Ruxolitinib enhances cytotoxic and apoptotic effects of temozolomide on glioblastoma cells by regulating WNT signaling pathway-related genes.

Although temozolomide is the primary chemotherapeutic agent in glioblastoma, current studies have focused on its combinational applications to overcome resistance by targeting multiple pathways. JAK/STAT and WNT are among the essential cancer-related signaling pathways. Ruxolitinib, the first approved JAK1/2 inhibitor, has promise in glioblastoma with its blood-brain barrier (BBB) crossing ability. The mentioned study aims to evaluate the anti-cancer potential of ruxolitinib individually and in combination with temozolomide on glioblastoma cells, brain cancer stem cells (BCSCs), and BBB-forming healthy cells. It also intends to determine the effects of JAK inhibitor treatment in combination with temozolomide on WNT signaling, which is known to cross-talk with the JAK/STAT pathway. The U87MG, BCSC, and HBMEC cell lines were the in vitro models. The cytotoxic and apoptotic effects of ruxolitinib and the combination were determined by the WST-1 test and Annexin V assay, respectively. The expression level changes of WNT signaling pathway genes caused by ruxolitinib and the combination treatments were defined by the qRT-PCR method. Network analysis of significantly upregulated and downregulated genes was performed via the GO KEGG pathway enrichment module of the String V11.5 database. The IC50 value of the ruxolitinib on U87MG glioblastoma cells was determined as 94.07 µM at 24th h. The combination of temozolomide and ruxolitinib had a synergistic effect on U87MG cells at 24th h. The combination index (CI) was determined as 0.796, and ED60 values of ruxolitinib and temozolomide were determined as 89.75 and 391.48 µM, respectively. Ruxolitinib improves the apoptotic effect of temozolomide on glioblastoma cells and brain cancer stem cells. Ruxolitinib regulates the WNT signaling pathway both individually and in combination with temozolomide. Our study indicates the potential of ruxolitinib to increase the cytotoxic and apoptotic activity of temozolomide in glioblastoma cells, also considering CSCs and healthy BBB-forming cells. As supported by gene expression and network analyses, the BBB-crossing agent ruxolitinib promises the potential to increase the efficacy of temozolomide in glioblastoma by affecting multiple signaling pathways in both cancer cells and CSCs.

Investigating the Effects of a Synthetic Cannabinoid on the Pathogenesis of Leukemia and Leukemic Stem Cells: A New Therapeutic Approach.

The popularity and usage of synthetic cannabinoids (SCs) are increasing due to their easy accessibility and psychoactive effects worldwide. Studies on cannabinoids on leukemic stem cells (LSC) and hematopoietic stem cells (HSCs), which are the precursors of leukemia cells, generally depend on the natural cannabinoid delta-9-THC. As there is only a limited number of studies focusing on the results of SC applications, the reflections upon LSCs have to be clarified. In this study, biological responses and antileukemic effects of JWH-018-one of the first produced and widely used SCs-were evaluated upon leukemia cells. Whether JWH-018 exhibited a preventive effect on both leukemic and HSCs was evaluated by presenting a therapeutic approach for the first time in the literature. Cells were analyzed in case of cell proliferation, apoptosis, and transcriptional expression profiling of some significant JAK/STAT and AKT/mTOR pathways, apoptotic, cell cycle regulation, and epigenetic chromatin remodeling-related genes following JWH-018 treatment. In conclusion, however, further studies are still needed upon both HSCs and LSCs to illuminate the effects of SCs on leukemogenesis on chronic myeloid leukemia (CML) more clearly; we consider that the JWH-018 can provide a therapeutic effect on the pathogenesis of leukemia and particularly upon LSCs and SCs might have therapeutic potential in addition to current therapy.

The effects of Epigallocatechin-3-gallate and Dabrafenib combination on apoptosis and the genes involved in epigenetic events in anaplastic thyroid cancer cells.

