Saswati Bhattacharya

Y Box-Binding Factor Promotes Eosinophil Survival by Stabilizing Granulocyte-Macrophage Colony-Stimulating Factor mRNA

Short-lived peripheral blood eosinophils are recruited to the lungs of asthmatics after allergen challenge, where they become long-lived effector cells central to disease pathophysiology. GM-CSF is an important cytokine which promotes eosinophil differentiation, function, and survival after transit into the lung. In human eosinophils, GM-CSF production is controlled by regulated mRNA stability mediated by the 3′ untranslated region, AU-rich elements (ARE). We identified human Y box-binding factor 1 (YB-1) as a GM-CSF mRNA ARE-specific binding protein that is capable of enhancing GM-CSF-dependent survival of eosinophils. Using a transfection system that mimics GM-CSF metabolism in eosinophils, we have shown that transduced YB-1 stabilized GM-CSF mRNA in an ARE-dependent mechanism, causing increased GM-CSF production and enhanced in vitro survival. RNA EMSAs indicate that YB-1 interacts with the GM-CSF mRNA through its 3′ untranslated region ARE. In addition, endogenous GM-CSF mRNA coimmunoprecipitates with endogenous YB-1 protein in activated eosinophils but not resting cells. Thus, we propose a model whereby activation of eosinophils leads to YB-1 binding to and stabilization of GM-CSF mRNA, ultimately resulting in GM-CSF release and prolonged eosinophil survival.

Resveratrol: Challenges in Translation to the Clinic — A Critical Discussion

Low cancer survival rates and the serious side effects often associated with current chemotherapeutics highlight the need for new and effective nontoxic anticancer agents. Since 1997 when Jang and colleagues first described resveratrols ability to inhibit carcinogenesis, it has consistently proven effective at tumor inhibition in diverse human cancer models. This finding has raised the hope that resveratrol would pioneer a novel class of nontoxic chemotherapeutics. As a consequence of initial basic and preclinical studies, resveratrol is now being extensively promoted in the unregulated nutraceutical sector. However, some fundamental aspects of resveratrols action need to be understood before it can be developed into a clinically viable anticancer drug. These areas pertain to the key mechanism(s) by which resveratrol potentiates its antitumor effects. Current research suggests that these mechanisms might be through novel pathways, requiring an understanding of cellular uptake, sentinel targets, and in vivo biological networks. The metabolism of resveratrol and its bioavailablity also warrant further consideration in light of recent in vitro and in vivo studies. Finally, we need to appreciate the sorts of information about resveratrol that may translate between different disease entities. We present a critical discussion of these issues and suggest important experiments that could pave the way to the successful translation of resveratrol to the clinic.Clin Cancer Res; 16(24); 5942–8. ©2010 AACR.

Resveratrol modulates the malignant properties of cutaneous melanoma through changes in the activation and attenuation of the antiapoptotic protooncogenic protein Akt/PKB.

Resveratrol, a nontoxic natural product, exhibits multifaceted biological effects including antimutagenic and anticancer properties. We examined the effect of resveratrol on the expression and activation of Akt/protein kinase B and its impact on melanoma cell migration and invasiveness. We also explored the use of resveratrol as an antimalignant treatment option against skin melanoma in mouse models of the disease. Akt expression and activity were determined by a combination of real-time PCR and western blot analysis. Cell lines stably expressing Akt or a dominant negative variant were used to further establish the role of Akt during the response to resveratrol. Wound healing and transwell assays were used as in-vitro correlates of melanoma cell migration and invasiveness. The efficacy of resveratrol in the treatment of melanoma was assessed in two syngeneic mouse models. Resveratrol downregulated and inactivated Akt in B16F10 and B16BL6 melanoma cells. Resveratrol also inhibited the migratory and invasive properties of these highly malignant cells. The reduction of cell migration and invasion, however, was reversed in cell lines overexpressing Akt or after cotreatment with pharmacological inhibitors that blocked Akt degradation. Dominant-negative Akt cells were more sensitive to resveratrol and had diminished migratory properties. Oral treatment with resveratrol reduced primary tumor volume, Akt expression, and the propensity for metastasis in syngeneic mouse models of melanoma. These results suggest that resveratrol can reduce the malignant properties of highly invasive melanoma cells by inactivating Akt. The nontoxic targeting of Akt by resveratrol makes it an attractive treatment option for melanoma.

Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells.

Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death.

HuR mRNA ligands expressed after seizure

HuR is a ubiquitously expressed AU-rich element (ARE)-binding protein that interacts with and stabilizes selective early response gene (ERG) mRNAs after cell activation or stress. To date, approximately 20 mRNAs have been unambiguously defined as HuR ligands. Given the discordance between the large number of ERG mRNAs and those few defined as ligands, we applied in vitro selection to isolate a broad range of HuR mRNA ligands using postseizure mouse hippocampal tissue. Selected mRNAs were converted into cDNA libraries and sequenced. Using this approach, we have identified over 600 novel, putative HuR mRNA ligands. These genes code for a variety of proteins, including transcription factors, signaling molecules, and kinases, but many have unknown function. Consistent with the means of their selection, several of these HuR ligands are differentially expressed in hippocampus after seizure. These results demonstrate a biochemical approach to identify and characterize the diverse repertoire of ligands for HuR and other regulatory mRNA-binding proteins.

Targeted Molecular Radiotherapy of Pediatric Solid Tumors Using a Radioiodinated Alkyl-Phospholipid Ether Analog

External-beam radiotherapy plays a critical role in the treatment of most pediatric solid tumors. Particularly in children, achieving an optimal therapeutic index to avoid damage to normal tissue is extremely important. Consequently, in metastatic disease, the utility of external-beam radiotherapy is limited. Molecular radiotherapy with tumor-targeted radionuclides may overcome some of these challenges, but to date there exists no single cancer-selective agent capable of treating various pediatric malignancies independently of their histopathologic origin. We tested the therapeutic potential of the clinical-grade alkyl-phospholipid ether analog CLR1404, 18-(p-iodophenyl)octadecyl phosphocholine, as a scaffold for tumor-targeted radiotherapy of pediatric malignancies. Methods: Uptake of CLR1404 by pediatric solid tumor cells was tested in vitro by flow cytometry and in vivo by PET/CT imaging and dosimetry. The therapeutic potential of 131I-CLR1404 was evaluated in xenograft models. Results: In vitro, fluorescent CLR1404-BODIPY showed significant selective uptake in a variety of pediatric cancer lines compared with normal controls. In vivo tumor-targeted uptake in mouse xenograft models using 124I-CLR1404 was confirmed by imaging. Single-dose intravenous injection of 131I-CLR1404 significantly delayed tumor growth in all rodent pediatric xenograft models and extended animal survival while demonstrating a favorable side effect profile. Conclusion: 131I-CLR1404 has the potential to become a tumor-targeted radiotherapeutic drug with broad applicability in pediatric oncology. Because 131I-CLR1404 has entered clinical trials in adults, our data warrant the development of pediatric clinical trials for this particularly vulnerable patient population.

Abstract 1236: Novel androgen receptor inhibitors in triple negative breast cancer

