Bently P. Doonan

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.

Regulation of Th1/Th17 cytokines and IDO gene expression by inhibition of calpain in PBMCs from MS patients.

Multiple sclerosis (MS) pathology is marked by the massive infiltration of myelin-specific T cells into the central nervous system (CNS). During active disease, pro-inflammatory Th1/Th17 cells predominate over immunoregulatory Th2/Treg cells. Here, we show that calpain inhibition downregulates Th1/Th17 inflammatory cytokines and mRNA in MS patient peripheral blood mononuclear cells (PBMCs) activated with anti-CD3/28 or MBP. Interestingly, calpain inhibition elevated IDO gene expression in MS PBMCs, which was markedly decreased in calpain expressing cells. Functional assay showed that incubation of MS patient PBMCs with calpain inhibitor or recombinant IDO attenuates T cell proliferation. These results suggest that calpain inhibition may attenuate MS pathology and augment the efficacy of standard immunomodulatory agents used to treat this disease.

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.

Ganoderic Acid DM: An Alternative Agent for the Treatment of Advanced Prostate Cancer

Prostate cancer is the most commonly diagnosed cancer in men and accounts for significant morbidity and mortality in the western world. While traditional therapies are effective at clearing early stage cancer, they often fail to treat late stage metastatic disease. Thus, an effective therapy that targets prostate tumor growth and metastasis is desired for alleviating the disease and improving patient outcomes. Natural extracts have been the focus of recent investigation, particularly those with reduced cellular toxicity to healthy tissue. In this review, we discuss one potential candidate, ganoderic acid, an extract from the Ganoderma lucidum mushroom that has been tested in multiple cancer models. Interestingly, ganoderic acid DM (GA-DM) has shown toxicity to both androgen-dependent and independent prostate cancer cells with reduced osteoclastogenesis in late stage metastatic disease. This review will discuss the current knowledge on this GA-DM extract and the potential benefit in treating advanced prostate cancer. We will also provide an overview on the targeted delivery of GA-DM through nanoparticles that would reduce bystander toxicity and improve the drugs effectiveness. An improved understanding of this drug and its uses will advance the field of natural chemotherapeutics, particularly in treating advanced prostate cancer.

HLA Class II Antigen Presentation in Prostate Cancer Cells: A Novel Approach to Prostate Tumor Immunotherapy.

Prostate cancer is a deadly disease that is in drastic need of new treatment strategies for late stage and metastatic prostate cancer. Immunotherapy has emerged as a viable option to fill this void. Clinical trials have been conducted that induce tumor clearance through cytotoxic T lymphocyte (CTL) activation, these studies have had mixed outcomes with the overlying problem being the lack of a complete immune response with sustained killing and the formation of tumor specific memory cells. To overcome this, we have outlined the need for activating the HLA class II pathway in inducing a sustained CD8+ T cell response and the development of effective memory. We have also discussed the ability of prostate cancer cells to express stable HLA class II molecules that can be manipulated for tumor antigen (Ag) processing and presentation. This review also sets to outline new directions that exist for the use of class II-restricted Ags/peptides in devising cancer vaccines as well as combined chemoimmunotherapy. A better understanding of these concepts will improve future cancer vaccine studies and further the field of cancer immunobiology.

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.

Prostate Cancer Immunotherapy: Exploiting the HLA Class II Pathway in Vaccine Design

Prostate cancer is the second most diagnosed cancer in men and current treatment of advanced prostate cancer is ineffective. Immunotherapy has emerged as a promising treatment option for metastatic prostate cancer but its clinical application is still in the early stages of development. In order to treat metastatic prostate tumors, new directions must be taken to improve current immunotherapeutic strategies. These include the identification of effective tumor antigens (Ags), the induction of the HLA class II pathway for Ag processing and CD4+ T cell activation, and the ability of tumor cells to act like Ag presenting cells. In this review, we suggest a model for tumor Ag selection, epitope modification and self-processing for presentation by class II proteins as a means of restoring immune activation and tumor clearance. We also outline the importance of a Gamma-IFN-inducible Lysosomal Thiol reductase (GILT) in Ag and modified peptide processing by tumor cells, generation of functional epitopes for T cell recognition, and inclusion of immune checkpoint blockers in cancer immunotherapy. Taken together, this review provides a framework for the future development of novel cancer vaccines and the improvement of existing immunotherapeutics in prostate cancer.

