Debanjan Bhattacharya

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.

T11TS inhibits glioma angiogenesis by modulation of MMPs, TIMPs, with related integrin αv and TGF-β1 expressions

During glioma development, angiogenesis plays a crucial role in growth and vascularization of primary brain tumors. T11 target structure (T11TS), a bioactive molecule, has been documented as an anti-neoplastic agent in glioma-induced rats and also in human glioma in vitro. This novel molecule induces apoptosis of tumor cells by way of immune potentiation and impairs the glioma cell cycle, but its role in glioma angiogenesis has not been worked out in detail. Matrix metalloproteinases (MMPs) are enzymes promoting tumor angiogenesis by enzymatically remodeling the extracellular matrix and altering surface protein expression such as integrin αv and the matrix-bound proteins like TGF-β1. The present study was formulated to assess the efficacy of T11TS in the modulations of MMP-2 and −9 and their endogenous inhibitors (TIMP-1 and TIMP-2) as well as modulations of integrin αv and TGF-β1 in glioma-induced rats and also on the phenotypic markers of endothelial cells (CD31 and CD34). The parameters used were zymography, western blot, and flow cytometric analyses. It was observed that T11TS administration significantly downregulates the expression of matrix metalloproteinase-2 and −9 along with its ligand integrin αv and upregulates TIMP-1 and TIMP-2. In situ immunofluorescence and FACS results revealed that T11TS administration decreased the expression of the phenotypic markers (CD31/PECAM1, CD34), inhibiting the cell grip and also downregulating TGF-β1 expression (ELISA) from microglia cells in the glioma microenvironment. These results suggest that T11TS suppresses the expression of positive angiogenic growth factors and potentiates the expression of negative regulators in glioma-associated endothelial cells (ECs), resulting in an anti-angiogenic effect on glioma-induced angiogenesis.

The novel immunotherapeutic molecule T11TS modulates glioma-induced changes of key components of the immunological synapse in favor of T cell activation and glioma abrogation

T-cell-mediated immune responses are typically low in conditions of malignant glioma which has been known to cause marked immunesuppression and dysregulate major T-cell signaling molecules. Thus, T-cell-based immunotherapies are currently in vogue in the treatment of malignant glioma. The novel glycopeptide, T11TS/S-LFA-3/S-CD58 has previously been shown by our group to be highly efficacious in glioma abrogation in in vivo and in vitro conditions. This glycopeptide ligands to the costimulatory CD2 molecule on T-cells, causing profound immune stimulation leading to glioma abrogation, suggesting probable involvement of T11TS in modulation of the T-cell signaling pathway. The present study offers a multi-targeted approach towards repair of some of the key components of the immunological synapse at the T-cell-APC interface and is therefore the first of its kind to offer a holistic model of restoration of immunological synapse components so as to trigger T-cells towards activation against glioma. The study thus indicates that the totally dysregulated molecular events at the immunological synapse in glioma are restored back to normal levels with the administration of T11TS, which finally culminates in glioma abrogation. The present study thus delineates an important T-cell signaling approach whereby T11TS acts as an anti-neoplastic agent, thus helping to chart out newer avenues in the fight against gliomas.

Specific allergen immunotherapy attenuates allergic airway inflammation in a rat model of Alstonia scholaris pollen induced airway allergy

Pollen grains are well established to be an important cause of respiratory allergy. Current pharmacologic therapies for allergic asthma do not cure the disease. Allergen specific immunotherapy is the only treatment method which re-directs the immune system away from allergic response leading to a long lasting effect. The mechanism by which immunotherapy achieves this goal is an area of active research world-wide. The present experimental study was designed to develop an experimental model of allergic lung inflammation based on a relevant human allergen, Alstonia scholaris pollen, and to establish the immunological and cellular features of specific allergen immunotherapy using this same pollen extract. Our results revealed that Alstonia scholaris pollen sensitization and challenge causes eosinophilic airway inflammation with mucin hypersecretion. This is associated with increased total IgE, increased expression of FcɛRI on lung mast cells and increased levels of IL-4, IL-5 & IL-13 as confirmed by ELISA, in-situ immunofluorescence and FACS assay. Allergen specific immunotherapy reduced airway inflammation and also decreased total IgE level, FcɛRI expression, IL-4, IL-5 & IL-13 levels. It was further noted that the reduction of these levels was more by intra-nasal route than by intra-peritoneal route. Thus we present a novel animal model of Alstonia scholaris pollen allergic disease and specific allergen immunotherapy which will pave the way towards the development of better treatment modalities.

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.

