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.     

Arecoline-mediated inhibition of AMP-activated protein kinase through reactive oxygen species is required for apoptosis induction

Arecoline is the major alkaloid of areca nut (AN) and known to induce reactive oxygen species (ROS) production and apoptosis. The metabolic sensor AMP-activated protein kinase (AMPK), activated by ROS, also regulates apoptosis. This study used several types of cells as the experimental model to analyze the roles of ROS and AMPK in arecoline-induced apoptosis. We found that arecoline dose-dependently increased intracellular ROS level, and two antioxidants, N-acetyl-L-cysteine (NAC) and glutathione, attenuated arecoline-induced apoptotic cell death. Interestingly, arecoline dose- and time-dependently inhibited rather than stimulated AMPK-Thr(172) phosphorylation, and both NAC and glutathione relieved this inhibition. The AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), also restored the phosphorylation level of AMPK-Thr(172) and attenuated apoptotic cell death under arecoline insult. In contrast, the AMPK inhibitor, compound C, and RNA interference of AMPK expression increased the cytotoxicity of arecoline. Collectively, these results suggest that arecoline may inhibit AMPK through intracellular ROS, responsible for the execution of apoptosis.     

The expression of hypoxia-inducible factor 1alpha is a favorable independent prognostic factor in renal cell carcinoma.

PURPOSE: Renal cell carcinoma (RCC) is the most common malignancy of the kidney composed of specific tumor types. The sporadic conventional RCCs are, in contrast to the other RCC types, characterized by a high rate of von Hippel-Lindau (VHL) mutations and hypermethylation. The majority of these tumors lack functional VHL protein (pVHL) that leads to increased hypoxia-inducible factor 1alpha (HIF-1alpha) expression. The pVHL is the physiologic regulator of the activity of HIF-1alpha by targeting it to the proteasome for degradation under normoxia. Both pVHL and HIF-1alpha target other genes that are important for cancer survival and proliferation. Expression of HIF-1alpha has been linked to poor prognosis in different malignancies, although few studies have been done on the relation between HIF-1alpha and clinical variables in RCC. EXPERIMENTAL DESIGN: HIF-1alpha protein expression was analyzed in tumor tissue from 92 patients with RCC. HIF-1alpha was quantified by Western blot relative to a positive control. RESULTS: The HIF-1alpha protein was expressed as two bands which strongly correlated (r = 0.906, P < 0.001); therefore, they were added and the sum evaluated against clinicopathologic variables. There was no association between HIF-1alpha and gender, stage, grade, tumor size, or vein invasion. Conventional RCCs had significantly higher HIF-1alpha expression compared with papillary and chromophobe RCCs and kidney cortex. In conventional RCC, HIF-1alpha was an independent prognostic factor. CONCLUSION: HIF-1alpha levels varied significantly between the different RCC types. In conventional RCC, HIF-1alpha was an independent prognostic factor. These data indicate that HIF-1alpha is involved in tumorogenesis and progression of RCC. Evaluation of other HIF target gene products and correlation to angiogenesis seems warranted.     

雄激素非依赖性前列腺癌DU145细胞中TRPM8和TRPA1的表达及活性研究

目的 探讨TRPM8和TRPA1在雄激素非依赖性前列腺癌DU145细胞中是否表达,以及薄荷醇是否通过作用于TRPM8通道来影响DU145细胞的生物学行为.方法 采用Nested RT-PCR实验、Western Blot实验和免疫组织化学法从不同水平(mRNA水平、蛋白水平)以及直观观察层面检测TRPM8和TRPA1在DU145细胞中的表达;采用Ca2+成像实验观察薄荷醇能否诱导DU145细胞内Ca2+浓度的变化;采用MTT法检测梯度浓度的薄荷醇对DU145细胞增殖能力的影响.结果 Nested RT-PCR实验、Western Blot实验和免疫组织化学法结果证明,在DU145细胞中检测到TRPM8表达,而未检测到TRPA1表达.Ca2+成像实验结果显示,在37℃环境下细胞荧光非常弱,加入薄荷醇后细胞内Ca2+荧光强度急剧升高.MTT法检测结果显示,与未经薄荷醇处理的对照组相比不同浓度(25、50、75、100μmol/L)薄荷醇处理组的细胞增殖率均下降(P﹤0.05).经BCTC预处理20 min,薄荷醇所致的细胞增殖抑制被部分恢复(P﹤0.05).结论 雄激素非依赖性前列腺癌DU145细胞中有大量的具有生物活性的TRPM8通道蛋白表达,而可同被薄荷醇激活的TRPA1却未被检测到表达.薄荷醇可激活DU145细胞中的TRPM8通道诱导Ca2+荧光强度急剧升高,抑制DU145细胞的增殖.     

