Human Prostate Cancer Cell Lines DU145 Research Articles

Neuroendocrine Prostate Cancer Drivers SOX2 and BRN2 Confer Differential Responses to Imipridones ONC201, ONC206, and ONC212 in Prostate Cancer Cell Lines.

Prostate cancer (PCa) is the leading cause death from cancer in men worldwide. Approximately 30% of castrate-resistant PCa's become refractory to therapy due to neuroendocrine differentiation (NED) that is present in <1% of androgen-sensitive tumors. First-in-class imipridone ONC201/TIC10 has shown clinical activity against midline gliomas, neuroendocrine tumors and PCa. We explored the question of whether NED promotes sensitivity to imipridones ONC201 and ONC206 by inducible overexpression of SOX2 and BRN2, well-known neuroendocrine drivers, in human PCa cell lines DU145 or LNCaP. Slight protection from ONC201 or ONC206 with SOX2 and BRN2 overexpression was observed in the inducible LNCaP cells but not in the DU145 cells. At 2 months, there was an apparent increase in CLpP expression in LNCaP SOX2-overexpressing cells but this did not confer enhanced sensitivity to ONC201. DU145 SOX2-overexpressing cells had a significantly reduced ONC201 sensitivity than DU145 control cells. The results support the idea that treatment of castrate-resistant prostate cancer by imipridones may not be significantly impacted by neuroendocrine differentiation as a therapy-resistance mechanism. The results support further testing of imipridones across subtypes of androgen-sensitive and castrate-resistant prostate cancer.

LncRNA EMX2OS, Regulated by TCF12, Interacts with FUS to Regulate the Proliferation, Migration and Invasion of Prostate Cancer Cells Through the cGMP-PKG Signaling Pathway.

BackgroundLncRNA EMX2OS (EMX2 opposite strand/antisense RNA) is notably downregulated in prostate cancer (PCa) tissues and may be regarded as a potential molecular biomarker for diagnosis and prognosis. However, its exact role in regulating the development of PCa is obscure.MethodsThe EMX2OS expression was assessed in PCa tissues, paracancer tissues, PCa cells and normal prostate epithelial cells by qPCR. Gain- and loss-of-function experiments were performed to investigate the role of EMX2OS and FUS in cGMP-PKG (cyclic guanosine monophosphate-dependent protein kinase)-mediated proliferation, invasion, and migration in human PCa cell lines DU145 and PC3. Then, the interaction of transcription factor 12 (TCF12) with EMX2OS promoter was confirmed by using the dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. RNA binding protein immunoprecipitation and RNA pull-down assays were used to verify the interaction between EMX2OS and FUS protein. Finally, the role of EMX2OS and FUS in tumor growth in vivo was validated in a xenograft nude mouse model.ResultsTCF12 and EMX2OS were both downregulated in PCa tissues and cells, and they negatively regulated cell proliferation, migration and invasion, and activated cGMP-PKG pathway in DU145 and PC3 cells. TCF12 was a transcription factor of EMX2OS. TCF12 and EMX2OS overexpression both down-regulated cell proliferation, migration and invasion, and activated cGMP-PKG pathway in DU145 and PC3 cells. Furthermore, EMX2OS directly bound with FUS protein and had a synergy effect with FUS protein on cGMP-PKG-mediated cell functions, which could be suppressed by (D)-DT-2 (a cGMP-PKG inhibitor). In addition, the overexpression of FUS or EMX2OS individually markedly decreased the volume and weight of tumors in vivo, and co-overexpression of them further inhibited tumor growth.ConclusionEMX2OS, transcriptionally regulated by TCF12, played a synergy role with FUS protein in regulating the proliferation, migration and invasion of PCa cells by activating the cGMP-PKG pathway.

