Phosphorylation of mitogen-activated protein kinase is inhibited by calcitonin in DU145 prostate cancer cells

One of the causes of insensitivity to androgen ablation therapy in prostate cancer is thought to be attributable to elevated neuropeptides secreted by neuroendocrine cells in the tumor mass. Calcitonin (CT), one of these neuropeptides, is reported to be associated with the growth of prostate cancer. There is an increase in mitogen-activated protein (MAP) kinase activation as prostate cancer progresses to a more advanced and androgen-independent disease. We examined the effect of CT on signal transduction and the relation between CT and early-response genes in the human androgen-insensitive prostate cancer cell line, DU145. The basal phosphorylation level of extracellular signal-regulated kinase 1/2, which is a key kinase in the mediation of growth factor-induced mitogenesis in prostate cancer cells, was constitutively up-regulated. N-[2-(4-bromocinnamyl) aminoethyl]-5-isoquinoline-sulfonamide (H89), a specific inhibitor of protein kinase A, potentiated the effects of more increased phosphorylation of extracellular signal-regulated kinase 1/2. CT induced the inhibition of this MAP kinase phosphorylation, and this effect was completely abolished by pretreatment with H89. Our findings demonstrate that CT caused the inhibition of constitutive MAP kinase phosphorylation in a protein kinase A-dependent manner in DU145. The transient increase of c-fos expression was detected after CT treatment, whereas expression of c-jun RNA was down-regulated after CT treatment. These results suggest that CT may regulate early-response genes, c-fos and c-jun, via a MAP kinase cascade. In conclusion, these findings suggest that DU145 might be a useful model as a therapeutic approach of neuropeptides in androgen-independent prostatic carcinoma.     

Adipocyte-fatty acid binding protein induces apoptosis in DU145 prostate cancer cells

Adipocyte-fatty acid binding protein (A-FABP) is a 14-15 kDa cytoplasmic protein that binds unesterified fatty acids (FA). It is believed that A-FABP is present in normal cells and disappears in cancer cells. Prostate cancer DU145 cells lack expression of A-FABP. Here, we report that transfection of A-FABP blocked growth of DU145 cells suggesting its role as a tumor suppressor. A-FABP transfected- prostate cancer DU145 cells underwent apoptosis when induced to overexpress A-FABP using an ecdysone-controlled expression system. DU145 cell cultures in complete medium exhibited a maximum of approximately 28% of apoptotic cells after 96 h of exposure to an ecdysone analog, Ponasterone A. We found that the possible mechanisms leading to the observed apoptotic effect may be due, in part, to an overexpression of tumor necrosis factor-alpha (TNF-alpha) and a moderate downregulation of transforming growth factor-alpha (TGF-alpha) in DU145 cells overexpressing A-FABP. The epidermal growth factor receptor (EGFR)/phosphatidyl inositol 3-kinase (PI 3-kinase) signaling pathway was not altered in these cells, suggesting that A-FABP may cause apoptosis by inducing downregulation of essential autocrine growth factors and/or upregulation of pro-apoptotic ones.     

FK228阻断细胞生存信号通路诱导前列腺癌细胞凋亡

目的：研究组蛋白去乙酰化酶（HDAC）抑制剂FK228诱导前列腺癌细胞系DU145凋亡的作用机制。方法：MTT比色法测定FK228抑制DUl45细胞增殖及其对细胞的杀伤效应；瑞氏-姬姆萨染色观察细胞形态的变化；流式细胞术分析细胞周期的改变；蛋白印迹实验检测细胞内蛋白表达水平的变化。结果：FK228明显抑制DU145细胞体外增殖，并介导细胞死亡；12．5ng／ml FK228作用细胞48h后，细胞存活率降至63．7％；伴有细胞形态改变及细胞周期阻滞于G0／G1期；细胞内多种重要的激酶蛋白，包括EGFR、Her2、Raf-1、Src、Cdk4、Akt及凋亡抑制蛋白Survivin均发生了不同程度的降解，细胞内两条重要的生存信号通路Raf-MEK—ERK及P13K／Akt被阻断，细胞发生凋亡。结论：FK228可以通过清除细胞内重要信号蛋白、阻断细胞生存信号通路来诱导DU145细胞发生凋亡。     

