C-kit Protein Research Articles

Loss of c-KIT expression in breast cancer correlates with malignant transformation of breast epithelium and is mediated by KIT gene promoter DNA hypermethylation

KIT Proto-Oncogene Receptor Tyrosine Kinase (KIT) is a transmembrane receptor tyrosine kinase which plays an important role in regulation of cell proliferation, survival and migration. Interestingly, the role of c-KIT in malignant transformation seems to be highly tissue-specific and it can act either as an oncogene or tumor suppressor gene. Here we analyzed the expression of c-KIT in normal breast tissues and tissues from different stages encompassing major steps of breast tumor development. Our study showed, that the c-KIT protein expression is gradually lost during the process of breast tissue transformation. The analysis of previously published datasets revealed that c-KIT expression in breast malignancies was downregulated at mRNA level. Because sequencing studies did not identify any recurrent mutations or copy number alterations, we proposed a potential epigenetic mechanism for the downregulation of c-KIT expression. In-silico analysis of the KIT promoter revealed the presence of CpG islands, therefore we performed bisulfite sequencing of normal breast epithelial tissues as well as breast tumor samples. We found, that KIT promoter is hypermethylated in breast tumors compared to normal breast tissues. Furthermore, treatment of breast cancer cell lines, that lack the expression of c-KIT, with methyltransferase inhibitor 5-Azacytidine (5Aza-2dC) resulted in increased expression of c-KIT mRNA. Collectively, our studies demonstrate that c-KIT expression is epigenetically downregulated during breast epithelium transformation and cancer development via KIT promoter hypermethylation.

A naphthalene diimide G-quadruplex ligand inhibits cell growth and down-regulates BCL-2 expression in an imatinib-resistant gastrointestinal cancer cell line

Gastro-intestinal tumours (GISTs) are driven by aberrant expression of the c-KIT oncoprotein. They can be effectively treated by the kinase inhibitor imatinib, which locks the c-KIT kinase domain into an inactive conformation. However resistance to imatinib, driven by active-site mutations, is a recurrent clinical challenge, which has been only partly met by the subsequent development of second and third-generation c-KIT inhibitors.It is reported here that a tetra-substituted naphthalene diimide derivative, which is a micromolar inhibitor of cell growth in a wild-type patient-derived GIST cell line, has a sub-micromolar activity in two distinct patient-derived imatinib-resistant cell lines. The compound has been previously shown to down-regulate expression of the c-KIT protein in a wild-type GIST cell line. It does not affect c-KIT protein expression in a resistant cell line to the same extent, whereas it profoundly down-regulates the expression of the anti-apoptopic protein BCL-2. It is proposed that the mechanism of action involves targeting quadruplex nucleic acid structures, and in particular those in the BCL-2 gene and its RNA transcript. The BCL-2 protein is up-regulated in the GIST-resistant cell line, and is strongly down-regulated after treatment. The compound strongly stabilises a range of G-quadruplexes including a DNA one from the BCL-2 promoter and an RNA quadruplex from its 5′-UTR region. A reporter assay construct incorporating the 5′-UTR quadruplex sequence demonstrates down-regulation of BCL-2 expression.

The role of HCN channels in peristaltic dysfunction in human ureteral tuberculosis

ObjectiveTo explore the role of HCN channels in ureteral peristaltic dysfunction by comparing the changes in HCN channel levels between normal and tuberculous ureters.MethodsA total of 32 specimens of human upper ureters were collected by nephrectomy from patients with renal tumor (control group, n = 16) or from patients with renal tuberculosis (experimental group, n = 16); the two groups did not receive radiotherapy, chemotherapy, immunotherapy, or any other special treatment before the surgical procedure. An experimental study on smooth muscle strips of human upper ureters showed variation in contraction amplitude and frequency after adding ZD7288, a specific blocker of HCN channels. The expression of HCN channels in the ureter was confirmed by Western blot (WB) and by confocal analysis of double immunostaining for c-kit and HCN channel proteins.ResultsBefore the addition of ZD7288, the experimental and control groups showed significant differences in the frequency and amplitude of the spontaneous contraction of isolated ureteral smooth muscle strips. After ZD7288 was added, the frequency and amplitude of the contractions of the ureteral smooth muscle strips were significantly lower in both groups. The differences observed before and after ZD7288 treatment in each group were significant (P < 0.001), and the difference in contraction amplitude observed between the two groups before ZD7288 was also significantly different (P < 0.001). By using WB technology, we showed that the expression of HCN channels was present in normal human ureters, with the expression of HCN4 and HCN1 being the highest; the expression of HCN4 and HCN1 in the control and experimental groups were both statistically significant (P < 0.001). HCN4 and HCN1 were expressed in the mucosal and smooth muscle layers of human control ureters and tuberculous ureters, as revealed by a confocal analysis of double immunostaining for c-kit and HCNs proteins; there were significant differences between the two groups (P < 0.001).ConclusionFour HCN channels are expressed in the ureter, mainly HCN4 and HCN1, suggesting that HCN channels are involved in the peristaltic contraction of ureteral ICCs, which may be an important reason for peristaltic dysfunction in ureteric tuberculosis.

