The future of cancer therapy: exploring the potential of patient-derived organoids in drug development.

Cancer therapy is on the brink of a significant transformation with the inclusion of patient-derived organoids (PDOs) in drug development. These three-dimensional cell cultures, directly derived from a patient's tumor, accurately replicate the complex structure and genetic makeup of the original cancer. This makes them a promising tool for advancing oncology. In this review, we explore the practical applications of PDOs in clinical drug screening and pharmacognostic assessment, as well as their role in refining therapeutic strategies. We provide insights into the latest advancements in PDO technology and its implications for predicting treatment responses and facilitating novel drug discoveries. Additionally, we address the operational challenges associated with incorporating PDOs into the drug development process, such as scaling up organoid cultures, ensuring consistent results, and addressing the ethical use of patient-derived materials. Aimed at researchers, clinicians, and key stakeholders in oncology, this article aims to succinctly present both the extraordinary potential and the obstacles to integrating PDOs, thereby shedding light on their prospective impact on the future of cancer treatment.

Investigation of cytotoxic and apoptotic effects of disodium pentaborate decahydrate on ovarian cancer cells and assessment of gene profiling.

After revealing the anti-cancer properties of boron, which is included in the category of essential elements for human health by the World Health Organization, the therapeutic potential of boron compounds has been begun to be evaluated, and its molecular effect mechanisms have still been among the research subjects. In ovarian cancer, mutations or amplifications frequently occur in the PI3K/Akt/mTOR pathway components, and dysregulation of this pathway is shown among the causes of treatment failure. In the present study, it was aimed to investigate the anti-cancer properties of boron-containing DPD in SKOV3 cells, which is an epithelial ovarian cancer model, through PI3K/AKT/mTOR pathway. The cytotoxic activity of DPD in SKOV3 cells was evaluated by WST-1 test, apoptotic effect by Annexin V and JC-1 test. The gene expressions associated with PI3K/AKT/mTOR pathway were determined by real-time qRT-PCR. In SKOV3 cells, the IC50 value of DPD was found to be 6.7 mM, 5.6 mM, and 5.2 mM at 24th, 48th and 72nd hour, respectively. Compared with the untreated control group, DPD treatment was found to induce apoptosis 2.6-fold and increase mitochondrial membrane depolarization 4.5-fold. DPD treatment was found to downregulate PIK3CA, PIK3CG, AKT2, IGF1, IRS1, MAPK3, HIF-1, VEGFC, CAB39, CAB39L, STRADB, PRKAB2, PRKAG3, TELO2, RICTOR, MLST8, and EIF4B genes and upregulate TP53, GSK3B, FKBP8, TSC2, ULK1, and ULK2 genes. These results draw attention to the therapeutic potential of DPD, which is frequently exposed in daily life, in epithelial ovarian cancer and show that it can be a candidate compound in combination with chemotherapeutics.

Photothermal effect of albumin-modified gold nanorods diminished neuroblastoma cancer stem cells dynamic growth by modulating autophagy.

