c-Myc Metabolic Addiction in Cancers Counteracted by Resveratrol and NQO2

Transcription factor c-myc is frequently amplified/overexpressed in human cancers. One event c-myc controls is metabolic reprogramming or the addiction for glucose and/or glutamine as nutrients. Rewiring of metabolic circuitry provides cancer cells with a gain-of-survival advantage. Accordingly, the aversion of two types of oncogenic-distinct metabolic addictions via c-myc control offers an anti-tumorigenic approach. Resveratrol reportedly inhibits the uptake/transport of glucose or glutamine and reduces c-myc expression in cancer cells. Whether c-myc control by resveratrol involves quinone reductase NQO2 is unknown. NQO2 expressing (shRNA08) and knockdown (shRNA25) CWR22Rv1 prostate cancer cells were generated and used to study the role of NQO2 in growth and cell cycle control. Immunoblot analyses were used to evaluate the changes of cell cycle-associated proteins. NQO2 in mediating degradation of cyclin D1 via AKT/GSK-3β by resveratrol was tested by determining AKT and chymotrypsin-like proteasome activities. Molecular modeling and pull-down/deletion assays were used to evaluate the interaction between NQO2 and AKT. Resveratrol interacts with NQO2, a quinone reductase that plays a key role in resveratrol-induced AKT/GSK3β-mediated degradation of cyclin D1. In this chapter, we unravel control of expression and stability of c-myc by the resveratrol-NQO2 axis as an approach to overcome c-myc-mediated metabolic reprogramming

Combined Metformin and Resveratrol Confers Protection Against UVC-Induced DNA Damage in A549 Lung Cancer Cells via Modulation of Cell Cycle Checkpoints and DNA Repair

Aging in humans is a multi-factorial cellular process that is associated with an increase in the risk of numerous diseases including diabetes, coronary heart disease and cancer. Aging is linked to DNA damage, and a persistent source of DNA damage is exposure to ultraviolet (UV) radiation. As such, identifying agents that confer protection against DNA damage is an approach that could reduce the public health burden of age-related disorders. Metformin and resveratrol have both shown effectiveness in preventing several age-related diseases; using human A549 cells, we investigated whether metformin or resveratrol, alone or combined, prevent UVC-induced DNA damage. We found that metformin inhibited UVC-induced upregulation of p53, as well as downregulated the expression of two DNA damage markers: γH2AX and p-chk2. Metformin also upregulated DNA repair as evidenced by the increase in expression of p53R2. Treatment with metformin also induced cell cycle arrest in UVC-induced cells, in correlation with a reduction in the levels of cyclin E/cdk2/Rb and cyclin B1/cdk1. Compared to metformin, resveratrol as a single agent showed less effectiveness in counteracting UVC-elicited cellular responses. However, resveratrol displayed synergism when combined with metformin as shown by the downregulation of p53/γH2AX/p-chk2. In conclusion, the results of the present study validate the effectiveness of metformin, alone or with the addition of resveratrol, in reducing the risk of aging by conferring protection against UV-induced DNA damage

Upregulation of PDL1 Expression by Resveratrol and Piceatannol in Breast and Colorectal Cancer Cells Occurs Via HDAC3/p300-Mediated NFkappaB Signaling

