PKA-induced F-actin rearrangement requires phosphorylation of Hsp27 by the MAPKAP kinase MK5

Mitogen-activated protein kinase (MAPK) pathways can play a role in F-actin dynamics. In particular, the p38 MAPK/MAPK-activated protein kinase 2 (MK2)/heat shock protein 27 (Hsp27) pathway is involved in F-actin alternations. Previously, we showed that MK5 is implicated in F-actin rearrangement induced by the cAMP/cAMP-dependent protein kinase pathway in PC12 cells, while others found Hsp27 to be a good in vitro MK5 substrate. Here we demonstrate that MK5 can specifically interact with Hsp27 in vivo and can induce phosphorylation at serine residues 78 and 82 in cells. siRNA-mediated depletion of Hsp27 protein levels, as well as overexpression of the non-phosphorylatable Hsp27-3A mutant prevented forskolin-induced F-actin reorganization. While ectopic expression of a constitutive active MK5 mutant was sufficient to induce F-actin rearrangement in PC12 cells, co-expression of Hsp27-3A could ablate this process. Our results imply that MK5 is involved in Hsp27-controlled F-actin dynamics in response to activation of the cAMP-dependent protein kinase pathway. These findings render the MK5/Hsp27 connection into a putative therapeutic target for conditions with aberrant Hsp27 phosphorylation such as metastasis, cardiovascular diseases, muscle atrophy, autoimmune skin disease and neuropathology.     

Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3

Prostate carcinoma is one of the most common malignant tumors and has become a more common cancer in men. Previous studies demonstrated that evodiamine (EVO) exhibited anti-tumor activities on several cancers, but its effects on androgen-independent prostate cancer are unclear. In the present study, the action mechanisms of EVO on the growth of androgen-independent prostate cancer cells (DU145 and PC3 cells) were explored. EVO dramatically inhibited the growth and elevated cytotoxicity of DU145 and PC3 cells. The flow cytometric analysis of EVO-treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest was accompanied by elevated Cdc2 kinase activity, an increase in expression of cyclin B1 and phosphorylated Cdc2 (Thr 161), and a decrease in expression of phosphorylated Cdc2 (Tyr 15), Myt-1, and interphase Cdc25C. TUNEL examination showed that EVO-induced apoptosis was observed at 72 h. EVO elevated the activities of caspase 3, 8, and 9 in DU145 cells, while in PC3 cells only the activities of caspase 3 and 9 were elevated. EVO also triggered the processing of caspase 3 and 9 in both DU145 and PC3 cells. We demonstrate that roscovitine treatment result in the reversion of G2/M arrest in response to EVO in both DU145 and PC3. However, inhibitory effect of roscovitine on EVO-induced apoptosis could only be observed in DU145 rather than PC3. In DU145, G2/M arrest might be a signal for initiation of EVO-triggered apoptosis. Whereas EVO-triggered PC3 apoptosis might be independent of G2/M arrest. These results suggested that EVO inhibited the growth of prostate cancer cell lines, DU145 and PC3, through an accumulation at G2/M phase and an induction of apoptosis.     

Selective amino acid restriction targets mitochondria to induce apoptosis of androgen-independent prostate cancer cells

Relative specific amino acid dependency is one of the metabolic abnormalities of cancer cells, and restriction of specific amino acids induces apoptosis of prostate cancer cells. This study shows that restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), or methionine (Met), modulates Raf and Akt survival pathways and affects the function of mitochondria in DU145 and PC3, in vitro. These three restrictions inhibit energy production (ATP synthesis) and induce generation of reactive oxygen species (ROS). Restriction of Tyr/Phe or Met in DU145 and Met in PC3 reduces mitochondrial membrane potential (DeltaPsim) and induces caspase-dependent and -independent apoptosis. In DU145, Tyr/Phe or Met restriction reduces activity of Akt, mitochondrial distribution of phosphorylated Raf and apoptosis inducing factor (AIF), and increases mitochondrial distribution of Bak. Mitochondrial Bcl-XL is increased in Tyr/Phe-restricted but decreased in Met-restricted cells. Under Tyr/Phe or Met restriction, reduced mitochondrial Raf does not inactivate the pro-apoptotic function of Bak. Tyr/Phe restriction also inhibits Bcl-2 and Met restriction inhibits Bcl-XL in mitochondria. These comprehensive actions damage the integrity of the mitochondria and induce apoptosis of DU145. In PC3, apoptosis induced by Met restriction was not associated with alterations in intracellular distribution of Raf, Bcl-2 family proteins, or AIF. All of the amino acid restrictions inhibited Akt activity in this cell line. We conclude that specific amino acid restriction differentially interferes with homeostasis/balance between the Raf and Akt survival pathways and with the interaction of Raf and Bcl-2 family proteins in mitochondria to induce apoptosis of DU145 and PC3 cells.     

