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Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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L’infection par le VIH-1 est un problème majeur de la santé publique qui touche plus de 35 millions de personnes à l’échelle mondiale. La réplication du VIH-1 est déclenchée par l’activation du promoteur LTR, qui contient deux sites de liaison pour le facteur de transcription NF-κB. Ces sites de liaison sont hautement conservés dans le génome du VIH-1, illustrant ainsi l’importance de NF-κB dans l’activité transcriptionelle des gènes du VIH-1 et la production de nouvelles particules virales. La withaferin A (WA) est une substance bioactive extraite de la plante Withania somnifera, qui possède des propriétés pharmacologiques non négligeables dans la régulation de la réponse immunitaire. Des études récentes ont démontré que le potentiel anti-inflammatoire de la WA est dû principalement à l’inhibition de la voie de NF-κB. Le but de ce projet est de déterminer l’effet de la WA sur la réplication du VIH-1 dans les cellules T, qui sont les cibles principales du virus. Des essais de transfections transitoires de cellules T Jurkat E6.1 avec des plasmides contenant le promoteur du VIH-1 ayant différentes constructions de NF-κB, ont démontré que la WA peut réduire l’activité du promoteur d’une manière dépendante de NF-κB. Quant à la production de particules virales, des essais d’infection avec des virus pseudotypés démontrent que la WA diminue la production virale jusqu’à 90% dans des cellules T stimulées avec PMA/PHA et TNF-α, tandis que les mutants ayant des sites de liaison défective pour NF-κB ne sont pas affectés. Des essais de retardement sur gel ainsi que des immunobuvardages de type Western ont montré que la WA altère l’habilité de NF-κB à transloquer dans le noyau, ce qui se traduit par l’inhibition de la synthèse de l’IκB-α, protéine inhibitrice de NF-κB, phénomène sous contrôle étroite de ce facteur de transcription. Ces résultats suggèrent que la WA pourrait permettre une diminution de la réplication virale d’une manière dépendante de NF-κB et ainsi empêcher la propagation du virus aux cellules T chez les individus infectés.

Part I BCR-ABL fusion oncogene results fromt(9;22)(q34;q11) translocation of chromosome is the most common genetic abnormality found in chronic myeloid leukemia (CML) cells . The encoded protein of this fusion gene exhibits constitutively active tyrosinekinase activity which is required for the pathogenesis of CML. We addressed how BCR-ABL oncoprotein increased Skp2 expression. Treatment of Imatinib or LY294002 reduced Skp2mRNA in BCR-ABL-positive K562 cells. Knockdown of AKT by small hairpin RNAalso reduced Skp2 expression. We found that BCR-ABL up-regulated Skp2 via Sp1 because (1) the Sp1 site located at the −386/−380 promoter region was important for BCR-ABL-induced Skp2 promoter activity, (2) chromatin immunoprecipitation assay demonstrated that Imatinib inhibited the recruitment of p300 to the Sp1 site of Skp2 promoter and (3) knockdown of Sp1 reduced Skp2 expression in K562 cells. These results suggest that BCR-ABL controls Skp2 gene transcription via the PI3K/AKT/Sp1 pathway. In addition to transcriptional regulation of Skp2, Bcr-Abl also modulates Skp2 protein stability in these cells. Treatment of Bcr-Abl kinase inhibitor imatinib led to G1 growth arrest accompanied with reduced Skp2 expression. Interestingly, reduction of Skp2 protein occurred prior to down-regulation of Skp2 mRNA suggesting a post-translational control. The half-life of Skp2 protein was significantly attenuated in imatinib-treated cells. Knockdown of Bcr-Abl similarly caused Skp2 protein instability. The decrease of Skp2 was induced by increased protein degradation through the ubiquitin/ proteasome pathway. Our results demonstrated that imatinib treatment or Bcr-Abl knockdown reduced Emi1, an endogenous inhibitor of the E3 ligase APC/Cdh1 which mediated the degradation of Skp2 protein. We found that Emi1 stability was regulated by phosphorylation and mutation of tyrosine 142 significantly reduced the stability. Lines of evidence suggested Bcr-Abl-induced Emi1 phosphorylation was mediated by Src kinase. (1) Src inhibitor SU6656 inhibited Emi1 tyrosine phosphorylation in Bcr-Abl-positive K562 cells. (2) Transfection of v-Src rescued the reduction of Emi1 by imatinib. (3) Mutation of tyrosine 142 to phenylalanine (Y142F) abolished the phosphorylation of Emi1 by recombinant Src kinase. In addition, ectopic expression of wild type but not Y142F mutant Emi1 could counteract imatinib-caused G1 growth arrest. Collectively, our results suggest that Bcr-Abl fusion oncogene increases Emi1 phosphorylation and stability to prevent Skp2 protein degradation via APC/Cdh1-induced ubiquitination and to enhance proliferation of CML cells. Part II Although imatinib therapy of chronic myelogenous leukemia is effective, the resistance to imatinib challenges the treatment of this disease. Therefore, search of novel drugs to overcome imatinib resistance is