Correction: 2'-Hydroxyflavanone effectively targets RLIP76-mediated drug transport and regulates critical signaling networks in breast cancer.

[This corrects the article DOI: 10.18632/oncotarget.24720.].

2'-Hydroxyflavanone effectively targets RLIP76-mediated drug transport and regulates critical signaling networks in breast cancer.

Breast cancer (BC) is the most common cancer in women. Estrogen, epidermal growth factor receptor 2 (ERBB2, HER2), and oxidative stress represent critical mechanistic nodes associated with BC. RLIP76 is a major mercapturic acid pathway transporter whose expression is increased in BC. In the quest of a novel molecule with chemopreventive and chemotherapeutic potential, we evaluated the effects of 2'-Hydroxyflavanone (2HF) in BC. 2HF enhanced the inhibitory effects of RLIP76 depletion and also inhibited RLIP76-mediated doxorubicin transport in BC cells. RNA-sequencing revealed that 2HF induces strong reversal of the gene expression pattern in ER+MCF7, HER2+ SKBR3 and triple-negative MDA-MB-231 BC cells with minimal effects on MCF10A normal breast epithelial cells. 2HF down regulated ERα and enhanced inhibitory effects of imatinib mesylate/Gleevec in MCF7 cells. 2HF also down regulated ERα and HER2 gene networks in MCF7 and SKBR3 cells, respectively. 2HF activated TP53 and inhibited TGFß1 canonical pathway in MCF7 and MDA-MB-231 BC cells. 2HF also regulated the expression of a number of critical prognostic genes of MammaPrint panel and their upstream targets including TP53, CDKN2A and MYC. The collective findings from this study provide a comprehensive, direct and integrated evidence for the benefits of 2HF in targeting major and clinically relevant mechanistic regulators of BC.

Oxidative stress and dietary phytochemicals: Role in cancer chemoprevention and treatment.

Several epidemiological observations have shown an inverse relation between consumption of plant-based foods, rich in phytochemicals, and incidence of cancer. Phytochemicals, secondary plant metabolites, via their antioxidant property play a key role in cancer chemoprevention by suppressing oxidative stress-induced DNA damage. In addition, they modulate several oxidative stress-mediated signaling pathways through their anti-oxidant effects, and ultimately protect cells from undergoing molecular changes that trigger carcinogenesis. In several instances, however, the pro-oxidant property of these phytochemicals has been observed with respect to cancer treatment. Further, in vitro and in vivo studies show that several phytochemicals potentiate the efficacy of chemotherapeutic agents by exacerbating oxidative stress in cancer cells. Therefore, we reviewed multiple studies investigating the role of dietary phytochemicals such as, curcumin (turmeric), epigallocatechin gallate (EGCG; green tea), resveratrol (grapes), phenethyl isothiocyanate (PEITC), sulforaphane (cruciferous vegetables), hesperidin, quercetin and 2'-hydroxyflavanone (2HF; citrus fruits) in regulating oxidative stress and associated signaling pathways in the context of cancer chemoprevention and treatment.

Proteomic analysis of signaling network regulation in renal cell carcinomas with differential hypoxia-inducible factor-2α expression.

BACKGROUND: The loss of von Hippel-Lindau (VHL) protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2α (HIF2α) instead of hypoxia-inducible factor-1α (HIF1α). METHODS AND PRINCIPAL FINDINGS: We used renal carcinoma cell lines that recapitulate the differences between mutant VHL and wild-type VHL genotypes. Utilizing a method relying on extracted peptide intensities as a label-free approach for quantitation by liquid chromatography-mass spectrometry, our proteomics study revealed regulation of key proteins important for cancer cell survival, proliferation and stress-resistance, and implicated differential regulation of signaling networks in VHL-mutant renal cell carcinoma. We also observed upregulation of cellular energy pathway enzymes and the stress-responsive mitochondrial 60-kDa heat shock protein. Finding reliance on glutaminolysis in VHL-mutant renal cell carcinoma was of particular significance, given the generally predominant dependence of tumors on glycolysis. The data have been deposited to the ProteomeXchange with identifier PXD000335. CONCLUSIONS AND SIGNIFICANCE: Pathway analyses provided corroborative evidence for differential regulation of molecular and cellular functions influencing cancer energetics, metabolism and cell proliferation in renal cell carcinoma with distinct VHL genotype. Collectively, the differentially regulated proteome characterized by this study can potentially guide translational research specifically aimed at effective clinical interventions for advanced VHL-mutant, HIF2α-over-expressing tumors.

