A novel gene on human chromosome 2p24 is differentially expressed between androgen-dependent and androgen-independent prostate cancer cells

Identification of genes involved in the transition from androgen-dependent to androgen-independent prostate cancer is important to extend our current knowledge of the disease. Using differential display RT-PCR analysis between androgen-dependent and androgen-independent prostate cancer cells, we have identified a novel gene, designated GC109. GC109 harbours a putative Cys-His cluster, a nuclear localisation signal, a leucine zipper and a ret finger protein (rfp)-like domain. GC109 mRNA expression in normal human tissues was found not to be restricted to the prostate. However, using a variety of 15 human cancer cell lines, GC109 mRNA was preferentially expressed in androgen-dependent LNCaP-FGC, compared with androgen-independent LNCaP-LNO, DU145 and PC3 human prostate cancer cells. Finally, the GC109 gene was mapped on human chromosome 2p24. Based on its protein domain structure and chromosomal localisation, we hypothesise that GC109 may be involved in chromosomal rearrangements in prostate cancer.     

Enhanced sensitivity to androgen withdrawal due to overexpression of interleukin-6 in androgen-dependent human prostate cancer LNCaP cells

Background:

The objective of this study was to investigate the effects of interleukin-6 (IL-6) overexpression in androgen-dependent prostate cancer LNCaP cells on their phenotype under an androgen-deprived condition.

Methods:

We established IL-6-overexpressing LNCaP (LNCaP/IL-6) by introducing the expression vector containing IL-6 cDNA. Changes in the phenotype in LNCaP/IL-6 were compared with that in LNCaP transfected with control vector alone (LNCaP/Co).

Results:

In vitro, the growth of LNCaP/IL-6 was significantly inferior to that of LNCaP/Co under an androgen-deprived condition. Similarly, LNCaP/IL-6 tumour in nude mice rapidly regressed after castration; however, LNCaP/Co tumour growth was transiently inhibited after castration and then continuously accelerated. After androgen withdrawal, expression levels of phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and Akt in LNCaP/IL-6 were markedly upregulated compared with those in LNCaP/Co; however, additional treatment with specific inhibitor of the MAPK or Akt signalling pathway significantly inhibited the growth of LNCaP/IL-6 compared with that of LNCaP/Co. Furthermore, gene microarray analyses showed that androgen deprivation resulted in differential expression of genes involved in growth, apoptotsis and tumorigenesis between LNCaP/Co and LNCaP/IL-6.

Conclusion:

Excessive secretion of IL-6 by LNCaP cells in an autocrine manner may have a suppressive function in their growth and acquisition of androgen-independent phenotype under an androgen-deprived condition.     

Antiproliferative activity of novel imidazopyridine derivatives on castration-resistant human prostate cancer cells

Metastatic prostate cancer (mPCa) relapses after a short period of androgen deprivation therapy and becomes the castration-resistant prostate cancer (CR PCa); to which the treatment is limited. Hence, it is imperative to identify novel therapeutic agents towards this patient population. In the present study, antiproliferative activities of novel imidazopyridines were compared. Among three derivatives, PHE, AMD and AMN, examined, AMD showed the highest inhibitory activity on LNCaP C-81 cell proliferation, following dose- and time-dependent manner. Additionally, AMD exhibited significant antiproliferative effect against a panel of PCa cells, but not normal prostate epithelial cells. Further, when compared to AMD, its derivative DME showed higher inhibitory activities on PCa cell proliferation, clonogenic potential and in vitro tumorigenicity. The inhibitory activity was apparently in part due to the induction of apoptosis. Mechanistic studies indicate that AMD and DME treatments inhibited both AR and PI3K/Akt signaling. The results suggest that better understanding of inhibitory mechanisms of AMD and DME could help design novel therapeutic agents for improving the treatment of CR PCa.     

Evidence for clonal outgrowth of androgen-independent prostate cancer cells from androgen-dependent tumors through a two-step process.

