Proliferation Of Prostate Cancer PC-3 Cells Research Articles

Identification of a druggable pocket of the calcium-activated chloride channel TMEM16A in its open state

The calcium-activated chloride channel TMEM16A is a potential drug target to treat hypertension, secretory diarrhea, and several cancers. However, all reported TMEM16A structures are either closed or desensitized, and direct inhibition of the open state by drug molecules lacks a reliable structural basis. Therefore, revealing the druggable pocket of TMEM16A exposed in the open state is important for understanding protein-ligand interactions and facilitating rational drug design. Here, we reconstructed the calcium-activated open conformation of TMEM16A using an enhanced sampling algorithm and segmental modeling. Furthermore, we identified an open-state druggable pocket and screened a potent TMEM16A inhibitor, etoposide, which is a derivative of a traditional herbal monomer. Molecular simulations and site-directed mutagenesis showed that etoposide binds to the open state of TMEM16A, thereby blocking the ion conductance pore of the channel. Finally, we demonstrated that etoposide can target TMEM16A to inhibit the proliferation of prostate cancer PC-3cells. Together, these findings provide a deep understanding of the TMEM16A open state at an atomic level and identify pockets for the design of novel inhibitors with broad applications in chloride channel biology, biophysics, and medicinal chemistry.

Mechanism of Yishen Tonglong Decoction inhibiting TLR4/p38 MAPK/NF-κB signaling pathway against prostate cancer via upregulating miR-145-5p

ObjectiveTo investigate the mechanism of Yishen Tonglong Decoction (益肾通癃汤, YSTLD) inhibiting the toll-like receptor 4/p38 mitogen activated protein kinases/nuclear factor kappa-B (TLR4/p38 MAPK/NF-κB) signaling pathway against prostate cancer by up-regulating miR-145-5p.Methods miRNA microarray technology was used to detect the changes of miRNA expression profile in prostate cancer PC-3 cells treated with YSTLD, and miRNAs with marked differences in miRNA microarray results were screened and validated by real-time polymerase chain reaction (qRT-PCR). Lentiviral transfection of miR-145-5p into prostate cancer PC-3 cells, Cell Counting Kit-8 (CCK8) assay, and scratch assay were adopted to detect the effects of miR-145-5p on prostate cancer PC-3 cell proliferation and migration. qRT-PCR and Western blot were employed to detect the effects of miR-145-5p on TLR4/p38 MAPK/NF-κB signaling pathway and the expression levels of apoptosis-related genes caspase3, tumor necrosis factor-α (TNF-α), Bax, and Bcl-2. qRT-PCR and Western blot were used to detect the effects of serum containing YSTLD on miR-145-5p, TLR4/p38 MAPK/NF-κB signaling pathway, and the expression levels of apoptosis-related genes caspase3, TNF-α, Bax, and Bcl-2. ResultsThe expression levels of 35 miRNAs in prostate cancer PC-3 cells treated with YSTLD were significantly different from those in the control group, with miR-145-5p being the most significantly different; qRT-PCR validation revealed that the miR-145-5p levels in prostate cancer PC-3 cells treated with YSTLD were significantly higher than those in the DMSO control group (P < 0.05). After lentiviral transfection of miR-145-5p into prostate cancer PC-3 cells, miR-145-5p was found to inhibit the proliferation and migration of prostate cancer PC-3 cells. Overexpression of miR-145-5p up-regulated expression levels of caspase3, TNF-α, and Bax mRNA, and down-regulated expression levels of p38 MAPK, p65 NF-κB, and Bcl-2 mRNA in prostate cancer PC-3 cells (P < 0.05), while up-regulated caspase3 protein expression levels in prostate cancer PC-3 cells and down-regulated expression levels of TLR4, p38 MAPK, and p65 NF-κB protein (P < 0.05). Serum containing YSTLD could up-regulate the expression levels of caspase3, TNF-α, and Bax mRNA, and down-regulate the mRNA expression levels of p38 MAPK, p65 NF-κB, Bcl-2, and TNF receptor-associated factor 1 (TRAF1) in prostate cancer PC-3 cells after intervening prostate cancer PC-3 cells (P < 0.05). Simultaneously, it up-regulated the expression levels of caspase3 protein and down-regulated the protein expression levels of TLR4, p38 MARK, p65 NF-κB, and TRAF1 in prostate cancer PC-3 cells (P < 0.05). ConclusionYSTLD can promote apoptosis of prostate cancer PC-3 cells by up-regulating the expression level of miR-145-5p and inhibiting TLR4/p38 MAPK/NF-κB signaling pathway, which may be an important mechanism of YSTLD against prostate cancer.

