Inhibition of cell growth and induction of apoptosis in human prostate cancer cell lines by 6-aminoquinolone WM13

Fluoroquinolones affect the proliferation and apoptotic cell death of several human malignancies. Therefore, we investigated whether new 6-aminoquinolone derivatives, initially synthesized as anti-HIV agents, could affect the proliferation and apoptotic cell death of human prostate cancer cell lines. PC3 and LNCaP cell lines were used as models of androgen-resistant and androgen-responsive prostate cancer, and proliferation of PC3 and LNCaP cells was strongly inhibited by 6-aminoquinolone WM13. Cytotoxicity, which was more pronounced in LNCaP, was accompanied by morphological changes, DNA damage, arrest at the S/G(2)/M phase of the cell cycle, and an increase of the sub-G(1) population. Molecular mechanism underlying WM13-induced cell death involved caspase-8 and -3 and modulation of the expression of apoptotic genes, as well as cleavage of poly-ADP ribose polymerase. Cell death following the treatment of human prostate cancer cell lines with WM13 can be attributed to apoptosis which, depending on the cell line, proceeds through different pathways.     

PAR, a novel androgen regulated gene, ubiquitously expressed in normal and malignant cells

During our work on the mechanism of hormone resistance of prostatic carcinomas, a novel gene that we called PAR (prostate androgen regulated) was isolated from an androgen resistant subline (LNCaP-OM) using a modified representational difference analysis. The complete sequence of the gene cDNA has 1029 nucleotides with a continuous reading frame of 438 bases encoding for 146 amino acids. Its deduced amino acid sequence has motifs for myristoylation and phosphorylation by protein kinase C. The PAR gene was overexpressed in all prostatic carcinoma cell lines studied (LNCaP, DU145, PC3 and LNCaP-OM) compared to the normal prostatic tissue. Furthermore, its expression was higher in androgen resistant prostate cancer lines DU145, PC3 and LNCaP-OM, in comparison to androgen sensitive LNCaP cells. The expression of this gene was down regulated by androgens in androgen sensitive prostate cells, but not in the hormone resistant cell lines. The PAR mRNA was detected in all 29 normal human tissues studied and overexpressed in most (67%) of their malignant counterparts. The PAR expression was higher in MCF7 and T47D breast cancer cell lines, as well as in all primary breast tumors studied compared to their normal tissue counterparts. The biological function of this gene is still unknown, but its ubiquitous expression in normal tissues and its overexpression in some malignancies suggest the PAR involvement in certain basic cellular processes and possibly, in malignant transformation.     

Apoptotic conversion: evidence for exchange of genetic information between prostate cancer cells mediated by apoptosis.

Changes in the outer membrane of apoptotic cells can induce neighboring cells to become phagocytic. Using genetically marked prostate cancer cell lines, we explored the possibility that genetic information might be transferred from an apoptotic cell to a phagocytic neighbor. Neomycin-resistant LNCaP cells that overexpress bcl-2 (LNCaP(bcl-2/neo-r)) were cocultured with hygromycin-resistant LNCaP cells (LNCaP(hygr-r)). The cocultures were then transiently exposed to serum starvation to induce apoptosis of LNCaP(hygr-r) cells. Surviving cells were then coselected in medium containing both antibiotics. Whereas monocultures of LNCaP(bcl-2/neo-r) or LNCaP(hygr-r) treated this way yielded no colonies, cocultures yielded dual-antibiotic-resistant clones at a frequency of approximately 1 in 10(5). Pre-exposure to an apoptotic agent was required; cocultures not exposed to serum starvation yielded no dual-selectable colonies. Analysis of DNA extracted from a dual-resistant clone demonstrated that the restriction endonuclease pattern of the neo-r gene was unaltered when compared with the parental LNCaP(bcl-2/neo-r). However the hygr-r gene demonstrated an altered restriction endonuclease pattern in the dual-resistant derivative compared with the parental LNCaP(hygr-r) cell line. This is evidence that genetic information can be transferred from one prostate cancer cell to another through the process of apoptosis, and we term this form of genetic transfer "apoptotic conversion."     