LEDGF binds to heat shock and stress-related element to activate the expression of stress-related genes

We have investigated the mechanism by which LEDGF protects cells against environmental stress. Our earlier report showed that a low level of LEDGF was present in the nucleus of most cell types and significant elevation of LEDGF level was induced by heat and oxidative stress. The cells overexpressing LEDGF-activated expression of heat shock proteins and enhanced survival of many cell types. Here we show that LEDGF binds to heat shock element (HSE) and stress-related regulatory element (STRE) to activate the expression of stress-related genes (Hsp27 and alphaB-crystallin). Apparently, HSE and STRE are present in promoters of many stress-related genes. Elevation of many stress-related proteins (STRPs) induced by LEDGF may protect cells against environmental stress. In yeast, it has been demonstrated that a single stress can activate the expression of multiple STRPs. This is known as "cross-protection," and now similar mechanism has been found in mammalian cells and LEDGF plays a vital role in it.     

Emodin-Induced Necroptosis in Prostate Cancer Cells via the Mitochondrial Fission HSP90/MLKL/PGAM Pathway

Currently, prostate cancer is one of the major malignant tumors in males. Recurrence and metastasis are the main obstacles that prevent the effective treatment of prostate cancer. In the present study, we aimed to evaluate emodin (EG) against human prostate cancer PC3 and DU145 cells. Our study showed that EG significantly decreased the cell viability of PC3 and DU145 cells and strikingly induced non-apoptotic cell death via necroptosis that was visualized through colony formation assay, Hoechst 33258 staining, and TEM analysis. Furthermore, RNA-sequencing and KEGG functional enrichment analysis revealed that the necroptosis-related pathway was activated upon EG treatment in PC3 cells. mRNA and protein expression of necroptosis markers were analyzed by qPCR and immunoblotting, which implied that EG-induced cell necroptosis via enhancing the expression of MLKL and HSP90AA1 activating PGAM pathway which is considered as a key mediator of mitochondrial fission and leading to ROS generation in PC3 and DU145 cells. Thus, our findings suggested that EG is a new small molecule agonist that induced necroptosis in prostate cancer cells via the mitochondrial fission HSP90/MLKL/PGAM pathway.     

Focal adhesion kinase controls aggressive phenotype of androgen-independent prostate cancer

Overexpression of focal adhesion kinase (FAK) has been well correlated with tumor development and/or the maintenance of tumor phenotype. In addition, inappropriate activation of the extracellular regulated kinase (ERK) signaling pathway is common to many human cancers. In the present study, we investigated the interplay between FAK and ERK in androgen-independent prostate cancer cells (PC3 and DU145 cells). We observed that suppression of FAK expression using small interfering RNA-mediated knockdown decreased the clonogenic activity, whereas overexpression of FAK increased it. We also observed that detachment of PC3 and DU145 cells from their substrate induced tyrosine phosphorylation of FAK. ERK knockdown diminished FAK protein levels and tyrosine phosphorylation of FAK as well as FAK promoter-reporter activity. We also tested the effect of MEK inhibitors and small interfering RNA-mediated knockdown of ERK1 and/or ERK2 on cell proliferation, invasiveness, and growth in soft agar of PC3 and DU145 cells. Inhibition of ERK signaling grossly impaired clonogenicity as well as invasion through Matrigel. However, inhibition of ERK signaling resulted in only a modest inhibition of 3H-thymidine incorporation and no effect on overall viability of the cells or increased sensitivity to anoikis. Taken together, these data show, for the first time, a requirement for FAK in aggressive phenotype of prostate cancer cells; reveal interdependence of FAK and ERK1/2 for clonogenic and invasive activity of androgen-independent prostate cancer cells; suggest a role for ERK regulation of FAK in substrate-dependent survival; and show for the first time, in any cell type, the regulation of FAK expression by ERK signaling pathway.     

