Combination treatment of prostate cancer cell lines with bioactive soy isoflavones and perifosine causes increased growth arrest and/or apoptosis.

PURPOSE: To determine whether targeting the androgen receptor (AR) and Akt pathways using a combination of genistein combined polysaccharide (GCP) and perifosine is more effective at inducing growth arrest/apoptosis in prostate cancer cells compared with treatment with GCP or perifosine as single agents. EXPERIMENTAL DESIGN: The effect of GCP and perifosine treatment was assessed in five prostate cancer cell lines: LNCaP (androgen sensitive), LNCaP-R273H, C4-2, Cds1, and PC3 (androgen insensitive). A clonogenic assay assessed the long-term effects on cell growth and survival. Flow cytometry and Western blot analysis of poly(ADP)ribose polymerase cleavage were used to assess short-term effects. Preliminary studies to investigate mechanism of action included Western blot for P-Akt, Akt, P-p70S6K, p70S6K, p53, and p21; prostate-specific antigen analysis; and the use of myristoylated Akt and AR-specific small interfering RNA. RESULTS: Combination treatment with GCP and perifosine caused a decrease in clonogenic potential in all cell lines. In short-term assays, growth arrest was observed in the majority of cell lines, as well as increased inhibition of Akt activity and induction of p21 expression. Increased apoptosis was only observed in LNCaP. Knockdown of AR caused a further increase in apoptosis. CONCLUSION: Combination treatment with GCP and perifosine targets the Akt pathway in the majority of the prostate cancer cell lines and causes increased inhibition of cell growth and clonogenicity. In LNCaP, combination treatment targets both the Akt and AR pathways and causes increased apoptosis. These data warrant clinical validation in prostate cancer patients.     

Antitumor effects of a novel phenylbutyrate-based histone deacetylase inhibitor, (S)-HDAC-42, in prostate cancer.

PURPOSE: To assess the antitumor effects of a novel phenylbutyrate-derived histone deacetylase (HDAC) inhibitor, (S)-HDAC-42, vis-à-vis suberoylanilide hydroxamic acid (SAHA) in in vitro and in vivo models of human prostate cancer. EXPERIMENTAL DESIGN: The in vitro effects of (S)-HDAC-42 and SAHA were evaluated in PC-3, DU-145, or LNCaP human prostate cancer cell lines. Cell viability, apoptosis, and indicators of HDAC inhibition were assessed. Effects on Akt and members of the Bcl-2 and inhibitor of apoptosis protein families were determined by immunoblotting. Immunocompromised mice bearing established s.c. PC-3 xenograft tumors were treated orally with (S)-HDAC-42 (50 mg/kg q.o.d. or 25 mg/kg q.d.) or SAHA (50 mg/kg q.d.) for 28 days. In vivo end points included tumor volumes and intratumoral changes in histone acetylation, phospho-Akt status, and protein levels of Bcl-xL and survivin. RESULTS: (S)-HDAC-42 was more potent than SAHA in suppressing the viability of all cell lines evaluated with submicromolar IC50 values. Relative to SAHA, (S)-HDAC-42 exhibited distinctly superior apoptogenic potency, and caused markedly greater decreases in phospho-Akt, Bcl-xL, and survivin in PC-3 cells. The growth of PC-3 tumor xenografts was suppressed by 52% and 67% after treatment with (S)-HDAC-42 at 25 and 50 mg/kg, respectively, whereas SAHA at 50 mg/kg suppressed growth by 31%. Intratumoral levels of phospho-Akt and Bcl-xL were markedly reduced in (S)-HDAC-42-treated mice, in contrast to mice treated with SAHA. CONCLUSIONS: (S)-HDAC-42 is a potent orally bioavailable inhibitor of HDAC, as well as targets regulating multiple aspects of cancer cell survival, which might have clinical value in prostate cancer chemotherapy and warrants further investigation in this regard.     

Enhanced radiation sensitivity in prostate cancer by inhibition of the cell survival protein clusterin.