Direct bone marrow injection of human bone marrow-derived stromal cells into mouse femurs results in greater prostate cancer PC-3 cell proliferation, but not specifically proliferation within the injected femurs

BackgroundWhile prostate cancer (PCa) cells most often metastasize to bone in men, species-specific differences between human and mouse bone marrow mean that this pattern is not faithfully replicated in mice. Herein we evaluated the impact of partially humanizing mouse bone marrow with human bone marrow-derived stromal cells (BMSC, also known as "mesenchymal stem cells") on human PCa cell behaviour.MethodsBMSC are key cellular constituents of marrow. We used intrafemoral injection to transplant 5 × 105 luciferase (Luc) and green fluorescence protein (GFP) expressing human BMSC (hBMSC-Luc/GFP) into the right femur of non-obese diabetic (NOD)-severe combined immunodeficiency (scid) interleukin (IL)-2γ−/− (NSG) mice. Two weeks later, 2.5 × 106 PC-3 prostate cancer cells expressing DsRed (PC-3-DsRed) were delivered into the mice via intracardiac injection. PC-3-DsRed cells were tracked over time using an In Vivo Imaging System (IVIS) live animal imaging system, X-ray and IVIS imaging performed on harvested organs, and PC-3 cell numbers in femurs quantified using flow cytometry and histology.ResultsFlow cytometry analysis revealed greater PC-3-DsRed cell numbers within femurs of the mice that received hBMSC-Luc/GFP. However, while there were overall greater PC-3-DsRed cell numbers in these animals, there were not more PC-3-DsRed in the femurs injected with hBMSC-Luc/GFP than in contralateral femurs. A similar proportion of mice in with or without hBMSC-Luc/GFP had bone lessions, but the absolute number of bone lesions was greater in mice that had received hBMSC-Luc/GFP.ConclusionPC-3-DsRed cells preferentially populated bones in mice that had received hBMSC-Luc/GFP, although PC-3-DsRed cells not specifically localize in the bone marrow cavity where hBMSC-Luc/GFP had been transplanted. hBMSC-Luc/GFP appear to modify mouse biology in a manner that supports PC-3-DsRed tumor development, rather than specifically influencing PC-3-DsRed cell homing. This study provides useful insights into the role of humanizing murine bone marrow with hBMSC to study human PCa cell biology.

Salmon Protein Hydrolysate Potentiates the Growth Inhibitory Effect of Bicalutamide on Human Prostate Cancer Cell Lines LNCaP and PC3 by Modulating Iron Homeostasis.

Prostate cancer is a common cause of cancer death in men. In advanced stages of prostate cancer, androgen deprivation therapy (ADT) is initiated. Despite ADT, prostate cancers invariably progress to become androgen independent. A growing body of evidence implicates iron dysmetabolism in prostate cancer progression. A bioactive peptide-rich salmon protein hydrolysate (SPH) has previously been demonstrated to modulate iron homeostatic mechanisms. In the present study, the anticancer effect of SPH and bicalutamide co-treatment on LNCaP and PC3 prostate cancer cell proliferation was investigated. Our results found that SPH potentiates the anti-proliferative effect of bicalutamide in a dose-dependent manner for both cell lines. In the presence of 160 µg/mL SPH, co-treatment with 1.0 µM bicalutamide decreased LNCaP cells’ relative colony survival from 25% (1.0 µM bicalutamide monotreatment) to 2% after culturing for 12 days. For PC3 cells, the relative colony survival diminished from 52% (10.0 µM bicalutamide) to 32% at an SPH concentration of 160 µg/mL. Gene expression profiling, employing quantitative real-time PCR, revealed that the inhibitory effects were related to significant FTH1 up-regulation with a concomitant TFRC down-regulation. In conclusion, our results provide in vitro evidence that SPH potentiates the growth inhibitory effect of bicalutamide on prostate cancer cells by modulating iron homeostasis mechanisms.

