Silibinin inhibits expression of HIF-1α through suppression of protein translation in prostate cancer cells

Silibinin is a polyphenolic flavonoid isolated from the milk thistle (Silybum marianum) and is reported to exhibit anticancer properties. Recently, it has been reported that silibinin inhibits hypoxia-inducible factor-1α (HIF-1α) expression in cancer cells. However, the precise mechanism by which silibinin decreases HIF-1 expression is not fully understood. In this study, silibinin inhibited basal and hypoxia induced expression levels of HIF-1α protein in LNCaP and PC-3 prostate cancer cells, while the rate of HIF-1α protein degradation and mRNA levels were not affected. We found that the decrease in HIF-1 protein by silibinin correlated with suppression of de novo synthesis of HIF-1α protein. Silibinin inhibited global protein synthesis coincided with reduction of eIF4F complex formation and induction of phosphorylation of the translation initiation factor 2α (eIF-2α) which can cause inhibition of general protein synthesis. These results suggest that silibinin’s activity to inhibit HIF-1α protein expression is associated with the suppression of global protein translation.     

Different effect of beta-carotene on proliferation of prostate cancer cells

It was shown that high doses of beta-carotene (>30 microM) decrease proliferation of prostate cancer cells in vitro. However, it is rather doubtful whether such concentration of beta-carotene is really accessible at cellular level. We studied the effect of 3 and 10 microM beta-carotene on proliferation and gene expression in LNCaP and PC-3 prostate cancer cell lines. Beta-carotene--more efficiently absorbed from medium by androgen-sensitive LNCaP cells--increased proliferation of LNCaP cells whereas it had weaker effect on PC-3 cells. Initial global analysis of expression of genes in both cell lines treated with 10 microM beta-carotene (Affymetrix HG-U133A) showed remarkable differences in number of responsive genes. Their recognition allows for conclusion that differences between prostate cancer cell lines in response to beta-carotene treatment are due to various androgen sensitivities of LNCaP and PC-3 cells. Detailed analysis of expression of selected genes in beta-carotene treated LNCaP cells at the level of mRNA and protein indicated that the observed increase of proliferation could have been the result of slight induction of a few genes affecting proliferation (c-myc, c-jun) and apoptosis (bcl-2) with no significant effect on major cell cycle control genes (cdk2, RB, E2F-1).     

Up-regulation of glyceraldehyde-3-phosphate dehydrogenase gene expression by HIF-1 activity depending on Sp1 in hypoxic breast cancer cells

Hypoxia up-regulates the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a cell type-specific manner. It is unknown whether this occurs in breast cancer. Here, we report that hypoxia up-regulates the GAPDH gene expression through breast cancer-specific molecular mechanisms in MCF-7 cells. Mutation analysis identified a novel hypoxia response element (HRE), in addition to the HRE found previously in prostate cancer LNCaP cells. Knockdown and overexpression of hypoxia-inducible factor (HIF)-1α indicated that HIF-1 contributed to the up-regulation of GAPDH gene expression by hypoxia. Although chromatin immunoprecipitation (ChIP) and plasmid immunoprecipitation analyses revealed the presence of HIF-1α on the novel HRE in both hypoxic cell lines, a mutation in either the novel HRE or its 3′-flanking GC-box resulted in a reduction of hypoxia-increased GAPDH promoter activity only in MCF-7 cells. ChIP analysis showed that Sp1 bound to the GC-box in MCF-7 cells, but not in LNCaP cells, in normoxia and hypoxia. Knockdown of Sp1 reduced hypoxia-increased promoter activity and expression level of GAPDH in MCF-7 cells. These results indicate that in MCF-7 cells, the activation of HIF-1 on the novel HRE contributes to the breast cancer-specific hypoxic induction of GAPDH gene expression and absolutely depends on the presence of Sp1 on the GC-box.     

Androgen Suppresses the Proliferation of Androgen Receptor-Positive Castration-Resistant Prostate Cancer Cells via Inhibition of Cdk2, CyclinA,and Skp2

The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3^AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3^AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27^Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3^AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3^AR cells partially blocked accumulation of p27^Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.     