Here, we investigated the photothermal effect of gold nanorods (GNRs) on human neuroblastoma CD133+ cancer stem cells (CSCs) via autophagic cell death. GNRs were synthesized using Cetyltrimethylammonium bromide (CTAB), covered with bovine serum albumin (BSA). CD133+ CSCs were enriched from human neuroblastoma using the magnetic-activated cell sorting (MACS) technique. Cells were incubated with GNRs coated with BSA and exposed to 808-nm near-infrared laser irradiation for 8 min to yield low (43 °C), medium (46 °C), and high (49 °C) temperatures. After 24 h, the survival rate and the percent of apoptotic and necrotic CSCs were measured using MTT assay and flow cytometry. The expression of different autophagy-related genes was measured using polymerase chain reaction (PCR) array analysis. Protein levels of P62 and LC3 were detected using an enzyme-linked immunosorbent assay (ELISA). The viability of CSC was reduced in GNR-exposed cells compared to the control group (p < 0.05). At higher temperatures (49 °C), the percent of apoptotic CSCs, but not necrotic cells, increased compared to the lower temperatures. Levels of intracellular LC3 and P62 were reduced and increased respectively when the temperature increased to 49 °C (p < 0.05). These effects were non-significant at low and medium temperatures (43 and 46 °C) related to the control CSCs (p > 0.05). The clonogenic capacity of CSC was also inhibited after photothermal therapy (p < 0.05). Despite these changes, no statistically significant differences were found in terms of CSC colony number at different temperatures regardless of the presence or absence of HCQ. Based on the data, the combination of photothermal therapy with HCQ at 49 °C can significantly abort the CSC clonogenic capacity compared to the control-matched group without HCQ (p < 0.0001). PCR array showed photothermal modulation of CSCs led to alteration of autophagy-related genes and promotion of co-regulator of apoptosis and autophagy signaling pathways. Factors related to autophagic vacuole formation and intracellular transport were significantly induced at a temperature of 49 °C (p < 0.05). We also note the expression of common genes belonging to autophagy and apoptosis signaling pathways at higher temperatures. Data showed tumoricidal effects of laser-irradiated GNRs by the alteration of autophagic response and apoptosis.

Interaction of alginate with nano-hydroxyapatite-collagen using strontium provides suitable osteogenic platform.

BACKGROUND: Hydrogels based on organic/inorganic composites have been at the center of attention for the fabrication of engineered bone constructs. The establishment of a straightforward 3D microenvironment is critical to maintaining cell-to-cell interaction and cellular function, leading to appropriate regeneration. Ionic cross-linkers, Ca2+, Ba2+, and Sr2+, were used for the fabrication of Alginate-Nanohydroxyapatite-Collagen (Alg-nHA-Col) microspheres, and osteogenic properties of human osteoblasts were examined in in vitro and in vivo conditions after 21 days. RESULTS: Physicochemical properties of hydrogels illustrated that microspheres cross-linked with Sr2+ had reduced swelling, enhanced stability, and mechanical strength, as compared to the other groups. Human MG-63 osteoblasts inside Sr2+ cross-linked microspheres exhibited enhanced viability and osteogenic capacity indicated by mineralization and the increase of relevant proteins related to bone formation. PCR (Polymerase Chain Reaction) array analysis of the Wnt (Wingless-related integration site) signaling pathway revealed that Sr2+ cross-linked microspheres appropriately induced various signaling transduction pathways in human osteoblasts leading to osteogenic activity and dynamic growth. Transplantation of Sr2+ cross-linked microspheres with rat osteoblasts into cranium with critical size defect in the rat model accelerated bone formation analyzed with micro-CT and histological examination. CONCLUSION: Sr2+ cross-linked Alg-nHA-Col hydrogel can promote functionality and dynamic growth of osteoblasts.

Propolis Extract Regulates microRNA Expression in Glioblastoma and Brain Cancer Stem Cells.

