Dynamic Interaction Between Organellas In the Management of Cytosolic Calcium Huvecs Exposed to 22 mM Glucose With Different Period Exposure

Background.In our previous research, when cell culture were exposed to high glucose, this will cause the increase of H2O2. At the exposure to 22 mM glucose on 3rd day, the increase of H2O2 that induced the activation of Phospholipase C (PLC) have caused 1P3 (Inositol tri-phosphate) mobilizing the release of Ca²+ from the depo Endoplasmic reticulum (ER). Thus, causing the increase of cytosolic Ca²+. Giving thapsigargin (TG) will cause significant increase in Cytosolic Ca²+ so that the most contribution to the increasing of Cytosolic Ca²+ derives from the ER . On the 7th day exposure, H2O2 played the same role as TG, causing direct incease in Cytosolic Ca²+ and an addition of Ca²+ free/buffer ethyleneglyco bis (ßaminoethyl ether).&NNN’N’– tetraacetic acid (EGTA) caused significant decrease of cytosolic Ca²+ basal and the greatest contribution to the increase of cytosolic Ca²+ on the 7th day, comes from extracellular. Administrating Cyclosporin A (CSA) 10 µM on the 9th day, caused significant decreasing on cytosolic Ca²+ basal, the ability of CSA in decreasing Ca²+ basal concentration was less than the 3rd and 7th days. At a high glucose condition with different length of exposure, a change of new cytosolic Ca²+ homeostatic regulation occurred and this enable a change in the dynamic interaction among ER, extracellular and mitochondria.Method.HUVECs culture exposed to 22 mM glucose for 3, 7 and 9 days. The cells were incubated with FURA2-AM. The evaluation of fluorescence cytosolic Ca²+ was done by epifluorescence Nikon digital camera-computerized analyser. To measure the cytosolic Ca²+ concentration we use Histogram Image Corel Draw Photo Paint 12.Result. Exposure to glucose 22mM on the 3rd day (65.4 ± 12.2) it showed the increase of cytosolic Ca²+ by giving Ca²+ free/EGTA 1 mM and CSA 10 mM caused the decrease of cytosolic Ca²+ (33.2 ± 4.47) TG1µM and CSA caused the decrease of cytosolic Ca²+ basal (53.07 ± 2.75) and Ca²+ -free/EGTA, TG and CSA (68.59 ± 5.71). On the 7th day exposure (92.74 ± 7.66) the decrease of cyto -solic Ca²+ basal occurred at the giving of Ca²+ -free/EGTA, TG (50.52 ± 9.23). EGTA and CSA (45.59 ±6.2). TG and CSA (73.55 ± 7.30), Ca²+ -free/EGTA and TG much more decrease the concentrate of cytosolic Ca²+ basal (17.58 ±4.5). On the 9th day of exposure to glucose (72.32 ±7.46), the giving of Ca²+ -free/EGTA, TG and CSA(35.76 ± 5.25) have caused the decrease of cytosolic Ca²+ basal. Conclusion.HUVECs culture exposed to 22mM glucose will cause the increase in H2O2and cytosolic Ca²+ basal. ER, mitochondria and extracellular regulate the Cytosolic Ca²+ and a dynamic interaction occurred among them to obtain a new homeostatic

Effects of Conditioned Medium of Co-Culture IL-2 Induced NK Cells and Human Whartonâs Jelly Mesenchymal Stem Cells (hWJMSCs) on Apoptotic Gene Expression in a Breast Cancer Cell Line (MCF-7)

Breast cancer (BC) is the most prevalent type of cancer among women and one of the major causes of cancer mortality in women. Metastasis in breast cancer (BC) occurs due to immunosurveillance deficiency, including impairment of natural killer (NK) cell maturation. Conditioned medium (CM) from human Wharton's jelly mesenchymal stem cells (hWJMSC-CM) is known to possess anticancer activity. The CM of co-culture of human recombinant IL-2 treated NK cells and hWJMSCs is expected to boost anticancer activity toward BC cells which can be analyzed from the effect of CM towards secretion of effector molecules and expression of BC cell apoptosis-related genes, and cytotoxic granules in human recombinant IL-2 treated NK (IL-2 NK) and hWJMSCs (IL-2 hWJMSCs). TNF-α, IFN-γ, perforin, granzyme were measured by ELISA, while the inhibition of cell proliferation was measured by MTS assay and BC cell apoptosis by flow cytometry and apoptotic gene expression by RTPCR. CM from co-cultured hWJMSCs and IL-2 NK cells inhibited NK and BC cell proliferation, increased expression of Bax and p53 and decreased the number of Bcl-2 in BC cells. In conclusion, CM of co-culture IL-2 treated NK cells and hWJMSCs induce apoptosis in BC cells as indicated by increased Bax and p53 expression and decreased Bcl-2 expression.

Direct and Indirect Effect of TNFα and IFNγ Toward Apoptosis in Breast Cancer Cells

Background: Breast cancer (BC) is the leading cause of death cancer in women. Cancer therapies using TNFα and IFNγ have been recently developed by direct effects and activation of immune responses. This study was performed to evaluate the effects of TNFα and IFNγ directly, and TNFα and IFNγ secreted by Conditioned Medium-human Wharton’s Jelly Mesenchymal Stem Cells (CM-hWJMSCs) toward apoptosis of BC cells (MCF7).Materials and Methods: BC cells were induced by TNFα and IFNγ in 175 and 350ng/mL, respectively. CM-hWJMSCs were produced by co-culture hWJMSCs and NK cells that secreted TNFα, IFNγ, perforin (Prf1), granzyme B (GzmB) for treating BC cells. The BC cells were treated with CM-hWJMSCs in 50%. The expression of apoptotic genes Bax, p53, and the antiapoptotic gene Bcl-2 were determined using RT-PCR.Results: TNFα and IFNγ at concentration of 350 ng/mL induced higher Bax expression compared to 175 ng/mL. TNFα and IFNγ 350 ng/mL, 175 ng/mL induced p53 expression, whilst TNFα and IFNγ at 350 ng/mL decreased Bcl-2 expression. Perf1, GzmB, TNFα and IFNγ-containing CM-hWJMSCs induced significantly apoptosis percentage, induced Bax expression, but did not effect p53, Bcl-2 expression.Conclusion: TNFα and IFNγ directly induce Bax, p53, decrease Bcl-2 gene expression. The Prf1, GzmB, TNFα, IFNγ-containing CM-hWJMSCs induce apoptosis and Bax expression.Keywords: breast cancer, Wharton’s Jelly mesenchymal stem cells, TNFα, IFN