Effects of ADAM17 siRNA and doxorubicin on proliferation and apoptosis of prostate cancer PC-3 cells

Objective To investigate the effects of ADAM17 siRNA combined with adriamycin (ADM) on proliferation and apoptosis of human prostate cancer cell line PC-3. Methods PC-3 cells stably transfected with ADAM17 siRNA expression plasmid were treated with ADM. The proliferation rate was observed by MTT colorimetry, the apoptosis rate was detected by acridine orange staining and flow cytometry (FCM), and the activity of apoptotic protein caspase-3 was analyzed. Results Both ADAM17 siRNA and ADM alone and in combination could decrease the proliferation rate and increase the apoptosis rate of PC-3 cells in varying degrees. FCM data showed that the apoptosis rates of ADAM17 siRNA and ADM alone were(6.2±1.4)% and (17.2±1.9)% respectively, which were significantly different from (36.4±3.7)% of ADAM17 siRNA and ADM alone (P<0.01). Both of them also had a good synergistic effect on inducing caspase-3 activity. Conclusions ADAM17 siRNA and ADM have different effects on the proliferation and apoptosis of human prostate cancer PC-3, but the combination of ADAM17 siRNA and ADM is more effective. It is suggested that gene therapy combined with chemotherapy may be an important method to improve the curative effect and reduce the risk of recurrence and metastasis of prostate cancer. Key words: Prostatic Neoplasms; Cells, Cultured; RNA, Small Interfering; Doxorubicin; Cell Proliferation; Apoptosis

Semisynthetic aurones inhibit tubulin polymerization at the colchicine-binding site and repress PC-3 tumor xenografts in nude mice and myc-induced T-ALL in zebrafish

Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model.

Inhibition of Prostate Cancer Cell Proliferation and Survival by Rosemary Extract

Prostate cancer is the most commonly diagnosed cancer in men. Despite many advances in diagnosis and treatment, prostate cancer is the second‐leading cause of cancer related death amongst men in North America and therefore there is an urgent need to find novel chemicals that can be used effectively towards the treatment of prostate cancer patients. Prostate cancers are typically characterized by the expression of androgen receptor (AR) and prostate‐specific antigen (PSA). Prostate cancers that are AR positive can be treated with hormonal therapy, however those which are AR negative are more aggressive and do not respond to hormone therapy, thus this subtype of prostate cancer requires targeted treatments. Many chemotherapeutic drugs have been discovered by screening natural products from plants. Polyphenols derived from the plant rosemary, have been found to have antioxidant and anti‐inflammatory effects and are proposed to have anticancer activity. In previous studies, rosemary extract (RE) has been found to reduce cell proliferation, viability, survival, and increase apoptosis in various cancer cell lines. However, there is very limited evidence examining RE's effects in androgen receptor negative prostate cancer cells. In the present study, we examine the effects of rosemary extract on PC‐3 prostate cancer cell proliferation, survival, migration and apoptosis. RE significantly inhibited PC‐3 cell proliferation and survival in a dose dependent manner. Furthermore, RE reduced cell migration and enhanced the cleavage of PARP, a marker of apoptosis. Our findings indicate that RE may have potent anti‐cancer properties against AR negative prostate cancer and supports the need for further investigation into its role in modulating key signaling pathways involved in cell proliferation, survival, and migration.Support or Funding InformationSupported by the Prostate Cancer Fight Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Efficient Synthesis of Aurone Mannich Bases and Evaluation of their Antineoplastic Activity in PC-3 Prostate Cancer Cells.

