LNCaP Human Prostate Cancer Cells Research Articles

310 The evaluation of anti-cancer activity of three novel carbocyclic pyrrolo[2,3-d]pyrimidine analogs in human prostate cancer LNCaP cells

310 The evaluation of anti-cancer activity of three novel carbocyclic pyrrolo[2,3-d]pyrimidine analogs in human prostate cancer LNCaP cells

Cyanidin induces apoptosis and differentiation in prostate cancer cells.

Several natural antioxidants, including anthocyanins, have been reported to have chemotherapeutic activity in vivo and in vitro. The aim of the present study was to delineate the anti-proliferative activity and the cytodifferentiation properties mediated by cyanidin-3-O-β-glucopyranoside (C3G) treatment in the DU145 and LnCap human prostatic cancer cell lines. C3G produced anti-proliferative effects through activation of caspase-3 and induction of p21 protein expression. The reduced cell viability was associated with a clear increase of DNA fragmentation in both cell lines after C3G treatment. Since LnCap and DU145 exhibited differences in sensitivity to C3G treatment, the redox state of these cells was further investigated by estimating the levels of ROS and GSH. C3G antioxidant activity was confirmed only in DU145 cell line. Treatment with C3G increased the levels of tumor suppressor P75NGFR, indicating a possible role of C3G in the acquisition of a normal-like cell phenotype. Results reported in the present study demonstrate that C3G, the most abundant anthocyanin in diet, may represent a new approach and highly effective strategy in reducing carcinogenesis. C3G may be considered a new therapeutic agent with both anti-proliferative and pro-differentiation properties.

Functional upregulation of the H2S/Cav3.2 channel pathway accelerates secretory function in neuroendocrine-differentiated human prostate cancer cells

Neuroendocrine-differentiated prostate cancer cells may contribute to androgen-independent proliferation of surrounding cells through Ca2+-dependent secretion of mitogenic factors. Human prostate cancer LNCaP cells, when neuroendocrine-differentiated, overexpress Cav3.2 T-type Ca2+ channels that contribute to Ca2+-dependent secretion. Given evidence for the acceleration of Cav3.2 activity by hydrogen sulfide (H2S), we examined the roles of the H2S/Cav3.2 pathway and then analyzed the molecular mechanisms of the Cav3.2 overexpression in neuroendocrine-differentiated LNCaP cells. LNCaP cells were differentiated by dibutyryl cyclic AMP. Protein levels and T-type Ca2+ channel-dependent currents (T-currents) were measured by immunoblotting and whole-cell pacth-clamp technique, respectively. Spontaneous release of prostatic acid phosphatase (PAP) was monitored to evaluate secretory function. The differentiated LNCaP cells exhibited neurite outgrowth, androgen-independent proliferation and upregulation of mitogenic factors, and also showed elevation of Cav3.2 expression or T-currents. Expression of cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), H2S-forming enzymes, and spontaneous secretion of PAP increased following the differentiation. The augmented T-currents were enhanced by H2S donors and suppressed by inhibitors of CSE, but not CBS. The PAP secretion was reduced by inhibition of CSE or T-type Ca2+ channels. During differentiation, Egr-1 and REST, positive and negative transcriptional regulators for Cav3.2, were upregulated and downregulated, respectively, and Egr-1 knockdown prevented the Cav3.2 overexpression. Our data suggest that, in neuroendocrine-differentiated LNCaP cells, H2S formed by the upregulated CSE promotes the activity of the upregulated Cav3.2, leading to the elevated secretory functions. The overexpression of Cav3.2 appears to involve upregulation of Egr-1 and downregulation of REST.

