Repolarization of glioblastoma macrophage cells using non-agonistic Dectin-1 ligand encapsulating TLR-9 agonist: plausible role in regenerative medicine against brain tumor.

AIM OF THE STUDY: Glioblastoma multiforme (GBM) is the most severe forms of brain cancer, eventually becoming the leading cause of brain cancer-related death worldwide. Owing to the bleak surgical interventions and resistance to the different treatment regime, GBM is a parlous disease demanding newer therapeutical perspective for its treatment. Toll-like receptors (TLRs) are well-known members of pathogen recognition receptors (PRRs) and have been extensively explored for their therapeutic and prophylactic potential in an array of disease including cancer. Recent trends in drug delivery research has shown shift towards delivering short DNA sequences (CpG DNA) to endosomal TLR9 within immune cells (macrophages, dendritic cells, etc.) for the activation of desired inflammatory response using non-agonistic ß-glucan particles; a well-known ligand for Dectin-1 receptors. Our study is therefore focused to explore the role of nano-encapsulated CpG ODN as critical players in polarizing M2 scavenging to much desired pro-inflammatory type. MATERIALS AND METHODS: The nanoparticles entrapping CpG ODN 1826 were prepared by using a fungal polymer Schizophyllan (SPG). The constructed nanoparticles were characterized and assessed for their efficacy on rat glioblastoma cells (C6). RESULTS: The constructed Schizophyllan (SPG) nanoparticles entrapping CpG ODN 1826 (95.3%) were of 25.49 nm in diameter and thus capable of crossing blood-brain barrier. The rat glioblastoma (C6) cells evaluated for intracellular oxidative burst and cytokine levels pre- and post-incubation with nanoparticles exhibited marked elevation in the expression of intracellular ROS and IFN-γ as well as IL-1ß post treatment. CONCLUSION: The findings indicate towards potentiality of repolarizing the M2 macrophages to much desired M1 phase by inducing higgh levels of oxidative burst and inflammatory cytokines. Consequently, the apoptosis was induced in glioblastoma cells establishing the suitablity of CpG ODN carrying nanoformulations as emerging therapeutic intervention for GBM.

Leishmanial CpG DNA nanovesicles: A propitious prophylactic approach against visceral leishmaniasis.

Unmethylated CpG motifs with phosphothioate backbone trigger TLR9 to elicit innate immune response characterized by the production of Th1 cytokines. The use of CpG DNA as an adjuvant has established its role in potentiating the humoral and cell mediated vaccine specific immune response. However, none of the synthetic oligodeoxynucleotides (ODNs) know and used till date are associated with the parasite itself. Our group identified a novel CG rich sequence of 14 base pairs from Leishmania donovani genome (Ld CpG ODN) and established it as a TLR9 agonist. The present study was designed to ascertain the adjuvanticity of Ld CpG ODN with soluble leishmanial antigen in experimental model of L. donovani. During the study Schizophyllan (SPG), a fungal polymer was used for encapsulating Ld CpG ODN for efficient endosomal delivery. The synthesized nanovehicles were of nearly 100 nm and localized within endosomes as confirmed by confocal microscopy. Immunization studies displayed the superior ability of synthesized nanovehicles co-administered with parasite antigen in augmenting innate immune response in comparison to ODN, nanoparticles or soluble antigen alone. The response included generation of ROS, NO and iNOS expression followed by proinflammatory cytokine milieu with reduced parasitic load within liver, spleen and bone marrow. These immune-tailored particles in combination with parasitic antigens elicited significant generation of cell mediated response owing to the presence of high levels of CD8+ T-cells and lymphocyte proliferation. Moreover, vaccination regime with synthesized adjuvant also activated humoral immunity by escalating the levels of IgG2 followed by reduced levels of anti-leishmanial IgG and IgG1 antibodies. The findings support the efficacy of Ld CpG ODN as a potential adjuvant against visceral leishmaniasis.

Jab1-siRNA Induces Cell Growth Inhibition and Cell Cycle Arrest in Gall Bladder Cancer Cells via Targeting Jab1 Signalosome.

BACKGROUND: The aberrant alteration in Jab1 signalosome (COP9 Signalosome Complex Subunit 5) has been proven to be associated with the progression of several carcinomas. However the specific role and mechanism of action of Jab1 signalosome in carcinogenesis of gall bladder cancer (GBC) are poorly understood. OBJECTIVE: The main objective of our study was to elucidate the role and mechanism of Jab1 signalosome in gall bladder cancer by employing siRNA. METHODS: Jab1 overexpression was identified in gall bladder cancer tissue sample. The role of Jab1-siRNA approach in cell growth inhibition and apoptotic induction was then examined by RT-PCR, Western Blotting, MTT, ROS, Hoechst and FITC/Annexin-V staining. RESULTS: In the current study, we have shown that overexpression of Jab1 stimulated the proliferation of GBC cells; whereas downregulation of Jab1 by using Jab1-siRNA approach resulted incell growth inhibition and apoptotic induction. Furthermore, we found that downregulation of Jab1 induces cell cycle arrest at G1 phase and upregulated the expression of p27, p53 and Bax gene. Moreover, Jab1-siRNA induces apoptosis by enhancing ROS generation and caspase-3 activation. In addition, combined treatment with Jab1-siRNA and gemicitabine demonstrated an enhanced decline in cell proliferation which further suggested increased efficacy of gemcitabine at a very lower dose (5µM) in combination with Jab1-siRNA. CONCLUSION: In conclusion, our study strongly suggests that targeting Jab1 signalosome could be a promising therapeutic target for the treatment of gall bladder cancer.

