Androgen-independent Human Prostate Cancer Cells Research Articles

Abstract 4131: Blocking endogenous TBX2 abrogates prostate cancer bone metastasis through WNT signaling

Abstract Bone metastasis is a crucial turning point in the progression of PCa and is seen in > 90% of the lethal cases. Identifying factors that mediate the metastatic spread and subsequent remodeling of the bone is highly relevant to successful therapeutic intervention in advanced human prostate cancer (PCa). TBX2, a transcription factor that belongs to the T-box family negatively regulates p21 cell cycle inhibitor and is known to play critical roles during embryonic development and cancer progression. Interestingly, a recent report found TBX2 over-expression in the bone metastases from patients with castrate resistant prostate cancer. We found TBX2 over-expression in human prostate cancer specimens and in aggressive androgen independent human PCa cell lines. Further, we found TBX2 over-expression in bone metastases in multiple xenograft mouse models of human PCa progression. Blocking endogenous TBX2 in PC3 and ARCaPM PCa cells through the use of a dominant negative construct resulted in decreased proliferation, colony formation and invasion in vitro. Utilizing various pre-clinical xenograft mouse models, we found that blocking endogenous TBX2 in PCa cells leads to decreased invasion and abrogation of bone and soft tissue metastasis in vivo. Further, utilizing the intra-tibial mouse model of growth in the bone microenvironment, we found that blocking endogenous TBX2 in PCa cells dramatically reduces bone remodeling. Additionally we found that TBX2 acts upstream of the canonical WNT (WNT3A) signaling in PCa and positively regulates its transcription by binding to its promoter. Further, rescue of WNT3A levels in PCa cells in the context of blocking endogenous TBX2 rescues the metastatic ability of the cells in vivo. Taken together, our findings highlight a novel and critical role of TBX2 in PCa progression, metastasis and the subsequent bone remodeling, and that TBX2 function in PCa is mediated through the canonical WNT signaling. Citation Format: Srinivas Nandana, Manisha Tripathi, Peng Duan, Gina Chu, Haiyen E. Zhau, Robert J. Matusik, Leland W.K. Chung. Blocking endogenous TBX2 abrogates prostate cancer bone metastasis through WNT signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4131.

Docetaxel induces Bcl-2- and pro-apoptotic caspase-independent death of human prostate cancer DU145 cells.

Docetaxel is a useful chemotherapeutic agent for the first-line treatment of hormone-refractory prostate cancer. Abnormal expression of Bcl-2 is commonly found in cancer cells, which increases their anti-apoptotic potency and chemo-resistance. We investigated the effects of Bcl-2 expression status on the susceptibility of DU145 cells, an androgen-independent human prostate cancer cell line, to docetaxel and other anticancer agents. A panel of Bcl-2-expressing DU145 cell lines was established. Bcl-2 expression levels were unrelated to the susceptibility of DU145 cells to docetaxel. The sensitivity of DU145 cells to cisplatin fluctuated, and the sensitivity to tumor necrosis factor (TNF)-α was decreased by Bcl-2 overexpression. In a xenograft mouse model, overexpression of Bcl-2 drastically decreased the sensitivity of DU145 cells to cisplatin and TNF-α; however, there was no change in the response to docetaxel. Fluorescent microscopy revealed that Bcl-2-overexpression had no effect on the docetaxel-induced death of DU145 cells, but significantly decreased DU145 cell death induced by cisplatin or TNF-α. Interestingly, docetaxel hardly induced caspase-3/7 activation in control or Bcl-2-overexpressing DU145 cells, but did at a low level in LNCaP cells, another prostate cancer cell line. Moreover, in contrast to LNCaP cells, the reduced viabilities of docetaxel-treated control and Bcl-2-overexpressing DU145 cells were not restored by the addition of either a Bid inhibitor or a panel of pro-apoptotic caspase inhibitors. These findings indicate that the antitumor effects of docetaxel on DU145 cells are independent of both Bcl-2 and pro-apoptotic caspases.

The Anti-Proliferative Effect of Boron Neutron Capture Therapy in a Prostate Cancer Xenograft Model.