Abstract 4649: Apigenin increases maspin expression and suppresses invasiveness in prostate cancer cells

Abstract Acquisition of metastatic capability during prostate cancer progression leads to clinically incurable malignancy and accounts for the majority of deaths from this disease. Metastasis involves multiple processes, including invasion, intravasation, extravasation and tumor growth at the secondary site. Maspin, a unique member of the serpin (serine protease inhibitor) family, shown to regulate cell motility, invasion and contributes in tumor metastasis. Loss of maspin has been frequently identified in clinical prostate cancer specimens and prostate cancer cell lines, therefore novel therapeutic approaches to restore its expression are needed. Apigenin (4′, 5, 7-trihydroxyflavone), a plant flavone has shown to possess anticancer properties and alters pathways that regulate tumor cell invasion and metastasis, however molecular basis of these effects remains unclear. We demonstrate that apigenin enhances maspin expression in human prostate cancer cells through upregulation of p53 activity and downregulation of histone deacetylase (HDAC) activity. Treatment of human prostate cancer LNCaP cells (harboring wild type p53) with 1-40 μM apigenin for 72 h resulted in dose- and time- dependent decrease in cell invasion and migration with concurrent increase in maspin expression and increased its nuclear localization. Apigenin treatment also resulted in the inhibition of HDAC enzyme activity, increase in H3 and H4 acetylation and p53 activation, similar with the use of Class I HDAC inhibitor, trichostatin A. In human prostate cancer DU145 cells, harboring mutant p53, apigenin treatment resulted in an increase in maspin expression which correlated with upregulation of wild-type p53 activity, conversely trichostatin A was unable to upregulate maspin expression. Transfection of DU145 cells with maspin reporter plasmid increased the activity of maspin promoter, consistent with the results. In another approach, endogenous levels of mapsin was restored after transfection of cells with wild type p53 in prostate cancer PC-3 cells (p53 null), which was further increased by apigenin treatment. Furthermore, knockdown of HDAC1 did not affect maspin levels in human prostate cancer LNCaP and DU145 cell lines suggesting that HDAC1 inhibition does not play a role in maspin regulation, whereas it appears that apigenin-dependent global modulation of HDAC activity resulted in decreased cell invasion and proliferation. Taken together, our findings suggest that apigenin-mediated increase in maspin expression is regulated, in part, by p53 and its activation resulting in decreased invasiveness and migration capabilities of prostate cancer cells. Citation Format: Sanjeev Shukla, Ata Abbas, Sanjay Gupta. Apigenin increases maspin expression and suppresses invasiveness in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4649. doi:10.1158/1538-7445.AM2015-4649

Characterization of a New Allelic Variant of Triosephosphate Isomerase from the LNCaP Human Prostate Cancer Cell Line: Enzyme Inhibition and Spectroscopic Studies.

Background: The glycolytic pathway plays an important role in tumor cells. Triosephosphate isomerase (TIM) catalyzes the reversible isomerization of D-glyceraldehyde-3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) in the glycolysis. Proteomics of a human prostate adenocarcinoma cell line revealed the presence of the G233D TIM variant, a new allelic type whose biochemical properties have not been reported [1].Objective: Provide the first biochemical and biophysical characterization of the allelic variant G233D of TIM.Methods: The Michaelis-Menten curves using both substrates of TIM were obtained. Also the effect of the competitive inhibitor phosphoenolpyruvate (PEP) was assessed in presence of GAP and DHAP. The thermal stability in absence and presence of PEP was analyzed by circular dichroism spectroscopy. For comparison purposes, all the measurements were carried out on the wild type TIM and variant G233D.Results: The G233D variant exhibited a kcat value 4-fold lower than that of the WT enzyme in the GAP isomerization to DHAP, which is the reverse reaction of the glycolytic pathway. The G233D variant exhibited Ki and IC50 values of 120 μM and 356 μM in the presence of several concentrations of GAP and 0.3 mM DHAP, respectively. These inhibition parameters are similar to those exhibited by the WT enzyme. The thermal unfolding cooperativity of G233D variant was significantly increased upon PEP binding, suggesting that the ligand-bound enzyme was trapped in a rigid conformation.Conclusion: We suggest that the flow of GAP through glycolysis could be enhanced by the decreased activity of the G233D variant in the formation of DHAP.

