Sagar Acharya

Bax and Bid act in synergy to bring about T11TS-mediated glioma apoptosis via the release of mitochondrial cytochrome c and subsequent caspase activation.

The specific apoptotic role of T11TS has been well established in glioma animal models. T11TS specifically induces the glioma cells to die an apoptotic death via immune cross-talk with the two intracranial immune competent cells-microglia and the brain-infiltrating lymphocytes. To unearth the molecular cascades operative within the glioma cells and to some extent in the two interacting immunocytes, we had initiated studies where preliminary findings not only had indicated the involvement of death receptors but had also hinted to the involvement of other apoptotic regulators. Hence, to identify the molecular pathway of apoptosis involving other apoptotic regulators in the three cell types, the cells were studied for the intrinsic apoptotic death regulators that were engaged to maintain the mitochondrial membrane integrity. The proteins that were selected could be divided into three broad classes-the Bcl-2 family of proteins-Bid, Bax and Bcl-2; the guardian of the genome p53 and the proteins downstream of mitochondria-Apaf-1, cytochrome c, caspase-9 and caspase-3. Activated Bid as well as maximal p53 expression was observed in the first dose of T11TS thus dually activating the pro-apoptotic Bax in the first and second dose in the glioma cells. Concurrently, the pro-survival protein Bcl-2s expression level was very much down-regulated in the same two doses favoring the internal microenvironment to proceed for apoptosis. High expression of cytochrome c and Apaf-1 and the presence of active caspase-9 and active caspase-3 in all the T11TS-treated tumor-bearing groups further adjudicated apoptosis of the glioma cells with clear involvement of mitochondrial death pathway in the T11TS-treated animals. Even though expression of the apoptotic regulators remained more or less the same indicating the involvement of mitochondria in the two interacting immunocytes, the intensity of expression of these proteins was much lower than the tumor cells. The present work focuses on the mechanistic approach of how T11TS mediates apoptosis and hence is the first approach of its kind in the field of immunology where the immunotherapeutic molecules mode of action has been worked out.

T11TS impedes glioma angiogenesis by inhibiting VEGF signaling and pro-survival PI3K/Akt/eNOS pathway with concomitant upregulation of PTEN in brain endothelial cells

The crucial role of angiogenesis in malignant glioma progression makes it a potential target of therapeutic intervention in glioma. Previous studies from our lab showed that sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) has potent anti-neoplastic and immune stimulatory effects in rodent glioma model. In the present study we investigated the anti-angiogenic potential of T11TS and deciphered the underlying molecular mechanism of its anti-angiogenic action in malignant glioma. Vascular endothelial growth factor (VEGF) signaling is crucial for initiating tumor angiogenic responses. The present preclinical study was designed to evaluate the effect of T11TS therapy on VEGF and VEGFR-2 expression in glioma associated brain endothelial cells and to determine the effects of in vivo T11TS administration on expression of PTEN and downstream pro-survival PI3K/Akt/eNOS pathway proteins in glioma associated brain endothelial cells. T11TS therapy in rodent glioma model significantly downregulated expression of VEGF along with its receptor VEGFR-2 and inhibited the expression of pro-survival PI3K/Akt/eNOS proteins in glioma associated brain endothelial cells. Furthermore, T11TS therapy in glioma induced rats significantly upregulated brain endothelial cell PTEN expression, inhibited eNOS phosphorylation and production of nitric oxide in glioma associated brain endothelial cells. Taken together our findings suggest that T11TS can be introduced as an effective angiogenesis inhibitor in human glioma as T11TS targets multiple levels of angiogenic signaling cascade impeding glioma neovascularisation.

Induction of G1 arrest in glioma cells by T11TS is associated with upregulation of Cip1/Kip1 and concurrent downregulation of cyclin D (1 and 3).

