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[This corrects the article DOI: 10.3389/fonc.2018.00592.].
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Cytokines enhance tumour cell recognition via cytotoxic effector cells and are therefore effectively used in cancer immunotherapy. Mesenchymal stem cells have efficient homing potential and have been used to target and inhibit various types of cancer mediated by the release of soluble/bioactive factors. Initial evaluation of the human Wharton's jelly stem cell conditioned medium (hWJSC-CM) and cell lysate (hWJSC-CL) against an ovarian cancer cell line (OVCAR3) demonstrated their inhibitory effect in vitro. The secreted cytokine profile was then studied to understand whether the OVCAR3 inhibitory effect was mediated by the cytokines. Expression of cytokines in OVCAR3 following 48 h treatment with hWJSC extracts, namely the hWJSC-CM (50%) and hWJSC-CL (10 µg/ml), was evaluated using multiplex cytokine assay. Paclitaxel (5 nM) was used as a positive control. Cytokines tumour necrosis factor α, interleukin (IL)-4, IL-6, IL-8, IL-10, IL-13, IL-17, IL-1ß and granulocyte colony-stimulating factor, reported to be involved in tumour growth, invasion and migration, were significantly decreased. Cytokines with antitumour effects, namely IL-1 receptor antagonist (IL-1RA), IL-2, IL-2 receptor, IL-5, IL-7, IL-12, IL-15, interferon (IFN)-α and IFN-γ, were mildly increased or decreased. Only the increases in IL-1RA (with paclitaxel, hWJSC-CM and hWJSC-CL) and granulocyte-macrophage colony-stimulating factor (with hWJSC-CL) were statistically significant. The chemokines monocyte chemoattractant protein 1, macrophage inflammatory protein (MIP)-1α, MIP-1ß and Regulated Upon Activation, Normally T-Expressed, and Secreted were significantly decreased while monokine induced by IFN-γ, IFN-γ induced protein 10 and Eotaxin demonstrated mild decreases. The growth factors basic fibroblast growth factor, vascular endothelial growth factor and hepatocyte growth factor were significantly decreased. Heatmaps demonstrated differential fold changes in cytokines and hierarchical cluster analysis revealed 3 major and 7 minor sub-clusters of associated cytokines, chemokines and growth factors. In conclusion, the hWJSC extracts decreased the expression of oncogenic cytokines, chemokines and growth factors, which mediated the inhibition of OVCAR3 cells in vitro.
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Ovarian cancer is a highly lethal and the second highest in mortality among gynecological cancers. Stem cells either naïve or engineered are reported to inhibit various human cancers in both in-vitro and in-vivo. Herein we report the cancer inhibitory properties of human Wharton's jelly stem cell (hWJSC) extracts, namely its conditioned medium (hWJSC-CM) and cell lysate (hWJSC-CL) against two ovarian cancer cell lines (OVCAR3 and SKOV3) in-vitro. Cell metabolic activity assay of OVCAR3 and SKOV3 cells treated with hWJSC-CM (12.5, 25, 50, 75, 100%) and hWJSC-CL (5, 10, 15, 30, and 50 µg/ml) demonstrated concentration dependent inhibition at 24-72 h. Morphological analysis of OVCAR3 and SKOV3 cells treated with hWJSC-CM (50, 75, 100%) and hWJSC-CL (15, 30, and 50 µg/ml) for 24-72 h showed cell shrinkage, membrane damage/blebbings and cell death. Cell cycle assay demonstrated an increase in the sub-G1 and G2M phases of cell cycle following treatment with hWJSC-CM (50, 75, 100%) and hWJSC-CL (10, 15, and 30 µg/ml) at 48 h. Both OVCAR3 and SKOV3 cells demonstrated mild positive expression of activated caspase 3 following treatment with hWJSC-CM (50%) and hWJSC-CL (15 µg/ml) for 24 h. Cell migration of OVCAR3 and SKOV3 cells were inhibited following treatment with hWJSC-CM (50%) and hWJSC-CL (15 µg/ml) for 48 h. Tumor spheres (TS) of OVCAR3 and SKOV3 treated with hWJSC-CM (50, 75, 100%) and hWJSC-CL (10, 15, 30 µg/ml) for 48 h showed altered surface changes including vacuolations and reduction in size of TS. TS of OVCAR3 and SKOV3 also showed the presence of few ovarian cancer stem cells (CSCs) in minimal numbers following treatment with hWJSC-CM (50%) or hWJSC-CL (15 µg/ml) for 48 h. Real-time gene expression analysis of OVCAR3 and SKOV3 treated with hWJSC-CM (50%) or hWJSC-CL (15 µg/ml) for 48 h demonstrated decreased expression of cell cycle regulatory genes (cyclin A2, Cyclin E1), prostaglandin receptor signaling genes (EP2, EP4) and the pro-inflmmatory genes (IL-6, TNF-α) compared to untreated controls. The results indicate that hWJSC-CM and hWJSC-CL inhibit ovarian cancer cells at mild to moderate levels by inducing cellular changes, cell cycle arrest, apoptosis, decreasing the expression of CSC markers and related genes regulation. Therefore, the stem cell factors in hWJSCs extracts can be useful in cancer management.