Anaplastic thyroid cancer cases with poor prognosis are associated with epigenetic modifications such as abnormal DNA methylation. Epigallocathesin-3-gallate (EGCG) is a polyphenol compound of green tea that is still under investigation on its role in cancer prevention. EGCG is known as an epigenetic diet in DNA methyltransferase inhibitor. The cytotoxic effects of Dabrafenib, EGCG, and dabrafenib in combination with EGCG were assessed by using WST-8 assay; and also, Flow cytometry was utilized to identify cells undergoing apoptosis after treatments of the SW-1736 cells. We investigated the mRNA expression of genes involved in epigenetic events in SW-1736 cells by real-time qRT-PCR following the treatments. We demonstrated for the first time that the Dabrafenib-EGCG combination reduced cell viability significantly depending on concentration and induced apoptosis by 8.49-fold through investigating the additive effect together on SW-1736 cells. The IC50 doses of Dabrafenib and EGCG for 48 h were determined as 6.7 µM and 22.5 µM, respectively. The results of qRT-PCR demonstrated that the Dabrafenib-EGCG combination significantly caused the down-regulation of genes involved in epigenetic regulation. We suggest that the combination of Dabrafenib and EGCG following in vivo phase studies will contribute as an alternative treatment option for the treatment of ATC.

Origanum Sipyleum Methanol Extract in Combination with Ponatinib Shows Synergistic anti-Leukemic Activities on Chronic Myeloid Leukemia Cells.

Origanum sipyleum is used in folk medicine due to its anti-inflammatory, antimicrobial, and antioxidant properties. Ponatinib, an effective tyrosine kinase inhibitor in the treatment of chronic myeloid leukemia (CML), has severe side effects. Thus, we aimed to determine a novel herbal combination therapy that might not only increase the anti-leukemic efficacy but also reduce the dose of ponatinib in targeting CML cells. Origanum sipyleum was extracted with methanol (OSM), and secondary metabolites were determined by phytochemical screening tests. The cytotoxic effects of OSM on K562 cells were measured by WST-1 assay. Median-effect equation was used to analyze the combination of ponatinib and OSM (p-OSM). Apoptosis, proliferation, and cell-cycle were investigated by flow-cytometry. Cell-cycle-related gene expressions were evaluated by qRT-PCR. OSM that contains terpenoids, flavonoids, tannins, and anthracenes exhibited cytotoxic effects on K562 cells. The median-effect of p-OSM was found as synergistic; OSM reduced the ponatinib dose ∼5-fold. p-OSM elevated the apoptotic and anti-proliferative activity of ponatinib. Consistently, p-OSM blocked cell-cycle progression in G0/G1, S phases accompanied by regulations in TGFB2, ATR, PP2A, p18, CCND1, CCND2, and CCNA1 expressions. OSM enhanced the anti-leukemic activity of ponatinib synergistically via inducing apoptosis, suppressing proliferation, and cell-cycle. As a result, OSM might offer a potential strategy for treating patients with CML.

A drug-responsive multicellular human spheroid model to recapitulate drug-induced pulmonary fibrosis.

Associated with a high mortality rate, pulmonary fibrosis (PF) is the end stage of several interstitial lung diseases. Although many factors are linked to PF progression, initiation of the fibrotic process remains to be studied. Current research focused on generating new strategies to gain a better understanding of the underlying disease mechanism as the animal models remain insufficient to reflect human physiology. Herein, to account complex cellular interactions within the fibrotic tissue, a multicellular spheroid model where human bronchial epithelial cells incorporated with human lung fibroblasts was generated and treated with bleomycin (BLM) to emulate drug-induced PF. Recapitulating the epithelial-interstitial microenvironment, the findings successfully reflected the PF disease, where excessive alpha smooth muscle actin and collagen type I secretion were noted along with the morphological changes in response to BLM. Moreover, increased levels of fibrotic linked COL13A1, MMP2, WNT3 and decreased expression level of CDH1 provide evidence for the model reliability on fibrosis modelling. Subsequent administration of the Food and Drug Administration approved nintedanib and pirfenidone anti-fibrotic drugs proved the drug-responsiveness of the model.

