Sulforaphane and its metabolite mediate growth arrest and apoptosis in human prostate cancer cells

The relation between the consumption of cruciferous vegetables and reduced prostate cancer occurrence has been documented, although the responsible phytochemicals are unknown. The effects of sulforaphane (SFN) which occurs as the precursor glucosinolate in broccoli and other cruciferous vegetables, and its metabolite N-acetylcysteine conjugate (SFN-NAC) on prostate cancer cells were investigated. SFN and SFN-NAC were analyzed with the androgen-dependent human prostate cancer LNCaP cell line model. Cell growth and apoptosis were determined with the expression of androgen receptor and prostate specific antigen, DNA synthesis, cell cycle progression, DNA strand breaks and caspase activation to ascertain the effects and mechanism. SFN and SFN-NAC were demonstrated for the first time to mediate a dose-dependent apoptosis and growth arrest in the prostate cancer cells. Caspases were activated and DNA strand breaks were detected in apoptotic cells. The expression of phosphorylated and dephosphorylated androgen receptors, and the production of prostate specific antigen were attenuated. The expression of cyclin D1 and DNA synthesis were inhibited along with G1 cell cycle block, causing decreased cell density and growth. SFN and its metabolite SFN-NAC have similar activities to induce growth arrest and apoptosis, indicating that the effects of SFN are maintained through the metabolic processes. SFN as a dietary component of cruciferous vegetables active in the prevention of prostate cancer is discussed.     

Statin induces apoptosis and cell growth arrest in prostate cancer cells.

Statins are a class of low molecular weight drugs that inhibit the rate-limiting enzyme of the mevalonate pathway 3-hydroxy-3-methylglutaryl-CoA reductase. Statins have been approved and effectively used to control hypercholesterolemia in clinical setting. Recent study showed statin's antitumor activity and suggested a potential role for prevention of human cancers. In this study, we did cell viability, DNA fragmentation, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays to evaluate the action of statins on prostate cancer cells and used Western blotting and RhoA activation assay to investigate the underlying molecular mechanism of action. Our data showed that lovastatin and simvastatin effectively decreased cell viability in three prostate cancer cell lines (PC3, DU145, and LnCap) by inducing apoptosis and cell growth arrest at G(1) phase. Both lovastatin and simvastatin induced activation of caspase-8, caspase-3, and, to a lesser extent, caspase-9. Both statins suppressed expression of Rb, phosphorylated Rb, cyclin D1, cyclin D3, CDK4, and CDK6, but induced p21 and p27 expression in prostate cancer cells. Furthermore, lovastatin and simvastatin suppressed RhoA activation and c-JUN expression, but not cyclooxygenase-2 expression. Our data showed that the antitumor activity of statins is due to induction of apoptosis and cell growth arrest. The underlying molecular mechanism of statin's action is mediated through inactivation of RhoA, which in turn induces caspase enzymatic activity and/or G(1) cell cycle. Future studies should focus on examining statins and other apoptosis-inducing drugs (e.g., cyclooxygenase-2 inhibitors or curcumin) together to assess their efficacy in prevention of prostate cancer.     

Tumor‐suppressive microRNA‐145 induces growth arrest by targeting SENP1 in human prostate cancer cells

Prostate cancer (PCa) prevails as the most commonly diagnosed malignancy in men and the third leading cause of cancer‐related deaths in developed countries. One of the distinct characteristics of prostate cancer is overexpression of the small ubiquitin‐like modifier (SUMO)‐specific protease 1 (SENP1), and the upregulation of SENP1 contributes to the malignant progression and cell proliferation of PCa. Previous studies have shown that the expression of microRNA‐145 (miRNA‐145) was extensively deregulated in PCa cell lines and primary clinical prostate cancer samples. Independent target prediction methods have indicated that the 3′‐untranslated region of SENP1 mRNA is a potential target of miR‐145. Here we found that low expression of miR‐145 was correlated with high expression of SENP1 in PCa cell line PC‐3. The transient introduction of miR‐145 caused cell cycle arrest in PC‐3 cells, and the opposite effect was observed when miR‐145 inhibitor was transfected. Further studies revealed that the SENP1 3′‐untranslated region was a regulative target of miR‐145 in vitro. MicroRNA‐145 also suppressed tumor formation in vivo in nude mice. Taken together, miR‐145 plays an important role in tumorigenesis of PCa through interfering SENP1.     

