Ponatinib and STAT5 Inhibitor Pimozide Combined Synergistic Treatment Applications Potentially Overcome Drug Resistance via Regulating the Cytokine Expressional Network in Chronic Myeloid Leukemia Cells.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative hematological disease characterized by the chimeric breakpoint-cluster region/Abelson kinase1 (BCR::ABL1) oncoprotein; playing a pivotal role in CML molecular pathology, diagnosis, treatment, and possible resistance arising from the success and tolerance of tyrosine kinase inhibitor (TKI)-based therapy. The transcription factor STAT5 constitutive signaling, which is influenced by the cytokine signaling network, triggers BCR::ABL1-based CML pathogenesis and is also relevant to acquired TKI resistance. The unsuccessful therapeutic approaches targeting BCR::ABL1, in particular third-line therapy with ponatinib, still need to be further developed with alternative combination strategies to overcome drug resistance. As treatment with the STAT5 inhibitor pimozide in combination with ponatinib resulted in an efficient and synergistic therapeutic approach in TKI-resistant CML cells, this study focused on identifying the underlying amplification of ponatinib response mechanisms by determining different cytokine expression profiles in parental and ponatinib-resistant CML cells, in vitro. The results showed that expression of interleukin (IL) 1B, IL9, and IL12A-B was increased by 2-fold, while IL18 was downregulated by 2-fold in the ponatinib-resistant cells compared to sensitive ones. Importantly, ponatinib treatment upregulated the expression of 21 of the 23 interferon and IL genes in the ponatinib-resistant cells, while treatment with pimozide or a combination dose resulted in a reduction in the expression of 19 different cytokine genes, such as for example, inflammatory cytokines, IL1A-B and IL6 or cytokine genes associated with supporting tumor progression, leukemia stem cell growth or poor survival, such as IL3, IL8, IL9, IL10, IL12, or IL15. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that the genes were mainly enriched in the regulation of receptor signaling through the Janus kinase/signal transducer and activator of transcription pathway, cytokine-cytokine receptor interaction, and hematopoietic cell lineage. Protein-protein interaction analysis showed that IL2, IL6, IL15, IFNG, and others appeared in the top lists of pathways, indicating their high centrality and importance in the network. Therefore, pimozide could be a promising agent to support TKI therapies in ponatinib resistance. This research would help to clarify the role of cytokines in ponatinib resistance and advance the development of new therapeutics to utilize the STAT5 inhibitor pimozide in combination with TKIs.

Differentiation of Neurons, Astrocytes, Oligodendrocytes and Microglia From Human Induced Pluripotent Stem Cells to Form Neural Tissue-On-Chip: A Neuroinflammation Model to Evaluate the Therapeutic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells.

Advances in stem cell (SC) technology allow the generation of cellular models that recapitulate the histological, molecular and physiological properties of humanized in vitro three dimensional (3D) models, as well as production of cell-derived therapeutics such as extracellular vesicles (EVs). Improvements in organ-on-chip platforms and human induced pluripotent stem cells (hiPSCs) derived neural/glial cells provide unprecedented systems for studying 3D personalized neural tissue modeling with easy setup and fast output. Here, we highlight the key points in differentiation procedures for neurons, astrocytes, oligodendrocytes and microglia from single origin hiPSCs. Additionally, we present a well-defined humanized neural tissue-on-chip model composed of differentiated cells with the same genetic backgrounds, as well as the therapeutic potential of bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles to propose a novel treatment for neuroinflammation derived diseases. Around 100 nm CD9 + EVs promote a more anti-inflammatory and pro-remodeling of cell-cell interaction cytokine responses on tumor necrosis factor-α (TNF-α) induced neuroinflammation in neural tissue-on-chip model which is ideal for modeling authentic neural-glial patho-physiology.

Relationship between miRNA-21, miRNA-155, and miRNA-182 expression and inflammatory factors in cerebrospinal fluid from patients with multiple sclerosis.