BACKGROUND: Grade IV gliomas are classified as glioblastoma (GBM), which is the most malignant brain cancer type. Various genetic and epigenetic mechanisms play a role in the initiation and progression of GBM. MicroRNAs (miRNAs) are small, non-coding RNA molecules that belong to the main epigenetic regulatory RNA class that plays different roles in either physiological or pathological conditions, including GBM pathogenesis regulating expression levels of the target genes. Brain Cancer Stem Cells (BCSCs) are responsible for poor prognosis, including therapy resistance and relapse. Epigenetic regulation mediated by miRNAs is also a critical component of BCSC selfrenewal and differentiation properties. Propolis is a resinous substance collected by honey bees from various plant sources. The flavonoid content of propolis varies depending on the collection region and the extraction method. Although there are studies that include the effects of different originated-propolis on the miRNA expression levels of the glioblastoma cells, the impact on the BCSCs has not been studied yet. OBJECTIVE: This study aims to evaluate the effects of propolis obtained from Aydin, a city in western Turkey, on miRNA expression levels of BCSCs and GBM cells. METHODS: Aydin propolis was dissolved in 60% ethanol, and after evaporation, distilled water was added to prepare the propolis stock solution. The flavonoids content of the Aydin propolis was determined by MS Q-TOF analysis. Commercially obtained U87MG and BCSCs were used as in-vitro brain cancer models. Cytotoxic and apoptotic effects of Aydin propolis were determined via WST-1 assay and Annexin V test, respectively. The miRNA expression profile was investigated using the real-time qRT-PCR method. The fold changes were calculated by the2-ΔΔCt method. The miRNA-mRNA-pathway interactions, including significantly altered miRNAs, were determined using different bioinformatics tools and databases. RESULTS: Quercetin 3-methyl ether was the main component of the Aydin propolis. Aydin propolis did not show significant cytotoxic and apoptotic effects on both GBM and BCSCs up to 2mg/ml concentration. Aydin propolis treatment decreased the expression of nine miRNAs in the U87MG and five miRNAs in the BCSCs. Moreover, ten miRNAs have upregulated from 2.22 to 10.56 folds in propolis treated GBM cells compared to the control group significantly (p<0.05). In the study, the potential roles of two new miRNAs, whose regulations in glioma were not previously defined, were identified. One of them was miR-30d-5p, a novel potential oncomiR in GBM, which was 2.46 folds downregulated in Aydin propolis treated GBM cells. The other one is miR-335-5p, which is a potential tumor suppressor miR in GBM, that was 5.66 folds upregulated in Aydin propolis treated GBM cells. FOXO pathway, its upstream and downstream regulators, and critically neuronal developmental regulators, NOTCH and WNT pathways, were determined as the most deregulated pathways in Aydin propolis treated cells. CONCLUSION: The determination of the anti-cancer effect of Aydin propolis on the miRNA expression of GBM, especially on cancer stem cells, may contribute to the elucidation of brain cancer genetics by supporting further analyses.

In Vitro Effects of Propofol on Cytotoxic, Apoptotic and PI3K-Akt Signaling Pathway Genes on Brain Cancer Cells.

AIM: The study aimed to determine the cytotoxic and apoptotic effect of propofol on glioma cells. BACKGROUND: Propofol [2,6-diisopropylphenol] is a commonly used intravenous anesthetic. Propofol is known to have a mechanism of action on the PI3K-AKT pathway. OBJECTIVE: This study aimed to evaluate the effect of propofol on the proliferation and apoptosis of human glioma cells, as well as to investigate changes in expression levels of the PI3K-AKT signaling pathway genes. MATERIALS AND METHODS: The cytotoxic effect of propofol on the U-87 MG cell line was determined by WST-1 method. Annexin V-FITC and Mitoprobe JC-1 assay were used to measure apoptosis by flow cytometry. The expression levels of genes in the PI3K-AKT signaling pathway were investigated by qRT-PCR. RESULTS: We have shown that propofol induced apoptosis in U-87 MG cells by 17.1 fold compared to the untreated control. Furthermore, significant differences were found in the expression levels of the PI3K-AKT signaling pathway genes. CONCLUSION: As a result of our study, it was found that propofol caused differences in expression levels of PI3K-AKT signaling pathway genes and it was suggested that these differences may be related to apoptosis induction.

Antileukemic Effects of Anti-miR-146a, Anti-miR-155, Anti-miR-181a, and Prednisolone on Childhood Acute Lymphoblastic Leukemia.