PurposeBoron neutron capture therapy (BNCT) is a selective radiation treatment for tumors that preferentially accumulate drugs carrying the stable boron isotope, 10B. BNCT has been evaluated clinically as an alternative to conventional radiation therapy for the treatment of brain tumors, and more recently, recurrent advanced head and neck cancer. Here we investigated the effect of BNCT on prostate cancer (PCa) using an in vivo mouse xenograft model that we have developed.Materials and MethodsMice bearing the xenotransplanted androgen-independent human PCa cell line, PC3, were divided into four groups: Group 1: untreated controls; Group 2: Boronophenylalanine (BPA); Group 3: neutron; Group 4: BPA-mediated BNCT. We compared xenograft growth among these groups, and the body weight and any motility disturbance were recorded. Immunohistochemical (IHC) studies of the proliferation marker, Ki-67, and TUNEL staining were performed 9 weeks after treatment.ResultsThe in vivo studies demonstrated that BPA-mediated BNCT significantly delayed tumor growth in comparison with the other groups, without any severe adverse events. There was a significant difference in the rate of freedom from gait abnormalities between the BPA-mediated BNCT group and the other groups. The IHC studies revealed that BNCT treatment significantly reduced the number of Ki-67-positive cells in comparison with the controls (mean±SD 6.9±1.5 vs 12.7±4.0, p<0.05), while there was no difference in the number of apoptotic cells, suggesting that BPA-mediated BNCT reduced PCa progression without affecting apoptosis at 9 weeks post-treatment.ConclusionsThis study has provided the first preclinical proof-of-principle data to indicate that BPA-mediated BNCT reduces the in vivo growth of PCa. Although further studies will be necessary, BNCT might be a novel potential treatment for PCa.

A new class of hybrid anticancer agents inspired by the synergistic effects of curcumin and genistein: Design, synthesis, and anti-proliferative evaluation.

Inspired by the synergistic effects of dietary natural products with different scaffolds on the inhibition of cancer cell proliferation, incorporation of central (1E,4E)-1,4-penta-dien-3-one linker (an optimal substitute for the central metabolically unstable diketone linker of curcumin), 1-alkyl-1H-imidazol-2-yl (a promising bioisostere of terminal aryl group in curcumin), and chromone (the common pharmacophore in genistein and quercetin) into one chemical entity resulted in ten new hybrid molecules, 3-((1E,4E)-5-(1-alkyl-1H-imidazol-2-yl)-3-oxopenta-1,4-dien-1-yl)-4H-chromen-4-ones. They were synthesized through a three-step transformation using acid-catalyzed aldol condensation as key step. The WST-1 cell proliferation assay showed that they have greater anti-proliferative potency than curcumin, quercetin, and genistein on both androgen-dependent and androgen-independent human prostate cancer cells.

Molecular cloning of canine co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) and investigation of its ability to suppress androgen receptor signalling in androgen-independent prostate cancer

Although the morbidity of canine prostate cancer is low, the majority of cases present with resistance to androgen therapy and poor clinical outcomes. These pathological conditions are similar to the signs of the terminal stage of human androgen-independent prostate cancer. The co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) is known to be overexpressed in human androgen-independent prostate cancer. However, there is little information about the structure and function of canine SGTA. In this study, canine SGTA was cloned and analysed for its ability to suppress androgen receptor signalling.The full-length open reading frame (ORF) of the canine SGTA gene was amplified by RT-PCR using primers designed from canine-expressed sequence tags that were homologous to human SGTA. The canine SGTA ORF has high homology with the corresponding human (89%) and mouse (81%) sequences. SGTA dimerisation region and tetratricopeptide repeat (TPR) domains are conserved across the three species. The ability of canine SGTA to undergo homodimerisation was demonstrated by a mammalian two-hybrid system and a pull-down assay. The negative impact of canine SGTA on androgen receptor (AR) signalling was demonstrated using a reporter assay in androgen-independent human prostate cancer cell lines. Pathological analysis showed overexpression of SGTA in canine prostate cancer, but not in hyperplasia. A reporter assay in prostate cells demonstrated suppression of AR signalling by canine SGTA. Altogether, these results suggest that canine SGTA may play an important role in the acquisition of androgen independence by canine prostate cancer cells.

Immature Colon Carcinoma Transcript 1 Is Essential for Prostate Cancer Cell Viability and Proliferation.