An efficient method for regioselective synthesis of C-7 Mannich bases of 6-hydroxyaurones was accomplished by the N,N-dialkylaminomethylation using aminals prepared from dimethylamine, dipropylamine, bis(2-methoxyethyl)amine, N-methylbutylamine, N-methylbenzylamine, morpholine, piperidine, and 1-methylpiperazine. Further transformation of 7-(N,N-dialkylamino)methyl group in these aurones led to formation of C-7 acetoxymethyl and methoxymethyl derivatives of 6-hydroxyaurones, some of which showed promising inhibition of PC-3 prostate cancer cell proliferation in the high nanomolar to low micromolar range that exceeded that of cisplatin.

Dental pulp stem cells (DPSCs) increase prostate cancer cell proliferation and migration under in vitro conditions.

Cancer as a multistep and complicated disease is regulated by several molecular and cellular events. Cancer treatment could be managed at the early stages when the tumor is confined in the tissue. However, disseminated cancer cells metastasize to other body parts and generate new tumors resulting in mortality. Mesenchymal stem cells (MSCs) are found in different body parts and helps adult tissue regeneration. The role of MSCs in cancer progression has emerged as one of the important aspects in cancer biology and is the aim of interest in recent years. In the current study, effects of Dental Pulp Stem Cells (DPSCs) on PC-3 prostate cancer cell proliferation and migration were conducted by cell proliferation, apoptosis, gene expression and cell migration analysis in vitro. Condition medium (CM) obtained from DPSCs increased cell proliferation of PC-3 cells and decreased apoptosis. Either administration of CM or trans well co-culture of DPSCs increased cell migration in scratch assay, confirmed by gene expression analysis of migratory genes including fibronectin, laminin and collagen type I (Col I). Furthermore, DPSCs participated in a self-organized structure with PC-3 cells in co-culture conditions. Overall, results indicated that DPSCs could promote PC-3 cancer cell proliferation and metastasis in co-culture conditions in vitro.

MicroRNA delivery system inhibits proliferation of androgen-independent prostate cancer

Objective To prepare a microRNA(miRNA)delivery system using the branched polyethyleneimine(BPEI),linear polyethylenimine(LPEI)and polyamidoamine dendrimers(PAMAM)loaded with miRNA-15 aand miRNA-16-1,which can inhibit prostate cancer PC3 cell proliferation,and to examine the zeta potential,intracellular uptake,and the inhibition effect on PC3 cells of the three constructed nano-complexes.Methods Particle size analyzer was used to determine the size and potential of the three kinds of nano-complexes,and the miRNA affinity capability of them was determined by agarose gel electrophoresis retardation assay.The uptake efficiency of the nano-complexes by PC3 cells was examined by NC-miRNA labeled with FAM.CCK8 method was used to determine the inhibitory effect of the nano-complexes loaded with miRNA-15 a and miRNA-16-1against PC3 cells,and PCR was used to analyze their inhibitory effect on expression of Bcl-2,Cylin D1 and Wnt3agene in PC3 cells.Results BPEI,LPEI and PAMAM loaded with miRNA could form stable nano-complexes.When N/P=5,the intracellular uptake of BPEI/miRNA-FAM by PC3 cells was significantly higher than that of LPEI/miRNA-FAM and PAMAM/miRNA-FAM(P0.05).BPEI,LPEI and PAMAM could all carry miRNA-15 aand miRNA-16-1into PC3 cells and block Bcl-2,Cylin D1 and Wnt3aexpression in PC3 cells.Conclusion BPEI,LPEI and PAMAM loaded with miRNA-15 aand miRNA-16-1can suppress proliferation of prostate cancer PC3 cells and block Bcl-2,Cylin D1 and Wnt3aexpression.