Aberrant sphingolipid signaling is involved in the resistance of prostate cancer cell lines to chemotherapy

Activation of the apoptosis program has been implicated in the response of cancer cells to chemotherapy. Therefore, we postulated that chemotherapy-resistant prostate cancer has developed a lesion in the apoptosis signal transduction cascade. In this study, we investigated the mechanism underlying the resistance of apoptosis-insensitive prostate cancer cells to apoptosis. We approached this by comparing the response of the androgen-sensitive LNCaP cell line and the androgen-insensitive PC3 cell line to treatment with the topoisomerase I inhibitor, camptothecin. We demonstrated that LNCaP cells are susceptible to camptothecin-induced cell death, and PC3 cells are resistant. Additional studies confirmed that the mode of cell death in the LNCaP cells was by apoptosis. We then determined that a component of the resistance to death in the apoptosis-insensitive cells involved a defect in the generation of ceramide, a key lipid mediator of apoptosis. Specifically, we demonstrated that PC3 cells are unable to elevate ceramide in response to treatment with camptothecin. In contrast, elevations in ceramide levels occur in LNCaP cells in response to the same treatment. Significantly, additional studies showed that treatment with exogenous ceramide overcomes the lesion in the PC3 cells and induces apoptosis. In attempting to gain preliminary insight into the nature of the lesion in ceramide formation in the apoptosis-resistant cells, we established that generation of ceramide in LNCaP cells is independent of the de novo pathway. These studies present novel insights into the mechanism by which prostate cancer cells may be resistant to induction of apoptosis. The significance of this study lies in the fact that an understanding of the biological and molecular events contributing to the resistance of prostate cancer to therapy is crucial to the development of more effective regimens for advanced disease.     

Doxazosin induces apoptosis in LNCaP prostate cancer cell line through DNA binding and DNA-dependent protein kinase down-regulation

Doxazosin is a quinazoline-based compound acting as an alpha-1-adrenergic inhibitor shown to induce apoptosis in prostate cancer cell lines via an alpha-1-adrenergic receptor-independent mechanism. To better understand the mechanism of doxazosin-induced apoptosis in prostate cancer, we performed cDNA microarray to analyze gene expression changes produced by doxazosin in the androgen-dependent human prostate cancer cell line, LNCaP. We found that 70 and 92 genes were deregulated after 8 and 24 h of doxazosin treatment, respectively. These genes are involved in several cellular processes such as cell-cycle regulation, cell adhesion and signal transduction pathways. Strikingly, we found that doxazosin induces deregulation of genes implicated in DNA replication and repair, such as GADD45A, XRCC5 and PRKDC. These facts, together with the demonstration of the ability of doxazosin to bind DNA, allowed us to propose a novel mechanism of action for doxazosin in prostate cancer cells that implies DNA-damage mediated apoptosis by down-regulation of XRCC5 and PRKDC genes.     

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)     

Identification and characterization of prostein, a novel prostate-specific protein

In this report, we describe the application of a systematic, genome-based approach to identify prostein, a novel prostate-specific protein expressed in normal and malignant prostate tissues. Characterization of the prostein gene shows that prostein cDNA encodes a 553-amino acid protein. The protein is predicted to be a type IIIa plasma membrane protein with a cleavable signal peptide and 11 transmembrane-spanning regions. The prostein gene is located on chromosome 1 at the WI-9641 locus between q32 and q42. Prostein mRNA is shown to be uniquely expressed in normal and cancerous prostate tissues using Northern blot, eDNA microarray, and real-time PCR analyses. Furthermore, prostein mRNA expression does not appear to be prostate tumor grade related and is restricted exclusively to prostate cell lines. Immunohistochemical staining using a mouse monoclonal antibody generated against prostein demonstrates that this protein is specifically detected in prostate tissues both at the plasma membrane and in the cytoplasm. Prostein expression is androgen responsive because treatment of LNCaP cells with androgen up-regulates prostein message and protein expression levels. These results validate prostein as a prostate-specific marker with potential utility in the diagnosis and treatment of prostate cancer.     

