Insulin enhances apoptosis induced by cisplatin in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy

Background Chemoresistance is common among patients with esophageal squamous cell carcinoma （ESCC）.We investigated the effect and mechanism of insulin on enhancing anticancer functions of cisplatin in human esophageal cancer cell line EC9706.Methods The viability of EC9706 cells exposed to cisplatin was assessed using MTT assay.The times T1,when the number of living cells reached a plateau and T2,when the number of living cells reached a new plateau after the addition of insulin were found.T1 and T2 plateau cells were stained by Annexin V-FITC/PI and monodansylcadaverin （MDC）.Fluorescent microscopy was used to observe the expression of apoptosis and autophagy intuitively.Apoptotic ratio and fluorescent intensity were analysed by flow cytometry （FCM） quantitatively.Western blotting analysis was used to estimate the protein expression levels of AKT,mTOR,PI3K,PTEN,autophage related indicator LC3-Ⅱ and autophage related protein Beclin1 changes that occurred in the course of treatment.Results A larger number of typical autophagosomes were detected in EC9706 cells exposed to cisplatin.Insulin can increase the apoptosis induced by cisplatin.Apoptotic ratio of T1 plateau cells （（32.6±4.3）％） is significantly less than T2 plateau （（47.5±5.6）％）.MDC fluorescent intensity at T1 plateau （104.9±13.2） was significantly higher than intensity at T2 plateau （82.6±10.3）.After cotreatment with insulin,the expression level of LC3-Ⅱ,Beclin1 and PTEN in T2 plateau cells were significantly downregulated,but AKT,mTOR and PI3K expressions significantly upregulated compared with T1 plateau.Conclusions Insulin could enhance cisplatin-induced apoptosis in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy.The activation of PI3K/Akt/mTOR signaling pathway induced by insulin resulted in the suppression of autophagy in EC9706 cells,which may be attributed to the anticancer effects of cisplatin.     

c-erbB-2、bcl-2蛋白在宫颈鳞癌细胞中的表达及其意义

目的　研究c erbB 2、bcl 2基因在宫颈鳞癌中的表达及其在宫颈癌发生、发展中的作用及意义。方法　采用免疫组织化学S P法分别检测 4 5例宫颈鳞癌组织、10例宫颈上皮内瘤变和 10例正常宫颈鳞状上皮中c erbB 2、bcl 2蛋白的表达。结果　c erbB 2、bcl 2蛋白在宫颈鳞癌中的阳性表达率分别为 5 5 .5 6 %、2 8.2 9% ;在CIN中的阳性表达率分别为 0、10 .0 0 % ;在NE组均无表达。c erbB 2蛋白在宫颈鳞癌中阳性表达与在CIN、NE中的表达差异有显著性 (P <0 .0 1) ;bcl 2蛋白在宫颈鳞癌中阳性表达与在CIN中的表达及在NE中的不表达差异均无显著性 ( P >0 .0 5 ) ;c erbB 2与bcl 2之间在宫颈鳞癌组织中的阳性表达呈负相关 ( P <0 .0 5 )。结论　c erbB 2、bcl 2基因参与了宫颈鳞癌的发生、发展过程。c erbB 2基因蛋白的过表达是宫颈组织恶性变的重要生物学标志。联合检测c erbB 2、bcl 2基因蛋白对判断宫颈癌预后有一定意义     

汉族、维吾尔族宫颈癌相关基因谱对比分析研究

目的：探讨汉族、维吾尔族官颈癌组织中基因表达的差异及子宫颈癌发病机理。方法：应用含有2048条人类全长基因cDNA表达谱芯片，对临床手术切除的3例汉族及3例维吾尔族（维族）宫颈鳞癌组织及自身部分正常宫颈组织标本的基因表达谱进行对比分析。站杲：3例汉族宫颈癌组织中有2条共同差异表达的基因，均为表达减少（下调）的基因。3例维吾尔族宫颈癌组织中有共同差异表达的基因64条。6例宫颈癌组织中无共同差异表达的基因，但在5例宫颈鳞癌组织中共同差异表达的基因3条，4例宫颈癌组织中共同差异表达基因49条。结论；宫颈癌的发生与多种肿瘤相关基因有关。汉、维族宫颈癌组织中有基因表达的差异，推测汉、维族宫颈癌的生存在差异。     

