In-vitro analysis on the potential use of dental pulp mesenchymal stem cells on arecoline-induced oral epithelial cells.

To assess the protective role of the secretome of dental pulp mesenchymal stem cells on arecoline-induced epithelial-mesenchymal transition and senescence on epithelial cells of the oral mucosa. Effect of varying concentrations of arecoline extract and dental pulp mesenchymal stem cell condition media (DPSC-CM) were noted on oral mucosal epithelial cells. MTT assay, Annexin V-FITC/PI assay, and the quantitative gene expressions of BCL2, PUMA, BAD, BAX, CASP3, CASP9, CASP12, TGFB1, CST3, COL1A2, COL3A1, TIMP1, TIMP2, CDH1, and CDH2 were assessed. Oral mucosal epithelial cells exposed only to the arecoline were the control. 50% and 100% DPSC-CM decreased apoptosis-related gene expression in the cells exposed with 25 µM arecoline compared to the control. 50% DPSC-CM attenuated the expression of all fibrotic genes and EMT-related genes. 20% and 100% DPSC-CM showed differential effects on fibrotic and EMT-related genes. DPSC-CM inhibited apoptosis, and attenuated expression of fibrotic and EMT-related genes on arecoline treated human oral epithelial cells.

CD44-positive cancer stem cells from oral squamous cell carcinoma exhibit reduced proliferation and stemness gene expression upon adipogenic induction.

We proposed to assess adipogenic differentiation and its effect on the proliferation and stemness markers in CD44 + OSCC CSCs. D44 + CSCs were sorted by magnetic sorting from the single-cell suspension of the OSCC tumor. Adipogenic differentiation was induced by an adipogenic induction medium. Lipid droplet formation was confirmed by oil red O staining. The expression of the cell surface marker was analyzed by flow cytometry. Real-time qPCR was performed to examine the gene expression activity. CD44 + OSCC CSCs can differentiate into adipocytes and adipogenesis in these cells decrease their proliferation and stemness gene expression. Adipogenic induction can make the cancer stem cells from OSCC tumors lose their stemness potential. Oral cancer, especially OSCC, is a huge burden worldwide. Similar to other stem cells, cancer stem cells can differentiate into other lineage cells. Our study shows that the proliferation and stemness gene expression in the CSCs from OSCC tumors can be thwarted by inducing them to differentiate into adipocytes, which could be advantageous to find out new clinical approaches in the treatment of cancers, like OSCC.

The Growth Factors and Cytokines of Dental Pulp Mesenchymal Stem Cell Secretome May Potentially Aid in Oral Cancer Proliferation.

BACKGROUND: Growth factors and cytokines responsible for the regenerative potential of the dental pulp mesenchymal stem cell secretome (DPMSC-S) are implicated in oral carcinogenesis. The impact and effects of these secretory factors on cancer cells must be understood in order to ensure their safe application in cancer patients. OBJECTIVE: We aimed to quantify the growth factors and cytokines in DPMSC-S and assess their effect on oral cancer cell proliferation. MATERIALS AND METHODS: DPMSCs were isolated from patients with healthy teeth (n = 5) that were indicated for extraction for orthodontic reasons. The cells were characterized using flow cytometry and conditioned medium (DPMSC-CM) was prepared. DPMSC-CM was subjected to a bead-based array to quantify the growth factors and cytokines that may affect oral carcinogenesis. The effect of DPMSC-CM (20%, 50%, 100%) on the proliferation of oral cancer cells (AW123516) was evaluated using a Ki-67-based assay at 48 h. AW13516 cultured in the standard growth medium acted as the control. RESULTS: VEGF, HCF, Ang-2, TGF-α, EPO, SCF, FGF, and PDGF-BB were the growth factors with the highest levels in the DPMSC-CM. The highest measured pro-inflammatory cytokine was TNF-α, followed by CXCL8. The most prevalent anti-inflammatory cytokine in the DPMSC-CM was IL-10, followed by TGF-ß1 and IL-4. Concentrations of 50% and 100% DPMSC-CM inhibited Ki-67 expression in AW13516, although the effect was non-significant. Moreover, 20% DPMSC-CM significantly increased Ki-67 expression compared to the control. CONCLUSIONS: The increased Ki-67 expression of oral cancer cells in response to 20% DPMSC-CM indicates the potential for cancer progression. Further research is needed to identify their effects on other carcinogenic properties, including apoptosis, stemness, migration, invasion, adhesion, and therapeutic resistance.

