Cytotoxic effects of Phenformin on ovarian cancer cells: expression of HIF-1α and PDK1 in the hypoxic microenvironment.

Today, many anticancer drugs are used clinically for ovarian cancer, one of the leading causes of cancer-related deaths in women. Phenformin is an antidiabetic drug of the biguanide class. It improves the antiproliferative activity in cancer cells. Hypoxia is an important component associated with ovarian cancer and its tumor microenvironment. The aim of this study was to investigate the anticancer effects of Phenformin in SKOV-3 human ovarian cancer cells under hypoxic conditions. SKOV-3 human ovarian cancer cells treated with different doses of Phenformin (0.5 mM, 1 mM, 2 mM, 5 mM) for 24 hours were subjected to WST-1 cell viability assay and Annexin V apoptosis assay. A dose-dependent decrease in cell viability with Phenformin treatment was observed. In addition, Phenformin activated percentage of apoptotic SKOV-3 cancer cells in a dose-dependent manner. In this study, Cobalt(II) chloride (CoCl2) treatment leads to increased hypoxia-inducible factor-1alpha (HIF-1α) expression and Phenformin can recover hypoxic condition. HIF-1α protein expression was significantly correlated with cell viability assay and apoptosis assay. We also found that Phenformin inhibits expression of phosphoinositide-dependent kinase 1 (PDK1) in SKOV-3 ovarian cancer cells. The ability to migrate to cancer cells was significantly reduced in a dose-dependent manner with Phenformin. This data demonstrates that Phenformin treatment can induce apoptosis and inhibit proliferation in ovarian cancer cells under hypoxic conditions. The findings reveal that HIF-1α is a new target for the treatment of ovarian cancer.

Comparison of innate lymphoid cells from tissue and blood in chronic tonsillitis and tonsillar hypertrophy.

OBJECT: Recurrent tonsillitis and tonsillar hypertrophy are two common diseases in children for which tonsillectomy is the definitive solution. The underlying causes of both diseases are not fully known. The aim of this study was to identify the predominant innate lymphoid cells in both diseases of the palatine tonsils, which are known to play an important role in the immune system. METHODS: Children who underwent tonsillectomy were divided into two groups as recurrent tonsillitis and tonsillar hypertrophy according to the indication for surgery. The proportions of innate lymphoid cell (ILC) groups and IFN-gamma, IL-10 and IL-17 secreting T lymphocyte cells were determined in tonsil and blood samples obtained during surgery. Local and peripheral immune responses were evaluated. Innate immune responses and acquired immune responses were compared. RESULTS: The results of our study showed that the proportions of the innate lymphoid cell 1 group (ILC1) were similar in tonsil tissue in patients with recurrent tonsillitis and tonsil hypertrophy, with no statistically significant difference. It was observed that the innate lymphoid cell 2 group (ILC2) was the predominant group in tonsil hypertrophy, the innate lymphoid cell 3 group (ILC3) was the predominant innate lymphoid cell group in recurrent tonsillitis, and the proportion of IL-17 secreting T lymphocytes in blood and tonsillar mononuclear cells was higher in recurrent tonsillitis patients than in tonsil hypertrophy patients. CONCLUSION: With the results obtained, the predominant innate lymphoid cells in the pathogenesis of both diseases were identified and local and peripheral responses were compared. These findings may be a guide for possible medical treatments for both diseases in the future.

Synovial fluid niche promoted differentiation of dental follicle mesenchymal stem cells toward chondrogenesis in rheumatoid arthritis.

