Immunologic changes during desensitization with cow's milk: How it differs from natural tolerance or nonallergic state?

BACKGROUND: Oral immunotherapy (OIT) is a novel allergen-specific treatment for food allergies. OBJECTIVE: To investigate the effect of OIT on blocking antibodies, T cell regulation, and cytokine response during immunoglobulin (Ig)E-mediated cow's milk allergy (CMA) treatment. METHODS: A total of 59 children with IgE-mediated CMA who were followed in pediatric allergy outpatient clinic and 18 healthy children were included. The children were evaluated in the following 4 groups: OIT group, elimination group (patients receiving dairy elimination diet), tolerance group (patients who developed tolerance), and healthy control group. Milk-specific IgE, IgG4, and IgA levels, cow's milk induration diameters in skin prick test, CD4 + CD25 + FoxP3 + Treg cell percentages, messenger RNA (mRNA) expressions, and interleukin (IL)-10, transforming growth factor-beta (TGF-ß), IL-2, IL-4, and IL-13 cytokine levels were compared between the groups. RESULTS: The mean age of the patients was 42.6 ± 39 (6-201) months, and 63.6% (n = 49) of the patients were girls. We observed an increase in total IgE levels (P = .02), a decrease in cow's milk sIgE (P = .08, NS), and an increase in cow's milk component (ß-lactoglobulin and casein) specific IgA (P < .05) and IgG4 (P < .001) levels at 2 months after the maintenance phase of OIT. In addition, the immune response after OIT treatment, which had a 100% clinical success rate, was notable for similar CD4 + CD25 + FoxP3 + cell percentages (P = .8), and increased IL-10 (P = .04) levels and increased but statistically nonsignificant TGF-ß levels (P = .17) compared with those before treatment. FoxP3 mRNA expression was similar to that of patients who developed natural tolerance. Pretreatment and post-treatment FoxP3 mRNA-FoxP3 flow cytometric expressions were positively correlated with TGF-ß concentrations in the OIT group. CONCLUSION: A successful immune response to OIT was found, possibly through the blockage of IgE-mediated allergen presentation by blocking antibodies, marked IL-10 cytokine response, and TGF-ß response. FoxP3 mRNA expression was similar to the natural tolerance mechanism, but more studies are needed.

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.

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.

PI3K/mTOR dual-inhibition with VS-5584 enhances anti-leukemic efficacy of ponatinib in blasts and Ph-negative LSCs of chronic myeloid leukemia.

Ponatinib is used for advanced treatment of chronic myeloid leukemia (CML), although low doses to prevent side effects do not suppress survival pathways and eradicate leukemia stem cells (LSCs). We evaluated the potential of ponatinib and PI3K/mTOR dual-inhibitor VS-5584 combination (PoVS) therapy to increase the anti-leukemic effects of ponatinib and investigated the underlying mechanisms at the molecular level. We measured the cytotoxicities of ponatinib, VS-5584, and PoVS (CCK-8 assay), and used the median-effect equation for combination analyses. We investigated the effects of inhibitory concentrations on apoptosis, cell viability and cell-cycle regulation (flow cytometry), protein levels (ELISA, Western blot), transcriptional activities (dual-luciferase reporter assay), gene expressions (qRT-PCR). VS-5584 exerted selective cytotoxic effects against CML and LSC cell lines. VS-5584 inhibited the PI3K/Akt/mTOR pathway, resulting in reduced cell viability, slightly induced caspase-independent apoptosis, prominent G0/G1 cell-cycle blockade that is not a consequence of quiescence. Normal hematopoietic stem cell line was the least affected. Moreover, ponatinib and VS-5584 mediated synergistic anti-leukemic effects on leukemic cells. VS-5584 reduced the ponatinib dose required to target leukemic cells. PoVS treatment inhibited PI3K/Akt/mTOR pathway more consistently than either of the two agents alone through reducing p-Akt, p-mTOR, p-S6K, p-PRAS40, p-S6. The subsequent downstream effects were an increase in C/EBP transcriptional activity and decreases in activities of E2F/DP1, Myc/Max, CREB, STAT3, NFκB, AP-1, Elk-1/SRF. Transcriptional regulation resulted in alterations in the expression levels of target mRNAs. Our results highlight PoVS can be a promising treatment strategy for eliminating CML cells and LSCs selectively, with the reduced ponatinib doses.

