Koren K. Mann

Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture

Epidemiological evidence suggests tea (Camellia sinensis L.) has chemopreventive effects against various tumors. Green tea contains many polyphenols, including epigallocatechin‐3 gallate (EGCG), which possess anti‐oxidant qualities. Reduction of chemically induced mammary gland carcinogenesis by green tea in a carcinogen‐induced rat model has been suggested previously, but the results reported were not statistically significant. Here we have tested the effects of green tea on mammary tumorigenesis using the 7,12‐dimethylbenz(a)anthracene (DMBA) Sprague‐Dawley (S‐D) rat model. We report that green tea significantly increased mean latency to first tumor, and reduced tumor burden and number of invasive tumors per tumor‐bearing animal; although, it did not affect tumor number in the female rats. Furthermore, we show that proliferation and/or viability of cultured Hs578T and MDA‐MB‐231 estrogen receptor‐negative breast cancer cell lines was reduced by EGCG treatment. Similar negative effects on proliferation were observed with the DMBA‐transformed D3‐1 cell line. Growth inhibition of Hs578T cells correlated with induction of p27Kip1 cyclin‐dependent kinase inhibitor (CKI) expression. Hs578T cells expressing elevated levels of p27Kip1 protein due to stable ectopic expression displayed increased G1 arrest. Thus, green tea had significant chemopreventive effects on carcinogen‐induced mammary tumorigenesis in female S‐D rats. In culture, inhibition of human breast cancer cell proliferation by EGCG was mediated in part via induction of the p27Kip1 CKI. J. Cell. Biochem. 82:387–398, 2001.

Cul3 overexpression depletes Nrf2 in breast cancer and is associated with sensitivity to carcinogens, to oxidative stress, and to chemotherapy

Nrf2 is the key transcription factor for cytoprotective gene programs. Nrf2 is normally maintained at very low concentrations by proteasomal degradation, through its interaction with the adapter protein Keap1 and the Cul3 E3 ligase. Increased Nrf2 concentration resulting from loss of function Keap1 mutations has been described in chemoresistant non–small cell lung cancer. Previous studies in breast cancer showed low levels of some Nrf2-regulated detoxification genes, but the mechanism has not been systematically examined. We found that half of the breast cancer cell lines examined have decreased concentration of Nrf2 compared with normal mammary epithelial cell lines, associated with variable but detectable levels in Keap1 levels, and consistently increased Cul3 mRNA and protein. Immunochemistry showed that 7 of 10 breast cancer specimens examined also have low Nrf2 levels and increased Cul3. Keap1 protein levels are variable. We found no C23Y mutation in Keap1 of any of the cell lines. Using siRNA, we silenced Cul3 in MCF-7 breast cancer cells, and microarray analysis reveals the induction of GCL, NQO1, AKR1C1, UGDH, and TXN by at least 2-fold. The Nrf2-regulated ABCC1 drug transporter was also found to be increased. These Cul3-silenced MCF7 cells are highly resistant to oxidative stress induced by H2O2, to the carcinogen benzo(a)pyrene, and to both Doxorubicin and Paclitaxel. This high Cul3/low Nrf2 signature may be key to cellular sensitivity to both chemical carcinogeneic stimuli as well as to cytotoxicity of commonly used chemotherapeutic drugs in established breast cancers. [Mol Cancer Ther 2009;8(8):2432–40]

Peroxisome Proliferator-Activated Receptor γ-Mediated NF-κB Activation and Apoptosis in Pre-B Cells

