Troglitazone suppresses telomerase activity independently of PPARγ in estrogen-receptor negative breast cancer cells

Background  

Breast cancer is one the highest causes of female cancer death worldwide. Many standard chemotherapeutic agents currently used to treat breast cancer are relatively non-specific and act on all rapidly dividing cells. In recent years, more specific targeted therapies have been introduced. It is known that telomerase is active in over 90% of breast cancer tumors but inactive in adjacent normal tissues. The prevalence of active telomerase in breast cancer patients makes telomerase an attractive therapeutic target. Recent evidence suggests that telomerase activity can be suppressed by peroxisome proliferator activated receptor gamma (PPARγ). However, its effect on telomerase regulation in breast cancer has not been investigated.     

Withaferin a suppresses estrogen receptor-α expression in human breast cancer cells

We have shown previously that withaferin A (WA), a promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits growth of MCF-7 and MDA-MB-231 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo by causing apoptosis. However, the mechanism of WA-induced apoptosis is not fully understood. The present study was designed to systematically determine the role of tumor suppressor p53 and estrogen receptor-α (ER-α) in proapoptotic response to WA using MCF-7, T47D, and ER-α overexpressing MDA-MB-231 cells as a model. WA treatment resulted in induction as well as increased S15 phosphorylation of p53 in MCF-7 cells, but RNA interference of this tumor suppressor conferred modest protection at best against WA-induced apoptosis. WA-mediated growth inhibition and apoptosis induction in MCF-7 cells were significantly attenuated in the presence of 17β-estradiol (E2). Exposure of MCF-7 cells to WA resulted in a marked decrease in protein levels of ER-α (but not ER-β) and ER-α regulated gene product pS2, and this effect was markedly attenuated in the presence of E2. WA-mediated down-regulation of ER-α protein expression correlated with a decrease in its nuclear level, suppression of its mRNA level, and inhibition of E2-dependent activation of ERE2e1b-luciferase reporter gene. Ectopic expression of ER-α in the MDA-MB-231 cell line conferred partial but statistically significant protection against WA-mediated apoptosis, but not G2/M phase cell cycle arrest. Collectively, these results indicate that WA functions as an anti-estrogen, and the proapoptotic effect of this promising natural product is partially attenuated by p53 knockdown and E2-ER-α.     

Troglitazone, a PPAR agonist, inhibits human prostate cancer cell growth through inactivation of NFκB via suppression of GSK-3β expression

PPARγ ligands have been reported to reduce proliferation of human prostate cancer cells. However, the molecular mechanism of PPARγ agonist-induced cell growth inhibition of prostate cancer cells is not clear. GSK-3β expression and NFκB activity have important roles in prostate cancer development. To investigate the mechanisms of the PPARγ agonist-induced prostate cancer cell growth inhibition, we examined the effect of troglitazone on the expression of PPARγ, GSK-3β and activity of NFκB as well as on the prostate cancer cell growth. Troglitazone induced the expression of PPARγ in the nuclear of PC-3 cells, but not in LNCaP cells. Troglitazone (0-16 uM) inhibited cancer cell growth in a similar extend between both cells accompanied by the induction of cell cycle arrest in G(0)/G(1) phase and an increased in the similar extent of apoptotic cell death in concentration dependent manner. Troglitazone inhibited the constitutive expression of GSK-3β and activation of NFκB. Co-treatment of troglitazone with a GSK-3β inhibitor (AR-a014418) or GSK-3β siRNA significantly augmented the inhibitory effect of troglitazone on the NFκB activity and on prostate cancer cell growth inhibition and apoptotic cell death. However, overexpression of GSK-3β hindered troglitazone-induced cell growth inhibition and NFκB inactivation. These results suggest that PPARγ agonist, troglitazone, inhibits prostate cancer cell growth through inactivation of NFκB via suppression of GSK-3β expression.     

