Expression of CD44s in lung cancer and its significance

AIM: To investigate expression of CD44s in lung cancer and it's clinical significance. METHODS: A total of 117 primary lung cancer from patients were examined for CD44s expression by immunohistochemical staining. RESULTS: CD44s mostly expressed in non-small cell lung cancer (NSCLC) but not in small ecll lung cancer (SCLC), and squamous cell carcinoma(SCC) showed much stronger expression of CD44s than adenocarcinoma(ADC)(P＜0.05). In comparison of the lung cancer with/ without lymph node metastasis, the latter showed stronger expression of CD44s(P＜0.01). According to TNM, there was a distinct statistic difference between early stage and advanced stage(P＜0.05). CONCLUSION: CD44s might be a better indicant in histological classification of lung cancer, lymph node metastasis, clinical stage and prognosis.     

Effects of p27kip1 gene on DNA and protein synthesis in esophageal carcinoma cells

AIM: To investigate the effects of p27kip1 gene on DNA replication and protein synthesis in esophageal carcinoma cells. METHODS: Recombinant adenovirus Ad-p27kip1 was constructed and transfected into esophageal carcinoma cell EC-9706, and its effects on DNA replication, protein synthesis and the growth of esophageal carcinoma cells were explored by means of cell growth count, [3H]-TdR, [3H]-Leucine incorporation method and flow cytometry. RESULTS: The growth of esophageal carcinoma cells was inhibited obviously. The radioactive intensity of -TdR and -Leucine in Ad-p27kip1 group decreased significantly than that in control group after EC-9706 transfection (P<0.01), while the multiplication of esophageal carcinoma cell was inhibited obviously. CONCLUSION: Ad-p27kip1 inhibited the multiplication of the esophageal carcinoma cells, which may be related to its suppressive function for DNA replication and protein synthesis.     

Influence of hypoxia-inducible factor-1 alpha on lung cancer cell A549 growth in vitro and in vivo

AIM: To study the role of hypoxia-inducible factor-1alpha(HIF-1α) on lung cancer cells A549 growth in vitro and in vivo. METHODS: To observe the growth rate of A549 cells after HIF-1α transfected, A549 cells (1×10⁶/mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups: the control group (group A, n=10), the HIF-1α transfected group(group B, n=10). The weights of subcutaneous tumor were detected. The resected specimens were made into paraffin-embedded sections. The proliferating cell nuclear antigen (PCNA) was identified by immunohistochemistry(ISH). The expressions of HIF-1α、 apoptosis-related protein survivin and bcl-2 were analyzed by Western blot. RESULTS: The growth rates of the HIF-1α transfected lung cancer cells A549 were significantly increased, and more importantly, the HIF-1α transfected lung cancer cells A549 was able to enhance lung cancer growth in nude mice(P＜0.05). The PCNA were increased significantly in group B, compared with group A. The expressions of HIF-1α, survivin and bcl-2 in group B were increased significantly than that of group A. CONCLUSIONS: HIF-1α increases lung cancer cells A549 growth in vitro and in vivo and its mechanism may be due to promotion of proliferation and inhibition of apoptosis.     

The study on the relationship between transforming growth factor-β1 and lung carcinoma

AIM: To explore the relationship between the expression of transforming growth factor-β1 (TGF-β1) and the pathogenesis of lung carcinoma, and the influence of radiotherapy on plasma TGF-β1 level of patients with lung carcinoma. METHODS: By immunohistochemical method, the expression of TGF-β1 was examined. An enzyme-linked immunoadsorbent assay (ELISA) was used to quantify the plasma TGF-β1 levels in different time as before radiotherapy, at the end of radiotherapy, and at the time of follow-up 6 months after radiotherapy, respectively. The changes of quantity of TGF-β1 in different time above were analysed statistically. RESULTS: There was a significant increase in TGF-β1 expression in the carcinoma compared with normal lung tissue. The mean TGF-β1 level in the 39 lung carcinoma patients before radiotherapy was (11.0±1.5) μg/L, which was significantly higher than control group (3.8±0.2 μg/L) (P＜0.05); but the plasma TGF-β1 level after radiotherapy was significantly lower (5.6±0.5 μg/L) compared with the level before radiotherapy (P＜0.05), and there was not significant differences compared with control group (P＞0.05). At the time of follow-up 6 months, the patients of lung carcinoma had a significantly higher plasma TGF-β1 level (11.3±1.2 μg/L) compared with the level at the end of radiotherapy (P＜0.05), but there was not significant differences compared with before radiotherapy (P＞0.05). Not significant difference was found in TGF-β1 levels among different histologic types. CONCLUSION: These data demonstrated that TGF-β1 was associated with the pathogenesis of lung carcinoma, and it may be a useful tumor marker in patients with lung carcinoma.     