Prostate cancer is the second leading cause of cancer-related death among men in the United States. More recently, immature colon carcinoma transcript 1 (ICT1) has been reported to be overexpressed in various kinds of cancer cells. However, the role of ICT1 in human prostate cancer has not yet been determined. The authors selected two ICT1-specific short hairpin RNA (shRNA) sequences to block its endogenous expression in human androgen-independent prostate cancer cell lines DU145 and PC-3. Decreased ICT1 expression by either specific shRNA significantly inhibited cell viability and proliferation. Moreover, compared to controls, ICT1-silenced cells were more inclined to redistribute in the G2/M phase, leading to cell cycle arrest. Flow cytometry and Annexin V-APC/7-AAD double staining confirmed that knockdown of ICT1 increased late apoptotic cells. Furthermore, they found that ICT1 knockdown restricting G2-M transition may be partly through suppression of CDK1 and Cyclin B1. Knockdown of ICT1 induced apoptosis through activation of poly ADP-ribose polymerase and caspase 3, upregulation of Bax expression, and downregulation of Bcl-2 expression in DU145 cells. In conclusion, this study highlights the crucial role of ICT1 in promoting prostate cancer cell proliferation in vitro. The depletion of ICT1 by lentivirus-mediated shRNA or small molecular inhibitor may provide a novel therapeutic approach for the treatment of prostate cancer.

Anti-proliferative effect and induction of apoptosis in androgen-independent human prostate cancer cells by 1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one.

Curcumin has poor in vivo absorption and bioavailability, highlighting a need for new curcumin analogues with better characteristics in these aspects. The aim of this study is to determine the anti-cancer properties of four selected curcumin analogues, on the cytotoxicity, proliferative and apoptotic effects on androgen-independent human prostate cancer cells (PC-3 and DU 145). Initial cytotoxicity screening showed MS17 has the highest cell inhibitory effect, with EC50 values of 4.4 ± 0.3 and 4.1 ± 0.8 µM, followed by MS13 (7.5 ± 0.1 and 7.4 ± 2.6 µM), MS49 (14.5 ± 1.2 and 12.3 ± 2.3 µM) and MS40E (28.0 ± 7.8 and 30.3 ± 1.9 µM) for PC-3 and DU 145 cells, respectively. Time-dependent analysis also revealed that MS13 and MS17 displayed a greater anti-proliferative effect than the other compounds. MS17 was chosen based on the high selectivity index value for further analysis on the morphological and biochemical hallmarks of apoptosis. Fluorescence microscopy analysis revealed apoptotic changes in both treated prostate cancer cells. Relative caspase-3 activity increased significantly at 48 h in PC-3 and 12 h in DU 145 cells. Highest enrichment of free nucleosomes was noted at 48 h after treatment with MS17. In conclusion, MS17 demonstrated anti-proliferative effect and induces apoptosis in a time and dose-dependent manner suggesting its potential for development as an anti-cancer agent for androgen-independent prostate cancer.

Corn silk maysin induces apoptotic cell death in PC-3 prostate cancer cells via mitochondria-dependent pathway

AimsDespite recent advances in prostate cancer diagnostics and therapeutics, the overall survival rate still remains low. This study was aimed to assess potential anti-cancer activity of maysin, a major flavonoid of corn silk (CS, Zea mays L.), in androgen-independent human prostate cancer cells (PC-3). Main methodsMaysin was isolated from CS of Kwangpyeongok, a Korean hybrid corn, via methanol extraction and preparative C18 reverse phase column chromatography. Maysin cytotoxicity was determined by either monitoring cell viability in various cancer cell lines by MTT assay or morphological changes. Apoptotic cell death was assessed by annexin V-FITC/PI double staining, depolarization of mitochondrial membrane potential (MMP), expression levels of Bcl-2 and pro-caspase-3 and by terminal transferase mediated dUTP-fluorescein nick end labeling (TUNEL) staining. Underlying mechanism in maysin-induced apoptosis of PC-3 cells was explored by evaluating its effects on Akt and ERK pathway. Key findingsMaysin dose-dependently reduced the PC-3 cell viability, with an 87% reduction at 200μg/ml. Maysin treatment significantly induced apoptotic cell death, DNA fragmentation, depolarization of MMP, and reduction in Bcl-2 and pro-caspase-3 expression levels. Maysin also significantly attenuated phosphorylation of Akt and ERK. A combined treatment with maysin and other known anti-cancer agents, including 5-FU, etoposide, cisplatin, or camptothecin, synergistically enhanced PC-3 cell death. SignificanceThese results suggested for the first time that maysin inhibits the PC-3 cancer cell growth via stimulation of mitochondria-dependent apoptotic cell death and may have a strong therapeutic potential for the treatment of either chemo-resistant or androgen-independent human prostate cancer.