Nutlin-3 enhances sorafenib efficacy in renal cell carcinoma.

The renal cell carcinoma (RCC) is one of the top 10 cancers in USA. The renal tumors are highly angiogenic and are resistant to conventional interventions, particularly radiotherapy. The advent of multi-specific tyrosine kinase inhibitor sorafenib has improved the progression-free survival in RCC, but overall survival in recurrent and metastatic RCC is still a concern that has lead to characterization of combinatorial regimens. Hence, we studied the effect of combination of nutlin-3, an MDM2 inhibitor, which increases p53 levels, and sorafenib in RCC. Sorafenib along with nutlin-3 synergistically inhibited the cell survival and enhanced caspase-3 cleavage leading to apoptosis in RCC. Nutlin-3 and sorafenib were more effective in reducing the migration of RCC, in combination than as single agents. Sorafenib and nutlin-3 decreased the phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) and ERK along with inducing p53 activity. The sorafenib and nutlin-3 co-treatment lead to enhanced levels of p53, p-p53, and increase in the levels of p53 pro-apoptotic effector PUMA, Bax, and decrease in the anti-apoptotic Bcl-2 levels. Importantly, our studies revealed that sorafenib alone can activate p53 in a concentration dependent manner. Thus, co-treatment of nutlin-3 with sorafenib leads to increased half-life of p53, which in turn can be activated by sorafenib, to induce downstream pro-apoptotic and anti-proliferative effects. This is the first report showing the synergistic effect of sorafenib and nutlin-3 while providing a strong clinical-translational rationale for further testing of sorafenib and nutlin-3 combinatorial regimen in human RCC.

RLIP76 regulates PI3K/Akt signaling and chemo-radiotherapy resistance in pancreatic cancer.

PURPOSE: Pancreatic cancer is an aggressive malignancy with characteristic metastatic course of disease and resistance to conventional chemo-radiotherapy. RLIP76 is a multi-functional cell membrane protein that functions as a major mercapturic acid pathway transporter as well as key regulator of receptor-ligand complexes. In this regard, we investigated the significance of targeting RLIP76 on PI3K/Akt pathway and mechanisms regulating response to chemo-radiotherapy. RESEARCH DESIGN AND METHODS: Cell survival was assessed by MTT and colony forming assays. Cellular levels of proteins and phosphorylation was determined by Western blot analyses. The impact on apoptosis was determined by TUNEL assay. The anti-cancer effects of RLIP76 targeted interventions in vivo were determined using mice xenograft model of the pancreatic cancer. The regulation of doxorubicin transport and radiation sensitivity were determined by transport studies and colony forming assays, respectively. RESULTS: Our current studies reveal an encompassing model for the role of RLIP76 in regulating the levels of fundamental proteins like PI3K, Akt, E-cadherin, CDK4, Bcl2 and PCNA which are of specific importance in the signal transduction from critical upstream signaling cascades that determine the proliferation, apoptosis and differentiation of pancreatic cancer cells. RLIP76 depletion also caused marked and sustained regression of established human BxPC-3 pancreatic cancer tumors in nude mouse xenograft model. RLIP76 turned out to be a major regulator of drug transport along with contributing to the radiation resistance in pancreatic cancer. CONCLUSIONS/SIGNIFICANCE: RLIP76 represents a mechanistically significant target for developing effective interventions in aggressive and refractory pancreatic cancers.

Didymin induces apoptosis by inhibiting N-Myc and upregulating RKIP in neuroblastoma.