A novel regulation of VEGF expression by HIF-1α and STAT3 in HDM2 transfected prostate cancer cells

On the basis of increasing roles for HDM2 oncoprotein in cancer growth and progression, we speculated that HDM2 might play a major role in hypoxia-induced metastatic process. For verification of this hypothesis, wild-type LNCaP prostate cancer cells and HDM2 transfected LNCaP-MST (HDM2 stably transfected) cells were studied. The data obtained from our experiments revealed that the HDM2 transfected LNCaP-MST cells possessed an ability to multiply rapidly and show distinct morphological features compared to non-transfected LNCaP cells. During exposures to hypoxia HDM2 expression in the LNCaP and LNCaP-MST cells was significantly higher compared to the normoxic levels. The LNCaP-MST cells also expressed higher levels of HIF-1α (hypoxia-inducible factor-1α) and p-STAT3 even under the normoxic conditions compared to the non-transfected cells. The HIF-1α and p-STAT3 expressions were increased several fold when the cells were subjected to hypoxic conditions. The HIF-1α and p-STAT3 protein expressions observed in HDM2 transfected LNCaP-MST cells were 20 and 15 folds higher, respectively, compared to the non-transfected wild-type LNCaP cells. These results demonstrate that HDM2 may have an important regulatory role in mediating the HIF-1α and p-STAT3 protein expression during both normoxic and hypoxic conditions. Furthermore, the vascular endothelial growth factor (VEGF) expression that is typically regulated by HIF-1α and p-STAT3 was also increased significantly by 136% (P < 0.01) after HDM2 transfection. The overall results point towards a novel ability of HDM2 in regulating HIF-1α and p-STAT3 levels even in normoxic conditions that eventually lead to an up-regulation of VEGF expression.     

PC-1基因在前列腺癌细胞系LNCaP和C4-2不同细胞周期的表达

目的:检测PC-1基因在前列腺癌细胞周期中各时间点的表达变化。方法:用200 ng/mL诺可唑(nocoda-zole)处理前列腺癌细胞系LNCaP和C4-2,16 h后使细胞处于G2/M期,在不同时间点收获细胞,分别进行流式分析和Western印迹,检测PC-1基因的表达。结果:流式分析和Western印迹结果显示,在G2/M期,LNCaP和C4-2前列腺癌细胞系中PC-1基因高表达。结论:PC-1基因的表达与前列腺癌细胞的细胞周期有关,提示PC-1可能在细胞周期调控中发挥作用。     

A20 gene expression is regulated by TNF, Vitamin D and androgen in prostate cancer cells

A20 is a TNF-inducible primary response gene and its product, a zinc finger protein, has antiapoptotic function in several cancer cells. We studied A20 gene expression in the Vitamin D- and TNF-sensitive LNCaP cell line and in the Vitamin D- and TNF-resistant PC-3 cell line. The results of the quantitative real-time RT-PCR analyses demonstrated that the basal level of A20 mRNA production in PC-3 cells was considerably higher than in LNCaP cells that is associated with the resistance of PC-3 cells. TNF induced A20 gene expression in both cell lines, but with different effect. A20 mRNA expression was down-regulated by 10nM calcitriol within 3-9h after treatment and up-regulated by androgen reaching maximal values by 6h after stimulation in LNCaP cells. We conclude that A20 may be involved in the regulation of cell proliferation by TNF, Vitamin D, and androgen in prostate cancer.     

Coumestrol suppresses hypoxia inducible factor 1α by inhibiting ROS mediated sphingosine kinase 1 in hypoxic PC-3 prostate cancer cells

Among many signals to regulate hypoxia inducible factor 1α (HIF-1α), sphingosine kinase 1 (SPHK1) is also involved in various biological activities such as cell growth, survival, invasion, angiogenesis, and carcinogenesis. Thus, in the present study, molecular mechanisms of coumestrol were investigated on the SPHK1 and HIF-1α signaling pathway in hypoxic PC-3 prostate cancer cells. Coumestrol significantly suppressed SPHK1 activity and accumulation of HIF-1α in a time- and concentration-dependent manner in hypoxic PC-3 cells. In addition, coumestrol inhibited the phosphorylation status of AKT and glycogen synthase kinase-3β (GSK 3β) signaling involved in cancer metabolism. Furthermore, SPHK1 siRNA transfection, sphigosine kinase inhibitor (SKI), reactive oxygen species (ROS) enhanced the inhibitory effect of coumestrol on the accumulation of HIF-1α and the expression of pAKT and pGSK 3β in hypoxic PC-3 cells by combination index. Overall, our findings suggest that coumestrol suppresses the accumulation of HIF-1α via suppression of SPHK1 pathway in hypoxic PC-3 cells.     

