Human Prostate Adenocarcinoma Cell Line Research Articles

Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-trasformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki = 49 µM). Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC50 = 48 and 66 µM, respectively) without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1β, IRKβ) activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells. In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis.

The HDAC Inhibitor FK228 Enhances Adenoviral Transgene Expression by a Transduction-Independent Mechanism but Does Not Increase Adenovirus Replication

The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected.

O-Prenylated Acridone Alkaloids from the Stems ofBalsamocitrus paniculata(Rutaceae)

Two new O-prenylated acridone alkaloids, balsacridone A (1) and B (2), together with eighteen known compounds were isolated from the methanol extract from the stems of Balsamocitrus paniculata, a Cameroonian medicinal plant. The structures of all compounds were determined by comprehensive analyses of their 1D and 2D NMR, mass spectral (EI and ESI) data, and chemical reactions. N-methyl-6-methoxybenzoxazolinone (16) was isolated for the first time from a natural source while compounds 13, 14, and 15 for the first time from this genus. Pure compounds were tested for their activity against bacteria, fungi, and plant pathogen oomycetes, using the paper disk agar diffusion assay. The agar diffusion test delivered low to missing antimicrobial activities, corresponding to MICs > 1 mg/mL. However, compounds 1-15 exhibited a strong suppressive effect on phagocytosis response upon activation with serum opsonized zymosan in the range of IC50 = 0.5-7.2 μM, and the acridone alkaloids (1-5), N-trans-p-coumaroyltyramine (13), and N-trans-pcoumaroyloctopamine (14) displayed weak cytotoxic activity against the human Caucasian prostate adenocarcinoma cell line PC-3, with IC₅₀ values ranging from 69.8 to 99.0 μM.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce cell cycle arrest and apoptosis in prostate cancer cells

Advanced hormone-refractory prostate cancer is associated with poor prognosis and limited treatment options. Members of the pyrrolo-1,5-benzoxazepine (PBOX) family of compounds exhibit anti-cancer properties in cancer cell lines (including multi-drug resistant cells), ex vivo patient samples and in vivo mouse tumour models with minimal toxicity to normal cells. Recently, they have also been found to possess anti-angiogenic properties in vitro. However, both the apoptotic pathways and the overall extent of the apoptotic response induced by PBOX compounds tend to be cell-type specific. Since the effect of the PBOX compounds on prostate cancer has not yet been elucidated, the purpose of this study was to investigate if PBOX compounds induce anti-proliferative effects on hormone-refractory prostate cancer cells. We examined the effect of two representative PBOX compounds, PBOX-6 and PBOX-15, on the androgen-independent human prostate adenocarcinoma cell line, PC3. PBOX-6 and -15 displayed anti-proliferative effects on PC3 cells, mediated initially through a sustained G2/M arrest. G2/M arrest, illustrated as DNA tetraploidy, was accompanied by microtubule depolymerisation and phosphorylation of anti-apoptotic proteins Bcl-2 and Bcl-xL and the mitotic spindle checkpoint protein BubR1. Phosphorylation of BubR1 is indicative of an active mitotic checkpoint and results in maintenance of cell cycle arrest. G2/M arrest was followed by apoptosis illustrated by DNA hypoploidy and PARP cleavage and was accompanied by degradation of BubR1, Bcl-2 and Bcl-xL. Furthermore, sequential treatment with the CDK1-inhibitor, flavopiridol, synergistically enhanced PBOX-induced apoptosis. In summary, this in vitro study indicates that PBOX compounds may be useful alone or in combination with other agents in the treatment of hormone-refractory prostate cancer.

Multifaceted preventive effects of single agent quercetin on a human prostate adenocarcinoma cell line (PC-3): implications for nutritional transcriptomics and multi-target therapy

The aim of the present study is to evaluate the effects of quercetin, a dietary flavonoid, on human prostate adenocarcinoma PC-3 cells. Lactate dehydrogenase (LDH) release, microculture tetrazolium test (MTT assay) and real-time PCR array were employed to evaluate the effects of quercetin on cell cytotoxicity, cell proliferation and expression of various genes in PC-3 cell line. Quercetin inhibited cell proliferation and modulated the expression of genes involved in DNA repair, matrix degradation and tumor invasion, angiogenesis, apoptosis, cell cycle, metabolism and glycolysis. No cytotoxicity of quercetin on PC-3 cells was observed. Taken together, as shown by the issues of the current study, the manifold inhibitory effects of quercetin on PC-3 cells may introduce quercetin as an efficacious anticancer agent in order to be used in the future nutritional transcriptomic investigations and multi-target therapy to overcome the therapeutic impediments against prostate cancer.