Gonadotropin-releasing hormone induces apoptosis of prostate cancer cells: role of c-Jun NH2-terminal kinase, protein kinase B, and extracellular signal-regulated kinase pathways

A standard therapy used today for prostate cancer is androgen ablation by gonadotropin-releasing hormone analogs (GnRH-a). Although most patients respond to androgen ablation as an initial systemic therapy, nearly all cases will develop androgen resistance, the management of which is still a major challenge. Here, we report that GnRH-a can directly induce apoptosis of the androgen-independent prostate cancer-derived DU145 and PC3 cell lines. Using specific inhibitors, we found that the apoptotic effect of GnRH-a is mediated by c-Jun NH2-terminal kinase (JNK) and inhibited by the phosphatidylinositol 3'-kinase (PI3K)-protein kinase B (PKB) pathway. Indeed, in DU145 cells, GnRH-a activates the JNK cascade in a c-Src- and MLK3-dependent manner but does not involve protein kinase C and epidermal growth factor receptor. Concomitantly, GnRH-a reduces the activity of the PI3K-PKB pathway, which results in the dephosphorylation of PKB mainly in the nucleus. The reduction of PKB activity releases PKB-induced inhibition of MLK3 and thus further stimulates JNK activity and accelerates the apoptotic effect of GnRH-a. Interestingly, extracellular signal-regulated kinase is also activated by GnRH-a, and this occurs via a pathway that involves matrix metalloproteinases and epidermal growth factor receptor, but its activation does not affect JNK activation and the GnRH-a-induced apoptosis. Our results support a potential use of GnRH-a for the treatment of advanced prostate cancer and suggest that the outcome of this treatment can be amplified by using PI3K-PKB inhibitors.     

Bone morphogenetic protein 7 protects prostate cancer cells from stress-induced apoptosis via both Smad and c-Jun NH2-terminal kinase pathways

We reported earlier that exposure to exogenous bone morphogenetic protein 7 (BMP7) could strongly inhibit serum starvation-induced apoptosis to C4-2B cell line, a variant of the LNCaP human prostate cancer cell line with propensity for bone metastasis. Whereas serum starvation suppressed the expression of survivin, a member of the inhibitor of apoptosis protein family, its expression was sustained in the presence of BMP7. In this study, we present evidence that BMP7 exposure up-regulated survivin promoter activity, an effect that was associated with activation of Smad, and could be repressed by dominant-negative Smad5. Additionally, serum starvation-induced suppression of c-jun NH2-terminal kinase (JNK) activity in C4-2B cells could be mostly restored by BMP7, and a JNK inhibitor could counteract the antiapoptotic effect of BMP7, without a significant effect on the level of survivin expression. Thus, we identified JNK pathway as another signaling mode for the antiapoptotic function of BMP7. To test the effect of endogenous up-regulation of BMP7, we genetically modulated the C4-2B cell line to overexpress BMP7 protein. Not only was this altered cell line resistant to serum starvation-induced apoptosis but it also exhibited patterns of Smad activation, survivin up-regulation, and JNK activation similar to those of the parental C4-2B cells exposed to exogenous BMP7. Consistent with these in vitro findings of BMP7 action, we acquired correlative results of Smad activation, survivin expression, and JNK activation in the progression of prostate cancer in the conditional Pten deletion mouse model, in which we first obtained the evidence of BMP7 overexpression.     

环王巴明诱导DU145细胞凋亡的机制研究

目的:研究环王巴明诱导DU145细胞凋亡作用及其对Gli-1和Bcl-2 mRNA表达的影响,探讨环王巴明诱导DU145细胞凋亡的机制.方法:不同浓度环王巴明处理DU145细胞后,吖啶橙染色,荧光显微镜下观察细胞形态变化.流式细胞术观察细胞凋亡率变化,RT-PCR检测5、10μmol/L环王巴明作用72h后的实验组和对照组Gli-1、Bcl-2 mRNA表达水平差异.结果:当环王巴明浓度大于5μmol/L作用72h后,荧光显微镜下可见细胞核固缩或碎裂,核仁变形等典型的凋亡形态学改变.环王巴明5μmol/L组Gli-1、Bcl-2mRNA表达减弱,但同对照组差异无统计学意义(P>0.05),与空白及DMSO对照组相比10μmol/L环王巴明可以显著下调Gli-1、Bcl-2 mRNA表达(P<0.01).结论:环王巴明可诱导人前列腺癌DU145细胞株凋亡,其作用机制可能与下调Gli-1和Bcl-2mRNA表达,活化细胞凋亡的线粒体途径有关.     