Anti-tumor activities of Panax quinquefolius saponins and potential biomarkers in prostate cancer

BackgroundProstate carcinoma is the second most common cancer among men worldwide. Developing new therapeutic approaches and diagnostic biomarkers for prostate cancer (PC) is a significant need. The Chinese herbal medicine Panax quinquefolius saponins (PQS) have been reported to show anti-tumor effects. We hypothesized that PQS exhibits anti-cancer activity in human PC cells and we aimed to search for novel biomarkers allowing early diagnosis of PC. MethodsWe used the human PC cell line DU145 and the prostate epithelial cell line PNT2 to perform cell viability assays, flow cytometric analysis of the cell cycle, and FACS-based apoptosis assays. Microarray-based gene expression analysis was used to display specific gene expression patterns and to search for novel biomarkers. Western blot and quantitative real-time PCR were performed to demonstrate the expression levels of multiple cancer-related genes. ResultsOur data showed that PQS inhibited the viability of DU145 cells and induced cell cycle arrest at the G1 phase. A significant decrease in DU145 cell invasion and migration were observed after 24 h treatment by PQS. PQS up-regulated the expression levels of p21, p53, TMEM79, ACOXL, ETV5, and SPINT1 while it down-regulated the expression levels of bcl2, STAT3, FANCD2, DRD2, and TMPRSS2. ConclusionPQS promoted cells apoptosis and inhibited the proliferation of DU145 cells, which suggests that PQS may be effective for treating PC. TMEM79 and ACOXL were expressed significantly higher in PNT2 than in DU145 cells and could be novel biomarker candidates for PC diagnosis.

Transcriptome analysis of differentially expressed genes and pathways associated with mitoxantrone treatment prostate cancer.

The global physiological function of specifically expressed genes of mitoxantrone (MTX)‐resistant prostate cancer (PCa) is unclear. In this study, gene expression pattern from microarray data was investigated for identifying differentially expressed genes (DEGs) in MTX‐resistant PCa xenografts. Human PCa cell lines DU145 and PC3 were cultured in vitro and xenografted into severe combined immunodeficiency (SCID) mice, treated with MTX intragastrically, three times a week until all mice relapsed. Gene expression profiles of the xenografts from castrated mice were performed with Affymetrix human whole genomic oligonucleotide microarray. The Cytoscape software was used to investigate the relationship between proteins and the signalling transduction network. A total of 355 overlapping genes were differentially expressed in MTX‐resistant DU145R and PC3R xenografts. Of these, 16 genes were selected to be validated by quantitative real‐time PCR (qRT‐PCR) in these xenografts, and further tested in a set of formalin‐fixed, paraffin‐embedded and optimal cutting temperature (OCT) clinical tumour samples. Functional and pathway enrichment analyses revealed that these DEGs were closely related to cellular activity, androgen synthesis, DNA damage and repair, also involved in the ERK/MAPK, PI3K/serine‐threonine protein kinase, also known as protein kinase B, PKB (AKT) and apoptosis signalling pathways. This exploratory analysis provides information about potential candidate genes and may bring new insights into the molecular cascade involvement in MTX‐resistant PCa.

Selection and characterization of DNA aptamer for metastatic prostate cancer recognition and tissue imaging

Prostate cancer (PCa) is the second leading cause of death and most prevalent cancer in men. The absence of curative options for castration-resistant metastatic prostate cancer and biomarkers able to discriminate between indolent and aggressive tumors contribute to these statistics. In this study, a DNA aptamer termed DML-7 was successfully selected against human PCa cell line DU145 by using the cell-based systematic evolution of ligands by exponential enrichment (SELEX) method. The selected aptamer DML-7 was found to internalize into target cells in a temperature-dependent manner and exhibit high binding affinity for target cells with dissociation constants in the nanomolar range. Binding analysis further revealed that DML-7 only binds to DU145 and PC-3 cells with metastatic potential, but not to LNCaP or 22Rv1 cells with low or nonmetastatic potential, demonstrating that DML-7 has excellent selectivity for the recognition of the metastatic PCa cells. Clinical tissue imaging further confirmed these results. Therefore, both high binding affinity and specificity to metastatic PCa cells and tissues afford DML-7 with the potential for development into a novel tool for diagnosis and targeted drug delivery against metastatic prostate cancer.

