Azim Hossain

A possible cross-talk between autophagy and apoptosis in generating an immune response in melanoma

Melanoma is the most aggressive form of skin cancer, responsible for the majority of skin cancer related deaths. Thus, the search for natural molecules which can effectively destroy tumors while promoting immune activation is essential for designing novel therapies against metastatic melanoma. Here, we report for the first time that a natural triterpenoid, Ganoderic acid DM (GA-DM), induces an orchestrated autophagic and apoptotic cell death, as well as enhanced immunological responses via increased HLA class II presentation in melanoma cells. Annexin V staining and flow cytometry showed that GA-DM treatment induced apoptosis of melanoma cells, which was supported by a detection of increased Bax proteins, co-localization and elevation of Apaf-1 and cytochrome c, and a subsequent cleavage of caspases 9 and 3. Furthermore, GA-DM treatment initiated a possible cross-talk between autophagy and apoptosis as evidenced by increased levels of Beclin-1 and LC3 proteins, and their timely interplay with apoptotic and/or anti-apoptotic molecules in melanoma cells. Despite GA-DM’s moderate cytotoxicity, viable cells expressed high levels of HLA class II proteins with improved antigen presentation and CD4+ T cell recognition. The antitumor efficacy of GA-DM was also investigated in vivo in murine B16 melanoma model, where GA-DM treatment slowed tumor formation with a significant reduction in tumor volume. Taken together, these findings demonstrate the potential of GA-DM as a natural chemo-immunotherapeutic capable of inducing a possible cross-talk between autophagy and apoptosis, as well as improved immune recognition for sustained melanoma tumor clearance.

Elevation of c-MYC Disrupts HLA Class II–Mediated Immune Recognition of Human B Cell Tumors

Elevated levels of the transcription factor c-myc are strongly associated with various cancers, and in particular B cell lymphomas. Although many of c-MYC’s functions have been elucidated, its effect on the presentation of Ag through the HLA class II pathway has not been reported previously. This is an issue of considerable importance, given the low immunogenicity of many c-MYC–positive tumors. We report in this paper that increased c-MYC expression has a negative effect on the ability of B cell lymphomas to functionally present Ags/peptides to CD4+ T cells. This defect was associated with alterations in the expression of distinct cofactors as well as interactions of antigenic peptides with class II molecules required for the presentation of class II–peptide complexes and T cell engagement. Using early passage Burkitt’s lymphoma (BL) tumors and transformed cells, we show that compared with B lymphoblasts, BL cells express decreased levels of the class II editor HLA-DM, lysosomal thiol-reductase GILT, and a 47-kDa enolase-like protein. Functional Ag presentation was partially restored in BL cells treated with a c-MYC inhibitor, demonstrating the impact of this oncogene on Ag recognition. This restoration of HLA class II–mediated Ag presentation in early passage BL tumors/cells was linked to enhanced HLA-DM expression and a concurrent decrease in HLA-DO in BL cells. Taken together, these results reveal c-MYC exerts suppressive effects at several critical checkpoints in Ag presentation, which contribute to the immunoevasive properties of BL tumors.

Enhancement of HLA class II-restricted CD4+ T cell recognition of human melanoma cells following treatment with bryostatin-1.

The majority of melanoma cells express detectable levels of HLA class II proteins, and an increased threshold of cell surface class II is crucial for the stimulation of CD4+ T cells. Bryostatin-1, a protein kinase C (PKC) activator, has been considered as a potent chemotherapeutic agent in a variety of in vitro tumor models. Little is known about the role of bryostatin-1 in HLA class II Ag presentation and immune activation in malignant tumors, especially in melanoma. In this study, we show that bryostatin-1 treatment enhances CD4+ T cell recognition of melanoma cells in the context of HLA class II molecules. We also show that bryostatin-1 treatment of melanoma cells increases class II protein levels by upregulating the class II transactivator (CIITA) gene. Flow cytometry and confocal microscopic analyses revealed that bryostatin-1 treatment upregulated the expression of costimulatory molecules (CD80 and CD86) in melanoma cells, which could prolong the interaction of immune cells and tumors. Bryostatin-1 also induced cellular differentiation in melanoma cells, and reduced tumorigenic factors such as pro-cathepsins and matrix-metalloproteinase-9. These data suggest that bryostatin-1 could be used as a chemo-immunotherapeutic agent for reducing tumorigenic potential of melanoma cells while enhancing CD4+ T cell recognition to prevent tumor recurrence.