In our laboratory, a novel therapeutic probe, T11TS, a membrane glycoprotein, was isolated which had antineoplastic activity against experimental glioma. Development of a novel therapeutic strategy with T11TS has unearthed a newer dimension of its mechanism of action: modulation of the cell cycle. In this study, we have presented evidence to support the finding that T11TS induces G1 cell cycle arrest of rat glioma cells. Results of flow cytometric studies showed that the treatment produced a marked increase in the proportion of cells in the G1 phase. Flow cytometry, immunoblotting, immunoprecipitation, and kinase assays were performed for investigating the involvement of G1 cell cycle regulators. T11TS induces downregulation of the cyclin-D (1 and 3) expression with the concurrent upregulation of p21Cip1 and p27Kip1 and their concomitant association with cyclin-dependent kinase 4, proliferating cell nuclear antigen and cyclin E respectively leading to a decrease in cyclin-dependent kinase 4 kinase activity. A transient rise in retinoblastoma protein level and coordinated binding of retinoblastoma protein with E2F coincided with the accumulation of cells in G1 phase. Thus, our observations have uncovered an antiproliferative pathway for T11TS, causing retardation of glioma cell cycle.

Immunomodulatory role of TIITS in respect to cytotoxic lymphocytes in four grades of human glioma

T11 target structure (T11TS), a membrane glycoprotein has been documented with antineoplastic activity in animal model in our lab. Previously, in animal study we have documented T11TS induced cytotoxic abrogation of tumor cells. Encouraged by these established findings by our group and as prerequisite for clinical trial, this study has been designed to assess the cytotoxic potential of the patients lymphocytes in in vitro study of autologous human glioma as modulated by T11TS. Meningioma samples were chosen as disease control group. The data produced indicates T11TS induced up regulation of cytotoxicity of T lymphocytes in grade I and II glioma. Significant enhancement of cytotoxic protein, perforin and granzyme suggest cytotoxic death of T11TS induced target tumor. Also, T11TS downregulates the TGF-β secretion in grade I and II tumor cells. These preliminary findings may help in pushing this molecule into pharmaceutical domain.

T11TS/SLFA-3 Differentially Regulate the Population of Microglia and Brain Infiltrating Lymphocytes to Reduce Glioma by Modulating Intrinsic Bcl-2 Expression rather than p53

T11TS/SLFA-3, the glycoprotein isolated from sheep erythrocyte membrane, acts as an antineoplastic agent causing apoptotic elimination of glioma cells through cell mediated immune response. Therefore, elucidation of the proper balance in proliferation and apoptosis of neuroimmune components viz. microglia and brain infiltrating lymphocytes with the neoplastic glial cells was the fundamental issue to establish the efficacy of T11TS as a therapeutic agent in glioma. To decipher its effectivity on proliferation rate of glioma cells, expression of GFAP and cell cycle phase distribution was analyzed with propidium iodide (PI) staining. The apoptotic regulation of interacting immuno-competent microglia, lymphocytes entering into the brain and target glioma cells were elucidated by cytoplasmic DNA fragmentation assay and phosphatidylserine (PS) externalization along with intrinsic p53 and Bcl-2 modulation of the cells. With the reduction of cellular proliferation rate, sharp increase of apoptosis in consecutive doses of T11TS showed the regression of glioma, where an increase of cytosolic p53 and a decrease of Bcl-2 with doses in these neoplastic cells facilitate the process. Resident microglia, the chief immunomodulator of brain, was found to show a low and steady level of proliferation and apoptosis, furnishing it as a stable pool of cells capable of controlling immune reaction in brain compartment. However, microglia showed higher basal level of p53 compared to the other cells in study and being modulated with T11TS dose, it was found to possess a steady level of Bcl-2 that aided to maintain low rate of apoptosis. Brain infiltrating lymphocytes showed increased apoptosis in tumorigenic condition and initial treatment phase mostly due to immuno-suppressive milieu and deprivation of microglial restimulation. But the second dose of T11TS showed reduced apoptosis, enhanced activation of lymphocytes and final dose acting as a regulatory dose, which reduce the infiltrating lymphocytes by apoptotic elimination. Wide fluctuation of cytosolic p53 was observed in these lymphocytes, but anti-apoptotic Bcl-2 was found to modulate apoptosis in the cells. Thus, T11TS was found to differentially regulate the population of immune effector cells against glioma to exert effective effector function in eradication of neoplastic cells where Bcl-2 constitutively suppresses pro-apoptotic function of p53. Regulation of the direction of these balances of cellular life and death in favor of glioma killing and also maintaining homeostasis in brain tissue after reaction established T11TS as an effective therapeutic probe against glioma.