Comparative effect of imatinib and ponatinib on autophagy and miRNome in chronic myeloid leukemia.

BCR-ABL tyrosine kinase inhibitors (TKIs) are selective therapies for the patients with Chronic Myeloid Leukemia (CML). Imatinib and ponatinib have remarkable long-term efficacy on a major molecular response. Although TKI related induction of cytotoxicity and apoptosis have been clearly investigated in molecular levels, their comparative effect on autophagy and miRNome are largely unknown. This study aimed to investigate the involvement of alterations of miRNA expressions in CML progression, and how imatinib and ponatinib affect this process, by comparing CML, imatinib-resistant CML and leukemia stem cells (LSC). Cytotoxicity analysis was conducted by WST-1, apoptosis was evaluated by AnnexinV, autophagy was analyzed by Tb/GFP TR-FRET LC3B assay and changes in miRNomes were evaluated with microarray method. Ponatinib showed higher cytotoxicity and apoptosis at far fewer concentrations than imatinib. Both imatinib and ponatinib was able to trigger autophagy in imatinib-resistant K562ima3 cell line but not in LSC. We pointed that imatinib and ponatinib caused significant miRNA profile alterations, especially in the expressions of miR-214-pre, miR-218, miR-19a-5p, miR-19b-1-5p, miR-27b-pre, miR-23b-pre, miR-320e, miR-200a-pre, miR-508-3p, miR-33-pre and miR-766. This study is the first comparative miRNome analysis of CML, resistant CML and LSCs following the imatinib or ponatinib treatment and may guide to identify new markers for diagnosis, follow-up of the disease and to develop novel therapeutic strategies if supported by preclinical studies.

The effect of tomatine on metastasis related matrix metalloproteinase (MMP) activities in breast cancer cell model.

Breast cancer is one of the most common malignancies in women and metastasis is the cause of morbidity and mortality in patients. In the development of metastasis, the matrix metalloproteinase (MMP) family has a very important role in tumor development. MMP-2 and MMP-9 work together for extracellular matrix (ECM) cleavage to increase migration. Tomatine is a secondary metabolite that has a natural defense role against plants, fungi, viruses and bacteria that are synthesized from tomato. In addition, tomatine is also known that it breaks down the cell membrane and is a strong inhibitor in human cancer cells. In this study, it was aimed to evaluate the effect of tomatine on cytotoxicity, apoptosis and matrix metalloproteinase inhibition in MCF-7 cell lines. Human breast cancer cell line (MCF-7) was used as a cell line. In MCF-7 cells, the IC50 dose of tomatine was determined to be 7.07µM. According to the control cells, apoptosis increased 3.4 fold in 48thh. Activation of MMP-2, MMP-9 and MMP-9\NGAL has been shown to decrease significantly in cells treated with tomatine by gelatin zymography compared to the control. As a result, matrix metalloproteinase activity and cell proliferation were suppressed by tomatine and this may provide support in treatment methods.

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

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

Genistein-induced mir-23b expression inhibits the growth of breast cancer cells.

AIM OF THE STUDY: Genistein, an isoflavonoid, plays roles in the inhibition of protein tyrosine kinase phosphorylation, induction of apoptosis, and cell differentiation in breast cancer. This study aims to induce cellular stress by exposing genistein to determine alterations of miRNA expression profiles in MCF-7 cells. MATERIAL AND METHODS: XTT assay and trypan blue dye exclusion assays were performed to examine the cytotoxic effects of genistein treatment. Expressions of miRNAs were quantified using Real-Time Online RT-PCR. RESULTS: The IC50 dose of genistein was 175 µM in MCF-7 cell, line and the cytotoxic effect of genistein was detected after 48 hours. miR-23b was found to be up-regulated 56.69 fold following the treatment of genistein. It was found that miR-23b was upregulated for MCF-7 breast cancer cells after genistein treatment. CONCLUSIONS: Up-regulated ex-expression of miR-23b might be a putative biomarker for use in the therapy of breast cancer patients. miR-23b up-regulation might be important in terms of response to genistein.