Reduction of myeloid-derived suppressor cells and lymphoma growth by a natural triterpenoid.

Lymphoma is a potentially life threatening disease. The goal of this study was to investigate the therapeutic potential of a natural triterpenoid, Ganoderic acid A (GA‐A) in controlling lymphoma growth both in vitro and in vivo. Here, we show that GA‐A treatment induces caspase‐dependent apoptotic cell death characterized by a dose‐dependent increase in active caspases 9 and 3, up‐regulation of pro‐apoptotic BIM and BAX proteins, and a subsequent loss of mitochondrial membrane potential with release of cytochrome c. In addition to GA‐As anti‐growth activity, we show that lower doses of GA‐A enhance HLA class II‐mediated antigen (Ag) presentation and CD4+ T cell recognition of lymphoma cells in vitro. The therapeutic relevance of GA‐A treatment was also tested in vivo using the EL4 syngeneic mouse model of metastatic lymphoma. GA‐A‐treatment significantly prolonged survival of EL4 challenged mice and decreased tumor metastasis to the liver, an outcome accompanied by a marked down‐regulation of STAT3 phosphorylation, reduction myeloid‐derived suppressor cells (MDSCs), and enhancement of cytotoxic CD8+ T cells in the host. Thus, GA‐A not only selectively induces apoptosis in lymphoma cells, but also enhances cell‐mediated immune responses by attenuating MDSCs, and elevating Ag presentation and T cell recognition. The demonstrated therapeutic benefit indicates that GA‐A is a candidate for future drug design for the treatment of lymphoma. J. Cell. Biochem. 116: 102–114, 2015.

Mechanisms regulating enhanced human leukocyte antigen class II-mediated CD4 + T cell recognition of human B-cell lymphoma by resveratrol

Abstract Malignant B-cells express measurable levels of human leukocyte antigen (HLA) class II proteins, but often escape immune recognition by CD4 + T cells. Resveratrol (Resv) has been the focus of numerous investigations due to its potential chemopreventive and anti-cancer effects, but it has never been tested in the regulation of immune components in B-cell tumors. Here, we show for the first time that Resv treatment enhances HLA class II-mediated immune detection of B-cell lymphomas by altering immune components and class II presentation in tumor cells. Resv treatment induced an up-regulation of both classical and non-classical HLA class II proteins (DR and DM) in B-lymphoma cells. Resv also altered endolysosomal cathepsins (Cat S, B and D) and a thiol reductase (GILT), increasing HLA class II-mediated antigen (Ag) processing in B-cell lymphomas and their subsequent recognition by CD4 + T cells. Mechanistic study demonstrated that Resv treatment activated the recycling class II pathway of Ag presentation through up-regulation of Rab 4B protein expression in B-lymphoma cells. These findings suggest that HLA class II-mediated immune recognition of malignant B-cells can be improved by Resv treatment, thus encouraging its potential use in chemoimmunotherapy of B-cell lymphoma.

HLA class II defects in Burkitt lymphoma: bryostatin-1-induced 17 kDa protein restores CD4+ T-cell recognition.

While the defects in HLA class I-mediated Ag presentation by Burkitt lymphoma (BL) have been well documented, CD4+ T-cells are also poorly stimulated by HLA class II Ag presentation, and the reasons underlying this defect(s) have not yet been fully resolved. Here, we show that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. The observed defect was not associated with low levels of BL-expressed costimulatory molecules, as addition of external co-stimulation failed to result in BL-mediated CD4+ T-cell activation. We further demonstrate that BL cells express the components of the class II pathway, and the defect was not caused by faulty Ag/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Treatment of BL with broystatin-1, a potent modulator of protein kinase C, led to significant improvement of functional class II Ag presentation in BL. The restoration of immune recognition appeared to be linked with an increased expression of a 17 kDa peptidylprolyl-like protein. These results demonstrate the presence of a specific defect in HLA class II-mediated Ag presentation in BL and reveal that treatment with bryostatin-1 could lead to enhanced immunogenicity.