PURPOSE: The purpose of this study is to evaluate the role of the cell survival gene clusterin in radiation-induced cell death in human LNCaP and PC-3 prostate cancer models. Experimental Design: Radiation sensitivities were compared in parental and clusterin-overexpressing LNCaP cells and in PC-3 cells and tumors treated with antisense or mismatch clusterin oligonucleotides. RESULTS: Clusterin-overexpressing LNCaP cells were less sensitive to irradiation with significantly lower cell death rates (23% after 8 Gy) compared with parental LNCaP cells (50% after 8 Gy) 3 days after irradiation. Clusterin expression in PC-3 cells after radiation was found to be up-regulated in a dose-dependent manner in vitro by 70% up to 12 Gy and in vivo by 84% up to 30 Gy. Inhibition of clusterin expression in PC-3 cells using antisense oligonucleotides (ASOs) occurred in a sequence- and dose-dependent manner and significantly enhanced radiation-induced apoptosis and decreased PC-3 cell growth rate and plating efficiency. Compared with mismatch control oligonucleotide treatment, clusterin ASO treatment enhanced radiation therapy and significantly reduced PC-3 tumor volume in vivo by 50% at 9 weeks. In addition, TUNEL staining revealed increased number of apoptotic cells in clusterin ASO-treated and irradiated PC-3 tumors, compared with treatment with mismatch control oligonucleotides plus radiation. CONCLUSIONS: These findings support the hypothesis that clusterin acts as a cell survival protein that mediates radioresistance through the inhibition of apoptosis. In vivo results further suggest that inactivation of clusterin using ASO technology might offer a novel strategy to improve results of radiation therapy for prostate cancer patients.     

Dual effects of glutathione-S-transferase pi on As2O3 action in prostate cancer cells: enhancement of growth inhibition and inhibition of apoptosis

To determine the effects of glutathione-S-transferase pi (GSTpi) on the actions of As2O3, As2O3-induced growth inhibition and apoptosis was studied in three prostate cancer cell lines: DU-145, PC-3 and LNCaP cells. As2O3 inhibited cell proliferation of DU-145 and PC-3 cells (both cells express GSTpi), but not of LNCaP cells (which lack GSTpi expression) at concentrations below 1 microM. LNCaP cells stably transfected and expressed GSTpi (LNCaP/GSTpi) became sensitive to As2O3 growth inhibition. As2O3 arrested cell growth of DU-145, PC-3 and LNCaP/GSTpi cells in the G2/M phase of the cell cycle at low concentrations (<2 microM), but did not induce apoptosis. At higher concentrations (10-20 microM), As2O3 induced apoptosis in LNCaP cells, but not in DU-145 or PC-3 cells. The apoptosis induction due to As2O3 treatment of LNCaP cell correlated with the activation of JNK and p38 and induction of p53 protein. LNCaP/GSTpi cells became insensitive to As2O3-induced apoptosis with reduced JNK activition. These data indicate that GSTpi increases growth inhibition due to As2O3 treatment and prevents As2O3-induced apoptosis in prostate cancer cells. Therefore, it appears that As2O3 inhibits cell growth and induces apoptosis through different mechanisms.     

Growth of human prostate cancer cells is significantly suppressed in vitro with sodium butyrate through apoptosis