Adipogenic Stimulation and Pyrrolidine Dithiocarbamate Induced Osteogenic Inhibition of Dental Pulp Stem Cells Is Countered by Cordycepin.

BACKGROUND: dental pulp-derived stem cells are easy to access and collect and are an excellent source of stem cells for regenerative therapy. These cells can interact with many biomolecules and scaffolds and can pass on the instructive signals to the sites of regeneration where they are used. In this regard cordycepin, a potential biomolecule derived from medicinal mushrooms with a spectrum of bioactive properties such as antioxidant, anti-inflammatory, and anticancer has not yet been tested for its effect on human dental pulp stem cells. OBJECTIVE: the objective of the present study was to assess the in vitro adipogenic and osteogenic differentiation potential of human dental pulp stem cells with or without induction after administration of cordycepin. MATERIALS AND METHODS: human dental pulp stem cells DPSCs were isolated from a healthy permanent tooth extracted for orthodontic purposes after obtaining informed consent. Flow cytometry technique was used to assess the surface markers of these cells such as CD73, CD90, and CD105, CD34, CD45, and HLA-DR. Further, an MTT assay was performed on the cells after subjecting them to various concentrations of cordycepin. Following this, the adipogenic and osteogenic potential of the dental pulp stem cells was assessed with or without induction under the influence/absence of 5 µM of cordycepin. The results obtained were statistically analyzed and documented. RESULTS: it was found that the dental pulp stem cells showed strong positive expression for CD73, CD90, and CD105 and faint expression of CD34, CD45, and HLA-DR. MTT assay revealed that 5 µM was the optimum concentration of cordycepin for all the assays. Concerning adipogenesis experiments, there was a statistically significant lowering of all the 4 adipogenesis-related genes PPARγ, FABP4, LPL, and C/EBPα following cordycepin treatment in the presence of induction compared to the only induction group and untreated control cells (p < 0.05). In connection with osteogenesis, was found that there was a statistically significant increase in the expression of RUNX2, COL1A1, OSX and OCN genes along with the increase in alkaline phosphatase and alizarin red staining in the DPSC treated with cordycepin along with the presence of induction and simultaneous addition of PDTC compared to the control untreated cells and cells treated with induction and simultaneous addition of PDTC (p < 0.05). CONCLUSION: cordycepin can be exploited for its osteopromotive properties and can be used as a bioactive molecule alongside the administration of dental pulp stem cells in the area of regenerative biology and medicine.

Angiogenesis induction in breast cancer: A paracrine paradigm.

Breast cancer is the most prevalent type of cancer among women globally. Angiogenesis contributes significantly to breast cancer progression and dissemination. Neovascularization is concurrent with the progression and growth of breast cancer. Breast cancer cells control angiogenesis by secreting pro-angiogenic factors like fibroblast growth factor, vascular endothelial growth factor, interleukin, transforming growth factor-ß, platelet-derived growth factor and several others. These pro-angiogenic factors trigger neovascularization, and thereby lead to breast cancer development and metastasis. The hypoxia-inducible factor (HIF)-regulated angiogenesis cascade is a crucial underlying factor in breast cancer growth and metastasis. To that end, several efforts have been made to identify druggable targets within the HIF-angiogenesis components. However, escape pathways are a major hindrance for targeted therapies against angiogenesis. Thus, understanding the key factors that trigger breast cancer angiogenesis is critical in elucidating ways to inhibit breast cancer. The current review provides an overview of the key growth factors that trigger breast cancer angiogenesis.

Allicin May Promote Reversal of T-Cell Dysfunction in Periodontitis via the PD-1 Pathway.