Protective effect of selenium supplementation on the genotoxicity of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate treatment in LNCaP cells

Selenium is an essential cofactor in the key enzymes involved in cellular antioxidant defense. It plays a critical role in testis and reproduction and regulates DNA damage within the prostate. Phthalates are ubiquitous environmental contaminants that cause alterations in endocrine and spermatogenic functions in animals. The objective of this study was to investigate the cytotoxicity and genotoxicity potentials of di(2-ethylhexyl)phthalate (DEHP), the most widely used phthalate and its primary toxic metabolite mono(2-ethylhexyl)phthalate (MEHP), and their effects on the antioxidant balance in the LNCaP human prostate adenocarcinoma cell line. Protection by selenium supplementation with either sodium selenite (SS, 30 nM) or selenomethionine (SM, 10 μM) was also investigated. Both DEHP (3 mM) and MEHP (3 µM) caused significant decreases in cell viability; altered antioxidant status, particularly decreasing the GPx1 activity; and induced DNA damage as measured by the alkaline comet assay. Selenium supplementation was highly protective against cytotoxicity, partially prevented genotoxicity, and restored the antioxidant status. The results of this study suggested that the underlying mechanism of cytotoxicity and resulting disturbances produced by DEHP or MEHP was an an oxidative stress process and/or an effect on the expression of antioxidant enzymes, and accentuated the importance of selenium status, particularly with respect to the high probability of phthalate exposures and their adverse effects.

Übergewicht als Risikofaktor für das Prostatakarzinom: Rolle der Adipozytokine und Beteiligung der Tyrosinkinasen

Obesity is considered to be a risk factor for prostate cancer. Mitogenic actions of leptin, an adipocyte-derived hormone in a variety of cancer cell types have been identified. We have investigated the proliferative effects of leptin on human prostate cancer cells and assessed the role of tyrosine kinase signalling in mediating these actions. Two human androgen-resistant prostate cancer cell lines and one androgen-sensitive human prostate adenocarcinoma cell line were treated with leptin (5-100 ng / mL) for up to 48 hours. Under serum-free conditions, cell proliferation was measured using an enzyme-linked colorimetric assay. Furthermore, phosphorylation of a downstream component of MAPK (ERK1 / 2) was detected by Western blotting and a specific inhibitor of MAPK (PD98059; 40 microM) was used to evaluate the role of this signalling pathway. Leptin dose-dependently increased the cell number in both androgen-resistant cell lines after 24 h and 48 h of incubation (percent of control: DU145 = 194.6 +/- 5.9 %, PC-3 = 177.9 +/- 6.8 %; 100 ng / mL leptin; 48 h; p < 0.001). Conversely, leptin's proliferative effect on the androgen-sensitive cell line was less pronounced (percent of control: LNCaP = 112.3 +/- 6.1 %; 100 ng / mL leptin; 48 h). Leptin also caused dose-dependent ERK1 / 2 phosphorylation in both androgen-resistant cell lines. In addition, pre-treatment with PD98059 inhibited these responses and attenuated leptin's mitogenic action. Data from this in vitro study suggest an association between obesity-associated hyperleptinemia and an increased risk for prostate cancer. Further investigations are necessary to clarify whether these data have clinical relevance regarding the use as a prognostic marker for predicting the timing of the occurrence of androgen resistency.