Objectives: In this study, we aimed to investigate the differentiation potential of dental follicle mesenchymal stem cells (MSCs) in the synovial fluid (SF) niche of early-onset or end-stage rheumatoid arthritis (RA). Patients and methods: Between May 2020 and January 2021, six patients (1 male, 5 females; mean age: 57.5±11.2 years; range, 49 to 65 years) who were diagnosed with RA with the indication of SF aspiration were included in the study. The third passage dental follicle stem cells (DFSCs) were cocultured with fresh SF samples of end-stage or early-onset RA patients in micromass culture system for 21 days. SF samples were analyzed for secreted cytokines. Chondrogenic markers (CD49e, CD49f) were analyzed in DFSCs, gene expression analysis was performed for the expressions of Col I, Col II, Aggrecan and Sox-9, and histochemical analysis was performed by staining three-dimensional pellets with anti-collagen II antibody. The neutralization assay was performed with anti-interleukin (IL)-6, anti-interferon-gamma (IFN-g), and anti-IL-1beta(b). Results: The high levels of IL-1b and IL-6 were observed in end-stage RA patients' SF samples compared to the early-onset patients (p<0.05). The CD49e and CD49f expressions in DFSCs were significantly higher in the SF samples of end-stage RA patients (p<0.05). Also, the Col II, Sox-9 and Aggrecan messenger ribonucleic acid (mRNA) expressions increased in the DFSCs, when cultured with end-stage RA patients' SF samples (p<0.01). Collagen-II expression in histochemical analysis of micromass pellets was higher in the DFSCs cultured with end-stage RA patients' SF samples. The neutralization of IL-6 significantly decreased the CD49e and CD49f expressions (p<0.05). Conclusion: The high levels of IL-6 in SF niche of end-stage RA patients were found to differentiate DFSCs toward chondrogenesis. Based on these findings, DFSCs can be used as a new cell-based treatment in RA patients for the cartilage damage.

Dental follicle mesenchymal stem cells ameliorated glandular dysfunction in Sjögren's syndrome murine model.

OBJECTIVE: Dental mesenchymal stem cells (MSCs) are potential for use in tissue regeneration in inflammatory diseases due to their rapid proliferating, multilineage differentiation, and strong anti-inflammatory features. In the present study, immunoregulatory and glandular tissue regeneration effects of the dental follicle (DF)MSCs in Sjögren's Syndrome (SS) were investigated. METHODS: Dental follicle (DF) tissues were obtained from healthy individuals during tooth extraction, tissues were digested enzymatically and DFMSCs were cultured until the third passage. DFMSCs were labeled with Quantum dot 655 for cell tracking analysis. The induction of the SS mouse model was performed by the injection of Ro60-273-289 peptide intraperitoneally. DFMSCs were injected intraperitoneally, or into submandibular, or lacrimal glands. Splenocytes were analyzed for intracellular cytokine (IFN-γ, IL-17, IL-10) secretion in T helper cells, lymphocyte proliferation, and B lymphocyte subsets. Histologic analysis was done for submandibular and lacrimal glands with hematoxylin-eosin staining for morphologic examination. RESULTS: The systemic injection of DFMSCs significantly reduced intracellular IFN-γ and IL-17 secreting CD4+ T cells in splenocytes (p<0.05), and decreased inflammatory cell deposits and fibrosis in the glandular tissues. DFMSCs differentiated to glandular epithelial cells in submandibular and lacrimal injections with a significant reduction in lymphocytic foci. The results showed that few amounts of DFMSCs were deposited in glandular tissues when applied intraperitoneally, while high amounts of DFMSCs were located in glandular tissues and differentiated to glandular epithelial cells when applied locally in SS murine model. CONCLUSION: DFMSCs have the potential for the regulation of Th1, Th17, and Treg balance in SS, and ameliorate glandular dysfunction. DFMSCs can be a beneficial therapeutic application for SS.

Immunoregulatory effects of dental mesenchymal stem cells on T and B lymphocyte responses in primary Sjögren's syndrome.

Background: In this article, the authors investigate the modulatory effects of dental mesenchymal stem cells (MSCs) on lymphocyte responses in primary Sjögren's syndrome (pSS), which is an autoimmune disease resulting from keratoconjunctivitis sicca and xerostomia. Methods: Mononuclear cells isolated from pSS patients cultured with or without dental MSCs and analyzed for lymphocyte responses via flow cytometry. Results: Dental-follicle (DF)- and dental-pulp (DP)-MSCs downregulated CD4+ T lymphocyte proliferation by increasing Fas-ligand expression on T lymphocytes and FoxP3 expressing Tregs, and decreasing intracellular IFN-γ and IL-17 secretion in pSS patients. DF-MSCs decreased the plasma B cell ratio in the favor of naive B cell population in pSS patients' mononuclear cells. Conclusion: DF- and DP-MSCs can be the new cellular therapeutic candidates for the regulation of immune responses in pSS.

