Endoplasmic reticulum stress, autophagic and apoptotic cell death, and immune activation by a natural triterpenoid in human prostate cancer cells.

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC50 s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 + T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.

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.

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.

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-A's 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.

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.

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.

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.

Development of immunoglobulin M memory to both a T-cell-independent and a T-cell-dependent antigen following infection with Vibrio cholerae O1 in Bangladesh.

Vibrio cholerae O1 can cause severe watery diarrhea that can be life-threatening without treatment. Infection results in long-lasting protection against subsequent disease. Development of memory B cells of the immunoglobulin G (IgG) and IgA isotypes to V. cholerae O1 antigens, including serotype-specific lipopolysaccharide (LPS) and the B subunit of cholera toxin (CTB), after cholera infection has been demonstrated. Memory B cells of the IgM isotype may play a role in long-term protection, particularly against T-cell-independent antigens, but IgM memory has not been studied in V. cholerae O1 infection. Therefore, we assayed acute- and convalescent-phase blood samples from cholera patients for the presence of memory B cells that produce cholera antigen-specific IgM antibody upon polyclonal stimulation in in vitro culture. We also examined the development of serological and antibody-secreting cell responses following infection. Subjects developed significant IgM memory responses by day 30 after infection, both to the T-cell-independent antigen LPS and to the T-cell-dependent antigen CTB. No significant corresponding elevations in plasma IgM antibodies or circulating IgM antibody-secreting cells to CTB were detected. In 17 subjects followed to day 90 after infection, significant persistence of elevated IgM memory responses was not observed. The IgM memory response to CTB was negatively correlated with the IgG plasma antibody response to CTB, and there was a trend toward negative correlation between the IgM memory and IgA plasma antibody responses to LPS. We did not observe an association between the IgM memory response to LPS and the vibriocidal titer.

Enolase inhibition alters metabolic hormones and inflammatory factors to promote neuroprotection in spinal cord injury.

Enolase inhibition is a potential therapeutic strategy currently being investigated for treatment of spinal cord injury (SCI) as it reduces pro-inflammatory cytokines and chemokines, alters metabolic factors, and reduces gliosis in acute SCI. Herein, the role of enolase in SCI has been examined to better understand the effects of this enzyme on inflammation, metabolic hormones, glial cell activation, and neuroprotection under these shorter injury conditions. Immunohistochemical analyses of inflammatory markers vimentin, Cox-2, and caspase-1 indicated that enolase inhibition attenuated the elevated levels of inflammation seen following SCI. Iba1, GFAP, NFP, and CSPG staining indicated that enolase inhibition with prolonged administration of ENOblock reduced microglia/astrocyte activation and lead to enhanced neuroprotection in SCI. An analysis of metabolic hormones revealed that ENOblock treatment significantly upregulated plasma concentrations of peptide YY, glucagon-like peptide 1, glucose-dependent insulinotropic peptide, glucagon, and insulin hormones as compared to vehicle-treated controls (Mann-Whitney, p ≤ 0.05). ENOblock did not have a significant effect on plasma concentrations of pancreatic polypeptide. Interestingly, ENOblock treatment inhibited chondroitin sulfate proteoglycan (CSPG), which is produced by activated glia and serves to block regrowth of axons across the lesion site following injury. An increased level of NeuN and MBP with reduced caspase-1 was detected in SCI tissues after ENOblock treatment, suggesting preservation of myelin and induction of neuroprotection. ENOblock also induced improved motor function in SCI rats, indicating a role for enolase in modulating inflammatory and metabolic factors in SCI with important implications for clinical consideration.

Calpain mediated expansion of CD4+ cytotoxic T cells in rodent models of Parkinson's disease.