a critical issue in clinic. Withaferin A (WA), an extract of Withania somniferia, exhibits anti-cancer activity on a number of solid tumors. In this study, we investigate the effect of WA on imatinib-sensitive and -resistant CML cells. WA at low concentrations induced autophagy in imatinib-sensitive K562 cells. Co-treatment of chloroquine suppressed autophagy and switched WA-treated K562 cells to apoptosis. This data indicated that autophagy protected K562 cells from apoptosis induced by WA. However, we found that WA triggered caspase activation and apoptosis in imatinib-resistant T315I-positive cells and this effect was associated with down-regulation of Akt activity. Treatment of the AKT inhibitor LY294002 also caused apoptosis in imatinib-resistant T315I-positive cells. Ectopic expression of constitutively active Akt reversed WA-induced apoptosis and caspase activation in imatinib-resistant T315I-positive cells. Molecular study demonstrates that WA repressed the Akt signaling pathway by decreasing Akt expression. We found that WA abolished formation of the hsp90/cdc37/Akt complex to cause Akt degradation through the ubiquitin- and proteasome-dependent pathway. More importantly, WA also induced AKT down-regulation and apoptosis in primary CML cells. Taken together, our results suggested that imatinib-resistant T315I-positive cells were more addicted to Akt-dependent survival pathway and were more sensitive to WA. Therefore, WA could be useful for the treatment of imatinib-resistant CML. Part III Suberoylanilide hydroxamic acid (SAHA) is undergoing clinical trial for the treatment of various cancers including chronic myeloid leukemia (CML). We study the potential miRNAs which involved in the anti-cancer effect of SAHA. Microarray analysis revealed that the expression of 57 and 63 miRNAs was significantly changed in K562 cells treated with SAHA for 8h and 24h respectively. Five miRNAs(miR-92a, miR-199b-5p, miR-223, miR-627 and miR-675) were highly expressed in K562 cells and continuously repressed by SAHA. miR-92a and miR-223 known to play important roles in normal and hematopoisis were further characterized. Up-regulation of miR-92a was found in K562 cells and in primary CML cells. Inhibition of miR-92a with SAHA led to increase of the tumor suppressor Fbxw7. Conversely, ectopic expression of pri-miR-92a reversed SAHA-induced apoptosis of K562 cells, increase of Fbxw7 3¡¦-UTR reporter activity and up-regulation of Fbxw7. Collecively, miR-92a is up-regulated in CML cells, and SAHA downregulated the expression of miR-92a to result in apoptosis of CML cells.

Chaperone proteins such as heat-shock proteins 70, 90 and 110, glucose-related protein 94 and calreticulin have been reported to be effective anti-tumor vaccines when purified from a tumor source. We have developed a procedure utilizing a free-solution-isoelectric focusing technique to obtain vaccines from tumor or normal tissue sources that are rich in multiple immunogenic chaperone proteins, called Chaperone-Rich Cell Lysate (CRCL). Tumor-associated peptides are presumed to be the currency of T-cell mediated anti-cancer immunity, and tumor-derived chaperone vaccines are believed to be purveyors of such peptides. As a novel anti-cancer strategy, we have examined the extent to which the peptide repertoire of CRCL can be manipulated. Here, we explored the concept of creating a designer CRCL, utilizing the adjuvant properties and the carrying capacity of CRCL to deliver exogenous antigenic peptides for DC-based presentation and ultimately demonstrate the anti-tumor efficacy of the designer vaccine in vivo. Designer CRCL allows for the development of personalized vaccines to those afflicted with cancer expressing known antigens.Growing evidence indicates that the stress response, specifically involving HSPs, has a profound impact on tumor immunogenicity. Enhancement of T-cell-mediated immunogenicity correlates with the expression of inducible heat shock protein 70 (iHSP70), the major heat-inducible member of the HSP70 family. In addition, studies have shown tumor-specific cell surface localization of iHSP70 correlates with an increased sensitivity to lysis mediated by human natural killer (NK) cells. Given these findings, investigating novel and effective means of modulating the heat shock response within tumor cells may bear great therapeutic potential and result in potent anti-tumor immune activity. Withaferin A (WA) is a compound isolated from the plant Withania somnifera that has been shown to induce a robust transcriptional heat shock response. In our studies, we found that WA treatment resulted in increased surface expression of iHSP70 in several tumor types leading to significant immunostimulatory effects. These findings indicated that WA-dependent modulation of the heat shock response may enhance tumor immunogenicity. Given the potent immunomodulatory and anti-tumor effects of WA as well as the adjuvanticity and specificity of peptide-complexed CRCL against tumors, these therapies individually have shown profound anti-cancer activity.