BACKGROUND: The impact of microRNAs (miRNAs) on the differentiation and function of inflammatory cells is well-established. MiRNAs play a crucial role in modulating the expression of pro-inflammatory genes in neuronal cells as well. With this knowledge in mind, our study aimed to explore the relationship between the expression of miRNAs and inflammatory markers in the cerebrospinal fluid (CSF) of patients diagnosed with multiple sclerosis (MS). By investigating this relationship, we aimed to gain insights into the potential involvement of miRNAs in the regulation of inflammation in the context of MS. MATERIALS AND METHODS: The expression levels of miRNA-21, miRNA-155, and miRNA-182 in cerebrospinal fluid (CSF) samples from multiple sclerosis (MS) patients and controls were determined by RT-PCR. CSF levels of the inflammatory cytokines IL-1ß, IL-6, and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). In addition, high-sensitivity C-reactive protein (hs-CRP) levels were measured by quantitative turbidimetry. RESULTS: The expression levels of microRNAs and inflammatory factors were found to be significantly higher in the CSF of MS patients compared to controls (P < 0.05). Receiver operating characteristics (ROC) analysis revealed that miRNA-21, miRNA-182, and miRNA-155 had a high area under the curve (AUC) in discriminating MS patients, with AUC values of 0.97 (P < 0.0001) for miRNA-21, 0.97 (P < 0.0001) for miRNA-182, and 0.96 (P < 0.0001) for miRNA-155. Notably, CSF miRNA-155 showed the highest accuracy in correctly diagnosing MS. Furthermore, a statistically significant relationship was observed between inflammatory cytokines and miRNA-21, miRNA-155 and miRNA-182. CONCLUSION: Our results demonstrated that cerebrospinal fluid (CSF) levels of IL-1ß, IL-6, TNF-α, hs-CRP and specific miRNAs serve as biomarkers for assessing central nervous system (CNS) inflammation and neurodegenerative processes in patients with multiple sclerosis (MS).

Evaluation of the anticancer effect of telomerase inhibitor BIBR1532 in anaplastic thyroid cancer in terms of apoptosis, migration and cell cycle.

Anaplastic thyroid cancer (ATC) represents the type with the worst prognosis among thyroid cancers. In ATC with a highly invasive phenotype, selective targeting of TERT with BIBR1532 may be a goal-driven approach to preserving healthy tissues. In present study, it was aimed to investigate the effects of treatment of SW1736 cells with BIBR1532 on apoptosis, cell cycle progression, and migration. The apoptotic effect of BIBR1532 on SW1736 cells was examined using the Annexin V method, the cytostatic effect using cell cycle test, migration properties using wound healing assay. Gene expression differences were determined by real-time qRT-PCR and differences in protein level by ELISA test. BIBR1532-treated SW1736 cells had 3.1-fold increase in apoptosis compared to their untreated counterpart. There was 58.1% arrest in the G0/G1 phase and 27.6% arrest in the S phase of the cell cycle in untreated group, treatment with BIBR1532 increased cell population in G0/G1 phase to 80.9% and decreased in S phase to 7.1%. Treatment with the TERT inhibitor resulted in a 50.8% decrease in cell migration compared to the untreated group. After BIBR1532 treatment of SW1736 cells, upregulation of BAD, BAX, CASP8, CYCS, TNFSF10, CDKN2A genes, and downregulation of BCL2L11, XIAP, CCND2 genes were detected. BIBR1532 treatment resulted in an increase in BAX and p16 proteins, and a decrease in concentration of BCL-2 protein compared to untreated group. Targeting TERT with BIBR1532 as a mono drug or using of BIBR1532 at "priming stage" prior to chemotherapy treatment in ATC may present a novel and promising treatment strategy.

The effects of PKI-402 on breast tumor models' radiosensitivity via dual inhibition of PI3K/mTOR.