Expression of Toll‐like receptor 4 in the prostate gland and its association with the severity of prostate cancer

Chronic inflammation has been postulated to be an important driving force to prostate carcinoma. Toll-like receptors (TLRs) compose a family of receptors mainly expressed on immune cells. Recently, functional TLRs have been shown to be also expressed in numerous cancer cells, but their significance has only recently begun to be explored. The purpose of this study was to investigate the putative role of TLR4 expression in prostate carcinoma. To determine if there is an association between TLR4 expression and the malignancy of the tumor, 35 prostate carcinoma samples showing different Gleason grades were analyzed by immunohistochemistry. Also, to explore the functionality of the receptors expressed on the epithelium, we analyzed the type of cytokine response elicited and the signaling pathways involved after TLR4 triggering in the human prostate adenocarcinoma cell line, DU-145. TLR4 is expressed in the normal prostate gland in both stroma and epithelium. TLR4 expression significantly drops to negative values as the Gleason grade augments in both, stroma and epithelium. Moreover, DU-145 cells also exhibit TLR4 expression and respond to TLR4 agonists, activating the transcription factor NF-kappaB and increasing the expression of pro-inflammatory mediators. Inhibition of the molecular adaptors MyD88 and MAL by overexpression of dominant-negative mutants diminished LPS-induced activation of NF-kappaB, showing that DU-145 cells activate the NF-kappaB through MyD88-dependent signaling pathways. We hypothesize that TLR4 in prostate cells could synergize with innate immune cells contributing to an eventual inflammatory process, which in genetically prone individuals could promote carcinogenesis. Prostate 69: 1387-1397, 2009. (c) 2009 Wiley-Liss, Inc.

Microrheology of the pericellular matrix

A membrane-coupled pericellular matrix (PCM) is expressed by a number of cell types and is in most cases associated with cell proliferation and migration. The PCM can be micrometers thick with the glycosaminoglycan hyaluronan (HA) as its backbone. For such an extended layer, however, the presence of hyaldherins like aggrecan or versican is a prerequisite. The latter, like HA, is associated with cancer progression and metastasis. As a model for a HA producing cell we used the human prostate adenocarcinoma cell line PC3. To probe the mechanical properties of the PCM we used microrheology, based on an optical trap equipped with far-field interferometry. We were able to probe the PCM at different distances from the membrane surface and found a soft (< 1 Pa) layer with a thickness of ∼ 1 μm in the absence of, and of ∼ 3 μm in the presence of exogenously added aggrecan. Furthermore, in the presence of aggrecan, part of the cells expressed long (<∼ 10 μm) microvilli extending from the surface. Probing in between the microvilli, we found again a soft (< 1 Pa) PCM. Both the viscoelastic PCM and the microvilli were absent on cells treated with the HA diminishing enzyme hyaluronidase, showing the structural importance of HA in the PCM.

Targeting Protein Kinase C (PKC) and Telomerase by Phenethyl Isothiocyanate (PEITC) Sensitizes PC-3 Cells Towards Chemotherapeutic Drug-Induced Apoptosis

Prostate cancer is the leading cause of cancer-related death in men, incidences of which are increasing gradually in India. Protein kinase C (PKC), an enzyme, gets overexpressed in prostate cancer and results in a resistance to chemotherapy. Telomerase, a reverse transcriptase, is highly activated in prostate cancer cells. Both of these enzymes can be considered as potential molecular markers for prostate cancer. The present study investigates the effects of natural isothiocyanate phenethyl isothiocyanate (PEITC) in modulating the activities of PKC and telomerase in the androgen-independent human prostate adenocarcinoma (PC-3) cell line. We observed that PEITC downregulated the antiapoptotic isoforms (PKC alpha and epsilon) efficiently and zeta moderately. Basal level of PKC delta, a proapoptotic form, was very poor and its modulation was not significant. PEITC also inhibited the activity of telomerase. Studies were conducted to measure the degree of apoptotic cell death induced either by PEITC alone or in combination with adriamycin or etoposide. Apoptosis was evident from the release of mitochondrial cytochrome c, apoptotic index, and by the induction of caspases 3 and 8. PEITC exhibited remarkable efficacy in sensitizing PC-3 cells to undergo cell death by adriamycin and etoposide, which might prove to be of considerable value in synergistic therapy of cancer.