Mitogen-activated protein kinase antisense oligonucleotide inhibits the growth of human lung cancer cells

Mitogen-activated protein kinase (MAPK) pathway is proposed to be a therapeutic target for cancer cells. In order to find the potential therapeutic usefulness of MAPK for cancer cells, the effect of EAS1, an antisense oligonucleotide for an MAPK, on cancer-cell-growth were investigated in vitro. EAS1 effectively inhibited the growth of several human lung cancer cell lines such as PC-14 cells upon exposure to 10-0-10-1 microM of EAS1 determined dye-formation (MTT) assay. The ED50 values were comparable to those obtained for the inhibition of MAPK activity, DNA synthesis. EAS1 arrested the PC-14 cells at the G2/M phase of cell cycle followed by apoptosis in a dose-dependent manner. In order to determine the factors which influence the cellular sensitivity against MAPK inhibition, the effect of EAS1 on H-ras-transformed murine fibroblast cells were compared with that on parental cells. The NIH3T3 cells transformed by the H-ras gene (PT22-3) showed higher sensitivity against the effects of EAS1. Because MAPK activity was activated by H-ras gene transfection in PT22-3, the status of the MAPK cascade in cells was the determining factor for the efficacy of EAS1. In addition, cell permeabilization by digitonin enhanced the growth inhibitory effect of EAS1. Penetration of the cell membrane by EAS1 is also crucial for the growth inhibitory effect of EAS1. In conclusion, MAPK is an important target for cancer treatment and MAPK antisense oligonucleotide is a potentially significant antitumor oligonucleotide.     

Basic fibroblast growth factor in human prostate cancer cells.

To increase our understanding of the potential role of basic fibroblast growth factor (bFGF) in malignant progression of prostate cancer, we determined the production of bFGF, the expression of FGF receptor (flg), and the response to exogenous bFGF in LNCaP, DU 145, and PC 3 cells. We observed that these three prostate cancer cell lines, which differed in their dependence on androgens for growth in vitro and in their in vivo behavior in nude mice, could be distinguished as follows: (a) androgen-sensitive LNCaP cells, which do not metastasize in nude mice, did not produce measurable amounts of bFGF, expressed small but measurable amounts of FGF receptor mRNA, and did respond to exogeneous bFGF; (b) androgen-insensitive, moderately metastatic DU 145 cells did produce measurable amounts of biologically active bFGF, expressed large amounts of FGF receptor mRNA, and responded to exogeneous bFGF and the heparin-binding fractions from DU 145 cell extracts; (c) androgen-insensitive and highly metastatic PC3 cells also produced measurable amounts of bFGF but did not demonstrate a growth response to either the heparin-binding fractions from PC3 cell extracts or exogenous bFGF, even though large amounts of FGF receptor mRNA were expressed in PC 3 cells. These results suggest the possibility that differences in production of, and response to, bFGF may be associated with different biological behavior.     