Acute Cholecystitis Reduces Interstitial Cells of Cajal in Porcine Gallbladder Through Decreased mRNA Synthesis

Background/Aims: Acute cholecystitis is a common gastrointestinal disorder, often characterized by acute cholecystitis with gallbladder motility disorder. Interstitial cells of Cajal (ICCs) are the pacemaker cells of gut motility in the gastrointestinal tract. Disruption of ICC function is related to motility disorders. The aim of this study was to explore the cellular and molecular mechanisms of ICCs in acute cholecystitis and after the resolution of acute inflammation. Materials and Methods: Fifty adult guinea pigs were randomly divided into five groups: a sham-administered group (control group); two groups that were intraperitoneally administered an anti-polyclonal neutrophil (PMN) antibody 24 h before common bile duct ligation (CBDL); and two groups of guinea pigs that were subjected to CBDL without receiving the PMN antibody. Guinea pigs that underwent CBDL were held for 24 h or 48 h after surgery before being subjected to laparotomy and cholecystectomy. Immunohistochemistry, TUNEL assays, western blotting, and real-time PCR were performed to determine ICC morphology and density, to detect ICC apoptosis, and to examine stem cell factor (SCF) and c-kit protein expression and SCF and c-kit mRNA levels, respectively. Results: Both hematoxylin-eosin staining and histological inflammation scores in the PMN groups were lower than those in the control groups (P < 0.01). No differences were observed in ICC morphology between groups. During acute cholecystitis, ICCs numbers were reduced. Conversely, the density of ICCs increased after inflammation was relieved (P < 0.01). In addition, SCF and c-kit protein and mRNA expression levels decreased during acute cholecystitis (P < 0.05) and increased after inflammation was relieved (P < 0.05). Furthermore, ICC apoptosis increased during acute cholecystitis and decreased after resolution of acute cholecystitis (P < 0.01). Conclusions: In acute cholecystitis, ICC injury may be related to gallbladder motility disorder.

Effect of bisacodyl on rats with slow transit constipation.

The effect of bisacodyl on the treatment of rats with slow transit constipation (STC) was studied. Forty-five female Wister rats were divided into control group, STC group, and STC bisacodyl group. The immunohistochemical method was used to determine interstitial cells of Cajal (ICC) and the expression of c-Kit protein. Body mass and the number of defecations were significantly decreased in the STC group compared with the control group on the 100th day after diphenoxylate administration, while dry weight of feces was significantly increased and the intestinal transit time was prolonged. There were significant differences in the number of defecations, dry weight of feces, and intestinal transit time among the three groups. The number of defecations was higher, dry weight of feces was lower, and intestinal transit time was shorter in the STC bisacodyl group compared to the STC group. In addition, ICC basement membrane dissolution occurred in the colon wall of the STC group. The connection between ICC and surrounding cells was destroyed, and the nucleus shrunken to different degrees. Moreover, c-Kit expression in the STC group was significantly lower than the control group. The connection between ICC and surrounding cells in the STC bisacodyl group was significantly stronger than the STC group, and the number of ICC and the expression of c-Kit were increased. Bisacodyl could reduce the severity of STC in rats by increasing the number of ICC and the expression of c-Kit.

Stage-specific expression of DDX4 and c-kit at different developmental stages of the porcine testis

Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis.

C-Kit receptor and tryptase expressing mast cells correlate with angiogenesis in breast cancer patients.