Autophagy-dependent crosstalk between GILT and PAX-3 influences radiation sensitivity of human melanoma cells

Melanoma represents an ever‐increasing problem in the western world as incidence rates continue to climb. Though manageable during early stages, late stage metastatic disease is highly resistant to current intervention. We have previously shown that gamma‐interferon‐inducible lysosomal thiol‐reductase (GILT) enhances HLA class II antigen processing and immune detection of human melanoma cells. Here we report that GILT expression inhibits a potential target, paired box‐3 (PAX‐3) protein, in late stage human metastatic melanoma. We also show that GILT transfection or induction by IFN‐γ, decreases PAX‐3 protein expression while upregulating the expression of Daxx, which is also a repressor of PAX‐3. Confocal microscopic analysis demonstrated that GILT co‐localizes with PAX‐3 protein, but not with Daxx within melanoma cells. Immunoprecipitation and immunoblotting studies suggest that GILT expression negatively regulates PAX‐3 through the autophagy pathway, potentially resulting in increased susceptibility to conventional treatment in the form of chemotherapy or radiotherapy. While high‐dose radiation is a common treatment for melanoma patients, our data suggest that GILT expression significantly increased the susceptibility of melanoma cells to low‐dose radiation therapy via upregulation of tumor suppressor protein p53. Overall, these data suggest that GILT has multiple roles in inducing human melanoma cells as better targets for radiation and immunotherapy.

Neuron specific enolase is a potential target for regulating neuronal cell survival and death: implications in neurodegeneration and regeneration

Enolase is a multifunctional enzyme primarily involved in catalyzing the conversion of 2phosphoglycerate to phosphoenolpyruvate during glycolysis and the reverse reaction during gluconeogenesis[1–4]. Though typically expressed in the cytosol, enolase has been shown to migrate to the cell surface upon inflammatory signal[3]. It then enhances antigen presentation for the invasion of host cells via plasminogen binding and subsequent plasmin activation, leading to degradation of the extracellular matrix. Cell surface expression of enolase, possibly due to an association with the urokinase-type plasminogen activator (uPA)/uPA receptor complex, additionally induces the production of reactive oxygen species, nitric oxide, and pro-inflammatory cytokines [tumor necrotic factor (TNF)-α, interleukin (IL)-1β, interferon-γ, and transforming growth factor-β] and chemokines [monocyte chemotactic protein 1 and macrophage inflammatory protein (MIP)-1α] to augment neurodegenerative response[3,5]. Lysosomal proteases, especially cathepsins (e.g. Cathepsin X or Cat X), are instrumental in processing several neuronal proteins that generate either

Enolase and Acute Spinal Cord Injury

Rachel Polcyn1, Mollie Capone1,2, Azim Hossain1, Denise Matzelle2,3, Naren L. Banik1,2,3 and Azizul Haque1* 1Department of Microbiology and Immunology, Hollings Cancer Center, Medical University of South Carolina, Charleston, USA 2Department of Neurosurgery, Medical University of South Carolina, Charleston, USA 3Department of Immunology, Ralph H. Johnson Veterans Administration Medical Center, Charleston, SC, USA *Corresponding author: Azizul Haque, Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Avenue, BSB-201, Charleston, SC 29425, USA, Tel: 843-792-9466; Fax: 843-792-2464; E-mail: haque@musc.edu

Abstract 1653: Regulation of autophagy and apoptosis by a natural triterpenoid in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL), a subtype of non-Hodgkin9s lymphoma, is the most common lymphoid malignancy in the western world. Treatment of DLBCL has been greatly improved in recent years with the addition of the monoclonal antibody Rituximab to the gold standard CHOP chemotherapy regimen, but these treatments are often ineffective in patients with highly aggressive disease or in patients of advanced age. A real problem given the population of DLBCL patients is largely 60 years of age or older, these chemotherapeutics also present the issue of severe toxicity to the patient(s). Thus, studies have focused lately on natural products that can selectively kill malignant lymphomas while displaying a reduced host toxicity profile. Here, we report that a natural extract from the Ganoderma lucidum mushroom, GA-DM, induces apoptosis in human DLBCL cell lines and primary tumors, and activates an orchestrated autophagy/apoptosis pathway in the murine B-cell lymphoma cell line A20. Antitumor efficacy of GA-DM was also investigated in vivo in a murine B-cell lymphoma model using the A20 cell line, where GA-DM (50mg/kg) treatment reduced both the number of tumor metastases and the overall tumor burden in diseased mice. These findings support the potential use of GA-DM as a novel chemotherapeutic in the treatment of DLBCL and could improve the treatment of higher risk patients with advanced disease who can9t tolerate current chemotherapy treatment. Citation Format: Bently Doonan, Azim Hossain, Faisal Radwan, Azizul Haque. Regulation of autophagy and apoptosis by a natural triterpenoid in diffuse large B-cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1653. doi:10.1158/1538-7445.AM2013-1653