Programmed cell death ligand 1 (PDL1) expressed in cancer cells interacting with its receptor programmed cell death 1 (PD1) expressed in immune cells represents a regulatory axis linked to the suppression and evasion of host immune functions. The blockade of PD1/PDL1 interaction using monoclonal antibodies has emerged as an effective therapy for several solid tumors; however, durable response has been observed in a subset of patients with PDL1-positive tumors. Thus, the understanding of the mechanisms responsible for the expression of PDL1 in tumor cells may help to improve the response to PDL1 blockade therapies. In this study, we investigated whether resveratrol, a grape-derived stilbenoid with immunoregulatory activity, modulates the expression of PDL1 in breast and colorectal cancer cells. The surface expression of PDL1 was determined by flow cytometry in cancer cells treated with resveratrol and/or piceatannol. Each stilbenoid alone induced PDL1 and when used in combination, elicited a synergistic upregulation of PDL1 in some cell lines. The induction of PDL1 by the combined use of stilbenoids was most pronounced in the Cal51 triple-negative breast cancer (TNBC) and SW620 colon cancer cells. The observed induction of PDL1 was transcriptionally mediated by nuclear factor (NF)-kappaB, as shown by NFkappaB reporter assays, the nuclear accumulation of the p65 subunit of NFkappaB, inhibition by the IKK inhibitor, BMS345541, and histone the modification inhibitors, resminostat, entinostat or anacardic acid. Combined treatment with resveratrol and piceatannol also decreased tumor cell survival as indicated by the upregulation of the DNA damaging marker, gammaH2AX, the cleavage of caspase 3, the downregulation of the survival markers, p38-MAPK/cMyc, and G1-to-S cell cycle arrest

AKT/mTOR as Novel Targets of Polyphenol Piceatannol Possibly Contributing to Inhibition of Proliferation of Cultured Prostate Cancer Cells

The polyphenol piceatannol has shown inhibition against tyrosine and serine/threonine kinases. Whether piceatannol also exerts activity on the mammalian target of rapamycin (mTOR), a kinase involved in growth control of eukaryotic cells, is not known. In this study, we tested the effects of piceatannol on proliferation of androgen-dependent (AD) LNCaP and androgen-independent (AI) DU145 and PC-3 prostate cancer (CaP) cells. Suppression of AD and AI CaP cell growth by piceatannol was accompanied by cell cycle blockade in G1/S and S phases for LNCaP and PC-3 and induction of apoptosis in DU145 cells. Induction of apoptosis by piceatannol in DU145 cells was evident by reduced expression of poly(ADP-ribose) polymerase (PARP), cleavage of caspase 3 and apoptosis inducing factor AIF, and an increase in cytochrome c. The apoptotic changes occurred in concordance with DNA damage, supported by increased phosphorylated histone H2AX. Immunoblot analyses showed that exposure of different-stage CaP cells to piceatannol also resulted in cell-type-specific downregulation of mTOR and its upstream and downstream effector proteins, AKT and eIF-4E-BP1. We propose that the observed AKT and mTOR changes are new targets of piceatannol possibly contributing to its inhibitory activities on proliferation of CaP cells

Application of Open-Access Databases to Determine Functional Connectivity Between Resveratrol-Binding Protein QR2 and Colorectal Carcinoma

Colorectal cancer (CRC) is a major cause of cancer-associated deaths worldwide. Recently, oral administration of resveratrol (trans-3,5,4\u27-trihydroxystilbene) has been reported to significantly reduce tumor proliferation in colorectal cancer patients, however, with little specific information on functional connections. The pathogenesis and development of colorectal cancer is a multistep process that can be categorized using three phenotypic pathways, respectively, chromosome instability (CIN), microsatellite instability (MSI), and CpG island methylator (CIMP). Targets of resveratrol, including a high-affinity binding protein, quinone reductase 2 (QR2), have been identified with little information on disease association. We hypothesize that the relationship between resveratrol and different CRC etiologies might be gleaned using publicly available databases. A web-based microarray gene expression data-mining platform, Oncomine, was selected and used to determine whether QR2 may serve as a mechanistic and functional biotarget within the various CRC etiologies. We found that QR2 messenger RNA (mRNA) is overexpressed in CRC characterized by CIN, particularly in cells showing a positive KRAS (Kirsten rat sarcoma viral oncogene homolog) mutation, as well as by the MSI but not the CIMP phenotype. Mining of Oncomine revealed an excellent correlation between QR2 mRNA expression and certain CRC etiologies. Two resveratrol-associated genes, adenomatous polyposis coli (APC) and TP53, found in CRC were further mined, using cBio portal and Colorectal Cancer Atlas which predicted a mechanistic link to exist between resveratrol→QR2/TP53→CIN. Multiple web-based data mining can provide valuable insights which may lead to hypotheses serving to guide clinical trials and design of therapies for enhanced disease prognosis and patient survival. This approach resembles a BioGPS, a capability for mining web-based databases that can elucidate the potential links between compounds to provide correlations of these interactions with specific diseases