Tyrosine protein kinase activity of human hyperplastic prostate and carcinoma cell lines PC3 and DU145

Using the substrate poly[Glu80Na,Tyr20] [poly(GT)] and the autoradiographic detection of alkali-resistant phosphoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tyrosine protein kinase (TPK) has been evidenced in human hyperplastic prostates (BPH) and the prostatic carcinoma cell lines PC3 and DU145. The enzyme was mainly found in the soluble fractions from hyperplastic tissues and in Triton extracts from the cell lines. However, its specific activity in tissues was 1.5- to 4.5-fold times higher in particulate than in soluble fractions and it was of the same order of magnitude as that of neoplastic cells. Under these conditions, no activity was detected in human seminal plasma and in sera from normal adult males or patients with BPH and/or prostatic carcinoma. On the other hand, some TPK activity was associated with human spermatozoa, with a specific activity 4- to 6-fold lower than in BPH tissue fractions and a total activity, per 10(6) cells, 5- to 20-fold lower than that in prostatic carcinoma cells. The activity of prostatic TPK was dependent upon the presence of the divalent cations Mn2+ or Mg2+ and it was completely abolished by heat denaturation. Angiotensin II, casein, and histone H2B were poor substrates compared to poly(GT). The TPK activities towards poly(GT) as well as endogenous proteins were not stimulated by epidermal growth factor and insulin or by dihydrotestosterone and estradiol. The autoradiography of alkali-resistant phosphoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several bands in both BPH tissues and neoplastic cells (molecular weight ranging from 17,000 to 200,000). Preliminary characterization of TPK by gel filtration on Sephacryl S-300 showed that the soluble enzyme from BPH tissues had a molecular weight of 50,000, while the particulate-associated TPK, when assayed on poly(GT), eluted with proteins of Mr 210,000. When these peak fractions were used for endogenous phosphorylation, several major alkali-resistant phosphoproteins in the range of Mr 40,000-60,000 were evidenced, together with a Mr 110,000 band phosphorylated by the particulate TPK of Mr 210,000. In similar conditions, the TPK solubilized from rat liver membranes and partially purified by gel filtration was associated with a Mr 170,000 alkali-resistant phosphoprotein. Thus, TPKs are expressed in BPH tissues and carcinoma cell lines. In BPH tissues, two forms of TPK are expressed and the predominant enzyme is soluble and of low molecular weight (Mr 40,000-60,000).     

trans-3,4,5'-Trihydroxystibene inhibits hypoxia-inducible factor 1alpha and vascular endothelial growth factor expression in human ovarian cancer cells.

trans-3,4,5'-Trihydroxystibene (resveratrol) is a natural product commonly found in the human diet and has been shown recently to have anticancer effects on various human cancer cells. However, the molecular basis for its anticancer action remains to be elucidated. In this study, we investigated the effect of resveratrol on hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) expression in human ovarian cancer cells A2780/CP70 and OVCAR-3. We found that although resveratrol did not affect HIF-1alpha mRNA levels, it did dramatically inhibit both basal-level and growth factor-induced HIF-1alpha protein expression in the cells. Resveratrol also greatly inhibited VEGF expression. Mechanistically, we demonstrated that resveratrol inhibited HIF-1alpha and VEGF expression through multiple mechanisms. First, resveratrol inhibited AKT and mitogen-activated protein kinase activation, which played a partial role in the down-regulation of HIF-1alpha expression. Second, resveratrol inhibited insulin-like growth factor 1-induced HIF-1alpha expression through the inhibition of protein translational regulators, including M(r) 70,000 ribosomal protein S6 kinase 1, S6 ribosomal protein, eukaryotic initiation factor 4E-binding protein 1, and eukaryotic initiation factor 4E. Finally, we showed that resveratrol substantially induced HIF-1alpha protein degradation through the proteasome pathway. Our data suggested that resveratrol may inhibit human ovarian cancer progression and angiogenesis by inhibiting HIF-1alpha and VEGF expression and thus provide a novel potential mechanism for the anticancer action of resveratrol.     