Inhibition of tumor growth and histopathological changes following treatment with a chemokine receptor CXCR4 antagonist in a prostate cancer xenograft model

The stromal derived factor-1 (SDF-1)/CXCR4 axis is associated with tumor aggressiveness and metastasis in prostate cancer. The present study aimed to explore the potential therapeutic effects of a CXCR4 antagonist in prostate cancer. The effect of SDF-1 and a CXCR4-specific antagonist, AMD3100, on human prostate cancer PC-3 cell proliferation and protein kinase B (Akt) signaling was assessed. Moreover, a PC-3 tumor xenograft model was used to evaluate the effect of AMD3100 on tumor growth and to identify the histopathological changes and immunohistochemical differences between AMD3100-treated and untreated groups. Cell proliferation was not significantly affected by SDF-1 or AMD3100 treatment in vitro. Western blot analysis revealed that SDF-1 stimulation enhanced the expression of phosphorylated Akt in the PC-3 cells, but that the SDF-1-induced expression of phosphorylated Akt was abrogated in the AMD3100-treated PC-3 cells. In the PC-3 tumor xenograft model, AMD3100 significantly inhibited tumor growth, while AMD3100-treated PC-3 tumors had lower levels of microvessel formation and a lower immunoreactivity for the proliferation marker Ki-67 and the anti-apoptotic marker Bcl-2 compared to control tumors in vivo. The CXCR4-specific antagonist inhibits SDF-1-induced CXCR4/Akt signal transduction, and effectively suppresses tumor growth in the PC-3 xenograft model. The present study indicates that CXCR4 targeting may represent a novel strategy for the treatment of castration-resistant prostate cancer (CRPC).

Rad9 Protein Contributes to Prostate Tumor Progression by Promoting Cell Migration and Anoikis Resistance

Rad9 as part of the Rad9-Hus1-Rad1 complex is known to participate in cell cycle checkpoint activation and DNA repair. However, Rad9 can act as a sequence-specific transcription factor, modulating expression of a number of genes. Importantly, Rad9 is up-regulated in prostate cancer cell lines and clinical specimens. Its expression correlates positively with advanced stage tumors and its down-regulation reduces tumor burden in mice. We show here that transient down-regulation of Rad9 by RNA interference reduces DU145 and PC3 prostate cancer cell proliferation and survival in vitro. In addition, transient or stable down-regulation of Rad9 impairs migration and invasion of the cells. Moreover, stable reduction of Rad9 renders DU145 cell growth anchorage-dependent. It also decreases expression of integrin β1 protein and sensitizes DU145 and LNCaP cells to anoikis and impairs Akt activation. On the other hand, stable expression of Mrad9, the mouse homolog, in DU145/shRNA Rad9 cells restores migration, invasion, anchorage-independent growth, integrin β1 expression, and anoikis resistance with a concomitant elevation of Akt activation. We thus demonstrate for the first time that Rad9 contributes to prostate tumorigenesis by increasing not only tumor proliferation and survival but also tumor migration and invasion, anoikis resistance, and anchorage-independent growth.

In vitro and in vivo double-enhanced suicide gene therapy mediated by generation 5 polyamidoamine dendrimers for PC-3 cell line

BackgroundOne of the most frequently used and efficient suicide gene therapies for prostate cancer is HSV-TK/GCV system, but its application has been limited due to lack of favorable gene vector and the reduction of "bystander effect". We investigated the effect of a novel combination of HSV-TK/GCV fused with Cx43 and gemcitabine using non-viral vector generation 5 polyamidoamine dendrimers (G5-PAMAM-D) on PC-3 cells.MethodsRT-PCR and Western blot were used to detect TK and Cx43 expression. Cell viability and proliferation were measured by using MTT assay. Cell apoptosis was detected with double-staining of Annexin V-FITC and propidium iodide (PI) by flow cytometry. Nude mice models were established to evaluate the therapeutic effect in vivo.ResultsG5-PAMAM-D efficiently delivered recombinant plasmids into PC-3 cells and HSV-TK and Cx43 could be expressed successfully. With gemcitabine, G5-PAMAM-D mediated HSV-TK and Cx43 expression effectively inhibited prostate cancer PC-3 cell proliferation, leading to more cellular apoptosis and inhibiting PC-3 tumor growth in nude mice models.ConclusionsThis study illustrates that this new suicide gene system mediated by G5-PAMAM-D is effective in decreasing PC-3 cell proliferation and inducing cell apoptosis, and inhibiting tumor growth in vivo. In a word, our study could provide a potential approach for gene therapy of prostate cancer.