环王巴明诱导DU145细胞凋亡的机制研究

目的：研究环王巴明诱导DU145细胞凋亡作用及其对Gli-1和Bcl-2 mRNA表达的影响,探讨环王巴明诱导DU145细胞凋亡的机制。方法：不同浓度环王巴明处理DU145细胞后,吖啶橙染色,荧光显微镜下观察细胞形态变化。流式细胞术观察细胞凋亡率变化,RT—PCR检测5、10μmol／L环王巴明作用72h后的实验组和对照组Gli-1、Bcl-2mRNA表达水平差异。结果：当环王巴明浓度大于5μmol／L作用72h后,荧光显微镜下可见细胞核固缩或碎裂,核仁变形等典型的凋亡形态学改变。环王巴明5μmol／L组Gli-1、Bcl-2 mRNA表达减弱,但同对照组差异无统计学意义（P〉0．05）,与空白及DMSO对照组相比10μmol／L环王巴明可以显著下调Gli—1、Bcl-2 mRNA表达（P〈0．01）。结论：环王巴明可诱导人前列腺癌DU145细胞株凋亡,其作用机制可能与下调Gli-1和Bcl-2 mRNA表达,活化细胞凋亡的线粒体途径有关。     

Activation of p38 mitogen-activated protein kinase is necessary for gemcitabine-induced cytotoxicity in human pancreatic cancer cells

BACKGROUND: Gemcitabine is a pyrimidine nucleoside analog that is clinically active against pancreatic cancer. We have recently demonstrated that p38 MAPK is specifically activated by gemcitabine and that pharmacological blockade of p38 MAPK signaling prevented gemcitabine-induced apoptosis in human pancreatic cancer cells. In this study, we further investigated the implication of p38 MAPK in the cytotoxic action of gemcitabine. MATERIALS AND METHODS: Cells expressing a dominant-negative mutant of p38 MAPK were generated. Clonogenic assays were used to assess the long-term effect on cancer cell viability in the human pancreatic cancer cells, PK1 and PCI43. The p38 MAPK activation level was assessed using an antibody specific to the phosphorylated form. RESULTS: Gemcitabine increased the activation level of p38 MAPK in a dose-dependent manner and induced apoptosis in the two tested human pancreatic cancer cell lines. The selective p38 MAPK inhibitors, SB203580 and SB202190, reduced gemcitabine-induced activation of p38 MAPK, prevented the gemcitabine-induced apoptosis and increased long-term clonogenic survival. Overexpression of a dominant-negative p38 mutant in cells resulted in the reduction of gemcitabine-induced p38 MAPK activation and apoptosis, and increases in clonogenic survival. CONCLUSION: These results strongly suggest that the activation of p38 MAPK signaling is necessary for gemcitabine-induced cell death in human pancreatic cancer cells. Based upon these results, we suggest that molecules of p38 MAPK signaling pathways should be listed as novel targets for gemcitabine-based therapy.     