Neuroblastomas arise from the neural crest cells and represent the most common solid tumors outside the nervous system in children. The amplification of N-Myc plays a primary role in the pathogenesis of neuroblastomas, whereas acquired mutations of p53 lead to refractory and relapsed cases of neuroblastomas. In this regard, dietary compounds which can target N-Myc and exert anticancer effects independent of p53 status acquire significance in the management of neuroblastomas. Hence, we investigated the anticancer properties of the flavonoid didymin in neuroblastomas. Didymin effectively inhibited proliferation and induced apoptosis irrespective of p53 status in neuroblastomas. Didymin downregulated phosphoinositide 3-kinase, pAkt, Akt, vimentin, and upregulated RKIP levels. Didymin induced G(2)/M arrest along with decreasing the levels of cyclin D1, CDK4, and cyclin B1. Importantly, didymin inhibited N-Myc as confirmed at protein, mRNA, and transcriptional level by promoter-reporter assays. High-performance liquid chromatography analysis of didymin-treated (2 mg/kg b.w.) mice serum revealed effective oral absorption with free didymin concentration of 2.1 µmol/L. Further in vivo mice xenograft studies revealed that didymin-treated (2 mg/kg b.w.) animals had significant reductions in tumors size compared with controls. Didymin strongly inhibited the proliferation (Ki67) and angiogenesis (CD31) markers, as well as N-Myc expression, as revealed by the histopathologic examination of paraffin-embedded section of resected tumors. Collectively, our in vitro and in vivo studies elucidated the anticancer properties and mechanisms of action of a novel, orally active, and palatable flavonoid didymin, which makes it a potential new approach for neuroblastoma therapy (NANT) to target pediatric neuroblastomas.

Anti-cancer effects of novel flavonoid vicenin-2 as a single agent and in synergistic combination with docetaxel in prostate cancer.

The present study was conducted to determine the efficacy of novel flavonoid vicenin-2 (VCN-2), an active constituent of the medicinal herb Ocimum Sanctum Linn or Tulsi, as a single agent and in combination with docetaxel (DTL) in carcinoma of prostate (CaP). VCN-2 effectively induced anti-proliferative, anti-angiogenic and pro-apoptotic effect in CaP cells (PC-3, DU-145 and LNCaP) irrespective of their androgen responsiveness or p53 status. VCN-2 inhibited EGFR/Akt/mTOR/p70S6K pathway along with decreasing c-Myc, cyclin D1, cyclin B1, CDK4, PCNA and hTERT in vitro. VCN-2 reached a level of 2.6±0.3µmol/l in serum after oral administration in mice which reflected that VCN-2 is orally absorbed. The i.v. administration of docetaxel (DTL), current drug of choice in androgen-independent CaP, is associated with dose-limiting toxicities like febrile neutropenia which has lead to characterization of alternate routes of administration and potential combinatorial regimens. In this regard, VCN-2 in combination with DTL synergistically inhibited the growth of prostate tumors in vivo with a greater decrease in the levels of AR, pIGF1R, pAkt, PCNA, cyclin D1, Ki67, CD31, and increase in E-cadherin. VCN-2 has been investigated for radioprotection and anti-inflammatory properties. This is the first study on the anti-cancer effects of VCN-2. In conclusion, our investigations collectively provide strong evidence that VCN-2 is effective against CaP progression along with indicating that VCN-2 and DTL co-administration is more effective than either of the single agents in androgen-independent prostate cancer.

Targeting p53-null neuroblastomas through RLIP76.