Study of low-frequency ultrasound suppress invasion of human prostate cancer cell PC-3

Objective To study whether ultrasound combined with microbubbles induces suppress invasion in androgen-independent prostate cancer cells PC-3 and to identify the probable mechanism.Methods Ultrasound with a frequency of 21 kHz and intensity of 46 mW/cm2 in continuous wave mode was used.Ultrasound combined with microbubbles (200 μl SonoVue) was used to treat androgen-independent human prostate cancer PC-3 cells for 30 seconds.PC-3 cells were divided into three groups:control group,ultrasound group and ultrasound combined with microbubbles group.Twelve hours after treatment,cell growth curve we drawn,and transwell chamber model was used to do cell invasion experiments in vitro.Real-time PCR was used to measure the expression matrix metalloproteinase-2 and matrix metalloproteinase-9 messenger ribonucleic acid.Results Twelve hours after treatment,cell growth was not significant difference among three groups (P ＞ 0.05).Twelve hours after treatment,ultrasound combined with microbubbles could significantly inhibit the invasion of human prostate cancer cells (P ＜0.05).Treatment with ultrasound combined with microbubbles down-regulated the expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 messenger ribonucleic acid.Conclusions Ultrasound combined with microbubbles inhibited the invasion in human androgen-independent prostate cancer cells through down-regulation of matrix metalloproteinase-2 and matrix metalloproteinase-9. Key words: Sonication; Microbubbles ; Prostate neoplasms ; Neoplasm invasiveness

Effect of lycopene isolated from Chlorella marina on proliferation and apoptosis in human prostate cancer cell line PC-3.

Even though the role of lycopene from tomato (trans form) in controlling prostate cancer was reported, lycopene (cis and trans 60:40) isolated from green algae Chlorella marina was not reported so far. The present study aimed to assess the anti-proliferative and apoptotic effect of lycopene from a new source and to compare the activity with available trans lycopene by using androgen-independent human prostate cancer cell lines. Exposure of PC-3 and DU-145 cell lines to algal lycopene (AL) at a dose of 20 and 50 μM significantly inhibited the growth and colony formation, and the percentage of inhibition was higher than tomatal lycopene (TL)-treated groups. The stability of AL in cell culture medium was high, when compared to TL under standard cell culture conditions. The level of lycopene was not detected in PC-3 cell lines cultured in medium lacking lycopene. Staining cells with acridine orange and ethidium bromide, the PC-3 control cells showed largely non-fragmented intact nucleoid. Stronger apoptosis signal was induced with higher concentrations (50 μM) of algal lycopene. Increased DNA damage was observed in AL- and TL-treated cells which appear as comet during single-cell gel electrophoresis. Flow cytometry results revealed that AL caused PC-3 cells to accumulate in the G0/G1 phase and to undergo apoptosis. The effect was higher in AL groups than TL-treated groups. Algal lycopene showed very significant anti-proliferative and apoptotic effect in human prostate cancer cell lines. Therefore, algal lycopene from C.marina would be recommended for the treatment of prostate cancer.

Growth hormone-releasing hormone antagonists abolish the transactivation of human epidermal growth factor receptors in advanced prostate cancer models.

Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in a variety of cellular phenotypes related with tumorigenesis process. Human epidermal growth factor receptor family members (HER) such as EGFR and HER2 are involved in mitogenic signaling pathways implicated in the progression of prostate cancer. We analyzed the cross-talk between GHRH and EGF receptors in prostate cancer. The effects of GHRH in HER signaling were evaluated on human androgen-independent PC3 prostate cancer cells in vitro and GHRH antagonist in vitro and in nude mice xenografts of PC3 prostate cancer. Time-course studies indicated that GHRH had a stimulatory activity on both the expression of EGFR and HER2. GHRH analogues, JMR-132 and JV-1-38, endowed with antagonistic activity for GHRH receptors, abrogated the response to GHRH in PC3 cells. GHRH stimulated a rapid ligand-independent activation of EGFR and HER2 involving at least cAMP/PKA and Src family signaling pathways. GHRH also stimulated a slow ligand-dependent activation of EGFR and HER2 involving an extracellular pathway with an important role for ADAM. Preliminary results also revealed an increase of mRNA for GHRH and GHRH receptor induced by EGF. The inhibition of tumor growth, in vivo, was associated with a substantial reduction in the expression of mRNA and protein levels of EGFR and HER2 in the tumors. GHRH antagonist JV-1-38, significantly decreased the phosphorylated Src levels. The cross-talk between HER and GHRH-R may be impeded by combining drugs acting upon GHRH receptors and HER family members in human advanced prostate cancer.