T11TS Treatment Augments Apoptosis of Glioma Associated Brain Endothelial Cells, Hint towards Anti‐Angiogenic Action in Glioma

Malignant glioma continues to be a clinical challenge with an urgent need for developing curative therapeutic intervention. Apoptosis induction in tumor‐associated endothelial cells represent a central mechanism that counteracts angiogenesis in glioma and other solid tumors. We previously demonstrated that intraperitoneal administration of sheep erythrocyte membrane glycopeptide T11‐target structure (T11TS) in rodent glioma model inhibits PI3K/Akt pathway and Raf/MEK/ERK signaling in glioma‐associated brain endothelial cells. In the present study, we investigated whether T11TS treatment influence apoptosis signaling in vivo in glioma‐associated brain endothelial cells. Annexin‐V/PI staining showed that T11TS treatment in glioma‐induced rats increases apoptosis of glioma‐associated endothelial cells within glioma milieu compared to brain endothelial cells in glioma induced and control groups. Flowcytometric JC‐1 assay revealed that T11TS administration triggers loss of mitochondrial membrane potential in glioma‐associated brain endothelial cells. Flowcytometry, immunoblotting, and in situ immunofluoresecnt imaging were employed to investigate the effect of T11TS on apoptotic regulatory proteins in brain endothelial cells. T11TS treatment‐upmodulated expression of p53, Bax, Fas, FasL, and FADD in glioma associated endothelial cells and downregulated Bcl‐2 protein. T11TS therapy induced cytochrome‐c release into cytosol, activated caspase −9, 8, 3, and cleaved Bid in glioma associated brain endothelial cells. The study demonstrates that T11TS induces apoptosis in glioma‐associated brain endothelial cells via p53 accumulation and activation of intrinsic as well as Fas‐dependent extrinsic pathway. The pro‐apoptotic action of T11TS on glioma‐associated endothelial cells provides crucial insight into how T11TS exerts its anti‐angiogenic function in glioma. J. Cell. Physiol. 232: 526–539, 2017.

T11TS IMMUNOTHERAPY REPAIRS PI3K-AKT SIGNALING IN T-CELLS: CLUES TOWARDS ENHANCED T-CELL SURVIVAL IN RAT GLIOMA MODEL†

Malignant glioma is the most fatal of astrocytic lineage tumors despite therapeutic advances. Onset and progression of gliomas is accompanied by severe debilitation of T‐cell defense and T‐cell survival. One of the chief contributors to T‐cell survival downstream of activation is the PI3K‐AKT pathway. Our prior studies showed that the novel immunotherapeutic molecule T11‐target structure (T11TS) blocks T‐cell apoptosis in glioma. We also showed activation of immunological synapse components and calcineurin‐NFAT pathway following T11TS immunotherapy of glioma‐bearing rats. This lead to investigations whether such T‐cell activation upon T11TS therapy translates into activation of downstream PI3K/AKT signals which may be related to observed blockade of T‐cell apoptosis. For the purpose, we assessed by flowcytometry and immunoblotting, expressions of PI3K, PDK1, AKT, p‐AKT, and PTEN in splenic T‐cells of normal, experimentally‐induced glioma‐bearing rats and glioma‐bearing rats receiving first, second and third doses of T11TS. We also determined comparative nuclear translocation of NF‐κB across groups. We found significant increases in T‐cell expressions of PDK1, PI3K, and p‐AKT in T11TS‐treated animal groups compared to sharp downregulations in glioma. AKT levels remained unchanged across groups. PTEN levels declined sharply after T11TS immunotherapy. T11TS also caused enhanced NF‐κB translocation to the T‐cell nucleus compared to glioma group. Results showed heightened activation of the PI3K‐AKT pathway in glioma‐bearing rats following T11TS immunotherapy. These results illustrate the novel role of T11TS immunotherapy in ameliorating the PI3K pathway in T‐cells in glioma‐bearing animals to enhance T‐cell survival, according greater defense against glioma. The study thus has far‐reaching clinical outcomes.

Abstract B17: ADGRB3 is a novel tumor suppressor epigenetically silenced in WNT medulloblastoma

