Expression of a novel apoptosis inhibitor-survivin in colorectal carcinoma

AIM: To investigate the role of survivin expression in the pathogenesis of colorectal carcinoma.METHODS: Immunohistochemistry S-P method and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) were used to detect the expression of survivin and apoptotic cell in situ in colorectal cancerous tissues, para-cancerous tissues and normal tissues of 48 cases of colorectal carcinoma.RESULTS: The survivin positive unit (PU) was higher in cancerous tissues (38.76±5.14)than in para-cancerous (25.17±7.26) or normal tissues (0.57±0.03) (P<0.05).The apoptosis index (AI) of para-cancerous tissues was(7.51±2.63%) higher than cancerous tissues (4.65±1.76%).The expression of survivin was associated with pathological grade, lymph node metastasis and Dukes stage of colorectal carcinoma.CONCLUSION: Survivin expression may play an important role in carcinogenesis of colorectal carcinoma and may be associated with malignant biological behaviors of colorectal carcinoma.

LY294002 potentiates the anti-cancer effect of oxaliplatin for gastric cancer via death receptor pathway

AIM:To examine the effects of combined treatment of oxaliplatin and phosphatidylinositol 3'-kinase inhibitor,2-(4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one(LY294002) for gastric cancer. METHODS:Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay.Apoptotic cells were detected by flow cytometric analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay.Western blotting and immuno-precipitation were used to examine protein expression and recruitment,respectively.Nuclear factorκB(NFκB) binding activities were investigated using electrophoretic mobility shift assay.Nude mice were used to investigate tumor growth. RESULTS:Treatment with combined oxaliplatin and LY294002 resulted in increased cell growth inhibi-tion and cell apoptosis in vitro,and increased tumor growth inhibition and cell death in the tumor mass in vivo.In MKN45 and AGS cells,oxaliplatin treatment promoted both protein kinase B(Akt) and NFκB activation,while pretreatment with LY294002 significantly attenuated oxaliplatin-induced Akt activity and NFκB binding.LY294002 promoted oxaliplatin-induced Fas ligand(FasL) expression,Fas-associated death domain protein recruitment,caspase-8,Bid,and caspase-3 activation,and the short form of cellular caspase-8/FLICEinhibitory protein(c-FLIPS) inhibition.In vivo,LY294002 inhibited oxaliplatin-induced activation of Akt and NFκB,and increased oxaliplatin-induced expression of FasL,inhibition of c-FLIPS,and activation of caspase-8,Bid,and caspase-3. CONCLUSION:Combination of oxaliplatin and LY294002 was therapeutically promising for gastric cancer treatment.The enhanced sensitivity of the combined treatment was associated with the activation of the death receptor pathway.

Aberrant expression of ether à go-go potassium channel in colorectal cancer patients and cell lines

AIM: To study the expression of ether à go-go (Eag1) potassium channel in colorectal cancer and the relation- ship between their expression and clinico-pathological features. METHODS: The expression levels of Eag1 protein were determined in 76 cancer tissues with paired non- cancerous matched tissues as well as 9 colorectal adenoma tissues by immunohistochemistry. Eag1 mRNA expression was detected in 13 colorectal cancer tissues with paired non-cancerous matched tissues and 4 colorectal adenoma tissues as well as two colorectal cancer cell lines (LoVo and HT-29) by reverse transcription PCR. RESULTS: The frequency of positive expression of Eag1 protein was 76.3% (58/76) and Eag1 mRNA was 76.9% (10/13) in colorectal cancer tissue. Expression level of Eag1 protein was dependent on the tumor size, lymphatic node metastasis, other organ metastases and Dukes’ stage (P < 0.05), while not dependent on age, sex, site and degree of differentiation. Eag1 protein and mRNA were negative in normal colorectal tissue, and absolutely negative in colorectal adenomas except that one case was positively stained for Eag1 protein. CONCLUSION: Eag1 protein and mRNA are aberrantly expressed in colorectal cancer and occasionally expressed in colorectal adenoma. The high frequency of expression of Eag1 in tumors and the restriction of normal expression to the brain suggest the potential of this protein for diagnostic, prognostic and therapeutic purposes.