PURPOSE: PI3K/Akt/mTOR pathway activation causes relapse and resistance after radiotherapy in breast cancer (BC). We aimed to radiosensitize BC cell lines to irradiation (IR) by PKI-402, a dual PI3K/mTOR inhibitor. METHODS: We performed cytotoxicity, clonogenicity, hanging drop, apoptosis and double-strand break detection, and phosphorylation of 16 essential proteins involved in the PI3K/mTOR pathway. RESULTS: Our findings showed that PKI-402 has cytotoxic efficiency in all cell lines. Clonogenic assay results showed that PKI-402 plus IR inhibited the colony formation ability of MCF-7 and breast cancer stem cell lines. Results showed that PKI-402 plus IR causes more apoptotic cell death than IR alone in the MCF-7 cells but did not cause significant changes in the MDA-MB-231. γ-H2AX levels were increased in MDA-MB-231 in PKI-402 plus IR groups, whereas we did not observe any apoptotic and γ-H2AX induction in BCSCs and MCF-10A cells in all treatment groups. Some pivotal phosphorylated proteins of the PI3K/AKT pathway decreased, several proteins increased and others did not change. CONCLUSION: In conclusion, if the combined use of PKI-402 with radiation is supported by in vivo studies, it can contribute to the treatment options and the course of the disease.

Spatio-temporal dynamics enhance cellular diversity, neuronal function and further maturation of human cerebral organoids.

The bioengineerined and whole matured human brain organoids stand as highly valuable three-dimensional in vitro brain-mimetic models to recapitulate in vivo brain development, neurodevelopmental and neurodegenerative diseases. Various instructive signals affecting multiple biological processes including morphogenesis, developmental stages, cell fate transitions, cell migration, stem cell function and immune responses have been employed for generation of physiologically functional cerebral organoids. However, the current approaches for maturation require improvement for highly harvestable and functional cerebral organoids with reduced batch-to-batch variabilities. Here, we demonstrate two different engineering approaches, the rotating cell culture system (RCCS) microgravity bioreactor and a newly designed microfluidic platform (µ-platform) to improve harvestability, reproducibility and the survival of high-quality cerebral organoids and compare with those of traditional spinner and shaker systems. RCCS and µ-platform organoids have reached ideal sizes, approximately 95% harvestability, prolonged culture time with Ki-67 + /CD31 + /ß-catenin+ proliferative, adhesive and endothelial-like cells and exhibited enriched cellular diversity (abundant neural/glial/ endothelial cell population), structural brain morphogenesis, further functional neuronal identities (glutamate secreting glutamatergic, GABAergic and hippocampal neurons) and synaptogenesis (presynaptic-postsynaptic interaction) during whole human brain development. Both organoids expressed CD11b + /IBA1 + microglia and MBP + /OLIG2 + oligodendrocytes at high levels as of day 60. RCCS and µ-platform organoids showing high levels of physiological fidelity a high level of physiological fidelity can serve as functional preclinical models to test new therapeutic regimens for neurological diseases and benefit from multiplexing.

Distribution and Phylogenetic Analysis of Subtypes and Alleles of Blastocystis sp. in the Stool Samples Collected from Patients with Gastrointestinal Complaints in Izmir, Turkey.

PURPOSE: Blastocystis sp. is one of the most prevalent intestinal protozoa found in humans and many other animals. The present study aimed to examine the distribution and genetic diversity of Blastocystis sp. in stool samples from patients with gastrointestinal complaints in Izmir, Turkey. METHODS: All stool samples of 439 patients with gastrointestinal complaints were examined by native-Lugol and trichrome staining. To investigate the presence of Blastocystis sp. in stool samples, DNA was isolated, and PCR was performed with the barcode region in the SSU rRNA gene. PCR positive samples were sequenced to identify subtypes and alleles of Blastocystis sp. RESULTS: The prevalence of Blastocystis sp. was found to be 16.6% (73/439) in patients with gastrointestinal complaints in Izmir, Turkey. Three different Blastocystis sp. subtypes were identified. ST3 (28/55; 51.0%) was the most common subtype followed by ST2 (19/55; 34.5%) and ST1 (8/55; 14.5%). Itching and diarrhea were the most prominent clinical symptoms in Blastocystis sp. positive patients. When clinical symptoms and subtypes were compared, diarrhea was found in 62.5%, 47.4%, and 46.4% of patients with ST1, ST2, and ST3 subtypes, respectively. In addition, itching was found in 37.5%, 32.1%, and 21.1% of patients with ST1, ST3, and ST2, respectively. Six distinct alleles were identified by allele analysis of Blastocystis 18S rRNA gene: allele 4 for ST1, alleles 9, 11, and 12 for ST2, and alleles 34 and 36 for ST3. In this study, Blastocystis sp. was detected in 16 of 21 districts, including the central and rural districts of Izmir. Although ST1 was detected in central districts, it was not found in rural districts. CONCLUSION: This study provides comprehensive data on the prevalence and molecular epidemiology of the genetic diversity at the level of subtypes and alleles of Blastocystis sp. in different districts of Izmir province in Turkey. To the best of our knowledge, this is the first study which evaluates the distribution of subtypes and alleles of Blastocystis sp. according to PCR and SSU rRNA gene sequencing in patients with gastrointestinal complaints in different districts of Izmir province in Turkey.