Anticancer Activities of Resveratrol in Colorectal Cancer

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a dietary polyphenolic phytochemical that has demonstrated health benefits such as cardioprotection, the prevention of neurodegeneration and chemoprevention. Resveratrol has shown great potential in the prevention and treatment of carcinomas and clinical trials support resveratrol as anticancer compound in colorectal carcinoma. Colorectal cancer remains a major cause of cancer-related deaths for both men and women in industrialized countries. Because of this widespread prevalence, identifying major risk factors and initiating colorectal screening procedures provide the distinct advantage for recognizing early disease and addressing treatable forms of CRC. Epidemiological studies of fruit and vegetable consumption in relationship to developing CRC have led to the notion that safe and inexpensive chemopreventive agents might be a valuable tool in diminishing the morbidity and mortality of CRC. While clinical trials and in vivo data show positive effects of resveratrol in CRC, the mechanism of action is relatively unclear. In this review, we will evaluate the current literature on the actions of resveratrol in CRC and provide a more mechanistic view of resveratrol in relationship with CRC

Activation of NQO1 in NQO1*2 Polymorphic Human Leukemic HL-60 Cells by Diet-Derived Sulforaphane

BACKGROUND: The NAD(P)H: quinone oxidoreductase (NQO1) confers protection against semiquinones and also elicits oxidative stress. The C609T polymorphism of the NQO1 gene, designated NQO1*2, significantly reduces its enzymatic activity due to rapid degradation of protein. Since down regulation of NQO1 mRNA expression correlates with increased susceptibility for developing different types of cancers, we investigated the link between leukemia and the NQO1*2 genotype by mining a web-based microarray dataset, ONCOMINE. Phytochemicals prevent DNA damage through activation of phase II detoxification enzymes including NQO1. Whether NQO1 expression/activity in leukemia cells that carry the labile NQO1*2 genotype can be induced by broccoli-derived phytochemical sulforaphane (SFN) is currently unknown. METHODS AND RESULTS: The ONCOMINE query showed that: (1) acute lymphoblastic leukemia and chronic myelogenous leukemia are associated with reduced NQO1 levels, and (2) under-expressed NQO1 was found in human HL-60 leukemia cell line containing the heterozygous NQO1*2 polymorphism. We examined induction of NQO1 activity/expression by SFN in HL-60 cells. A dose-dependent increase in NQO1 level/activity is accompanied by upregulation of the transcription factor, Nrf2, following 1-10 μM SFN treatment. Treatment with 25 µM SFN drastically reduced NQO1 levels, inhibited cell proliferation, caused sub-G1 cell arrest, and induced apoptosis, and a decrease in the levels of the transcription factor, nuclear factor-κB (NFκB). CONCLUSIONS: Up to 10 μM of SFN increases NQO1 expression and suppresses HL-60 cell proliferation whereas ≥ 25 μM of SFN induces apoptosis in HL-60 cells. Further, SFN treatment restores NQO1 activity/levels in HL-60 cells expressing the NQO1*2 genotype

NRH:quinone Oxidoreductase 2 (NqO2) and Glutaminase (GLS) Both Play a Role in Large Extracellular Vesicles (LEV) Formation in Preclinical LNCaP-C4-2B Prostate Cancer Model of Progressive Metastasis