Molecular typing and resistance mechanisms of carbapenem resistant Pseudomonas aeruginosa isolated from a Chinese surgical intensive care unit

Background Carbapenems are an important class of drugs for the treatment of Pseudomonas aeruginosa （P.aeruginosa） infections. However, carbapenem resistance has been commonly observed in nonfermenter species ofbacteria. The purpose of this study was to investigate the molecular epidemiology and carbapenem resistant mechanismsof R aerucfinosa isolated from a sur.clical intensive care unit （SICU） in China.     

Identification of Max binding protein as a novel binding protein of Nck1 and characterization of its role in inhibiting human liver cancer SK-HEP-1 cells

Background  The tendency of tumor cells to disperse throughout the liver is a distinct feature of hepatocellular carcinoma (HCC). Nck family adaptor proteins function to regulate actin cytoskeletal reorganization that leads to cell motility. We previously found that Max binding protein (MNT) was differentially expressed in HCC, and interacted with Nck1 by 2-DE. MNT is a protein member of the Myc/Max/Mad network which plays roles in cell proliferation, differentiation, and death. We investigated the effects of MNT on migration of human liver cancer SK-HEP-1 cells to study the migration regulatory role of MNT in HCC cells.

Methods  Interaction between MNT and Nck1 was further validated in hepatoma cells by GST-pull down assay and immunoprecipitation. siRNAs specific to MNT (MNT siRNA) were used to knockdown MNT expression. Western blotting, transwell assay were used to determine the migration potential of cells. 

Results  Interaction between MNT and Nck1 was validated in hepatoma cells. MNT knockdown promoted the migration of human liver cancer SK-HEP-1 cells (P <0.01).

Conclusion  The results suggest that MNT, via interaction with Nck1, inhibits hepatoma cell migration.

     

Genome-wide study reveals an important role of spontaneous autoimmunity, cardiomyocyte differentiation defect and anti-angiogenic activities in gender-specific gene expression in Keshan disease

Background Keshan disease （KD） is an endemic cardiomyopathy in China.The etiology of KD is still under debate and there is no effective approach to preventing and curing this disease.Young women of child-bearing age are the most frequent victims in rural areas.The aim of this study was to determine the differences between molecular pathogenic mechanisms in male and female KD sufferers.Methods We extracted RNA from the peripheral blood mononuclear cells of KD patients （12 women and 4 men） and controls （12 women and 4 men）.Then the isolated RNA was amplified,labeled and hybridized to Agilent human 4×44k whole genome microarrays.Gene expression was examined using oligonucleotide microarray analysis.A quantitative polymerase chain reaction assay was also performed to validate our microarray results.Results Among the genes differentially expressed in female KD patients we identified：HLA-DOA,HLA-DRA,and HLA-DQA1 associated with spontaneous autoimmunity; BMP5 and BMP7,involved in cardiomyocyte differentiation defect; and ADAMTS 8,CCL23,and TNFSF15,implicated in anti-angiogenic activities.These genes are involved in the canonical pathways and networks recognized for the female KD sufferers and might be related to the pathogenic mechanism of KD.Conclusion Our results might help to explain the higher susceptibility of women to this disease.     

Overexpression of interleukin-l7 in tumor-associated macrophages is correlated with the differentiation and angiogenesis of laryngeal squamous cell carcinoma

Background  Interleukin-l7 (IL-17), which exerts strong pro-inflammatory effects, has emerged as an important mediator in inflammation-associated cancer. The aim of this study was to clarify the relationship between IL-17 and tumor associated macrophages (TAMs), and the correlation of the microvessel density in the development of laryngeal squamous cell carcinoma (LSCC).