Cancer stem cells in human colon cancer

Cancer is composed of heterogeneous cell populations.Not every cell in the tumor has the capacity to initiate and sustain tumor growth.It has been postulated that only a subset of cells,the so-called cancer stem cells (CSCs),are able to initiate and propagate tumor development.In two papers published on Nov.19,2006 issue of Nature(doi:10.1038/nature 05372,and doi:10.1038/nature 05384),CSCs are shown to exist in human colon cancer.CSCs have the capacity to regenerate themselves and produce non-CSC progeny.To examine whether colon     

Serum estrogen levels and prostate cancer risk in the prostate cancer prevention trial: a nested case�Ccontrol study

Objective

Finasteride reduces prostate cancer risk by blocking the conversion of testosterone to dihydrotestosterone. However, whether finasteride affects estrogens levels or change in estrogens affects prostate cancer risk is unknown.

Methods

These questions were investigated in a case?Ccontrol study nested within the prostate cancer prevention trial (PCPT) with 1,798 biopsy-proven prostate cancer cases and 1,798 matched controls.

Results

Among men on placebo, no relationship of serum estrogens with risk of prostate cancer was found. Among those on finasteride, those in the highest quartile of baseline estrogen levels had a moderately increased risk of Gleason score < 7 prostate cancer (for estrone, odds ratio [OR] = 1.51, 95% confidence interval [CI] = 1.06?C2.15; for estradiol, OR = 1.50, 95% CI = 1.03?C2.18). Finasteride treatment increased serum estrogen concentrations; however, these changes were not associated with prostate cancer risk.

Conclusion

Our findings confirm those from previous studies that there are no associations of serum estrogen with prostate cancer risk in untreated men. In addition, finasteride results in a modest increase in serum estrogen levels, which are not related to prostate cancer risk. Whether finasteride is less effective in men with high serum estrogens, or finasteride interacts with estrogen to increase cancer risk, is uncertain and warrants further investigation.     

Suppression of growth and invasive behavior of human prostate cancer cells by ProstaCaid™: mechanism of activity

Since the use of dietary supplements as alternative treatments or adjuvant therapies in cancer treatment is growing, a scientific verification of their biological activity and the detailed mechanisms of their action are necessary for the acceptance of dietary supplements in conventional cancer treatments. In the present study we have evaluated the anti-cancer effects of dietary supplement ProstaCaid? (PC) which contains mycelium from medicinal mushrooms (Ganoderma lucidum, Coriolus versicolor, Phellinus linteus), saw palmetto berry, pomegranate, pumpkin seed, green tea [40% epigallocatechin-3-gallate (EGCG)], Japanese knotweed (50% resveratrol), extracts of turmeric root (BCM-95?), grape skin, pygeum bark, sarsaparilla root, Scutellaria barbata, eleuthero root, Job's tears, astragalus root, skullcap, dandelion, coptis root, broccoli, and stinging nettle, with purified vitamin C, vitamin D3, selenium, quercetin, citrus bioflavonoid complex, β sitosterolzinc, lycopene, α lipoic acid, boron, berberine and 3.3'-diinodolymethane (DIM). We show that PC treatment resulted in the inhibition of cell proliferation of the highly invasive human hormone refractory (independent) PC-3 prostate cancer cells in a dose- and time-dependent manner with IC50 56.0, 45.6 and 39.0 μg/ml for 24, 48 and 72 h, respectively. DNA-microarray analysis demonstrated that PC inhibits proliferation through the modulation of expression of CCND1, CDK4, CDKN1A, E2F1, MAPK6 and PCNA genes. In addition, PC also suppresses metastatic behavior of PC-3 by the inhibition of cell adhesion, cell migration and cell invasion, which was associated with the down-regulation of expression of CAV1, IGF2, NR2F1, and PLAU genes and suppressed secretion of the urokinase plasminogen activator (uPA) from PC-3 cells. In conclusion, the dietary supplement PC is a promising natural complex with the potency to inhibit invasive human prostate cancer.     