The role of the WNT signaling pathway in the maxillary sinus squamous cell carcinoma.

Paranasal sinus tumors are a rare type of cancer. Most of these tumors are of epithelial origin and 80% of them are maxillary sinus squamous cell carcinoma. The WNT signaling pathway is an essential embryonic regulatory pathway known to play an important role in many cancers, including head and neck cancers. However, the effect of this pathway in maxillary sinus tumors has not been studied before. The aim of the study was to determine the changes in the regulatory genes of the WNT signaling pathway in maxillary sinus tumors. For this purpose, total RNA was isolated from the pathological preparations of 85 patients who had previously been operated on for squamous cell maxillary sinus tumor, and gene expression changes were evaluated by real-time RT-qPCR. The interactions among proteins encoded by genes, whose expression levels were found to be decreased and increased, were determined by protein-protein interaction (PPI) network analysis using string database, and signaling pathways that they are involved in were examined by Reactome database. A significant decrease in the expression of 28 genes compared to the control (fold change < 2.00 and p-value < 0.05) and a significant increase in the expression of 23 genes (fold change < 2.00 and p-value < 0.05) were detected. According to in silico analysis results, Signal Transduction (REACTOME:R-HSA-162582) and Signaling by WNT (REACTOME:R-HSA-195721) pathways were determined as most regulated pathways and FZD4-LRP5 and BCL9-CTNNB1 were determined as the strongest interactions. The current study contributes to illuminating the genetic regulation of maxillary sinus carcinoma in which genetic knowledge is limited. Our findings take attention to the dysregulations of the WNT signaling pathway that may support maxillary sinus carcinogenesis. The results will pave the way for further studies that investigate the therapy target potential of the WNT signaling pathway in this rare cancer.

Comparative expression analysis of dasatinib and ponatinib-regulated lncRNAs in chronic myeloid leukemia and their network analysis.

LncRNAs are associated with malignancies with their tumor suppressor/oncogenic properties. Although many studies are conducted related to the mechanism of action for dasatinib and ponatinib in chronic myeloid leukemia (CML), their comparative effects on lncRNA expressions are largely unknown. Hence, we aimed to define the lncRNAs involved in the treatment of CML with dasatinib and ponatinib. We measured the cytotoxicities of dasatinib/ponatinib with CCK-8 assay and identified differentially expressed lncRNAs (DEL) by qRT-PCR. We determined the principal functions of DELs by Ingenuity Pathway Analysis (IPA) and performed gene ontology (GO) analysis for apoptosis and anti-proliferation-related lncRNAs. Apoptotic and anti-proliferative activities of dasatinib/ponatinib were confirmed by flow-cytometry. In K562 cells, dasatinib/ponatinib re-regulated lncRNAs which were dysregulated in leukemia. DELs after treatment (forty with dasatinib, thirty-seven with ponatinib) were related to increased cell death; decreased cell viability, proliferation, tumor growth, invasion, migration. Dasatinib-mediated network was related to cancer, hematological disease while ponatinib-mediated network was associated with cancer, cell death/survival, cell-to-cell signaling/interaction. Both treatments predicted activation of IFNγ, IL1ß, TNF as upstream regulators, specially this effect was higher in dasatinib. Comparison analysis showed that ponatinib was predicted more effective in cell death of tumor cell line than dasatinib. We confirmed that ponatinib was more potent than dasatinib to induce apoptosis and inhibit proliferation of CML cells, in consensus with IPA and GO analysis results. LncRNAs are specifically involved in anti-leukemic activities of dasatinib and ponatinib. Our findings will contribute to understanding signalization occurring in CML cells after standard treatments.