Role of intracellular prostaglandin E2 in cancer-related phenotypes in PC3 cells

Prostaglandin E₂ (PGE₂) and hypoxia-inducible factor-1α (HIF-1α) affect many mechanisms that have been shown to play a role in prostate cancer. In PGE₂-treated LNCaP cells, up-regulation of HIF-1α requires the internalization of PGE₂, which is in sharp contrast with the generally accepted view that PGE₂ acts through EP receptors located at the cell membrane. Here we aimed to study in androgen-independent PC3 cells the role of intracellular PGE₂ in several events linked to prostate cancer progression. To this end, we used bromocresol green, an inhibitor of prostaglandin uptake that blocked the immediate rise in intracellular immunoreactive PGE₂ following treatment with 16,16-dimethyl-PGE₂. Bromocresol green prevented the stimulatory effect of 16,16-dimethyl-PGE on cell proliferation, adhesion, migration and invasion and on HIF-1α expression and activity, the latter assessed as the HIF-dependent activation of (i) a hypoxia response element-luciferase plasmid construct, (ii) production of angiogenic factor vascular endothelial growth factor-A and (iii) in vitro angiogenesis. The basal phenotype of PC3 cells was also affected by bromocresol green, that substantially lowered expression of HIF-1α, production of vascular endothelial growth factor-A and cell proliferation. These results, and the fact that we found functional intracellular EP receptors in PC3 cells, suggest that PGE₂-dependent intracrine mechanisms play a role in prostate cancer Therefore, inhibition of the prostaglandin uptake transporter might be a novel therapeutic approach for the treatment of prostate cancer.     

Cytosolic phospholipase A2α sustains pAKT,pERK and AR levels in PTEN-null/mutated prostate cancer cells

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog (PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A₂α (cPLA₂α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA₂α led to an increase in pAKT, pGSK3β and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA₂α expression with siRNA decreased pAKT, pGSK3β and cyclin D1 levels in both PC-3 (PTEN deletion) and LNCaP cells. Silencing of cPLA₂α decreased pERK and AR protein levels. The inhibitory effect of cPLA₂α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA₂α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA₂α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.     

Differential effects on growth, cell cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines.

Epidemiologic studies have suggested that nutrition plays an important role in carcinogenesis and that 30% of cancer morbidity and mortality can potentially be prevented with proper adjustment of diets. Resveratrol, a polyphenol present in red wines and a variety of human foods, has recently been reported to exhibit chemopreventive properties when tested in a mouse skin cancer model system. In this study, we investigated the effects of resveratrol on growth, induction of apoptosis, and modulation of prostate-specific gene expression using cultured prostate cancer cells that mimic the initial (hormone-sensitive) and advanced (hormone-refractory) stages of prostate carcinoma. Androgen-responsive LNCaP and androgen-nonresponsive DU-145, PC-3, and JCA-1 human prostate cancer cells were cultured with different concentrations of resveratrol (2. 5 x 10(-5)-10(-7) M). Cell growth, cell cycle distribution, and apoptosis were determined. Addition of 2.5 x 10(-5) M resveratrol led to a substantial decrease in growth of LNCaP and in PC-3 and DU-145 cells, but only had a modest inhibitory effect on proliferation of JCA-1 cells. Flow cytometric analysis showed resveratrol to partially disrupt G1/S transition in all three androgen-nonresponsive cell lines, but had no effect in the androgen-responsive LNCaP cells. In difference to the androgen-nonresponsive prostate cancer cells however, resveratrol causes a significant percentage of LNCaP cells to undergo apoptosis and significantly lowers both intracellular and secreted prostate-specific antigen (PSA) levels without affecting the expression of the androgen receptor (AR). These results suggest that resveratrol negatively modulates prostate cancer cell growth, by affecting mitogenesis as well as inducing apoptosis, in a prostate cell-type-specific manner. Resveratrol also regulates PSA gene expression by an AR-independent mechanism.     