Prostate cancers require androgen for growth but progress to an androgen-independent stage under the selective pressure of androgen ablation therapy. Here we describe a novel human prostate cancer xenograft (LAPC-9) propagated by serial passage in male severe combined immunodeficient mice that expresses prostate-specific antigen and wild-type androgen receptor. In response to castration, LAPC-9 cells undergo growth arrest and persist in a dormant, androgen-responsive state for at least 6 months. After prolonged periods of androgen deprivation, spontaneous androgen-independent outgrowths develop. Thus, prostate cancers progress to androgen independence through two distinct stages, initially escaping dependence on androgen for survival and, subsequently, for growth. Through the use of serial dilution and fluctuation analysis, we provide evidence that the latter stage of androgen independence results from clonal expansion of androgen-independent cells that are present at a frequency of about 1 per 10(5)-10(6) androgen-dependent cells. We conclude that prostate cancers contain heterogeneous mixtures of cells that vary in their dependence on androgen for growth and survival and that treatment with antiandrogen therapy provides selective pressure and alters the relative frequency of these cells, thereby leading to outgrowths of androgen-independent cancers.     

新型棉酚衍生物对体外前列腺癌LNCaP细胞自噬影响的研究

目的:研究新型棉酚衍生物Apogossypolone(ApoG2)在体外对前列腺癌LNCaP细胞的自噬作用.方法:采用MTT、AO染色、透射电镜、流式细胞术和免疫组化方法,研究ApoG2对前列腺癌LNCaP细胞的生长抑制及诱导自噬的作用.结果:当ApoG2浓度＞2.5,μg/mL时,对前列腺癌LNCaP细胞有明显的增殖抑制,且有时间与剂量依赖性,ApoG2作用LNCaP细胞72 h时的IC50值为4.43 μg/mL.AO染色观察LNCaP细胞有自噬特征;透射电镜观察细胞内有自噬泡形成;流式细胞术检测细胞内出现大量酸性液泡细胞器(AVOs);免疫组化结果显示,ApoG2作用于LNCaP细胞后可以使细胞内Beclin 1的表达增高.结论:ApoG2在体外可以诱导前列腺癌LNCaP细胞的自噬,抑制前列腺癌LNCaP细胞的生长.     

Valproic acid activity in androgen-sensitive and -insensitive human prostate cancer cells

Histone deacetylase (HDAC) inhibitors are currently undergoing clinical trials in cancer patients on the basis of their effect on proliferation, differentiation and apoptosis demonstrated in vitro. The goal of this study was to assess the effects of an HDAC inhibitor, valproic acid (VA), on proliferation, androgen-sensitivity, androgen receptor levels and E-cadherin (E-cad) expression in human prostate cancer cells. The effects of VA were evaluated in androgen-sensitive, LNCaP and -insensitive PC-3 human prostate cancer cell lines. Proliferation was assayed by cell counts and protein expression by Western blot analysis. Morphological changes were analysed under an inverted phase contrast microscope. High VA concentrations (1-25 mM) induced a very strong reduction in cell numbers ( approximately 90% with respect to control) of the two cell lines due to drug cytotoxicity. A low concentration (0.45 mM VA) slightly reduced (14%) LNCaP cell proliferation and abolished the response to androgen. In the PC-3 cells, the same concentration of VA had a more pronounced (40%) inhibitory effect and induced a response to dihydrotestosterone in terms of an enhancement in cell growth. These events were associated with morphological changes, an absence of cytotoxicity, an increase in androgen receptor levels, and, in PC-3 cells, an enhancement in E-cad expression which may be ascribed to VA differentiative action. Our findings, obtained with a VA dose (0.45 mM), which is consistent with plasma concentrations reached under oral administration of therapeutical doses in patients treated for different diseases, suggest that VA might have clinical value in prostate cancer therapy in androgen-sensitive and -insensitive tumors.     