Histone deacetylase inhibitors (HDACis) have shown significant antiproliferative and apoptotic properties in various types of cancer cells, including prostate cancer cells, and are therefore being evaluated as a treatment modality. However, the mechanism by which sodium butyrate (SB) induces apoptosis is not completely understood. We focused on SB which exists in the intestine and is therefore expected to have less adverse effects. In this study, three prostate cancer cell lines (LNCaP, DU145 and PC-3) were treated in vitro with different concentrations of SB. Cell proliferation was studied by the XTT assay; cell cycle analysis and induction of apoptosis were studied by laser scanning cytometry. Western blot analysis was used to study p21, p27, CDK2, CDK4, CDK6, caspase-3, caspase-7, Fas, FADD, TRADD, Bcl-2 and Bax protein expression. SB inhibited cell growth and induced apoptosis in a concentration-dependent manner in human prostate cancer cells (LNCaP, DU145 and PC-3). Western blot analysis showed dose-dependent increases of p21 levels in DU145 and PC-3 cells, and dose-dependent decreases of CDK2, CDK4, CDK6 and procaspase-3 protein levels in all three prostate cancer cell lines. Bcl-xL was significantly down-regulated in DU145 cells, and Bcl-2 was significantly down-regulated in PC-3 and LNCaP cells. No significant changes were observed in procaspase-7, TRADD and Bax expression, although slight decreases in Fas and FADD expression were seen in all three prostate cancer cell lines. Analysis of cell morphology using laser scanning microscopy detected condensed and fragmented nuclei. In conclusion, SB induces G1 and G2 arrest by increasing p21 expression resulting in CDK2, CDK4 and CDK6 down-regulation. SB potently induced apoptosis, which was accompanied by DNA fragmentation, down-regulated Bcl-2 in LNCaP and PC-3 cells, Bcl-xL in DU145 cells, and down-regulated procaspase-3, but not procaspase-7, in these human prostate cancer cell lines. These results suggest that SB may serve as a new modality for the treatment of hormone refractory prostate cancer.     

A potent indole-3-carbinol derived antitumor agent with pleiotropic effects on multiple signaling pathways in prostate cancer cells

Indole-3-carbinol has emerged as a promising chemopreventive agent due to its in vivo efficacy in various animal models. However, indole-3-carbinol exhibits weak antiproliferative potency and is unstable in acidic milieu. Thus, this study was aimed at exploiting indole-3-carbinol to develop potent antitumor agents with improved chemical stability. This effort culminated in OSU-A9 {[1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol}, which is resistant to acid-catalyzed condensation, and exhibits 100-fold higher apoptosis-inducing activity than the parent compound. Relative to indole-3-carbinol, OSU-A9 displays a striking qualitative similarity in its effects on the phosphorylation or expression of multiple signaling targets, including Akt, mitogen-activated protein kinases, Bcl-2 family members, survivin, nuclear factor-kappaB, cyclin D1, p21, and p27. The ability of OSU-A9 to concurrently modulate this broad range of signaling targets underscores its in vitro and in vivo efficacy in prostate cancer cells. Nevertheless, despite this complex mode of mechanism, normal prostate epithelial cells were less susceptible to the antiproliferative effect of OSU-A9 than PC-3 and LNCaP prostate cancer cells. Treatment of athymic nude mice bearing established s.c. PC-3 xenograft tumors with OSU-A9 at 10 and 25 mg/kg i.p. for 42 days resulted in a 65% and 85%, respectively, suppression of tumor growth. Western blot analysis of representative biomarkers in tumor lysates revealed significant reductions in the intratumoral levels of phosphorylated (p-) Akt, Bcl-xL, and RelA, accompanied by robust increases in p-p38 levels. In conclusion, the ability of OSU-A9 to target multiple aspects of cancer cell survival with high potency suggests its clinical value in prostate cancer therapy.     

RWJ-241947 (MCC-555), a unique peroxisome proliferator-activated receptor-gamma ligand with antitumor activity against human prostate cancer in vitro and in beige/nude/ X-linked immunodeficient mice and enhancement of apoptosis in myeloma cells induced by arsenic trioxide.

