Bakiye Goker Bagca

Type 2 Diabetes Inhibited Human Mesenchymal Stem Cells Angiogenic Response by Over-Activity of the Autophagic Pathway

The current study aimed to address the impact of serum from type 2 diabetes patients on the angiogenic properties of human bone marrow mesenchymal stem cells and its relationship to autophagy signaling. Human primary stem cells were enriched and incubated with serum from diabetic and normal subjects for 7 days. Compared to data from the control group, diabetic serum was found to induce a higher cellular death rate (P < 0.001) and apoptotic changes (P < 0.01). We also showed that diabetic condition significantly abolished angiogenesis tube formation on Matrigel substrate, decreased cell chemotaxis (P < 0.01) in response to SDF‐1α, and inhibited endothelial differentiation rate (P < 0.0001). Western blotting showed autophagic status by high levels of P62 (P < 0.0001), beclin‐1 (P < 0.0001), and increase in LC3II/I ratio (P < 0.001). In vivo Matrigel plug assay revealed that supernatant conditioned media prepared from cells exposed to diabetic serum caused a marked reduction in the recruitment of VE‐cadherin‐ (P < 0.01) and α‐SMA‐positive (P < 0.0001) cells 7 days after subcutaneous injection. PCR expression array analysis confirmed the overexpression of autophagy and apoptosis genes in cultured cells in response to a diabetic condition (P < 0.05). Using bioinformatic analysis, we noted a crosstalk network between DM2, angiogenesis, and autophagy signaling. DM2 could potently modulate angiogenesis by the interaction of IL‐1β with downstream insulin receptor and upstream androgen receptor. Corroborating to data, diabetic serum led to abnormal regulation of P62 during the angiogenic response. These data demonstrate that diabetic serum decreased human mesenchymal stem cell angiogenic properties directly on angiogenesis pathways or by the induction of autophagy signaling. J. Cell. Biochem. 118: 1518–1530, 2017.

Functional convergence of Akt protein with VEGFR-1 in human endothelial progenitor cells exposed to sera from patient with type 2 diabetes mellitus

Diabetes mellitus type 2 predisposes patients to various microvascular complications. In the current experiment, the potent role of diabetes mellitus was investigated on the content of VEGFR-1, -2, Tie-1 and -2, and Akt in human endothelial progenitor cells. The gene expression profile of mTOR and Hedgehog signaling pathways were measured by PCR array. The possible crosstalk between RTKs, mTOR and Hedgehog signaling was also studied by bioinformatic analysis. Endothelial progenitor cells were incubated with serum from normal and diabetic for 7days. Compared to non-treated cells, diabetic serum-induced cell apoptosis (~2-fold) and prohibited cell migration toward bFGF (p<0.001). ELISA analysis showed that diabetes exposed cells had increased abundance of Tie-1, -2 and VEGFR-2 and reduced amount of VEGFR-1 (p<0.0001) in diabetic cells. Western blotting showed a marked reduction in the protein level of Akt after cells exposure to serum from diabetic subjects (p<0.0001). PCR array revealed a significant stimulation of both mTOR and Hedgehog signaling pathways in diabetic cells (p<0.05). According to data from bioinformatic datasets, we showed VEGFR-1, -2 and Tie-2, but not Tie-1, are master regulators of angiogenesis. There is a crosstalk between RTKs and mTOR signaling by involving P62, GABARAPL1, and HTT genes. It seems that physical interaction and co-expression of Akt decreased the level of VEGFR-1 in diabetic cells. Regarding data from the present experiment, diabetic serum contributed to uncontrolled induction of both mTOR and Hedgehog signaling in endothelial progenitor cells. Diabetes mellitus induces mTOR pathway by involving receptor tyrosine kinases while Hedgehog stimulation is independent of these receptors.