We evaluated the role of allicin in periodontitis using an in silico and in vitro design. An in silico docking analysis was performed to assess the plausible interactions between allicin and PD-L1. The cytokine profile of gingival crevicular fluid (GCF) samples obtained from periodontitis patients was estimated by cytometric bead array. CD3+ lymphocytes isolated from the peripheral blood were sorted and characterized using immunomagnetic techniques. Cultured and expanded lymphocytes were treated with the GCF samples to induce T-cell exhaustion. Optimum concentrations of allicin were added to exhausted lymphocytes to compare the expression of TIM-3 and LAG-3 gene expression at baseline and post-treatment. Allicin was found to bind to the PD-L1 molecule as revealed by the in-silico experiment, which is possibly an inhibitory interaction although not proven. GCF from periodontitis patients had significantly higher concentrations of TNF-α, CCL2, IL-6, IFN-γ, and CXCL8 than controls. GCF treatment of CD3+ lymphocytes from the periodontitis patients significantly increased expression of T-cell exhaustion markers TIM-3 and LAG-3. Allicin administration with GCF treatment resulted in significant lowering of the expression of exhaustion markers. Allicin may exert an immunostimulatory role and reverse immune-destructive mechanisms such as T-cell exhaustion.

Tinospora cordifolia (Thunb.) Miers (Giloy) inhibits oral cancer cells in a dose-dependent manner by inducing apoptosis and attenuating epithelial-mesenchymal transition.

BACKGROUND: Tinospora cordifolia (Thunb.) Miers (Giloy) has been applied successfully as an anti-inflammatory, anti-diabetic, and even as an anti-cancer agent. Yet, to date, the application of Giloy has not been explored concerning oral cancer. OBJECTIVES: To assess the effect of T cordifolia (Thunb.) Miers (Giloy) extract (TcE) on an oral cancer cell line. METHODS: AW13516 (oral cancer cell line) cells were treated with the prepared aqueous extract of TcE for 24 h at various concentrations ranging between 5 µg/ml and 100 µg/ml and compared with control (cells without treatment). Thee effect of the extracts on apoptosis was assessed by through Annexin V flow cytometry assay and Luminometry based assessment of Caspase 8, 9 and caspase 3/7 activity. RNA was isolated from treated cells and gene expression of selected metastatic genes (MMP1, MMP10, and CXCL8); epithelial-mesenchymal stem cell genes (TWIST1, SNAIL, ZEB1, Oct4) and stemness related genses (Nanog, Sox2) were analyzed by using a quantitative real-time PCR system. The experiments were performed in triplicates. RESULTS: Aqueous extract of TcE was found to induce apoptosis inducer in AW13516 cells in a concentration-dependent manner and was potent even at a low concentration of 5 µg/ml. The apoptosis induction was confirmed with the caspase activity assay. Treatment of the cells with the extract for 24 h exhibited a significant decrease in the expression of EMT genes in a dose-dependent manner without an effect on the metastatic genes. CONCLUSION: Aqueous extract of TcE induces apoptosis-mediated cell death in the oral cancer cell line AW13516 while attenuating its potential for epithelial mesenchymal transition.

Gingival crevicular fluid infiltrating CD14+ monocytes promote inflammation in periodontitis.

Periodontitis is a condition that occurs because of inflammation-mediated tissue degeneration. Many studies have been conducted to identify inflammatory molecules in periodontitis, but the well-defined role of cells from the immune system in the progression of periodontitis as well as in gingival tissue degeneration has not been appropriately established. The objective of the present study was to characterize the monocytes isolated from the gingival crevicular fluid (GCF) in patients with periodontitis. GCF was obtained from periodontitis patients and healthy controls. Cytokine levels of CCL2 were evaluated by ELISA in GCF samples. CD14+ monocytes were separated using magnetic sorting from GCF. RT-qPCR was performed to assess the gene expression. Cytometric bead array analysis was performed to analyze the levels of cytokines and chemokines in the secretome of cells. CD14+ monocytes from GCF secreted higher levels of CCL2 and showed elevated expression of genes responsible for monocyte migration. Additionally, upon lipopolysaccharide stimulation, these monocytes secreted higher levels of inflammatory cytokines and chemokines. This investigation aids in understanding the inflammatory microenvironment of periodontitis by characterizing GCF in terms of infiltrated CD14+ monocytes, cytokines, and molecules secreted by these monocytes, which are specific for cellular differentiation.