PURPOSE: RWJ-241947 (MCC-555) is a novel peroxisome proliferator-activated receptor-gamma ligand of the thiazolidinedione class that was recently developed as an antidiabetic drug with unique properties. Some thiazolidinediones have anticancer activity against solid and hematological malignancies; the anticancer potency of RWJ-241947 has not been examined. We, therefore, investigated these effects in vitro and in vivo either alone or in combination with other compounds. EXPERIMENTAL DESIGN: Tumor growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, soft agar colony assay in vitro, and xenografts in nude mice. Its effects on cell cycle, differentiation, and apoptosis were examined. RESULTS: In vitro studies using various solid and hematological tumor cell lines showed that RWJ-241947 had antiproliferative activity against prostate cancer cells, with the strongest effect against the androgen-independent PC-3 prostate cancer cells. It increased expression of cyclin-dependent kinase inhibitor p21(WAF1), deceased cyclin E, and induced apoptosis in PC-3 cells. It increased E-cadherin and lowered protein expression of prostate-specific antigen without down-regulating the androgen receptor in androgen-dependent LNCaP prostate cancer cells. Reporter gene assays showed that this peroxisome proliferator-activated receptor-gamma ligand inhibited androgen activation of the androgen receptor response elements of the prostate-specific antigen gene. Remarkably, in vivo treatment of male beige/nude/X-linked immunodeficient (BNX) mice with RWJ-241947 profoundly suppressed growth of PC-3 prostate cancer xenografts with prominent apoptosis, as well as fibrosis, including inflammatory and giant cell reaction in the remaining tumor tissue. Notably, the experimented mice had a significantly decreased cholesterol. In addition, we studied the combination of arsenic trioxide (As2O3), which is used in the treatment of multiple myeloma, and RWJ-241947; these two reagents together prominently inhibited proliferation and caused apoptosis of multiple myeloma cells. CONCLUSIONS: RWJ-241947 has surprisingly potent antiproliferative effects against prostate cancer cells in vivo, and it enhances the antitumor activity of As2O3 against myeloma cells. Small, well-defined clinical studies using RWJ-241947 are in order for these cancers.     

Effects of soybean isoflavones on cell growth and apoptosis of the human prostatic cancer cell line LNCaP

BACKGROUND: Epidemiological studies have suggested that soybean isoflavones are associated with a lower risk of prostate cancer. However, the mechanisms of prostate cancer prevention by soybean isoflavones have yet to be fully clarified. METHODS: Two soybean isoflavones (genistein and daidzein) and their glucosides (genistin and daidzin) were tested for their effects on cell growth and apoptosis of the LNCaP human prostatic cancer cell line. RESULTS: Among these isoflavones, genistein was found to inhibit the growth of LNCaP most effectively, with an IC50 value of 40 microM. The inhibition of cell growth by genistein was accompanied by the suppression of DNA synthesis and the induction of apoptosis. Expression of prostate-specific antigen (PSA) in LNCaP was also significantly reduced by the treatment with genistein. CONCLUSIONS: The results suggest that genistein might primarily influence human prostate cancer development by reducing tumor growth.      

Allyl isothiocyanate,a constituent of cruciferous vegetables,inhibits growth of PC-3 human prostate cancer xenografts in vivo

We have shown previously that allyl isothiocyanate (AITC), a constituent of cruciferous vegetables, significantly inhibits survival of PC-3 and LNCaP human prostate cancer cells in culture, whereas proliferation of a normal prostate epithelial cell line is minimally affected by AITC even at concentrations that are highly cytotoxic to the prostate cancer cells. The present studies were designed to test the hypothesis that AITC administration may retard growth of human prostate cancer xenografts in vivo. Bolus i.p. injection of 10 micromol AITC, three times per week (Monday, Wednesday and Friday) beginning the day of tumor cell implantation, significantly inhibited the growth of PC-3 xenograft (P < 0.05 by two-way ANOVA). For example, 26 days after tumor cell implantation, the average tumor volume in control mice (1025 +/- 205 mm3) was approximately 1.7-fold higher compared with AITC-treated mice. Histological analysis of tumors excised at the termination of the experiment revealed a statistically significant increase in number of apoptotic bodies with a concomitant decrease in cells undergoing mitosis in the tumors of AITC-treated mice compared with that of control mice. Western blot analysis indicated an approximately 70% reduction in the levels of anti-apoptotic protein Bcl-2 in the tumor lysate of AITC-treated mice compared with that of control mice. Moreover, the tumors from AITC-treated mice, but not control mice, exhibited cleavage of BID, which is known to promote apoptosis. Statistically significant reduction in the expression of several proteins that regulate G2/M progression, including cyclin B1, cell division cycle (Cdc)25B and Cdc25C (44, 45 and 90% reduction, respectively, compared with control), was also observed in the tumors of AITC-treated mice relative to control tumors. In conclusion, the results of the present study indicate that AITC administration inhibits growth of PC-3 xenografts in vivo by inducing apoptosis and reducing mitotic activity.     