Methods  Histopathological observations and immunohistochemistry staining for IL-17, CD68, and CD34 were performed on 72 specimens (32 cases of LSCC, 20 cases of adjacent tissues of carcinoma as controls, and 20 cases of chronic hypertrophic laryngitis). Double immunohistochemical staining was done to determine which cells expressed IL-17. Real-time quantitative PCR determined the mRNA expression of IL-17. ELISA was used to detect the expression of the serum level of IL-17 in the three groups.

Results  The inflammation response had increased in LSCC. Overexpression of IL-17 and CD68 protein were seen in LSCC (P <0.01). The expression of IL-17 was different between well and poorly differentiated LSCC (P <0.01). The IL-17 expressing cells were mainly located in macrophages (CD68⁺/IL17⁺) as demonstrated by double immunohistochemical staining. IL-17 expression significantly correlated with high microvessel density (CD34⁺) in LSCC (P <0.05). Relatively higher mRNA expression levels of IL-17 were seen in LSCC compared to the controls (P <0.05). The serum expression of IL-17 was similar among the three groups (P >0.05).

Conclusion  IL-17 was expressed by TAMs, and IL-17 may significantly correlate to the differentiation and angiogenesis in the development of LSCC.

     

Value of frozen section examination in diagnosis and treatment of high-grade cervical intraepithelial neoplasia

Background  Invasive cancer of the cervix is considered a preventable disease because it has a long pre-invasive state, cervical cytology screening programs are currently available, and treatment of pre-invasive lesions is effective. We tested the accuracy of frozen section examination (FSE) of cone specimens to identify the endocervical margin and rule out invasion in patients with high-grade cervical intraepithelial neoplasia (CIN).

Methods  For 320 consecutive patients with a preoperative biopsy result of CIN stage 2/3, cold-knife conization (CKC) was performed followed by FSE. The results from analyses of permanent paraffin sections (PS) were compared with the FSE findings.

Results  The accuracy of FSE was 87% (278/320). For all of the seven patients with an invasive squamous cell carcinoma of the cervix identified by FSE, the diagnosis was confirmed by PS analysis. For one patient, the FSE result was cervicitis, whereas PS ananlysis showed microinvasive carcinoma. Appropriate surgery was performed for all patients based on the FSE and biopsy results. The FSE and PS results were not significantly different (P=0.000). Definitive examination of margin status using PS was concordant with FSE findings in all cases.

Conclusions  FSE is a rapid and reliable method for evaluating CKC specimens. It can identify frank invasion, permit adequate treatment in a one-stage procedure, and reliably detect clear resection margins. Since discrepancies do exist and may result in inappropriate treatment, further research is required to decrease these discrepancies and avoid missing even one case.

     

Uterine artery embolization in cesarean scar pregnancy: safe and effective intervention

Background Cesarean scar pregnancy （CSP） is a very rare but life-threatening entity and there is no optimal management strategy.Here we report a successfully conservative treatment of CSP.Methods We retrospectively analyzed the clinical data of 54 women with CSP,who underwent uterine artery embolization between January 2007 and September 2012 at the Peking University People＇s Hospital.We evaluated the clinical outcomes,the technique and the complications of uterine artery embolization.Results Of the 54 patients,2 patients with hemorrhage after induced abortion received bilateral uterine artery embolization treatment alone,and 52 patients underwent suction curettage after bilateral uterine artery embolization.All 54 women were successfully cured,without any severe complications,and uterine function was restored.During the follow-up,one patient had accidental normal interuterine pregnancy and received induced abortion during the first trimester.Conclution Uterine artery embolization combined with suction curettage is an effective and safe conservative treatment for cesarean scar pregnancy.     