C-Kit protein is a transmembrane tyrosine kinase (TK) receptor (c-KitR-TK), which is predominantly expressed on mast cells (MCs) playing a role in tumor angiogenesis. It could be also expressed on epithelial breast cancer cells (EBCCs), but no data have been published regarding the correlation between mast cells positive to c-KitR (MCs-c-KitR), EBCCs positive to c-KitR (EBCCs-c-KitR), BC angiogenesis in terms of microvessel density (MVD) and the main clinic-pathological features. This study aims to evaluate the above parameters and their correlations in a series of selected 121 female early BC patients. It has been found a strong correlation between MVD and MCDPT, and MCs-c-KitR, MVD and MCs density positive to tryptase (MCDPT), and MCs-c-KitR and MCDPT by Pearson correlation. These data suggest an involvement of both MCDPT and MCs-c-KitR in BC tumor angiogenesis. Furthermore, BC tissue expressing c-KitR could be a putative predictive factor to c-KitR-TK inhibitors. In this way, selected patients with higher MCs-c-KitR could be candidate to receive c-KitR-TK inhibitors (e.g. masitinib, sunitinib) or tryptase inhibitors (e.g. nafamostat mesilate, gabexate mesilate).

183 In Vitro Generation and Characterization of Putative Primordial Germ Cells Derived from Induced Pluripotent Stem Cells in Cattle

Embryonic pluripotent stem cells (ESC) and induced pluripotent stem cells (iPSC) were reported capable of differentiating into primordial germ cell-like (PGCL) and functional gametes in vitro in the murine model (Hikabe et al. 2016 Nature 539, 299-303). The in vitro generation of primordial germ cells (PGC) and gametes from farm animals would greatly contribute to enhance animal production technologies and to the creation of adequate models for several disorders. The present study aimed at the generation of PGC in vitro (iPGC) from iPSC in cattle and their characterisation through pluripotency and germ cell markers. For that, bovine iPSC previously generated and characterised (Bressan et al. 2015 Reprod. Fertil. Dev. 27, 254) were submitted to in vitro differentiation into epiblast-like cells (EpiLC) and iPGC by the protocol adapted from mice (Hayashi et al. 2011 Cell 146, 519-532). The biPS cells were induced into EpiLC by culture in fibronectin-coated (16.7 µg mL−1) 6-well plates in N2B27 culture medium supplemented with 20 ng mL−1 activin A, 12 ng mL−1 basic fibroblast growth factor (bFGF), and 1% knockout serum replacement (KSR) for 48 h and further differentiated into iPGC by non-adherent culture (Agreewell plates, StemCell Technologies, Vancouver, BC, Canada) with GK15 medium (GMEM supplemented with 15% KSR, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 2 mm l-glutamine, and 1% antibiotics) in the presence of 500 ng mL−1 BMP4, 100 ng mL−1 SCF, 500 ng mL−1 BMP8b, and 50 ng mL−1 epidermal growth factor for 4 days. The cells were then characterised regarding morphology, detection of alkaline phosphatase, immunofluorescence for OCT4, DDX4, VASA, and c-Kit proteins, and transcripts of pluripotency-related genes OCT4 and SOX2, as well as of imprinted genes (H19, SNRPN) and imprinted-related (DNMT1, DNMT3B) genes were analysed through RT-qPCR and compared with constitutive genes GAPDH, NAT1, and ACTB. Alkaline phosphatase and immunofluorescence analysis were positive for all specific markers. Interestingly, although OCT4 and SOX2 expression was present in iPS, EpiLC, and iPGC, this last group presented greater OCT4 and lesser SOX2 transcript amounts compared with other groups, suggesting, as expected, that PGC are still pluripotent but may already be differentiating into germ-cell lineages. The expression of H19 was increased in iPGC, whereas the expression of SNRPN was decreased only in the fibroblast group, potentially indicating epigenetic reprogramming process in these cells. Expression of DNMT1 and DNMT3B was not different between pluripotent groups but subtly increased when compared with that in fibroblasts. The results obtained herein represent an important first step in the in vitro generation of PGC and gametes from domestic farm animals, an unprecedented and desirable tool for enhancing new reproductive technologies and providing new understanding of cellular reprogramming and pluripotent germ cell biology. Financially supported by FAPESP grants 2013/08135-2, 2013/13686-8, 2015/26818-5; CNPq 482163/2013-5.

Investigation of c-KIT and Ki67 expression in normal, preneoplastic and neoplastic canine prostate

Backgroundc-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines.ResultsEleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive.ConclusionThe present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer.

Putative genomic characteristics of BRAF V600K versus V600E cutaneous melanoma.