Se-methylselenocysteine suppresses the growth of prostate cancer cell DU145 through connexin 43-induced apoptosis

Se-methylselenocysteine (MSC), as a chemopreventive agent, shows antitumor effects in some cancer models, but its mechanism is still unclear. This study is to explore whether MSC induces apoptosis in prostate cancer (PCa) cells DU145 through connexin 43 (Cx43) activation. The experiment was performed in a PCa cell line model DU145 and using a series of biological assay methods to investigate the regulating pathway from MSC through Cx43 to downstream molecules, demonstrating an important role of Cx43 in PCa development and as a potential treatment target. The human PCa cell line DU145 was used as a model. The effects of MSC on Cx43 expression were examined by reverse transcription-polymerase chain reaction, western blot; effects on cell growth and proliferation were determined by WST-1 and colony formation assay; small interfering ribonucleic acid was used to evaluate the direct contribution of Cx43 to cancer cell apoptosis. Student's t-test was used to calculate the difference between the groups in SPSS software. Results showed that MSC inhibited the growth and colony formation of the DU145 cells; MSC induced cell apoptosis by increasing Cx43 expression at messenger ribonucleic acid and protein levels; MSC decreased B-cell lymphoma-2 (Bcl-2) and increased bad levels of DU145 cells. As a conclusion, MSC exerts pro-apoptosis effects through increasing Cx43 expression, which in turn down-regulates Bcl-2 and up-regulates bad expression.

1685P - Prostate Cancer Stem Cells Display Lowered Activity of the mTOR Pathway and Resistance against mTOR Inhibition Caused by Elevated Hypoxic Signaling

ABSTRACT Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSCs), were recently identified and characterized in Prostate cancer (PCa). We theorized that the PI3K/AKT pathway and mTOR, one of its main effectors, play a role in CSC maintenance. Moreover, hypoxia was known to be involved in CSC maintenance and to have a regulating effect on mTOR signaling. In our study, we therefore evaluated the effects of mTOR inhibitors on proliferation, assessed the activity of the mTOR pathway and measured the mitochondrial activity as well as levels of radical oxygen species (ROS) of human as well as murine CSCs in vitro. Further, we examined basal levels of mediators of hypoxic signaling such as HIF1α and its effectors.We isolated CSC like cells from the human PCa cell line DU145 and the murine PCa cell line TRAMPC1 using FACS according to their expression of the stem cell markers CD44, CD49f and Sca-1. We used sphere formation assays to confirm stem and progenitor cell properties. Next, we treated the CSC and non-CSC cell populations with mTOR inhibitors under normoxic and hypoxic conditions in order to determine their cell viability at time points up to 72h. Mitochondrial activity was measured using extracellular flux (XF) analysis and levels of ROS were obtained using ROS-specific fluorescent dyes and flow cytometry. Our results suggest that CSC like PCa cells are more resistant to mTOR inhibitors when compared to their non-CSC counterparts. Most interestingly, while the non-CSC population displays higher sensitivity to mTOR inhibitors under hypoxic conditions, the viability of CSCs remains unaffected. Immunoblotting uncovers generally lower mTOR activity in CSCs compared to non-CSCs. Hypoxia leads to a decrease in mTOR activity in both subpopulations, though this effect is stronger in CSC like subpopulations through higher HIF1α-mediated negative feedback in both cell lines studied. At the same time we do not observe alterations in signaling through AR or PI3K downstream effectors besides mTOR, such as AKT. We propose that prostate CSCs might be resistant against mTOR inhibition and that inhibitors targeting multiple kinases along the PI3K/AKT/mTOR axis would be more effective. Our findings could be helpful to assess the impact of mTOR targeted therapy in prostate cancer in light of ongoing clinical trials. Disclosure All authors have declared no conflicts of interest.

