Osteogenic and Adipogenic Differentiation Potential of Oral Cancer Stem Cells May Offer New Treatment Modalities.

(1) Treatment failure of oral squamous cell carcinoma (OSCC) is generally due to the development of therapeutic resistance caused by the existence of cancer stem cells (CSCs), a small cell subpopulation with marked self-renewal and differentiation capacity. Micro RNAs, notably miRNA-21, appear to play an important role in OSCC carcinogenesis. Our objectives were to explore the multipotency of oral CSCs by estimating their differentiation capacity and assessing the effects of differentiation on stemness, apoptosis, and several miRNAs' expression. (2) A commercially available OSCC cell line (SCC25) and five primary OSCC cultures generated from tumor tissues obtained from five OSCC patients were used in the experiments. Cells harboring CD44, a CSC marker, were magnetically separated from the heterogeneous tumor cell populations. The CD44+ cells were then subjected to osteogenic and adipogenic induction, and the specific staining was used for differentiation confirmation. The kinetics of the differentiation process was evaluated by qPCR analysis of osteogenic (Bone Morphogenetic Protein-BMP4, Runt-related Transcription Factor 2-RUNX2, Alkaline Phosphatase-ALP) and adipogenic (Fibroblast Activation Protein Alpha-FAP, LIPIN, Peroxisome Proliferator-activated Receptor Gamma-PPARG) markers on days 0, 7, 14, and 21. Embryonic markers (Octamer-binding Transcription Factor 4-OCT4, Sex Determining Region Y Box 2-SOX2, and NANOG) and micro RNAs (miRNA-21, miRNA-133, and miRNA-491) were also correspondingly evaluated by qPCR. An Annexin V assay was used to assess the potential cytotoxic effects of the differentiation process. (3) Following differentiation, the levels of markers for the osteo/adipo lineages showed a gradual increase from day 0 to day 21 in the CD44+ cultures, while stemness markers and cell viability decreased. The oncogenic miRNA-21 also followed the same pattern of gradual decrease along the differentiation process, while tumor suppressor miRNA-133 and miRNA-491 levels increased. (4) Following induction, the CSCs acquired the characteristics of the differentiated cells. This was accompanied by loss of stemness properties, a decrease of the oncogenic and concomitant, and an increase of tumor suppressor micro RNAs.

Acetylsalicylic-acid (ASA) regulation of osteo/odontogenic differentiation and proliferation of human dental pulp stem cells (DPSCs) in vitro.

OBJECTIVE: The study aimed to investigate acetylsalicylic acid (ASA) effects on osteo/odontogenic differentiation and proliferation of dental pulp stem cells (DPSCs) in vitro and the potential involvement of adenosine monophosphate-activated protein kinase (AMPK) pathway in these processes. DESIGN: DPSCs were isolated from third molars pulp tissues of five patients and grown in osteogenic medium alone or supplemented with ASA. Expression of DPSCs markers was tested by flow-cytometry. Cytotoxicity of ASA at concentrations of 10, 50 and 100 µg/ml was tested by MTT and NR assays. Osteo/odontogenic differentiation was analyzed via alizarin red staining and ALP activity. Quantitative PCR (qPCR) was used for osteo/odontogenic markers' (DSPP, BMP2, BMP4, BSP, OCN and RUNX2) and c-Myc expression analysis. AMPK inhibition of ASA-induced osteo/odontogenesis was tested by qPCR of selected markers (DSPP, OCN and RUNX2). RESULTS: Cytotoxicity assays showed that only the highest ASA dose decreased cell viability (89.1 %). The smallest concentration of ASA applied on DPSCs resulted in a remarkable enhancement of osteo/odontogenic differentiation, as judged by increased mineralized nodules' formation, ALP activity and gene expression of analyzed markers (increase between 2 and 30 folds), compared to untreated cells. ASA also increased DPSCs proliferation. Interestingly, AMPK inhibition per se upregulated DSPP, OCN and RUNX2; the gene upregulation was higher when ASA treatment was also included. c-Myc expression level decreased in cultures treated with ASA, indicating undergoing differentiation processes. CONCLUSIONS: Low concentrations of ASA (corresponding to the standard use in cardiovascular patients), were shown to stimulate osteo/odontogenic differentiation of dental pulp stem cells.