Approximately 50% of all cutaneous melanomas harbor activating BRAF V600 mutations; among, these 10-30% carry the V600K mutation. Clinically, patients with V600K tumors experience distant metastases sooner and have an increased risk of relapse and shorter survival than patients with V600E tumors. Despite the clinical and other histopathological differences between these BRAF tumor subtypes, little is known about them at the genomic level. Herein, we systematically compared BRAF V600E and V600K skin cutaneous melanoma (SKCM) samples from the Cancer Genome Atlas (TCGA) for differential protein, gene, and microRNA expression genome-wide using the Mann-Whitney U-test. Our analyses showed that elements of energy-metabolism and protein-translation pathways were upregulated and that proapoptotic pathways were downregulated in V600K tumors compared with V600E tumors. We found that c-Kit protein and KIT gene expressions were significantly higher in V600K tumors than in V600E tumors, concurrent with significant downregulation of several KIT-targeting microRNAs (mir) including mir-222 in V600K tumors, suggesting KIT and mir-222 might be key genomic contributors toward the clinical differences observed. The relationship that we uncovered among KIT/c-Kit expression, mir-222 expression, and growth and prosurvival signals in V600 tumors is intriguing. We believe that the observed clinical aggressiveness of V600K tumors compared to V600E tumors may be attributable to the increased energy metabolism, protein translation and prosurvival signals compared with V600E tumors. If confirmed using larger numbers of V600K tumors, our results may prove useful for designing clinical management and targeted chemotherapeutical interventions for BRAF V600K-positive melanomas. Finally, the small sample size in V600K tumors is a major limitation of our study.

Changes in the interstitial cells of Cajal and neuronal nitric oxide synthase positive neuronal cells with aging in the esophagus of F344 rats.

The aging-associated cellular and molecular changes in esophagus have not been established, yet. Thus we evaluated histological structure, interstitial cells of Cajal (ICCs), neuronal nitric oxide synthase (nNOS)-positive cells, and contractility in the esophagus of Fischer 344 rat at different ages (6-, 31-, 74-weeks, and 2-years). The lamina propria thickness and endomysial area were calculated. The immunoreactivity of c-Kit, nNOS and protein gene product (PGP) 9.5 was counted after immunohistochemistry. Expression of c-Kit, stem cell factor (SCF), nNOS and PGP 9.5 mRNA was measured by real-time PCR, and expression of c-Kit and nNOS protein was detected by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The lamina propria thickness increased (6 week vs 2 year, P = 0.005) and the endomysial area of longitudinal muscle decreased with aging (6 week vs 2 year, P<0.001), while endomysial area of circular muscle did not significantly decrease. The proportions of NOS-immunoreactive cells and c-Kit-immunoreactive areas declined with aging (6 week vs 2 year; P<0.001 and P = 0.004, respectively), but there was no significant change of PGP 9.5-immunopositiviy. The expressions of nNOS, c-Kit and SCF mRNA also reduced with aging (6 week vs 2 year; P = 0.006, P = 0.001 and P = 0.006, respectively), while the change of PGP 9.5 mRNA expression was not significant. Western blot showed the significant decreases of nNOS and c-Kit protein expression with aging (6 week vs 2 year; P = 0.008 and P = 0.012, respectively). The EFS-induced esophageal contractions significantly decreased in 2-yr-old rat compared with 6-wk-old rats, however, L-NG-Nitroarginine methylester did not significantly increase the spontaneous and EFS-induced contractions in the 6-wk- and 2-yr-old rat esophagus. In conclusion, an increase of lamina propria thickness, a decrease of endomysial area, c-Kit, SCF and NOS expression with preserved total enteric neurons, and contractility in aged rat esophagus may explain the aging-associated esophageal dysmotility.

Correlation between acute gastrointestinal injury severity and intestinal microenvironment in rabbits with severe multiple trauma