eNOS protects prostate cancer cells from TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent anti-cancer agent because it induces apoptosis of most tumor cells with little or no effect on normal cells. In this study, we investigated the effect of TRAIL on human prostate normal and cancer cell lines, and found that the prostate cancer cell lines PC-3, ALVA-31, DU 145 and TSU-Pr1 were sensitive to TRAIL-induced apoptosis, while normal PrEC cells and cancer cell line LNCaP were resistant. No correlation was found between the sensitivity of cells to TRAIL and the expression of TRAIL receptors DR4 and DR5, and pro-apoptotic proteins Bax and Bak. However, LNCaP cells displayed a high Akt activity. Furthermore, we found that endothelial nitric oxide synthase (eNOS), one of the Akt substrates, was highly expressed in LNCaP but not in other cells. Inhibition of eNOS activity by NOS inhibitor sensitized LNCaP cells to TRAIL. Moreover, PC-3 cell clones stably expressing eNOS were resistant to TRAIL-induced apoptosis. Taken together, these results indicate that eNOS can regulate the sensitivity of prostate cancer cells to TRAIL, and down-regulation of eNOS activity may sensitize prostate cancer cells to TRAIL-based therapy.     

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.     

A novel bis-indole destabilizes microtubules and displays potent in vitro and in vivo antitumor activity in prostate cancer

Purpose

Microtubules are one of the most useful subcellular targets in chemotherapy. We identified a novel indole, (3-(1H-indol-2-yl)phenyl)(1H-indol-2-yl)methanone (15), that inhibits tubulin action and exhibits potent antitumor activity in various preclinical models.

Methods

In vitro cancer cell growth inhibition was measured by SRB or MTT assay in human cancer cell lines. Apoptosis induced by 15 was examined in LNCaP and PC-3 cells. Effects of 15 on cell cycle distribution and tubulin were investigated via in vitro models. In vivo toxicity and xenograft efficacy studies were conducted in mice.

Results

Indole 15 inhibited the in vitro growth of a number of human cancer cell lines, including drug-resistant cell lines that over-express P-glycoprotein, multidrug resistance-associated proteins, and breast cancer resistance protein with IC₅₀ values in the range of 34?C162?nM. Nanomolar concentrations of the compound caused down-regulation of bcl-2, induced PARP cleavage, and induced apoptosis in both LNCaP and PC-3 prostate cancer cells, as confirmed by anti-histone ELISA and DNA laddering. In vitro studies revealed that the compound inhibited polymerization of purified tubulin and induced a strong and concentration-dependent G₂M arrest in PC-3 cells. In vivo studies in immunodeficient mice bearing PC-3 tumor xenografts showed that the compound effectively inhibited tumor growth.

Conclusions

The potent in vitro and in vivo antitumor activities of this novel indole suggest that drugs with this novel chemical scaffold might be developed for treatment of drug-resistant prostate cancer.     

Perifosine induces differentiation and cell death in prostate cancer cells

We analyzed the mechanism of action for perifosine (D-21266), a new synthetic alkylphospholipid Akt inhibitor, using LNCaP and PC-3 prostate cancer cells. Perifosine treatment of PC-3 cells resulted in cytostatic and cytotoxic effects. Cytostatic effects were characterized by cell growth arrest, cell cycle block, and morphological changes, such as a cell enlargement and granulation, hallmarks of differentiating PC-3 cells. Specific differentiation markers including prostasomal, secretory and plasma membrane proteins, and keratins were induced by perifosine. Among them, we detected strong induction and secretion of CEACAM5 protein. In contrast, perifosine strongly reduced caveolin-1 RNA levels. Cytotoxic effects included para-apoptosis, apoptosis, and necrosis. To pursue the mechanisms responsible for these activities we focused on signaling pathways that lie downstream of Akt. Perifosine-triggered GSK-3beta activation in PC-3 and LNCaP cells resulted in the expression of GSK-3beta-related differentiation markers. This expression was reduced in the presence of specific siRNA for GSK-3beta or for its target CREB protein. The use of the GSK-3beta inhibitor lithium chloride indicated that GSK-3beta partially protects prostate cancer cells from the cytotoxic effects of perifosine. Together, these findings indicate that perifosine induces GSK-3beta-related differentiation and caspase-independent cell death in prostate cancer PC-3 cells. In addition our results identify specific biomarkers for perifosine therapy.     