Parkinson's disease (PD), a debilitating progressive degenerative movement disorder associated with loss of dopaminergic (DA) neurons in the substantia nigra (SN), afflicts approximately one million people in the U.S., including a significant number of Veterans. Disease characteristics include tremor, rigidity, postural instability, bradykinesia, and at a cellular level, glial cell activation and Lewy body inclusions in DA neurons. The most potent medical/surgical treatments do not ultimately prevent disease progression. Therefore, new therapies must be developed to halt progression of the disease. While the mechanisms of the degenerative process in PD remain elusive, chronic inflammation, a common factor in many neurodegenerative diseases, has been implicated with associated accumulation of toxic aggregated α-synuclein in neurons. Calpain, a calcium-activated cysteine neutral protease, plays a pivotal role in SN and spinal cord degeneration in PD via its role in α-synuclein aggregation, activation/migration of microglia and T cells, and upregulation of inflammatory processes. Here we report an increased expression of a subset of CD4+ T cells in rodent models of PD, including MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mice and DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride]/6-hydroxydopamine rats, which produced higher levels of perforin and granzyme B - typically found in cytotoxic T cells. Importantly, the CD4+ cytotoxic subtype was attenuated following calpain inhibition in MPTP mice, suggesting that calpain and this distinct CD4+ T cell subset may have critical roles in the inflammatory process, disease progression, and neurodegeneration in PD.

Antigen-specific memory B-cell responses to Vibrio cholerae O1 infection in Bangladesh.

Cholera, caused by Vibrio cholerae, is a noninvasive dehydrating enteric disease with a high mortality rate if untreated. Infection with V. cholerae elicits long-term protection against subsequent disease in countries where the disease is endemic. Although the mechanism of this protective immunity is unknown, it has been hypothesized that a protective mucosal response to V. cholerae infection may be mediated by anamnestic responses of memory B cells in the gut-associated lymphoid tissue. To characterize memory B-cell responses to cholera, we enrolled a cohort of 39 hospitalized patients with culture-confirmed cholera and evaluated their immunologic responses at frequent intervals over the subsequent 1 year. Memory B cells to cholera antigens, including lipopolysaccharide (LPS), and the protein antigens cholera toxin B subunit (CTB) and toxin-coregulated pilus major subunit A (TcpA) were enumerated using a method of polyclonal stimulation of peripheral blood mononuclear cells followed by a standard enzyme-linked immunospot procedure. All patients demonstrated CTB, TcpA, and LPS-specific immunoglobulin G (IgG)and IgA memory responses by day 90. In addition, these memory B-cell responses persisted up to 1 year, substantially longer than other traditional immunologic markers of infection with V. cholerae. While the magnitude of the LPS-specific IgG memory B-cell response waned at 1 year, CTB- and TcpA-specific IgG memory B cells remained significantly elevated at 1 year after infection, suggesting that T-cell help may result in a more durable memory B-cell response to V. cholerae protein antigens. Such memory B cells could mediate anamnestic responses on reexposure to V. cholerae.

Memory T-cell responses to Vibrio cholerae O1 infection.

Vibrio cholerae O1 can cause diarrheal disease that may be life-threatening without treatment. Natural infection results in long-lasting protective immunity, but the role of T cells in this immune response has not been well characterized. In contrast, robust B-cell responses to V. cholerae infection have been observed. In particular, memory B-cell responses to T-cell-dependent antigens persist for at least 1 year, whereas responses to lipopolysaccharide, a T-cell-independent antigen, wane more rapidly after infection. We hypothesize that protective immunity is mediated by anamnestic responses of memory B cells in the gut-associated lymphoid tissue, and T-cell responses may be required to generate and maintain durable memory B-cell responses. In this study, we examined B- and T-cell responses in patients with severe V. cholerae infection. Using the flow cytometric assay of the specific cell-mediated immune response in activated whole blood, we measured antigen-specific T-cell responses using V. cholerae antigens, including the toxin-coregulated pilus (TcpA), a V. cholerae membrane preparation, and the V. cholerae cytolysin/hemolysin (VCC) protein. Our results show that memory T-cell responses develop by day 7 after infection, a time prior to and concurrent with the development of B-cell responses. This suggests that T-cell responses to V. cholerae antigens may be important for the generation and stability of memory B-cell responses. The T-cell proliferative response to VCC was of a higher magnitude than responses observed to other V. cholerae antigens.

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

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.