Objective To investigate the correlation between acute gastrointestinal injury severity and intestinal microenvironment in rabbits with severe multiple trauma. Methods A total of 60 New Zealand white rabbits were enrolled and randomly assigned into the experimental group (48 rabbits) and control group (12 rabbits). In experimental group, the models of traffic-induced injuries were successful made by using self-made small gravity traction colliders. There were nine rabbits with craniocerebral injury combined with damage of liver and spleen, three with four extremity fractures combined with damage of liver and spleen, 12 with rib fractures combined with damage of lungs and pleural effusion, 11 with epidural hematoma, contusion and laceration of brain as well as fractures of four extremities and pelvis, and nine with multiple fractures. The injuries had met the criteria of multiple severe traumas according to the injury severity score (ISS). The control group had similar condition with experimental group except for participation in injury model. The parameters at time points of 6, 12, 24 and 48 h after injury were observed. The parameters were: (1)Enzyme linked immunosorbent assay (ELISA) and high pressure liquid chromatography (HPLC) were used to detect the levels of diamine oxidase (DAO) and the ratio of lactulose to mannitol in urine in order to evaluate the permeability of intestinal mucosal barrier. (2)The small intestinal propulsive rate was detected by carbon pushing method. The interstitial cells of Cajal in the small intestine wall were observed by transmission electron microscopy. The expression of C-kit was detected by Western blot. By these means, the dynamic function of intestinal mucosal barrier was evaluated. (3)The damage degree of intestinal mucosal barrier was evaluated by pathological observation and Chiu score in the end of small intestine and colon. (4)The intestinal bacterial translocation was evaluated by intestinal microflora culture, mesenteric lymph nodes checking as well as translocation examination of liver and spleen. Results (1)The level of DAO plasma and urine lactulose/mannitol ratio at 6 h after injury increased to varied degrees, and reached the peak during 12-24 h, the correlation analysis of which showed that plasma DAO levels were positively correlated with AGI grade at 6, 12 and 24 h (r=0.486 3, 0.493 3, 0.477 6, P<0.05). The ratio of urinary lactulose/mannitol excretion was only positively correlated with AGI grade at 6 and 12 h (r=0.478 5, 0.497 2, P<0.05). (2)The gastrointestinal transmission rate in the experimental group was (48.2±5.2)%, and that of the control group was (60.3±3.0)%. The gastrointestinal transmission rate of model group rabbits was significantly longer than that of control group (P<0.05). (3)The interstitial cells of Cajal decreased dramatically during 12-24 h after the injury, and the expression level of C-kit protein decreased consistently with the increase of AGI grade. (4)The intestinal and colonic mucosa tissues of the experimental group showed obvious necrosis at 12-24 h after injury, and the Chiu score increased gradually with the increase of AGI grade under light microscope. (5)The Enterobacter and Enterococcus in the experimental group increased significantly compared with that in the control group. But the numbers of Bifidobacterium, Lactobacillus, Lactobacillus, Bacteroides as well as the ratio of Bifidobacterium to Enterobacteriaceae significantly decreased(P<0.05). (6)The organ bacterial translocation rates were 24%, 42% and 62% after injury in experimental group (P<0.05). Conclusions Acute gastrointestinal injury may occur early after severe multiple trauma, and the injury severity is closely related to the change of intestinal barrier function and bacterial translocation. Early attention and active correction of the change of intestinal microenvironment are of great importance for treatment of multiple trauma. Key words: Multiple traumas; Intestinal mucosa; Bacterial translocation; Acute gastrointestinal injury

N-Acetylcysteine Compared to Metformin, Improves The Expression Profile of Growth Differentiation Factor-9 and Receptor Tyrosine Kinase c-Kit in The Oocytes of Patients with Polycystic Ovarian Syndrome.

BackgroundParacrine disruption of growth factors in women with polycystic ovarian syndrome (PCOS) results in production of low quality oocyte, especially following ovulation induction. The aim of this study was to investigate the effects of metformin (MET), N-acetylcysteine (NAC) and their combination on the hormonal levels and expres- sion profile of GDF-9, BMP-15 and c-kit, as hallmarks of oocyte quality, in PCOS patients.Materials and MethodsThis prospective randomized, double-blind, placebo controlled trial aims to study the effects of MET, NAC and their combination (MET+NAC) on expression of GDF-9, BMP-15 and c-kit mRNA in oocytes [10 at the germinal vesicle (GV) stage, 10 at the MI stage, and 10 at the MII stage from per group] derived following ovulation induction in PCOS. Treatment was carried out for six weeks, starting on the third day of previous cycle until oocyte aspiration. The expression of GDF9, BMP15 and c-kit were determined by quantitative real time polymerase chain reaction (RT-qPCR) and western blot analysis. Data were analyzed with one-way ANOVA.ResultsThe follicular fluid (FF) level of c-kit protein significantly decreased in the NAC group compared to the other groups. Significant correlations were observed between the FF soluble c-kit protein with FF volume, androstenedione and estradiol. The GDF-9 expression in unfertilized mature oocytes were significantly higher in the NAC group com- pared to the other groups (P<0.001). Similar difference was not observed between the MET, NAC+MET and control groups. The c-kit expression in unfertilized mature oocytes were significantly lower in the NAC group compared to the other groups (P<0.001). Similar difference was not observed between the MET, NAC+MET and control groups (Registration number: IRCT201204159476N1).ConclusionWe concluded that NAC can improve the quality of oocytes in PCOS.