Triple negative breast cancer (TNBC), which lacks expression of hormone receptors and human epidermal growth factor receptor 2, has the worst prognosis of all breast cancer subtypes, due to inherent chemo-resistance and a lack of therapeutic targets. A subset of TNBC has been shown to express androgen receptor (AR) but despite promising pre-clinical data, clinical trials evaluating the use of approved anti-androgens (bicalutamide, enzalutamide) in AR-positive TNBC has shown modest efficacy to date. In prostate cancer, bicalutamide binds to AR with low affinity and in the presence of AR gene amplification or mutation, can exhibit partial agonist activity leading to resistance. In order to increase affinity for AR and to avoid AR antagonist to agonist conversion synthesized 7-substituted umbelliferones (UMB) derivatives with a distinct scaffold modified from available AR antagonists (Kandil et al Bioorganic & Medicinal Chemistry Letters, 2016). These agents exhibited inhibitory activity in human prostate cancer cells lines at sub-micro molar level, a 50-fold and 30-fold improvement over bicalutamide and enzalutamide, respectively. We hypothesized that these novel AR antagonists would be effective in inhibiting the growth of AR-positive TNBC cells. Methods: TNBC AR negative mesenchymal MDA-MB-231 (as control) and AR positive MDA-MB-231 cells were utilized for determination of the efficacy of the UMB derivatives. For proliferation assays, viability of the cells was monitored over a period of 72 hours with redox dye Cell Titer-Blue. Standard Western blotting techniques were used for protein detection. For apoptosis analysis, standard flow cytometry reagents, FITC labelled Annexin V and Propidium Iodine(PI) mix were utilized. Results: UMB derivatives decreased the expression of AR in AR-positive MDA-MB-453 in a dose dependent manner. UMB derivatives, at micro Molar concentration decreased cell proliferation of AR-positive and interestingly AR-negative cell lines by 20 to 50%, also in a dose dependent manner. UMB derivatives induced apoptosis in both cell lines. Interestingly, UMB derivatives induced a change from a mesenchymal to epithelial phenotype in AR-negative MDA-MB-231 cells, which was confirmed by decreased expression of vimentin and other mesenchymal markers. Conclusions: Novel UMB derivatives inhibit growth and cause apoptosis in TNBC cells and induce mesenchymal-to-epithelial transition in an AR-negative mesenchymal TNBC cell line. Targeting AR with novel AR antagonists seem to be a promising therapeutic approach for AR-positive TNBC and further evaluation of AR antagonists in AR-negative mesenchymal TNBC is warranted. Citation Format: Saswati Bhattacharya, Yuanqi Cai, Steven Kurina, Pamela Westmark, Benjamin Hokanson, Matthew Anderson, Ruth O’Regan. Novel androgen receptor inhibitors in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1236. doi:10.1158/1538-7445.AM2017-1236

Abstract 4214: Resveratrol: Effect on polyamine metabolism and synergistic interactions with DFMO and DenSpm in breast cancer and uveal melanoma cell lines

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Resveratrol, (3,5,4’-trihydroxy-trans-stilbene), was first reported to act as a chemo-preventative anticancer compound in mice by Jang et al. in 1997. It was subsequently shown to inhibit the growth and initiation of cancer cells in vitro and tumorigenesis in mouse cancer models. Recent work has focused on the role of the mitochondria in the resveratrol-induced death of the cancer cells, with resveratrol promoting apoptosis in both cancer cell lines and human tumors growing in host mice. In a colon cancer cell line (Caco), resveratrol has also been shown to affect the metabolism of polyamines, a group of compounds essential for the growth and proliferation of cells. Perturbations of polyamine levels and enzymes involved in their metabolism have long been targets of potential anti-cancer treatments. Difluormethylornithine (DFMO), a suicide inhibitor of ODC (the rate limiting biosynthetic enzyme), and N1, N11-diethylnorspermine (DenSpm), a polyamine analog leading to the induction of the polyamine catabolic enzyme SSAT, have been shown to reduce growth of cancer cells in several models and have been tested in clinical trials. In addition to reducing viability and inducing apoptosis, resveratrol has also been reported to reduce ODC activity and induce SSAT activity in human colon cancer lines. The objectives of the current study were to extend the findings of earlier reports to uveal melanoma (C918) and breast cancer cells (MCF-7), examining whether or not resveratrol affects the activity of the enzymes in these cancer cell lines. Potential synergistic interactions between resveratrol and the inhibitors DFMO and DenSpm were also investigated. In our studies, resveratrol was found to reduce the expression of ODC and significantly induce the expression of SSAT as determined by real-time PCR. This is the first report of the effect of resveratrol on these enzymes in cells other than colon cancer cells. Resveratrol, DFMO and DenSpm each reduced cell viability and proliferation over a three-day course of treatment as determined by the conversion of resazurin to resorufin by viable cells (Cell Titer Blue Assay®). Combinations of resveratrol and DFMO or DenSpm affected proliferation to a greater degree than any of the compounds used alone. The ability of these compounds to induce apoptosis was tested by measuring the activity of the executioner caspases 3/7 in treated cells using the fluorogenic substrate Ac-DEVD-AMC. As was found in viability studies, the synergistic effects of the compounds were also observed with caspase activity, where the combinations of resveratrol and DFMO or DenSpm led to enhanced activation of the enzymes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4214. doi:10.1158/1538-7445.AM2011-4214