PCOTH, a novel gene overexpressed in prostate cancers, promotes prostate cancer cell growth through phosphorylation of oncoprotein TAF-Ibeta/SET

Through genome-wide cDNA microarray analysis coupled with microdissection of prostate cancer cells, we identified a novel gene, prostate collagen triple helix (PCOTH), showing overexpression in prostate cancer cells and its precursor cells, prostatic intraepithelial neoplasia (PIN). Immunohistochemical analysis using polyclonal anti-PCOTH antibody confirmed elevated expression of PCOTH, a 100-amino-acid protein containing collagen triple-helix repeats, in prostate cancer cells and PINs. Knocking down PCOTH expression by small interfering RNA (siRNA) resulted in drastic attenuation of prostate cancer cell growth, and concordantly, LNCaP derivative cells that were designed to constitutively express exogenous PCOTH showed higher growth rate than LNCaP cells transfected with mock vector, suggesting the growth-promoting effect of PCOTH on prostate cancer cell. To investigate the biological mechanisms of this growth-promoting effect, we applied two-dimensional differential gel electrophoresis (2D-DIGE) to analyze the phospho-protein fractions in LNCaP cells transfected with PCOTH. We found that the phosphorylation level of oncoprotein TAF-Ibeta/SET was significantly elevated in LNCaP cells transfected with PCOTH than control LNCaP cells, and these findings were confirmed by Western blotting and in-gel kinase assay. Furthermore, knockdown of endogenous TAF-Ibeta expression by siRNA also attenuated viability of prostate cancer cells as well. These findings suggest that PCOTH is involved in growth and survival of prostate cancer cells thorough, in parts, the TAF-Ibeta pathway, and that this molecule should be a promising target for development of new therapeutic strategies for prostate cancers.     

Role of fibroblast growth factor receptor signaling in prostate cancer cell survival.

BACKGROUND: Expression of fibroblast growth factors (FGFs) is increased in a substantial fraction of human prostate cancers in vivo and in prostate cancer cell lines. Altered FGF signaling can potentially have a variety of effects, including stimulating cell proliferation and inhibiting cell death. To determine the biologic significance of altered FGF signaling in human prostate cancer, we disrupted signaling by expression of a dominant-negative (DN) FGF receptor in prostate cancer cell lines. METHODS: PC-3, LNCaP, and DU145 prostate cancer cells were stably transfected with DN FGFR constructs, and LNCaP and DU145 cells were infected with a recombinant adenovirus expressing DN FGFR-1. The effect of DN FGFR-1 expression was assessed by colony-formation assays, cell proliferation assays, flow cytometry, and cytogenetic analysis. Key regulators involved in the G(2)-to-M cell cycle transition were assessed by western blotting to examine cyclin B1 expression and by in vitro kinase assay to assess cdc2 kinase activity. RESULTS: Stable transfection of the DN FGFR-1 construct inhibited colony formation by more than 99% in all three cell lines. Infection of LNCaP and DU145 prostate cancer cells with adenovirus expressing DN FGFR-1 led to extensive cell death within 48 hours. Flow cytometry and cytogenetic analysis revealed that the DN FGFR-1 receptor led to arrest in the G(2) phase of the cell cycle before cell death. Cyclin B1 accumulated in DN FGFR-1-infected LNCaP cells, but cdc2 kinase activity was decreased. CONCLUSIONS: These findings reveal an unexpected dependence of prostate cancer cells on FGF receptor signal transduction to traverse the G(2)/M checkpoint. The mechanism for the G(2) arrest is not clear. Our results raise the possibility that FGF-signaling antagonists might enhance the cell death induced by other prostate cancer therapies.     

Prostate-specific antigen (PSA) promoter-driven androgen-inducible expression of sodium iodide symporter in prostate cancer cell lines

Currently, no curative therapy for metastatic prostate cancer exists. Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) would enable those cells to concentrate iodide from plasma and might offer the ability to treat prostate cancer with radioiodine. Therefore, the aim of our study was to achieve tissue-specific expression of full-length human NIS (hNIS) cDNA in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP and in subcell lines C4, C4-2, and C4-2b in vitro. For this purpose, an expression vector was generated in which full-length hNIS cDNA coupled to the prostate-specific antigen (PSA) promoter has been ligated into the pEGFP-1 vector (NIS/PSA-pEGFP-1). The PSA promoter is responsible for androgen-dependent expression of PSA in benign and malignant prostate cells and was therefore used to mediate androgen-dependent prostate-specific expression of NIS. In addition, two control vectors were designed, which consist of the pEGFP-1 vector containing the PSA promoter without NIS cDNA (PSA-pEGFP-1) and NIS cDNA without the PSA promoter (NIS-pEGFP-1). Prostate cancer cells were transiently transfected with each of the above-described expression vectors, incubated with or without androgen (mibolerone) for 48 h, and monitored for iodide uptake activity. In addition, stably transfected LNCaP cell lines were established for each vector. Prostate cells transfected with NIS/PSA-pEGFP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in a range comparable to that observed in control cell lines transfected with hNIS cDNA. Perchlorate-sensitive iodide uptake was not observed in cells transfected with NIS/PSA-pEGFP-1 and treated without androgen or in cells transfected with the control vectors. In addition, prostate cancer cell lines without PSA expression (PC-3 and DU-145) did not show iodide uptake activity when transfected with NIS/PSA-pEGFP-1. Western blotting of LNCaP and C4-2b cell membranes transfected with NIS/PSA-pEGFP-1 using a monoclonal antibody that recognizes the COOH-terminus of hNIS revealed a band with a molecular weight of 90,000 that was not detected in androgen-deprived cells or in cells transfected with the control vectors, as well as a minor band at Mr 150,000 in transiently transfected LNCaP cell membranes. In conclusion, tissue-specific androgen-dependent iodide uptake activity has been induced in prostate cancer cells by PSA promoter-directed NIS expression. This study represents an initial step toward therapy of prostate cancer with radioiodine.     