Abstract 2431: Co-targeting ROS and CLU-mediated stress response using SMIP004 (a novel inducer of ROS and cancer cell selective apoptosis) and OGX-011 in MDV3100-resistant, castrate-resistant prostate cancer

Abstract Prostate cancer is the most common male carcinoma in North America and the second leading cause of cancer related death in men. While new drugs targeting Androgen Receptor (AR), like abiraterone and MDV3100, significantly increase survival of patients with castration resistant prostate cancer (CRPC), many patients still die of metastatic resistant disease. Continued therapeutic improvements towards prolonging the chronicity of CRPC will require characterization of innate and stress-activated survival responses, and rational combinatorial co-targeting strategies designed to abrogate them. OGX-011 is a second generation antisense oligonucleotide targeting Clusterin (CLU) currently under evaluation in phase III clinical trials. CLU is a molecular chaperone involved in endoplasmic reticulum (ER) stress, protein homeostasis and in many signaling and transcriptional survival networks. It is cytoprotective under stress conditions and correlated with treatment resistance in prostate and other cancers. SMIP004 is a novel drug that modulates cell cycle and ER stress and unfolded protein responses (UPR). Some of its fuctions have been correlated to Reactive Oxygen Species (ROS) modulation. However, the exact mechanisms of action of SMIP004 are not completely understood. Cell survival of LNCaP (castration sensitive), C-42 (castration resistant), M42D, M49F (MDV3100 resistant) and PC3 (AR negative) human prostate cancer cell lines was assessed by crystal violet assay. CLU silencing was obtained by transfecting cells with short interference RNA or OGX011. Western blotting analysis was used to detect proteins expression and RT-QPCR for mRNA levels. ROS were evaluated in live cells after incubation with 2′,7′-DCFDA by FACS Canto II. Consistent with induction of proteotoxic stress, SMIP004 significantly up-regulated CLU mRNA and protein levels in prostate cancer cell lines sensitive and resistant to MDV3100. SMIP004 as well as MDV3100 were able to increase ROS production. Interestingly CLU silencing, through siRNA or OGX011, inhibited ROS while CLU overexpression, after SMIP004 and/or MDV3100, significantly induced ROS production in MDV3100 sensitive and resistant cells. Moreover SMIP004 efficiently controlled cell survival of MDV3100 sensitive and resistant prostate cancer cells but the combination with clusterin inhibitors had a synergistic effect in these cells. Interestingly, SMIP004 reduced AR variant-7, a AR splicing variant associated with abiraterone and MDV3100 resistance, in both MDV3100 sensitive and resistant cells. Clusterin plays a central role in ROS tolerance in prostate cancer cells. Clusterin inhibition in combination with SMIP004 is able to control cell survival of MDV-3100 sensitive and resistant cells also trough a modulation of oxydative stress. Citation Format: Lucia Nappi, Eliana Beraldi, Fan Zhang, Dieter Wolf, Martin Gleave. Co-targeting ROS and CLU-mediated stress response using SMIP004 (a novel inducer of ROS and cancer cell selective apoptosis) and OGX-011 in MDV3100-resistant, castrate-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2431. doi:10.1158/1538-7445.AM2015-2431

Abstract 1913: Rhamnetin inhibits prostate cancer progression in an autochthonous mouse prostate cancer model

