Natarajan Bhaskaran

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.

Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1

The pi‐class glutathione S‐transferase (GSTP1) actively protect cells from carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells. In this study we investigated whether loss of GSTP1 contributes to increased DNA damage that may predispose men to a higher risk of prostate cancer. We found significantly elevated (103%; P < 0.0001) levels of 8‐oxo‐2′‐deoxogunosine (8‐OHdG), an oxidative DNA damage marker, in adenocarcinomas, compared to benign counterparts, which positively correlated (r = 0.2) with loss of GSTP1 activity (34%; P < 0.0001). Silencing of GSTP1 using siRNA approach in normal human prostate epithelial RWPE1 cells caused increased intracellular production of ROS and higher susceptibility of cells to H2O2‐mediated oxidative stress. Additionally, human prostate carcinoma LNCaP cells, which contain a silenced GSTP1 gene, were genetically modified to constitutively express high levels of GSTP1. Induction of GSTP1 activity lowered endogenous ROS levels in LNCaP‐pLPCX‐GSTP1 cells, and when exposed to H2O2, these cells exhibited significantly reduced production of ROS and 8‐OHdG levels, compared to vector control LNCaP‐pLPCX cells. Furthermore, exposure of LNCaP cells to green tea polyphenols caused reexpression of GSTP1, which protected the cells from H2O2‐mediated DNA damage through decreased ROS production compared to nonexposed cells. These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress‐induced DNA damage, may be an important target for primary prevention of prostate cancer.

Plant Flavone Apigenin Binds to Nucleic Acid Bases and Reduces Oxidative DNA Damage in Prostate Epithelial Cells

Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it’s binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2′ deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities.

High-fat diet activates pro-inflammatory response in the prostate through association of Stat-3 and NF-κB

Signal transducer and activator of transcription (Stat)‐3 and nuclear factor‐kappa B (NF‐κB) are important signaling pathways constitutively activated during inflammation. We previously reported that high‐fat diet (HFD) intake induces oxidative stress in the prostate through elevated expression of NADPH oxidase subunits causing NF‐κB activation. We sought to determine whether Stat‐3 is involved in the activation of NF‐κB in the prostate as a result of HFD feeding, leading to inflammation.

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.

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress

AIMS Protection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms. MAIN METHODS The cytoprotective effect of chamomile was examined on H(2)O(2)-induced cellular stress in RAW 264.7 murine macrophages. KEY FINDINGS RAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H(2)O(2). Treatment with 50μM H(2)O(2) for 6h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10-20μg/mL for 16h followed by H(2)O(2) treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus. SIGNIFICANCE These molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties.

Deregulation of FoxO3a accelerates prostate cancer progression in TRAMP mice

Forkhead box, class “O” (FoxO) transcription factors are involved in multiple signaling pathways and possess tumor suppressor functions. Loss of PTEN and activation of PI3K/Akt is frequently observed in prostate cancer, which may potentially inactivate FoxO activity. We therefore investigated the role of FoxO transcription factors in prostate cancer progression, in particular FoxO3a, in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, which mimics progressive forms of human disease.

Isolation of T cells from mouse oral tissues.

BackgroundUtilizing mouse models provides excellent immunological and experimental tools to study oral immune responses. However for functional assays, isolating T lymphocytes from the oral tissues has proved to be challenging due to the absence of reliable methods that yield viable cells with consistency. To study adaptive immune cell interactions in the oral mucosal tissues, it is necessary to isolate T cells with a good viability and study them at the single cell level.FindingsWe have established an improved method to isolate immune cells, including Tregs and Th17 cells from intra-epithelial niches and lamina propria of the tongue, gingival and palatal tissues in the oral mucosa of mice.ConclusionThis new method of isolating immune cells from oral tissues will enable us to further our understanding of oral tissue immune cells and their role during oral infections and oral inflammation.

TLR-2 Signaling Promotes IL-17A Production in CD4+CD25+Foxp3+ Regulatory Cells during Oropharyngeal Candidiasis.

Recent studies show that CD4+CD25+Foxp3+ regulatory cells (Tregs) produce effector cytokines under inflammatory conditions. However, the direct role of microbial agents that serve as toll-like receptor (TLR) ligands in the induction of effector cytokines in Tregs is less clear. Here we show that CD4+Foxp3+Tregs produce the effector cytokine IL-17A during oropharyngeal candidiasis (OPC) and inflammatory bowel disease in a TLR-2/Myd88 signaling dependent manner. TLR-2 ligands promote proliferation in Tregs in the presence and absence of TCR signals and inflammatory cytokines in vitro. The proliferation is directly dependent on TLR-2 expression in Tregs. Consistent with this, Tlr2−/− mice harbor fewer thymically derived Tregs and peripheral Tregs under homeostatic conditions in vivo. However, under Th17 inducing conditions, IL-6 and TLR-2 signaling both in Tregs as well as antigen presenting cells (APC) are critical for maximal ROR-γt and IL-17A up-regulation in Foxp3+ Tregs. The minimal and transient loss of Foxp3 expression and suppressive properties are due to the presence of IL-6 in the milieu, but not the direct effect of TLR-2 signaling in Tregs. Taken together, our data reveal that TLR-2 signaling promotes not only proliferation, but also IL-17A in Tregs, depending on the cytokine milieu. These IL-17A producing Tregs may be relevant in mucosal infections and inflammation.

Chamomile Confers Protection against Hydrogen Peroxide-Induced Toxicity through Activation of Nrf2-Mediated Defense Response

Oxidative stress plays an important role in the development of various human diseases. Aqueous chamomile extract is used as herbal medicine, in the form of tea, demonstrated to possess antiinflammatory and antioxidant properties. We demonstrate the cytoprotective effects of chamomile on hydrogen peroxide (H2O2)‐induced cellular damage in macrophage RAW 264.7 cells. Pretreatment of cells with chamomile markedly attenuated H2O2‐induced cell viability loss in a dose‐dependent manner. The mechanisms by which chamomile‐protected macrophages from oxidative stress was through the induction of several antioxidant enzymes including NAD(P)H:quinone oxidoreductase, superoxide dismutase, and catalase and increase nuclear accumulation of the transcription factor Nrf2 and its binding to antioxidant response elements. Furthermore, chamomile dose‐dependently reduced H2O2‐mediated increase in the intracellular levels of reactive oxygen species. Our results, for the first time, demonstrate that chamomile has protective effects against oxidative stress and might be beneficial to provide defense against cellular damage. Copyright