基因芯片分析前列腺癌细胞系中转移相关基因

目的:通过基因芯片技术检测具有不同转移潜能的前列腺癌细胞系LNCaP和C4-2中表达差异的基因,寻找前列腺癌转移相关基因.方法:采用TRIzol一步法提取LNCaP和C4-2细胞的总RNA,并纯化mRNA,反转录合成荧光分子标记的cDNA探针,与基因芯片杂交.采用Genepix Pro 6.0图像分析软件进行芯片图像分析,把图像信号转化为数字信号,然后以差异大于等于2倍的标准来确定差异表达基因.结果:在LNCaP与C4-2细胞中,表达差异的基因共有417个,上调基因301个,下调基因116个.分析显示差异表达基因分别与细胞增殖、代谢、细胞因子、细胞转移等相关,其中发现7个基因与肿瘤细胞的转移相关,分别为EGFR、EGF、PIK3R1、Cyclin E、HYAL1、GNAI1、AZGP1.结论:筛选出LNCaP与C4-2细胞系中7个转移相关基因,为进一步研究前列腺癌转移机制奠定了基础.     

Pancratistatin induces apoptosis and autophagy in metastatic prostate cancer cells

The Amaryllidaceae alkaloid pancratistatin has been proven to selectively induce apoptotic cell death in a variety of human cancer cells with an insignificant effect on non-cancerous cells. In this study we report, for the first time, the effects of pancratistatin (PST) on models of metastatic prostate cancer. The effects of pancratistatin on prostate cancer DU145 and LNCaP cell lines was assessed by microscopy, enzymatic activity assays and Western blotting. Apoptosis was determined by nuclear condensation and caspase activation, and autophagy was observed by MDC staining and LC3 expression levels. Human prostate xenografts were used to test the potential therapeutic efficacy of intra-tumor administration of pancratistatin in vivo. Pancratistatin treatment reduced cell viability and induced apoptosis in androgen-responsive (LNCaP) and androgen-refractory (DU145) prostate cancer cell lines in a dose- and time-dependent manner, but with an insignificant effect on normal human fibroblast (NHF) cells at the effective dose. Increased reactive oxygen species production and collapse of mitochondrial membrane potential resulted from treatment with pancratistatin in both cancer cell lines. This study presents the novel finding that pancratistatin treatment caused decreased migration capacity and increased autophagy levels in metastatic prostate cancer cells. Importantly, in this proof-of-concept study, pancratistatin reduced the volume of xenograft tumors compared to control-treated animals, and was well-tolerated. Our results highlight the potential of pancratistatin for clinical development as a selective therapeutic for treatment of metastatic prostate cancer.     

Induction of apoptosis in prostate cancer cell lines by the green tea component, (−)-epigallocatechin-3-gallate

Green tea components exert many biological effects, including antitumor and cancer preventive activities. In the search for anticancer agents for prostate cancer the inhibitory effects of green tea components were tested on the prostate cancer cell lines LNCaP, PC-3 and DU145. (−)-Epigallocatechin-3-gallate (EGCG) proved to be the most potent catechin at inhibiting cell growth. The inhibition induced by EGCG was found to occur via apoptotic cell death as shown by changes in nuclear morphology and DNA fragmentation. Thus, we report the first evidence that EGCG is the active component in green tea and induces apoptosis in human prostate cancer cells.