Abstract Aging increases oxidative stress and cancer risk. Age-regulated genes viz. FoxO3a and Klotho are stimulated by reactive oxygen species (ROS) due to increased oxidative stress. Klotho, an antiaging gene, modulates stress-induced senescence, and decreases with advancing age, whereas forkhead transcription factor, FoxO3a is known to involve in a paradigm that engages in antioxidant, anti-proliferative and tumor suppressor functions. We have previously demonstrated that FoxO3a is deregulated in human prostate adenocarcinoma and in transgenic adenocarcinoma of the mouse prostate (TRAMP) model that mimics progressive forms of human disease. Here we demonstrate the anticancer effects of rhamnetin, a dietary flavonoid present in fruits and vegetables, in modulating Klotho and FoxO3a signaling by suppressing ROS. Per-oral feeding of rhamnetin at 10- and 20-μg/mouse/day (gavaged in 0.2 ml vehicle containing 0.5% methyl cellulose and 0.025% Tween 20) to TRAMP mice for six days per week for 16 weeks, starting at 8 weeks of age, exhibited marked reduction in tumor growth with no signs of metastasis. Rhamnetin feeding to TRAMP resulted in a significant dose-dependent reduction in FoxO3a, in the dorso-lateral prostates. FoxO3a protein was redistributed more in nuclear than the cytosolic compartments after rhamnetin feeding. Simultaneously, decreased FoxO3a phosphorylation (Ser-253) was observed in the dorso-lateral prostate, which correlated with decreased cytoplasmic retention of FoxO3a with no significant changes in 14-3-3 chaperone protein in the cytosol after rhamnetin feeding. Additionally, decreased IGF-1 protein expression with subsequent decrease in p-Akt (Ser-473) and p-Erk1/2 (Thr202/Tyr204) was observed after rhamnetin feeding demonstrating that the decreased nuclear presence of p-Akt (Ser-473) may restrict FoxO3a phosphorylation and its retention in the nucleus along with decreased p-Erk1/2 levels in the dorso-lateral prostate. Furthermore, rhamnetin intake led to increased levels of SOD2 and significantly enhanced the expression of the thioredoxin/peroxiredoxin (Trx/Prx) in the dorso-lateral prostate. Similar findings were recorded in cell culture studies where rhamnetin treatment to human prostate cancer LNCaP and PC-3 cells resulted in the restoration of Klotho and FoxO3a in the nucleus. This increase in Klotho and FoxO3a protein expressions in the nucleus may be a decisive factor for increase resistance to oxidative stress and cancer progression. Our finding suggests that rhamnetin has potential to modulate Klotho and FoxO signaling cascades and could be represented as therapeutic targets in prostate cancer. Citation Format: Christine Oak, Natarajan Bhaskaran, Sanjay Gupta, Sanjeev Shukla. Rhamnetin inhibits prostate cancer progression in an autochthonous mouse prostate cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1913. doi:10.1158/1538-7445.AM2015-1913

Oncolytic virus carrying shRNA targeting SATB1 inhibits prostate cancer growth and metastasis.

Recent studies suggest that SATB1 is a promising therapeutic target for prostate cancer. To develop novel SATB1-based therapeutic agents for prostate cancer, in this study, we aimed to construct ZD55-SATB1, an oncolytic adenovirus ZD55 carrying shRNA targeting SATB1, and investigate its effects on the inhibition of prostate cancer growth and metastasis. ZD55-SATB1 was constructed and used to infect human prostate cancer cell lines DU145 and LNCaP. The inhibitory effect of ZD55-SATB1 on SATB1 expression was evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The cytotoxicity of ZD55-SATB1 was detected by MTT assay. Cell invasion was detected by Matrigel invasion assay. The in vivo antitumor activities of ZD55-SATB1 were evaluated in xenograft mouse model. We found that ZD55-SATB1 selectively replicated and significantly reduced SATB1 expression in DU145 and LNCaP cells. ZD55-SATB1 effectively inhibited the viability and invasion of DU145 and LNCaP cells in vitro and inhibited prostate cancer growth and metastasis in xenograft nude mice. In conclusion, replicative oncolytic adenovirus armed with SATB1 shRNA exhibits effective antitumor effect in human prostate cancer. Our study provides the basis for the development of ZD55-SATB1 for the treatment of prostate cancer.

Combination of Quercetin and 2-Methoxyestradiol Enhances Inhibition of Human Prostate Cancer LNCaP and PC-3 Cells Xenograft Tumor Growth.

Quercetin and 2-Methoxyestradiol (2-ME) are promising anti-cancer substances. Our previous in vitro study showed that quercetin synergized with 2-Methoxyestradiol exhibiting increased antiproliferative and proapoptotic activity in both androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cell lines. In the present study, we determined whether their combination could inhibit LNCaP and PC-3 xenograft tumor growth in vivo and explored the underlying mechanism. Human prostate cancer LNCaP and PC-3 cells were inoculated subcutaneously in male BALB/c nude mice. When xenograft tumors reached about 100 mm3, mice were randomly allocated to vehicle control, quercetin or 2-Methoxyestradiol singly treated and combination treatment groups. After therapeutic intervention for 4 weeks, combination treatment of quercetin and 2-ME i) significantly inhibited prostate cancer xenograft tumor growth by 46.8% for LNCaP and 51.3% for PC-3 as compared to vehicle control group, more effective than quercetin (28.4% for LNCaP, 24.8% for PC3) or 2-ME (32.1% for LNCaP, 28.9% for PC3) alone; ii) was well tolerated by BALB/c mice and no obvious toxic reactions were observed; iii) led to higher Bax/Bcl-2 ratio, cleaved caspase-3 protein expression and apoptosis rate; and iv) resulted in lower phosphorylated AKT (pAKT) protein level, vascular endothelial growth factor protein and mRNA expression, microvascular density and proliferation rate than single drug treatment. These effects were more remarkable compared to vehicle group. Therefore, combination of quercetin and 2-ME can serve as a novel clinical treatment regimen owning the potential of enhancing antitumor effect on prostate cancer in vivo and lessening the dose and side effects of either quercetin or 2-ME alone. These in vivo results will lay a further solid basis for subsequent researches on this novel therapeutic regimen in human prostate cancer.