Brain-derived neurotrophic factor activation of TrkB induces vascular endothelial growth factor expression via hypoxia-inducible factor-1alpha in neuroblastoma cells

The extent of angiogenesis and/or vascular endothelial growth factor (VEGF) expression in neuroblastoma tumors correlates with metastases, N-myc amplification, and poor clinical outcome. Recently, we have shown that insulin-like growth factor-I and serum-derived growth factors stimulate VEGF expression in neuroblastoma cells via induction of hypoxia-inducible factor-1alpha (HIF-1alpha). Because another marker of poor prognosis in neuroblastoma tumors is high expression of brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor, TrkB, we sought to evaluate the involvement of BDNF and TrkB in the regulation of VEGF expression. VEGF mRNA levels in neuroblastoma cells cultured in serum-free media increased after 8 to 16 hours in BDNF. BDNF induced increases in VEGF and HIF-1alpha protein, whereas HIF-1beta levels were unaffected. BDNF induced a 2- to 4-fold increase in VEGF promoter activity, which could be abrogated if the hypoxia response element in the VEGF promoter was mutated. Transfection of HIF-1alpha small interfering RNA blocked BDNF-stimulated increases in VEGF promoter activity and VEGF protein expression. The BDNF-stimulated increases in HIF-1alpha and VEGF expression required TrkB tyrosine kinase activity and were completely blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) pathways. These data indicate that BDNF plays a role in regulating VEGF levels in neuroblastoma cells and that targeted therapies to BDNF/TrkB, PI3K, mTOR signal transduction pathways, and/or HIF-1alpha have the potential to inhibit VEGF expression and limit neuroblastoma tumor growth.     

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.     

Overexpression of human papillomavirus type 16 oncoproteins enhances hypoxia-inducible factor 1 alpha protein accumulation and vascular endothelial growth factor expression in human cervical carcinoma cells.

PURPOSE: Human papillomavirus (HPV)-16 oncoproteins, E6 and E7, are associated with enhanced tumor angiogenesis in human cervical cancers. The purpose of this study was (a) to investigate whether expression of HPV-16 E6 and E7 oncoproteins induces hypoxia-inducible factor 1 alpha (HIF-1 alpha) and vascular endothelial growth factor expression in cervical cancer cells; and (b) to assess the effect of resveratrol on 16 E6- and E7-induced HIF-1 alpha and VEGF gene expression. EXPERIMENTAL DESIGN: Human cervical cancer cell lines C-33A and HeLa were transiently cotransfected with pSG5-HPV-16 E6 or 16 E7 constructs along with HIF-1 alpha small interfering RNA (siRNA) or nonspecific siRNA. The expression of HIF-1 alpha/VEGF was measured using real-time PCR, Western blot analysis, or ELISA. The in vitro angiogenic activity induced by 16 E6- and E7-transfected cells was examined. The effect of resveratrol on oncoprotein-induced HIF-1 alpha/VEGF expression and in vitro angiogenesis was investigated. RESULTS: HPV-16 E6- and E7-transfected cervical cancer cells express increased HIF-1 alpha protein and VEGF expression. These stimulatory effects were abrogated by cotransfection with either HIF-1 alpha siRNA or treatment with resveratrol. Blocking extracellular signal-regulated kinase 1/2 (ERK 1/2) and phosphoinositide-3-kinase by PD98059 and LY294002, respectively, abolished 16 E6- and E7-induced HIF-1 alpha and VEGF expression. Functionally, we showed that HPV-16 E6- and E7-transfected cervical cancer cells stimulated in vitro capillary or tubule formation, and these angiogenic effects could be abolished either by cotransfection with HIF-1 alpha siRNA or by treatment with resveratrol. CONCLUSION: HPV-16 oncoproteins contribute to enhanced angiogenesis in cervical cancer cells via HIF-1 alpha-dependent VEGF expression. Resveratrol suppresses 16 E6- and E7-induced HIF-1 alpha-mediated angiogenic activity and, thus, is a promising chemotherapeutic agent for human cervical cancer.     