Medulloblastoma (MB) is the most aggressive primary malignant brain tumor in children and arises from neural progenitor cells in the developing cerebellum. Although some subtypes of MB show a favorable prognosis with treatment, still one third of patients succumb to this disease and the children who survive suffer from long-term side effects of the aggressive treatments. ADGRB3 (formerly called BAI3) is a member of the ADGRB1-3 subfamily of adhesion GPCR transmembrane proteins, which are highly expressed in the brain specially in cerebellum and hippocampal neurons. The ADGRB3 gene was recently found to undergo somatic mutations in several cancers, but nothing is known about the functional role of ADGRB3 in medulloblastoma. Our recent findings based on analysis of a published microarray dataset and RT-PCR experiments showed that ADGRB3 mRNA expression was selectively repressed in WNT-MB tumor tissue compared to other three molecular subgroups and normal human cerebellar tissue. Using bisulfite sequencing we have detected hypermethylation of the ADGRB3 promoter exclusively in WNT-MB subgroup of human MB tissue but not in the other three molecular subgroups and normal human cerebellar tissue. These results clearly indicate that ADGRB3 is epigenetically silenced in WNT-MB via promoter hypermethylation. In vitro experiments showed that restoration of ADGRB3 expression in ADGRB3-silenced MB cell lines slows cell growth and inhibits WNT signaling targets. We further analyzed whether methyl CpG binding proteins and histone modifications modulate transcriptional repression of ADGRB3 in WNT-MB. ChIP assays revealed enrichment of repressive MBD2 protein in the ADGRB3 promoter region. siRNA mediated knockdown of ADGRB3 in ADGRB3-silent MB cell line (UW288-1) was found to reactivate ADGRB3 expression. We found that in vitro treatment with KCC-07, a recently discovered MBD2 inhibitor, reactivates ADGRB3 mRNA expression in ADGRB3-silent but not expressing MB cells. We are currently investigating whether ADGRB3 can negatively regulate the WNT/beta-catenin pathway or acts via an independent pathway that synergizes with it to facilitate transformation. Altogether, our findings define an epigenetic mechanism for ADGRB3 silencing in WNT-MB and indicate a novel role of ADGRB3 as a potent suppressor of oncogenic cerebellar transformation. Our findings also highlight the potential of epigenetic reactivation of ADGRB3 as a less toxic therapeutic intervention for the children suffering from WNT-MB. Citation Format: Debanjan Bhattacharya, Dan Zhu, Narra Devi, Erwin G. Van Meir. ADGRB3 is a novel tumor suppressor epigenetically silenced in WNT medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B17.

Abstract C5: T11TS arrests angiogenic signaling in malignant glioma by attenuating brain endothelial cell angiopoietin-1/Tie2 expression and inducing apoptosis in glioma associated brain endothelial cells.

Malignant gliomas are highly aggressive primary brain tumors resistant to conventional treatments mainly because of sustained uncontrolled angiogenesis. Limited efficacies of currently available treatments necessitate development of multi-level targeted anti-angiogenic therapy for treatment of malignant glioma. Previously we reported that sheep erythrocyte membrane glycopeptide T11 target structure (T11TS) therapy in a rodent glioma model inhibits brain endothelial cell VEGF signaling and downstream PI3K/Akt/eNOS pathway. The angiopoietin-1/Tie2 signaling operative in brain endothelial cells is implicated in glioma angiogenesis but the role of T11TS therapy on pro-angiogenic Ang-1/Tie2 axis has not been determined yet. Induction of endothelial cell apoptosis is a common mechanism of action of many anti-angiogenic agents that restrict tumor progression. The purpose of the present study is to investigate the effect of T11TS therapy in regulation of angiopoietin-1/Tie2 signaling in glioma associated brain endothelial cells and to identify whether T11TS therapy triggers apoptosis in glioma associated brain endothelial cells. Additionally, the study evaluated the apoptotic pathways and proteins involved in T11TS induced apoptosis of brain endothelial cells. Primary brain endothelial cells isolated from the cerebral cortices of glioma induced rats before and after T11TS therapy were used for experiments. Expression of angiopoietin-1, Tie2, phospho-Tie2, and apoptotic pathway regulator proteins and caspases in brain endothelial cells were determined by flowcytometry, immunoblotting, immunofluorescent imaging, and immunohistochemical staining of rat cerebral sections. Flow cytometric Annexin V-PI assay and JC-1 assay were employed for detection of apoptosis in glioma associated brain endothelial cells. Our findings revealed that T11TS therapy significantly downregulates angiopoietin-1 and Tie2 receptor expression and activation in glioma associated brain endothelial cells. T11TS therapy induced apoptotic death of glioma associated brain endothelial cells as evidenced from membrane phosphatidylserine translocation and reduction of mitochondrial membrane potential. T11TS therapy stimulated both mitochondrial and extrinsic pathways of apoptosis by activating caspases 9, 3, and 8, triggering cleavage of Bid and modulating expression of apoptotic regulator proteins like p53, Bax, Bcl-2, Fas, and FasL in brain endothelial cells. Blockage of Ang-1/Tie2 signaling in brain endothelial cells and induction of intrinsic and extrinsic pathway mediated apoptosis in glioma associated brain endothelial cells are the underlying mechanistic events contributing to the anti-angiogenic action of T11TS therapy in malignant glioma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C5. Citation Format: Debanjan Bhattacharya, Swapna Chaudhuri. T11TS arrests angiogenic signaling in malignant glioma by attenuating brain endothelial cell angiopoietin-1/Tie2 expression and inducing apoptosis in glioma associated brain endothelial cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C5.