Combination of resveratrol and BIBR1532 inhibits proliferation of colon cancer cells by repressing expression of LncRNAs.

Colorectal cancer (CRC) is the third most common cancer worldwide. The development of tumor drug resistance is observed in the treatment of CRC. Combinations of anticancer agents are attracting considerable interest in order to overcome drug resistance in CRC. This study aims to investigate the effect of resveratrol and BIBR1532, either alone or in combination, on the cell viability as well as on expression of long non-coding RNAs (LncRNAs) for HT-29 colon adenocarcinoma cells. The cytotoxic effects of resveratrol and BIBR1532 on HT-29 cells were determined using WST-1 test. Flow cytometry was used to determine apoptotic cell death after treatments. Real-Time PCR was used to identify expression of LncRNAs after treatments. LncExpDB and GEPIA2 were used to evaluate expression profiles of LncRNAs, whose expression levels were decreased in HT-29 cells after treatments, in normal tissues and colon adenocarcinoma tumors. IC50 concentrations of BIBR1532 and resveratrol were found to be 50.81 µM at 48 h and 86.23 µM at 72 h, respectively. Combination index value was 1.07617. BIBR1532, resveratrol, or their combination reduced the cell viability of HT-29 cells. CCAT1, CRNDE, HOTAIR, PCAT1, PVT1, SNHG16 were down-regulated after treatments. In silico analysis revealed that LncRNAs whose expression levels were decreased after treatments were associated with CRC. Resveratrol, BIBR1532, or their combination may have anti-proliferative effect on colorectal cancer cells through repressing expression of LncRNAs that are involved in progression of CRC.

Temozolomide treatment combined with AZD3463 shows synergistic effect in glioblastoma cells.

Temozolomide (TMZ) is used in the standard therapy regimen for patients with glioblastoma (GBM). However, some GBM patients do not respond to TMZ therapy. The combining therapeutic agents in GBM treatment are attracting considerable interest due to TMZ resistance. This study aims to identify the combinatorial effect of TMZ and AZD3463 on the viability of the T98G GBM cells. The cytotoxic effects of compounds were determined by using WST-8 assay. Flow cytometry was used to determine apoptosis and cell cycle profiles after treatments. Real-time PCR was used to identify mRNA expression of genes in the PI3K/AKT signaling pathway after treatments. IC50 concentrations of TMZ and AZD3463 were found to be 1.54 mM and 529 nM after incubation for 48 h, respectively. The combination treatment showed a synergistic effect on reducing the viability of GBM cells. Each one of TMZ, AZD3463, and combination treatments induced apoptosis. Treatments, either alone or the combination of these agents, caused the cell cycle arrest in distinct phases. TMZ and AZD3463 treatments, either alone or in combination, downregulated mRNA expression of genes in the PI3K/AKT signaling pathway. The combination of TMZ with AZD3463 may increase the efficacy of single TMZ treatment in GBM cells due to decreased expression of genes in the PI3K/AKT signaling pathway that is responsible for drug resistance and intratumoral heterogeneity.

Telomerase inhibition regulates EMT mechanism in breast cancer stem cells.

BACKROUND: CSCs having the common features of high telomerase activity and high migration and invasion capabilities play a vital role as the initiators of metastasis. Small molecule BIBR1532 has been shown to target cancer cells by inhibiting telomerase. Recent studies have suggested that telomerase activity is associated with epithelial mesenchymal transition (EMT). EMT program, which causes epithelial cells to acquire a mesenchymal morphology, is known to play a significant role in cancer metastasis. METHODS: The hypothesis of our study was that suppression of telomerase in breast cancer and cancer stem cells would interrupt EMT mechanism. Cytotoxicity of BIBR1532 was evaluated using WST-1 assay in all cell lines and the effects of BIBR1532 on apoptosis were investigated with Annexin V. Migration rate of the cells was examined by wound healing assay and sphere forming capacities were observed by hanging drop test. Finally, the expression of 84 EMT-related genes was analyzed by real-time qPCR. RESULTS: The IC50 values for the MDA-MB-231 and breast epithelial stem cells of BIBR1532 were analyzed as 18.04 and 38.71 µl at 72 h, respectively. Interestingly, apoptosis was only induced in stem cells. In hanging drop test, sphere areas were reduced in stem cells treated with BIBR1532. In wound healing assay, BIBR1532 decreased the migration rate of stem cells. Together with this, expression of EMT-related genes were regulated in stem cells towards a epithelial phenotype. CONCLUSION: Our obtained results indicated that telomerase inhibition affects the EMT mechanism. The targeted elimination of breast cancer stem cells by a telomerase inhibitor in cancer treatment may limit the mobility and stemness of cancer cells interrupting the EMT mechanism, thus may prevent metastasis.