Association of fucosyltransferase 2 gene variants with ulcerative colitis in Han and Uyghur patients in China

AIM:To investigate the contribution of fucosyltransferase 2 (FUT2) variants to the genetic susceptibility and clinical heterogeneity of ulcerative colitis (UC) between Han and Uyghur patients in Xinjiang, China. METHODS:A total of 102 UC patients (53 Han patients including 22 men and 31 women, and 49 Uyghur patients including 25 men and 24 women; aged 48 ± 16 years) and 310 age-and sex-matched healthy controls were enrolled from January 2010 to May 2011 in Xinjiang People's Hospital of China. UC was diagnosed based on the clinical, endoscopic and histological findings following Lennard-Jones criteria. Blood samples were collected and genomic DNA was extracted by the routine laboratory methods. Polymerase chain reaction-sequence-based typing method was used to identify FUT2 variants rs281377, rs1047781, rs601338 and rs602662. Genotypic and allelic frequencies were documented and compared between the UC patients and the healthy controls. Genotypic frequencies were also compared between Han and Uyghur patients. Potential association of genetic variation and UC between Han and Uyghur patients was examined. RESULTS: rs281377 was found significantly associated with UC in the Han population as compared with the controls (P = 0.011) while rs281377 was not associated with UC in the Uyghur population (P = 0.06). TT homozygous rs281377 frequencies were higher in the UC groups than in the controls (88.7% vs 68.7% and 55.1% vs 50.3%). rs1047781 was specifically associated with UC in the Uyghur population (P = 0.001), but not associated with UC in the Han population (P = 0.13). TT homozygous rs1047781 frequencies were lower in the UC groups than in the controls (9.5% vs 11.8% and 4.0% vs 6.7%). rs601338 was statistically related to UC in both populations (Han, P = 0.025; Uyghur, P = 8.33 × 10 -5 ). AA homozygous rs601338 frequencies were lower in the UC groups than in the controls (0% vs 1.8% and 12.2% vs 13.4%). No association was found between rs602662 and UC in both Han and the Uyghur populations. Allelic analysis showed that rs281377 allele was significantly associated with UC in the Han population as compared with the controls [P = 0.001, odd ratio (OR) = 0.26], however, it was not associated with UC in the Uyghur population (P = 0.603, OR = 1.14), and rs1047781 allele was associated with UC in the Uyghur population (P = 0.001, OR = 0.029) while it was not associated with UC in the Han population (P = 0.074, OR = 0.62). Moreover, rs601338 was associated with UC in both Han (P = 0.005, OR = 0.1) and Uyghur pop- ulations (P = 0.002, OR = 0.43). Meta analysis showed that rs1047781 and rs601338 conferred risk of UC as compared with the controls [P = 0.005, OR = 0.47; P = 0.0003, OR = 0.35; 95% confidence interval (CI) = 0.31-0.72 and 0.21-0.58], but rs281377 and rs602662 showed no statistically significant differences betweenpatients with UC and controls (P = 0.10, OR = 0.71; P = 0.68, OR = 0.09; 95% CI = 0.47-1.07 and 0.56-1.47). CONCLUSION:Functionally relevant FUT2 gene variants are associated with UC, suggesting that they play a potential role in the pathogenesis of UC and may contribute to the clinical heterogeneity of UC between Han and Uyghur patients.

Role of mast cell-mi R-490-5p in irritable bowel syndrome

AIM To determine the functional role of mi R-490-5p in mast cell proliferation and apoptosis,and in the mast cell tryptase/PAR-2 signal pathway.METHODS The 3rd generation of lentivirus vector systems containing enhanced green fluorescent protein(EGFP)(Ruisai Inc.,Shanghai,China),which acts as a reporter gene was used to construct the mmu-mi R-490-5p lentivirus expression vector p EGFP-antagomi R-490-5p,and the lentivirus vector p EGFP-negative was used as a negative control.The stably transfected mast cell line p815 was then constructed.GFP positive cells were successfully transfected cells.We determined the expression of mi R-490-5p in p815 mast cells before and after transfection using quantitative real-time PCR(q RT-PCR).In addition,after transduction with the lentivirus vectors,the role of mi R-490-5p in mast cell proliferation and apoptosis was investigated using the CCK-8 assay and flow cytometry,respectively.The m RNA levels of tryptase and PAR-2 were detected by q RT-PCR and the protein levels were detected by Western blot.RESULTS The inhibition of mi R-490-5p expression promoted apoptosis and inhibited proliferation of p815 mast cells.The m RNA levels of tryptase and PAR-2 were significantly increased after transfection comparedwith the control group,tryptase(P=0.721,normal vs null;P=0.001,si RNA vs normal;P=0.002,si RNA vs null)and PAR-2(P=0.027,si RNA vs null;P=0.353,normal vs null;P=0.105,si RNA vs normal).The protein levels of tryptase and PAR2 were slightly higher in the si RNA group than those in the control group,but the difference was not statistically significant(P>0.05).CONCLUSION mi R-490-5p plays a vital role in the pathogenesis of irritable bowel syndrome by affecting mast cell proliferation and apoptosis;with down-regulation of mi R-490-5p,the m RNA level of mast cell tryptase and PAR-2 increased,and the protein level increased,but the difference was not statistically significant.

