Investigating the potential therapeutic role of targeting STAT3 for overcoming drug resistance by regulating energy metabolism in chronic myeloid leukemia cells.

Objectives: STATs are one of the initial targets of emerging anti-cancer agents due to their regulatory roles in survival, apoptosis, drug response, and cellular metabolism in CML. Aberrant STAT3 activity promotes malignancy, and acts as a metabolic switcher in cancer cell metabolism, contributing to resistance to TKI nilotinib. To investigate the possible therapeutic effects of targeting STAT3 to overcome nilotinib resistance by evaluating various cellular responses in both sensitive and nilotinib resistant CML cells and to test the hypothesis that energy metabolism modulation could be a mechanism for re-sensitization to nilotinib in resistant cells. Materials and Methods: By using RNAi-mediated STAT3 gene silencing, cell viability and proliferation assays, apoptotic analysis, expressional regulations of STAT mRNA transcripts, STAT3 total, pTyr705, pSer727 protein expression levels, and metabolic activity as energy metabolism was determined in CML model K562 cells, in vitro. Results: Targeting STAT3 sensitized both parental and especially nilotinib resistant cells by decreasing leukemic cell survival; inducing leukemic cell apoptosis, and decreasing STAT3 mRNA and protein expression levels. Besides, cell energy phenotype was modulated by switching energy metabolism from aerobic glycolysis to mitochondrial respiration in resistant cells. RNAi-mediated STAT3 silencing accelerated the sensitization of leukemia cells to nilotinib treatment, and STAT3-dependent energy metabolism regulation could be another underlying mechanism for regaining nilotinib response. Conclusion: Targeting STAT3 is an efficient strategy for improving the development of novel CML therapeutics for regaining nilotinib response, and re-sensitization of resistant cells could be mediated by induced apoptosis and regulation in energy metabolism.

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.

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.

Effect of Different Dentin Conditioning Agents on Growth Factor Release, Mesenchymal Stem Cell Attachment and Morphology.

INTRODUCTION: EDTA has been considered the gold standard in regenerative endodontic treatments. The aim of this study was to evaluate the effects of different dentin conditioning agents other than EDTA on released growth factors, mesenchymal stem cell attachment, and morphology. METHODS: Transforming growth factor beta 1, vascular endothelial growth factor, bone morphogenetic protein 2, and fibroblast growth factor 2 release from prepared dentin discs conditioned with 17% EDTA, 10% citric acid, 1% phytic acid (IP6), or 37% phosphoric acid were quantified using the enzyme-linked immunosorbent assay after final irrigation and after 3 days of adipose-derived mesenchymal stem cell (adMSC) seeding. Forty root fragments were prepared from extracted single-rooted teeth. The morphology and attachment of adMSCs on the conditioned root fragments were observed using a scanning electron microscope. Data for growth factor quantification were analyzed using 1-way analysis. RESULTS: The highest transforming growth factor beta 1 release was observed after citric acid treatment followed by phosphoric acid; there was no significant difference between them, but compared with EDTA and 1% IP6, there were significant differences observed. The enzyme-linked immunosorbent assay detected a very minor exposure of vascular endothelial growth factor and fibroblast growth factor 2 after dentin conditioning, but there were no significant differences between the groups. The greatest bone morphogenetic protein 2 release was observed in the 1% IP6 group, but there were no significant differences between the groups. Three days of adMSC seeding after dentin conditioning has made a dramatic increase in all of the growth factors, and phosphoric acid appeared to be the most effective agent with significant differences compared with the remaining groups. Scanning electron microscopic observations showed that none of the conditioning solutions had an adverse effect on stem cell proliferation and attachment to root dentin. Different cell morphologies like round, oblong, flat, and well-attached cells with developed filopodia were observed in the dentin-conditioned groups. CONCLUSIONS: Phosphoric acid conditioning could be useful and may have beneficial effects in regenerative endodontic treatments.

The role of EGFR overexpression on the recurrence of basal cell carcinomas with positive surgical margins.

BACKGROUND: Epidermal growth factor receptor (EGFR) expression may have role on recurrence of basal cell carcinoma (BCC) with positive surgical margin(s). OBJECTIVE: The aim was to investigate the role of genetic expression changes of EGFR on recurrence rates in patients in follow up with surgically excised BCC with positive surgical margin(s). METHODS: Thirty-four surgical margin-positive BCC lesions that were closely followed up without an immediate reoperation were included in this study. Real-time polymerase chain reaction (PCR) was performed from the both healthy and tumoral tissue samples. RESULTS: EGFR was expressed at a significantly higher rate in tumoral tissues compared to healthy tissues (p < 0,05). In patients with recurrence lesions, EGFR expression was 6,66 times higher compared to patients with non-recurrent. Also, there was statistically significant difference EGFR expression for infiltrative subtypes (p < 0,05). CONCLUSION: Our study focuses on the role of EGFR overexpression specifically and outcomes for recurrent and infiltrative subtyped lesions are significant for both clinic and pathogenesis of BCC. Similar studies have to be performed with high numbered patient groups.

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.