Abstract 3554: The fungal metabolite galiellalactone inhibits ALDH positive prostate cancer cells

Abstract Introduction: Tumor stem cells have shown resistance to conventional cancer therapy and may be a cause for recurrent cancer, making tumor stem cell targeted therapy attractive. The fungal metabolite galiellalactone is suggested to be a Stat3 specific inhibitor and we have previously shown that galiellalactone inhibits prostate cancer (PCa) cell growth, both in vitro and in vivo, in PCa cells expressing phosphorylated Stat3 (Hellsten et al, 2008). Aldehyde dehydrogenase (ALDH) has shown promise as a marker of cancer stem cells and in this study we investigated the ALDH activity in human PCa cell lines and the effects of galiellalactone on ALDH+ PCa cells. Methods: The human PCa cell lines DU145, LNCaP, and long-term interleukin-6 stimulated LNCaP (LNCaP-IL6) cells were used. Galiellalactone was prepared by synthesis (Johansson &amp; Sterner, 2002). PCa cells were subjected to Aldefluor assay followed by flow cytometry to detect ALDH+ subpopulations. WST-1 proliferation assay was performed to study the effect of galiellalactone on proliferation of ALDH+ and ALDH- cells sorted from LNCaP-IL6 cells. Results: In DU145 cells 2 ± 0.25% of the cells were ALDH+ and in LNCaP-IL6 cells, 2 ± 0.36% were ALDH+. No ALDH+ cells were detected in LNCaP cells. Treatment with 5, 10, 25 or 50 µM galiellalactone for 24h decreased the ALDH+ cell population in DU145 and LNCaP-IL6 cells in a dose dependent manner: 25 µM galiellalactone decreased the ALDH+ population in DU145 cells by 40% and by 75% in LNCaP-IL6 cells. In LNCaP-IL6 cells a dose dependent decrease in ALDH+ cells was observed after 72h (65% decrease at 25 µM galiellalactone). However, in DU145 cells no difference in the relative amount of ALDH+ cells was observed in galiellalactone treated DU145 cells compared to untreated control cells after 72h. The proliferation was decreased by galiellalactone in a dose dependent manner in all cell populations isolated from LNCaP-IL6 cells. The proliferation of ALDH+ cells isolated from LNCaP-IL6 cells was inhibited by 75%, ALDH- cells by 75% and unsorted LNCaP-IL6 cells by 80% after treatment with 25 µM galiellalactone for 48h. Conclusions: ALDH+ subpopulations, possibly representing tumor stem cell populations were detected in human PCa cell lines, and were clearly inhibited by galiellalactone. ALDH is associated with cell growth and drug resistance, and targeting ALDH+ PCa cells with galiellalactone may be a novel potent treatment against therapy resistant PCa. The effect and mechanism of galiellalactone treatment of ALDH+ PCa cells will be further investigated. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3554.

Inhibition of proliferation, invasion, and migration of prostate cancer cells by downregulating elongation factor-1alpha expression.

Overexpression of elongation factor-1alpha (EF-1alpha) has been reported to contribute to the development and progression of various cancers. However, its role in prostate cancer (PCa) still remains poorly understood. In the present study, we investigate the influence of EF-1alpha in Du145, a high-grade metastatic PCa cell line, and demonstrate that EF-1alpha plays an essential role in cellular properties associated with tumor progression, namely cell proliferation, invasion, and migration. In this study, EF-1alpha expression in human PCa cell line Du145 was reduced by RNA interference (RNAi) technology, and the proliferation, invasion, and migration of EF-1alpha-reduced Du145 cells were examined. We also detected an EF-1alpha expression pattern in 20 pairs of primary PCa samples and their corresponding normal tissues. Expression of EF-1alpha was detectable in four PCa cell lines (22RV1, LnCap, Du145, and PC3), indicating its possible role in pathogenesis of PCa. RNAi-mediated knockdown of EF-1alpha expression in Du145 cells, which expressed the highest level of EF-1alpha among four PCa cell lines, led to a decrease in proliferation. Similarly, suppression of EF-1alpha inhibited Du145 cell migration and invasion through a basement membrane substitute. Furthermore, we found that the normal prostate tissues showed a relatively low level of EF-1alpha expression, whereas PCa tissues demonstrated significantly higher expression levels of EF-1alpha (P < 0.001). Taken together, these findings support the hypothesis that EF-1alpha affects multiple processes involved in tumor progression, and identify EF-1alpha as a potential therapeutic target.