Prednisolone has been used frequently in the treatment of acute lymphoblastic leukemia. However, to overcome the challenges of the treatment, the development of additional therapies is of great importance. Small, non-protein-coding RNAs, namely, microRNAs (miRNAs), are critical epigenetic regulators with physiological and pathological importance. This study is aimed at determining the effects of miR-146a, miR-155, and miR-181a inhibition with their corresponding anti-miRs on both leukemic and healthy cells, individually and with prednisolone. Leukemic (SUP-B15) and healthy B-lymphocyte (NCI-BL 2171) cell lines were used in this study. A total of 12 experimental groups included individual and combinational silenced ALL-associated miRNAs (hsa-miR-155, hsa-miR-146a, and hsa-miR-181a) and their combination with prednisolone. Cytotoxicity, proliferation, cell cycle, and apoptosis analyses were performed by using WST-1, trypan blue, APC-BrdU, Annexin V, and JC-1 methods in each study group, respectively. To control the effectiveness of anti-miR transfection and prednisolone application, miRNA expression analysis was performed from all groups. Anti-miR application was effective on the viability, proliferation, cell cycle, and apoptosis of leukemia cells, and this effect was increased with prednisolone administration. In addition, this activity was found to be very low on healthy cells. In conclusion, anti-miR applications may have the potential for clinical use of adjuvant to or as an alternative to conventional therapies for childhood acute lymphoblastic leukemia.

Effect of valproic acid on miRNAs affecting histone deacetylase in a model of anaplastic thyroid cancer.

BACKGROUND: Thyroid cancer is the most common malignant tumor of the endocrine system seen in the thyroid gland. More than 90% of thyroid cancers comprise papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Although anaplastic thyroid carcinoma (ATC) accounts for less than 2% of thyroid cancer. But patients' lifespan after diagnosis is about 6 months. Surgical interventions, radioactive iodine use, and chemotherapy are not sufficient in the treatment of ATC, so alternative therapies are needed. METHODS AND RESULTS: The WST-1 assay test was performed to evaluate the anti-proliferative effects of Valproic acid (VPA). Also, the effect of VPA on miRNAs affecting histone deacetylase was determined by Quantitative RT-PCR. In the SW1736 cell line, IC50 dose for VPA was found 1.6 mg/ml. In our study, the level of oncogenic genes expression in cells treated with VPA, including miR-184, miR-222-5p, miR-124-3p, and miR-328-3p, decreased. Also, the expression of tumor inhibitory genes including miR-323-5p, miR-182-5p, miR-138-5p, miR-217, miR-15a-5p, miR-29b-3p, miR-324-5p and miR-101-5p increased significantly. CONCLUSIONS: VPA can ad-just countless gene expression patterns, including microRNAs (miRNAs), by targeting histone deacetylase (HDAC). However, further studies are required for more accurate results.

Sonic hedgehog signaling is associated with resistance to zoledronic acid in CD133high/CD44high prostate cancer stem cells.

Cancer stem cells (CSCs) are a unique population that has been linked to drug resistance and metastasis and recurrence of prostate cancer. The sonic hedgehog (SHH) signal regulates stem cells in normal prostate epithelium by affecting cell behavior, survival, proliferation, and maintenance. Aberrant SHH pathway activation leads to an unsuitable expansion of stem cell lineages in the prostate epithelium and the transformation of prostate CSCs (PCSCs). Zoledronic acid (ZOL), one of the third-generation bisphosphonates, effectively prevented bone metastasis and treated advanced prostate cancer despite androgen deprivation therapy. Despite strong evidence for the involvement of the SHH in human PCSCs survival and drug resistance, the roles of SHH in the PCSCs-related resistance to ZOL remain to be fully elucidated. The present study aimed to investigate the role of the SHH pathway in ZOL resistance of PCSCs in 2D and three 3D cell culture conditions. For this purpose, we isolated CD133high/ CD44high PCSCs using a flow cytometer. Following ZOL treatment, mRNA and protein expressions of the components of the SHH signaling pathway in PCSCs and non-CSCs were analyzed using qRT-PCR and Immunofluorescence staining, respectively. Our finding suggested that SHH signaling may be activated by different mechanisms that lead to avoidance of the inhibition effect of ZOL. Thereby, SHH pathways may be associated with the resistance to ZOL developed by prostate CSCs. Inhibition of CSCs-related SHH signaling along with ZOL treatment should be considered to achieve improvement in survival or delayed treatment failure and prevention of the CSCs-related drug resistance.