Knock-down of calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells

Calcitonin (CT) and its receptor (CTR) are expressed only in basal epithelium of benign prostate and in whole epithelium of malignant prostates. Also, CT and CTR mRNA levels in prostate cancers increase with an increase in tumor grade. We tested the role of the CT/CTR autocrine axis on the tumorigenicity of prostate cancer cells. We enforced the expression of CTR in CT-positive/CTR-deficient PC-3 cells. In contrast, we knocked down CTR expression in CT/CTR-positive PC-3M cells. The effect of CTR modulation on the oncogenicity was evaluated by the rate of cell proliferation, invasion, colony formation and in vivo growth in nude mice. Up-regulation of CTR in PC-3 cells and its down-regulation in PC-3M cells significantly altered their tumorigenicity. Intratumorally administered CTR RNAi in preexisting PC-3M xenografts markedly attenuated their further growth. This treatment also led to a remarkable decrease in endothelial cell populations in the tumors and increase in apoptotic, PCNA-negative cell populations. Tumors receiving CTR RNAi treatment displayed markedly lower levels of urokinase-type plasminogen activator, phospho-Akt and survivin, suggesting CTR activates uPA-uPAR axis and PI-3-kinase-Akt-survivin pathway. These results suggest an important role for CT-CTR autocrine axis in the progression of localized prostate tumor to a metastatic phenotype, and offer a potential therapeutic option for invasive cancers.     

Suppression of human prostate cancer cell growth by ciprofloxacin is associated with cell cycle arrest and apoptosis

For hormone resistant prostate cancer (HRPC), chemotherapy is used but the mortality is 100% with a mean survival time of 7-8 months. Our previous studies have shown the chemotherapeutic effect of ciprofloxacin in bladder cancer. At doses 50-400 micro g/ml ciprofloxacin, the concentrations that are normally achieved at doses currently used for the treatment of anti-bacterial infections, inhibited bladder cancer cell growth and induced S/G2M arrest with modulation of key cell cycle regulatory genes and ultimately activated apoptotic processes. In this study, we investigated the effect of ciprofloxacin on androgen independent prostate carcinoma, PC3 cells and compared our results with non-tumorigenic prostate epithelial cells. The main advantage of this fluroquinolone antibiotic is its relative non-toxicity as compared to current chemotherapy, which is not very effective, for the treatment of advanced hormone resistant prostate cancer. PC3 cells as well as normal prostate epithelial cells (MLC8891) were treated with 25-400 micro g/ml ciprofloxacin, and cell counting was done during 3 days of treatment. The cell death was determined using DAPI staining of cell nuclei, 7AAD-staining followed by flow cytometric analysis as well as by activation of caspase-3, a member of the ICE family of enzymes involved in the apoptotic cascade. The cell lysates were analyzed by immunoblotting techniques for the expression of key genes targeted by ciprofloxacin (p21WAF1, Bax and Bcl-2). Translocation of bax was visualized using a fluorescence staining procedure followed by laser confocal microscopic imaging. Treatment of prostate cancer cells with ciprofloxacin resulted in a dose- and time-dependent inhibition of cell growth (70-100% with 50-400 micro g/ml of the drug). There was a concomitant induction of cell cycle arrest at the S and G2/M phases of the cell cycle as well as induction of apoptosis. The CDK inhibitor p21WAF1 was down-regulated as early as 12 h following ciprofloxacin treatment (100-200 micro g/ml for 12-24 h). There was a significant increase in the Bax/Bcl-2 ratio with translocation of Bax, a pro-apoptotic protein, to mitochondria with concomitant activation of caspase 3. These results suggest the potential usefulness of the fluroquinolone, ciprofloxacin as a chemotherapeutic agent for advanced prostate cancer. The fluroquinolone ciprofloxacin showed anti-proliferative and apoptosis inducing activity on prostate cancer cells but not on non-tumorigenic prostate epithelial cells. These effects of ciprofloxacin were mediated by cell cycle arrest at S-G2/M phase of the cell cycle, Bax translocation to mitochondrial membrane and by increasing the Bax/Bcl-2 ratio in PC3 prostate cancer cells. Based on our in vitro results, further in-depth in vivo animal or human investigations are warranted.     