Evaluation of the miRNA profiling and effectiveness of the propolis on B-cell acute lymphoblastic leukemia cell line

Acute lymphoblastic leukemia (ALL) is one of the most frequent causes of death from cancer. Since the discovery of chemotherapeutic agents, ALL has become a model for improvement of survival. In parallel to this, serious side effects were observed and new natural therapeutic options has been discussed. One of these substances is called propolis which is a resinous substance gathered by honeybees. In the molecular era, miRNAs have been shown to play crucial roles in the development of many clinical conditions. The aim of this study is to evaluate the effect of Aydın propolis on 81 human miRNA activity in CCRF-SB leukemia cell line. Apoptotic effects of propolis on cell lines were also evaluated and apoptosis were found to be induced 1.5 fold in B-cell leukemia cells. The expression of 63 miRNAs (46 miRNAs were downregulated, 19 miRNAs were upregulated) in propolis treated leukemia cells have changed significantly (p<0.05). In conclusion propolis has changed expression of miRNAs which have epigenetic effects on leukemic cells. It is thought that it can be a promising agent for ALL treatment for future studies.

Alginate-gelatin encapsulation of human endothelial cells promoted angiogenesis in in vivo and in vitro milieu†

Up to present, many advantages have been achieved in the field of cell‐based therapies by applying sophisticated methodologies and delivery approaches. Microcapsules are capable to provide safe microenvironment for cells during transplantation in a simulated physiological 3D milieu. Here, we aimed to investigate the effect of alginate‐gelatin encapsulation on angiogenic behavior of human endothelial cells over a period of 5 days. Human umbilical vein endothelial cells were encapsulated by alginate‐gelatin substrate and incubated for 5 days. MTT and autophagy PCR array analysis were used to monitor cell survival rate. For in vitro angiogenesis analysis, cell distribution of Tie‐1, Tie‐2, VEGFR‐1, and VEGFR‐2 were detected by ELISA. In addition to in vitro tubulogenesis assay, we monitored the expression of VE‐cadherin by Western blotting. The migration capacity of encapsulated HUVECs was studied by measuring MMP‐2 and MMP‐9 via gelatin zymography. The in vivo angiogenic potential of encapsulated HUVECs was analyzed in immune‐compromised mouse implant model during 7 days post‐transplantation. We demonstrated that encapsulation promoted HUVECs cell survival and proliferation. Compared to control, no significant differences were observed in autophagic status of encapsulated cells (p > 0.05). The level of Tie‐1, Tie‐2, VEGFR‐1, and VEGFR‐2 were increased, but did not reach to significant levels. Encapsulation decreased MMP‐2, ‐9 activity and increased the VE‐cadherin level in enclosed cells (p < 0.05). Moreover, an enhanced in vivo angiogenic response of encapsulated HUVECs was evident as compared to non‐capsulated cells (p < 0.05). These observations suggest that alginate‐gelatin encapsulation can induce angiogenic response in in vivo and in vitro conditions.

Cancer Stem Cells and Autophagy: Present Knowledge and Future Perspectives

Macroautophagy, commonly referred to as autophagy, is a recycling process involving lysosomal degradation of the cell components such as proteins and organelles. This process prevents damage to the cell through the degradation of nonfunctional cellular components and provides raw material and energy which are required to realize biosynthesis reactions. Since autophagy has evolutionarily conserved complex molecular mechanisms, the relationship between autophagy and cancer is multifaceted. There are some insights in which autophagy, also referred to as type 2 cell death, has been suggested as an alternative approach to kill cancer cells have defected apoptosis mechanism. On the other hand, it has also been shown in recent studies that autophagy mechanism, especially in cancer stem cells, may be responsible for obtaining epithelial–mesenchymal transition, invasion, metastasis, drug resistance and recurrence. This chapter focuses on the role of autophagy mechanisms on cancer stem cells and its place in future treatment approaches.

Docosahexaenoic acid attenuates the detrimental effect of palmitic acid on human endothelial cells by modulating genes from the atherosclerosis signaling pathway: NOVINBAHADOR et al.

