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

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

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

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

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

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

Inhibitory effects of the HDAC inhibitor valproic acid on prostate cancer growth are enhanced by simultaneous application of the mTOR inhibitor RAD001

AimsTo analyze the combined impact of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell growth.Main methodsPC-3, DU-145 and LNCaP cells were treated with RAD001, VPA or with an RAD001–VPA combination for 3 or 5 days. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT assay, flow cytometry and Western blotting, respectively. Effects of drug treatment on cell signaling pathways were determined.Key findingsSeparate application of RAD001 or VPA distinctly reduced tumor cell growth and impaired cell cycle progression. Significant additive effects were evoked when both drugs were used in concert. Particularly, the cell cycle regulating proteins cdk1, cdk2, cdk4 and cyclin B were reduced, whereas p21 and p27 were enhanced by the RAD001–VPA combination. Signaling analysis revealed deactivation of EGFr, ERK1/2 and p70S6k. Phosphorylation of Akt was diminished in DU-145 but elevated in PC-3 and LNCaP cells.SignificanceThe RAD001–VPA combination exerted profound antitumor properties on a panel of prostate cancer cell lines. Therefore, simultaneous blockage of HDAC and mTOR related pathways should be considered when designing novel therapeutic strategies for treating prostate carcinoma.     

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

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

Natural antibodies against nerve growth factor inhibit in vitro prostate cancer cell metastasis

Prostate cancer is a major cause of death in older men, and bone metastasis is the primary cause of morbidity and mortality in prostate cancer. Prostate is an abundant source of nerve growth factor (NGF) that is secreted by malignant epithelial cells and utilized as an important autocrine factor for growth and metastasis. We previously showed that intravenous gammaglobulin (IVIg) contains natural antibodies against NGF, which inhibit growth and differentiation of the NGF-dependent cell line PC-12. In the present study, we examined the effects of these natural antibodies on in vitro migration or metastasis of two prostate cancer cell lines namely DU-145 and PC-3. Cancer cell migration was assessed using these cell lines in the upper chambers of Matrigel invasion chambers. The effects of IVIg and affinity-purified anti-NGF antibodies on cell migration through membrane into the lower chamber were assessed in dose/response experiments by a colorimetric method. Affinity-purified natural IgG anti-NGF antibody inhibited DU-145 migration by 38% (p = 0.01) and PC-3 migration by 25% (p = 0.02); whereas, a monoclonal anti-NGF antibody inhibited DU-145 migration by 40% (p = 0.01) and PC-3 migration by 37% (p = 0.02), at the same concentration. When IVIg was depleted of NGF-specific IgG by affinity chromatography, there was no significant inhibition of migration of the DU-145 and PC-3 cells at a concentration of 1 mg/well. Removal of the NGF-specific antibody from the IVIg was also demonstrated by a lack of effect on PC-12 cell differentiation. Therefore, IVIg is able to inhibit the migration of prostate cancer cell lines, through Matrigel chambers in vitro, only when the natural NGF-specific antibodies actively are present in IVIg.     

Cholestane-3β, 5α, 6β-triol Suppresses Proliferation,Migration, and Invasion of Human Prostate Cancer Cells

Oxysterols are oxidation products of cholesterol. Cholestane-3β, 5α, 6β-triol (abbreviated as triol) is one of the most abundant and active oxysterols. Here, we report that triol exhibits anti-cancer activity against human prostate cancer cells. Treatment of cells with triol dose-dependently suppressed proliferation of LNCaP CDXR-3, DU-145, and PC-3 human prostate cancer cells and reduced colony formation in soft agar. Oral administration of triol at 20 mg/kg daily for three weeks significantly retarded the growth of PC-3 xenografts in nude mice. Flow cytometric analysis revealed that triol treatment at 10–40 µM caused G1 cell cycle arrest while the TUNEL assay indicated that triol treatment at 20–40 µM induced apoptosis in all three cell lines. Micro-Western Arrays and traditional Western blotting methods indicated that triol treatment resulted in reduced expression of Akt1, phospho-Akt Ser473, phospho-Akt Thr308, PDK1, c-Myc, and Skp2 protein levels as well as accumulation of the cell cycle inhibitor p27^Kip. Triol treatment also resulted in reduced Akt1 protein expression in PC-3 xenografts. Overexpression of Skp2 in PC-3 cells partially rescued the growth inhibition caused by triol. Triol treatment suppressed migration and invasion of DU-145, PC-3, and CDXR-3 cells. The expression levels of proteins associated with epithelial-mesenchymal transition as well as focal adhesion kinase were affected by triol treatment in these cells. Triol treatment caused increased expression of E-cadherin protein levels but decreased expression of N-cadherin, vimentin, Slug, FAK, phospho-FAK Ser722, and phospho-FAK Tyr861 protein levels. Confocal laser microscopy revealed redistribution of β-actin and α-tubulin at the periphery of the CDXR-3 and DU-145 cells. Our observations suggest that triol may represent a promising therapeutic agent for advanced metastatic prostate cancer.     