宫颈鳞癌组织转移相关基因的初步筛查

目的: 应用基因芯片检测盆腔淋巴结转移组和无转移组宫颈鳞癌组织中差异表达基因,从中筛选肿瘤转移相关基因。方法: 利用基因芯片技术对4例盆腔淋巴结转移组和6例无转移组宫颈鳞癌组织标本进行差异表达基因检测,并用Real-time PCR对部分差异表达基因进行验证。通过基因库检索、PUBMED文献检索和基因本体分析从差异表达基因中筛选肿瘤转移相关基因。结果: 从盆腔淋巴结转移组及无转移组宫颈鳞癌组织中筛选出表达差异明显的基因329个,通过Real-time PCR验证,基因芯片结果可靠。从差异表达基因中筛选出与肿瘤转移相关基因44个。结论: 通过基因芯片筛查,初步建立了宫颈鳞癌转移相关差异基因表达谱,说明宫颈鳞癌的转移过程受多基因调控。     

Performance of the microscopic observation drug susceptibility assay in pyrazinamide susceptibility testing for Mycobacterium tuberculosis

Background Drug susceptibility assay is very important in tuberculosis therapy. Pyrazinamide is a first line antituberculosis drug and diagnosis of its resistance in Mycobacterium tuberculosis （M. tuberculosis） is difficult and time consuming by conventional methods. In this study, we aimed to evaluate the performance of the microscopic observation drug susceptibility （MODS） assay in the detection of pyrazinamide resistance in M. tuberculosis relative to the conventional Wayne assay and Lowenstein-Jensen （L J） proportion method. Methods M. tuberculosis clinical isolates （n=132） were tested by the MODS and the Wayne assay： the results were compared with those obtained by the LJ proportion method. Mutations in the gene were identified by direct sequencing of the pncA genes of all isolates in which pyrazinamide resistance was detected by any of the three methods. Results Compared to the LJ results, the sensitivity and specificity of the MODS assay were 97.8% and 96.5% respectively; the sensitivity and specificity of the Wayne assay were 87.0% and 97.7% respectively. Mutations in the pncA gene were found in 41 of 46 strains that were pyrazinamide resistant （3 tests）, in 1 of the 4 strains （LJ only）, in 42 of 48 strains （at least I test）, but no mutations in 1 strain sensitive according to the MODS assay only. The MODS assay, Wayne assay and LJ proportion method provided results in a median time of 6, 7 and 26 days respectively. Conclusions MODS assay offers a rapid, simple and reliable method for the detection of pyrazinamide resistance in M. tuberculosis and is an optimal alternative method in resource limited countries.     

Overexpression of glucosylceramide synthase and its significance in the clinical outcome of non-small cell lung cancer

Background Glucosylceramide synthase （GCS）,an enzyme responsible for ceramide glycosylation,plays an important role in multidrug resistance （MDR） in some tumors in vitro; however,its expression and clinicopathological significance in non-small cell lung cancer （NSCLC） remains unclear.Methods We evaluated GCS expression in 116 paired tumor and adjacent non-cancerous tissues and 50 frozen tissues from patients with NSCLC using immunohistochemistry and western blotting,and explored the correlation between GCS and NSCLC clinicopathological characteristics and prognosis.We observed the association between GCS and the MDR proteins P-glycoprotein （P-gp） and lung resistance-related protein （LRP） to determine the link between GCS and MDR at the histological level.Results GCS expression was significantly upregulated in NSCLC tumors compared with non-cancerous tissue.There was high GCS expression in 75/116 tumor specimens （64.7％） and 16/116 non-cancerous specimens （13.8％）.High GCS expression was significantly associated with poor differentiation （P=0.01）,lymph node metastasis （P=0.004）,recurrence/ distant metastasis （P=0.006）,and chemotherapy resistance （P=0.025）.Multivariate analysis demonstrated that GCS immunopositivity was an independent risk factor for survival （P=0.018）.P-gp was expressed in 80/116 tumors （69.0％） and in 12/116 non-cancerous tissue specimens （10.3％; P=0.001）; LRP was expressed in 85/116 tumors （73.3％） and 19/116 non-cancerous tissue specimens （16.4％; P=0.001）.Importantly,the results demonstrated that increased GCS expression in NSCLC cancer specimens correlated with increased expression of P-gp and LRP,molecules known to stimulate cancer cell MDR （r=0.612 and 0.503,P=0.01 and 0.035,respectively）.Conclusion GCS upregulation might contribute to the development of NSCLC and could be a useful prognostic indicator and chemoresistance predictor for NSCLC patients.     

