Akt Phosphorylation Orchestrates T11TS Mediated Cell Cycle Arrest in Glioma Cells.

The novel anti-neoplastic glycopeptide T11TS retards glioma both in in-vitro clinical samples and in-vivo models. This study investigates the correlation between altering the glioma microenvironment with glioma arrest and death. Flow cytometry, immunoblotting, ELISA, and co-immunoprecipitation were employed to investigate glioma cell arrest and death. Results include a decline in phosphorylation of Akt and attenuation of p21 phosphorylation (Thr145,Ser146) and disassociation of p-Akt-Mdm2 and p-Akt-BAD facilitating death by Akt>BAD. T11TS influence phosphorylation patterns in two focal axes Akt>p21 and Akt>Mdm2>p53. The current article provides crucial insight in deciphering the mechanism of T11TS induced glioma cell arrest and death.

Phytochemical composition analysis and evaluation of in vitro medicinal properties and cytotoxicity of five wild weeds: A comparative study.

Background: Medicinal plants are a source of phytochemicals and they are used for the treatment of several oxidative stress-related or other diseases for their effectiveness, low toxicity and easy availability. Five traditionally used and less characterized herbaceous weeds of West Bengal, India, namely, Heliotropium indicum, Tridax procumbens, Cleome rutidosperma, Commelina benghalensis and Euphorbia hirta, were investigated for the current research study. Methods: Aqueous and 70% ethanolic extracts of the leaves were analyzed for estimation of essential phytochemicals and to evaluate their in vitro antioxidant status, medicinal properties and cytotoxic effects. To the best of our knowledge, several assays and comparative evaluations using these herbs are reported for the first time. For quantitative study, UV-vis spectrophotometry and high-performance liquid chromatography with diode array detector HPLC-DAD techniques were used. Antibacterial properties were investigated using the Kirby-Bauer disc diffusion method. For in vitro anti-lithiatic study, a titration method was used. The cell viability assay was done using peripheral blood mononuclear cells. Results: The aqueous extract exhibits higher content of polyphenols, flavonoids, tannins and inhibition percentage values for free radical scavenging assays, whereas the 70% ethanolic extract exhibits higher content of alkaloids and cardiac glycosides. HPLC-DAD analysis of 70% ethanolic extracts led us to identify 10 predominant phenolic constituents. Euphorbia hirta extracts showed minimum cytotoxicity (cell death ~2.5% and 4% in water and 70% ethanolic extract, respectively ), whereas Cleome rutidosperma and Tridax procumbens' 70% ethanolic extracts showed higher cell death (~13% and 28%, respectively), compared with the control (cell death ~10-12%). Conclusions: The study concluded that of all the medicinal weeds selected for the current study, Euphorbia hirta possesses the highest amount of bioactive compounds and hence exhibits the highest in vitro antioxidant activity and promising in vitro medicinal properties.

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.

L-Theanine healed NSAID-induced gastric ulcer by modulating pro/antioxidant balance in gastric ulcer margin.

L-Theanine is a unique non-protein-forming amino acid present in tea [Camellia sinensis (L.) O. Kuntze]. In the present work, we evaluated the healing effect of L-theanine on NSAID (indomethacin)-induced gastric ulcer. Histology of the stomach tissues revealed maximum ulceration on the third day after indomethacin administration (18 mg/kg, single dose p.o.) which was accompanied by increased lipid peroxidation; protein carbonylation; Th1 cytokine synthesis, and depletion of thiol, mucin, prostaglandin (PG) E, Th2 cytokine synthesis; and total antioxidant status in mice. L-Theanine healed gastric ulcer at a dose of 10 mg/kg b.w. but aggravated the ulcerated condition at a higher dose of 40 mg/kg b.w. At 10 mg/kg b.w., L-theanine significantly alleviated the adverse oxidative effect of indomethacin through enhanced synthesis of PGE2 by modulation of cyclo-oxygenase-1 and 2 [COX-1 and COX-2] expression, Th1/Th2 cytokine balance, and restoration of cellular antioxidant status at the gastric ulcer margin. The present study revealed for the first time the dose-dependent biphasic effect of a natural neuroprotective agent, L-theanine, on gastric ulcer disease.

Significant modulation of macrophages associated cytokines TNF-α, VEGF and apoptotoic protein Bax, Bcl2 abrogates tumor cells.

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 grades I and II glioma. There was significant up regulation in TNF-α and significant down regulation in VEGF expression in T11TS treated macrophages in grade I and II glioma. We also attempted to know any possible apoptotic role of T11TS in tumor cells by comparing Bax and Bcl2 in treated and untreated tumor cells of all four grades. We found significant up regulation in Bax expression and down regulation in Bcl2 expression of grades I and II glioma. The outcome may help in pushing this molecule into pharmaceutical domain.

Polysaccharide-rich fraction of Termitomyces eurhizus accelerate healing of indomethacin induced gastric ulcer in mice.