S-allylcysteine Improves Blood Flow Recovery and Prevents Ischemic Injury by Augmenting Neovasculogenesis

Studies suggest that a low level of circulating human endothelial progenitor cells (EPCs) is a risk factor for ischemic injury and coronary artery disease (CAD). Consumption of S-allylcysteine (SAC) is known to prevent CAD. However, the protective effects of SAC on the ischemic injury are not yet clear. In this study, we examined whether SAC could improve blood flow recovery in ischemic tissues through EPC-mediated neovasculogenesis. The results demonstrate that SAC significantly enhances the neovasculogenesis of EPCs in vitro. The molecular mechanisms for SAC enhancement of neovasculogenesis include the activation of Akt/endothelial nitric oxide synthase signaling cascades. SAC increased the expression of c-kit, β-catenin, cyclin D1, and Cyclin-dependent kinase 4 (CDK4) proteins in EPCs. Daily intake of SAC at dosages of 0.2 and 2 mg/kg body weight significantly enhanced c-kit protein levels in vivo. We conclude that dietary consumption of SAC improves blood flow recovery and prevents ischemic injury by inducing neovasculogenesis in experimental models.

Abstract 463: MicroRNA-7 suppresses RB1 expression leading to chromosomal instability in leukemia cells harboring c-Kit mutation

Abstract C-Kit mutation D816V is a well-known indicator of poor prognosis in acute myeloid leukemia (AML) harboring t(8;21) chromosomal translocations. However, the mechanism the D816V mutation promotes therapeutic resistance is still under investigation. C-Kit V814 mutation is a murine counterpart of the human D816V. We utilized a murine IL-3 dependent cell line Ba/F3 with or without the c-Kit V814 mutation to investigate its downstream signaling pathway and the effect on the expression of bcl-2 family members and critical granulocytic transcription factors. The c-Kit V814 and wild type (WT) c-Kit were retrovirally transduced into Ba/F3 cells. Unexpectedly, Gata1 mRNA was significantly downregulated to one-tenth in V814+Ba/F3 cells (p &amp;lt; 0.01), contrary to the previous reports that WT c-Kit signaling induces Gata1 expression. When V814+Ba/F3 cells were treated with several c-Kit signaling pathway inhibitors, mRNA and protein expression levels of Gata1 recovered to normal levels only with MEK 1/2 inhibitor (PD325901) treatment. We then hypothesized that miRNAs might control Gata1 transcription. We used an array to identify differences in miRNA expression between Ba/F3 cells and V814+Ba/F3 cells, and between V814+Ba/F3 cells treated with or without PD325901. Only miR-7a-5p and miR-706 were significantly upregulated in V814+Ba/F3 cells compared with Ba/F3 without the V814 mutation, and downregulated (&amp;gt; 2-fold) in cells treated with PD325901 compared with controls. We focused our further analysis on miR-7 as it is highly conserved in vertebrates. The differences in miR-7 expression were confirmed using qRT-PCR (10.5-fold change, p &amp;lt; 0.01). In silico analysis implicated Rb1 as a candidate miR-7 target that may regulate Gata1 transcription. Luciferase reporter constructs containing murine and human Rb1 miR-7 target sequence exhibited 0.51 and 0.49 times lower luciferase activity than the controls, respectively (p &amp;lt; 0.01). To further determine the biological significance of the c-Kit mutation that would transform the cells to more aggressive phenotype, we focused on DNA and chromosomal instability caused by RB1 deterioration. We exposed WT c-Kit Ba/F3 and V814+Ba/F3 cells to 4 Gy irradiation and then used immunohistochemistry to analyze the frequency of γH2AX foci in the cells. The percentage of cells with more than 10 γH2AX foci was significantly higher in V814+Ba/F3 cells than WT Ba/F3 cells (27% vs. 14%, p &amp;lt; 0.05), Here, we clearly show that a single mutation of c-Kit is sufficient to regulate miR-7 expression leading to RB1 translational suppression. Since c-Kit mutation requires only a single step to inhibit RB1, this mechanism is more likely to facilitate oncogenesis than bi-allelic RB1 chromosomal and/or gene alterations in leukemia cells. Taken together, we identified miR-7 as a suppressor of RB1 in V814 mutation-positive cells, which might reflect the responsibility of D816V to refractory feature in AML. Citation Format: Keiji Kurata, Shinichiro Kawamoto, Ryota Masutani, Kimikazu Yakushijin, Katsuya Yamamoto, Hiroshi Matsuoka, Takayuki Takubo, Hironobu Minami. MicroRNA-7 suppresses RB1 expression leading to chromosomal instability in leukemia cells harboring c-Kit mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 463. doi:10.1158/1538-7445.AM2017-463

The Dome Wall of Bladder Acts as a Pacemaker Site in Detrusor Instability in Rats.