Transforming growth factor-ß suppresses metastasis in a subset of human colon carcinoma cells

ABSTRACT: BACKGROUND: TGFbeta signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy. However, we present evidence here for an anti-metastatic role of TGFbeta signaling. METHODS: To test the importance of TGFbeta signaling to cell survival and metastasis we compared human colon carcinoma cell lines that are either non-tumorigenic with TGFbeta response (FET), or tumorigenic with TGFbeta response (FETalpha) or tumorigenic with abrogated TGFbeta response via introduction of dominant negative TGFbetaRII (FETalpha/DN) and their ability to metastasize. Metastatic competency was assessed by orthotopic transplantation. Metastatic colony formation was assessed histologically and by imaging. RESULTS: Abrogation of TGFbeta signaling through introduction of a dominant negative TGFbeta receptor II (TGFbetaRII) in non-metastatic in FETalpha human colon cancer cells permits metastasis to distal organs, but importantly does not reduce invasive behavior at the primary site. Loss of TGFbeta signaling in FETalpha-DN cells generated enhanced cell survival capabilities in response to cellular stress in vitro. We show that enhanced cellular survival is associated with increased AKT phosphorylation and cytoplasmic expression of inhibitor of apoptosis (IAP) family members (survivin and XIAP) that elicit a cytoprotective effect through inhibition of caspases in response to stress. To confirm that TGFbeta signaling is a metastasis suppressor, we rescued TGFbeta signaling in CBS metastatic colon cancer cells that had lost TGFbeta receptor expression due to epigenetic repression. Restoration of TGFbeta signaling resulted in the inhibition of metastatic colony formation in distal organs by these cells. These results indicate that TGFbeta signaling has an important role in the suppression of metastatic potential in tumors that have already progressed to the stage of an invasive carcinoma. CONCLUSIONS: The observations presented here indicate a metastasis suppressor role for TGFbeta signaling in human colon cancer cells. This raises the concern that therapies targeting inhibition of TGFbeta signaling may be imprudent in some patient populations with residual TGFbeta tumor suppressor activity.     

Androgen receptor expression in prostate cancer stem cells: is there a conundrum?

Androgen deprivation therapy (ADT) is standard frontline therapy for metastatic prostate cancer. However, prostate cancer progresses to a castrate-resistant state. The response of prostate cancer to androgen deprivation is mediated by the androgen receptor (AR). Castrate-resistant disease is marked by a gain-of-function in AR and AR reactivation. The stem cell hypothesis of cancer would therefore predict that AR should be expressed in the prostate cancer stem cell, since genetic selection for gain-of-function changes in AR, such as AR gene amplification, should occur at the level of the stem cell population. Initial reports characterizing prostate cancer stem cells suggest that AR is not expressed in this population, which is an apparent conundrum. Here, we examined the CD44+/24- LNCaP putative stem cell population by in-cell Western and show that AR is expressed at the protein level. Our findings suggest that at least a subset of prostate cancers express AR in the putative stem cell population.     

PPARγ potentiates anticancer effects of gemcitabine on human pancreatic cancer cells

In order to improve the prognosis of patients with unresectable pancreatic cancer, there is an urgent need for enhancement of the anticancer effect of gemcitabine (Gem), a first-line drug for the disease. Here, we demonstrated that ligands for peroxisome proliferator-activated receptor γ (PPARγ) such as pioglitazone (Pio) and rosiglitazone potentiated the cytotoxic action of Gem on human pancreatic cancer cells in a dosage-dependent manner. Notably, the synergistic effect was PPARγ-dependent, since the effect was augmented by PPARγ overexpression and was attenuated by both a PPARγ inhibitor (GW9662) and PPARγ-specific siRNA. To further increase the collaborative effect, the histone deacetylase (HDAC) inhibitor valproic acid (VPA), a known potentiator for PPARγ function, was added to the combinatorial treatment, robustly inducing apoptosis mediated by highly expressed death receptors, including Fas/CD95 and DR5. In xenograft tumor experiments in nude mice, Gem plus Pio significantly suppressed tumor growth as compared with the control treatment, while Gem-only treatment did not. Triple treatment with Gem, Pio, and VPA failed to demonstrate a significant antitumor effect when compared with Gem plus Pio in the current setting. Considered together, Gem plus PPARγ ligands, including Pio, may have therapeutic advantage in the treatment of advanced pancreatic cancer. Since Pio is widely used in the treatment of diabetes mellitus, it may become a feasible partner of Gem-based chemotherapy, fine-tuning the strength of the therapy in a dosage-dependent fashion.     

