Melissa A. Babcook

Apigenin inhibits prostate cancer progression in TRAMP mice via targeting PI3K/Akt/FoxO pathway

Forkhead box O (FoxO) transcription factors play an important role as tumor suppressor in several human malignancies. Disruption of FoxO activity due to loss of phosphatase and tensin homolog and activation of phosphatidylinositol-3 kinase (PI3K)/Akt are frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits antiproliferative and anticarcinogenic activities through mechanisms, which are not fully defined. In the present study, we show that apigenin suppressed prostate tumorigenesis in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice through the PI3K/Akt/FoxO-signaling pathway. Apigenin-treated TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate as well as completely abolished distant organ metastasis. Apigenin treatment resulted in significant decrease in the weight of genitourinary apparatus (P < 0.0001), dorsolateral (P < 0.0001) and ventral prostate (P < 0.028), compared with the control group. Apigenin-treated mice showed reduced phosphorylation of Akt (Ser473) and FoxO3a (Ser253), which correlated with its increased nuclear retention and decreased binding of FoxO3a with 14-3-3. These events lead to reduced proliferation as assessed by Ki-67 and cyclin D1, along with upregulation of FoxO-responsive proteins BIM and p27/Kip1. Complementing in vivo results, similar observations were noted in human prostate cancer LNCaP and PC-3 cells after apigenin treatment. Furthermore, binding of FoxO3a with p27/Kip1 was markedly increased after 10 and 20 μM apigenin treatment resulting in G0/G1-phase cell cycle arrest, which was consistent with the effects elicited by PI3K/Akt inhibitor, LY294002. These results provide convincing evidence that apigenin effectively suppressed prostate cancer progression, at least in part, by targeting the PI3K/Akt/FoxO-signaling pathway.

Plant Phytochemicals as Epigenetic Modulators: Role in Cancer Chemoprevention

In recent years, “nutri-epigenetics,” which focuses on the influence of dietary agents on epigenetic mechanism(s), has emerged as an exciting novel area in epigenetics research. Targeting of aberrant epigenetic modifications has gained considerable attention in cancer chemoprevention research because, unlike genetic changes, epigenetic alterations are reversible and occur during early carcinogenesis. Aberrant epigenetic mechanisms, such as promoter DNA methylation, histone modifications, and miRNA-mediated post-transcriptional alterations, can silence critical tumor suppressor genes, such as transcription factors, cell cycle regulators, nuclear receptors, signal transducers, and apoptosis-inducing and DNA repair gene products, and ultimately contribute to carcinogenesis. In an effort to identify and develop anticancer agents which cause minimal harm to normal cells while effectively killing cancer cells, a number of naturally occurring phytochemicals in food and medicinal plants have been investigated. This review highlights the potential role of plant-derived phytochemicals in targeting epigenetic alterations that occur during carcinogenesis, by modulating the activity or expression of DNA methyltransferases, histone modifying enzymes, and miRNAs. We present in detail the epigenetic mode of action of various phytochemicals and discuss their potential as safe and clinically useful chemopreventive strategies.

Synergistic Simvastatin and Metformin Combination Chemotherapy for Osseous Metastatic Castration-Resistant Prostate Cancer

Docetaxel chemotherapy remains a standard of care for metastatic castration-resistant prostate cancer (CRPC). Docetaxel modestly increases survival, yet results in frequent occurrence of side effects and resistant disease. An alternate chemotherapy with greater efficacy and minimal side effects is needed. Acquisition of metabolic aberrations promoting increased survival and metastasis in CRPC cells includes constitutive activation of Akt, loss of adenosine monophosphate-activated protein kinase (AMPK) activity due to Ser-485/491 phosphorylation, and overexpression of 3-hydroxy-3-methylglutaryl–Coenzyme A reductase (HMG-CoAR). We report that combination of simvastatin and metformin, within pharmacologic dose range (500 nmol/L to 4 μmol/L simvastatin and 250 μmol/L to 2 mmol/L metformin), significantly and synergistically reduces C4-2B3/B4 CRPC cell viability and metastatic properties, with minimal adverse effects on normal prostate epithelial cells. Combination of simvastatin and metformin decreased Akt Ser-473 and Thr-308 phosphorylation and AMPKα Ser-485/491 phosphorylation; increased Thr-172 phosphorylation and AMPKα activity, as assessed by increased Ser-79 and Ser-872 phosphorylation of acetyl-CoA carboxylase and HMG-CoAR, respectively; decreased HMG-CoAR activity; and reduced total cellular cholesterol and its synthesis in both cell lines. Studies of C4-2B4 orthotopic NCr-nu/nu mice further demonstrated that combination of simvastatin and metformin (3.5–7.0 μg/g body weight simvastatin and 175–350 μg/g body weight metformin) daily by oral gavage over a 9-week period significantly inhibited primary ventral prostate tumor formation, cachexia, bone metastasis, and biochemical failure more effectively than 24 μg/g body weight docetaxel intraperitoneally injected every 3 weeks, 7.0 μg/g/day simvastatin, or 350 μg/g/day metformin treatment alone, with significantly less toxicity and mortality than docetaxel, establishing combination of simvastatin and metformin as a promising chemotherapeutic alternative for metastatic CRPC. Mol Cancer Ther; 13(10); 2288–302. ©2014 AACR.