Effect of stromal interaction molecular 1 gene on proliferation and apoptosis of prostate cancer PC-3 cells

Objective To evaluate the effects of stromal interaction molecular 1 (STIM1) on proliferation and apoptosis of prostate cancer PC-3 cells.Methods STIMI siRNA sequence was designed and constructed into plasmid pGCSIL-GFP vector.The sequence inserted was confirmed by sequence test and polymerase chain reaction (PCR).The efficiency of plasmid pGCSIL-GFP-STIM1 was confirmed by ectopic cotransfection test.Chromic virus STIM1-RNAi-LV was packed.After it was transfected into prostate cancer PC-3 cells,the proliferation,apoptosis and cell cycle of PC-3 cells were evaluated.Results The plasmid vector pGCSIL-GFP-STIMI was confirmed to be correct and effective by sequence examination,PCR and ectopic target test.After the Chromic virus STIM1-RNAi-LV was transfected into PC-3 cells,the proliferation ability in experimental group was apparently reduced as compared with that in control group.After transfection for 5 days,the apoptosis rate in experimental group was ( 29.85 ± 0.86 ) ％,and that in control group was (2.92 ± 0.14) ％ ( P ＜ 0.05 ).At the same time,the number of cells in the stage G1 and S was reduced and that in stage G2/M was increased in experimental group as compared with control group ( P ＜0.05).Conclusion When STIM1 gene was inhibited in prostate cancer PC-3 cells,the proliferation was reduced,the number of apoptotic was increased and the cell cycle statues were changed,indicating that STIM1 may have important role in proliferation and apoptosis of PC-3 cells. Key words: Prostate neoplasm; Stromal interaction molecular 1; Apoptosis

Hypoxia-inducible factor -1α in prostate cancer: Expression and significance

To study the expression and significance of hypoxia-inducible factor -1 alpha (HIF-1α) in prostate cancer, successfully established recombinant expression plasmids pcDNA3.1-HIF-1α and pll3.7-siHIF-1α were transfected into PC-3 prostate cancer cells using Fermentas transfection reagents. Stable HIF-1α expression cell lines were selected using G418 selective culture. Protein and mRNA expressions of HIF-1α, MMP-2 and MMP-9 were assayed by Western blot and RT-PCR. Cell growth was evaluated using the MTT assay growth curve. The ability of invasion of prostate cancer cells was assayed in Transwell chambers. Compared to other four groups, HIF-1α mRNA expression was not obviously upregulated in pcDNA3.1-HIF-1α-PC-3 cells, while it was obviously downregulated in pll3.7-siHIF-1α-PC-3 cells. MMP-2 and MMP-9 mRNA were significantly upregulated in pcDNA3.1-HIF-1α-PC-3 cells. HIF-1α, MMP-2 and MMP-9 protein were upregulated in pcDNA3.1-HIF-1α-PC-3 cells by Western blot, while they were downregulated in pll3.7-siHIF-1α-PC-3 cells. The abilities of proliferation and invasion of prostate cancer cells were statistically significantly enhanced in pcDNA3.1-HIF-1α-PC-3 cells (Pi€¼0.05). Proliferation and invasion of PC-3 prostate cancer cells are promoted by HIF-1α, which provides a foundation for the research on HIF-1α in the pathogenesis of prostate cancer and suggests that HIF-1α gene may be a good target for anti-tumor therapy. Key words: Hypoxia-inducible factor-1alpha, prostate cancer, expression, significance.