Novel Pharmacologic Targeting of Tight Junctions and Focal Adhesions in Prostate Cancer Cells

Cancer cell resistance to anoikis driven by aberrant signaling sustained by the tumor microenvironment confers high invasive potential and therapeutic resistance. We recently generated a novel lead quinazoline-based Doxazosin® derivative, DZ-50, which impairs tumor growth and metastasis via anoikis. Genome-wide analysis in the human prostate cancer cell line DU-145 identified primary downregulated targets of DZ-50, including genes involved in focal adhesion integrity (fibronectin, integrin-α6 and talin), tight junction formation (claudin-11) as well as insulin growth factor binding protein 3 (IGFBP-3) and the angiogenesis modulator thrombospondin 1 (TSP-1). Confocal microscopy demonstrated structural disruption of both focal adhesions and tight junctions by the downregulation of these gene targets, resulting in decreased cell survival, migration and adhesion to extracellular matrix (ECM) components in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. Stabilization of cell-ECM interactions by overexpression of talin-1 and/or exposing cells to a fibronectin-rich environment mitigated the effect of DZ-50. Loss of expression of the intracellular focal adhesion signaling effectors talin-1 and integrin linked kinase (ILK) sensitized human prostate cancer to anoikis. Our findings suggest that DZ-50 exerts its antitumor effect by targeting the key functional intercellular interactions, focal adhesions and tight junctions, supporting the therapeutic significance of this agent for the treatment of advanced prostate cancer.

Design, synthesis, and evaluation of novel heteroaromatic analogs of curcumin as anti-cancer agents

To improve the potential of curcumin to treat advanced hormone-refractory prostate cancer, three series (A–C) of heteroaromatic analogs (thirty two compounds) with different monoketone linkers have been synthesized and evaluated for cytotoxicity against two human androgen-independent prostate cancer cell lines (PC-3 and DU-145). Among them, thirty analogs are more potent than curcumin against PC-3 cells, and twenty one analogs are more cytotoxic towards DU-145 cells relative to curcumin. The most potent compounds (44, 45, 51, and 52) also showed impressive cytotoxicity against three other metastatic cancer cell lines (MDA-MB-231, HeLa, and A549), with IC50 values ranging from 50 nM to 390 nM. All four most potent analogs exhibited no apparent cytotoxicity towards the MCF-10A normal mammary epithelial cells. Taken together, selective enhancement of cell death in prostate cancer cell lines and other aggressive cancer cell lines suggests that nitrogen-containing heteroaromatic rings are promising bioisosteres of the substituted phenyl ring in curcumin.

The study on the effects of apigenin on the proliferation in human androgen-independent prostate cancer cell PC-3 and the role Caspase-3 involved in

Objective To observe the effect of apigenin (API) on the proliferation of human androgen-independent prostate cancer cell line PC-3 and molecular mechanisms.Methods Clutures of PC-3 cell were exposured to API at the different concentrationa of 0,10,20 and 40 μmol/L respectively.The effect of API on growth inhibition were determinated with morphometry,cell counting kit-8 (CCK-8) assay.The expression of Caspase-3 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR).Results CCK-8 assay showed that the absorbance were 1.48 ±0.04,0.81 ±0.04,0.69 ± 0.03 and 0.33 ± 0.03 respectively when 0,10,20,40 μmol/L API treated PC-3 cells for 48 h,compared to the control group,10,20,40 μmol/L groups significantly inhibited the proliferation on PC-3 cells,the inhibition rates were 44.7％,53.5％,77.4％ (P ＜ 0.01).The Caspase-3 mRNA expression of 0,10,20,40 μmol/L group were 0.230 ± 0.036,0.410 ± 0.080,0.480 ± 0.100,0.730 ± 0.150,there was statistically significant differences between experimental and control groups (P ＜ 0.01).Conclusion The growth inhibition could be taken by API in PC-3 in a dose-dependent manner.The mechanism was that high expression level of Caspase-3 was caused by API,which led to the apoptosis of PC-3. Key words: Apigenin; Androgen-independent prostate cancer; Apoptosis; Caspase-3