17β-estradiol regulates giant vesicle formation via estrogen receptor-alpha in human breast cancer cells

A significant proportion of the genes regulated by 17-beta-estradiol (E2) via estrogen receptor alpha (ERα) have roles in vesicle trafficking in breast cancer. Intracellular vesicle trafficking and extracellular vesicles have important roles in tumourigenesis. Here we report the discovery of giant (3-42μm) intracellular and extracellular vesicles (GVs) and the role of E2 on vesicle formation in breast cancer (BC) cell lines using three independent live cell imaging techniques. Large diameter vesicles, GVs were also identified in a patient-derived xenograft BC model, and in invasive breast carcinoma tissue. ERα-positive (MCF-7 and T47D) BC cell lines demonstrated a significant increase in GV formation after stimulation with E2 which was reversed by tamoxifen. ERα-negative (MDA-MB-231 and MDA-MB-468) BC cell lines produced GVs independently of E2 and tamoxifen. These results indicate the existence of both intracellular and extracellular vesicles with considerably larger dimensions than generally recognised with BC cells and suggest that the GVs are regulated by E2 via ERα in ERα-positive BC but by E2-independent mechanisms in ER-ve BC.     

Increased expression of enolase α in human breast cancer confers tamoxifen resistance in human breast cancer cells

Enolase-α (ENO-1) is a key glycolytic enzyme that has been used as a diagnostic marker to identify human lung cancers. To investigate the role of ENO-1 in breast cancer diagnosis and therapy, the mRNA levels of ENO-1 in 244 tumor and normal paired tissue samples and 20 laser capture-microdissected cell clusters were examined by quantitative real-time PCR analysis. Increased ENO-1 mRNA expression was preferentially detected in estrogen receptor-positive (ER+) tumors (tumor/normal ratio >90-fold) when compared to ER-negative (tumor/normal ratio >20-fold) tumor tissues. The data presented here demonstrate that those patients whose tumors highly expressed ENO-1 had a poor prognosis with greater tumor size (>2 cm, *P = .017), poor nodal status (N > 3, *P = .018), and a shorter disease-free interval (≦1 year, *P < .009). We also found that higher-expressing ENO-1 tumors confer longer distance relapse (tumor/normal ratio = 82.8–92.4-fold) when compared to locoregional relapse (tumor/normal ratio = 43.4-fold) in postsurgical 4-hydroxy-tamoxifen (4-OHT)-treated ER+ patients (*P = .014). These data imply that changes in tumor ENO-1 levels are related to clinical 4-OHT therapeutic outcome. In vitro studies demonstrated that decreasing ENO-1 expression using small interfering RNA (siRNA) significantly augmented 4-OHT (100 nM)-induced cytotoxicity in tamoxifen-resistant (Tam-R) breast cancer cells. These results suggest that downregulation of ENO-1 could be utilized as a novel pharmacological approach for overcoming 4-OHT resistance in breast cancer therapy.     

Disruption of ERα signalling pathway by PPARγ agonists: evidences of PPARγ-independent events in two hormone-dependent breast cancer cell lines

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that can be activated by natural ligands such as 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ(2)) as well as synthetic drugs such as thiazolidinediones. The treatment of human breast cancer cell lines with PPARγ agonists is known to have antiproliferative effects but the role of PPARγ activation in the process remains unclear. In the present study, we investigated the effects of four PPARγ agonists, Rosiglitazone (RGZ), Ciglitazone (CGZ), Troglitazone (TGZ) and the natural agonist 15d-PGJ(2), on estrogen receptor alpha (ERα) signalling pathway in two hormone-dependent breast cancer cell lines, MCF-7 and ZR-75-1. In both of them, TGZ, CGZ and 15d-PGJ(2) induced an inhibition of ERα signalling associated with the proteasomal degradation of ERα. ZR-75-1 cells were more sensitive than MCF-7 cells to these compounds. Treatments that induced ERα degradation inhibited cell proliferation after 24 h. In contrast, 24 h exposure to RGZ, the most potent activator of PPARγ disrupted neither ERα signalling nor cell proliferation. 9-cis retinoic acid never potentiated the proteasomal degradation of ERα. PPARγ antagonists (T0070907, BADGE and GW 9662) did not block the proteolysis of ERα in MCF-7 and ZR-75-1 cells treated with TGZ. ERα proteolysis still occurred in case of PPARγ silencing as well as in case of treatment with the PPARγ-inactive compound Δ2-TGZ, demonstrating a PPARγ-independent mechanism. The use of thiazolidinedione derivatives able to trigger ERα degradation by a PPARγ-independent pathway could be an interesting tool for breast cancer therapy.     