Combination effect of PectaSol and Doxorubicin on viability, cell cycle arrest and apoptosis in DU-145 and LNCaP prostate cancer cell lines

The effect of PectaSol on Dox (Doxorubicin) cytotoxicity in terms of apoptosis and cell cycle changes in PCa (prostate cancer) cell lines (DU‐145 and LNCaP) has been investigated. Combination of PectaSol and Dox resulted in a viability of 29.4 and 32.6% (P<0.001) in DU‐145 and LNCaP cells. The IC₅₀ values decreased 1.5‐fold and 1.3‐fold in the DU‐145 and LNCaP cells respectively. In the DU‐145 cells, combination of PectaSol and Dox resulted in a reduction in p27 gene and protein expression (P<0.001). In LNCaP cells, this combination increased p53, p27 and Bcl‐2 expression. Treatment with both drugs in DU‐145 cells led to an increase in sub‐G₁ arrest (54.6% compared with 12.2% in Dox). In LNCaP cells, combination of the drugs led to an increased in G₂/M arrest (61.7% compared with 53.6% in Dox). Based on these findings, progressive cytotoxicity effect of Dox and PectaSol together rapidly induce cell death in DU‐145 through apoptosis and in LNCaP cells through cell cycle arrest (G₂/M arrest).     

IQGAP2, A candidate tumour suppressor of prostate tumorigenesis

Loss of IQGAP2 contributes to the tumorigenesis of hepatocellular carcinoma and gastric cancer. However, whether IQGAP2 also suppresses prostate tumorigenesis remains unclear. We report here that IQGAP2 is a candidate tumour suppressor of prostate cancer (PC). Elevated IQGAP2 was detected in prostatic intraepithelial neoplasia (PIN), early stages of PCs (Gleason score ≤3), and androgen-dependent LNCaP PC cells. However, IQGAP2 was expressed at substantially reduced levels not only in prostate glands and non-tumorigenic BPH-1 prostate epithelial cells but also in advanced (Gleason score 4 or 5) and androgen-independent PCs. Furthermore, xenograft tumours that were derived from stem-like DU145 cells displayed advanced features and lower levels of IQGAP2 in comparison to xenograft tumours that were produced from non stem-like DU145 cells. Collectively, these results suggest that IQGAP2 functions in the surveillance of prostate tumorigenesis. Consistent with this concept, ectopic IQGAP2 reduced the proliferation of DU145, PC3, and 293T cells as well as the invasion ability of DU145 cells. While ectopic IQGAP2 up-regulated E-cadherin in DU145 and PC3 cells, knockdown of IQGAP2 reduced E-cadherin expression. In primary PC and DU145 cells-derived xenograft tumours, the majority of tumours with high levels of IQGAP2 were strongly-positive for E-cadherin. Therefore, IQGAP2 may suppress PC tumorigenesis, at least in part, by up-regulation of E-cadherin. Mechanistically, overexpression of IQGAP2 significantly reduced AKT activation in DU145 cells and inhibition of AKT activation upregulated E-cadherin, suggesting that IQGAP2 increases E-cadherin expression by inhibiting AKT activation. Taken together, we demonstrate here that IQGAP2 is a candidate tumour suppressor of PC.     

Mitostatin is down-regulated in human prostate cancer and suppresses the invasive phenotype of prostate cancer cells

MITOSTATIN, a novel putative tumor suppressor gene induced by decorin overexpression, is expressed in most normal human tissues but is markedly down-regulated in advanced stages of mammary and bladder carcinomas. Mitostatin negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27. In this study, we demonstrated that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells not only induced a significant reduction in cell growth, but also inhibited migration and invasion. Moreover, Mitostatin inhibited colony formation in soft-agar of PC3 and LNCaP cells as well as tumorigenicity of LNCaP cells in nude mice. Conversely, targeting endogenous Mitostatin by siRNA and anti-sense strategies in PC3 and DU145 prostate cancer cells enhanced the malignant phenotype in both cell lines. In agreement of these anti-oncogenic roles, we discovered that Mitostatin was absent in ∼35% (n = 124) of prostate tumor samples and its overall reduction was associated with advanced cancer stages. Collectively, our findings indicate that MITOSTATIN may acts as a tumor suppressor gene in prostate cancer and provide a novel cellular and molecular mechanism to be further exploited and deciphered in our understanding of prostate cancer progression.     