Naftopidil, a selective alpha-1 adrenoceptor antagonist, inhibits growth of human prostate cancer cells by G1 cell cycle arrest

Alpha-1 adrenoceptor antagonists are generally prescribed for benign prostate hyperplasia with lower urinary tract symptoms. Naftopidil, a selective alpha-1 adrenoceptor antagonist, is frequently used in Japan because it has fewer side effects. Here we demonstrate for the first time that naftopidil has growth inhibitory effect in androgen-sensitive and -insensitive human prostate cancer cell lines. The concentrations causing 50% inhibition (IC50) of cancer cell growth were 22.2 +/- 4.0 microM in androgen-sensitive LNCaP cells and 33.2 +/- 1.1 microM in androgen-insensitive PC-3 cells. FACS analysis revealed that cell growth inhibition by naftopidil was due to the arrest of the G1 cell cycle. Expressions of p27(kip1) and p21(cip1) were significantly increased in LNCaP cells treated with naftopidil. In PC-3 cells, naftopidil induced p21(cip1) but not p27(kip1). In vivo, oral administration of naftopidil to nude mice inhibited the growth of PC-3 tumors as compared to vehicle-treated controls. These results suggest that naftopidil may be useful in the chemoprevention of prostate cancer and the intervention of hormone refractory prostate cancer.     

Modulation of growth of human prostate cancer cells by the N-acetylcysteine conjugate of phenethyl isothiocyanate

There is growing evidence that thiol conjugates of isothiocyanates present in cruciferous vegetables are effective cancer chemopreventive and potentially active therapeutic agents. The effects of the N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) on tumor cell growth were analyzed in human prostate cancer cell lines LNCaP, androgen-dependent, and DU-145, androgen-independent. Exposure of the cells to PEITC-NAC at high concentrations caused cytolysis, while at lower concentrations PEITC-NAC mediated a dose-dependent growth modulation, with reduction of DNA synthesis and growth rate, inhibition of clonogenicity and induction of apoptosis in both types of prostate cancer cells. PEITC-NAC decreased cells in S and G2M phases of cell cycle, blocking cells entering replicating phases. In parallel, a significant enhancement of cells expressing the cell cycle regulator p21 as well as its intensity was determined using a fluorescent antibody technique. The action of PEITC-NAC was time-dependent, with the magnitude of inhibition increasing to 50-65% after PEITC-NAC exposure for several days. Interaction of tumor cells with dissociation products of PEITC-NAC, PEITC and NAC, are proposed as the mechanism of growth regulation.     

Mechanism of 2-methoxyestradiol-induced apoptosis and growth arrest in human breast cancer cells

2-Methoxyestradiol, a well-known nonpolar endogenous metabolite of 17beta-estradiol, has been shown to selectively induce apoptosis in a number of cancer cell lines, but not in normal cells. The mechanism of 2-methoxyestradiol-induced apoptosis appears to vary considerably in different cell lines examined. In the present study, we systematically analyzed the mechanisms of 2-methoxyestradiol-induced apoptosis in the estrogen receptor-negative MDA-MB-435s human breast cancer cells. We found that 2-methoxyestradiol induced the activation of JNK, ERK, and p38 MAPKs. 2-methoxyestradiol-induced JNK activation was associated with the induction of apoptosis through the mitochondrial pathways as a result of increased phosphorylation (inactivation) of the anti-apoptotic Bcl-2 and Bcl-xL proteins. In comparison, 2-methoxyestradiol-induced activation of ERK and p38 in these cells was found to have a protective effect against 2-MeO-E(2)-induced apoptosis. Consistent with this observation, the presence of pharmacological inhibitor of ERK or p38 enhanced 2-methoxyestradiol-induced apoptosis. Mechanistically, inhibition of ERK and p38 activity was associated with activation of various caspases and PARP cleavage, and it also stabilized the pro-apoptotic proteins Bax and Bim, thereby preventing them from degradation during 2-methoxyestradiol treatment. These results suggest that ERK and p38 MAPKs may serve as viable targets for the sensitization of human breast cancer cells to 2-methoxyestradiol-induced apoptosis.     