Nucleostemin gene expression in human breast tumor tissues and its relation with clinical pathology

AIM: To study the expression of nucleostemin (NS) gene in human breast tumor tissues and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor.METHODS: Total RNA was isolated from human breast tumor tissue.The methods of electrophoresis and RT-PCR were used in measuring NS gene expression level,and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor were analyzed.RESULTS: The results indicated that there was no NS gene expression detected in normal breast tissues,and NS gene expression in malignant breast tumor tissues (P<0.01) was higher than that in the benign breast tumor tissues.The higher histological grades of the breast cancer showed the stronger NS gene expression (P<0.01),the higher TNM stages of the breast cancer showed the stronger NS gene expression (P<0.01),and the level of NS gene expression had not correlation with the histological types (P>0.05).CONCLUSION: It is suggested that there is no relation of NS gene expression level with histological types of the breast cancer,but there is a marked correlation of NS gene expression level with the histological grades and TNM stages.     

Effect of interleukin 18 gene modification on anti-tumor activity induced by lung cancer cell-derived exosomes

AIM: To study the effect of interleukin 18( IL-8 ) gene modification on anti-tumor activity induced by lung cancer cell-derived exosomes.METHODS: Exosomes isolated from the supernatants of IL-18 gene-modified NCI-H460 lung cancer cells (IL-18/H460), pcDNA3.1⁺ vector-modified cancer cells (DNA3.1/H460) and non-modified NCI-H460 lung cancer cells (NCI-H460) were observed under transmission electron microscope.The expression of heat-shock protein 70(HSP70),human leukocyte antigen(HLA) and IL-18 were determined by Western blotting.T lymphocytes were activated by exosomes or exosome-pulsed dendritic cells(DCs).The activity of T cells for killing lung cancer cells were detected by lactate dehydrogenase (LDH) method.The killing rates were calculated and compared.RESULTS: Exosomes showed typical morphous under transmission electron microscope.The protein levels of HSP70 and HLA were detected in the exosomes of all 3 groups, and IL-18 protein was only observed in IL-18/H460 group.The killing rates of exosome-activated T cells in IL-18/H460 group with the ratio of effector cell to target cell at 25∶ 1, 10∶ 1 and 5∶ 1 were (38.45±5.42)%, (25.17±3.94)% and (11.75±3.22)%, respectively.The killing rates of exosome-pulsed DC-activated T cells in this group were (89.05±4.06)%, (64.97±6.02)% and (40.16±4.98)%, respectively.The killing rates in IL-18/H460 group were higher than those in DNA3.1/H460 group and NCI-H460 group.The anti-tumor efficacy of exosome-pulsed DC－activated T cells was stronger than that of exosome-activated T cells.CONCLUSION: IL-18 gene modification enhances the anti-tumor activity induced by NCI-H460 lung cancer cell-derived exosomes.     

Analysis on relationship of lung cancer with length polymorphisms of microsatellite D3S1234 and D3S1300 located on FHIT gene

AIM:To analyze the relationship between lung cancer and length polymorphisms of microsatellite D3S1234 and D3S1300 located on fragile histidine triad(FHIT) gene. METHODS:The case-control study was conducted among 54 subjects of cancer and 131 healthy subjects. The length polymorphism was detected with the methods of multiplex PCR and urea-polyacrylamide gel electrophoresis. Allele frequency was calculated and the difference of allele frequency between cancer subjects and healthy persons was analyzed by the method of Chi-square. The relationship between lung cancer and length polymorphism of D3S1234 and D3S1300 was studied by the method of Binary Logistic analysis.RESULTS:No obvious difference in allele frequency of D3S1234 was observed between cancer subjects and healthy persons，but significant difference in allele frequency of D3S1300 was found between two groups. The results of Binary Logistic analysis showed that smoking was related with an increased risk of lung cancer. When the effect of smoking was excluded,length polymorphisms of D3S1234 and D3S1300 were found to be associated with higher risk of lung cancer. Meanwhile,length polymorphisms of D3S1234 and D3S1300 and smoking were proved to have interactive effects on the risk of lung cancer. CONCLUSION:The results suggest that there are statistic associations between lung cancer and length polymorphisms of D3S1234 and D3S1300,which may be beneficial to diagnose lung cancer early and explore its pathogenesis.     