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.

Epigenetic signatures in gastric cancer: current knowledge and future perspectives.

INTRODUCTION: Gastric cancer (GC) is the fifth most common malignancy in the world and accounts for 7.7% of all cancer-related deaths. Early diagnosis of GC is critical in terms of prognosis, and aberrations at the molecular level, especially epigenetic alterations, manifest much earlier than histological findings. In recent years, there has been a great deal of research on the epigenomic profile of GC, and epigenetic alterations seem to play a more important role than genetic factors. With the introduction of epigenetic drugs into clinical use in the last decade, the importance of the epigenetic background of GC has increased considerably. AREAS COVERED: In this review, we summarize the role of methylation changes, histone modifications, and non-coding RNAs in the pathogenesis of GC and how these signatures can be used as diagnostic and therapeutic targets in clinical management. EXPERT OPINION: Epigenetic alterations take place before most genetic aberrations observed in GC and may have an initiating role in the pathogenesis of GC. They can be used as biomarkers in risk calculation, early diagnosis, and evaluation of prognosis of GC, as well as treatment targets.

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.

Chk1/2 inhibitor AZD7762 enhances the susceptibility of IDH-mutant brain cancer cells to temozolomide.

The IDH mutation initially exhibits chemosensitive properties, progression-free survival cannot be achieved in the later grades, and malignant transformation occurs as a result of TMZ-induced hypermutation profile and adaptation to this profile. In this study, we evaluated the potential of the combination of TMZ and AZD7762 at molecular level, to increase the anticancer activity of TMZ in IDH-mutant U87-mg cells. We used the WST-1 test to evaluate cytotoxic effect of TMZ and AZD7762 combination with dose-effect and isobologram curves. The effects of the inhibitory and effective concentrations of the combination on apoptosis, cell cycle and γ-H2AX phosphorylation were analyzed with flow cytometry. The expression of genes responsible for the DNA damage response was analyzed with qRT-PCR. The combination showed a synergistic effect with high dose reduction index. Single and combined administrations of TMZ and AZD7762 increased in G2/M arrest from 24 to 48 h, and cells in the G2/M phase shifted towards octaploidy at 72 h. While no double-strand breaks were detected after TMZ treatment, AZD7762 and combination treatments caused a significant increase in γ-H2AX phosphorylation and increased apoptotic stimulation towards 72 h although TMZ did not cause apoptotic effect in IDH-mutant U87-mg cells. The genes controlling the apoptosis were determined to be upregulated in all three groups, and genes regarding cell cycle checkpoints were downregulated. Targeting Chk1/2 with AZD7762 simultaneously with TMZ may be a potential therapeutic strategy for both increasing the sensitivity of IDH-mutant glioma cells to TMZ and reducing the dose of TMZ. In IDH-mutant glioma cells, AZD7762, the Chk1/2 inhibitor, can increase the efficacy of Temozolomide by (i) increasing mitotic chaos, and (ii) inhibiting double-strand break repair, (iii) thereby inducing cell death.

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.

Molecular perspective on targeted therapy in breast cancer: a review of current status.