Galiellalactone is a novel therapeutic candidate against hormone‐refractory prostate cancer expressing activated Stat3

Signal transducer and activator of transcription 3 (Stat3) is constitutively active (phosphorylated) in several forms of cancer, including prostate cancer (PCa). Stat3 signaling may be an interesting target for cancer therapy since inhibition of this pathway mediates growth inhibition and apoptosis of these cells. In this study we investigated the in vitro and in vivo effects of the fungal metabolite galiellalactone, a direct inhibitor of Stat3, on PCa cells. The human PCa cell lines DU145, PC-3, and LNCaP were used. Nude mice with subcutaneous PCa cell xenografts were subjected to daily intraperitoneal injections of galiellalactone for 3 weeks. The effect of galiellalactone on the induction of apoptosis of cultured PCa cells was investigated by Western blot analysis, immunocytochemistry, and annexin V staining. Effects of galiellalactone on Stat3 signaling were investigated by a luciferase reporter gene assay. Expression of Stat3 associated proteins and mRNA was investigated by Western blot and real-time quantitative PCR analysis. Galiellalactone induced apoptosis of p-Stat3 positive PCa cells (androgen-insensitive DU145 and PC-3) but not in cells lacking p-Stat3 (androgen-sensitive LNCaP). Galiellalactone inhibited Stat3-mediated luciferase activity (IC(50) approximately 5 microM) and reduced the expression of Bcl-2, Bcl-x(L), c-myc, and cyclin D1. Furthermore, galiellalactone significantly suppressed DU145 xenograft growth in vivo (42% growth reduction; P<0.002) and reduced the relative mRNA expression of Bcl-x(L) and Mcl-1. Galiellalactone induced growth inhibition and apoptosis in androgen-insensitive PCa cells expressing p-Stat3. We suggest that galiellalactone is a potential anti-tumor lead against hormone-refractory PCa with constitutively active Stat3.

The vitamin D analogue BXL-628 inhibits growth factor-stimulated proliferation and invasion of DU145 prostate cancer cells

Suppression of the invasive phenotype is essential in developing new therapeutic tools to treat advanced prostate cancer (PC) indicating that androgen-independent prostate cancer (AI-PC) is characterized by increased metastatic potential. In the present study, we have investigated the effect of the nonhypercalcemic vitamin D analogue BXL-628 on proliferation and invasive properties of the human PC cell line DU145. In particular, the effect of the analogue was tested following stimulation with a potent growth factor, keratinocyte growth factor (KGF), which stimulates both proliferation and invasion of these cells. We have also evaluated the effect of the analogue on KGF stimulation of PI3K/AKT signaling pathway. Cell proliferation was determined by cell counting. Invasion through Matrigel was evaluated using Boyden chambers. PI3K activity was measured by immunokinase assay and AKT phosphorylation was evaluated by western blot analysis. Keratinocyte growth factor receptor (KGFR) autotransphosphorylation was evaluated by western blot after immunoprecipitation of the receptor. BXL-628 is able to inhibit both proliferation and invasion of DU145 cells in basal conditions and in response to KGF. Following stimulation with KGF, the inhibition is due to suppression of KGFR autotransphosphorylation and downstream PI3K/AKT activation, both achieved following a brief (5 min) incubation with the analogue. This effect on KGFR autophosphorylation was still present when cells were treated with the alpha-amanitin, an inhibitor of RNA transcription, indicating a rapid, nongenomic effect. Our results demonstrate that the vitamin D analogue BXL-628 is able to suppress KGF-induced proliferation and invasion of AI-PC cells in vitro, prospecting a possible use of the drug, which is currently in phase II clinical studies for benign prostatic hyperplasia, in the treatment of advanced prostate cancer.