6-Shogaol Exhibits Anti-viral and Anti-inflammatory Activity in COVID-19-Associated Inflammation by Regulating NLRP3 Inflammasomes.

Recent global health concern motivated the exploration of natural medicinal plant resources as an alternative target for treating COVID-19 infection and associated inflammation. In the current study, a phytochemical, 6-shogaol [1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one; 6-SHO] was investigated as a potential anti-inflammatory and anti-COVID-19 agent. In virus release assay, 6-SHO efficiently (94.5%) inhibited SARS-CoV2 replication. When tested in the inflammasome activation model, 6-SHO displayed mechanistic action by regulating the expression of the inflammasome pathway molecules. In comparison to the existing drugs, remdesivir and hydroxy-chloroquine, 6-SHO was not only found to be as effective as the standard anti-viral drugs but also much superior and safe in terms of predicted physicochemical properties and clinical toxicity. Comparative molecular dynamics simulation demonstrated a stable interaction of 6-SHO with NLRP3 (the key inflammasome regulator) in the explicit water environment. Overall, this study provides important cues for further development of 6-SHO as potential anti-inflammatory and anti-viral therapeutic agents.

Synthesis, biological evaluation, and molecular docking analysis of phenstatin based indole linked chalcones as anticancer agents and tubulin polymerization inhibitors.

A library of new phenstatin based indole linked chalcone compounds (9a-z and 9aa-ad) were designed and synthesized. Of these, compound 9a with 1-methyl, 2- and 3-methoxy substituents in the aromatic ring was efficacious against the human oral cancer cell line SCC-29B, spheroids, and in a mouse xenograft model of oral cancer AW13516. Compound 9a exhibited anti-cancer activity through disrupting cellular integrity and affecting glucose metabolism-which is a hallmark of cancer. The cellular architecture was affected by inhibition of tubulin polymerization as observed by an immunofluorescence assay on 9a-treated SCC-29B cells. An in vitro tubulin polymerization kinetics assay provided evidence of direct interaction of 9a with tubulin. This physical interaction between tubulin and compound 9a was further confirmed by Surface Plasmon Resonance (SPR) analysis. Molecular docking experiments and validations revealed that compound 9a interacts and binds at the colchicine binding site of tubulin and at active sites of key enzymes in the glucose metabolism pathway. Based on in silico modeling, biophysical interactions, and pre-clinical observations, 9a consisting of phenstatin based indole-chalcone scaffolds, can be considered as an attractive tubulin polymerization inhibitor candidate for developing anti-cancer therapeutics.

Myeloid-derived suppressor cells impede T cell functionality and promote Th17 differentiation in oral squamous cell carcinoma.

Oral tumor microenvironment is characterized by chronic inflammation signified with infiltrating leukocytes and soluble mediators which cause immune suppression. However, how immunosuppressive cells like myeloid-derived suppressor cells (MDSCs) maintain the immunosuppressive tumor microenvironment and influence T cell function in oral squamous cell carcinoma (OSCC) patients remains poorly understood. In the present study, we found that percentages of MDSCs were higher in oral cancer patients compared to healthy individuals and correlated with cancer stage. Monocytic MDSCs (M-MDSCs) were prevalent in the periphery, while granulocytic/polymorphonuclear subset dominated the tumor compartment. M-MDSCs suppressed the lymphocyte proliferation and decreased the CD3-ζ (zeta) chain expression and interferon gamma production. The percentage of M-MDSCs in peripheral blood correlated inversely with CD3-ζ chain expression in T cells of these patients. Interleukin 6 (IL-6)-induced phosphorylated STAT3-regulated programmed cell death ligand 1, CCAAT/enhancer-binding proteins alpha and beta and Interleukin 10 expression in MDSCs. MDSCs inhibited TGF-ß-driven generation of induced regulatory T cells in vitro. M-MDSCs secreted interleukins IL-6, IL-1ß, IL-23 and PGE2 and facilitated T-helper 17 (Th17) cell differentiation which utilizes nitric oxide synthase and cyclooxygenase 2 enzyme activity. Interestingly, OSCC patients showed increased levels of Th17 cells in peripheral blood and tumor tissue. Thus, increased frequency of MDSCs, Th17 cells and decreased expression of CD3-ζ chain portray T cell tolerance and chronic inflammatory state facilitating tumor growth.