Initial development of a cytotoxic amino-seco-CBI warhead for delivery by prodrug systems

Cyclopropabenzaindoles (CBIs) are exquisitely potent cytotoxins which bind and alkylate in the minor groove of DNA. They are not selective for cancer cells, so prodrugs are required. CBIs can be formed at physiological pH by Winstein cyclisation of 1-chloromethyl-3-substituted-5-hydroxy-2,3-dihydrobenzo[e]indoles (5-OH-seco-CBIs). Corresponding 5-NH2-seco-CBIs should also undergo Winstein cyclisation similarly. A key triply orthogonally protected intermediate on the route to 5-NH2-seco-CBIs has been synthesised, via selective monotrifluoroacetylation of naphthalene-1,3-diamine, Boc protection, electrophilic iodination, selective allylation at the trifluoroacetamide and 5-exo radical ring-closure with TEMPO. This intermediate has potential for introduction of peptide prodrug masking units (deactivating the Winstein cyclisation and cytotoxicity), addition of diverse indole-amide side-chains (enhancing non-covalent binding prior to alkylation) and use of different leaving groups (replacing the usual chlorine, allowing tuning of the rate of Winstein cyclisation). This key intermediate was elaborated into a simple model 5-NH2-seco-CBI with a dimethylaminoethoxyindole side-chain. Conversion to a bio-reactive entity and the bioactivity of this system were confirmed through DNA-melting studies (ΔTm=13°C) and cytotoxicity against LNCaP human prostate cancer cells (IC50=18nM).

Synthesis, crystal structure and effect of indeno[1,2-b]indole derivatives on prostate cancer in vitro. Potential effect against MMP-9

A highly regiospecific synthesis of a series of indenoindoles is reported, together with X-ray studies and their activity against human prostate cancer cells PC-3 and LNCaP in vitro. The most effective compound 7,7-dimethyl-5-[(3,4-dichlorophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9bhexahydro-indeno[1,2-b]indole-9,10-dione 7q reduced the viability in both cell lines in a time and dose-dependent manner. Inhibitory effects were also observed on the adhesion, migration, and invasion of the prostate cancer cells as well as on clonogenic possibly by inhibition of MMP-9 activity. Molecular docking of 7q and 6k into MMP-9 human active site was also performed to determine the probable binding mode.

Effects of special AT-rich binding protein-1 overexpression on growth of implanted human prostate cancer LNCaP cell carcinoma in nude mice and possible mechanism