BackgroundThe aim of this study was to confirm that the interstitial cells of Cajal (ICCs) in the dome wall of the bladder are pacemaker cells, and that the dome wall of the bladder acts as a pacemaker site in the detrusor instability (DI) rat model.Material/MethodsThe model of DI in Wistar rats was established and urodynamic studies measuring the bladder volume and pressure were performed. The detrusor excitability was investigated using the amplitude and frequency of phasic contraction of strips. The localization and quantity of ICCs was identified by immunohistochemistry and c-KIT protein expression in the rat bladder. PCR assay and Western blot were used to assess the expression of HCN2 and Cx43.ResultsThe bladder capacity, residual volume, voiding volume, and maximum voiding pressure were significantly increased in the DI group. The contraction frequency and amplitude of the strips from the dome of the bladder in the DI group were higher than the triangle, body, and base parts. Both the concentration of c-KIT positive ICCs cells and expression of the c-KIT protein in the dome wall were higher than in other parts of the bladder. The expression of HCN2 and Cx43 in each part of the DI rat group were obviously higher than each part in the control group. Compared to the body, base, and triangle parts, the expression of HCN2 and Cx43 in the dome wall were obviously higher in the DI group.ConclusionsThe quantity of ICCs was higher in the dome wall and the dome wall of bladder acts as a pacemaker site in the DI rat model.

Effect of acupuncture at points selected from different regions on SCF-kit signaling pathway in diabetic gastroparesis rats

To observe the effect of acupuncture at points selected from different regions on the positive expression of interstitial cells of Cajal (ICC) and the stem cell factor (SCF) in gastric antrum tissues in diabetic gastroparesis (DGP) rats, and to explore the influence of region-based point selection on the acupoint combination efficacy. Sixty Sprague-Dawley (SD) rats were randomly divided into a normal group (group A), a model group (group B), a group of Zusanli (ST 36) plus Zhongwan (CV 12) (group C), a group of Zusanli (ST 36) plus Neiguan (PC 6) (group D), and a group of Zusanli (ST 36) plus non-meridian non-acupoint points (group E), based on the random number table (n=12). DGP rat model was established by single intraperitoneal injection of 2% streptozotocin and common diet. After successful modeling, the rats were treated once a day for 4 weeks. Positive ICC and SCF expressions were measured by immunohistochemistry. Compared with group A, the gastrointestinal propulsion rate of group B showed a statistically significant decrease (P<0.05). Compared with group B, the gastrointestinal propulsion rate and the expression of ICC in the gastric antrum were significantly higher in group C, group D and group E, and the between-group differences were statistically significant (P<0.05); the expression of C-kit protein in group C was statistically significantly higher than that in group D and group E (P<0.05). The expression of SCF protein was significantly increased in group C than in group B, and the difference was statistically significant (P<0.05). Acupuncture can improve the symptoms of delayed gastric emptying in DGP model rats, and regulate the expression of ICC and SCF in gastric antrum tissues. The effect of Zusanli (ST 36) plus Zhongwan (CV 12) in the gastric region is superior to that of the Zusanli (ST 36) plus distal Neiguan (PC 6) or non-meridian non-acupoint point, indicating that region-based point selection is the key factor affecting the effect of acupoint combination.

Circulating-free DNA Mutation Associated with Response of Targeted Therapy in Human Epidermal Growth Factor Receptor 2-positive Metastatic Breast Cancer.