Bone microenvironment-related growth factors, zoledronic acid and dexamethasone differentially modulate PTHrP expression in PC-3 prostate cancer cells.

Bone metastasis microenvironment-related growth factors such as insulin-like growth factor 1 (IGF-1), transforming growth factor beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF) and interleukin 6 (IL-6) show survival factor activity, thereby inhibiting chemotherapy-induced apoptosis of PC-3 prostate cancer cells in vitro. Recently, zoledronic acid has been shown to induce apoptosis in PC-3 prostate cancer cells while overexpression of parathyroid hormone-related protein (PTHrP) inhibits serum deprivation-induced apoptosis in PC-3 cells. Consequently, we have investigated whether IGF-1, TGF-beta1, bFGF, IL-6, zoledronic acid and/or dexamethasone affect the expression of the PTHrP and type I PTH/PTHrP receptor (PTH.1R) in PC-3 prostate cancer cells using relative quantitative PCR and real-time PCR (expression at mRNA level) and immunocytochemical and immunofluorescence analysis (expression at protein level). Our data show that IGF-1, TGF-beta1, bFGF and IL-6 increase PTHrP mRNA expression and its perinuclear localization, while zoledronic acid (50 muM, 100 muM for 24 h and 48 h) and dexamethasone suppress PTHrP expression in PC-3 cells. We did not detect any appreciable change of the PTH.1R expression due to IGF-1, TGF- beta1, bFGF, IL-6, zoledronic acid or dexamethasone in PC-3 cells. Therefore, it is conceivable that bone metastasis microenvironment-related survival factor/anti-apoptotic activity and zoledronic acid anticancer action/pro-apoptotic activity on PC-3 cells is mediated, at least in part, by differential modulation of PTHrP expression.     

Activated eosinophils upregulate the metastasis suppressor molecule E-cadherin on prostate tumor cells.

Cell adhesion molecules (CAMs) play an important role in cancer metastasis by facilitating attachment to vascular endothelia, invasion and spread into secondary tissue sites. We have shown that activated eosinophils (EosA) inhibited the growth of prostate cancer (Pca) cells in vitro. In the present study, we examined the ability of EosA 24 hr conditioned supernatants (EosAcs) to modulate the expression of ICAM-1, VCAM-1, ELAM-1, E-cadherin and N-cadherin expression on human Pca cell lines, Du-145 and PC-3 by flow cytometry. TNF-alpha, IL-10 and IL-12 were also evaluated. ICAM-1, expressed on PC-3 and DU 145 cells, was enhanced by TNF-alpha and IL-10. ELAM-1 was present on DU 145 cells but absent on PC-3. TNF-alpha and IL-10 enhanced ELAM-1 on DU 145, but EosA 24 hr supematants failed to do so. All three cytokines, namely IL-10, IL-12 and TNF-alpha-induced ELAM-1 on PC-3 tumor cells. Although VCAM-1 was absent on DU 145 and PC-3 cells, it was expressed on DU-145 cells after exposure to EosA: tumor cell co-cultures, and was expressed on PC-3 following exposure to IL-10 and IL-12. N-cadherin and E-cadherin were both expressed on DU-145. While N-cadherin was expressed on PC-3 cells, E-cadherin was not. N-cadherin was enhanced on DU-145 and PC-3 cells following exposure to EosA co-culture and upregulated on PC-3 by IL-10 and EosA 24 hr supernatants, but decreased by IL-12. E-cadherin was up-regulated on DU 145 cells following co-culture with EosA and was induced on PC-3 by IL-10 and IL-12, but not by EosA co-culture and 24 hr supematants. In conclusion, inflammatory and non-inflammatory cytokines modulate CAM expression on Pca cells; EosA and EosA 24 hr supernatants also exerted modulatory activity of CAM expression. Most significantly, the metastasis suppressor molecule, E-cadherin was enhanced on DU 145 cells by EosA and induced on PC-3 by IL-10 and IL-12 both of which are produced by EosA. This suggests potential use of these cytokines in immunotherapeutic strategies for prostate cancer and its metastasis.     