Exploration of (hetero)aryl Derived Thienylchalcones for Antiviral and Anticancer Activities.

BACKGROUND: Search for new antiviral and anticancer agents are essential because of the emergence of drug resistance in recent years. In continuation of our efforts in identifying the new small molecule antiviral and anticancer agents, we identified chalcones as potent antiviral and anticancer agents. OBJECTIVE: With the aim of identifying the broad acting antiviral and anticancer agents, we discovered substituted aryl/heteroaryl derived thienyl chalcones as antiviral and anticancer agents. METHOD: A focused set of thienyl chalcone derivaties II-VI was screened for selected viruses Hepatitis B virus (HBV), Herpes simplex virus 1 (HSV-1), Human cytomegalovirus (HCMV), Dengue virus 2 (DENV2), Influenza A (H1N1) virus, MERS coronavirus, Poliovirus 1 (PV 1), Rift Valley fever (RVF), Tacaribe virus (TCRV), Venezuelan equine encephalitis virus (VEE) and Zika virus (ZIKV) using the National Institute of Allergy and Infectious Diseases (NIAID)'s Division of Microbiology and Infectious Diseases (DMID) antiviral screening program. Additionally, a cyclopropylquinoline derivative IV has been screened for 60 human cancer cell lines using the Development Therapeutics Program (DTP) of NCI. RESULTS: All thienyl chalcone derivatives II-VI displayed moderate to excellent antiviral activity towards several viruses tested. Compounds V and VI were turned out be active compounds towards human cytomegalovirus for both normal strain (AD169) as well as resistant isolate (GDGr K17). Particularly, cyano derivative V showed very high potency (EC50: <0.05 µM) towards AD169 strain of HCMV compared to standard drug Ganciclovir (EC50: 0.12 µM). Additionally, it showed moderate activity in the secondary assay (AD169; EC50: 2.30 µM). The cyclopropylquinoline derivative IV displayed high potency towards Rift Valley fever virus (RVFV) and Tacaribe virus (TCRV) towards Rift Valley fever virus (RVFV). The cyclopropylquinoline derivative IV is nearly 28 times more potent in our initial in vitro visual assay (EC50: 0.39 µg/ml) and nearly 17 times more potent in neutral red assay (EC50: 0.71 µg/ml) compared to the standard drug Ribavirin (EC50: 11 µg/ml; visual assay and EC50: 12 µg/ml; neutral red assay). It is nearly 12 times more potent in our initial in vitro visual assay (EC50: >1 µg/ml) and nearly 8 times more potent in neutral red assay (EC50: >1.3 µg/ml) compared to the standard drug Ribavirin (EC50: 12 µg/ml; visual assay and EC50: 9.9 µg/ml; neutral red assay) towards Tacaribe virus (TCRV). Additionally, cyclopropylquinoline derivative IV has shown strong growth inhibitory activity towards three major cancers (colon, breast, and leukemia) cell lines and moderate growth inhibition shown towards other cancer cell lines screened. CONCLUSION: Compounds V and VI were demonstrated viral inhibition towards Human cytomegalovirus, whereas cyclopropylquinoline derivative IV towards Rift Valley fever virus and Tacaribe virus. Additionally, cyclopropylquinoline derivative IV has displayed very good cytotoxicity against colon, breast and leukemia cell lines in vitro.

IGF-1 and somatocrinin trigger islet differentiation in human amniotic membrane derived mesenchymal stem cells.

AIM: To induce differentiation of human amniotic membrane derived mesenchymal stem cells (hAMMSCs) into insulin producing cells (IPCs) by treating with somatocrinin or growth hormone releasing hormone (GHRH) and Insulin-like growth factor-1 (IGF-1). MAIN METHOD: In this investigation, we cultivated and characterized hAMMSCs and then treated with IGF-1 and somatocrinin to find out whether this combination gives better yield of insulin producing cells. We showed that hAMMSCs can give rise to IPCs on exposure to serum-free defined media containing specific growth factors and differentiating agents in presence of IGF-1 and somatocrinin. KEY FINDING: A combination of IGF-1 and somatocrinin lead to differentiation of large number of IPCs from hAMMSCs. These IPCs were found to be positive for dithizone indicating their insulin secretory mechanism. Moreover these cells were also found to be positive for C-peptide. IPCs released insulin in response to glucose challenge. Gene expression analysis exhibited significant up-regulation of pancreatic transcription factor GLUT2 and Insulin. SIGNIFICANCE: Our data thus demonstrates for the first time that somatocrinin and IGF-1 synergistically enhance the differentiation of hAMMSCs into IPCs.