Green Tea Polyphenols Induce p53-Dependent and p53- Independent Apoptosis in Prostate Cancer Cells through Two Distinct Mechanisms

Inactivation of the tumor suppressor gene p53 is commonly observed in human prostate cancer and is associated with therapeutic resistance. We have previously demonstrated that green tea polyphenols (GTP) induce apoptosis in prostate cancer cells irrespective of p53 status. However, the molecular mechanisms underlying these observations remain elusive. Here we investigated the mechanisms of GTP-induced apoptosis in human prostate cancer LNCaP cells stably-transfected with short hairpin-RNA against p53 (LNCaPshp53) and control vector (LNCaPshV). GTP treatment induced p53 stabilization and activation of downstream targets p21/waf1 and Bax in a dose-dependent manner specifically in LNCaPshV cells. However, GTP-induced FAS upregulation through activation of c-jun-N-terminal kinase resulted in FADD phosphorylation, caspase-8 activation and truncation of BID, leading to apoptosis in both LNCaPshV and LNCaPshp53 cells. In parallel, treatment of cells with GTP resulted in inhibition of survival pathway, mediated by Akt deactivation and loss of BAD phosphorylation more prominently in LNCaPshp53 cells. These distinct routes of cell death converged to a common pathway, leading to loss of mitochondrial transmembrane potential, cytochrome c release and activation of terminal caspases, resulting in PARP-cleavage. GTP-induced apoptosis was attenuated with JNK inhibitor, SP600125 in both cell lines; whereas PI3K-Akt inhibitor, LY294002 resulted in increased cell death prominently in LNCaPshp53 cells, establishing the role of two distinct pathways of GTP-mediated apoptosis. Furthermore, GTP exposure resulted in inhibition of class I HDAC protein, accumulation of acetylated histone-H3 in total cellular chromatin, resulting in increased accessibility of transcription factors to bind with the promoter sequences of p21/waf1 and Bax, regardless of the p53 status of cells, consistent with effects elicited by an HDAC inhibitor, trichostatin A. These results demonstrate that GTP induces prostate cancer cell death by two distinct mechanisms regardless of p53 status, thus identifying specific well-defined molecular mechanisms that may be targeted by chemopreventive and/or therapeutic strategies.

Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necrosis in C4-2B osseous metastatic castration-resistant prostate cancer cells