Molecular biological investigation of temozolomide and KC7F2 combination in U87MG glioma cell line.

Glioblastom Multiforme (GBM) is the most invasive and malignant member of the IV grade of the subclass Astrocytoma according to the last assessment of the 2016 WHO report. Due to the resistance to treatment and weak response, as well as the topographical structure of the blood brain barrier, the treatment is also difficult due to the severe clinical manifestation, and new treatment methods and new therapeutic agents are needed. Temozolomide (TMZ) is widely used in the treatment of glioblastoma and is considered as the primary treatment modality. TMZ, a member of the class of cognitive agents, is currently considered the most effective drug because it can easily pass through the blood brain barrier. Glucose metabolism is a complex energy producing machine that, a glucose molecule produces 38 molecules of ATP after full glycolytic catabolism. According to Otto Warburg's numerous studies cancer cells perform the first glycolytic step without entering the mitochondrial step. These cells produce lactic acid and make the micro-media more acidic even in aerobic conditions. This phenomenon is attributed to the Warburg hypothesis and either as aerobic glycolysis. Although glycolysis enzymes are the primary actors of this phenotypic expression, some genetic and epigenetic factors are no exception. We experimentally used KC7F2 active ingredient to target cancer metabolism. In our study, we evaluated cancer metabolism in combination with the effect of TMZ chemotherapeutic agent, examining the effect of two different agents separately and in combination to observe the effects of cancer cell proliferation, survival, apoptosis and expression of metabolism genes on expression. We observed that the combined effect of reduced the effective dose of the TMZ alkylating agent and that the effect was increased and the effect of the combined teraphy is assessed from a metabolic point of view and that it suppresses aerobic glycolysis.

Targeting TdT gene expression in Molt-4 cells by PNA-octaarginine conjugates.

Peptide nucleic acid (PNA) is an amide based structural nucleic acid mimic with potential applications in gene therapeutic drug discovery. In the present study, we evaluated and compared the effects on gene expression, cell viability and apoptosis of two antisense PNA-d-octaarginine conjugates, targeting sequences at the AUG translation start site or the 5'-UTR of the TdT (terminal deoxynucleotidyl transferase) gene, as well as a sense oligomer corresponding to the 5'-UTR-antisense, in Molt-4 cells. The protein level of TdT was determined by flow cytometry, and qPCR was used for mRNA expression analysis. Mismatch PNAs were used as control to address the sequence/target spcifity of the biological effects. The results showed that treatment with the AUG- and to slightly lesser extent with the 5'-UTR-antisense PNAs reduced the TdT mRNA as wel as the protein level, whereas only very low effect was observed for the 5'-UTR-sense PNA. A parallel effect was observed on reduced cell survival and increased rate of apoptosis. Our findings suggest that antisense PNAs can inhibit expression of the TdT gene and induce apoptosis in Molt-4 cells.

High Glucose Content Abrogated the Normal Activity of Heat Shock Protein Signaling Pathway in Human Neuroblastoma Cells.

BACKGROUND: Detrimental effects of high glucose content (HGC) were proved in different tissues such as the central nervous system. It seems that diabetic conditions could also alter the functional behavior of stem cells residing in the context of the nervous system. METHODS: The possible effects of 40 and 70 mmol glucose were examined on HSP70 signaling pathways with a specific focus on protein translation, folding values of human neuroblastoma cell line SHSY-5Y after 72 h. Human neuroblastoma cells were exposed to 5, 40 and 70 mmol glucose doses. The transcription level of genes related to HSP70 signaling was also evaluated by PCR array. RESULTS: The data from PCR array showed high glucose especially 70 mmol could potentially modulate the normal function of protein folding, endoplasmic reticulum derived protein folding and synthesis in neuroblastoma cells (p <0.05). CONCLUSIONS: Data showed that high glucose condition makes neuroblastoma cells prone to biochemical insufficiency by affecting the function of HSP70 signaling pathway and protein synthesis.