Effect of PKB/Akt signal pathway in mifepristone-induced apoptosis in prostate cancer cell line PC-3

Objective To investigate the cell apoptosis induced by mifepristone in prostate carcinoma cell line PC-3 and the mechanisms involved. Methods Western blotting analysis was used to detect Akt phosphorylation and associated protein expression in different prostate cancer cell line cells and PC-3 cells treated with mifepristone. Results In PC-3 cells, Akt phosphorylation, that was p-Akt (Thr308) and p-Akt ( Ser473 ) , was significantly higher than the hormone-dependent prostate cancer LNCaP cells, and their expression levels were respectively 0. 27 ±0. 05 and 0. 22 ±0.07, 0.46 ±0.09 and 0. 37 ±0. 10 (P ＜0.05). The p-Akt (Thr308) and p-Akt (Ser473) phosphorylation in PC-3 cells treated with 10 μmol/L mifepristone was significantly decreased. The p-Akt ( Thr308) phosphorylation was particularly marked, its expression was respectively 0. 24 ± 0. 08, 0. 11 ± 0. 03, 0. 05 ± 0. 01 and 0. 01 ± 0. 00 during 4-24 h, and declined rapidly as compared with the p-Akt ( Ser473) phosphorylation which expression levels were respectively 0. 56 ± 0. 17, 0.42 ± 0. 11, 0. 28 ± 0. 09 and 0. 12 ± 0. 05 during 4-24 h. Caspase-9 and caspase-3 were activated into cleavage fragment in the PC-3 cells treated with mifepristone. Conclusion Akt kinase plays an important role in prostate cancer growth, development and hormone-dependent to hormone-independent transformation. The p-Akt ( Thr ) phosphorylation, which plays a dominant role in Akt phosphorylation , is necessary in the course of Akt activation. Mifepristone inhibits prostate cancer PC-3 cell proliferation and induces apoptosis by suppressing the Akt signal transduction pathway and activating caspase. Key words: Prostate carcinoma; Mifepristone; Apoptosis; Akt/PKB signal pathway

5α‐androstane‐3α,17β‐diol supports human prostate cancer cell survival and proliferation through androgen receptor‐independent signaling pathways: Implication of androgen‐independent prostate cancer progression

Androgen and androgen receptor (AR) are involved in growth of normal prostate and development of prostatic diseases including prostate cancer. Androgen deprivation therapy is used for treating advanced prostate cancer. This therapeutic approach focuses on suppressing the accumulation of potent androgens, testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), or inactivating the AR. Unfortunately, the majority of patients with prostate cancer eventually advance to androgen-independent states and no longer respond to the therapy. In addition to the potent androgens, 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), reduced from 5alpha-DHT through 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), activated signaling may represent a novel pathway responsible for the progression to androgen-independent prostate cancer. Androgen sensitive human prostate cancer LNCaP cells were used to compare 5alpha-DHT and 3alpha-diol activated androgenic effects. In contrast to 5alpha-DHT, 3alpha-diol regulated unique patterns of beta-catenin and Akt expression as well as Akt phosphorylation in parental and in AR-silenced LNCaP cells. More significantly, 3alpha-diol, but not 5alpha-DHT, supported AR-silenced LNCaP cells and AR negative prostate cancer PC-3 cell proliferation. 3alpha-diol-activated androgenic effects in prostate cells cannot be attributed to the accumulation of 5alpha-DHT, since 5alpha-DHT formation was not detected following 3alpha-diol administration. Potential accumulation of 3alpha-diol, as a result of elevated 3alpha-HSD expression in cancerous prostate, may continue to support prostate cancer growth in the presence of androgen deprivation. Future therapeutic strategies for treating advanced prostate cancer might need to target reductive 3alpha-HSD to block intraprostatic 3alpha-diol accumulation.