Elucidation of the Chemopreventive Role of Stigmasterol Against Jab1 in Gall Bladder Carcinoma.

BACKGROUND: Plant sterols have proven a potent anti-proliferative and apoptosis inducing agent against several carcinomas including breast and prostate cancers. Jab1 has been reported to be involved in the progression of numerous carcinomas. However, antiproliferative effects of sterols against Jab1 in gall bladder cancer have not been explored yet. OBJECTIVE: In the current study, we elucidated the mechanism of action of stigmasterol regarding apoptosis induction mediated via downregulation of Jab1 protein in human gall bladder cancer cells. METHODS: In our study, we performed MTT and Trypan blue assay to assess the effect of stigmasterol on cell proliferation. In addition, RT-PCR and western blotting were performed to identify the effect of stigmasterol on Jab1 and p27 expression in human gall bladder cancer cells. We further performed cell cycle, Caspase-3, Hoechst and FITC-Annexin V analysis, to confirm the apoptosis induction in stigmasterol treated human gall bladder cancer cells. RESULTS: Our results clearly indicated that stigmasterol has up-regulated the p27 expression and down-regulated Jab1 gene. These modulations of genes might occur via mitochondrial apoptosis signaling pathway. Caspase-3 gets activated with the apoptotic induction. Increase in apoptotic cells and DNA were confirmed through annexin V staining, Hoechst staining, and cell cycle analysis. CONCLUSION: Thus, these results strongly suggest that stigmasterol has the potential to be considered as an anticancerous therapeutic agent against Jab1 in gall bladder cancer.

Hesperidin Induces ROS-Mediated Apoptosis along with Cell Cycle Arrest at G2/M Phase in Human Gall Bladder Carcinoma.

A natural predominant flavonoid hesperidin rich in citrus fruits exhibits multifunctional medicinal properties. The anticancerous potential of hesperidin has been widely explored; however, the gall bladder carcinoma (GBC) still remains untouched due to the unavailability of efficient experimental model. The aim of our study was to identify the apoptotic and antiproliferative potential of hesperidin in GBC. The promising efficacy of hesperidin was assessed through the generation of reactive oxygen species (ROS), cellular apoptosis, and loss of mitochondrial membrane potential (MMP) in the primary cells generated from surgically removed cancerous gall bladder tissues. Moreover, cell cycle analysis and caspases-3 activity were performed to confirm the apoptosis inducing potential of hesperidin. Results revealed that hesperidin exposure for 24 h at a dose of 200 µM reduced the cell proliferation of GBC cells significantly. In addition, hesperidin treatment further resulted in an increased ROS generation and nuclear condensation at the same dose. Caspase-3 activation and cell cycle arrest at G2/M phase were also accelerated in a dose-dependent manner. Together, these results suggest that hesperidin can be considered as a potential anticancerous compound for the treatment of GBC. Furthermore, evaluation of the pharmacological aspects of hesperidin is desirable for drug development.

In silico analysis of human Toll-like receptor 7 ligand binding domain.

Toll-like receptors recognizing pathogen-associated molecular patterns are preface actors for innate immunity. Among them TLR7 is a transmembrane protein playing very crucial role in the signaling pathways involved in innate immunity by recognizing viral ssRNA and specific small molecule agonists. The unavailability of experimental 3D structure of this receptor till date hampers the focused exploration of TLR7 interaction with its ligands. However, several proteins possessing high homology domain enabled us to construct a reliable 3D model of hTLR7 ECD, which was employed to generate the homodimer model using protein-protein docking strategy. Further molecular docking studies between developed homodimer model and ligands were performed to explore the most preferred site of hTLR7 ECD interacting with ligands. The comparative analysis of docking energies and protein-ligand interactions of all the ligands revealed resiquimod as the prominent agonist. Furthermore, molecular interactions between protein-ligand complexes suggested LRR15 and LRR16 region of hTLR7 ECD as the most preferential site for ligand binding. The Ser434 and Gly437 of LRR15 region of hTLR7 were found to be conserved with Drosophila Toll protein. The obtained complex model may lead to a better understanding of TLR7 functioning along with its inheritance from invertebrates to mammals.