Breast cancer is categorized at the molecular level according to the status of certain hormone and growth factor receptors, and this classification forms the basis of current diagnosis and treatment. The development of resistance to treatment and recurrence of the disease have led researchers to develop new therapies. In recent years, most of the research in the field of oncology has focused on the development of targeted therapies, which are treatment methods developed directly against molecular abnormalities. Promising advances have been made in clinical trials investigating the effect of these new treatment modalities and their combinations with existing therapeutic treatments in the treatment of breast cancer. Monoclonal antibodies, tyrosine kinase inhibitors, antibody-drug conjugates, PI3K/Akt/mTOR pathway inhibitors, cyclin-dependent kinase 4/6 inhibitors, anti-angiogenic drugs, PARP inhibitors are among the targeted therapies used in breast cancer treatment. In this review, we aim to present a molecular view of recently approved target agents used in breast cancer.

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.

Genotyping and Molecular Identification of Acanthamoeba Genotype T4 and Naegleria fowleri from Cerebrospinal Fluid Samples of Patients in Turkey: Is it the Pathogens of Unknown Causes of Death?

PURPOSE: This study was aimed to investigate the presence of pathogenic free-living amoebae (FLA) in suspected cases of meningoencephalitis with unknown causes of death in Turkey. METHOD: A total of 92 patients, who were diagnosed as meningoencephalitis, were enrolled. All cerebrospinal fluid (CSF) samples were directly microscopically examined and cultured. Acanthamoeba, N. fowleri and B. mandrillaris were further investigated using molecular diagnostic tools including real-time PCR, sequencing, and phylogenetic analyses. RESULTS: The examined CSF samples were not found positive for the presence of FLA by microscopic examination and culture method. However, two CSF samples were detected positive by real-time PCR assay. Of the positive CSF samples, one was identified as Acanthamoeba genotype T4 and the second positive sample was identified as N. fowleri belonging to genotype II. Furthermore, the pathogens diagnoses was verified through Sanger sequencing. CONCLUSION: This study was significant to report the presence of Acanthamoeba genotype T4 and N. fowleri genotype II in CSF samples by real-time PCR assay. The present study shows the significance of primary amoebic meningoencephalitis (PAM) and granulomatous amoebic encephalitis (GAE) as one of the differential diagnoses to be considered by clinicians during the evaluation of suspected meningoencephalitis or cases of unknown cause in Turkey. Using real-time PCR, this has made the rapid detection, in a short time-frame, of Acanthamoeba and N. fowleri in CSF samples from patients. The problems with qPCR is that it is not available in every laboratory, reagents are expensive, and it requires skilled and expert personnel to set up these assays.

Targeting UPR signaling pathway by dasatinib as a promising therapeutic approach in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disease that mediated by BCR/ABL oncogenic signaling. CML can be targeted with the imatinib, dasatinib, and nilotinib TKI inhibitors, the latter two of them have been approved for imatinib-resistant or -intolerant CML patients. The TKIs resistance occurs by different molecular mechanisms, including overexpression of BCR-ABL, mutations in the TKI binding site of BCR/ABL, and ER-stress. Unfolded protein responses (UPR) is a cytoprotective mechanism which is activated by ER-stress. The IRE1, PERK, and ATF6 are three main arms of the UPR mechanism and are activated by a common mechanism involving the dissociation of the ER-chaperone BiP/GP78. There is a correlation between ER-stress, CML progression, and response to TKI treatment. In the present study, we aimed to determine alterations of the expression levels of genes related to UPR pathway signaling after treatment with dasatinib in K562 chronic myeloid leukemia cell line by quantitative RT-PCR relatively. The array-data revealed that treatment with dasatinib significantly decreased the UPR mechanism-related genes (including HSPA1B, HSPA2, HSPA4L, ATF6, ATF6B, CEBPB, PERK, TRIB3, DNAJB, ERN1, and UHRF1) in K562 cells. In conclusion, the results showed that dasatinib regulates the UPR mechanism that plays a significant role in cancer progression and therapy resistance in CML. Thus, dasatinib-induced dysfunction of the UPR mechanism may promise encouraging therapy for CML.

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 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.

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.