Objective To investigate the effects of overexpressed special AT- rich binding protein- 1 (SATB1)on the growth of implanted human prostate cancer LNCaP cell carcinoma in nude mice and possible mechanism. Methods The pcDNA3.1- SATB1, and pcDNA3.1 were transfected into human prostate cancer LNCaP cells by applying LipofectamineTM 2000. The xenografts prostate cell carcinoma cancer model was established by subcutaneously injecting 2×107 untransfected LNCaP, pcDNA3.1- SATB1 transfected LNCaP and pcDNA3.1 transfected LNCaP cells into the right flank of BALB/c nude mice. Immunohistochemistry and Western blotting were used to detect the expression of SATB1. The volume of tumor was firstly measured at the 7th day after the injection and then measured every four days.All mice were sacrificed at the 27th day. Td T- mediated d UTP nick end labeling(TUNEL) was used to assay the apoptosis of tumor cells in each group. Immunohistochemistry was used to detect the expression of Vimentin, E- cadherin and matrix metalloproteinase (MMP)- 2 in each group. Results Immunohistochemistry and Western blotting revealed in the pcDNA3.1- transfected group, SATB1 was stably expressed in nude mice, and tumor models of human prostate cancer LNCaP cells were constructed successfully.Final volume(mm3) of tumor in pcDNA3.1- transfected LNCaP cells group was 2 242.0±259.2, significantly greater than in control groups(P<0.05).TUNEL assay showed the average rate of apoptosis in the pcDNA3.1- transfected group was(31.3±8.9)%, significantly higher than in the control groups(P< 0.05). The average IOD values of Vimentin, MMP- 2 and E- cadherin expression in the pcDNA3.1- transfected group was 417.9±18.2, 539.1±41.6, and 156.4±11.9, and there were statistically significant difference in comparison with the control group(P< 0.05). Conclusion The tumor models of human prostate cancer LNCaP cells overexpressing SATB1 gene were successfully constructed. SATB1 can promote the growth and proliferation, and inhibit apoptosis of prostate cancer cells probably by regulating E- cadherin, Vimentin, and MMP- 2, which provides the basis for research on the metastasis mechanism of prostate cancer. Key words: Special AT-rich binding protein-1; Prostate cancer; Model,animal

Effects of treatment with androgen receptor ligands on microRNA expression of prostate cancer cells.

Post-transcriptional regulation by microRNA (miRNA) is an important aspect of androgen receptor (AR) signalling in prostate cancer cells. However, the global profiling of miRNA expression in prostate cancer cells following treatment with AR ligands has not been reported so far. In this study we examined the effect of treatment with two AR agonists (mibolerone (MIB) and dihydrotestosterone (DHT)) and an AR antagonist (bicalutamide (BIC)) on miRNA expression in the human androgen-dependent LNCaP prostate cancer cell line using microarray technology and verification of selected miRNA using quantitative real-time PCR (qRT-PCR). No miRNA was identified as differentially expressed following treatment with the AR antagonist BIC. In contrast, a number of common and compound-specific alterations in miRNA expression were observed following treatment with AR agonists. Unexpectedly it was found that treatment with the AR agonists resulted in the repression of miR-221, a miRNA previously established to be involved with prostate cancer development. This observation indicates that this miRNA may have a more complex role in prostate cancer development than considered previously. Treatment with MIB led to an induction of miR-210 expression, a hypoxia-related miRNA. This miRNA is reported to be involved in cell adaptation to hypoxia and thus induction in conditions of normoxia may be important in driving metabolic changes observed in prostate cancer. Thus examining the effect of AR agonists and antagonists on miRNA expression can provide novel insights into the response of cells to AR ligands and subsequent downstream events.

Label‐Free Quantitative Proteomics Identify Global Protein Interactors for the Androgen Receptor Associated Cochaperone SGTA

Molecular chaperones facilitate proper folding and regulation of steroid hormone receptors (SHRs). Upon proper folding, SHRs bind ligand with high affinity, translocate to the nucleus and initiate gene expression accordingly. A novel cochaperone, human small glutamine rich TPR (tetratricopeptide repeat) containing protein alpha (SGTA), is a down‐regulator of androgen (AR), glucocorticoid (GR), and progesterone (PR) receptors. It binds to heat shock protein (Hsp) 70 and 90 kDa. Additionally, SGTA plays a role in cellular processes such as cell cycle progression and apoptosis. Therefore, label‐free quantitative proteomics was utilized to determine novel interactors in LNCaP human prostate cancer cells. Unknown protein interactions were purified using a Nickel resin and further analyzed with liquid chromatography mass spectrometry (LC‐MS/MS). To determine if interactions were transient or strong, a comparison of a 3‐hour versus an overnight incubation was used. Preliminary studies using rigorous statistical analysis have identified a broad range of proteins involved in different pathways other than the chaperoning pathway. Thus, future studies aim to validate these interactions with SGTA using nickel purification and assess their functional relevance, which will contribute to the understanding of the role SGTA plays within the chaperoning pathway and any cross‐talk with other pathways.

The in vitro and in vivo anti-cancer activities of a standardized quassinoids composition from Eurycoma longifolia on LNCaP human prostate cancer cells.