The potential of JAK/STAT pathway inhibition by ruxolitinib in the treatment of COVID-19.

Ruxolitinib is the first approved JAK1 and JAK2 inhibitor, and is known to interfere with the JAK / STAT signaling pathway, one of the critical cellular signaling pathways involved in the inflammatory response. This review presents an overview of SARS-CoV-2 and the COVID-19 pandemic, and then focuses on the potential efficacy of ruxolitinib in this infection. The potential targets of ruxolitinib were determined by using genetic alterations that have been reported in COVID-19 patients. The potential effectiveness of ruxolitinib is suggested by evaluating the interactions of these potential targets with ruxolitinib or JAK/STAT pathway.

PI3K/AKT/mTOR pathway and autophagy regulator genes in paranasal squamous cell carcinoma metastasis.

Although there are many studies on the role of PI3K/AKT/mTOR pathway and autophagy genes in the mechanism of head and neck cancer formation and prognostic significance, there is no study investigating the role of the genes in paranasal sinus carcinomas. The aim of the study was to assess the role of the PI3K/AKT/mTOR pathway and autophagy related gene expression changes in squamous cell carcinoma of paranasal sinuses with and without neck metastasis. Eight paranasal squamous cell carcinoma patients (five without and three with neck metastasis) were included. Tissues were obtained during the surgery. Total RNA was isolated from the tissues and cDNA synthesis was performed. Expression levels of the genes were determined using qRT-PCR method. The results were evaluated using the 2-∆∆Ct method, and fold changes of the gene expression levels in primary tumor and neck metastasis tissues were calculated according to the normal tissue. Expression levels of both PI3K/AKT/mTOR pathway and positive regulators of autophagy were significantly increased in metastasis-related two groups, especially in neck metastasis tissues. The increase in PI3K/AKT/mTOR pathway and autophagy related gene expression levels may support the metastatic character in paranasal squamous cell carcinomas. This is the first study to assess autophagy related genes in paranasal sinus cancer at transcriptome-level. Support of the transcriptome-level findings by the further protein analyses will contribute to the illumination of the rare paranasal sinus cancer molecular biology.

Epigenetic modifications in chronic myeloid leukemia cells through ruxolitinib treatment.

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.

Effects of telomerase inhibitor on epigenetic chromatin modification enzymes in malignancies.

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.

Docosahexaenoic acid reversed atherosclerotic changes in human endothelial cells induced by palmitic acid in vitro.