The formation of atherosclerotic changes leads to dysfunction in numerous cell types, especially endothelial cells. In the current experiment, we aimed to show the therapeutic effect of Docosahexaenoic acid on palmitic‐induced atherosclerotic changes in the human endothelial lineage. Human Umbilical Vein Endothelial cells were incubated with 1 mM palmitic acid for 48 hours and then exposed to 40 µM docosahexaenoic acid for next 24 hours. Cellular atherosclerosis and lipid removal were confirmed by the application of Oil red O solution. The cell survival rate was studied by using MTT assay and flow cytometry analysis of Annexin V. We also measured the protein level of tumor necrosis factor‐α and granulocyte‐macrophage colony‐stimulating factor by immunofluorescence imaging. The transcription level of genes participating in the atherosclerosis signaling pathway was monitored in atherosclerotic endothelial cells before and after treatment with docosahexaenoic acid. The viability of the cells was reduced after 48 hours incubation with palmitic acid. It is noteworthy that the number of viable endothelial cells was increased after exposure to docosahexaenoic acid. Compared with the cells that received palmitic acid, Oil red O staining showed a decrease in the cellular content of fatty acid after incubation with docosahexaenoic acid (P < 0.05). PCR array indicated that the modulation of key genes played a role in atherosclerosis and reached near‐control levels. These data support the notion that incubation of atherosclerotic human endothelial cells with docosahexaenoic acid could return the detrimental effects of palmitic acid by modulation of the atherosclerosis signaling pathway.

Effects of telomerase inhibitor on epigenetic chromatin modification enzymes in malignancies: Biray Avci et al.

Telomerase has a critical role in cell proliferation, tumor maintaining, and therapy resistance, which act by modifying many signaling pathways. 2‐[(E)‐3‐Naphtalen‐2‐yl‐but‐2‐enoylamino]‐benzoic acid (BIBR1532) is one of the most studied telomerase inhibitors, and it targets telomerase components TERC and TERT. In this novel study, we aimed to investigate the epigenetic effects of BIBR1532 on both hematologic malignancies and solid tumors. K‐562 human chronic myeloid leukemia cell line and U87MG glioblastoma cell line were compared with control groups without BIBR1532 treatment. Cytotoxic effects of BIBR1532 were determined by using WST‐1 assay. Apoptotic effects of BIBR1532 were detected by using annexin V method. To assess expression changes in the human epigenetic chromatin modification enzyme genes, total RNA was isolated from K‐562 and U87MG cells treated with BIBR1532 and untreated control cells. BIBR1532 induced 2.41‐fold apoptotic cell death in U87MG cell lines compared with control groups. Apoptosis was slightly induced in K‐562 cells with BIBR1532 treatment compared with control cells. We observed that BIBR1532 also regulates similar genes in both cell lines, and it is useful on epigenetic mechanisms. As a result, telomerase inhibitor BIBR1532 has a significant effect on both hematological malignancies and solid tumors.

Investigation of the effect of telomerase inhibitor BIBR1532 on breast cancer and breast cancer stem cells: DOĞAN et al.