Tissue prostate-specific antigen facilitates refractory prostate tumor progression via enhancing ARA70-regulated androgen receptor transactivation

Despite being well recognized as the best biomarker for prostate cancer, pathophysiologic roles of prostate-specific antigen (PSA) remain unclear. We report here that tissue PSA may be involved in the hormone-refractory prostate cancer progression. Histologic analyses show that the increased tissue PSA levels are correlated with lower cell apoptosis index and higher cell proliferation rate in hormone-refractory tumor specimens. By stably transfecting PSA cDNA into various prostate cancer cell lines, we found that PSA could promote the growth of androgen receptor (AR)-positive CWR22rv1 and high-passage LNCaP (hormone-refractory prostate cancer cells) but not that of AR-negative PC-3 and DU145 cells. Surprisingly, the protease activity of PSA is not crucial for PSA to stimulate growth and promote AR transactivation. We further showed that increased PSA could enhance ARA70-induced AR transactivation via modulating the p53 pathway that results in the decreased apoptosis and increased cell proliferation in prostate cancer cells. Knockdown of PSA in LNCaP and CWR22rv1 cells causes cell apoptosis and cell growth arrest at the G(1) phase. In vitro colony formation assay and in vivo xenografted tumor results showed the suppression of prostate cancer growth via targeting PSA expression. Collectively, our findings suggest that, in addition to being a biomarker, PSA may also become a new potential therapeutic target for prostate cancer. PSA small interfering RNA or smaller molecules that can degrade PSA protein may be developed as alternative approaches to treat the prostate cancer.     

雷公藤红素对人前列腺癌细胞凋亡及SENP1基因表达的影响

目的：研究雷公藤红素对人前列腺癌（PCa）细胞生长、凋亡及SUMO特异蛋白酶1（SUMO-specificproteases 1,SENP1）基因表达的影响。方法：对前列腺癌细胞PC-3和LNCaP进行雷公藤红素处理,通过测定细胞生长曲线,荧光染色,荧光显微镜观察,流式细胞仪分析和实时定量PCR,检测雷公藤红素对前列腺癌细胞生长、凋亡及SENP1 mRNA表达水平的影响。结果：雷公藤红素能够显著抑制PCa细胞的生长,并且抑制作用呈剂量依赖性;雷公藤红素能够诱导PCa细胞凋亡,1μmol/L雷公藤红素处理24h诱导14.8%的PC-3细胞和23.2%的LNCaP细胞发生凋亡或死亡;雷公藤红素还能够降低PCa细胞中SENP1 mRNA水平,尤其是PC-3细胞。结论：雷公藤红素能够显著抑制PCa细胞的生长并诱导细胞凋亡,表明雷公藤红素具有抗前列腺癌作用,雷公藤红素能够降低PCa细胞中SENP1 mRNA水平,揭示雷公藤红素可能通过SENP1相关信号通路达到抗前列腺癌作用。     

Enhanced antitumor activity of low-dose continuous administration schedules of topotecan in prostate cancer

Purpose

The objective of this study was to determine the antitumor effects of alternate dosing schedules of topotecan in prostate cancer.

Results

A concentration-dependent increase in cytotoxicity was observed in PC-3 and LNCaP cells after topotecan treatment using conventional and metronomic protocols. A significant increase in potency (2.4–18 fold, after 72 h) was observed following metronomic dosing compared with conventional dosing administration in both cell lines. Metronomic dosing also increased the percentage of PC-3 cells in the G₂/M, compared with control, but did not alter LNCaP cell cycle distribution. Metronomic dosing increased p21 protein expression in LNCaP and PC-3 cells compared with conventional dosing. The observed in vitro activity was confirmed using an in vivo model of human prostate cancer. Metronomic dosing and continuous infusion decreased tumor volume significantly (p ≤ 0.05) compared with control and conventional topotecan treatment, but had no effect on tumor vascular staining.

