COMMIPHORA MUKUL EXTRACT AND GUGGULSTERONE EXHIBIT ANTITUMOUR ACTIVITY THROUGH INHIBITION OF CYCLIN D1, NF-Κß AND INDUCTION OF APOPTOSIS IN ORAL CANCER CELLS

ABSTRACTComiphora mukul, a promising medicinal plant and its constituent Guggulsterone (GS) is used in Ayurveda since decades. This study was aimed toinvestigate the anticancer potential of C. mukul and GS on oral cancer cell lines (SCC-4, KB). MTT assay was used to determine tumour cell proliferation,propidium iodide labeling and annexin V- binding, followed by flow cytometry was used to determine cell cycle and apoptosis of tumor cells aftertreatment. Expression of regulatory proteins such as NF-κß, cyclin D1, p53 and vascular endothelial growth factor was determined by western blot.C. mukul and GS significantly inhibited tumor cell growth, caused cell cycle arrest and apoptosis in both tumor cells. Such activities appeared to bedue to inhibition of NF-κß, cyclin D1and restoration of p53. Overall our data suggests that C. mukul and GS may be developed as chemopreventive andchemotherapeutic drug for oral cancer.Keywords: Commiphora mukul, Oral cancer, Antitumor, Cell cycle, Apoptosis, NF-κß, Cyclin D1, P53

Resveratrol, cancer and cancer stem cells: A review on past to future

Cancer remains to be an unresolved medical challenge despite of tremendous advancement in basic science research and clinical medicine. One of the major limitations is due to the side effects of chemotherapy which remains to be palliative without offering any permanent cure for cancer. Cancer stem cells (CSCs) are the subpopulation of cells in tumors that remain viable even after surgery, chemo- and radio-therapy that eventually responsible for tumor relapse. Hence, by eliminating non-stem cancer cells and cancer stem cells from the patient, permanent cure is expected. Phytochemicals have been under the intensive study to target these CSCs effectively and permanently as they do not cause any side effects. Resveratrol (RSV) is one such compound attaining lot of interest in recent days to target CSCs either alone or in combination. RSV has been used by several researchers to target cancer cells in a variety of disease models, however its CSC targeting abilities are under intensive study at present. This review is to summarize the effects of RSV under in vitro and in vivo conditions along with advantages and disadvantages of its uses against cancer cells and cancer stem cells. From the first reports on phytochemical applications against cancer and cancer stem cells in 1997 and 2002 respectively followed by later reports, up to date observations and developments are enlisted from PubMed in this comprehensive review. RSV is shown to be a potential compound having impact on altering the signal transduction pathways in cancer cells. However, the effects are variable under in vitro and in vivo conditions, and also with its use alone or in combination with other small molecules. Past research on RSV is emphasizing the importance of in vivo experimental models and clinical trials with different prospective combinations, is a hope for future promising treatment regimen

Detection of Glutathione S-Transferase gene (GST2, GST3) during induction of somatic embryogenesis in grape (Vitis vinifera L.)

Glutathione S-transferases (GSTs) are an important group of multifunctional enzymes that belong to diverse multigene families. In plants these enzymes are involved in the detoxification of xenobiotic compounds, herbicide detoxification, and primary and secondary metaboplism and play an important role in plant growth and development. There are several reports that show that the expression of GST is linked to the developmental phases of somatic embryogenesis. This study highlights the detection of transcript abundances of glutathione S-transferase genes GST2 and GST3 in the process of somatic embryogenesis of Vitis vinifera, and the lack thereof in non-embryogenic tissue of leaf cultures of grape (control). These results indicate that the expression of GST2 and GST3 could be used as a molecular signal for the identification of embryogenic cultures during the early development of somatic embryos

Two-dimensional unsteady forced convection heat transfer in power-law fluids from a cylinder