Treatment of prostate cancer cells with adenoviral vector-mediated antisense RNA using androgen-dependent and androgen-independent promoters

The present study was designed to develop a novel antisense approach to prostate cancer therapy. We constructed ODC/AdoMetDC double antisense RNA recombinant adenovirus mediated by a prostate-specific androgen-dependent promoter (pADxsi-P2-AdoMetDC-ODC-PolyA) or a prostate-specific androgen-independent promoter (pADxsi-P1-AdoMetDC-ODC-PolyA). Western blot analysis was performed to detect the ODC and AdoMetDC protein levels. The growth curves for each group of cells were determined by MTT assay. Flow cytometry was conducted to detect cell apoptosis, proliferation, and cell cycle distribution in order to demonstrate the effects of the recombinant adenoviruses on the prostate cancer cells. RT-PCR, Western blotting, MTT, and tumor growth inhibition assay in nude mice demonstrated that pADxsi-P1-AdoMetDC-ODC-PolyA and pADxsi-P2-AdoMetDC-ODC-PolyA exhibited inhibitory effects on cell proliferation at both the gene and protein levels. Meanwhile, inhibiting effects of the pADxsi-P2-AdoMetDC-ODC-PolyA is more profound than those of the pADxsi-P1-AdoMetDC-ODC-PolyA vector. Both of the adenoviral vectors exhibited significant inhibitory effects on the growth of prostate tumors, and the inhibitory effects of the pADxsi-P2-AdoMetDC-ODC-PolyA vector were stronger than those of the pADxsi-P1-AdoMetDC-ODC-PolyA vector.     

Correlation between overexpression of EpCAM in prostate tissues and genesis of androgen-dependent prostate cancer

The objective of this study was to investigate the role of epithelial cell adhesion molecule (EpCAM) in the genesis and the progress of prostate cancer, especially of castration-resistant prostate cancer. Protein expression of EpCAM in ten pairs of prostate cancer tissues and normal adjacent tissues, plus three cell lines, was examined. Short hairpin RNA (shRNA) interference technique was employed to silence the expression of EpCAM in prostate cancer cell LNCaP and construct a stable transfected cell line. In vitro assay was conducted to analyze the effect of EpCAM expression on the expressions of Androgen receptor (AR), Prostate specific antigen (PSA), and cellular proliferation and invasion. EpCAM was found significantly expressed higher in prostate cancer tissues than in normal adjacent tissues. In three cell lines (DU-145, PC-3, and LNCaP), the expression of EpCAM in LNCaP, androgen-dependent prostate cancer cells, was significantly higher than that in the other two. As EpCAM was silenced in LNCaP, the expression levels of AR and PSA obviously descended, and cellular abilities of proliferation and invasion were obviously inhibited.The overexpression of EpCAM has correlation with the genesis of prostate cancer, especially androgen-dependent prostate cancer. As the expression of AR is facilitated, prostate cancer cells’ abilities to proliferate and invade are consequently enhanced.     

Androgens regulate Hedgehog signalling and proliferation in androgen-dependent prostate cells

Prostate cancer (PCa) is androgen sensitive in its development and progression to metastatic disease. Hedgehog (Hh) pathway activation is important in the initiation and growth of various carcinomas including PCa. We and others have observed aberrations of Hh pathway during the progression of PCa to the castration-resistant state. The involvement of androgen signalling in Hh pathway activation, however, remains largely elusive. Here we investigate the direct role of androgen signalling on Hh pathway. We examined the effect of Dihydrosterone (DHT), antiandrogen, bicalutamide, and Hh pathway inhibitor, KAAD-cyclopamine in four human prostate cell lines (two cancerous: LNCaP, VCaP, and two normal: PNT2 and PNT2-ARm which harbours a mutant version of androgen receptor (AR) that is commonly found in LNCaP). Cell proliferation as well as Hh pathway members (SHH, IHH, DHH, GLI, PTCH) mRNA expression levels were assessed. We showed that KAAD-cyclopamine decreased cell proliferation of DHT-stimulated LNCaP, VCaP and PNT2-ARm cells. SHH expression was found to be downregulated by DHT in all AR posititve cells. The negative effect of DHT on SHH expression was counteracted when cells were treated by bicalutamide. Importantly, KAAD-cyclopamine treatment seemed to inhibit AR activity. Moreover, bicalutamide as well as KAAD-cyclopamine treatments induced GLI and PTCH expression in VCaP and PNT2-ARm. Our results suggest that Hh pathway activity can be regulated by androgen signalling. Specifically, we show that the DHT-induced inhibition of Hh pathway is AR dependent. The mutual interaction between these two pathways might be important in the regulation of cell proliferation in PCa.     