Plain language summary In this article, the authors investigate the modulatory effects of dental mesenchymal stem cells (dental MSCs) on lymphocyte responses in primary Sjögren's syndrome (pSS), which is characterized by the infiltration of lymphocytes in exocrine glands. Lymphocyte proliferation, apoptosis, Tregs and total Bregs, intracellular cytokine secretion, total memory, plasma and naive B cell subsets were analyzed in pSS patients and compared them with healthy individuals. Dental follicle- and dental pulp-MSCs modulated CD4⁺ T lymphocyte responses in pSS patients' mononuclear cells by increasing Fas-ligand expression, enhancing FoxP3-expressing Tregs, and decreasing pro-inflammatory cytokine secretion. Our findings provide evidence for the potential role of dental-MSCs as a cellular therapy option in the treatment of pSS.

1,25-dihydroxyvitamin D3 regulates t helper and b lymphocyte responses substantially in drug-naive primary Sjögren's syndrome patients' mononuclear cells

Background/aim: A correlation between vitamin D deficiency and primary Sjögren's syndrome (pSS) has already been described. The limited data has been reported regarding the pathological relevance of vitamin D in primary Sjögren's syndrome. In this study, the peripheral blood mononuclear cells were cocultured with 1,25-dihydroxyvitamin D3 to determine the modulatory effect of vitamin D3 on T and B lymphocyte phenotypes in pSS. Materials and methods: Venous blood samples were collected from 11 patients in the treatment phase and 9 drug-naive pSS patients. Peripheral blood mononuclear cells (PBMC) were isolated and separately cultured in the presence and absence of 1,25-dihydroxyvitamin D3 (10 mM) for 5 days of culture period. Lymphocyte proliferation was analyzed for CFSE signaling via flow cytometry. CD3+CD4+ cells were analyzed for intracellular IFN- and IL-17 expressions. CD19+IgD cells were analyzed for CD38 and CD27 expressions to evaluate naive and total memory B cell subsets. Culture supernatants were analyzed for the IFN-, IL-17, and IL-10 cytokine secretions via flow cytometry. Results: 1,25-dihydroxyvitamin D3 significantly decreased Th lymphocyte proliferative responses in drug-naive (p < 0.005) and treated pSS patients (p < 0.05), and B lymphocyte proliferation in drug-naive pSS PBMC cultures (p < 0.01) compared to mononuclear cell cultures alone. 1,25-dihydroxyvitamin D3 significantly decreased IFN- and IL-17 secreting Th cells in both drug naive (p < 0.005 and p < 0.01, respectively) and treated subjects (p < 0.05 and p < 0.05, respectively) by increasing FoxP3 expressing CD4+CD25+ Treg cell frequency. Plasma B lymphocytes significantly reduced in the presence of 1,25-dihydroxyvitamin D3 in drug naive pSS (p < 0.001) and treated patients (p < 0.05) mononuclear cell cultures compared to PBMC cultures alone. Total memory B cell subsets significantly increased with 1,25-dihydroxyvitamin D3 in drug naive pSS when compared with PBMC cultures alone (p < 0.005). IFN- and IL-17 cytokine levels in culture supernatants significantly reduced (p < 0.05 and p < 0.01, respectively) in drug naive pSS patients' PBMC cultures with 1,25-dihydroxyvitamin D3, and IL-10 levels significantly enhanced in both drug-naive (p < 0.01) and treated pSS patients' PBMC cultures (p < 0.01) in the presence of 1,25-dihydroxyvitamin D3. Conclusion: In conclusion, 1,25-dihydroxyvitamin D3 regulated immune responses in both treated and drug-naive pSS patients, but have a more pronounced modulatory effect on mononuclear cell responses in drug-naive pSS patients.