The search for p53-independent mechanism of cancer cell killing is highly relevant to pediatric neuroblastomas, where successful therapy is limited by its transformation into p53-mutant and a highly drug-resistant neoplasm. Our studies on the drug-resistant p53-mutant as compared with drug-resistant p53 wild-type neuroblastoma revealed a novel mechanism for resistance to apoptosis: a direct role of p53 in regulating the cellular concentration of proapoptotic alkenals by functioning as a specific and saturable allosteric inhibitor of the alkenal-glutathione conjugate transporter, RLIP76. The RLIP76-p53 complex was showed by both immunoprecipitation analyses of purified proteins and immunofluorescence analysis. Drug transport studies revealed that p53 inhibited both basal and PKCα-stimulated transport of glutathione conjugates of 4HNE (GSHNE) and doxorubicin. Drug resistance was significantly greater for p53-mutant as compared with p53 wild-type neuroblastoma cell lines, but both were susceptible to depletion of RLIP76 by antisense alone. In addition, inhibition of RLIP76 significantly enhanced the cytotoxicity of cisplatin. Taken together, these studies provide powerful evidence for a novel mechanism for drug and apoptosis resistance in p53-mutant neuroblastoma, based on a model of regulation of p53-induced apoptosis by RLIP76, where p53 is a saturable and specific allosteric inhibitor of RLIP76, and p53 loss results in overexpression of RLIP76; thus, in the absence of p53, the drug and glutathione-conjugate transport activities of RLIP76 are enhanced. Most importantly, our findings strongly indicate RLIP76 as a novel target for therapy of drug-resistant and p53-mutant neuroblastoma.

2'-hydroxyflavanone inhibits proliferation, tumor vascularization and promotes normal differentiation in VHL-mutant renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the top ten cancers prevalent in USA. Loss-of-function mutations in the von Hippel-Lindau (VHL) gene constitute an established risk factor contributing to 75% of total reported cases of RCC. Loss-of-VHL leads to a highly vascularized phenotype of renal tumors. Intake of citrus fruits has been proven to reduce the risk of RCC in multicenter international studies. Hence, we studied the effect of 2'-hydroxyflavanone (2HF), an active anticancer compound from oranges, in RCC. Our in vitro investigations revealed that 2HF suppresses VHL-mutant RCC to a significantly greater extent than VHL-wild-type RCC by inhibiting epidermal growth factor receptor signaling, which is increased due to VHL mutations in RCC. Our results also revealed for the first time, that 2HF inhibits glutathione S-transferase pi activity. 2HF reduced cyclin B1 and CDK4 levels and induced G2/M phase arrest in VHL-mutant RCC. Importantly, 2HF inhibited the angiogenesis in VHL-mutant RCC by decreasing vascular endothelial growth factor expression. Our in vivo studies in mice xenografts confirmed our in vitro results as evident by decreased levels of proliferation marker, Ki67 and angiogenic marker, CD31, in 2HF-treated mice xenografts of VHL-mutant RCC. 2HF also increased the expression of E-cadherin in VHL-mutant RCC, which would be of significance in restoring normal epithelial phenotype. Collectively, our in vitro and in vivo results revealed the potent antiproliferative, anti-angiogenic and prodifferentiation properties of 2HF in VHL-mutant RCC, sparing normal cells, which could have significant implications not only in the specific management of VHL-mutant RCC but also towards other VHL syndromes.

The sensors and regulators of cell-matrix surveillance in anoikis resistance of tumors.

Normal cells continuously monitor the nature of their respective cellular microenvironment. They are equipped with an inherent molecular defense to detect changes that can precipitate and trigger an oncogenic cascade in the internal and external environment of cells. The process called anoikis unleashes many a characteristic molecular change in the cells which eventually program to cell death in response to cell detachment and inappropriate cellular attachment, both of which can otherwise potentiate the ability of cells to preferentially pursue a malignant course due to the release of molecular discipline which conforms them to a benign structural and functional spectrum. The initiation and propagation of signaling that serves as a switch to cell survival or cell death mediated by surveillance of cell microenvironment is comprised of many heterogeneous sets of molecules interacting mainly at the interface of cell-extracellular matrix. Transforming cells continuously reprogram their signaling characteristics in sensing and modulating the stimuli from cell surface molecules like integrins, cadherins and immunoglobulin family of cell adhesion molecules at adhesion complexes, which enables them to resist anoikis and metastasize to different organs. Actin cytoskeleton binds BIM and Bcl2 modifying factor (BMF), which are regulated by the adhesion status and consequent conformation of cytoskeleton in the cells. This review aims at an integrated synopsis of fundamental mechanisms of the critical interactions of cell surface molecules to facilitate a focused analysis of the differential regulation of signaling processes at cell-ECM junctions that collectively rein the anoikis resistance, which in turn impacts metastatic aggressiveness and drug resistance of tumors originating from respective organs.