Transforming growth factor ß1 induces apoptosis by suppressing FLICE‐like inhibitory protein in DU145 prostate epithelial cells

Transforming growth factor ß (TGFß) is a paracrine mediator of prostate epithelial cell apoptosis. In rodents, castration induces production of TGFβ by stromal cells, which leads to apoptosis of epithelial cells. To identify potential mediators of this cell death pathway, we developed a model using DU145 cells, a tumorigenic human prostate epithelial cell line. We discovered that at low density, in low mitogen media, DU145 cells apoptose when treated with TGFβ1. Prior to the onset of death, TGFβ1 treatment downregulated the expression of the caspase inhibitor FLICE‐like inhibitory protein (FLIP), at both the mRNA and protein level, suggesting a causal role between FLIP downregulation and cell death. To confirm the importance of FLIP in TGFβ1‐induced apoptosis, we employed small interfering RNA (siRNA) to silence FLIP expression. Doing so led to apoptosis, which is consistent with the hypothesis that FLIP prevents death in these cells. Furthermore, inhibition of caspase‐8 by siRNA knockdown partially rescued the apoptotic effects of TGFβ1, suggesting a role for death receptor signaling components in TGFß‐mediated death of prostate epithelial cells. © 2008 Wiley‐Liss, Inc.     

Caspase-3-dependent protein kinase C delta activity is required for the progression of Ginsenoside-Rh2-induced apoptosis in SK-HEP-1 cells

Ginsenoside-Rh2 (G-Rh2) has been shown to induce apoptosis in a variety of cell types. In this study, we show that G-Rh2-induced apoptosis is accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in the human hepatoma cell line, SK-HEP-1. Furthermore, protein kinase C delta (PKCδ) activity was markedly up-regulated in a lipid activator-independent manner with kinetics similar to those of PKCδ and PARP cleavages during the apoptotic progression. Pre-treatment of cells with the caspase-3 specific inhibitor (z-DEVD-fmk) effectively prevented the G-Rh2-induced proteolytic activation of PKCδ. Moreover, rottlerin, a specific PKCδ inhibitor blocked G-Rh2-induced proapoptotic effects on the cells including the release of cytochrome c, activation of caspase-3 activity, and proteolytic cleavage and activation of PKCδ. These results suggest that G-Rh2-induced apoptosis is functionally linked to mitochondrial dysfunction and caspase-3 activity is regulated by positive feedback with PKCδ via the mitochondrial pathway.     

竹节香附素A对HepG2细胞缺氧诱导因子-1α基因和蛋白表达的影响

目的:探讨竹节香附素A对缺氧条件下人肝癌细胞株(HepG2)细胞缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)基因和蛋白表达的影响.方法:取体外低氧环境下HepG2细胞进行不同干预:特定浓度(10 μg/ml)竹节香附素A不同作用时间(0 h、6 h、12 h、24 h);不同浓度(0、5、10、20 μg/ml)竹节香附素A特定作用时间(24 h)处理,通过半定量逆转录-聚合酶链反应(RT-PCR)和Western blot检测HepG2细胞HIF-1α mRNA和蛋白的表达变化情况.结果:体外低氧环境下HepG2细胞可见HIF-1α mRNA和蛋白强表达,特定浓度(10 μg/ml)竹节香附素A可显著抑制HIF-1α mRNA和蛋白的表达水平,药物作用0 h、6 h、12 h、24 h时HIF-1α mRNA 条带灰度值(A/A0)分别为0.73±0.08、0.44±0.12、0.24±0.09、0.07±0.03,差异具有统计学意义(P<0.01);HIF-1α蛋白A/A0比值分别为2.31±0.33、1.70±0.17、1.30±0.96、0.09±0.13,差异具有统计学意义(P<0.01).不同浓度竹节香附素A特定作用时间(24 h)处理可显著抑制HIF-1α mRNA和蛋白的表达水平,药物浓度0、5、10、20 μg/ml处理,HIF-1α mRNA A/A0比值分别为0.65±0.07、0.43±0.08、0.19±0.03、0.08±0.02,差异具有统计学意义(P<0.01);HIF-1α蛋白A/A0比值分别为0.84±0.08、0.39±0.06、0.28±0.04、0.08±0.03,差异具有统计学意义(P<0.01).结论:竹节香附素A对缺氧条件下肝癌HepG2细胞HIF-1α mRNA和蛋白的表达具有抑制作用,并存在时间-剂量依赖性效应.