Forced convection heat transfer characteristics of a cylinder (maintained at a constant temperature) immersed in a streaming power-law fluids have been studied numerically in the two-dimensional (2-D), unsteady flow regime. The governing equations, namely, continuity, momentum and thermal energy, have been solved using a finite volume method based solver (FLUENT 6.3) over wide ranges of conditions (power law index, 0.4≤n≤1.8; Reynolds number, 40≤Re≤140; Prandtl number, 1≤Pr≤100). In particular, extensive numerical results elucidating the influence of Reynolds number, Prandtl number and power-law index on the isotherm patterns, local and average Nusselt numbers and their evolution with time are discussed in detail. Over the ranges of conditions considered herein, the nature of flow is fully periodic in time. The heat transfer characteristics are seen to be influenced in an intricate manner by the value of the Reynolds number (Re), Prandtl number (Pr) and the power-law index (n). Depending upon the value of the power-law index (n), though the flow transits from being steady to unsteady somewhere in the range I33 < Re < 50, the fully periodic behavior is seen only beyond the critical value of the Reynolds number (Re). As expected, the average Nusselt number increases with an increase in the values of Reynolds and/or Prandtl numbers, irrespective of the value of the flow behavior index. A strong influence of the power-law index on both local and time-averaged Nusselt numbers was observed. Broadly, all else being equal, shear-thinning behavior (n < 1) promotes heat transfer whereas shear-thickening behavior (n > 1) impedes it. Furthermore, this effect is much more pronounced in shear-thinning fluids than that in shear-thickening fluids

Two-dimensional unsteady flow of power-law fluids over a cylinder

The unsteady flow of incompressible power-law fluids over an unconfined circular cylinder in cross-flow arrangement has been studied numerically. The two-dimensional (2-D) field equations have been solved using a finite volume method based solver (FLUENT 6.3). In particular, the effects of the power-law index (0.4≤n≤1.8) and Reynolds number (40≤Re≤140) on the detailed kinematics of the flow (streamline, surface pressure and vorticity patterns) and on the macroscopic parameters (drag and lift coefficients, Strouhal number) are presented in detail. The periodic vortex shedding and the evolution of detailed kinematics with time are also presented to provide insights into the nature of flow. The two-dimensional flow transits from steady to unsteady behaviour at a critical value of the Reynolds number ReI(40-50) and the von-Karman vortex street is observed beyond the critical Reynolds number (Re). Obviously, both the lift coefficient and Strouhal number values are zero for the steady flow, but their values increase with the increasing Reynolds number (Re) in the unsteady flow regime. For highly shear-thickening fluids (n=1.8), the flow becomes unsteady at Re=40 and unsteadiness in the flow appears at Re=50 for all values of power-law index (n). As expected, the evolution of the kinematics and vortex shedding show a complex dependence on the flow parameters near the transition in the flow. For a fixed value of the Reynolds number (Re), the drag coefficient increases and lift coefficient decreases with increasing value of the power-law index (n). For a fixed value of the power-law index (n), the drag coefficient gradually increases with the Reynolds number (Re). Similar to the drag coefficient, lift coefficient also shows a complex dependence on the power-law index (n) near the transition zone. The value of the Strouhal number (St) decreases with the increasing value of the power-law index (n) at a fixed value of the Reynolds number (Re)

The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8(+) T Cells

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+) and CD8(+) T cells secreting gamma interferon (IFN-gamma) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis. We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease

A Cancer Nanovaccine for Co-Delivery of Peptide Neoantigens and Optimized Combinations of STING and TLR4 Agonists

Immune checkpoint blockade (ICB) has revolutionized cancer treatment and led to complete and durable responses, but only for a minority of patients. Resistance to ICB can largely be attributed to insufficient number and/or function of antitumor CD8+ T cells in the tumor microenvironment. Neoantigen targeted cancer vaccines can activate and expand the antitumor T cell repertoire, but historically, clinical responses have been poor because immunity against peptide antigens is typically weak, resulting in insufficient activation of CD8+ cytotoxic T cells. Herein, we describe a nanoparticle vaccine platform that can overcome these barriers in several ways. First, the vaccine can be reproducibly formulated using a scalable confined impingement jet mixing method to coload a variety of physicochemically diverse peptide antigens and multiple vaccine adjuvants into pH-responsive, vesicular nanoparticles that are monodisperse and less than 100 nm in diameter. Using this approach, we encapsulated synergistically acting adjuvants, cGAMP and monophosphoryl lipid A (MPLA), into the nanocarrier to induce a robust and tailored innate immune response that increased peptide antigen immunogenicity. We found that incorporating both adjuvants into the nanovaccine synergistically enhanced expression of dendritic cell costimulatory markers, pro-inflammatory cytokine secretion, and peptide antigen cross-presentation. Additionally, the nanoparticle delivery increased lymph node accumulation and uptake of peptide antigen by dendritic cells in the draining lymph node. Consequently, nanoparticle codelivery of peptide antigen, cGAMP, and MPLA enhanced the antigen-specific CD8+ T cell response and delayed tumor growth in several mouse models. Finally, the nanoparticle platform improved the efficacy of ICB immunotherapy in a murine colon carcinoma model. This work establishes a versatile nanoparticle vaccine platform for codelivery of peptide neoantigens and synergistic adjuvants to enhance responses to cancer vaccines

Fine-Needle Aspiration-Based Patient-Derived Cancer Organoids

Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications

Epithelial Expressed B7-H4 Drives Differential Immunotherapy Response in Murine and Human Breast Cancer.