Long term explant culture for harvesting homogeneous population of human dental pulp stem cells.

Dental pulp stem cells have emerged as a preferred source of mesenchymal stem cells, because of its easy availability and high stem cell content. Dental pulp is a specific fibrous tissue that contains heterogeneous populations of odontoblasts, fibroblasts, pericytes, progenitors, stem cells, leukocytes and neuronal cells. In this study, we propose sustained explant culture as a simple, economical and efficient process to isolate dental pulp stem cells from human Dental pulp Tissue. Historically explant cultures were used to get fibroblast cells from embryonic chick heart using plasma clot cultures. The subculture was performed by lifting mother explant (original explant) and grafting it in a new plasma clot. We modified this age old technique to suit the modern times. Here we demonstrate for the first time that the mother explant (E0) of human dental pulp tissue could be sub-cultured consecutively seven times (E7) without displacement. This technique is highly reproducible and permits growth and proliferation of dental pulp stem cells yielding an enriched homogeneous mesenchymal stem cells population in the first passage itself as revealed by surface marker expression. These dental pulp stem cells exhibit differentiation into adipogenic, chondrogenic and osteogenic lineage revealing their mesenchymal stem cell nature. We propose that dental pulp stem cells isolated by sustained explant culture are phenotypically and functionally comparable to those obtained by enzymatic method. It is a simple, inexpensive and gentle method, which may be preferred over the conventional techniques for obtaining stem cells from other tissue sources as well especially in cases of limited starting material.

Identification of Novel 5,6-Dimethoxyindan-1-one Derivatives as Antiviral Agents.

BACKGROUND: Discovery of novel antiviral agents is essential because viral infection continues to threaten human life globally. Various heterocyclic small molecules have been developed as antiviral agents. The 5,6-dimethoxyindan-1-on nucleus is of considerable interest as this ring is the key constituent in a range of bioactive compounds, both naturally occurring and synthetic, and often of considerable complexity. OBJECTIVE: The main purpose of this research was to discover and develop small molecule heterocycles as broad-spectrum of antiviral agents. METHOD: A focused small set of 5,6-dimethoxyindan-1-one analogs (6-8) along with a thiopene derivative (9) was screened for selected viruses (Vaccinia virus - VACA, Human papillomavirus - HPV, Zika virus - ZIKV, Dengue virus - DENV, Measles virus - MV, Poliovirus 3 - PV, Rift Valley fever virus - RVFV, Tacaribe virus - TCRV, Venezuelan equine encephalitis virus - VEEV, Herpes simplex virus 1 -HSV-1 and Human cytomegalovirus - HCMV) using the National Institute of Allergy and Infectious Diseases (NIAID)'s Division of Microbiology and Infectious Diseases (DMID) antiviral screening program. RESULTS: These molecules demonstrated moderate to excellent antiviral activity towards variety of viruses. The 5,6-dimethoxyindan-1-one analog (7) demonstrated high efficacy towards vaccinia virus (EC50: <0.05 µM) and was nearly 232 times more potent than the standard drug Cidofovir (EC50: 11.59 µM) in primary assay whereas it demonstrated moderate activity (EC50: >30.00 µM) in secondary plaque reduction assay. The thiophene analog (9) has shown very good viral inhibition towards several viruses such as Human papillomavirus, Measles virus, Rift Valley fever virus, Tacaribe virus and Herpes simplex virus 1. CONCLUSION: Our research identified a novel 5,6-dimethoxyindan-1-one analog (compound 7), as a potent antiviral agent for vaccinia virus, and heterocyclic chalcone analog (compound 9) as a broad spectrum antiviral agent.