Correction to: Low-level laser irradiation at a high power intensity increased human endothelial cell exosome secretion via Wnt signaling.

After publication of this paper, the authors determined an error in Fig. 4. Below is the correct Fig. 4.

Role of Autophagy in Breast Cancer Development and Progression: Opposite Sides of the Same Coin.

The term "autophagy", which means "self (auto) - eating (phagy)", describes a catabolic process that is evolutionarially conserved among all eukaryotes. Although autophagy is mainly accepted as a cell survival mechanism, it also modulates the process known as "type II cell death". AKT/mTOR pathway is an upstream activator of autophagy and it is tightly regulated by the ATG (autophagy-related genes) signaling cascade. In addition, wide ranging cell signaling pathways and non-coding RNAs played essential roles in the control of autophagy. Autophagy is closely related to pathological processes such as neurodegenerative diseases and cancer as well as physiological conditions. After the Nobel Prize in Physiology or Medicine 2016 was awarded to Yoshinori Ohsumi "for his discoveries of mechanisms for autophagy", there was an explosion in the field of autophagy and molecular biologists started to pay considerable attention to the mechanistic insights related to autophagy in different diseases. Since autophagy behaved dualistically, both as a cell death and a cell survival mechanism, it opened new horizons for a deeper analysis of cell type and context dependent behavior of autophagy in different types of cancers. There are numerous studies showing that the induction of autophagy mechanism will promote survival of cancer cells. Since autophagy is mainly a mechanism to keep the cells alive, it may protect breast cancer cells against stress conditions such as starvation and hypoxia. For these reasons, autophagy was noted to be instrumental in metastasis and drug resistance. In this chapter we have emphasized on role of role of autophagy in breast cancer. Additionally we have partitioned this chapter into exciting role of microRNAs in modulation of autophagy in breast cancer. We have also comprehensively summarized how TRAIL-mediated signaling and autophagy operated in breast cancer cells.

Investigation of the effect of telomerase inhibitor BIBR1532 on breast cancer and breast cancer stem cells.

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.

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

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.

Low-level laser irradiation at a high power intensity increased human endothelial cell exosome secretion via Wnt signaling.

The distinct role of low-level laser irradiation (LLLI) on endothelial exosome biogenesis remains unclear. We hypothesize that laser irradiation of high dose in human endothelial cells (ECs) contributes to the modulation of exosome biogenesis via Wnt signaling pathway. When human ECs were treated with LLLI at a power density of 80 J/cm2, the survival rate reduced. The potential of irradiated cells to release exosomes was increased significantly by expressing genes CD63, Alix, Rab27a, and b. This occurrence coincided with an enhanced acetylcholine esterase activity, pseudopodia formation, and reduced zeta potential value 24 h post-irradiation. Western blotting showed the induction of LC3 and reduced level of P62, confirming autophagy response. Flow cytometry and electron microscopy analyses revealed the health status of the mitochondrial function indicated by normal ΔΨ activity without any changes in the transcription level of PINK1 and Optineurin. When cells exposed to high power laser irradiation, p-Akt/Akt ratio and in vitro tubulogenesis capacity were blunted. PCR array and bioinformatics analyses showed the induction of transcription factors promoting Wnt signaling pathways and GTPase activity. Thus, LLLI at high power intensity increased exosome biogenesis by the induction of autophagy and Wnt signaling. LLLI at high power intensity increases exosome biogenesis by engaging the transcription factors related to Wnt signaling and autophagy stimulate.

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.

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-1ß 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. © 2016 Wiley Periodicals, Inc.