Activation of the RalGEF/Ral pathway promotes prostate cancer metastasis to bone

A hallmark of metastasis is organ specificity; however, little is known about the underlying signaling pathways responsible for the colonization and growth of tumor cells in target organs. Since tyrosine kinase receptor activation is frequently associated with prostate cancer progression, we have investigated the role of a common signaling intermediary, activated Ras, in prostate cancer metastasis. Three effector pathways downstream of Ras, Raf/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase, and Ral guanine nucleotide exchange factors (RalGEFs), were assayed for their ability to promote the metastasis of a tumorigenic, nonmetastatic human prostate cancer cell line, DU145. Oncogenic Ras promoted the metastasis of DU145 to multiple organs, including bone and brain. Activation of the Raf/ERK pathway stimulated metastatic colonization of the brain, while activation of the RalGEF pathway led to bone metastases, the most common organ site for prostate cancer metastasis. In addition, loss of RalA in the metastatic PC3 cell line inhibited bone metastasis but did not affect subcutaneous tumor growth. Loss of Ral appeared to suppress expansive growth of prostate cancer cells in bone, whereas homing and initial colonization were less affected. These data extend our understanding of the functional roles of the Ral pathway and begin to identify signaling pathways relevant for organ-specific metastasis.     

Nuclear factor erythroid 2-related factor-2 activity controls 4-hydroxynonenal metabolism and activity in prostate cancer cells

4-Hydroxynonenal (HNE) is an end product of lipoperoxidation with antiproliferative and proapoptotic properties in various tumors. Here we report a greater sensitivity to HNE in PC3 and LNCaP cells compared to DU145 cells. In contrast to PC3 and LNCaP cells, HNE-treated DU145 cells showed a smaller reduction in growth and did not undergo apoptosis. In DU145 cells, HNE did not induce ROS production and DNA damage and generated a lower amount of HNE-protein adducts. DU145 cells had a greater GSH and GST A4 content and GSH/GST-mediated HNE detoxification. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a regulator of the antioxidant response. Nrf2 protein content and nuclear accumulation were higher in DU145 cells compared to PC3 and LNCaP cells, whereas the expression of KEAP1, the main negative regulator of Nrf2 activity, was lower. Inhibition of Nrf2 expression with specific siRNA resulted in a reduction in GST A4 expression and GS-HNE formation, indicating that Nrf2 controls HNE metabolism. In addition, Nrf2 knockdown sensitized DU145 cells to HNE-mediated antiproliferative and proapoptotic activity. In conclusion, we demonstrated that increased Nrf2 activity resulted in a reduction in HNE sensitivity in prostate cancer cells, suggesting a potential mechanism of resistance to pro-oxidant therapy.     

Genistein Suppresses Prostate Cancer Growth through Inhibition of Oncogenic MicroRNA-151

Genistein has been shown to suppress the growth of several cancers through modulation of various pathways. However, the effects of genistein on the regulation of oncogenic microRNA-151 (miR-151) have not been reported. In this study, we investigated whether genistein could alter the expression of oncogenic miR-151 and its target genes that are involved in the progression and metastasis of prostate cancer (PCa). Real-time RT-PCR showed that the expression of miR-151 was higher in PC3 and DU145 cells compared with RWPE-1 cells. Treatment of PC3 and DU145 cells with 25 μM genistein down-regulated the expression of miR-151 compared with vehicle control. Inhibition of miR-151 in PCa cells by genistein significantly inhibited cell migration and invasion. In-silico analysis showed that several genes (CASZ1, IL1RAPL1, SOX17, N4BP1 and ARHGDIA) suggested to have tumor suppressive functions were target genes of miR-151. Luciferase reporter assays indicated that miR-151 directly binds to specific sites on the 3'UTR of target genes. Quantitative real-time PCR analysis showed that the mRNA expression levels of the five target genes in PC3 and DU145 were markedly changed with miR-151 mimics and inhibitor. Kaplan-Meier curves and log-rank tests revealed that high expression levels of miR-151 had an adverse effect on survival rate. This study suggests that genistein mediated suppression of oncogenic miRNAs can be an important dietary therapeutic strategy for the treatment of PCa.     

