A natural BH3-mimetic, small molecule inhibitor of Bcl-2, (-)-gossypol, shows promise in ongoing Phase II-III clinical trials for human prostate cancer. Here we show that (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy via blocking Bcl-2—Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1 and activating the autophagic pathway. (-)-Gossypol-induced autophagy is Beclin1- and Atg5-dependent. Our results demonstrate for the first time that (-)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (-)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which would facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.

No abstract

The dopamine 3 (D3) subtype receptor has been implicated in several neurological conditions, and potent and selective D3 ligands may have therapeutic potential for the treatment of drug addiction, Parkinson's disease, and schizophrenia. In this paper, we report computational homology modeling of the D3 receptor based upon the high-resolution X-ray structure of rhodopsin, extensive structural refinement in the presence of explicit lipid bilayer and water environment, and validation of the refined D3 structural models using experimental data. We further describe the development, validation, and application of a hybrid computational screening approach for the discovery of several classes of novel and potent D3 ligands. This computational approach employs stepwise pharmacophore and structure-based searching of a large three-dimensional chemical database for the identification of potential D3 ligands. The obtained hits are then subjected to structural novelty screening, and the most promising compounds are tested in a D3 binding assay. Using this approach we identified four compounds with K(i) values better than 100 nM and eight compounds with K(i) values better than 1 microM out of 20 compounds selected for testing in the D3 receptor binding assay. Our results suggest that the D3 structural models obtained from this study may be useful for the discovery and design of novel and potent D3 ligands. Furthermore, the employed hybrid approach may be more effective for lead discovery from a large chemical database than either pharmacophore-based or structure-based database screening alone.

Antiapoptotic members of the Bcl-2 family proteins are overexpressed in prostate cancer and are promising molecular targets for modulating chemoresistance of prostate cancer. (-)-Gossypol, a natural BH3 mimetic, is a small-molecule inhibitor of Bcl-2/Bcl-xL/Mcl-1 currently in phase II clinical trials as an adjuvant therapy for human prostate cancer. Our objective is to examine the chemosensitization potential of (-

Musashi-1 (MSI1) is an RNA-binding protein that acts as a translation activator or repressor of target mRNAs. The best-characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin-dependent kinase inhibitor P21WAF-1. We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA-binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (–)-gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti-tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (–)-gossypol with the RNA binding pocket of MSI1. We further showed that (–)-gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (–)-gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (–)-gossypol as a potential small molecule inhibitor of MSI1-RNA interaction, and suggests that inhibition of MSI1's RNA binding activity may be an effective anti-cancer strategy.

Purpose: Bcl-x L overexpression is common in head and neck squamous cell carcinomas (HNSCC) and correlates with resistance to chemotherapy. Thus, a nonpeptidic, cellpermeable small molecule that mimics the BH3 domain of proapoptotic proteins may inhibit Bcl-x L function and have therapeutic potential for HNSCC by overcoming drug-resistance. (؊)-Gossypol, the levorotatory isomer of a natural product isolated from cottonseeds and roots, was recently discovered to bind to the BH3 binding groove of Bcl-x L and Bcl-2.Experimental Design: We investigated the in vitro effects of (؊)-gossypol on HNSCC cell lines as well as on fibroblast and keratinocyte cultures by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell survival assays and assessed the results with respect to Bcl-2 family protein expression.Results: We observed dose-dependent growth inhibition of 10 HNSCC cell lines at biologically achievable doses (2.5-10 mol/L). (؊)-Gossypol doses required to inhibit the growth of human fibroblast cell lines by 50% were 2-to 10-fold higher than for HNSCC cell lines. To inhibit human oral keratinocyte growth by 50%, (؊)-gossypol concentrations were 2-to 3-fold higher than for HNSCC cell lines.Conclusions: There is a direct correlation between Bclx L -to-Bcl-x S ratios and sensitivity to (؊)-gossypol. This agent induced apoptosis in a much higher proportion of cells with wild-type p53. Importantly, cell lines resistant to cisplatin were very sensitive to (؊)-gossypol. These results demonstrate that (؊)-gossypol has potent antitumor activity in HNSCC in vitro. This agent may be developed as a novel therapeutic agent for HNSCC, either alone or in combination with existing chemotherapeutic agents.

It has been suggested that endoplasmic reticulum (ER) stress facilitates fibrotic remodeling. Therefore, modulation of ER stress may serve as one of the possible therapeutic approaches to renal fibrosis. We examined whether and how activation of AMP-activated protein kinase (AMPK) suppressed ER stress induced by chemical ER stress inducers [tunicamycin (TM) and thapsigargin (TG)] and also nonchemical inducers in tubular HK-2 cells. We further investigated the in vivo effects of AMPK on ER stress and renal fibrosis. Western blot analysis, immunofluorescence, small interfering (si)RNA experiments, and immunohistochemical staining were performed. Metformin (the best known clinical activator of AMPK) suppressed TM- or TG-induced ER stress, as shown by the inhibition of TM- or TG-induced upregulation of glucose-related protein (GRP)78 and phosphorylated eukaryotic initiation factor-2α through induction of heme oxygenase-1. Metformin inhibited TM- or TG-induced epithelial-mesenchymal transitions as well. Compound C (AMPK inhibitor) blocked the effect of metformin, and 5-aminoimidazole-4-carboxamide-1β riboside (another AMPK activator) exerted the same effects as metformin. Transfection with siRNA targeting AMPK blocked the effect of metformin. Consistent with the results of cell culture experiments, metformin reduced renal cortical GRP78 expression and increased heme oxygenase-1 expression in a mouse model of ER stress-induced acute kidney injury by TM. Activation of AMPK also suppressed ER stress by transforming growth factor-β, ANG II, aldosterone, and high glucose. Furthermore, metformin reduced GRP78 expression and renal fibrosis in a mouse model of unilateral ureteral obstruction. In conclusion, AMPK may serve as a promising therapeutic target through reducing ER stress and renal fibrosis.

Ferumoxytol, which is originally intended for MRI and anemia treatment, is currently the only inorganic nanodrug approved by FDA for clinical application in vivo. Common ferumoxytol seems incapable of meeting the requirements for diverse applications. Thus, the development of a novel strategy based on co-precipitation to produce ferumoxytol with high quality is an imminent task. Herein, we proposed a physically assisted strategy, namely hydrocooling and magnetically internal heating co-precipitation, to optimize the properties of ferumoxytol and thus significantly enhance its magnetic performance. Magnetization of the newly developed ferumoxytol can reach 104-105 emu g-1 Fe, which is the highest value among the reported results. It has been found that the crystalline structures of the newly developed ferumoxytol have been greatly improved on the basis of pharmaceutical quality criteria.