Therapeutic potential of an anti-diabetic drug, metformin: alteration of miRNA expression in prostate cancer cells.

BACKGROUND AND AIMS: Prostate cancer is the most commonly diagnosed cancer in males in many populations. Metformin is the most widely used anti-diabetic drug in the world, and there is increasing evidence of a potential efficacy of this agent as an anti-cancer drug. Metformin inhibits the proliferation of a range of cancer cells including prostate, colon, breast, ovarian, and glioma lines. MicroRNAs (miRNAs) are a class of small, non- coding, single-stranded RNAs that downregulate gene expression. We aimed to evaluate the effects of metformin treatment on changes in miRNA expression in PC-3 cells, and possible associations with biological behaviour. MATERIALS AND METHODS: Average cell viability and cytotoxic effects of metformin were investigated at 24 hour intervals for three days using the xCELLigence system. The IC50 dose of metformin in the PC-3 cells was found to be 5 mM. RNA samples were used for analysis using custom multi-species microarrays containing 1209 probes covering 1221 human mature microRNAs present in miRBase 16.0 database. RESULTS: Among the human miRNAs investigated by the arrays, 10 miRNAs were up-regulated and 12 miRNAs were down-regulated in the metformin-treated group as compared to the control group. In conclusion, expression changes in miRNAs of miR-146a, miR-100, miR-425, miR-193a-3p and, miR-106b in metformin-treated cells may be important. This study may emphasize a new role of metformin on the regulation of miRNAs in prostate cancer.

In vitro evaluation of (99m)Tc-EDDA/tricine-HYNIC-Q-Litorin in gastrin-releasing peptide receptor positive tumor cell lines.

Bombesin and its derivatives exhibit a high affinity for gastrin-releasing peptide receptor (GRPr), which is over-expressed in a variety of human cancers (prostate, pancreatic, lung, etc.). The aim of this study was to investigate the in vitro potential of the hydrazinonicotinamide (HYNIC)-Q-Litorin. (99m)Tc labeling was performed by using different co-ligands: tricine and ethylenediamine diacetic acid (EDDA). The radiochemical stability of radiolabeled peptide conjugates was checked at room temperature and in cysteine solution up to 24 h. The in vitro cell uptake of (99m)Tc-EDDA-HYNIC-Q-Litorin and (99m)Tc-tricine-HYNIC-Q-Litorin were evaluated on pancreatic tumor and control cell lines. Optimum specific activity and incubation time were determined for all the cell lines. The results showed that the cell uptake of the radiolabeled peptide conjugates in tumor cell lines were higher than in the control cell line. The findings of this study indicated the need for further development of in vivo study as a radiopharmaceutical for pancreatic tumor imaging.

The expression of URGCP gene in prostate cancer cell lines: correlation with rapamycin.

Molecular targets in prostate cancer are continually being explored, for which there are currently few therapeutic options. Rapamycin (RPM) is an antifungal macrolide antibiotic isolated from Streptomyces hygroscopicus which can inhibit the G1 to S transition. URGCP (upregulator of cell proliferation) is a novel gene located on chromosome 7p13. We aimed to investigate the role of URGCP gene expression changes in PC3, DU145, and LNCAP cell lines with/out RPM. Average cell viability and cytotoxic effect of rapamycin were investigated at 24 h intervals for three days by using Trypan blue dye exclusion test and XTT assay. Cytotoxic effects of rapamycin in DU145, PC3 and LNCAP cells were detected in time and dose dependent manner with the IC(50) doses within the range of 1-100 nM. As the results were evaluated, IC(50) doses in the DU145, PC3, and LNCaP cells were detected as 10, 25, and 50 nM, respectively. The mean relative ratios of URGCP gene expression in DU145, LNCAP and PC3 cells were found as -1.48, 6.59 and -13.00, respectively, when compared to rapamycin-free cells. The False Discovery Rate adjusted p value in DU145, LNCAP and PC3 were 1.25 × 10(-5), 2.20 × 10(-8) and 6.20 × 10(-9), respectively. When the URGCP gene expression level is compared between the dose and control group, we found that URGCP gene expression was significantly decreased in dose groups of DU145 and PC3 cells.