursor in the absence of soluble RANKL, and this effect was not blocked by OPG. Addition of CM from DU145 cells increased expression of MMP-9 mRNA by osteoclast precursors. Our findings indicate that prostate cancer mediates osteoclastogenesis through induction of RANKL expression by osteoblasts and through direct actions on osteoclast precursors mediated by some factors other than RANKL.     

Altered response to thyroid hormones by prostate and breast cancer cells

Transferrin, an abundant bone marrow constituent, has been shown to be a potent mitogen in vitro in the prostate cancer cell line PC3. T4 (L-thyroxine) and T3 (3′,3,5-tri-iodo-L-thyronine) are regulators of cell metabolism. In this study, the effects of nonphysiological concentrations (about two orders of magnitude higher) of T4, T3, T2 (3,5-di-iodo-L-thyronine), RT3 (reverse T3, 3′,5′,3-tri-iodo-L-thyronine) and transferrin (about three orders of magnitude lower) were tested on the prostate cancer cell lines PC3, DU145 and LNCaP, and the breast cancer cell line MCF-7. In PC3 cells, increased proliferation by transferrin could be reversed by the addition of T3 or T4. T4 decreased proliferation in all cell lines tested, while transferrin increased proliferation in PC3 cells only. T3 decreased proliferation in PC3, LNCaP and MCF-7 cells but had no effect on DU145 cells. T4 and T3 gave two-state behavior in LNCaP cells. These results were combined to determine the essential iodines which produced the observed proliferative effects. Cell lines responded differently to T4, T3, T2, RT3 and transferrin suggesting a specific interaction among the compounds tested and the different cell lines. Finally, regulation of gene expression was demonstrated using DU145 cells. Upregulation of c-fos mRNA was observed in cultures at early time-points in the presence of T4, transferrin or both. Decreased expression was observed at later time-points with no expression at 4 h. An explanation for these results may be a change in thyroid hormone receptor/ligand affinity. Thus, the interactions between thyroid hormones and cancer cells may be different from those between thyroid hormones and normal cells. Received: 28 January 1999 / Accepted: 26 August 1999     

CD44 potentiates the adherence of metastatic prostate and breast cancer cells to bone marrow endothelial cells

The aim of this current study was to examine the significance of CD44 expression in mediating cancer cell adhesion to human bone marrow endothelial cell(s) (hBMEC). Differential CD44 expression on two metastatic prostate cancer cell lines, PC3 (CD44 +ve) and DU145 (CD44 -ve) and four breast cancer cell lines was confirmed by immunoblotting and immunocytochemistry. In cell adhesion assays, PC3 but not DU145 cells demonstrated a rapid adhesion to hBMECs. Treatment of PC3 cells with a neutralizing antibody against CD44 standard (CD44s) and CD44 splice variants decreased PC3 cell adhesion to hBMECs. Similarly, depletion of CD44 expression using RNA interference decreased the ability of PC3 cells and two CD44 +ve breast cancer cell lines (MDA-MB-231 and MDA-MB-157) to bind FITC-conjugated hyaluronan (FITC-HA) and to adhere to hBMECs. In contrast, transfection of DU145 cells or the T47D and MCF-7 breast cancer cell lines to express CD44s increased cell surface binding of FITC-HA and cell adherence to hBMECs. Treatment of PC3 and MDA-MD-231 cells but not hBMECs with hyaluronidase attenuated cell adhesion, suggesting that cell surface expression of CD44 on prostate and breast cancer cells may promote the retention of a HA coat that facilitates their initial arrest on bone marrow endothelium.     

Adenovirus-mediated IKKbetaKA expression sensitizes prostate carcinoma cells to TRAIL-induced apoptosis

Despite the fact that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in cancer cells, TRAIL resistance in cancer cells has challenged the use of TRAIL as a therapeutic agent. First, prostate carcinoma cell lines (DU145, LNCaP and PC3) were screened for sensitivity to adenovirus delivery of TRAIL (Ad5hTRAIL). As amplified Ikappa B kinase (IKK) activity is responsible for the constitutive nuclear factor-kappaB (NF-kappaB) activation leading to uncontrolled cell growth and metastasis, a dual vector approach using both an adenovirus vector (Ad) expressing the dominant-negative mutant of IKKbeta (AdIKKbetaKA) and Ad5hTRAIL was employed to determine if prostate cancer cells were sensitized to TRAIL in the setting of IKK inhibition. Inhibition of the NF-kappaB pathway through IKK blockade sensitized all three prostate cancer cell lines to TRAIL, regardless of NF-kappaB activation or decoy receptor gene expression. Moreover, a novel quantitative real-time RT-PCR assay and conventional flow cytometry analysis indicated that TRAIL-resistant DU145 and LNCaP cells, but not TRAIL-sensitive PC3 cells, expressed substantial amounts of TRAIL Decoy Receptor 4. In conclusion, TRAIL decoy receptor expression appeared to be the chief determinant of TRAIL resistance encountered in prostate carcinoma cell lines.     