Thapsigargin resistance in human prostate cancer cells

BACKGROUND: Thapsigargin (TG) is a potent inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCAs). TG-based prodrugs are being developed for the treatment of prostate cancer (PC). To develop optimal TG-based therapeutics it is important to understand the mechanisms of resistance to TG that may potentially occur in cancer cells. METHODS: DU145/TG and PC3/TG cells were derived from human PC DU145 and PC3 cells, respectively, by incremental exposure to TG. Growth assays, Western blot analyses, cDNA microarrays, semiquantitative and real-time polymerase chain reaction (PCR), Northern blot analyses, and immunohistochemistry were used to study these cells. RESULTS: DU145/TG cells are 1100-fold and PC3/TG cells are 1350-fold resistant to TG. Although expression of both SERCA and p-glycoprotein can mediate TG resistance in hamster cells, neither is modulated in DU145/TG cells. In contrast, in PC3/TG cells, SERCA, and not p-glycoprotein, is significantly overexpressed but cannot by itself account for the 1350-fold resistance to TG in these cells. Several genes not previously identified to be altered by TG selection are modulated in DU145/TG and PC3/TG cells. Furthermore, the spectrum of genes modulated in DU145/TG cells are distinct from that in PC3/TG cells, even though both cells are of prostate origin and share the same TG-resistant phenotype. CONCLUSIONS: PC cells can adapt to SERCA inhibition by TG. However, they demonstrate cell type-specific plasticity with respect to gene expression upon TG selection. Further, previously not described mechanisms of resistance appear to be recruited in the TG-resistant PC cells, which provide a novel model to study mechanisms of resistance and adaptation in PC on TG-mediated dysregulation of Ca2+ homeostasis.     

A new jasmonic acid stereoisomeric derivative induces apoptosis via reactive oxygen species in human prostate cancer cells

With the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, in the present study, we evaluated the effect of a (−)-jasmonic acid derivative, the 3-hydroxy-2(S)-(2Z-butenyl)-cyclopentane-1(S)-acetic acid, obtained by biotransformation, on cell growth in androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells. The results obtained show that the new compound was able to inhibit the growth of both prostate cancer cells. In addition, our data seem to indicate that the apoptosis evocated by this new molecule, at least in part, appears to be associated with an increase of reactive oxygen species (ROS) production.     

Inhibition of telomerase activity by a distamycin derivative: effects on cell proliferation and induction of apoptosis in human cancer cells

In this study, we evaluated the potential of the distamycin derivative MEN 10716 as a telomerase inhibitor. Exposure of human melanoma cell extracts to MEN 10716 induced a dose-dependent inhibition of telomerase activity, with an IC50 of 24+/-3 microM. When intact JR8 melanoma cells were chronically exposed to the drug (200 microM every other day for 50 days), a marked inhibition (>80%) of the enzyme's catalytic activity was consistently observed starting from day 1. At later points in time, MEN 10716 inhibited melanoma cell proliferation and induced apoptosis. Cells surviving MEN 10716 exposure were characterised by a higher melanin content and a greater expression of p16(INK4A) protein than control cells. The effects of MEN 10716 were subsequently evaluated in different tumour cell systems. In particular, even in the H460 non-small cell lung cancer cell line, chronic exposure of the cells to the drug (100 microM every other day for 50 days) induced a consistent inhibition (>85%) of telomerase activity, a reduction of cell proliferation potential, and apoptosis. Conversely, MEN 10716 treatment did not appreciably inhibit cell proliferation in the U2-OS telomerase-negative human osteogenic sarcoma cell line. Interestingly, no variation in the mean telomere length was observed in MEN 10716-treated JR8 melanoma cells, whereas an appreciable increase in the mean telomere length was found in H460 lung cancer cells after drug exposure. Overall, the results of the study indicate that MEN 10716 is a possible telomerase inhibitor and suggest that abrogation of telomerase activity can affect cell proliferation even through pathways that are not dependent on telomere erosion.     