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

Melanoma Immunomodulation: A War of Attrition

most common cancer, with a lifetime risk of 2.04% for men and 1.38% for women (Jemal et al., 2008; Jilaveanu et al., 2009). According to the American Cancer Society, in 2009 the number of new cases of melanoma rose to 68,720 with 8,650 new deaths attributed to the disease (ACS, 2010). There are many known factors that contribute to the formation of melanoma. One major factor influencing the increase in melanoma cases is increased exposure to ultra-violet radiation (UVR). Other risk factors include skin type, hair/eye color, the presence of dysplastic nevi and/or increased nevi, and a family history of melanoma (Jilaveanu et al., 2009). Also, mutations in BRAF, CDKN2A, and CDK4 genes have all been attributed to melanoma development. BRAF mutations have been found in 70% of all melanomas and greater than 90% of these mutations carry a single missense (single nucleotide change) mutation (Meyle & Guldberg, 2009). CDKN2A is involved in melanoma pathogenesis and is a germline mutation found in younger patients (Liu et al., 2007). CDK4 is involved in cell-cycle arrest and has been identified in 10% of melanomas (Bennett, 2008). These mutations are mostly detected in non-chronic sun-induced damage melanomas, while chronic sun-induced damage melanomas are more commonly observed. When melanoma arises, early diagnosis is crucial to survival. With early diagnosis, more than 80% of cases can be treated successfully through surgery (Zhu et al., 2009). This surgery includes excision of the tumor and surrounding tissue; lymph nodes near the tumor may also be removed if evidence of metastasis is present. Other treatments for melanoma include radiation and chemotherapy, used particularly in cases of highly aggressive and metastatic disease (ACS, 2010). Side-effects from these treatments include fatigue, malaise, and an increased susceptibility to non-melanoma cancers (Kamposioras et al., 2010). Also, these therapies are severely toxic to the patients, suggesting a need for improved, less toxic treatment options that specifically target melanoma tumors, like immunotherapy. The multiple ways that melanoma alters the immune system locally, at the site of the tumor, and systemically makes the disease difficult to treat but ideal for the study of immunomodulation (Berinstein, 2009). Immunomodulation, the alteration of the immune system or its function, is exploited in multiple forms by melanoma tumors from changes in the cellular and sub-cellular makeup of the tumor to changes in the tumor microenvironment that suppress localized and systemic attempts at disease reduction.

Abstract 5487: Anti-proliferative, apoptotic and immunological effects of ganoderic acid DM on prostate cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Natural extracts have been the focus of recent investigation in the design of novel chemotherapeutics. One potential natural therapeutic agent is ganoderic acid DM (GA-DM), an extract from the Ganoderma lucidum mushroom previously shown to inhibit 5-α-reductase activity and prevent binding of dihydrotestosterone to the androgen receptor in prostate cancer cells. Studies in our laboratory used nonradioactive cell proliferation and cytotoxicity assays to investigate GA-DMs effect on both androgen dependent and independent prostate cancer cell lines, revealing dose dependent killing and inhibition of proliferation by this natural extract. Microscopic analysis using Hoechst staining showed DNA breakdown consisted with apoptosis in GA-DM-treated prostate cancer cells. Annexin V staining confirmed low levels of apoptotic cell death when treated with GA-DM. Western blotting and biochemical analysis revealed that GA-DM induced apoptotic cell death that was independent of caspase activation, cytochrome c release, and Bax induction. Further investigation showed that GA-DM treatment activated HLA protein expression, implying some immunological involvement contributing to GA-DM-induced cell death. This study outlines a novel mechanism of GA-DM-induced inhibition of proliferation, induction of apoptosis and immune components in both androgen dependent and independent prostate cancer cells, the implications of which support GA-DMs potential use in multimodal chemoimmunotherapeutics. 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 5487. doi:10.1158/1538-7445.AM2011-5487