Quassinoids are a group of diterpenoids found in plants from the Simaroubaceae family. They are also the major bioactive compounds found in Eurycoma longifolia which is commonly used as traditional medicine in South East Asia to treat various ailments including sexual dysfunction and infertility. These uses are attributed to its ability to improve testosterone level in men. Chronic consumption of E. longifolia extracts has been reported to increase testosterone level in men and animal model but its effect on prostate growth remains unknown. Therefore, the present study investigates the effects of a standardized total quassinoids composition (SQ40) containing 40% of the total quassinoids found in E. longifolia on LNCaP human prostate cancer cell line. SQ40 inhibited LNCaP cell growth at IC50 value of 5.97 μg/mL while the IC50 on RWPE-1 human prostate normal cells was 59.26 μg/mL. SQ40 also inhibited 5α-dihydrotestosterone-stimulated growth in LNCaP cells dose-dependently. The inhibitory effect of SQ40 in anchorage-independent growth of LNCaP cells was also demonstrated using soft agar assay. SQ40 suppressed LNCaP cell growth via G0/G1 phase arrest which was accompanied by the down-regulation of CDK4, CDK2, Cyclin D1 and Cyclin D3 and up-regulation of p21Waf1/Cip1 protein levels. SQ40 at higher concentrations or longer treatment duration can cause G2M growth arrest leading to apoptotic cell death as demonstrated by the detection of poly(ADP-ribose) polymerase cleavage in LNCaP cells. Moreover, SQ40 also inhibited androgen receptor translocation to nucleus which is important for the transactivation of its target gene, prostate-specific antigen (PSA) and resulted in a significant reduction of PSA secretion after the treatment. In addition, intraperitoneal injection of 5 and 10 mg/kg of SQ40 also significantly suppressed the LNCaP tumor growth on mouse xenograft model. Results from the present study suggest that the standardized total quassinoids composition from E. longifolia promotes anti-prostate cancer activities in LNCaP human prostate cancer cells.

Synthesis and evaluation of naphthalene-based thiosemicarbazone derivatives as new anticancer agents against LNCaP prostate cancer cells

Fourteen new naphthalene-based thiosemicarbazone derivatives were designed as anticancer agents against LNCaP human prostate cancer cells and synthesized. MTT assay indicated that compounds 6, 8 and 11 exhibited inhibitory effect on LNCaP cells. Among these compounds, 4-(naphthalen-1-yl)-1-[1-(4-hydroxyphenyl)ethylidene)thiosemicarbazide (6), which caused more than 50% death on LNCaP cells, was chosen for flow cytometric analysis of apoptosis. Flow cytometric analysis pointed out that compound 6 also showed apoptotic effect on LNCaP cells. Compound 6 can be considered as a promising anticancer agent against LNCaP cells owing to its potent cytotoxic activity and apoptotic effect.

Cell-type-specific enrichment of risk-associated regulatory elements at ovarian cancer susceptibility loci.

Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10(-30)), OSECs (P = 2.4 × 10(-23)) and HMECs (P = 6.7 × 10(-15)) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer.

FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation, and migration in prostate cancer cells.

ELL-associated factor 2 (EAF2) is an androgen-regulated tumor suppressor in the prostate. However, the mechanisms underlying tumor suppressive function of EAF2 are still largely unknown. Identification of factors capable of modulating EAF2 function will help elucidate the mechanisms underlying EAF2 tumor suppressive function. Using eaf-1(the ortholog of EAF2) mutant C. elegans model, RNAi screen was used to identify factors on the basis of their knockdown to synergistically enhance the reduced fertility phenotype of the eaf-1 mutant C. elegans. In human cells, the interaction of EAF2 with FOXA1 and the effect of EAF2 on the FOXA1 protein levels were determined by co-immunoprecipitation and protein stability assay. The effect of EAF2 and/or FOXA1 knockdown on the expression of AR-target genes was determined by real-time RT-PCR and luciferase reporter assays. The effect of EAF2 and/or FOXA1 knockdown on LNCaP human prostate cancer cell proliferation and migration was tested using BrdU assay and transwell migration assay. RNAi screen identified pha-4, the C. elegans ortholog of mammalian FOXA1, on the basis of its knockdown to synergistically enhance the reduced fertility phenotype of the eaf-1 mutant C. elegans causing sterility. EAF2 co-immunoprecipitated with FOXA1. EAF2 knockdown enhanced endogenous FOXA1 protein level, whereas transfected GFP-EAF2 down-regulated the FOXA1 protein. Also, EAF2 knockdown enhanced the expression of AR-target genes, cell proliferation, and migration in LNCaP cells. However, FOXA1 knockdown inhibited the effect of EAF2 knockdown on AR-target gene expression, cell proliferation, and migration in LNCaP cells, suggesting that FOXA1 can modulate EAF2 regulation of AR transcriptional activation, cell proliferation, and migration. These findings suggest that regulation of the AR signaling pathway, cell proliferation, and migration through FOXA1 represents an important mechanism of EAF2 suppression of prostate carcinogenesis.