The role of peroxisome proliferator-activated receptor γ (PPARγ) in adipocyte physiology has been exploited for the treatment of diabetes. The expression of PPARγ in lymphoid organs and its modulation of macrophage inflammatory responses, T cell proliferation and cytokine production, and B cell proliferation also implicate it in immune regulation. Despite significant human exposure to PPARγ agonists, little is known about the consequences of PPARγ activation in the developing immune system. Here, well-characterized models of B lymphopoiesis were used to investigate the effects of PPARγ ligands on nontransformed pro/pre-B (BU-11) and transformed immature B (WEHI-231) cell development. Treatment of BU-11, WEHI-231, or primary bone marrow B cells with PPARγ agonists (ciglitazone and GW347845X) resulted in rapid apoptosis. A role for PPARγ and its dimerization partner, retinoid X receptor (RXR)α, in death signaling was supported by 1) the expression of RXRα mRNA and cytosolic PPARγ protein, 2) agonist-induced binding of PPARγ to a PPRE, and 3) synergistic increases in apoptosis following cotreatment with PPARγ agonists and 9-cis-retinoic acid, an RXRα agonist. PPARγ agonists activated NF-κB (p50, Rel A, c-Rel) binding to the upstream κB regulatory element site of c-myc. Only doses of agonists that induced apoptosis stimulated NF-κB-DNA binding. Cotreatment with 9-cis-retinoic acid and PPARγ agonists decreased the dose required to activate NF-κB. These data suggest that activation of PPARγ-RXR initiates a potent apoptotic signaling cascade in B cells, potentially through NF-κB activation. These results have implications for the nominal role of the PPARγ in B cell development and for the use of PPARγ agonists as immunomodulatory therapeutics.

Genetic Landscapes of Relapsed and Refractory Diffuse Large B-Cell Lymphomas

Purpose: Relapsed or refractory diffuse large B-cell lymphoma (rrDLBCL) is fatal in 90% of patients, and yet little is known about its biology. Experimental Design: Using exome sequencing, we characterized the mutation profiles of 38 rrDLBCL biopsies obtained at the time of progression after immunochemotherapy. To identify genes that may be associated with relapse, we compared the mutation frequency in samples obtained at relapse to an unrelated cohort of 138 diagnostic DLBCLs and separately amplified specific mutations in their matched diagnostic samples to identify clonal expansions. Results: On the basis of a higher frequency at relapse and evidence for clonal selection, TP53, FOXO1, MLL3 (KMT2C), CCND3, NFKBIZ, and STAT6 emerged as top candidate genes implicated in therapeutic resistance. We observed individual examples of clonal expansions affecting genes whose mutations had not been previously associated with DLBCL including two regulators of NF-κB: NFKBIE and NFKBIZ. We detected mutations that may be affect sensitivity to novel therapeutics, such as MYD88 and CD79B mutations, in 31% and 23% of patients with activated B-cell–type of rrDLBCL, respectively. We also identified recurrent STAT6 mutations affecting D419 in 36% of patients with the germinal center B (GCB) cell rrDLBCL. These were associated with activated JAK/STAT signaling, increased phospho-STAT6 protein expression and increased expression of STAT6 target genes. Conclusions: This work improves our understanding of therapeutic resistance in rrDLBCL and has identified novel therapeutic opportunities especially for the high-risk patients with GCB-type rrDLBCL. Clin Cancer Res; 22(9); 2290–300. ©2015 AACR.

Darinaparsin: a novel organic arsenical with promising anticancer activity.

Darinaparsin is an organic arsenical composed of dimethylated arsenic linked to glutathione, and is being investigated for antitumor properties in vitro and in vivo. While other arsenicals, including arsenic trioxide, have been used clinically, none have shown significant activity in malignancies outside of acute promyelocytic leukemia. Darinaparsin has significant activity in a broad spectrum of hematologic and solid tumors in preclinical models. Here, we review the literature describing the signaling pathways and mechanisms of action of darinaparsin and compare them to mechanisms of cell death induced by arsenic trioxide. Darinaparsin has overlapping, but distinct, signaling mechanisms. We also review the current results of clinical trials with darinaparsin (both intravenous and oral formulations) that demonstrate significant antitumor activity.