Castration-resistant prostate cancer (CRPC) cells acquire resistance to chemotherapy and apoptosis, in part, due to enhanced aerobic glycolysis and biomass production, known as the Warburg effect. We previously demonstrated that combination simvastatin (SIM) and metformin (MET) ameliorates critical Warburg effect-related metabolic aberrations of C4-2B cells, synergistically and significantly decreases CRPC cell viability and metastatic properties, with minimal effect on normal prostate epithelial cells, and inhibits primary prostate tumor growth, metastasis, and biochemical failure in an orthotopic model of metastatic CRPC, more effectively than docetaxel chemotherapy. Several modes of cell death activated by individual treatment of SIM or MET have been reported; however, the cell death process induced by combination SIM and MET treatment in metastatic CRPC cells remains unknown. This must be determined prior to advancing combination SIM and MET to clinical trial for metastatic CRPC. Treatment of C4-2B cells with combination 4 μM SIM and 2 mM MET (SIM+MET) led to significant G1-phase cell cycle arrest and decrease in the percentage of DNA-replicating cells in the S-phase by 24 h; arrest was sustained throughout the 96-h treatment. SIM+MET treatment led to enhanced autophagic flux in C4-2B cells by 72–96 h, ascertained by increased LC3B-II (further enhanced with lysosomal inhibitor chloroquine) and reduced Sequestosome-1 protein expression, significantly increased percentage of acidic vesicular organelle-positive cells, and increased autophagic structure accumulation assessed by transmission electron microscopy. Chloroquine, however, could not rescue CRPC cell viability, eliminating autophagic cell death; rather, autophagy was upregulated by C4-2B cells in attempt to withstand chemotherapy. Instead, SIM+MET treatment led to Ripk1- and Ripk3-dependent necrosis by 48–96 h, determined by propidium iodide-Annexin V flow cytometry, increase in Ripk1 and Ripk3 protein expression, necrosome formation, HMGB-1 extracellular release, and necrotic induction and viability rescue with necrostatin-1 and Ripk3-targeting siRNA. The necrosis-inducing capacity of SIM+MET may make these drugs a highly-effective treatment for apoptosis- and chemotherapy-resistant metastatic CRPC cells.

Obesity-initiated metabolic syndrome promotes urinary voiding dysfunction in a mouse model

Accumulating evidences suggests that obesity and metabolic syndrome (MetS) contribute towards lower urinary tract symptoms (LUTS) through alterations in the phenotype of bladder and prostate gland. Clinical studies indicate a link between MetS and LUTS. Nevertheless, there is lack of suitable animal model(s) which could illustrate an association linking obesity to LUTS. We examined the lower urinary tract function in an obesity‐initiated MetS mouse model.

Statin Use in Prostate Cancer: An Update.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, known as statins, are commonly prescribed for the treatment of hypercholesterolemia and cardiovascular disease. A systematic review was conducted using the keywords “statin and prostate cancer” within the title search engines including PubMed, Web of Science, and the Cochrane Library for relevant research work published between 2004 and December 2015. Although still premature, accumulating clinical evidence suggests that statin use may be beneficial in the prevention and/or treatment of prostate cancer. These human studies consist of meta-analyses of secondary endpoints obtained from randomized, controlled cardiovascular disease clinical trials of statins, patient database, observational studies, and a few, small case–control studies, directly addressing statin use on prostate cancer pathology and recurrence. This review summarizes and discusses the recent clinical literature on statins and prostate cancer with a recommendation to move forward with randomized, placebo-controlled clinical trials, investigating the use of statins. Additional preclinical testing of statins on prostate cancer cell lines and in vivo models is needed to elucidate pathways and determine its efficacy for prevention and/or treatment of prostate cancer, more specifically, the difference in the effectiveness of lipophilic versus hydrophilic statins in prostate cancer.

Abstract 16: Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necroptosis in C4-2B osseous metastatic castration-resistant prostate cancer cells