Correlation of epidermal growth factor receptor overexpression with increased epidermal growth factor receptor gene copy number in esophageal squamous cell carcinomas

Background  Esophageal squamous cell carcinoma (ESCC) is one of the most frequent malignancies in China and epidermal growth factor receptor (EGFR) is widely distributed in human epithelial cell membrane. The aim of this study was to investigate the protein overexpression and gene copy number of EGFR in ESCC, and help to identify patients who may benefit from EGFR targeted therapies.  

Methods  Immunohistochemistry (IHC) was performed to analyze the expression of EGFR in 105 cases of ESCC, 16 cases of squamous epithelial atypical hyperplasia, and 11 cases of normal esophageal tissue. Fluorescence in situ hybridization (FISH) was performed to analyze the gene copy number in 80 cases of ESCC, eight cases of squamous epithelial atypical hyperplasia, and eight samples of normal esophageal tissue.

Results  The IHC-positive rates of EGFR in 105 cases of ESCC, 16 cases of squamous epithelial atypical hyperplasia, and 11 normal esophageal tissues were 97% (102/105), 44% (7/16), and 18% (2/11) respectively. The difference in the expression of EGFR among different esophageal tissue groups had statistically significance (P <0.05). Among the 105 cases of ESCC, overexpression of EGFR was found in 90 cases (86%), of which 55 cases scored 3+ for EGFR staining and 35 cases scored 2+. In ESCC, the expression of EGFR was significantly correlated with depth of invasion and TNM stage (P <0.05), but not with other parameters. The FISH-positive rates of EGFR in 80 cases of ESCC, the eight cases of squamous epithelial atypical hyperplasia, and eight samples of normal esophageal tissue were 31.3% (25/80), 0 (0/8) and 0 (0/8) respectively. In ESCC, EGFR gene amplification was found in 17 (21%) cases, high polysomy in 8 (10%) cases, disomy in 34 cases, low trisomy in 17 cases, and high trisomy in four cases. EGFR FISH-positive was significantly correlated with depth of invasion and lymph node metastasis (P <0.05). EGFR FISH-positive was significantly associated with overexpression of EGFR.

Conclusion  Protein overexpression and/or increased gene copy number of EGFR is common in ESCC, and EGFR targeted therapy may be appropriate for ESCC patients.

     

Effect of c-Met inhibitor SU11274 on human colon cancer cell growth

Background Colon cancer is one of the major malignancies worldwide and it still remains resistant to much of the currently available chemotherapy. Downregulation of HGF/c-Met signaling pathway is an emerging therapy for cancer treatment.

Methods In this study, the inhibitory effects of c-Met phosphorylation were observed with SU11274 on different colon cancer cell lines in vitro.

Results The results revealed the significant inhibitory effects of SU11274 on cell proliferation and cell survival, in a time and dose-dependent manner. Furthermore, the inhibitory effects of SU11274 on different subgroups of colon cancer cells via the HGF/c-Met signaling pathway were implicated in this study.

Conclusion The results suggested the possible selective therapeutic effects of c-Met inhibitor on colon cancer.

     

MicroRNA 145 may play an important role in uveal melanoma cell growth by potentially targeting insulin receptor substrate-1