Effects of H pylori therapy on erythrocytic and iron parameters in iron deficiency anemia patients with H pylori-positive chronic gastristis

AIM:To elucidate the influences of H pylori infection on oral iron treatment for iron deficiency anemia(IDA). METHODS:A total of 86 patients were divided into two groups:group A,receiving ferrous succinate combined with triple therapy for H pylori eradication,and group B(control),treated with ferrous succinate only.During treatment of IDA,dynamic changes in hemoglobin(Hb) level,mean corpuscular volume(MCV),mean corpuscular hemoglobin(MCH),serum iron(SI),and serum ferritin (SF)were compared between the groups. RESULTS:Hb was slightly higher in group A at d 14 after the start of triple therapy for H pylori eradication (P>0.05).After the therapy,the increase of Hb in group A became significantly faster than that in group B (P<0.05).At d 56,the mean Hb in group A returned to the normal level,however,in group B,it was lower than that in group A(P<0.05)although it had also increased compared with that before oral iron treatment.The MCV and MCH in group A recovered to the normal level,and were much higher than those in group B(P<0.05)at d 21.In Group B,the MCV and MCH remained at lower than normal levels until d 42 after the start of therapy. And then,they reached a plateau in both groups and the differences disappeared(P>0.05).The SF in group A was higher than that in group B(P<0.05)28 d after the treatment and its improvement was quicker in group A (P<0.05),and the difference between the two groups was even more significant(P<0.01)at d 56.The SI in group A was higher than that in group B(P<0.05)at d 14 and this persisted until d 56 when the follow-up of this research was finished. CONCLUSION:Treatment of H pylori can enhance the efficacy of ferrous succinate therapy in IDA patients with H pylori-positive chronic gastritis.

Ca2+/calmodulin-dependent protein kinase II regulates colon cancer proliferation and migration via ERK1/2 and p38 pathways

AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of Ca MKⅡin tissue samples and MMP2,MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by q RTPCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and woundhealing assay.RESULTS We first demonstrated that CaMK Ⅱ was ove rexpressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116,the Ca MKII-specific inhibitor KN93,but not its inactive analogue KN92,decreased cancer cell proliferation. Furthermore,KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSION Our findings highlight Ca MKⅡ as a potential critical mediator in human colon tumor development and metastasis.

KN-93,a specific inhibitor of CaMKⅡ inhibits human hepatic stellate cell proliferation in vitro

AIM: To investigate the effects of KN-93, a CaMKⅡ selective inhibitor on cell proliferation and the expression of p53 or p21 protein in human hepatic stellate cells. METHODS: Human hepatic stellate cells (LX-2) were incubated with various concentrations (0-50 mmol/L) of KN-93 or its inactive derivative, KN-92. Cell proliferation was measured by CCK-8 assay, and the expression of two cell cycle regulators, p53 and p21, was determined by SDS-PAGE and Western blotting. RESULTS: KN-93 (5-50 mmol/L) decreased the proliferation of human hepatic stellate cells in a dose- dependent manner from 81.76% (81.76% ± 2.58% vs 96.63% ± 2.69%, P < 0.05) to 27.15% (27.15% ± 2.86% vs 96.59% ± 2.44%, P < 0.01) after 24 h treatment. Incubation of 10 mmol/L KN-93 induced the cell growth reduction in a time-dependent manner from 78.27% at 8 h to 11.48% at 48 h. However, KN-92, an inactive derivative of KN-93, did not inhibit cell proliferation effectively. Moreover, analysis of cell cycle regulator expression revealed that KN-93 rather than KN-92 reduced the expression of p53 and p21.