Abnormal activity of atherosclerotic endothelial cells paving luminal surface of blood vessels has been described in many diseases. It has been reported that natural polyunsaturated fatty acids such as docosahexaenoic acid exert therapeutic effects in atherosclerotic condition. Human umbilical vein endothelial cells were treated with 1mM palmitic acid for 48 hours and exposed to 40µM docosahexaenoic acid for the next 24 hours. Real-time polymerase chain reaction analysis was used to measure the expression of PTX3, iNOS, and eNOS. The level of nitric oxide was detected by Griess reagent. The transcription level of genes participating in coagulation and blood pressure was studied by polymerase chain reaction array. Docosahexaenoic acid improved the survival rate by reducing apoptosis rate (P < .05). Compared with that of the group given palmitic acid, attenuation of proinflammatory status was indicated by reduced interleukin-6 (P < .05) and prostaglandin E2 levels. All genes PTX3, iNOS, and eNOS were down-regulated after being exposed to docosahexaenoic acid. Nitric oxide contents were not changed in cells exposed to docosahexaenoic acid. Polymerase chain reaction array confirmed the reduction of LPA, PDGFß, ITGA2, SERPINE1, and FGA after exposure to docosahexaenoic acid for 24 hours (P < .05). Docosahexaenoic acid had potential to blunt atherosclerotic changes in the modulation of genes controlling blood coagulation, pressure, and platelet function. SIGNIFICANCE OF THE STUDY: The current experiment showed that docosahexaenoic acid could reverse atherosclerotic changes in human endothelial cells induced by palmitic acid. The increased levels of interleukin-6 and prostaglandin E2 in atherosclerotic cells were returned to near-to-normal status. Gene expression analysis showed a reduced activity of genes participating in atherosclerotic endothelial cells treated by docosahexaenoic acid. The expression of genes related to cell clotting activity was also similar to that of normal cells.

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.

Antagonistic Effect of Oxytocin and Tacrolimus Combination on Adipose Tissue - Derived Mesenchymal Stem Cells: Antagonistic effect of oxytocin and tacrolimus.

Tacrolimus (FK506) is a chemotherapeutic agent, which uses calcineurin pathway via inhibiting the stimulation of T cells to prevent the formation of immune response in the recipient individual in organ transplants. FK506 is mainly metabolized in the liver by cytochrome P450 enzyme system and is known that it has high toxic effects on different cells. Mesenchymal stem cells (MSCs) have recently gained importance since their potential to be used in cellular therapy and tissue regeneration. In some clinical cases, MSCs are transferred into the patient after the organ transplantations in order to support the treatment. Because of their immunomodulatory actions and assistance to the regeneration, popularity of MSCs have been increasing recently. However, since immunosuppressive agents have a potential cytotoxic and apoptotic effect on MSCs, researches have attempted to use it as a combination with an agent that alleviates these effects. Oxytocin (OT) is primarily acting as a neuromodulator in humans and is a peptide hormone secreted by the pituitary gland of the neurohypophysis. OT has such effects on cells as to confer resistance against oxidative stress on cells and to increase the proliferation and help regeneration. Studies on the active substance of FK506 were aimed to investigate the cytotoxic effects on human adipose tissue derived MSCs. The purpose of this study was to determine the cytotoxic, apoptotic and morphological effects of FK506, an immunosuppressive agent, on adipose tissue - derived MSC (ADMSC) which has the potential to be used for immune suppression. In addition, it was aimed to determine whether the agent could reduce the cytotoxic, apoptotic, and morphological effects on ADMSCs when used in combination with OT. For this purpose, the cytotoxic effects of the FK506 and OT on ADMSCs were determined by time and dose dependent manner by the WST-1 test. Isobologram analysis was evaluated using the WST-1 test according to IC50 values of FK506 and OT. The apoptotic effects of the agents on the ADMSCs were determined by the Annexin V method. Immunofluorescence staining was performed to determine morphological changes. Changes in the levels of oxidative stress markers were measured by colorimetric and flourometric methods using lipid peroxidation, superoxide dismutase activity, catalase activity and glutathione peroxidase assays. The IC50 values of FK506 and OT on ADMSCs were calculated as 17.44µM and 13.43µM, respectively.FK506 and OT were found to have antagonistic activity on ADMSCs (CI value of the combination was 1.24). The effects of the agents individually and in combination on the levels of apoptosis and oxidative stress markers have been evaluated. When the results obtained from the study are evaluated, the adipose- tissue derived mesenchymal stem cells used with takrolimus and oxytocin combination have a potential for novel treatment approaches.

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.