In the course of studies aimed at the role of oxidative stress in the development of metastatic potential in the LNCaP-C4-2B prostate cancer progression model system, we found a relative decrease in the level of expression of the cytoplasmic nicotinamide riboside: quinone oxidoreductase (NQO2) and an increase in the oxidative stress in C4-2B cells compared to that in LNCaP or its derivatives C4 and C4-2. It was also found that C4-2B cells specifically shed large extracellular vesicles (LEVs) suggesting that these LEVs and their cargo could participate in the establishment of the osseous metastases. The level of expression of caveolin-1 increased as the system progresses from LNCaP to C4-2B. Since NQO2 RNA levels were not changed in LNCaP, C4, C4-2, and C4-2B, we tested an altered cellular distribution hypothesis of NQO2 being compartmentalized in the membrane fractions of C4-2B cells which are rich in lipid rafts and caveolae. This was confirmed when the detergent resistant membrane fractions were probed on immunoblots. Moreover, when the LEVs were analyzed for membrane associated caveolin-1 as possible cargo, we noticed that the enzyme NQO2 was also a component of the cargo along with caveolin-1 as seen in double immunofluorescence studies. Molecular modeling studies showed that a caveolin-1 accessible site is present in NQO2. Specific interaction between NQO2 and caveolin-1 was confirmed using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Interestingly, whole cell lysate and mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B showed an increasing expression of glutaminase (GLS, kidney type). The extrusion of LEVs appears to be a specific property of the bone metastatic C4-2B cells and this process could be inhibited by a GLS specific inhibitor BPTES, suggesting the critical role of a functioning glutamine metabolism. Our results indicate that a high level of expression of caveolin-1 in C4-2B cells contributes to an interaction between caveolin-1 and NQO2 and to their packaging as cargo in the shed LEVs. These results suggest an important role of membrane associated oxidoreductases in the establishment of osseous metastases in prostate cancer

A MicroRNA-1280/JAG2 Network Comprises a Novel Biological Target in High-Risk Medulloblastoma

Over-expression of PDGF receptors (PDGFRs) has been previously implicated in high-risk medulloblastoma (MB) pathogenesis. However, the exact biological functions of PDGFRα and PDGFRβ signaling in MB biology remain poorly understood. Here, we report the subgroup specific expression of PDGFRα and PDGFRβ and their associated biological pathways in MB tumors. c-MYC, a downstream target of PDGFRβ but not PDGFRα, is involved in PDGFRβ signaling associated with cell proliferation, cell death, and invasion. Concurrent inhibition of PDGFRβ and c-MYC blocks MB cell proliferation and migration synergistically. Integrated analysis of miRNA and miRNA targets regulated by both PDGFRβ and c-MYC reveals that increased expression of JAG2, a target of miR-1280, is associated with high metastatic dissemination at diagnosis and a poor outcome in MB patients. Our study may resolve the controversy on the role of PDGFRs in MB and unveils JAG2 as a key downstream effector of a PDGFRβ-driven signaling cascade and a potential therapeutic target

Functional/Activity Network (FAN) Analysis of Gene-Phenotype Connectivity Liaised by Grape Polyphenol Resveratrol

Resveratrol is a polyphenol that has witnessed an unprecedented yearly growth in PubMed citations since the late 1990s. Based on the diversity of cellular processes and diseases resveratrol reportedly affects and benefits, it is likely that the interest in resveratrol will continue, although uncertainty regarding its mechanism in different biological systems remains.We hypothesize that insights on disease-modulatory activities of resveratrol might be gleaned by systematically dissecting the publicly available published data on chemicals and drugs. In this study, we tested our hypothesis by querying DTome (Drug-Target Interactome), a web-based tool containing data compiled from open-source databases including DrugBank, PharmGSK, and Protein Interaction Network Analysis (PINA). Four direct protein targets (DPT) and 219 DPT-associated genes were identified for resveratrol. The DPT-associated genes were scrutinized by WebGestalt (WEB-based Gene SeT Analysis Toolkit). This enrichment analysis resulted in 10 identified KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. Refined analysis of KEGG pathways showed that 2 - one linked to p53 and a second to prostate cancer - have functional connectivity to resveratrol and its four direct protein targets. These results suggest that a functional activity network (FAN) approach may be considered as a new paradigm for guiding future studies of resveratrol. FAN analysis resembles a BioGPS, with capability for mapping a Web-based scientific track that can productively and cost effectively connect resveratrol to its primary and secondary target proteins and to its biological functions