Primary study of leptin and human hepatocellular carcinoma in vitro

AIM: To investigate the expression level and effects of leptin in human hepatocellular carcinoma cells in vitro and to explore the correlation between them. METHODS: Human hepatocellular carcinoma cell line HepG2 was cultured in vitro, and (the expression level) mRNA of leptin and leptin receptors in HepG2 were assessed using reverse transcription polymerase chain reaction (RT-PCR). Effects of different concentrations of leptin (50 ng/mL, 100 ng/mL, 200 ng/mL) on HepG2 were detected with colorimetric assay by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) after incubation periods of 24 h, 48 h, and 72 h. Flow cytometry was performed to assess cell cycle progression of different concentrations of leptin as stated above after each 24 h incubation period. RESULTS: mRNA of leptin and leptin receptors (including short and long isoforms) were expressed in HepG2. The 72 h incubation of leptin at different concentrations (50 ng/mL, 100 ng/mL, 200 ng/mL) promoted proliferation of HepG2 in a concentration- and time- dependent manner. The experimental group shows significant statistical differences when compared to the controlled group which contained 0 ng/mL of leptin. As the concentration of leptin increases, significant fewer cells were detected in G0-G1 phase and more cells in S and G2-M phases. CONCLUSION: Leptin and leptin receptor are simultaneously expressed in human hepatocellular carcinoma cell line HepG2. Addition of leptin (0 ng/mL-200 ng/mL) in 72 h periods indicated there is a concentration- and time-dependent correlation in the stimulation of HepG2 cell proliferation. The effect of proliferation by leptin is due to promotion of DNA synthesis and enhancement of mitotic activity. The relationship between leptin and human hepatocellular carcinoma cells might indicate that adipokine could be associated with the progression of human hepatocellular carcinoma.

Possible mechanism for the regulation of glucose on proliferation, inhibition and apoptosis of colon cancer cells induced by sodium butyrate

AIM: To study the effect of glucose on sodium butyrate- induced proliferation inhibition and apoptosis in HT-29 cell line, and explored its possible mechanisms. METHODS: HT-29 cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum, and were allowed to adhere for 24 h, and then replaced with experimental medium. Cell survival rates were detected by MTT assay. Apoptosis was detected by TUNEL assay. Glucose transport protein 1 (GLUT1) and monocarboxylate transporter 1 (MCT1) mRNA expression was detected by RT-PCR. RESULTS: Low concentration of glucose induced apoptosis and regulated proliferation in HT-29 cell line, and glucose can obviously inhibit the effect of proliferation inhibition and apoptosis induced by sodium butyrate. Glucose also down-regulated the expression of MCT1mRNA (0.28 ± 0.07 vs 0.19 ± 0.10, P < 0.05), and decreased the expression of GLUT1mRNA slightly (0.18 ± 0.04 vs 0.13 ± 0.03, P < 0.05). CONCLUSION: Glucose can regulate the effect of proliferation inhibition and apoptosis induced by sodium butyrate and this influence may be associated with the intracellular concentration of glucose and sodium butyrate.

Habitual rapid food intake and ineffective esophageal motility

AIM:To study non-cardiac chest pain(NCCP) in relation to ineffective esophageal motility(IEM) and rapid food intake.METHODS:NCCP patients with a self-reported habit of fast eating underwent esophageal manometry for the diagnosis of IEM.Telephone interviews identified eating habits of additional IEM patients.Comparison of manometric features was done among IEM patients with and without the habit of rapid food intake and healthy controls.A case study investigated the effect of 6-mo gum chewing on restoration of esophageal motility in an IEM patient.The Valsalva maneuver was performed in IEM patients and healthy controls to assess the compliance of the esophagus in response to abdominal pressureincrease.RESULTS:Although most patients diagnosed with NCCP do not exhibit IEM,remarkably,all 12 NCCP patients who were self-reporting fast eaters with a main complaint of chest pain(75.0%) had contraction amplitudes in the mid and distal esophagus that were significantly lower compared with healthy controls [(23.45 mmHg(95%CI:14.06-32.85)vs 58.80 mmHg(95%CI:42.56-75.04),P < 0.01 and 28.29 mmHg(95%CI:21.77-34.81) vs 50.75 mmHg(95%CI:38.44-63.05),P < 0.01,respectively)].In 7 normal-eating IEM patients with a main complaint of sensation of obstruction(42.9%),the mid amplitude was smaller than in the controls [30.09 mmHg(95%CI:19.48-40.70) vs 58.80 mmHg(95%CI:42.56-75.04),P < 0.05].There was no statistically significant difference in manometric features between the fast-eating and normal-eating groups.One NCCP patient who self-reported fast eating and was subsequently diagnosed with IEM did not improve with proton-pump inhibition but restored swallow-induced contractions upon 6-mo gum-chewing.The Valsalva maneuver caused a markedly reduced pressure rise in the mid and proximal esophagus in the IEM patients.CONCLUSION:Habitual rapid food intake may lead to IEM.A prospective study is needed to validate this hypothesis.Gum-chewing might strengthen weakened esophageal muscles.