Progressive tumor features accompany epithelial–mesenchymal transition induced in mitochondrial DNA–depleted cells

The growth of LNCaP, a human prostate adenocarcinoma cell line, and MCF-7, a human breast adenocarcinoma cell line, is initially hormone dependent. We previously demonstrated that LNrho0-8 and MCFrho0, derived from LNCaP and MCF-7 by depleting mitochondrial DNA (mtDNA), exhibited hormone-independent growth that led to progressed phenotypes. Here, we demonstrate that LNrho0-8 and MCFrho0 have invasive characters as evaluated by the ability of invasion through the extracellular matrix (ECM) in vitro. In addition, the induction of vimentin and the repression of E-cadherin expression in rho0 cells indicate that they are mesenchymal cells. Since LNrho0-8 and MCFrho0 were derived from epithelial cancer cell lines, LNCaP and MCF-7 must have lost epithelial features and gained the mesenchymal phenotype by epithelial-mesenchymal transition (EMT) during the mtDNA depletion. In the rho0 cell lines, the Raf/MAPK signaling cascade was highly activated together with the expressions of transforming growth factor-beta (TGF-beta) and type I TGF-beta receptor (TGF-betaRI). EMT requires cooperation of TGF-beta signaling with activation of the Raf/MAPK cascade, suggesting that EMT was induced in mtDNA depleted cells resulting in the acquisition of progressive tumor features, such as higher invasiveness and loss of hormone dependent growth. Our results indicate that decreasing mtDNA content induces EMT, enabling the progressive phenotypes observed in cancer.

Evaluation of silibinin on the viability, migration and adhesion of the human prostate adenocarcinoma (PC‐3) cell line

Prostate cancer (PCA) is the most common cancer diagnosed in men and the second most common cause of death due to cancers after lung cancer. Metastasis of cancer cells involves multiple processes and various cytophysiological changes, including changed adhesion capability between cells and extracellular matrix (ECM) and damaged intercellular interaction. Silibinin, a naturally occurring flavonoid antioxidant found in the milk thistle, has recently been shown to have potent antiproliferative effect against various malignant cell lines. In the present study, PC-3 cells were incubated with various concentrations of silibinin for different times; then, cell cytotoxicity, cell adhesion and cell motility were assessed using MTT assay, cell-matrix adhesion assay and cell migration assay, respectively. The results showed that silibinin exerted a dose- and time-dependent inhibitory effect on the viability, motility and adhesion of highly metastatic PC-3 cells. These observations indicate that silibinin can probably inhibit metastasis in PCA.

Inhibition of expression of anti-apoptotic protein Bcl-2 and induction of cell death in radioresistant human prostate adenocarcinoma cell line (PC-3) by methyl jasmonate

Hormone refractory human prostate cancer cell lines are known to be radioresistant, a feature attributed to their ability to induce anti-apoptotic proteins of the Bcl-2 family when exposed to radiation. We investigated whether pro-apoptotic compounds such as methyl jasmonate, a plant stress hormone, can counteract the radiation-induced anti-apoptotic mechanism in a human prostate cancer cell line PC-3. Significant ( p < 0.05) increase in cytotoxicity was observed in the combined treatment groups compared to single treatments with methyl jasmonate or γ-radiation. Treatment of irradiated PC-3 cells with methyl jasmonate resulted in suppression of anti-apoptotic Bcl-2 protein and elevation of caspase-3 activity. Our results showed increased apoptosis in the combined treatment group as compared to the irradiated group or the untreated control. In summary, methyl jasmonate suppressed the radiation-induced Bcl-2 expression and enhanced the radiation sensitivity of human prostate cancer cells.

Formation of Hyaluronan- and Versican-rich Pericellular Matrix by Prostate Cancer Cells Promotes Cell Motility

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.