Methods

The cytotoxicity of topotecan after conventional or metronomic dosing was determined by examining cellular morphology, mitochondrial enzymatic activity (MTT), total cellular protein (SRB), annexin V and propidium iodine (PI) staining, cell cycle and protein gel blot analysis in human prostate cancer cell lines (PC-3 and LNCaP) and the effects metronomic or continuous infusion on tumor growth in an in vivo tumor xenograft model.

Conclusions

These data support the hypothesis that low-dose continuous administration of topotecan increases potency compared with conventional dosing in prostate cancer. These data also suggest the novel finding that the enhanced antitumor activity of topotecan following low-dose exposure correlates to alterations in cell cycle and increased p21 expression.     

The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level

Metformin is a widely used antidiabetic agent, which regulates glucose homeostasis through inhibition of liver glucose production and an increase in muscle glucose uptake. Recent studies suggest that metformin may reduce the risk of cancer, but its mode of action in cancer remains not elucidated. We investigated the effect of metformin on human prostate cancer cell proliferation in vitro and in vivo. Metformin inhibited the proliferation of DU145, PC-3 and LNCaP cancer cells with a 50% decrease of cell viability and had a modest effect on normal prostate epithelial cell line P69. Metformin did not induce apoptosis but blocked cell cycle in G(0)/G(1). This blockade was accompanied by a strong decrease of cyclin D1 protein level, pRb phosphorylation and an increase in p27(kip) protein expression. Metformin activated the AMP kinase pathway, a fuel sensor signaling pathway. However, inhibition of the AMPK pathway using siRNA against the two catalytic subunits of AMPK did not prevent the antiproliferative effect of metformin in prostate cancer cells. Importantly, oral and intraperitoneal treatment with metformin led to a 50 and 35% reduction of tumor growth, respectively, in mice bearing xenografts of LNCaP. Similar, to the in vitro study, metformin led to a strong reduction of cyclin D1 protein level in tumors providing evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent epidemiological studies.     

Guggulsterone-induced apoptosis in human prostate cancer cells is caused by reactive oxygen intermediate dependent activation of c-Jun NH2-terminal kinase

Guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, causes apoptosis in cancer cells but the sequence of events leading to cell death is poorly understood. We now show that guggulsterone-induced cell death in human prostate cancer cells is caused by reactive oxygen intermediate (ROI)-dependent activation of c-Jun NH(2)-terminal kinase (JNK). Exposure of PC-3 and LNCaP cells to apoptosis inducing concentrations of guggulsterone resulted in activation of JNK and p38 mitogen-activated protein kinase (p38 MAPK) in both cell lines and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in LNCaP cells. The guggulsterone-induced apoptosis in PC-3/LNCaP cells was partially but statistically significantly attenuated by pharmacologic inhibition (SP600125) as well as genetic suppression of JNK activation. On the other hand, pharmacologic inhibition of p38 MAPK activation in PC-3 or LNCaP cells (SB202190) and ERK1/2 activation in LNCaP cells (PD98059) did not protect against guggulsterone-induced cell death. The guggulsterone treatment caused generation of ROI in prostate cancer cells but not in a normal prostate epithelial cell line (PrEC), which was also resistant to guggulsterone-mediated JNK activation. The guggulsterone-induced JNK activation as well as cell death in prostate cancer cells was significantly attenuated by overexpression of catalase and superoxide dismutase. In addition, guggulsterone treatment resulted in a decrease in protein level and promoter activity of androgen receptor in LNCaP cells. In conclusion, the present study reveals that the guggulsterone-induced cell death in human prostate cancer cells is regulated by ROI-dependent activation of JNK and guggulsterone inhibits promoter activity of androgen receptor.