Stanniocalcin-2 is a HIF-1 target gene that promotes cell proliferation in hypoxia

Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1α dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.     

NKX3.1 potentiates TNF-α/CHX-induced apoptosis of prostate cancer cells through increasing caspase-3 expression and its activity

NKX3.1, a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as tumor suppressor gene. Previously we have demonstrated that forced expression of NKX3.1 reduced cell growth and invasion in prostate cancer cell line PC-3. Presently, we investigated the effect of NKX3.1 on the sensitivity of the prostate cancer cells to apoptosis inducer tumor necrosis factor-α (TNF-α) and cycloheximide (CHX). PC-3 cells were transfected with NKX3.1 expression plasmid (pcDNA3.1-NKX3.1) and LNCaP cells were transfected with siRNA expression plasmid (pRNAT-RNAi1) targeting NKX3.1. The cell morphology and apoptotic rate were analyzed by Hoechst 33342 staining and Flow Cytometry in absence or presence of TNF-α and CHX. The activity of caspase-3 was determined using DEVD-pNA as substrate. Simultaneously, the effect of NKX3.1 on caspase-3 expression was detected using RT-PCR and Western blot. The results showed that ectopic expression of NKX3.1 promoted TNF-α/CHX-induced apoptosis in PC-3 cells, whereas knockdown of NKX3.1 protected LNCaP cells from apoptosis induced by TNF-α/CHX. The pro-apoptosis activity of NKX3.1 might partially contribute to its elevation of caspase-3 expression and activity. Manipulating NKX3.1 expression should be a promising therapeutic strategy for treating both androgen-dependent and androgen-independent prostate cancer.     

miR-182在前列腺癌组织中的表达及其对前列腺癌细胞增殖和凋亡的影响

目的:观察前列腺癌组织及不同前列腺癌细胞系中miR-182的表达,并探讨下调其表达对前列腺癌细胞增殖和凋亡的影响及机制。方法:采用实时荧光定量PCR(q RT-PCR)检测30例前列腺癌组织和30例相应的癌旁组织以及前列腺正常上皮RWPE-1细胞、前列腺癌PC-3、LNCa P和DU145细胞中miR-182的表达,进一步采用Lipfectamine 2000脂质体转染miRNA-182 inhibitor和阴性对照miRNA于PC-3细胞后,通过噻唑蓝(MTT)比色法检测细胞增殖情况,流式细胞术检测细胞凋亡率,免疫印迹(Western blot)法检测转录因子FOXO1、血管内皮生长因子(VEGF)和抑癌基因p53蛋白的表达。结果:miR-182在前列腺癌组织中的表达明显高于癌旁组织(P0.05);miR-182在前列腺癌细胞系PC-3、LNCa P和DU145中的表达均高于前列腺正常上皮细胞RWPE-1(P0.05),其中PC-3细胞中miR-182表达水平最高。转染miRNA-182 inhibitor至PC-3细胞成功下调miR-182表达后,细胞的增殖能力明显受到抑制,细胞凋亡能力明显增强,FOXO1表达水平显著升高,VEGF和p53的表达明显降低,差异均具有统计学意义(P0.05)。结论:miR-182在前列腺癌组织及细胞中呈高表达,下调miR-182的表达可能通过增加FOXO1的表达并减少VEGF和p53的表达,抑制前列腺癌细胞增殖并诱导细胞凋亡。     

癌基因D52家族新基因PC-1在前列腺癌细胞系LNCaP和C4-2B的G2／M期高表达

目的：检测癌基因D52家族新基因船一,在细胞周期各时相的表达变化。方法：用1mmol／L羟基脲处理前列腺癌细胞系LNCaP和C4-2B 40h,使细胞同步化于G1／S期,在药物撤除后0-16h不同时间点收获细胞,分别进行流式分析和Westernblot检测。结果：流式分析和Westernblot检测在LNCaP和C4-2B细胞系中得到了趋势一致的结果,印PC-1基因在GVM期高表达。结论：PC-1基因的表达与前列腺癌细胞的细胞周期有关,表明PC-1蛋白可能在G2／M期发挥作用。