XMRV: a new virus in prostate cancer?

Several recent articles have reported the presence of a gammaretrovirus, termed "XMRV" (xenotropic murine leukemia virus-related virus) in prostate cancers (PCa). If confirmed, this could have enormous implications for the detection, prevention, and treatment of PCa. However, other articles report failure to detect XMRV in PCa. We tested nearly 800 PCa samples, using a combination of real-time PCR and immunohistochemistry (IHC). The PCR reactions were simultaneously monitored for amplification of a single-copy human gene, to confirm the quality of the sample DNA and its suitability for PCR. Controls showed that the PCR assay could detect the XMRV in a single infected cell, even in the presence of a 10,000-fold excess of uninfected human cells. The IHC used 2 rabbit polyclonal antisera, each prepared against a purified murine leukemia virus (MLV) protein. Both antisera always stained XMRV-infected or -transfected cells, but never stained control cells. No evidence for XMRV in PCa was obtained in these experiments. We discuss possible explanations for the discrepancies in the results from different laboratories. It is possible that XMRV is not actually circulating in the human population; even if it is, the data do not seem to support a causal role for this virus in PCa.     

Acetylbritannilactone suppresses growth via upregulation of krüppel-like transcription factor 4 expression in HT-29 colorectal cancer cells

Acetylbritannilactone (ABL) is a new active compound isolated from Inula Britannica L, a traditional Chinese medicinal herb. It has been reported that ABL can inhibit the proliferation of vascular smooth muscle cells (VSMCs) and neointima formation after balloon injury in rats. ABL also shows chemopreventive properties by inducing cell apoptosis in breast and ovarian cancers, but the antitumor activity and the molecular targets of ABL in colon cancer cells have not been determined. In this study, we showed that ABL inhibits the growth in dose- and time-dependent manners by inducing cell cycle arrest in G0/G1 phase of HT-29 human colon cancer cells. This suppression was accompanied by a strong decrease of cyclin E and CDK4 protein levels, and an increase in p21 protein expression in HT-29 cells. We also show that ABL-induced growth inhibition is associated with the upregulation of KLF4 expression. The overexpression of KLF4 by infection with pAd-KLF4 resulted in growth inhibition, with decrease in the protein levels of cyclin E and CDK4, and increase in the expression of p21, similarly to the effects of ABL. Conversely, knockdown of KLF4 using a specific siRNA impaired the ABL-induced growth inhibition in HT-29 cells. These results suggest that KLF4 as an important cellular target of ABL mediates the growth inhibition of HT-29 cells induced by ABL via upregulation of p21 expression.     

Tumor-suppressive microRNA-497 targets IKKβ to regulate NF-κB signaling pathway in human prostate cancer cells

Background: Prostate cancer (PCa) is one of the most prevalent malignant tumors, PCa-related death is mainly due to the high probability of metastasis. MicroRNAs (miRNAs) play an important role in cancer initiation, progression and metastasis by regulating their target genes. Methods: real-time PCR was used to detected the expression of microRNA-497. The molecular biological function was investigated by using cell proliferation assays, cell cycle assay, and migration and invasion assay. We used several Algorithms and confirmed that IKKβ is directly regulated by miR-497. Results: Here, we found miR-497 is downregulated in human prostate cancer (PCa) and inhibites the proliferation activity, migration and invasion of PC3-AR cells. Subsequently, IKKβ is confi rmed as a target of miR-497. Furthermore, knockdown of IKKβ expression resulted in decreased proliferation activity, migration and invasion. Finally, similar results was found after treatment with a novel IKK-β inhibitor (IMD-0354) in PC3-AR cells. CDK8, MMP-9, and PSA were involved in all these process. Conclusion: Taken together, our results show evidence that miR-497 may function as a tumor suppressor genes by regulating IKK-β in PCa, and may provide a strategy for blocking PCa metastasis.