Secreted factors from metastatic prostate cancer cells stimulate mesenchymal stem cell transition to a pro‐tumourigenic ‘activated’ state that enhances prostate cancer cell migration

Mesenchymal stem cells (MSCs) are a heterogeneous population of multipotent cells that are capable of differentiating into osteocytes, chondrocytes and adipocytes. Recently, MSCs have been found to home to the tumour site and engraft in the tumour stroma. However, it is not yet known whether they have a tumour promoting or suppressive function. We investigated the interaction between prostate cancer cell lines 22Rv1, DU145 and PC3, and bone marrow‐derived MSCs. MSCs were ‘educated’ for extended periods in prostate cancer cell conditioned media and PC3‐educated MSCs were found to be the most responsive with a secretory profile rich in pro‐inflammatory cytokines. PC3‐educated MSCs secreted increased osteopontin (OPN), interleukin‐8 (IL‐8) and fibroblast growth factor‐2 (FGF‐2) and decreased soluble fms‐like tyrosine kinase‐1 (sFlt‐1) compared to untreated MSCs. PC3‐educated MSCs showed a reduced migration and proliferation capacity that was dependent on exposure to PC3‐conditioned medium. Vimentin and α‐smooth muscle actin (αSMA) expression was decreased in PC3‐educated MSCs compared to untreated MSCs. PC3 and DU145 education of healthy donor and prostate cancer patient‐derived MSCs led to a reduced proportion of FAP+ αSMA+ cells contrary to characteristics commonly associated with cancer associated fibroblasts (CAFs). The migration of PC3 cells was increased toward both PC3‐educated and DU145‐educated MSCs compared to untreated MSCs, while DU145 migration was only enhanced toward patient‐derived MSCs. In summary, MSCs developed an altered phenotype in response to prostate cancer conditioned medium which resulted in increased secretion of pro‐inflammatory cytokines, modified functional activity and the chemoattraction of prostate cancer cells.     

Identification of genes linked to gefitinib treatment in prostate cancer cell lines with or without resistance to androgen: a clue to application of gefitinib to hormone-resistant prostate cancer

Understanding the molecular action of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, might allow us to perform more effective therapies for hormone-independent advanced prostate cancer. A DNA microarray study was undertaken to comprehensively analyze the alteration of levels of 1,081 genes after gefitinib treatment in androgen-independent PC3 and DU145 cells and androgen-dependent LNCaP cells. The proliferation of PC3, DU145 and LNCaP cells was significantly inhibited by 50.2%, 83.8% and 55.2%, respectively, 6 days after 10 microM gefitinib administration. Of the above 1,081 genes, we identified 23, 13 and 33 genes with significantly different expression in PC3, DU145 and LNCaP cells, respectively, 24 h after 10 microM-gefitinib exposure. Among the identified genes, only Quiescin Q6, a negative cell cycle regulator, was increased after gefitinib treatment in all three cell lines regardless of gefitinib sensitivity. Except for Quiescin Q6, there were no overlapping genes between PC3 and DU145 cells. However, levels of several oncogenes or proliferation-related genes were changed after gefitinib treatment in the 2 androgen-independent cell lines. We also identified 7 unique genes [glycyl-tRNA synthetase, interferon, alpha-inducible protein, stratifin, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, dual specificity phosphatase 9, guanine nucleotide binding protein (G protein) beta polypeptide 2, neural retina leucine zipper] whose levels were altered exclusively after gefitinib administration in gefitinib-resistant PC3 and LNCaP cells, but not in DU145 cells, suggesting that these 7 genes could be targets for overcoming gefitinib resistance. Collectively, our molecular profiling data will serve as a framework for understanding the molecular action of gefitinib for prostate cancer.     

Growth of human prostate cancer cells is significantly suppressed in vitro with sodium butyrate through apoptosis