Endothelin-1 Overexpression Exacerbates Atherosclerosis and Induces Aortic Aneurysms in Apolipoprotein E Knockout Mice

Objective—Endothelin (ET)-1 plays a role in vascular reactive oxygen species production and inflammation. ET-1 has been implicated in human atherosclerosis and abdominal aortic aneurysm (AAA) development. ET-1 overexpression exacerbates high-fat diet–induced atherosclerosis in apolipoprotein E−/− (Apoe−/−) mice. ET-1–induced reactive oxygen species and inflammation may contribute to atherosclerosis progression and AAA development. Approach and Results—Eight-week-old male wild-type mice, transgenic mice overexpressing ET-1 selectively in endothelium (eET-1), Apoe−/− mice, and eET-1/Apoe−/− mice were fed high-fat diet for 8 weeks. eET-1/Apoe−/− had a 45% reduction in plasma high-density lipoprotein (P<0.05) and presented ≥2-fold more aortic atherosclerotic lesions compared with Apoe−/− (P<0.01). AAAs were detected only in eET-1/Apoe−/− (8/21; P<0.05). Reactive oxygen species production was increased ≥2-fold in perivascular fat, media, or atherosclerotic lesions in the ascending aorta and AAAs of eET-1/Apoe−/− compared with Apoe−/− (P<0.05). Monocyte/macrophage infiltration was enhanced ≥2.5-fold in perivascular fat of ascending aorta and AAAs in eET-1/Apoe−/− compared with Apoe−/− (P<0.05). CD4+ T cells were detected almost exclusively in perivascular fat (3/6) and atherosclerotic lesions (5/6) in ascending aorta of eET-1/Apoe−/− (P<0.05). The percentage of spleen proinflammatory Ly-6Chi monocytes was enhanced 26% by ET-1 overexpression in Apoe−/− (P<0.05), and matrix metalloproteinase-2 was increased 2-fold in plaques of eET-1/Apoe−/− (P<0.05) compared with Apoe−/−. Conclusions—ET-1 plays a role in progression of atherosclerosis and AAA formation by decreasing high-density lipoprotein, and increasing oxidative stress, inflammatory cell infiltration, and matrix metalloproteinase-2 in perivascular fat, vascular wall, and atherosclerotic lesions.

Exposure to Moderate Arsenic Concentrations Increases Atherosclerosis in ApoE / Mouse Model

Arsenic is a widespread environmental contaminant to which millions of people are exposed worldwide. Exposure to arsenic is epidemiologically linked to increased cardiovascular disease, such as atherosclerosis. However, the effects of moderate concentrations of arsenic on atherosclerosis formation are unknown. Therefore, we utilized an in vivo ApoE(-/-) mouse model to assess the effects of chronic moderate exposure to arsenic on plaque formation and composition in order to facilitate mechanistic investigations. Mice exposed to 200 ppb arsenic developed atherosclerotic lesions, a lower exposure than previously reported. In addition, arsenic modified the plaque content, rendering them potentially less stable and consequently, potentially more dangerous. Moreover, we observed that the lower exposure concentration was more atherogenic than the higher concentration. Arsenic-enhanced lesions correlated with several proatherogenic molecular changes, including decreased liver X receptor (LXR) target gene expression and increased proinflammatory cytokines. Significantly, our observations suggest that chronic moderate arsenic exposure may be a greater cardiovascular health risk than previously anticipated.

Organotins are potent activators of PPARγ and adipocyte differentiation in bone marrow multipotent mesenchymal stromal cells.