Castration-resistant prostate cancer (CRPC) cells acquire resistance to chemotherapy and apoptosis in part due to enhanced aerobic glycolysis and biomass production, known as Warburg effect. We previously demonstrated that combination simvastatin (SIM) and metformin (MET) ameliorates critical Warburg effect-related metabolic aberrations of C4-2B cells, synergistically and significantly decreases CRPC cell viability and metastatic properties, with minimal effect on normal prostate epithelial cells, and inhibits primary prostate tumor growth, metastasis, and biochemical failure in an orthotopic model of metastatic CRPC, more effectively than docetaxel chemotherapy. Several modes of cell death activated by individual treatment of SIM or MET have been reported; however, the cell death process induced by combination SIM and MET treatment in metastatic CRPC cells remains unknown. This must be determined prior to advancing combination SIM and MET to clinical trial for metastatic CRPC. Treatment of C4-2B cells with combination 4μM SIM and 2mM MET (SIM + MET) led to significant G1-phase cell cycle arrest and decrease in percentage of DNA-replicating cells in S-phase by 24h; arrest was sustained throughout 96h treatment. SIM + MET treatment led to enhanced autophagic flux in C4-2B cells by 72-96h, ascertained by increased LC3B-II (further enhanced with lysosomal inhibitor chloroquine) and reduced Sequestosome-1 protein expression, significantly increased percentage acidic vesicular organelle-positive cells, and increased autophagic structure accumulation assessed by transmission electron microscopy. Chloroquine, however, could not rescue CRPC cell viability, eliminating autophagy as a mechanism of cell death; rather, autophagy was upregulated by C4-2B cells in attempt to withstand chemotherapy. Instead, SIM + MET treatment led to Ripk1- and Ripk3-dependent necroptosis by 48-96h, determined by propidium iodide-Annexin V flow cytometry, increase in Ripk1 and Ripk3 protein expression, necrosome formation, HMGB-1 extracellular release, and necroptotic-induction and viability rescue with necrostatin-1 (Ripk1 activity inhibitor) and Ripk3-targeting siRNA. The necroptosis-inducing capacity of SIM + MET may reinvent these drugs as highly-effective treatment for apoptosis- and chemotherapy-resistant metastatic CRPC cells. Citation Format: Melissa A. Babcook, R. Michael Sramkoski, Hisashi Fujioka, Firouz Daneshgari, Alexandru Almasan, Sanjeev Shukla, Sanjay Gupta. Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necroptosis in C4-2B osseous metastatic castration-resistant prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 16. doi:10.1158/1538-7445.AM2015-16

Abstract 5452: High-fat diet induces inflammation by increasing estrogen levels through Stat3, estrogen receptor alpha and aromatase in the mouse prostate.

High-fat diet (HFD), an unfortunate lifestyle choice common in the Western world, is associated with inflammation and thereby is regarded as a risk factor for various diseases including obesity, diabetes, and cancer. The molecular mechanism(s) responsible for HFD-induced inflammation in the prostate gland are not well understood. Estrogen receptor alpha (ER-α), signal transducer and activator of transcription (Stat)-3 and aromatase are important signaling molecules constitutively activated during inflammation and have been shown to be upregulated in prostate cancer. We have previously reported that HFD activates a pro-inflammatory response in the prostate through elevated expression of Stat-3 (Prostate 72:233-43, 2012). In the current study, we sought to investigate a possible link between intraprostatic inflammation, HFD feeding, signaling molecules involved in inflammation viz. ER-α, Stat-3, p-Stat3 (Ser727) and aromatase. C57BL/6 mice were fed either a regular diet (RD) or a HFD for 4, 8 and 12 weeks. Serum, visceral fat and prostate tissues were obtained for analysis. HFD intake caused an increase in fat and serum testosterone levels. No significant changes were observed in the testosterone levels in the prostate. A marked increase in estrogen levels was noted in the prostate and visceral fat at 8 weeks of HFD intake. HFD feeding resulted in a significant increase in the expression of aromatase, ER-α, Stat-3 and p-Stat3 (Ser727) at the message and protein levels. Immunoprecipitation and ChIP analysis demonstrated an increased association between aromatase, p-Stat-3 and ER-α in the HFD group. IHC analysis revealed higher expression of Stat-3, ER-α and aromatase in the prostate, accompanied by the morphologic evidence of increased intraprostatic inflammation in the HFD group. Taken together, our findings suggest that HFD increases estrogen levels by increased binding of ER-α and p-Stat-3 to the promoter of aromatase and their interaction is associated with increased intraprostatic inflammation which might contribute to the initiation and development of prostate cancer. Citation Format: Natarajan Bhaskaran, Sanjeev Shukla, Vijay S. Thakur, Melissa A. Babcook, Gregory T. MacLennan, Guiming Liu, Firouz Daneshgari, Sanjay Gupta. High-fat diet induces inflammation by increasing estrogen levels through Stat3, estrogen receptor alpha and aromatase in the mouse prostate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5452. doi:10.1158/1538-7445.AM2013-5452

Ser-486/491 phosphorylation and inhibition of AMPKα activity is positively associated with Gleason score, metastasis, and castration-resistance in prostate cancer: A retrospective clinical study

We previously demonstrated that adenosine monophosphate‐activated protein kinase (AMPKα) activity is significantly inhibited by Ser‐486/491 phosphorylation in cell culture and in vivo models of metastatic and castration‐resistant prostate cancer, and hypothesized these findings may translate to clinical specimens.