Visceral leishmaniasis (VL) causes fatal life-threatening disease, if left untreated. The current drugs have various limitations; hence, natural products from medicinal plants are being focused in search of new drugs to treat leishmaniasis. The aim of the present study was to evaluate the antileishmanial and immunomodulatory activities of F5 and F6 alcoholic fractions from Withania somnifera leaves and purified withaferin-A in Leishmania donovani-infected peritoneal macrophages and BALB/c mice. We observed that F5 (15 mu g/mL), F6 (10 mu g/mL), and withaferin-A (1.5 mu M) reduce amastigote count in peritoneal macrophages and induce reactive oxygen species and significant decrease in IL-10 mRNA expression compared to control upon treatment. Subsequently, in vivo study mice were treated with F5 (25 and 50 mg/kg b.wt.), F6 (25 and 50 mg/kg b.wt.) orally, and withaferin-A (2 mg/kg b.wt.) intraperitoneally for 10 consecutive days and a drastic reduction in parasite burden in both spleen and liver were observed. The treatment resulted in the reduction in IL-10, IL-4, and TGF-beta mRNA expression and a significant increase in IFN-gamma /IL-10 expression ratio in the treated group compared to control. The humoral response of these alcoholic fractions and withaferin-A shows increased IgG2a levels when compared with IgG1 in treated mice. Taken together, our result concludes that withanolides in alcoholic fractions demonstrate a potent antileishmanial and immunomodulatory activities in experimental VL.

Neurological disorders are the biggest concern globally and ageing contributes in worsening the disease scenarios. In AD or AD like diseases, there is abnormal accumulation of extracellular amyloid beta produced due to abnormal processing of the transmembrane amyloid precursor protein, by β and γ-secretases. It spreads in the cortical and limbic regions of the brain leading to neuronal toxicity, impairment in memory and neurological functions. Aβ deposition in the CNS is common in aging HIV patients. Neurotoxic protein Tat, results in increased Aβ in combination with drugs of abuse cocaine. We examined the role of Withaferin A, against Aβ induced neurotoxicity. Our in-vitro dose optimization study demonstrates that lower concentrations (0.5–2 μM) of WA significantly reduce the Aβ40, without inducing cytotoxicity in the APP plasmid transfected SH-SY5Y cells (SHAPP). We demonstrate that Aβ secretion is increased in the presence of Tat (50 ng/ml) and coc (0.1 μM), WA reduces the Tat and coc induced increase in Aβ40. Additionally, we studied the role of WA against NF-kB mediated neuroinflammation, and observed that WA inhibits the expression of NFkB2 and RELA transcription factors, which play a major role in the expression of inflammatory chemokines. Further, to address the issue of minimal drug bioavailability in the CNS, we developed the WA loaded liposomal nanoformulation (WA-LNF) and characterized its size (499+/-50nm), toxicity and drug binding efficacy (28%). Our in-vitro 3D BBB transmigration of WA-LNF demonstrated ~40% transmigration efficiency. Furthermore, it was imperative for us to understand the mechanism of action of WA, therefore we studied the molecular mechanism of interaction of WA with Aβ protein by in-silico molecular dynamics simulations. We demonstrated that WA binds to the middle region of Aβ protein and the amino acid motif involved were FAEDVGS highlighting the mid-region Aβ capture by WA. 3 Hydrogen bonds were formed between WA and the amino acids, ASN17, GLY15 and SER16. This study reports WA as a potent neuroprotectant against amyloid induced neurotoxicity. Our study may have an immense therapeutic potential to target Aβ in the CNS, in the ageing patients and/or PLWH and/or ageing drug abusers.