Background:The addition of anti-human epidermal growth factor receptor 2 (HER2)-targeted drugs, such as trastuzumab, lapatinib, and trastuzumab emtansine (T-DM1), to chemotherapy significantly improved prognosis of HER2-positive breast cancer patients. However, it was confused that metastatic patients vary in the response of targeted drug. Therefore, methods of accurately predicting drug response were really needed. To overcome the spatial and temporal limitations of biopsies, we aimed to develop a more sensitive and less invasive method of detecting mutations associated with anti-HER2 therapeutic response through circulating-free DNA (cfDNA).Methods:From March 6, 2014 to December 10, 2014, 24 plasma samples from 20 patients with HER2-positive metastatic breast cancer who received systemic therapy were eligible. We used a panel for detection of hot-spot mutations from 50 oncogenes and tumor suppressor genes, and then used targeted next-generation sequencing (NGS) to identify somatic mutation of these samples in those 50 genes. Samples taken before their first trastuzumab administration and subsequently proven with clinical benefit were grouped into sensitive group. The others were collected after disease progression of the trastuzumab-based therapy and were grouped into the resistant group.Results:A total of 486 single-nucleotide variants from 46 genes were detected. Of these 46 genes, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), proto-oncogene c-Kit (KIT), and tumor protein p53 (TP53) were the most common mutated genes. Seven genes, including epidermal growth factor receptor (EGFR), G protein subunit alpha S (GNAS), HRas proto-oncogene (HRAS), mutL homolog 1 (MLH1), cadherin 1 (CDH1), neuroblastoma RAS viral oncogene homolog (NRAS), and NOTCH1, that only occurred mutations in the resistant group were associated with the resistance of targeted therapy. In addition, we detected a HER2 S855I mutation in two patients who had persistent benefits from anti-HER2 therapy.Conclusion:Targeted NGS of cfDNA has potential clinical utility to detect biomarkers from HER2-targeted therapies.

Abdominal Manual Therapy Repairs Interstitial Cells of Cajal and Increases Colonic c-Kit Expression When Treating Bowel Dysfunction after Spinal Cord Injury.

Background This study aimed to evaluate the therapeutic effects of abdominal manual therapy (AMT) on bowel dysfunction after spinal cord injury (SCI), investigating interstitial cells of Cajal (ICCs) and related c-kit expression. Methods Model rats were divided as SCI and SCI with drug treatment (intragastric mosapride), low-intensity (SCI + LMT; 50 g, 50 times/min), and high-intensity AMT (SCI + HMT; 100 g, 150 times/min). After 14 days of treatment, weight, improved Basso-Beattie-Bresnahan (BBB) locomotor score, and intestinal movement were evaluated. Morphological structure of spinal cord and colon tissues were examined. Immunostaining, RT-PCR, and western blot were used to assess c-kit expression. Results In SCI rats, AMT could not restore BBB, but it significantly increased weight, shortened time to defecation, increased feces amounts, and improved fecal pellet traits and colon histology. AMT improved the number, distribution, and ultrastructure of colonic ICCs, increasing colonic c-kit mRNA and protein levels. Compared with the SCI + Drug and SCI + LMT groups, the SCI + HMT group showed better therapeutic effect in improving intestinal transmission function and promoting c-kit expression. Conclusions AMT is an effective therapy for recovery of intestinal transmission function. It could repair ICCs and increase c-kit expression in colon tissues after SCI, in a frequency-dependent and pressure-dependent manner.

Electroacupuncture at ST36 Protects ICC Networks via mSCF/Kit-ETV1 Signaling in the Stomach of Diabetic Mice.

Background. Electroacupuncture (EA) at ST36 has been used to regulate gastric motility and effectively improve gastric emptying in diabetic patients. Nevertheless, the specific mechanisms underlying the efficacy of this treatment remain unknown. The aim of this study was to assess the variations of interstitial cells of Cajal (ICC) and explore the changes in mSCF/KIT-ETV1 signaling in the antrum and corpus of diabetic mice after treatment with EA. Methods. Male C57BL/6 mice were randomized into five groups: control group, diabetic group (DM), diabetic-plus-sham EA group (SEA), diabetic-plus-low-frequency EA group (LEA), and diabetic-plus-high-frequency EA group (HEA). The expression levels of Ano1, c-Kit, and ETV1 were assessed by immunofluorescence in the antrum and corpus. Western blotting and PCR methods were further used to evaluate c-Kit, mSCF, and ETV1 expression. Results. (1) c-Kit and Ano1 were obviously decreased in the DM group, but c-Kit reduced much more than Ano1. (2) The mSCF, c-Kit, and ETV1 mRNA and protein levels were obviously decreased in the DM group in both the antrum and the corpus (P < 0.01), but they were significantly elevated in the LEA and HEA groups (P < 0.01). Conclusions. Ano1 is a reliable marker to detect ICC changes in diabetes; low- and high-frequency EA at acupoint ST36 can protect the networks of ICC possibly via normal activation of mSCF/KIT-ETV1 signaling.