Effect of compound Chinese traditional medicine PC-SPES Ⅱ in inhibiting proliferation of human prostate cancer cell LNCaP and on expressions of AR and PSA

To investigate the effect of compound Chinese traditional medicine PC-SPES II I in inhibiting proliferation of human prostate cancer cell LNCaP based on the androgen receptor (AR) signaling pathway. The effect of PC-SPES II on LNCaP cell proliferation was detected by MTT assay. According to the findings, at the mass concentration of 180-1 440 mg x L(-1), PC-SPES II significantly inhibited the proliferation of LNCaP cells; the IC50 of PC-SPES II at 24 h and 48 h were 311.48, 199.01 mg x L(-1), respectively. The flow Cytometry detection showed 240 mg x L(-1) PC-SPES II arrested cells in G2/M phase, and an obvious apoptotic peak appeared before G0/G1 peak and rose over time. Meanwhile, Hoechst 33258 staining revealed apoptotic cellular morphology. Annexin V-FITC/PI staining manifested an increase in apoptotic cell ratio at the PC-SPES II concentration of 480 mg x L(-1) in a dose dependent manner. The prostate specific antigen (PSA) secretion of LNCaP cells was tested by PSA ELISA kit. Besides, compared with 25 mg x L(-1) Bic, 480 mg x L(-1) PC-SPES II significantly reduced the cell secretion of PSA. The AR and PSA mRNA and protein expressions were detected by qRT-PCR and Western blot. According to the results, after the induction of LNCaP cells with synthetic androgen 25 μg x L(-1) R1881, 240-480 mg x L(-1) PC-SPES II notably down-regulated the AR and PSA mRNA and protein expressions and inhibited the translocation of AR from cytoplasm to nucleus. In summary, PC-SPES II significantly can inhibit the in vitro proliferation of LNCaP cells and arrest cell cycle arrest in G2/M phase. Its mechanism may be associated with the down-regulation of the AR and PSA expressions and the inhibition of AR nuclear translocation.

Expression of Prostate Specific Antigen(PSA) by Etoposide and Androgen in Human Prostate Cancer Cells

Prostate cancer(PrCa) is a leading cause of cancer-related death in man. Prostate specific antigen(PSA) is widely utilized as a marker for the diagnosis and monitoring of prostate cancer. This study examined effects of androgen 5α-dihydrotestosterone(DHT) vs DHEA and/or anticancer agent etoposide on proliferation and PSA expression of the prostate cancer LNCaP cells. A progressive increase in PSA secretion was observed at higher DHT concentration in case of 48 h treatment. compared to DHEA. Androgen played a significant role in the regulation of cellular proliferation and production of PSA in human prostate cancer LNCaP cells. In contrast with DHT, DHEA administration stimulated LNCaP cell proliferation in a time and dose-response manner, etoposide had a strong cytotoxic and apoptotic effect on LNCaP cells. Moreover, etoposide reduced cell growth and PSA expression induced by DHT and DHEA. These results demonstrate that etoposide could be a potential agent for the treatment of hormone refractory prostate cancer. Further studies of the mechanisms of DHEA will be required to discern the implication of DHEA supplementation on prostatic health because supplemental DHEA use may pose a cancer risk in patients with nascent or occult prostate cancer