Adipocyte differentiation in bone marrow is potentially deleterious to both bone integrity and lymphopoiesis. Here, we examine the hypothesis that organotins, common environmental contaminants that are dual ligands for peroxisome proliferator-activated receptor (PPAR) γ and its heterodimerization partner retinoid X receptor (RXR), are potent activators of bone marrow adipogenesis. A C57Bl/6-derived bone marrow multipotent mesenchymal stromal cell (MSC) line, BMS2, was treated with rosiglitazone, a PPARγ agonist, bexarotene, an RXR agonist, or a series of organotins. Rosiglitazone and bexarotene potently activated adipocyte differentiation; however, bexarotene had a maximal efficacy of only 20% of that induced by rosiglitazone. Organotins (tributyltin [TBT], triphenyltin, and dibutyltin) also stimulated adipocyte differentiation (EC₅₀ of 10-20 nM) but with submaximal, structure-dependent efficacy. In coexposures, both bexarotene and TBT enhanced rosiglitazone-induced adipogenesis. To investigate the contribution of PPARγ to TBT-induced adipogenesis, we examined expression of PPARγ2, as well as its transcriptional target FABP4. TBT-induced PPARγ2 and FABP4 protein expression with an efficacy intermediate between rosiglitazone and bexarotene, similar to lipid accumulation. A PPARγ antagonist and PPARγ-specific small hairpin RNA suppressed TBT-induced differentiation, although to a lesser extent than rosiglitazone-induced differentiation, suggesting that TBT may engage alternate pathways. TBT and bexarotene, but not rosiglitazone, also induced the expression of TGM2 (an RXR target) and ABCA1 (a liver X receptor target). The results show that an environmental contaminant, acting with the same potency as a therapeutic drug, induces PPARγ-dependent adipocyte differentiation in bone marrow MSCs. Activation of multiple nuclear receptor pathways by organotins may have significant implications for bone physiology.

Arsenic trioxide inhibits nuclear receptor function via SEK1/JNK-mediated RXRα phosphorylation

We have previously published that 2 proven treatments for acute promyelocytic leukemia, As2O3 and retinoic acid, can be antagonistic in vitro. We now report that As2O3 inhibits ligand-induced transcription of the retinoic acid receptor, as well as other nuclear receptors that heterodimerize with the retinoid X receptor alpha (RXRalpha). As2O3 did not inhibit transactivation of the estrogen receptor or the glucocorticoid receptor, which do not heterodimerize with RXRalpha. We further show that As2O3 inhibits expression of several target genes of RXRalpha partners. Phosphorylation of RXRalpha has been reported to inhibit nuclear receptor signaling, and we show by in vivo labeling and phosphoamino acid detection that As2O3 phosphorylated RXRalpha in the N-terminal ABC region exclusively on serine residues. Consistent with our previous data implying a role for JNK in As2O3-induced apoptosis, we show that pharmacologic or genetic inhibition of JNK activation decreased As2O3-induced RXRalpha phosphorylation and blocked the effects of As2O3 on RXRalpha-mediated transcription. A mutational analysis indicated that phosphorylation of a specific serine residue, S32, was primarily responsible for inhibition of RXRalpha-mediated transcription. These data may provide some insight into the rational development of chemotherapeutic combinations involving As2O3 as well as into molecular mechanisms of arsenic-induced carcinogenesis resulting from environmental exposure.

Arsenic trioxide decreases AKT protein in a caspase-dependent manner

Arsenic trioxide (As2O3) is used clinically to treat acute promyelocytic leukemia but is less successful in other malignancies. To identify targets for potential combination therapies, we have begun to characterize signaling pathways leading to As2O3-induced cytotoxicity. Previously, we described the requirement for a reactive oxygen species–mediated, SEK1/c-Jun NH2-terminal kinase (JNK) pathway to induce apoptosis. AKT inhibits several steps in this pathway; therefore, we postulated that As2O3 might decrease its activity. Indeed, As2O3 decreases not only AKT activity but also total AKT protein, and sensitivity to As2O3 correlates with the degree of AKT protein decrease. Decreased AKT expression further correlates with JNK activation and the release of AKT from the JNK-interacting protein 1 scaffold protein known to assemble the mitogen-activated protein kinase cascade. We found that As2O3 regulates AKT protein stability without significant effects on its transcription or translation. We show that As2O3 decreases AKT protein via caspase-mediated degradation, abrogated by caspase-6, caspase-8, caspase-9, and caspase-3 inhibitors but not proteosome inhibitors. Furthermore, As2O3 enhances the ability of a heat shock protein 90 inhibitor to decrease AKT expression and increase growth inhibition. This suggests that As2O3 may be useful in combination therapies that target AKT pathways or in tumors that have constitutively active AKT expression. [Mol Cancer Ther 2008;7(6):1680–7]