beta-Lapachone-induced apoptosis in human prostate cancer cells: involvement of NQO1/xip3.

beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells.     

Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is overexpressed in prostate cancer, but the mechanism by which MIF exerts effects on tumor cells remains undetermined. MIF interacts with its identified membrane receptor, CD74, in association with CD44, resulting in ERK 1/2 activation. Therefore, we hypothesized that increased expression or surface localization of CD74 and MIF overexpression by prostate cancer cells regulated tumor cell viability. Prostate cancer cell lines (LNCaP and DU-145) had increased MIF gene expression and protein levels compared with normal human prostate or benign prostate epithelial cells (p < 0.01). Although MIF, CD74, and CD44 variant 9 expression were increased in both androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells, cell surface of CD74 was only detected in androgen-independent (DU-145) prostate cancer cells. Therefore, treatments aimed at blocking CD74 and/or MIF (e.g., inhibition of MIF or CD74 expression by RNA interference or treatment with anti-MIF- or anti-CD74- neutralizing Abs or MIF-specific inhibitor, ISO-1) were only effective in androgen-independent prostate cancer cells (DU-145), resulting in decreased cell proliferation, MIF protein secretion, and invasion. In DU-145 xenografts, ISO-1 significantly decreased tumor volume and tumor angiogenesis. Our results showed greater cell surface CD74 in DU-145 prostate cancer cells that bind to MIF and, thus, mediate MIF-activated signal transduction. DU-145 prostate cancer cell growth and invasion required MIF activated signal transduction pathways that were not necessary for growth or viability of androgen-dependent prostate cells. Thus, blocking MIF either at the ligand (MIF) or receptor (CD74) may provide new, targeted specific therapies for androgen-independent prostate cancer.     

Synthesis and biological evaluation of novel cytotoxic phospholipids for prostate cancer

We describe herein synthesis, SAR, and biological evaluation of a novel series of cytotoxic serine amide phosphates (SAPs) for prostate cancer. These compounds were tested for their cytotoxicity in five human prostate cancer cell lines (DU-145, PC-3, LNCaP, PPC-1, and TSU), and in CHO and RH7777 cells (negative controls). Comparison of anticancer effects of these compounds with a standard chemotherapeutic agent 5-fluorouracil shows that they are very effective in killing prostate cancer cells with low micromolar cytotoxicity and provide us a new lead for the development of drugs for prostate cancer.     

Targeting apoptosis in the hormone- and drug-resistant prostate cancer cell line, DU-145, by gossypol/zoledronic acid combination

Possible synergistic cytotoxic and apoptotic effects of gossypol with zoledronic acid on DU-145 cells were explored, along with the rationale behind any observed synergism due to the different apoptotic proteins involved. XTT cell proliferation assay was used to assess the cytotoxicity, and DNA fragmentation and caspase 3/7 activity were measured to verify apoptosis. Human Apoptosis Array was used to evaluate apoptotic proteins. The synergistic cytotoxic combination treatment had a versatile effect on apoptotic proteins, through inhibition of anti-apoptotic proteins (including cIAP-1, cIAP-2, survivin, livin, claspin, p53, p21, PON-2 and heat shock proteins) and concurrently the induction of pro-apoptotic proteins (Bad, Bax, Fas, FADD, cleaved caspase-3 and p27). Both drugs had a minimal toxicity profile comparing to cytotoxic agents. Combination treatments targeting many pivotal apoptosis-related proteins may be a rationale option for treatment of prostate cancer.     