Leptin and adiponectin interact in the regulation of prostate cancer cell growth via modulation of p53 and bcl‐2 expression

To investigate the effects of leptin, full-length adiponectin (fAd) and globular adiponectin (gAd), alone and in combination, on LNCaP and PC3 prostate cancer cell proliferation, and on the expression of p53 and bcl-2 gene expression. LNCaP and PC3 prostate cancer cells were cultured and treated with the following: 0-100 nM leptin; 0-100 nM fAd +/- 100 nM leptin; 0-100 nM gAd +/- 100 nM leptin. Proliferation assays and quantitative reverse transcriptase-polymerase chain reaction for p53 tumour-suppressor gene and bcl-2 oncogene expression were performed on treated samples. Co-incubation of PC3 cells with 100 nM leptin and 1 or 100 nM fAd significantly decreased cell proliferation to approximately half of basal (P < 0.001); there was no significant effect in LNCaP cells. Leptin-induced inhibition of p53 expression in LNCaP cells was rescued by fAd. Leptin and fAd dose-dependently potentiated p53 expression in PC3 cells (P < 0.001); leptin and gAd also increased p53 expression (P < 0.05 and P < 0.001). fAd and gAd had no effect on bcl-2 expression in the presence of leptin in LNCaP cells. In PC3 cells, bcl-2 expression was inhibited to negligible levels in the presence of leptin. Leptin and adiponectin interact, resulting in the inhibition of prostate cancer cell proliferation, particularly in PC3 cells, via modulation of p53 and bcl-2 expression. Our findings support the notion that high leptin and low adiponectin levels may be important in driving obesity-related prostate cancer progression.

Decursin Suppresses Human Androgen-Independent PC3 Prostate Cancer Cell Proliferation by Promoting the Degradation of β-Catenin

Alterations in the Wnt/beta-catenin pathway are associated with the development and progression of human prostate cancer. Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, inhibits the growth of androgen-independent human prostate cancer cells, but little is known about its mechanism of action. Using a cell-based screen, we found that decursin attenuates the Wnt/beta-catenin pathway. Decursin antagonized beta-catenin response transcription (CRT), which was induced with Wnt3a-conditioned medium and LiCl, by promoting the degradation of beta-catenin. Furthermore, decursin suppressed the expression of cyclin D1 and c-myc, which are downstream target genes of beta-catenin and thus inhibited the growth of PC3 prostate cancer cells. In contrast, decursinol, in which the (CH3)2-C=CH-COO- side chain of decursin is replaced with -OH, had no effect on CRT, the level of intracellular beta-catenin, or PC3 cell proliferation. Our findings suggest that decursin exerts its anticancer activity in prostate cancer cells via inhibition of the Wnt/beta-catenin pathway.

Zyflamend®-mediated inhibition of human prostate cancer PC3 cell proliferation: Effects on 12-LOX and Rb protein phosphorylation

The multiherb anti-inflammatory product Zyflamend was investigated for its antiproliferative effects on PC3 human prostate cancer cells and eicosanoid metabolism in this prostate cancer cell line. Zyflamend produced a concentration-dependent inhibition of cloned COX-1, COX-2, and 5-LOX enzyme activities, with inhibition of 5-HETE production being greater than that of PGE2 formation. Applied to intact PC3 cells, Zyflamend was found to be most potent against 12-LOX, followed by 5-LOX and then COX activities. The concentration-dependent inhibition of PC3 cell proliferation was associated with a selective G2/M arrest of the cell cycle and induction of apoptosis, as evidenced by flow cytometric staining of PC3 cells with annexin V. Zyflamend also produced a concentration-dependent down-regulation of 5-LOX and 12-LOX expression. Determination of cell signal transduction proteins demonstrated that Zyflamend produced an increase in p21 phosphorylation but down-regulated phosphorylation of retinoblastoma (Rb) protein. The decrease in pRb protein was shown to be due to 12-LOX inhibition and a decline in 12-HETE levels in the cells. Replenishing 12-HETE in Zyflamend-treated cells overcame the ability of this multiple herb product to inhibit cell proliferation, and concordantly, 12-HETE blocked Zyflamend’s ability to down-regulate phosphorylation of Rb protein. We conclude that the effective control of human prostate cancer cell proliferation with Zyflamend is multi-mechanistic but, in part, involves regulation of aberrant tumor cell eicosanoid metabolism, especially on 5- and 12-LOX, as well as restoration of Rb tumor suppressor protein function through regulation of its phosphorylation status.