Synthesis and Biological Evaluation of New Asymmetrical Bisintercalators as Potential Antitumor Drugs

The good results obtained in the past decade with various types of potential bisintercalating agents, e.g., LU 79553, DMP 840, BisBFI, MCI3335, WMC-26, BisAC, BisPA, and the asymmetrical derivative WMC-79 (Chart 1), prompted us to investigate a new series of asymmetrical bisintercalators, compounds 1a-t (Chart 2), which can combine the potentiality of bisintercalation with a possible different mechanism of action due to two diverse chromophores. The DNA-binding properties of these compounds have been examined using fluorometric techniques: target compounds are excellent DNA ligands, with a clear preference for binding to AT-rich duplexes. In vitro cytotoxicity of these derivatives toward human hormone-refractory prostate adenocarcinoma cell line (PC-3) is described. Apoptosis assays of four selected compounds are also reported. Very potent cytotoxic compounds, some of them capable of inducing early apoptosis, have been identified.

Molecular Mechanism of Adaphostin-mediated G1 Arrest in Prostate Cancer (PC-3) Cells

Adaphostin (NSC680410), a small molecule congener of tyrphostin AG957, has been demonstrated previously to have significant anti-proliferative effects in several leukemia models. However, this effect of adaphostin in adherent cells/solid tumor models has not been examined. In this study, we investigated the anti-proliferative effects of adaphostin in the human prostate cancer cell line PC-3. Specifically, we explored the potential molecular mechanism(s) by which adaphostin elicits its anti-proliferative effect(s). We demonstrate that adaphostin inhibits the proliferation of PC-3 cells by inducing a G(1) phase cell cycle arrest. This adaphostin-induced G(1) arrest was associated with an increase in the expression of p21 and p27 and a decrease in the expression of G(1)-specific cyclins (cyclin A, D1, and D3) and cyclin-dependent kinases 4 and 6. Consequently, a dramatic decrease in the phosphorylation of retinoblastoma protein was also observed. Additionally, we found that adaphostin treatment induced a decrease in the phosphorylation of nucleophosmin, a major nuclear phosphoprotein, and that this decreased phosphorylation was a result of the p21- and p27-mediated inactivation of cyclin E-cyclin-dependent kinase 2 complex kinase activity. Furthermore, we have determined that the adaphostin-mediated cell cycle arrest of PC-3 cells is dependent upon activation of the p38 MAPK. We also demonstrate that the hepatocyte growth factor receptor-c-Met is involved in the adaphostin-mediated signaling events that regulate p38 MAPK. Taken together, these results identify for the first time a signaling cascade of adaphostin-mediated G(1) phase-specific cell cycle arrest in PC-3 cells. These findings suggest that the tyrphostin member has a broader spectrum of activity than originally predicted.

A Novel Serum Marker, Total Prostate Secretory Protein of 94 Amino Acids, Improves Prostate Cancer Detection and Helps Identify High Grade Cancers at Diagnosis

New biomarkers for prostate cancer are needed. We determined whether a novel serum marker, total PSP94 can be used to accomplish these goals. We conducted a case-control study of 1,212 men with no previous history of prostate cancer and who underwent a prostate biopsy from 1998 to 2000 because of an increased PSA or an abnormal DRE. Serum PSP94 levels were assessed using a sandwich enzyme-linked immunosorbent assay technique. Cases were patients with prostate cancer, and controls were patients who had no evidence of cancer. Multivariate logistic regression analysis was used to determine whether or not PSP94 levels improved the predictive value for prostate cancer. Of the 1,212 men 596 (49.2%) had cancer detected. The median PSP94 level was significantly lower among cases (2.60 ng/ml) than among controls (3.40 ng/ml, p <0.0001). The adjusted odds ratios for the presence of prostate cancer for patients with the lowest quartile of PSP94, compared to patients in the highest quartile was 2.70 (95% CI 1.8 - 4.0, p <0.0001). Among a subgroup of 649 men in whom PSA had a low predictive value (PSA less than 20 ng/ml, normal DRE and less than 70 years), 260 (40.1%) were found to have cancer. In this subgroup total PSP94 levels helped discriminate between patients with high grade disease (Gleason score 8 or more, median 1.90 ng/ml), moderate grade disease (Gleason score 7, median 2.34 ng/ml) and low grade disease (Gleason score 6 or less, median 2.60 ng/ml, p = 0.007). PSA and the FTPSA were not able to distinguish between patients with different grades in this group. Patients with low total PSP94 levels had a high probability for having prostate cancer detected at biopsy. The total PSP94 level was able to help identify patients with high grade disease among a subset of patients in whom PSA and FTPSA are least informative.