Comparative chemosensitivity of circulating human prostate cancer cells and primary cancer cells

The chemosensitivity of circulating PC-3 human prostate cancer cells, isolated from nude mice orthotopically implanted with PC-3, was compared to that of the parental PC-3 cells. PC-3 and circulating PC-3, both labeled with green fluorescent protein (GFP), were seeded in 96-well plates. The MTT assay was then performed at 24, 48, and 72 hours, comparing control cultures to cultures treated with cisplatin at 1, 2.5, 5 and 10 μm/ml, and docetaxel at 10, 20, 25 and 50 μm/ml at each time point. The circulating tumor cells (CTCs) exhibited a significantly increased sensitivity to both cisplatin and docetaxel when compared to PC-3 parental cells, with docetaxel having the greater efficacy. The future goal, based on these studies, is the culture of CTCs from cancer patients' peripheral blood for chemosensitivity testing, for improved individualized therapy.     

Androgen receptor binding activity in human prostate cancer

Androgen binding (cytosol and nucleus) was measured in tissue obtained from 223 untreated patients with proven prostate cancer (199 primary tumor, 24 malignant lymph nodes), 19 patients with hormone refractory cancer, and 46 patients with benign prostatic hyperplasia (BPH). The mean binding in both the cytosol and nucleus was significantly higher for patients with cancer than for those with BPH. Binding appeared to correlate with tumor stage. Androgen binding in malignant nodes can differ from that in the primary tissue and can vary from node to node in the same patient. Results obtained from an assay using a single saturating concentration of R1881 correlated well with those calculated from a full six-point Scatchard analysis when an adequate amount (500 mg) of tissue was available. However, binding results obtained from a single-point analysis performed on needle biopsy specimens (about 50 mg) obtained before complete surgical removal of the prostate correlated poorly with those derived from a full six-point analysis performed on tissue (500-1000 mg) removed from the center of the malignancy. Androgen binding in nuclear extracts of histologically benign tissue adjacent to the malignancy was significantly higher than in nuclear extracts of BPH tissue. Cytosolic androgen binding in tissue removed from patients who were refractory to hormonal therapy was higher than in tissue from untreated cancer patients. The binding of estradiol by extracts of benign and malignant prostate tissue was low or absent and, thus, did not appear to be a significant phenomenon.     

Antiproliferative action of melatonin on human prostate cancer LNCaP cells

Recent experimental evidence suggests that melatonin, the major pineal hormone, might possess oncostatic properties. The present experiments were performed to verify whether melatonin might modulate the growth of androgen-dependent prostate cancer cells (LNCaP) and to obtain information on its possible mechanism of action. We have shown that melatonin, when given in the nanomolar range, significantly inhibits the proliferation of LNCaP cells; moreover, the pineal gland hormone affects cell cycle distribution by inducing an accumulation of the cells in G0/G1 and a decrease in S phase. To investigate the mechanism of action of melatonin, by RT-PCR analysis we were able to demonstrate the expression, in prostate cancer cells, of a mRNA coding for the membrane Mel1a melatonin receptor. However, by radioreceptor assay, no detectable binding of 2-[125I]iodomelatonin could be observed in membrane preparations from these cells, suggesting that the levels of translation of the mRNA for Mel1a are possibly too low to mediate the antiproliferative action of the hormone. This hypothesis is further supported by the following observations: i) melatonin analogs, specifically acting through membrane receptors (i.e., 2-bromomelatonin), were completely ineffective in modulating prostate cancer cell proliferation; ii) melatonin failed to prevent forskolin-induced cAMP accumulation. These results indicate that melatonin, at nanomolar concentrations, exerts a direct antiproliferative action on androgen-dependent prostate cancer cells, significantly affecting their distribution throughout the cell cycle. Membrane receptors do not seem to be involved in the oncostatic action of the pineal gland hormone.