Background MicroRNAs （miRNAs） contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. In this study, we investigated the role of miR-145 in the pathogenesis of uveal melanoma. Methods Expression profiles of miRNAs in uveal melanoma were performed using Agilent miRNA array. Quantitative real-time polymerase chain reaction was used to screen the expression levels of miR-145 in normal uveal tissue, uveal melanoma tissue, and uveal melanoma cell lines. Lenti-virus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Cell proliferation, cell cycle, and cell apoptosis of these miR-145 overexpression cell lines were examined by MTT assay and flow cytometry respectively. The target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase reporter assay. The expression of insulin-like growth factor-1 receptor （IGF-1R）, insulin receptor substrate-1 （IRS-1） proteins was determined by Western blotting analysis. IRS- 1 was knocked down in OCM-1 cells. TUNEL, BrdU, and flow cytometry assay were performed in IRS-1 knocked down OCM-1 cell lines to analyze its function. Results Forty-seven miRNAs were up regulated in uveal melanoma and 61 were down regulated, miR-145 expression was significantly lower in uveal melanoma sample and the cell lines were compared with normal uveal sample. Overexpression of miR-145 suppressed cell proliferation by blocking the G1 phase entering S phase in uveal melanoma cells, and promoted uveal melanoma cell apoptosis. IRS-1 was identified as a potential target of miR-145 by dual luciferase reporter assay. Knocking down of IRS-1 had similar effect as overexpression of miR-145. Conclusion miR-145 might act as a tumor suppressor in uveal melanoma, and downregulation of the target IRS-1 might be a potential mechanism.     

miR-30b and miR-30c expression predicted response to tyrosine kinase inhibitors as first line treatment in non-small cell lung cancer

Background Aberrantly expressed microRNAs are a hallmark of cancer,and microRNA expression profiling is associated with tumor progression and response to chemotherapy,suggesting their potential application as prognostic and predictive biomarkers.The role of microRNAs in lung cancer remains elusive.It has been recently reported that epidermal growth factor receptor （EGFR） and hepatocyte growth factor receptor （MET） tyrosine kinase can regulate expression of specific microRNAs including miR-30b,miR-30c,miR-221,miR-222,miR-103 and miR-203,and induce tumorigenesis and gefitinib resistance in lung cancers.We intend to study the role of miR-30b and miR-30c expression in predicting response to tyrosine kinase inhibitors （TKIs） in non-small cell lung cancer （NSCLC）.Methods We have therefore retrospectively examined expression of miR-30b miR-30c in 41 formalin fixed paraffin embedded tissue samples from NSCLC patients when TKIs were used as first line therapy.Results We found a significant correlation between expression of miR-30b and miR-30c.Furthermore,miR-30b and miR-30c expression correlated with short-term response.Kaplan-Meier analysis further revealed that the expression of miR-30b and miR-30c predicted progression free survival and the overall survival rate in the examined cohort.Conclusion Our study identified miR-30b and miR-30c as useful prognostic predictors in NSCLC patients who underwent first line treatment with TKIs.     

Adjacent segment disease after anterior cervical decompression and fusion: analysis of risk factors on X-ray and magnetic resonance imaging

Background Adjacent segment disease （ASD） is common after cervical fusion.The aim of this study was to evaluate the risk factors for ASD on X-ray and magnetic resonance imaging （MRI）.Methods Patients included in this study had received revision surgeries after developing symptomatic ASD following anterior decompression and fusion.A control group that had not developed ASD was matched 1：1 by follow-up time and fusion segments.Plate-to-disc distances （PDDs）,developmental cervical canal stenosis on X-ray,cervical disc degeneration grading,and cervical disc bulge impingements on preoperative MRI were measured and compared between the ASD group and the control group.Results Thirty-four patients with complete radiographic data were included in the ASD group.The causative segments of ASD included nine cases of C3-4,18 cases of C4-5,three cases of C5-6,and four cases of C6-7.The ASD occurred at the upper adjacent segments in 26 patients and at the lower adjacent segments in eight patients.PDD distributions were similar between the ASD group and the control group.Developmental cervical canal stenosis was a risk factor for ASD,with an odd ratio value of 2.88.Preoperative cervical disc degenerations on MRI were similar between the ASD group and the control group.In the upper-level ASD group,the disc bulge impingement was （19.7±9.7）％,which was significantly higher than that of the control group of （11.8±4.8）％.Conclusions ASD was more likely to develop above the index level of fusion.Developmental cervical canal stenosis and greater disc bulge impingement may be risk factors for the development of ASD.