Application of multiple gene methylations in plasma for diagnosis of lung cancer

AIM: To determine the aberrant methylation status in the gene promoter regions of CDH13, RASSF1A, DLEC1, SEPT9and RUNX3by detecting the plasma specimens and the value of their combined detection for diagnosis of lung cancers. METHODS: Nest methylation specific PCR (nMSP) was used to detect the promoter methylation status of the 5 genes in the plasma from 106 normal controls， lung cancer tissues, lung benign tissues and the plasma from 106 patients with lung cancers. Multiple displacement amplification (MDA) was used to amplify modified genomic DNA to solve the problem of insufficient of plasma DNA template. RESULTS: The positive rates of promoter methylation of CDH13, RASSF1A, DLEC1, SEPT9and RUNX3in the lung cancer tissues were 51.9%, 44.3%, 54.7%, 36.8%, 24.5%, respectively, and those in the plasma were 46.2%, 41.5%, 50.9%, 31.1%, 19.8%, respectively. The results of the Kappa consistency check showed that the lung cancer tissues and the plasma had obviously coherence in the methylation status of the 5 gene promoter regions. Combination of DLEC1, CDH13, RASSF1A, and SEPT9 had a higher diagnostic efficiency than the others, as their ACC value was 0.8208 and youden index was 0.6415 (with the sensitivity of 81.13% and the specificity of 83.02%). CONCLUSION: Combination detection of promoter methylation of lung cancer-related genes in the plasma is expected to apply to the early diagnosis of lung cancer.     

Expression of PARP-1 in epithelial ovarian cancer and its relationship with epithelial-mesenchymal transition

AIM: To investigate the expression of poly(ADP-ribose) polymerase-1(PARP-1) in the epithelial ovarian cancer(EOC) and its relationship with epithelial-mesenchymal transition(EMT). METHODS: The expression of PARP-1, E-cadherin, vimentin and Snail was detected in the EOC and benign ovarian tumor tissues by immunohistochemical method and real-time PCR. The expression of PARP-1, E-cadherin, vimentin and Snail proteins in the SKOV3 cells treated with efficient PARP-1 inhibitor PJ34 was determined by Western blotting. RESULTS: The positive expression rates of PARP-1, vimentin and Snail were significantly higher in the EOC than that in the benign ovarian tumor tissues, whereas the positive expression rate of E-cadherin was the opposite(P<0.05). The expression of PARP-1, E-cadherin, vimentin and Snail in the EOC was associated with the histological grade, clinical stage and lymphatic metastasis(P<0.05), but no relationship with age and pathological types was observed. The expression of E-cadherin in the EOC was negatively co-related to that of PARP-1. In contrast, the expression of vimentin and Snail in the EOC was positively co-related to that of PARP-1. The relative mRNA expression of PARP-1, vimentin and Snail in the EOC was significantly higher than that in the benign ovarian tumor tissues(P<0.05), while the mRNA expression of E-cadherin in the EOC was remarkably lower than that in the benign ovarian tumor tissues(P<0.05). The protein expression of PARP-1, vimentin and Snail in the SKOV3 cells was significantly decreased(P<0.05), while E-cadherin protein was increased after treated with PJ34(P<0.05). CONCLUSION: PARP-1 may contribute to the onset of EMT in the EOC by regulating the expression of E-cadherin, vimentin and Snail. The role of PARP-1, which is relevant to EMT, might be important in the development of ovarian cancer.     

Expression of ERK5 in colorectal cancer tissues and its correlation with distant metastasis in colorectal cancer patients

AIM: To investigate the expression of extracellular signal-regulated kinase 5 (ERK5) in primary colorectal cancer (CRC) and adjacent normal mucosa, and to analyze the relationship between ERK5 expression and clinicopathological parameters for exploring the functions of ERK5 in the occurrence and development of CRC. METHODS: The expression of ERK5 in carcinoma tissues and normal mucosa was examined by a set of tissue microarrays and the method of immunohistochemistry. The potential relationship between ERK5 expression and clinicopathological features was also analyzed. RESULTS: ERK5 expression was significantly higher in CRC tissues (134/338, 39.6%) than that in normal tissues (21/80, 26.2%; P<0.05). Overexpression of ERK5 in CRC tissues was significantly correlated with distant metastasis (P<0.05). However, no correlation between ERK5 expression and age at surgery, sex, tumor location, the depth of invasion, lymph node metastasis, TNM staging or differentiation grade was found (P>0.05). According to the Kaplan-Meier analysis, there is no significant difference in 5-year overall survival between the patients with ERK5 expression at high level and at low level. CONCLUSION: ERK5 protein is highly expressed in CRC with distant metastasis. This may be a promotive factor in the process of distant metastasis.     