The current study aims to determine the healing activity of water soluble polysaccharide-rich fraction of a wild mushroom, Termitomyces eurhizus (TEps) against the indomethacin induced gastric ulceration in mice model. Gastric tissue histology, myeloperoxidase (MPO) activity, cyclooxygenases (COX) 1 and 2 expression, prostaglandin E2 (PGE2) synthesis, and modulation of pro/anti inflammatory cytokines expression were studied for this purpose. Histological study shows that TEps (20 mg/kg) effectively healed the gastric ulceration. Based on biochemical results, the healing capacities of TEps could be attributed to reduction of MPO activity and protection of mucosal mucin content. Enhanced synthesis of PGE2 by modulation of COX-1 and COX-2 expression and a prominent shift of cytokines expression from pro (TNF-α, IL-1ß) to anti inflammatory (IL-10) side are also held responsible for ulcer healing. The preliminary study highlights the anti-ulcerogenic property of polysaccharide-rich fraction of Termitomyces eurhizus and opens an alternative cure for NSAID induced gastroduodenal diseases.

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.

Gallic Acid Enriched Fraction of Phyllanthus emblica Potentiates Indomethacin-Induced Gastric Ulcer Healing via e-NOS-Dependent Pathway.

The healing activity of gallic acid enriched ethanolic extract (GAE) of Phyllanthus emblica fruits (amla) against the indomethacin-induced gastric ulceration in mice was investigated. The activity was correlated with the ability of GAE to alter the cyclooxygenase- (COX-) dependent healing pathways. Histology of the stomach tissues revealed maximum ulceration on the 3rd day after indomethacin (18 mg/kg, single dose) administration that was associated with significant increase in inflammatory factors, namely, mucosal myeloperoxidase (MPO) activity and inducible nitric oxide synthase (i-NOS) expression. Proangiogenic parameters such as the levels of prostaglandin (PG) E(2), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), von Willebrand Factor VIII, and endothelial NOS (e-NOS) were downregulated by indomethacin. Treatment with GAE (5 mg/kg/day) and omeprazole (3 mg/kg/day) for 3 days led to effective healing of the acute ulceration, while GAE could reverse the indomethacin-induced proinflammatory changes of the designated biochemical parameters. The ulcer healing activity of GAE was, however, compromised by coadministration of the nonspecific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), but not the i-NOS-specific inhibitor, L-N6-(1-iminoethyl) lysine hydrochloride (L-NIL). Taken together, these results suggested that the GAE treatment accelerates ulcer healing by inducing PGE(2) synthesis and augmenting e-NOS/i-NOS ratio.

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 patient's 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-ß secretion in grade I and II tumor cells. These preliminary findings may help in pushing this molecule into pharmaceutical domain.

Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation.

The mechanism of indomethacin-induced gastric ulcer healing by ellagic acid (EA) in experimental mice model is described in our study. Ulcer index (UI) and myeloperoxidase (MPO) activity of the stomach tissues showed maximum ulceration on the third day after indomethacin (18 mg/kg, single dose) administration. Preliminary observation of UI and MPO activity suggests that EA possesses ulcer-healing activity. Other anti-ulcer parameters such as the levels of prostaglandin E(2), cyclooxygenase (COX) 1 and 2 enzymes, anti-inflammatory cytokines [interleukin (IL)-4 and -5], pro-angiogenic factors, e.g. vascular endothelial growth factor, hepatocyte growth factor (HGF), and endothelial growth factor (EGF) were down-regulated by indomethacin. EA (7 mg/kg/day) treatment for 3 days shifted the indomethacin-induced pro-inflammatory biochemical parameters to the healing side. These activities were correlated with the ability of EA to alter the COX-2-dependent healing pathways. The ulcer-healing activity of EA was, however, compromised by pre-administration of the specific COX-2 inhibitor, celecoxib, and NS-398. Taken together, these results suggested that the EA treatment accelerates ulcer healing by inducing IL-4, EGF/HGF levels and enhances COX-2 expression.

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.

Immunological profile of arsenic toxicity: a hint towards arsenic-induced carcinogenesis.

Arsenic (a Group I carcinogen in humans) contamination and poisoning of human populations in different parts of Southeast and Eastern Asia, including West Bengal and Bangladesh, has become a major environmental concern. Arsenic intoxication affects diverse human organs including the lungs, liver, skin, bladder and kidney. This metalloid acts as a promoter of carcinogenesis, exerting toxic effects on the immune system. The present study was aimed at investigating arsenic-induced carcinogenesis and effects on the immune system in an animal model. Tumors were induced using ethylnitrosourea (ENU) and arsenic was used as a promoter. To investigate specific effects on the immune system, cytokine (TNF-α, IFNγ, IL4, IL6, IL10, IL12) production of lymphocytes was evaluated by FACS. The damaging consequences of treatment were assessed by evaluating the specific programmed cell death cascade in lymphocytes, assessed by FACS readings. The results revealed that under arsenic influence, and more so with arsenic+ENU, marked neoplastic changes were noted, which were corroborated with histological changes, cytokine modulation and apoptosis hinted at marked neoplastic changes.

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 p21 and p27 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.