IFN Unresponsiveness in LNCaP Cells Due to the Lack of JAK1 Gene Expression

We reported previously that 23% of human lung adenocarcinoma cell lines were unresponsive to IFN-gamma. To extend this finding to cancer cells derived from distinct tissues of origin, we assessed IFN-gamma receptor signaling in the LNCaP human prostate adenocarcinoma cell line, which in previous experiments by others failed to induce a range of IFN-dependent biological responses. In this report, we show that LNCaP cells fail to respond to either IFN-gamma or IFN-alpha because of an impairment in the proximal signaling events downstream of both IFN-gamma and IFN-alpha/beta receptors that lead to the activation of STAT1. Furthermore, we show that LNCaP insensitivity to the IFNs is a result of the absence of expression of the JAK1 kinase, an obligate component shared by both IFN-gamma and IFN-alpha/beta receptors. JAK1 was undetectable in LNCaP cells at both protein and message levels. Treatment of LNCaP cells with a combination of inhibitors of DNA methyltransferases and histone deacetylases induced expression of JAK1 message. These results identify the molecular basis for IFN insensitivity in the LNCaP cell line and suggest that epigenetic silencing of key immunologic signaling components may be one mechanism by which tumor cells evade immune detection and elimination.

Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells

The transactivation function of the human androgen receptor (AR) can be regulated by several coregulators that may be either positive or negative. Ubiquitous transcription factor Sp1 not only regulates the basal expression of the AR but also acts as its coregulator. Our previous study has shown that quercetin, one of the main polyphenols, can effectively inhibit the expression and function of the AR. The present study is to address if quercetin may affect Sp1's action on AR transactivation activity in human prostate adenocarcinoma cell lines, LNCaP and PC-3. First, we showed that indeed in transient transfections Sp1 could enhance transcriptional activity of the AR promoter and of androgen upregulated gene promoters, i.e. the prostate-specific antigen and the hK2 genes. Interestingly, the enhancing activity of Sp1 could be repressed by quercetin. The gel shift and western blot analyses indicated that the specific DNA motif binding activity of Sp1 and its protein levels were not altered by quercetin. However, the state of interaction of Sp1 with the AR treated by quercetin plus androgen was different from that by androgen treatment or none as demonstrated by coimmunoprecipitation experiments and glutathione S-transferase (GST) pull-down assays. Moreover, we showed that quercetin caused changes in post-translational modification of AR protein. The above findings strongly suggest that changes induced by quercetin in post-translational modification of the AR and in states of physical interaction of Sp1 with the AR may be critical for the attenuation of AR's function.

Synthesis and biological evaluation of indazolo[4,3- bc][1,5]naphthyridines (10-aza-pyrazolo[3,4,5- kl]acridines): a new class of antitumor agents

A series of potential DNA-binding antitumor agents, 2-[ω-(alkylamino)alkyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3- bc][1,5]naphthyridines ( 2a– f), 10-aza derivatives of PZA, has been prepared by condensation of 9-chloro-2-methoxy-6-nitro-5,10-dihydrobenzo[ b][1,5]naphthyridin-10-one ( 6) with the appropriate (ω-aminoalkyl)hydrazine in tetrahydrofuran/methanol. Compound 6 was obtained by heating at 100 °C in H 2SO 4 5, yielded by the condensation of 2,6-dichloro-3-nitrobenzoic acid ( 4) and 6-methoxy-3-pyridinamine ( 3). The non-covalent DNA-binding properties of 2 have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives against human hormone-refractory prostate adenocarcinoma cell line (PC-3) are described and compared to that of parent drug PZA. We selected the most cytotoxic target derivatives 2c, d, the in vitro inactive 2f, and reference compound PZA to investigate whether in vitro treatment with these drugs was able to induce necrotic and/or apoptotic cell death. To this purpose, we evaluated the percentage of apoptotic cells in PC-3 treated with the target compounds 2c, d, f and reference compound PZA, by Annexin V staining and Propidium iodide (PI)/Annexin V, biparametric flow cytometric analysis and agarose gel electrophoresis.