Expression of NK4 gene in human pancreatic cancer cells and its effect on growth of human vascular endothelial cells

AIM:The current study was designed to construct eukaryotic expression vector containing NK4 gene and transfect it into human pancreatic cancer cell lines.METHODS:The recombinant of pcDNA3/hNK4 was digested by restriction enzyme,the NK4 gene was cloned into a high effective eukaryotic expressing plasmid which contains CMV2 immediate early gene promoter and then transiently introduced into the pancreatic cancer cell line SW1990 by lipo fectamine and clonal cel lines that secrete high levels of NK4 protein were isolated.The expression of NK4 was observed by RT-PCR and Western blot,in vitro the vascular endothelial cell proliferation inhibiting activity of NK4 was examined by 3-[4,5-dimethylthiazolzyl]-2,5-diphenyl tetrazolium bromide(MTT)method.RESULTS:A specific expression of NK4 gene mRNA by lipofectamine-mediated transfer exhibited only in SW1990/NK4 cells,Western blot analysis demonstrated that there was positive expression of NK4 protein(50 kD).The NK4 inhibited proliferation of the vascular endothelial cellsin vitro.CONCLUSION:The recombinant of pRC/CMV2-hNK4 is a high effective expressing eukaryotic vector.The bio-engineering product of the NK4 is an angiogenesis inhibitor and may play an important role in the gene therapy for tumor.     

Expression and functional analysis of novel gene AngRem104 associated with glomerular sclerosis

AIM:To study the expression and function of novel gene AngRem104.METHODS:Northern blot was performed to detect the distribution of AngRem104 in human multiple normal tissues as well as the effect of AngⅡ and AT1R antagonist (losartan) on AngRem104 expression. The sense and antisense eukaryotic expression vectors of AngRem104 were constructed and transfected into human mesangial cells. RT-PCR was used to detect the expression of FN when AngRem104 was over-expressed. Primary sequence and motif analysis of AngRem104 protein were performed by on-line ExPasy predictive tools.RESULTS:AngRem104 was predicted to localize at the cellular nucleus. It was widely expressed in human heart, placenta, liver, muscle, kidney and pancreas. Moreover, the up-regulated expression of AngRem104 induced by AngⅡ was inhibited by losartan in a dose-dependent manner.CONCLUSION:AngRem104 is a novel nuclear protein related to the expression of fibronectin and could be up-regulated by AngⅡ in human MC.     

Relationship between genetic polymorphisms of GSTM1 as well as GSTT1 and lung cancer

AIM and METHODS:To examine the relationship of glutathione S-transferase M1(GSTM1) and T1(GSTT1) with the occurrence of lung cancer, The case-control study was conducted among 161 lung cancer and 165 healthy controls. The genetic polymorphisms of GSTM1 and GSTT1 were detected with the method of multiplex polymerase chain reaction. Logistic regression analyses were used to assess the interaction of between different genotypes as well as between null genotypes and smoking. RESULTS:The frequences of GSTM1 and GSTT1 null genotypes had no obvious difference between lung cancer and healthy controls. In non-smoking subjects, the frequence of GSTM1 null genotype was significantly different between lung cancer and healthy controls. Furthermore, GSTM1 null genotype was significantly overrepresented in adenocarcinoma patients aged 60 or over, compared with controls.The results from interaction analyses showed although smoking and GSTM1 deletion were associated with an increased risk of lung cancer, GSTM1 and GSTT1 null genotypes combined with smoking did not have interaction effect on the risk of lung cancer. The risk for adnocarcinoma in the individuals at the age of 60 or over and in nonsmokers without GSTM1 gene but with GSTT1 functional genotype decreased by 48.5% and 45.3%, respectively. CONCLUSION:Our results suggest that GSTM1 deletion is an important host risk for lung cancer, and imply that GSTT1 functional genotype protects the old (aged 60 or over) and nonsmokers who are lack of GSTM1 gene from the risk of adenocarcinoma.