Tyrphostin AG825 triggers p38 mitogen-activated protein kinase-dependent apoptosis in androgen-independent prostate cancer cells C4 and C4-2

Prostate cancer (PCa) progression is aided by abnormal autocrine growth factor loops. We screened for small cell-permeable inhibitors of receptor tyrosine kinases that could block their signaling and trigger cell death in PCa cell lines. We found that the human epidermal growth factor receptor (HER)-2/neu inhibitor tyrphostin AG825 is preferentially toxic to PCa cells that are phenotypically androgen independent. These effects were dose and time dependent in the human LNCaP, C4, and C4-2 cell line models of progression and correlated with the inhibition of HER-2/neu phosphoactivation and its down-regulation. In addition, we show that the inhibition of HER-2/neu signaling with AG825 triggers an imbalance between extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activation, which leads to p38-dependent apoptosis. Inhibition of HER-1 with Compound 56 had no effect. These findings suggest that the androgen-independent C4 and C4-2 cells can be killed by selectively inhibiting their HER-2/neu signaling pathway and provide insights into the mechanism of action of AG825 in PCa cells.     

p38 mitogen-activated protein kinase pathway is involved in protein kinase Calpha-regulated invasion in human hepatocellular carcinoma cells

Protein kinase Calpha (PKCalpha) has been suggested to play an important role in tumorigenesis, invasion, and metastasis. In this study, we investigated the signal pathways selectively activated by PKCalpha in human hepatocellular carcinoma (HCC) cells to determine the role of mitogen-activated protein kinases (MAPK) in PKCalpha-mediated HCC migration and invasion. A stable SK-Hep-1 cell clone (siPKCalpha-SK) expressing DNA-based small interfering RNA (siRNA) PKCalpha was established and was then characterized by cell growth, migration, and invasion. The expression of PKCalpha was decreased in siPKCalpha-SK, and cell growth, migration, and invasion were reduced. These changes were associated with the decrease in p38 MAPK phosphorylation level, but not in c-jun-NH(2)-kinase-1/2 (JNK-1/2) and extracellular signal-regulated kinase-1/2 (ERK-1/2). This phenomenon was confirmed in the SK-Hep-1 cells treated with antisense PKCalpha olignucleotide. The p38 MAPK inhibitor SB203580 or dominant negative p38 mutant plasmid (DN-p38) was used to evaluate the dependency of p38 MAPK in PKCalpha-regulated migration and invasion. Attenuation of cell migration and invasion was revealed in the SK-Hep-1 cells treated with the SB203580 or DN-p38, but not with ERK-1/2 inhibitor PD98059 or JNK-1/2 inhibitor SP600125. Overexpression of constitutively active MKK6 or PKCalpha may restore the inactivation of p38 and the attenuation of cell migration and invasion in siPKCalpha-SK. Similar findings were observed in the stable HA22T/VGH cell clone expressing siRNA PKCalpha. This study provides new insight into the role of p38 MAPK in PKCalpha-mediated malignant phenotypes, especially in PKCalpha-mediated cancer cell invasion, which may have valuable implications for developing new therapies for some PKCalpha-overexpressing cancers.     

Retinoid-related molecules induce cytochrome c release and apoptosis through activation of c-Jun NH(2)-terminal kinase/p38 mitogen-activated protein kinases.

Retinoid-related molecules have been described that induce apoptosis in a variety of cancer cell lines. Of particular interest is the apoptotic activity of the all-trans-retinoic acid receptor gamma-selective molecules MX2870-1 and MX3350-1. These compounds have been shown to be effective in vivo against lung cancer and could therefore serve as important leads for novel anticancer drugs. We analyzed the death signaling pathways activated by these molecules. We observed that apoptotic retinoid-related molecules (RRMs) cause the release of cytochrome c from the mitochondria and subsequent activation of caspases 9 and 3. This was preceded by a strong and sustained activation of c-Jun NH(2)-terminal kinase as well as p38 kinase, which was independent of caspase activity. Inhibition of p38 kinase activity by the specific inhibitor SB203580 did not affect the induction of apoptosis by MX2870-1. However, interference with the activation of c-Jun NH(2)-terminal kinase and p38 stress kinases by PD169316 completely blocked all signs of apoptosis, including caspase activity, DNA fragmentation, and phosphatidylserine externalization. PD169316 also prevented the cleavage of Bid and the release of cytochrome c induced by this class of RRMs. Furthermore, processing and activation of different caspases by MX2870-1 was completely inhibited by increasing concentrations of PD169316. Thus, the investigated RRMs induce a death pathway, which is independent of Fas ligand, that is also activated by UV radiation and other agents. Our findings open the possibility for the future use of this class of RRMs in combination therapies with other anticancer drugs.     