Ellagic Acid Inhibits Migration and Invasion by Prostate Cancer Cell Lines

Polyphenolic compounds from pomegranate fruit extracts (PFEs) have been reported to possess antiproliferative, pro-apoptotic, anti-inflammatory and anti-invasion effects in prostate and other cancers. However, the mechanisms responsible for the inhibition of cancer invasion remain to be clarified. In the present study, we investigated anti-invasive effects of ellagic acid (EA) in androgen-independent human (PC-3) and rat (PLS10) prostate cancer cell lines in vitro. The results indicated that non-toxic concentrations of EA significantly inhibited the motility and invasion of cells examined in migration and invasion assays. The EA treatment slightly decreased secretion of matrix metalloproteinase (MMP)-2 but not MMP-9 from both cell lines. We further found that EA significantly reduced proteolytic activity of collagenase/gelatinase secreted from the PLS-10 cell line. Collagenase IV activity was also concentration-dependently inhibited by EA. These results demonstrated that EA has an ability to inhibit invasive potential of prostate cancer cells through action on protease activity.

Abstract 2623: Snail 1 regulates invasion through uPA-uPAR signaling using the MAPK pathway in prostate cancer cells.

Abstract Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -β (TGF-β) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by urokinase (uPA) and its receptor (uPAR) activities. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including invasion. Androgen-dependent LNCaP and 22Rv1 and androgen-independent ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail1 which led to increased cell migration and invasion. Superarray analysis revealed an upregulation in uPAR expression in Snail-transfected cells as compared to Neo control. Next, the protein expression levels of Snail1, uPA, and uPAR were measured by Western Blot analysis. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. Also, uPAR was transiently silenced in ARCaP Snail-overexpressing cells using uPAR siRNA. Additionally, cells were treated with MAPK inhibitor, UO126, at set time points. The data suggests that overexpression of Snail1 increased uPA and uPAR protein levels, and uPA activity was elevated in conditioned media from LNCaP, 22Rv1 and ARCaP Snail-overexpressing cells, as compared to Neo controls. Additionally, the silencing of uPAR decreased cell invasion in ARCaP Snail-overexpressing cells. Finally, MAPK inhibitor, UO126, inhibited cell invasion and decreased uPA activity. Therefore, Snail-mediated cell invasion in human prostate cancer cells may occur via the regulation of the MAPK and uPA/ uPAR signaling pathway. Acknowledgements: These studies were supported by the: NIH/NCRR/RCMI grant G12RR03062 and NIH P20 grant 1P20MD002285. Citation Format: Diandra D. Randle, Valerie Marah. Snail 1 regulates invasion through uPA-uPAR signaling using the MAPK pathway in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2623. doi:10.1158/1538-7445.AM2013-2623

Characterization and sub-cellular localization of somatostatin receptors in DU-145 and PC-3 human androgen-independent prostate cancer cells: effect of mono- and bi-specific somatostatin analogs on cell growth

Characterization and sub-cellular localization of somatostatin receptors in DU-145 and PC-3 human androgen-independent prostate cancer cells: effect of mono- and bi-specific somatostatin analogs on cell growth

Effect of histone deacetylase and DNA methyltransferase inhibitors on the expression of the androgen receptor gene in androgen-independent prostate cancer cell lines

Androgen receptor(AR) expression in prostate cancer (CaP) cells varies due to the multiple changes including epigenetic modifications such as DNA methylation and histone deacetylation. DNA methyltransferase and histone deacetylase inhibitors are promising for the treatment of CaP. The aim of our study was to analyze the 5-aza-2'-deoxycytidine (Aza‑dC) and sodium butyrate (NaB) effects on CaP cells with modified AR gene expression. The androgen-independent human prostate cancer cell lines PC3 (lacking a functional AR) and DU145 (strongly limited expression due to methylations in the AR gene) were used. PCR of bisulfite-modified DNA and RT-PCR with bisulfite-sequencing were used for AR gene analysis of DU145 and PC3 cells following their treatment with Aza-dC and/or NaB. Re-acetylated histones around the AR gene were detected by conventional PCR of immunoprecipitated DNA obtained from treated cells. In both cell lines without the AR expression, the combined treatment was followed with significant decrease of cell viability. The co-treatment of DU145 cells caused site-specific demethylation in the AR promoter region followed by gene re-expression and increased acetylation in histones H3 and H4. The co-treatment with Aza-dC and NaB was the most effective in demethylation and re-expression of the AR gene. In the AR gene promoter, the location and density of deme-thylated CpGs indicated the existence of distinct promoter hot spot that could be a target of AR gene inactivation therapy of CaP patients during androgen deprivation.