It is emphasized that cancer stem cells (CSCs) forming the subpopulation of tumour cells are responsible for tumour growth, metastasis, and cancer drug resistance. Inadequate response to conventional therapy in breast cancer leads researchers to find new treatment methods and literature surveys that support CSC studies. A selective anticancer agent BIBR1532 inhibits the telomerase enzyme. Many of the chemotherapeutic drugs used in clinical trials have harmful effects, but the advantage of telomerase‐based inhibitors is that they are less toxic to healthy tissues. The phosphoinositide 3‐kinase (PI3K)/serine/threonine kinase (Akt)/mammalian target of rapamycin (mTOR) pathway is common in breast cancer, and the interaction between the mTOR pathway and human telomerase reverse transcriptase (hTERT) is essential for the survival of cancer cells. In our study, we treated MCF‐7, breast cancer stem cell (BCSC) and normal breast epithelial cell MCF10A with the BIBR1532 inhibitor. The IC 50 doses for the 48th hour of BIBR1532 treatment were detected as 34.59 μM in MCF‐7, 29.91 μM in BCSCs, and 29.07 μM in MCF10A. It has been observed that this agent induces apoptosis in the BCSC and MCF‐7 cell lines. According to the results of cell cycle analysis, G 2/M phase accumulation was observed in BCSC and MCF‐7 cell lines. It has also been shown that BIBR1532 suppresses telomerase activity in BCSC and MCF‐7. The effect of BIBR1532 on the mTOR signalling pathway has been investigated for the first time in this study. It is thought that the telomerase inhibitor may bring a new approach to the treatment and it may be useful in the treatment of CSCs.

Heat shock protein 70 modulates neural progenitor cells dynamics in human neuroblastoma SH-SY5Y cells exposed to high glucose content

In the current experiment, detrimental effects of high glucose condition were investigated on human neuroblastoma cells. Human neuroblastoma cell line SH‐SY5Y were exposed to 5, 40, and 70 mM glucose over a period of 72 h. Survival rate and the proliferation of cells were analyzed by MTT and BrdU incorporation assays. Apoptosis was studied by the assays of flow cytometry and PCR array. In order to investigate the trans‐differentiation capacity of the cell into mature neurons, we used immunofluorescence imaging to follow NeuN protein level. The transcription level of HSP70 was shown by real‐time PCR analysis. MMP‐2 and −9 activities were shown by gelatin Zymography. According to data from MTT and BrdU incorporation assay, 70 mM glucose reduced cell viability and proliferation rate as compared to control (5 mM glucose) and cells treated with 40 mM glucose (P < 0.05). Cell exposure to 70 mM glucose had potential to induced apoptosis after 72 h (P < 0.05). Our results also demonstrated the sensitivity of SH‐SY5Y cells to detrimental effects of high glucose condition during trans‐differentiation into mature neuron‐like cells. Real‐time PCR analysis confirmed the expression of HSP70 in cells under high content glucose levels, demonstrating the possible cell compensatory response to an insulting condition (pcontrol vs 70 mM group <0.05). Both MMP‐2 and ‐9 activities were reduced in cells being exposed to 70 mM glucose. High glucose condition could abrogate the dynamics of neural progenitor cells. The intracellular level of HSP70 was proportional to cell damage in high glucose condition.

Epigenetic modifications in chronic myeloid leukemia cells through ruxolitinib treatment: BIRAY AVCI et al.

Chronic myeloid leukemia is a clonal malignancy of hematopoietic stem cell that is characterized by the occurrence of t(9;22)(q34;q11.2) translocation, named Philadelphia chromosome. Ruxolitinib is a powerful Janus tyrosine kinase 1 and 2 inhibitor that is used for myelofibrosis treatment. DNA‐histone connection mediates a wide range of genes that code methylation, demethylation, acetylation, deacetylation, ubiquitination, and phosphorylation enzymes. Epigenetic modifications regulate chromatin compactness, which plays pivotal roles in critical biological processes including the transcriptional activity and cell proliferation as well as various pathological mechanisms, including CML. This study is aimed to determine the alterations of the expression levels of epigenetic modification–related genes after ruxolitinib treatment. Total RNA was isolated from K‐562 cells treated with the IC50 value of ruxolitinib and untreated K‐562 control cells. A reverse transcription procedure was performed for complementary DNA synthesis, and gene expressions were detected by real‐time polymerase chain reaction compared with the untreated cells. Ruxolitinib treatment caused a significant alteration in the expression levels of epigenetic regulation‐related genes in K‐562 cells. Our novel results suggested that ruxolitinib has inhibitor effects on epigenetic modification–regulator genes.