The expression of URGCP gene in prostate cancer cell lines: correlation with rapamycin

Molecular targets in prostate cancer are continually being explored, for which there are currently few therapeutic options. Rapamycin (RPM) is an antifungal macrolide antibiotic isolated from Streptomyces hygroscopicus which can inhibit the G1 to S transition. URGCP (upregulator of cell proliferation) is a novel gene located on chromosome 7p13. We aimed to investigate the role of URGCP gene expression changes in PC3, DU145, and LNCAP cell lines with/out RPM. Average cell viability and cytotoxic effect of rapamycin were investigated at 24?h intervals for three days by using Trypan blue dye exclusion test and XTT assay. Cytotoxic effects of rapamycin in DU145, PC3 and LNCAP cells were detected in time and dose dependent manner with the IC₅₀ doses within the range of 1–100?nM. As the results were evaluated, IC₅₀ doses in the DU145, PC3, and LNCaP cells were detected as 10, 25, and 50?nM, respectively. The mean relative ratios of URGCP gene expression in DU145, LNCAP and PC3 cells were found as ?1.48, 6.59 and ?13.00, respectively, when compared to rapamycin-free cells. The False Discovery Rate adjusted p value in DU145, LNCAP and PC3 were 1.25?×?10^?5, 2.20?×?10^?8 and 6.20?×?10^?9, respectively. When the URGCP gene expression level is compared between the dose and control group, we found that URGCP gene expression was significantly decreased in dose groups of DU145 and PC3 cells.     

西达本胺对人前列腺癌DU145及PC3细胞凋亡的影响

目的:研究西达本胺对前列腺癌DU145、PC3细胞生长抑制及凋亡调控的作用。方法:设置西达本胺浓度分别为4、8、16、32和64μmol/L的5个处理组和对照组(未加西达本胺),分别处理DU145、PC3细胞不同时间,采用MTT法检测细胞的增殖抑制情况,用倒置显微镜观察细胞形态,用流式细胞仪进行细胞凋亡分析,用免疫印迹法检测细胞内Bax、Bcl-2、caspase-3、caspase-9的表达水平。结果:与对照组比较,西达本胺处理组可使细胞明显变圆、体积缩小、脱壁细胞增多;MTT法检测显示,随着西达本胺浓度的增加和作用时间的延长,对DU145、PC3细胞的增殖抑制作用增强,并呈时间、剂量正相关(P<0.01);流式细胞术显示,对照组和16、64μmol/L西达本胺组细胞凋亡率分别为42.24%、50.23%;随着西达本胺浓度的增加,Bcl-2的表达呈下调趋势,Bax、caspase-3、caspase-9的表达呈上调趋势,呈剂量正相关。结论:西达本胺对前列腺癌DU145、PC3细胞生长具有明显的抑制作用,作用机制可能与Bax、Bcl-2、caspase-3及caspase-9凋亡信号通路有关。     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Elevated expression of ERK 2 in human tumor cells chronically treated with PD98059

We examined the effect of chronic exposure of tumor cells to a mitogen-activated protein kinase/extracellular signal-regulated kinases (ERK) kinase inhibitor, PD98059, on cell proliferation was investigated. Human renal carcinoma cells (ACHN) and prostatic carcinoma cells (DU145) were cultured in the presence of PD98059 for more than 4 weeks (denoted ACHN (PD) cells and DU145 (PD) cells, respectively) and proliferation and signal transduction pathways were examined. PD98059 significantly inhibited the proliferation of parental cells. However, PD98059 failed to inhibit proliferation of ACHN (PD) and DU145 (PD) cells significantly. Expression of ERK 1 and 2 was elevated in these cells. These phenotypes were reversible. Downregulation of ERK 2, but not ERK 1, by small interfering RNA significantly inhibited the proliferation of ACHN (PD) and DU145 (PD) cells. Taken together, chronic exposure of tumor cells to PD98059 induced elevated expression of ERK 2, which was associated with decreased sensitivity of cellular proliferation to PD98059.