Brefeldin A enhances docetaxel-induced growth inhibition and apoptosis in prostate cancer cells in monolayer and 3D cultures

Docetaxel is a commonly used chemotherapeutic drug for patients with late stage prostate cancer. However, serious side effect and drug resistance limit its clinical success. Brefeldin A is a 16-membered macrolide antibiotic from mangrove-derived Fungus Aspergillus sp. (9Hu), which exhibited potent cytotoxicity against human cancer cells. In the present study, we determined the effect of brefeldin A on docetaxel-induced growth inhibition and apoptosis in human prostate cancer PC-3 cells. Brefeldin A in combination with docetaxel inhibited the growth of PC-3 cells in monolayer and in three dimensional cultures. The combination also potently stimulated apoptosis in PC-3 cells as determined by propidium iodide staining and morphological assessment. Mechanistic studies showed that growth inhibition and apoptosis in PC-3 cells treated with brefeldin A and docetaxel were associated with decrease in the level of Bcl-2. The present study indicates that combined brefeldin A with docetaxel may represent a novel approach for improving the efficacy of docetaxel, and Bcl-2 may serve as a target for brefeldin A to enhance the effects of docetaxel chemotherapy.     

Pifithrin-μ, an Inhibitor of Heat-Shock Protein 70, Can Increase the Antitumor Effects of Hyperthermia Against Human Prostate Cancer Cells

Hyperthermia (HT) improves the efficacy of anti-cancer radiotherapy and chemotherapy. However, HT also inevitably evokes stress responses and increases the expression of heat-shock proteins (HSPs) in cancer cells. Among the HSPs, HSP70 is known as a pro-survival protein. In this study, we investigated the sensitizing effect of pifithrin (PFT)-μ, a small molecule inhibitor of HSP70, when three human prostate cancer cell lines (LNCaP, PC-3, and DU-145) were treated with HT (43°C for 2 h). All cell lines constitutively expressed HSP70, and HT further increased its expression in LNCaP and DU-145. Knockdown of HSP70 with RNA interference decreased the viability and colony-forming ability of cancer cells. PFT-μ decreased the viabilities of all cell lines at one-tenth the dose of Quercetin, a well-known HSP inhibitor. The combination therapy with suboptimal doses of PFT-μ and HT decreased the viability of cancer cells most effectively when PFT-μ was added immediately before HT, and this combination effect was abolished by pre-knockdown of HSP70, suggesting that the effect was mediated via HSP70 inhibition. The combination therapy induced cell death, partially caspase-dependent, and decreased proliferating cancer cells, with decreased expression of c-Myc and cyclin D1 and increased expression of p21^WAF1/Cip, indicating arrest of cell growth. Additionally, the combination therapy significantly decreased the colony-forming ability of cancer cells compared to therapy with either alone. Furthermore, in a xenograft mouse model, the combination therapy significantly inhibited PC-3 tumor growth. These findings suggest that PFT-μ can effectively enhance HT-induced antitumor effects via HSP70 inhibition by inducing cell death and arrest of cell growth, and that PFT-μ is a promising agent for use in combination with HT to treat prostate cancer.     

Phenylboronic acid is a more potent inhibitor than boric acid of key signaling networks involved in cancer cell migration

Previous studies from our lab have shown that both boric (BA) and phenylboronic acid (PBA) inhibit the migration of prostate cancer cell lines, as well as non-tumorigenic prostate cells. Our results indicate that PBA is more potent than BA in targeting metastatic and proliferative properties of cancer cells. Here we focus on the impact of BA and PBA on Rho family of GTP-binding proteins and their downstream targets. Treatment with 1 mM PBA and BA decreases activities of RhoA, Rac1 and Cdc42 in DU-145 metastatic prostate cancer cells, but not in normal RWPE-1 prostate cells. Furthermore, ROCKII activity and phosphorylation of myosin light chain kinase decrease as a result of either PBA or BA treatment in DU-145 cells, suggesting these compounds target actomyosin-based contractility.Key words: boric acid, phenylboronic acid, migration, prostate cancer, natural compounds, DU-145