UNLABELLED: Combinations of immune checkpoint inhibitors (ICI, including anti-PD-1/PD-L1) and chemotherapy have been FDA approved for metastatic and early-stage triple-negative breast cancer (TNBC), but most patients do not benefit. B7-H4 is a B7 family ligand with proposed immunosuppressive functions being explored as a cancer immunotherapy target and may be associated with anti-PD-L1 resistance. However, little is known about its regulation and effect on immune cell function in breast cancers. We assessed murine and human breast cancer cells to identify regulation mechanisms of B7-H4 in vitro. We used an immunocompetent anti-PD-L1-sensitive orthotopic mammary cancer model and induced ectopic expression of B7-H4. We assessed therapy response and transcriptional changes at baseline and under treatment with anti-PD-L1. We observed B7-H4 was highly associated with epithelial cell status and transcription factors and found to be regulated by PI3K activity. EMT6 tumors with cell-surface B7-H4 expression were more resistant to immunotherapy. In addition, tumor-infiltrating immune cells had reduced immune activation signaling based on transcriptomic analysis. Paradoxically, in human breast cancer, B7-H4 expression was associated with survival benefit for patients with metastatic TNBC treated with carboplatin plus anti-PD-L1 and was associated with no change in response or survival for patients with early breast cancer receiving chemotherapy plus anti-PD-1. While B7-H4 induces tumor resistance to anti-PD-L1 in murine models, there are alternative mechanisms of signaling and function in human cancers. In addition, the strong correlation of B7-H4 to epithelial cell markers suggests a potential regulatory mechanism of B7-H4 independent of PD-L1. SIGNIFICANCE: This translational study confirms the association of B7-H4 expression with a cold immune microenvironment in breast cancer and offers preclinical studies demonstrating a potential role for B7-H4 in suppressing response to checkpoint therapy. However, analysis of two clinical trials with checkpoint inhibitors in the early and metastatic settings argue against B7-H4 as being a mechanism of clinical resistance to checkpoints, with clear implications for its candidacy as a therapeutic target

FIGURE 4 from Epithelial Expressed B7-H4 Drives Differential Immunotherapy Response in Murine and Human Breast Cancer

Overexpression of B7-H4 in the EMT6 murine model induced resistance to anti-PD-L1 ICI. A, EMT6 cells were virally transduced with the pBabe-B7-H4 retroviral vector and overexpress exogenous murine B7-H4. B, EMT6 B7-H4+ tumors maintain high B7-H4 expression in vivo assessed by IHC. C, Animals were orthotopically injected with EMT6 cells ± B7-H4 and treated 1x per week with anti-PD-L1 at 200 µg (first dose) or 100 µg (subsequent doses) for 4 weeks, after tumors reached 100 mm3. D, EMT6-B7-H4+ tumors are significantly resistant to anti-PD-L1 immunotherapy compared with control tumors. Data were analyzed by one-way ANOVA of individual AUC values with Tukey post hoc test for multiple comparisons between EMT6 anti-PD-L1 and EMT6-B7-H4+ anti-PD-L1–treated groups (P = 0.0174, EMT6 Isotype n = 21, EMT6 anti-PD-L1 n = 23, EMT6-B7-H4+ Isotype n = 21, EMT6-B7-H4+ anti-PD-L1 n = 23. Data were collected from a total of three independent experiments). E and F, When tumor response is categorized into three groups, EMT6-B7-H4+ tumors have overall greater intrinsic resistance to treatment and reduced complete response compared with EMT6 control tumors (P = 0.035, χ2 = 6.683, df = 2, n = 23 mice for EMT6 parental tumors and n = 31 mice for B7-H4 tumors). G, EMT6-B7-H4+ tumors were stained by mIF. B7-H4 is expressed on CD45− tumor cells in vivo. Representative image shown. Scale bar 10 µm. H, Quantification of G, n = 3 mice. Data analyzed by unpaired t test. Data were analyzed in GraphPad Prism v10.</p