Experimental therapy of PC-3 and DU-145 human androgen-independent prostate cancers with targeted cytotoxic analog of somatostatin AN-162.

236 Background: Management of castration-resistant prostate cancer (CRPC) is challenging because of limitations in efficacy of current therapies. Somatostatin receptors are expressed in human CRPC. Here we tested targeted somatostatin AN-162 analog consisting of doxorubicin (DOX) conjugated to octapeptide RC-121, acting as a carrier, in human androgen-independent prostate cancer cell lines in vitro and in vivo. Methods: Expression of mRNA for the five subtypes of the somatostatin receptor in PC-3 and DU-145 human prostate cancer cell lines was evaluated by RT-PCR. Somatostatin receptor binding was measured with radioligand assays. The effect of AN-162 and DOX on the viability of PC-3 and DU-145 cells was assessed by MTS assay. Nude mice bearing PC-3 and DU-145 tumors were randomized to 5 groups (control, AN-162, DOX, somatostatin analog RC-160 as a control, and DOX + RC-160). Treatment consisted i.v. injections of AN-162, DOX, RC-160, DOX + RC-160, or vehicle once a week for 4 weeks. Tumor volume was measured every week; the study lasted 28 days. The doses of AN-162 were equivalent to 1.45 mg/kg DOX (2.5 μmol/kg). Results: The PC-3 and DU-145 cell lines were positive for the five subtypes of the somatostatin receptor. AN-162 and DOX (0.10–10 µM) inhibited the proliferation of PC-3 and DU-145 prostate cancer cells in a dose-dependent manner. AN-162 exerted a stronger inhibition of proliferation than DOX alone, but in vitro the difference was not significant. In vivo, AN-162 significantly inhibited growth of both tumor models’ compared with the controls and the groups given equimolar doses of doxorubicin, RC-160, or doxorubicin unconjugated to RC-160. Conclusions: Our work demonstrates potent inhibitory effects of AN-162 on somatostatin receptor positive androgen-independent prostate cancers, which were greater than any of the components of AN-162. The mechanisms of action of targeted cytotoxic analog of somatostatin AN-162 in CRPC should be explored. Our findings suggest the possible use of AN-162 in patients with CRPC.

Expression of Heat Shock Protein 27 in Prostate Cancer Cell Lines According to the Extent of Malignancy and Doxazosin Treatment

PurposeHeat shock protein 27 (HSP27) is known as the material that plays a role in apoptosis control in tumor and cell protection including the immune response, drug tolerance, and so on. In this study, HSP27 expression according to the prostate cancer malignancy level was evaluated, and HSP27 expression was also analyzed after inducing apoptosis by doxazosin treatment of the prostate cancer cell lines.Materials and MethodsReverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining of the HSP27 was implemented by the culture of RWPE-1, LNCaP, androgen-independent human prostate cancer cells (PC-3), and TSU-Pr1 cell lines. Analysis was separately conducted in the control group, control vector group treated by dimethyl sulfoxide, and groups treated with 10 µM or 25 µM doxazosin. The expression of HSP27 in RT-PCR and immunofluorescence staining was observed and evaluated after conversion to numerical values.ResultsIn the RT-PCR results, depending on the cell type, LNCaP, TSU-Pr1 showed the highest HSP27 expression followed by PC-3, LNCaP and RWPE-1 in sequence. After doxazosin treatment, the expression detected by RT-PCR was stronger at a 25-µM doxazosin concentration compared to that at a 10-µM concentration, and the result was similar by immunofluorescence staining.ConclusionsHSP27 expression increased depending on the prostate cancer cell line. This meant that HSP27 expression was related to the prostate cancer malignancy level. Additionally, the higher the treatment concentration in PC-3 was, the higher the HSP27 expression was. This result showed that doxazosin induced apoptosis of prostate cancer.