Targeting Cdc37 inhibits multiple signaling pathways and induces growth arrest in prostate cancer cells

Members of the 90-kDa heat shock protein (HSP90) family are known to bind and stabilize intermediates in a wide variety of cell signaling pathways and contribute to their dysregulation in cancer. An important intracellular cofactor for HSP90 is Cdc37, a protein with a broad role in fostering the activities of protein kinases. By targeting Cdc37 using RNA interference, we have shown that the loss of Cdc37 function induces irreversible growth arrest in androgen receptor-positive and -negative prostate carcinoma cells. In contrast to HSP90-directed agents, Cdc37 targeting seems to affect cancer cells through a distinct mechanism and does not significantly deplete the intracellular levels of most known HSP90 client proteins. Instead, Cdc37 depletion inhibits cellular kinase activity and flux through growth-promoting signal transduction cascades. We show that the loss of Cdc37 leads to reduced activity of the Erk, Akt, mTOR, and androgen-induced pathways. We have also discovered synergistic interactions between Cdc37 inactivation and the HSP90-inhibitory anticancer drug 17-(allylamino)-17-demethoxygeldanamycin (17AAG). These interactions involve enhanced degradation of proteins essential for growth and inhibition of 17AAG-induced expression of the antiapoptotic HSP70. Thus, Cdc37 is essential for maintaining prostate tumor cell growth and may represent a novel target in the search for multitargeted therapies based on the HSP90 chaperone system.     

Induced apoptosis in human prostate cancer cells by blocking of vascular endothelial growth factor by siRNA

Introduction

Vascular endothelial growth factor (VEGF) regulates several cell functions including; proliferation, differentiation, permeability, vascular tone, and the production of vasoactive molecules. The purpose of this study was to evaluate the potency of specific short-interfering RNA (siRNA) to suppress human VEGF expression by siRNA and investigate the effects of VEGF down-regulation on the cell proliferation and apoptosis of the human prostate cancer cell lines DU-145.

Methods

Transfection was performed using X-tremeGENE siRNA transfection reagent. At different time intervals, transfected cells were harvested and total RNA was extracted for RT-PCR. The VEGF content in supernatants were measured by ELISA. Inhibition of cell growth by hVEGF-siRNA was measured by using cell proliferation ELISA BrdU assay. Apoptotic cells were evaluated by using annexin-V-FITC apoptotic detection method.

Results

Transfection of hVEGF-siRNA resulted in statistically significant inhibition of hVEGF-mRNA that in turn caused a marked reduction in the expression of hVEGF. The cell growth was assessed every 24?h for 4?days after siRNA treatment resulted in a marked inhibition of cell proliferation as compared to scramble siRNA. The results of apoptosis showed that approximately 15?% of the cells treated with control-siRNA manifested evident apoptotic changes after 24?hpt, whereas DU-145 cells treated with hVEGF-siRNA significantly were positive, that is to say, 53?% at 72?hpt 23.9?±?2.78?% (P?<?0.001) and 13?±?1.57?% at 96?hpt.

Conclusion

Our findings indicate that siRNA are effective in eliciting the RNAi pathway in cancerous cells and that specific siRNA efficiently down-regulate VEGF expression. They could decrease VEGF production and induce apoptosis, which may also be linked to the inhibition of cancerous cell proliferation. Therefore, it can be concluded that siRNA-mediated suppression of VEGF represents a powerful tool against prostate cancer cell proliferation. VEGF down-regulation exerts a direct anti-apoptotic function in the DU-145 cell lines and promises the development of drugs for cancer therapy.     

Androgen signaling is required for the vitamin D-mediated growth inhibition in human prostate cancer cells

Epidemiological data on prostate cancer incidence has suggested that vitamin D deficiency may be a risk factor for prostate cancer. The antiproliferative activity of 1alpha, 25-dihydroxyvitamin D3 (1,25-VD) and its analogues has been demonstrated in many prostate cancer models, yet the detailed mechanisms underlying this protective effect of vitamin D remain to be determined. Here, we demonstrate that two androgen receptor (AR)-positive prostate cancer cell lines, LNCaP and CWR22R, are more sensitive to the growth inhibitory effects of 1,25-VD compared to the AR-negative prostate cancer cell lines, PC-3 and DU 145. 1,25-VD treatment inhibited cyclin-dependent kinase 2 (cdk2) activity and induced G0/G1 arrest. Interestingly, we also found that 1,25-VD treatment induced the expression of AR, and that the onset of the G0/G1 arrest in LNCaP and CWR22R cells is correlated with the onset of increasing expression of AR. This implies that the antiproliferative actions of 1,25-VD in AR-positive prostate cancer might be mediated through AR. Furthermore, a reduction in 1,25-VD-mediated growth inhibition was observed when AR signaling was blocked by antiandrogens, AR RNA interference, or targeted disruption of AR. Taken together, our data suggest that the androgen/AR signaling plays an important role in the antiproliferative effects of 1,25-VD and restoration of androgen responsiveness by 1,25-VD might be beneficial for the treatment of hormone-refractory prostate cancer patients.     