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Mesenchymal stem cells (MSCs) from various sources have been used in cartilage differentiation with variable success. Therefore, it is of interest to evaluate the in vitro differentiation potential of the hWJSCs derived from the human umbilical cords into chondrocytes at the stem cell research facility at the King Abdulaziz University. hWJSCs are an attractive choice for tissue engineering and regenerative medical applications including cartilage regeneration. We evaluated the hWJSCs using classical histological and cartilage related gene expression studies. Some of the known parameters were re-examined for consistency at the current laboratory conditions. Early passages (P1-P4) showed short fibroblastic morphology and high expression of MSC related surface markers namely CD29 (99.9%), CD44 (97.8%), CD73 (99.6%), CD90 (95.1%) and CD105 (98.9%). MTT assay showed time dependent increase in hWJSCs proliferation by 61.06% and 206.31% at 48h and 72h respectively. Toluidine blue histology showed that hWJSCs were successfully differentiated into chondrocytes in chondrocytic differentiation medium for 21 days. Differentiated hWJSCs also showed significantly increased expression of collagen type II, aggrecan and SOX9 compared to the undifferentiated control. It should be noted that the determination of the average cell yield, the population doubling time and histological staining wtih alcian blue and/or safronin O is required in future studies for improved evaluation of differentiation. Painless derivation, abundance of stem cells that are hypo-immunogenic and safety issues makes this method advantages to MSCs derived from other sources.
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Ovarian cancer is one of the most lethal gynaecological cancers. Its subtle onset and absence of symptoms in early stages are associated with poor prognosis and high mortality. Identification of early biomarkers would aid in ovarian cancer control. Mesenchmal stem cells (MSCs) and/or their secretory products are identified to have cancer inhibitory properties. Therefore, it is of interest to study the anticancer properties of human Wharton's jelly stem cells conditioned medium (hWJSCs-CM) on primary ovarian carcinoma cells in vitro. Primary cultures of epithelial ovarian carcinoma cells (EOCs) and hWJSCs were used in this study. EOCs were exposed to hWJSC-CM (100%) for 24h-72h and changes in mophology and cell proliferation were monitored. Treatment with hWJSC-CM showed altered morphological changes that resulted in death of EOCs. Colorimetric assay [MTT, (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide)] showed mean decreases in EOC proliferation by 16.21%, 23.89% and 40.08% at 24h, 48h and 72h respectively compared to control. Ingenuity Pathway Analysis (IPA, Igenuity Systems, USA) deduced important molecular pathways and signaling networks associated with cancer cell death and these correlated with significant expression of tumour suppresors and apoptotic genes in hWJSCs. Secretory products of hWJSC-CM induced cell death of EOCs via apoptosis. IPA identification of canonical genes/pathways involved in EOCs that overlap with hWJSCs tumour suppressors and apoptosis genes further support this hypotheis. Additional in vitro and in vivo studies are necessary to validate EOCs inhibition with hWJSC-extracts towards their mechanism of action.