Phenethyl isothiocyanate (PEITC) promotes G2/M phase arrest via p53 expression and induces apoptosis through caspase- and mitochondria-dependent signaling pathways in human prostate cancer DU 145 cells

Phenethyl isothiocyanate (PEITC), one of many compounds found in cruciferous vegetables, has been reported as a potential anticancer agent. In earlier studies, PEITC was shown to inhibit cell growth and induction of apoptosis in many cancer cell lines. However, no report has shown whether PEITC can induce apoptosis in human prostate cancer cells. Herein, we aimed to determine whether PEITC has anticancer activity in DU 145 human prostate cancer cells. As a result, we found that PEITC induced a dose-dependent decrease in cell viability through induction of cell apoptosis and cell cycle arrest in the G(2)/M phase of DU 145 cells. PEITC induced morphological changes and DNA damage in DU 145 cells. The induction of G(2)/M phase arrest was mediated by the increase of p53 and WEE1 and it reduced the level of CDC25C protein. The induction of apoptosis was mediated by the activation of caspase-8-, caspase-9- and caspase-3-depedent pathways. Results also showed that PEITC caused mitochondrial dysfunction, increasing the release of cytochrome c and Endo G from mitochondria, and led cell apoptosis through a mitochondria-dependent signaling pathway. This study showed that PEITC might exhibit anticancer activity and become a potent agent for human prostate cancer cells in the future.     

Role of mitogen-activated protein kinases in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells

The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in apoptosis induction by phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived cancer chemopreventive agent, with DU145 and LNCaP human prostate cancer cells as a model. The MAPK family of serine/threonine kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c-jun N-terminal kinase1/2/3 (JNK1/2/3), and p38 MAPK play an important role in cell proliferation and apoptosis in response to different stimuli. Exposure of DU145 and LNCaP cells to growth suppressive concentrations of PEITC resulted in activation of ERK1/2 and JNKs, but not p38 MAPK, in both cell lines. In DU145 cells, the apoptosis induction by PEITC was statistically significantly attenuated by pharmacological inhibition of JNKs with SP600125. Adenovirus-mediated overexpression of Flag-tagged JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), an inhibitor of JNK, also inhibited PEITC-induced apoptosis in DU145 cells. On the other hand, inhibition of ERK1/2 activation with MEK1 inhibitor PD98059 failed to offer protection against PEITC-induced apoptosis in DU145 cells. In LNCaP cells, the PEITC-induced cell death was not affected by either pretreatment with PD98059 or SP600125 or overexpression of JBD of JIP-1. These results indicate that involvement of MAPKs in apoptosis induction by PEITC in human prostate cancer cells is cell line-specific.     

Roles of p38- and c-jun NH2-terminal kinase-mediated pathways in 2-methoxyestradiol-induced p53 induction and apoptosis

As 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, has been established to cause apoptosis of prostate cancer cells, the downstream effectors of the signaling remain unclear. In the current study, we investigated molecular mechanisms by which 2-ME induces apoptosis in human prostate cancer cell line, LNCaP. It was found that 2-ME mediates apoptosis through p53 induction. Nuclear factor kappaB (NFkappaB) was activated by 2-ME and closely regulated by the mitogen-activated protein kinase, p38. Inhibition of p38 or NFkappaB resulted in suppression of p53 induction and apoptosis. Moreover, we demonstrated that 2-ME activates the c-jun NH2-terminal kinase (JNK)/activation protein (AP)-1 pathway. Interestingly, inhibition of JNK strongly reduced Bcl-2 phosphorylation by 2-ME as well as p53 induction, and almost completely suppressed 2-ME-induced apoptosis. Androgen stimulation with dihydrotestosterone, a major endogenous metabolite of testosterone, also significantly inhibited p38/NFkappaB and JNK/AP-1 activation and apoptosis. The results suggest that not only p53 induction through p38/JNK-dependent NFkappaB/AP-1 activation but also JNK-dependent Bcl-2 phosphorylation are required for 2-ME-induced apoptosis; moreover, inhibition of these pathways may be involved in androgen-mediated resistance to apoptosis.