Down-regulation of Jagged-1 induces cell growth inhibition and S phase arrest in prostate cancer cells

Notch is an ancient cell signaling system that regulates cell fate specification, stem cell maintenance and initiation of differentiation in many tissues. It has been reported that Jagged-1, a Notch ligand, is significantly over expressed in metastatic prostate cancer compared to localized prostate cancer or benign prostatic tissues. Therefore, deregulation of Jagged-1 protein levels may play a role in prostate cancer cell growth and progression. Hence, the aim of our current study was to investigate the mechanistic role of Jagged-1 in prostate cancer cell growth and cell cycle progression. Our results show, for the first time, that down-regulation of Jagged-1 induces cell growth inhibition and S phase cell cycle arrest in prostate cancer cells, with reduced CDK2 kinase activity and increased p27 expression. These results suggest that Jagged-1 could be a potential therapeutic target for the treatment of prostate cancer.     

High throughput screening of methylation status of genes in prostate cancer using an oligonucleotide methylation array

Recent work using high-throughput microarray technology has discovered altered expression of a large number of genes in prostate cancer. Many of these alterations may be the consequence of changes in methylation status in the CpG islands of promoter or exon 1 regions of these genes. In order to determine the methylation status of a large number of genes and ESTs we combined the principle of match/mismatch hybridization with the technique of whole genome labeling to develop a highly specific oligonucleotide-based methylation microarray. Using this array, we analyzed the methylation status of 105 genes and ESTs in three prostate cancer cell lines. Between 32 and 47% of these genes and ESTs were methylated in these cell lines. By correlating the methylation status of this array with the results of Affymetrix expression arrays of three prostate cancer cell lines, we determined that methylation of genes played a significant role (37%) in down-regulating the expression of certain genes in prostate cancer. We also tested this array on a number of primary prostate tissue samples. Our results indicated that a subset of genes in this microarray (25/105) were methylated in all prostate cancer samples but not in normal prostate, suggesting the potential significance of alterations in the methylation status of certain genes in the development of prostate cancer.     

3,3'-Diindolylmethane suppresses growth of human esophageal squamous cancer cells by G1 cell cycle arrest

3,3'-Diindolylmethane (DIM), an active metabolite of indole-3-carbinol, is thought to have antitumor effects in experimental animals and induce apoptosis in various cancer cells. However, the biological functions of DIM in human esophageal cancer cells are unknown. Thus, the purpose of this study was to investigate the cytotoxic effects of DIM in human esophageal squamous cell carcinoma (ESCC) cells to elucidate the molecular mechanism of cell death. Three human ESCC cell lines (TT, TE-8 and TE-12) were used to test the response to DIM. MTT, cell cycle and western blot analyses were conducted. DIM significantly inhibited the proliferation of ESCC cells in a dose- and time-dependent manner. The percentage of G1?phase cells increased 48?h after being treated with DIM. DIM also reduced cyclin?D1, cyclin?E2, cyclin-dependent kinase (CDK) 4 and CDK 6 activities, and increased p15 and p27 levels. Additionally, DIM diminished pro-caspase-9 protein expression levels and induced increased cleaved poly (ADP-ribose) polymerase levels. These results indicate that DIM leads to G1?phase cell cycle arrest and induces apoptosis by activating caspase-9 in ESCC cells.