Histone deacetylase inhibitors (HDACis) have shown significant antiproliferative and apoptotic properties in various types of cancer cells, including prostate cancer cells, and are therefore being evaluated as a treatment modality. However, the mechanism by which sodium butyrate (SB) induces apoptosis is not completely understood. We focused on SB which exists in the intestine and is therefore expected to have less adverse effects. In this study, three prostate cancer cell lines (LNCaP, DU145 and PC-3) were treated in vitro with different concentrations of SB. Cell proliferation was studied by the XTT assay; cell cycle analysis and induction of apoptosis were studied by laser scanning cytometry. Western blot analysis was used to study p21, p27, CDK2, CDK4, CDK6, caspase-3, caspase-7, Fas, FADD, TRADD, Bcl-2 and Bax protein expression. SB inhibited cell growth and induced apoptosis in a concentration-dependent manner in human prostate cancer cells (LNCaP, DU145 and PC-3). Western blot analysis showed dose-dependent increases of p21 levels in DU145 and PC-3 cells, and dose-dependent decreases of CDK2, CDK4, CDK6 and procaspase-3 protein levels in all three prostate cancer cell lines. Bcl-xL was significantly down-regulated in DU145 cells, and Bcl-2 was significantly down-regulated in PC-3 and LNCaP cells. No significant changes were observed in procaspase-7, TRADD and Bax expression, although slight decreases in Fas and FADD expression were seen in all three prostate cancer cell lines. Analysis of cell morphology using laser scanning microscopy detected condensed and fragmented nuclei. In conclusion, SB induces G1 and G2 arrest by increasing p21 expression resulting in CDK2, CDK4 and CDK6 down-regulation. SB potently induced apoptosis, which was accompanied by DNA fragmentation, down-regulated Bcl-2 in LNCaP and PC-3 cells, Bcl-xL in DU145 cells, and down-regulated procaspase-3, but not procaspase-7, in these human prostate cancer cell lines. These results suggest that SB may serve as a new modality for the treatment of hormone refractory prostate cancer.     

Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells

Impaired Ca²⁺ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na⁺ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca²⁺ entry (SOCE) by decreasing the driving force for Ca²⁺. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer.We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na⁺ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target.     

沉默CHD1L对前列腺癌PC3细胞恶性生物学行为的影响及其可能机制

目的:探讨染色质解旋酶DNA结合蛋白1样基因(chromodomain helicase/ATPase DNA binding protein 1-like gene,CHD1L)对前列腺癌细胞侵袭、迁移能力的影响及其可能的作用机制.方法:采用实时荧光定量PCR技术检测前列腺癌细胞株LNCAP、PC3、DU145以及前列腺上皮细胞株RWPE-1中CHD1L mRNA表达水平;转染siRNA干扰前列腺癌PC3细胞CHD1L的表达,并用Transwell侵袭实验和划痕实验分析沉默CHD1L对前列腺癌细胞侵袭和迁移能力的影响;Western blotting 检测PC3细胞MMP-9、N-钙黏蛋白和E-钙黏蛋白的表达水平.结果:CHD1L mRNA在前列腺癌细胞中的表达水平明显高于前列腺上皮细胞(P＜0.01),其中以前列腺癌PC3细胞的表达水平最高.侵袭实验中,干扰组的穿膜细胞数明显低于阴性对照组和空白对照组[(49.67 ±6.67)vs(113.67 ±5.69)和(112.00±12.49)个,P＜0.05).划痕实验中,干扰组48 h伤口愈合率也低于阴性对照组和空白对照组[(21.27 ±3.27)％ vs(48.47±5.72)％和(49.93±3.35)％,P＜0.05].干扰组细胞MMP-9和N-钙黏蛋白表达下调,E-钙黏蛋白表达上调.结论:沉默CHD1L可降低前列腺癌PC3细胞的侵袭迁移能力,该作用可能是通过调控MMP-9和EMT相关蛋白表达实现的.     

Suppression of telomerase, reexpression of KAI1, and abrogation of tumorigenicity by nerve growth factor in prostate cancer cell lines.

Nerve growth factor (NGF) is expressed in the prostate, where it appears to be involved in the control of epithelial cell growth and differentiation. NGF production is decreased in prostate tumors. However, the role of this neurotrophin in the control of proliferation and progression of prostate cancers is still a matter of investigation. Prostate adenocarcinomas are telomerase-positive tumors. Chronic exposure of DU145 and PC3 prostate tumor cell lines to NGF resulted in a dramatic down-regulation of telomerase activity. This effect was correlated in terms of concentrations and time with a remarkable down-regulation of cell proliferation both in vitro and in vivo but was not secondary to NGF-induced quiescence. No down-regulation of telomerase activity was, in fact, detectable during serum starvation-induced quiescence. LNCaP cells, which do not express NGF receptors, appear to be insensitive to the actions of NGF. DU145 and PC3 cells do not express the KAI1 metastasis suppressor gene, which is present in the prostate and is progressively lost during the progression of prostate cancers. Chronic NGF treatment strongly induced the reexpression of this gene in these cell lines, and this effect was correlated with the suppression of their invasive potential in vitro. The data presented here suggest that NGF reverts two metastatic prostate cancer cell lines to slowly proliferating, noninvasive phenotypes characterized by a very low telomerase activity and by the expression of the KAI1 metastasis suppressor gene.