Therapeutic Profile of T11TS vs. T11TS+MiADMSA: A Hunt for a More Effective Therapeutic Regimen for Arsenic Exposure

Arsenic exposure is a serious health hazard worldwide. We have previously established that it may result in immune suppression by upregulating Th2 cytokines while downregulating Th1 cytokines and causing lymphocytic death. Treatment modalities for arsenic poisoning have mainly been restricted to the use of chelating agents in the past. Only recently have combination therapies using a chelating agent in conjunction with other compounds such as anti-oxidants, micronutrients and various plant products, been introduced. In the present study, we used T11TS, a novel immune potentiating glycopeptide alone and in combination with the sulfhydryl-containing chelator, mono-iso-amyl-dimarcaptosuccinic acid (MiADMSA) as a therapeutic regimen to combat arsenic toxicity in a mouse model. Results indicated that Th1 cytokines such as TNF-α, IFNγ, IL12 and the Th2 cytokines such as IL4, IL6, IL10 which were respectively downregulated and upregulated following arsenic induction were more efficiently restored to their near normal levels by T11TS alone in comparison with the combined regimen. Similar results were obtained with the apoptotic proteins studied, FasL, BAX, BCL2 and the caspases 3, 8 and 9, where again T11TS proved more potent than in combination with MiADMSA in preventing lymphocyte death. The results thus indicate that T11TS alone is more efficient in immune re-establishment after arsenic exposureas compared to combination therapy with T11TS+MiADMSA.

Comparative evaluation of T11 target structure and its deglycosylated derivative nullifies the importance of glycan moieties in immunotherapeutic efficacy

Sheep red blood cell (SRBC), a non-specific biological response modifier that has long been used as a classical antigen, has been shown to exert an immunomodulatory and anti-tumor activities in experimental animals. The active component of SRBC, which is responsible for such effects, was found to be a cell surface acidic glycoprotein molecule, known as T11 target structure (T11TS). In the present study, T11TS was isolated and purified to homogeneity using a five-step protocol involving isolation of sheep erythrocyte membrane from packed cell volume, 20% ammonium sulfate cut of the crude membrane proteins mixture, immunoaffinity purification using mouse anti-sheep CD58 mAb (L180/1) tagged matrix, preparative gel electrophoresis, and gel electroelution process. Finally, the purity and identity of the proteins were confirmed by the matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis. The in silico glycosylation site analysis showed that the extracellular domain contained three N-glycosylation sites (N-12, N-62, and N-111) and one O-glycosylation site (T-107). However, the experimental analysis negated the presence of O-linked glycan moieties on T11TS. To investigate the role of glycan moieties in the current immunotherapeutic regime, T11TS and its deglycosylated form (dT11TS) were administered intraperitoneally (i.p.) in N-ethyl-N-nitrosourea-induced immune-compromised mice at 0.4 mg/kg body weight. It was observed that both the forms of T11TS could activate the compromised immune status of mice by augmenting immune receptor expression (CD2, CD25, CD8, and CD11b), T-helper 1 shift of cytokine network, enhanced cytotoxicity, and phagocytosis activity. Therefore, the results nullify the active involvement of the N-linked glycan moieties in immunotherapeutic efficacy of T11TS.

T11TS Induced Modulation of Macrophages Associated Cytokines TNF- α ,VEGF and Apoptototic Protein Bax, Bcl2 Abrogates Tumor Cells in In vitro Grade I,II Human Glioma

T11 target structure (T11TS), a membrane glycoprotein has been documented with anti-neoplastic activity in glioma bearing animal model in our lab. In this study, we have evaluated the phagocytic potential, expression of VEGF, TNF-α in T11TS treated and untreated macrophages in all four grades of glioma. The data indicates the significant enhancement of phagocytosis in T11TS treated macrophages of Grade I and II glioma. There was significant upregulation in TNF-α and significant down-regulationin VEGF expression in T11TS treated macrophages in grade I and II glioma.We alsoattempted to know any possible apoptotic role of T11TS in tumor cell by comparing Bax and Bcl2 in treated and untreated tumour cell of all four grades. We found significant up-regulation in Bax expression and down-regulation in Bcl2 expression of grade I and II glioma. The outcome may help in pushing this molecule into pharmaceutical domain.