Extracellular 2'5'-oligoadenylate synthetase 2 mediates T-cell receptor CD3-ζ chain down-regulation via caspase-3 activation in oral cancer.

Decreased expression of CD3-ζ chain, an adaptor protein associated with T-cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3-ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour-derived factors promote immune suppression by down-regulating CD3-ζ chain expression. 2'5'-Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3-ζ chain down-regulation and OAS2 stimulation. The surrogate situation was established by over-expressing OAS2 in a HEK293 cell line and cell-free supernatant was collected. These supernatants when incubated with T cells resulted in down-regulation of CD3-ζ chain, which shows that the secreted OAS2 is capable of regulating CD3-ζ chain expression. Incubation of T cells with cell-free supernatants of oral tumours or recombinant human OAS2 (rh-OAS2) induced caspase-3 activation, which resulted in CD3-ζ chain down-regulation. Caspase-3 inhibition/down-regulation using pharmacological inhibitor or small interfering RNA restored down-regulated CD3-ζ chain expression in T cells induced by cell-free tumour supernatant or rh-OAS2. Collectively these results show that OAS2 leads to impairment in CD3-ζ chain expression, so offering an explanation that might be applicable to the CD3-ζ chain deficiency observed in cancer and diverse disease conditions.

Mechanisms involved in the down-regulation of TCR zeta chain in tumor versus peripheral blood of oral cancer patients.

Immune dysfunction is the hallmark of patients with oral cancer. Down-regulation of T cell receptor (TCR) zeta chain expression was observed in T cells from patients with oral squamous cell carcinoma. In peripheral blood, the decrease in TCR zeta chain showed an inverse correlation with the tumor stage as demonstrated by western blotting, confocal microscopy and flow cytometry. The mechanism of TCR zeta chain degradation in the peripheral blood involves ubiquitination and subsequent targeting of TCR zeta for degradation in the lysosome. Decreased expression of PKC theta and the subsequent decrease of TCR zeta chain transcription factor Elf-1 and its binding to DNA may contribute to the decreased/or absent TCR zeta chain transcripts in the tumor infiltrating lymphocytes. Oral cancer patients exhibiting TCR zeta chain defect also showed impaired lymphocyte proliferation, cytokine profile and intracellular calcium release upon stimulation with anti CD3 mAb. Our data shows that posttranslational degradation is primarily responsible for decreased TCR zeta chain expression in the peripheral blood, while a transcriptional defect is observed in the tumor compartment. The down-regulation of TCR zeta chain culminates into impaired lymphocyte responses in these patients.

Impaired lymphocyte responses and their restoration in oral cancer patients expressing distinct TCR variable region.

We have analyzed TCR Vbeta gene usage and clonality of T cells in the peripheral blood of patients with oral cancer and healthy individuals. A large repertoire of clonal TCR Vbeta was observed in the peripheral blood of cancer patients, and this clonal expansion was dominantly represented in the CD8+ T cells. A marked decrease in the lymphocyte proliferative responses to mitogen and anti-CD3 MAb and spontaneous apoptosis was observed in lymphocytes. Appropriate co-stimulation of lymphocytes (anti-TCR Vbeta MAb and anti-CD28 MAb) restored the lymphocyte proliferative responses and CD3-zeta chain expression in these patients.

Heat shock protein induced TCR gammadelta gene rearrangements in patients with oral cancer.

We analyzed the T cell receptor (TCR) gammadelta gene repertoire in peripheral blood and tumor compartment of oral cancer (OC) patients before and after stimulation with heat shock proteins (hsp), which are known ligands for gammadelta T cells. Clonal TCR gamma and delta gene rearrangements in lymphocytes from tumor compartment and peripheral blood were studied using TCR Vgamma and Vdelta gene primers in PCR followed by heteroduplex analysis. Vgamma gene segments derived from VgammaI or VgammaII gene families were most dominantly expressed in peripheral blood lymphocytes (PBL) as compared to tumor infiltrating lymphocytes (TIL) of OC patients. Of the rearranged TCR delta alleles Vdelta1-Jdelta1 and Vdelta2-Jdelta1 gene rearrangements were the most predominant in PBL and TIL of OC patients respectively. Stimulation of gammadelta T cells with hsp 60/70 demonstrated a selective clonal expansion of Vgamma9-Vdelta2 (VgammaII family) subset indicating that, this expanded population of cells could be responsible for eliciting an immune response against oral tumor cells.