ADAM17 mediates hypoxia-induced drug resistance in hepatocellular carcinoma cells through activation of EGFR/PI3K/Akt pathway.

A disintegrin and metalloproteinase-17 (ADAM17) is a member of the metalloproteinase superfamily and involved in the cleavage of ectodomain of many transmembrane proteins. ADAM17 is overexpressed in a variety of human tumors, which is associated with tumor development and progression. In the present study, we sought to investigate the expression and function of ADAM17 in hypoxia-treated hepatocellular carcinoma (HCC) cells. Western blot analysis was used to measure the expression of ADAM17 in HCC cell lines (Hep3B and HepG2 cells). Annexin V/PI double staining was performed to analyze the effects of ADAM17 on hypoxia-mediated cisplatin resistance. ADAM17 expression was upregulated by hypoxia treatment in HCC cells at both mRNA and protein levels. Overexpression of ADAM17 reduced cisplatin-induced apoptosis in HCC cells, accompanies by less cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP). Forced expression of ADAM17 enhanced the phosphorylation of epidermal growth factor receptor (EGFR) and Akt without affecting the expression of total EGFR and Akt. Pretreatment with EGFR inhibitor AG1478 or phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 rescued ADAM17-mediated cisplatin resistance of HCC cells. ADAM17 silencing attenuated hypoxia-induced cisplatin resistance and enhanced the accumulation of cleaved caspase-3 and PARP. Western blot analysis showed that overexpression of hypoxia-inducible factor-1α (HIF-1α), a transcription factor, upregulated the expression of ADAM17 and HIF-1α silencing downregulated the expression of ADAM17 in hypoxia-treated HCC cells, indicating the regulation of ADAM17 by HIF-1α. Taken together, our results indicated that ADAM17 is upregulated by hypoxia and contributes to hypoxia-induced cisplatin resistance via EGFR/PI3K/Akt pathway.

[The application of surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in diagnosing dysplasia and chronic atrophic gastric-carditis in population with high risk of gastric-cardia adenocarcinoma].

OBJECTIVES: To evaluate the serum biomarkers for diagnosis of gastric cardia dysplasia (DYS) and chronic atrophic gastric-carditis (CAG) and to provide a novel screening method for high risk population of gastric-cardia adenocarcinoma (GCA). METHODS: Proteomic spectra were generated by surface-enhanced laser desorption/ionization-time of flight-mass spectra (SELDI-TOF-MS) and weak cation exchange protein chip system. A set of spectra derived from analysis of serum from 143 symptom-free subjects at high-risk area for GCA, including 63 cases with histologically normal gastric cardia epithelia, 57 of CAG and 23 of DYS, were analyzed by bioinformatics like decision tree classification algorithm. The sensitivity and the specificity for test group were performed by using 10-fold cross validation classification with the decision tree classification model. RESULTS: One protein spot with a ratio of mass to charge (M/Z) of M3894. 0 was selected to build a decision tree classification model to identify the case with DYS or normal. With this classification model, the sensitive rate for DYS identification was 87% (20/23). Two proteins with M/Z of M2942. 15 and M33316. 6 were used to build a decision tree classification model. With this model, the sensitivity for discriminating CAG from normal was 93% (53/57) and the specificity was 92 (58/63). CONCLUSIONS: The gastric cardia lesions of DYS and CAG could be identified by SELDI-TOF-MS technique specifically in symptom-free subjects at high incidence area for GCA. The present findings provide a new screening way for high-risk subjects with CGA.

Expression of p53, inducible nitric oxide synthase and vascular endothelial growth factor in gastric precancerous and cancerous lesions: correlation with clinical features.

BACKGROUND: The growth and metastasis of tumors depend on the development of an adequate blood supply via angiogenesis. Recent studies indicate that the inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF) and the tumor suppressor p53 are fundamental play-markers of the angiogenic process. Overexpression of iNOS and VEGF has been shown to induce angiogenesis in tumors. P53 suppresses angiogenesis by down-regulating VEGF and iNOS. The correlation of expression of p53, VEGF and iNOS and clinical features in gastric carcinogenesis, however, has not been well characterized. METHODS: The expression of p53, iNOS and VEGF in gastric precancerous and cancerous lesions and its relation with the clinical features was determined with immunohistochemistry (avidin-biotin-peroxidase complex method) on 55 randomly selected GC patients and 60 symptom-free subjects from the mass survey in the high-incidence area for GC in Henan, northern China. RESULTS: The positive immunostaining rates for p53, iNOS and VEGF in gastric carcinomas were 51%, 44% and 51%, respectively, and correlated well with TNM stages, but did not show significant difference among the groups with different degrees of gastric wall invasion depth by GC. A positive immunostaining reaction for the iNOS protein was significantly correlated with lymph node metastasis (p = 0.019; Spearman correlation coefficient). P53 protein accumulation was higher in the poorly-differentiated gastric carcinoma than in well-differentiated one. In gastric biopsies, no positive immunostaining was observed for p53, iNOS and VEGF in the histologically normal tissue and chronic superficial gastritis (CSG). However, p53, iNOS and VEGF positive immunostaining was observed in the tissues with different severities of lesions of chronic atrophic gastritis (CAG), intestinal metaplasia (IM) and dysplasia (DYS), and the positive rates increased with the lesion progression from CAG to IM to DYS. A high coincidental positive and negative immunostaining rate for p53, iNOS and VEGF was observed both in biopsy samples with CAG, IM and DYS from the symptom-free subjects and in gastric cancer tissue from the GC patients. CONCLUSIONS: The present results indicated that p53 protein accumulation and increased expression of iNOS and VEGF might be responsible for gastric carcinogenesis and tumor aggressiveness of gastric cancer.

Intervention and follow-up on human esophageal precancerous lesions in Henan, northern China, a high-incidence area for esophageal cancer.

BACKGROUND: Esophageal cancer (EC) remains a leading cause of cancer-related deaths in Linzhou (formerly Linxian) and Huixian of Henan province, northern China, which has been well recognized as the highest incidence area for EC. The lack of useful chemoprevention agents and early detection methods is the key factors for stable EC incidence in these areas. Human esophageal carcinogensis has been considered as a multistep progressive process. The natural history for EC, however is not very clear. METHODS: Follow-up studies with linear repeated biopsies and histopathological examination were performed on 778 subjects from Linzhou and Huixian. Of these subjects, 578 subjects were followed for 11 years (1989-2000), 400 subjects with different severity of esophageal precancerous lesions were randomly divided into 2 groups for intervention studies with calcium and decaffeinated green tea (DGT). Each group included 200 subjects (100 subjects for treatment, and 100 subjects for placebo). In calcium group, each subject received an oral supplementation of 1,200 mg of calcium daily for 11 months. In DGT group, each subject received 5 mg of DGT daily for 12 months. In placebo group, each subject received placebo pill for 11 months (calcium group) and 12 month (DGT group). At the entry and the end of the trial, esophageal biopsy specimens were taken at the middle and the lower thirds of the esophagus and from macroscopic lesions, if only, of each subject. RESULTS: DGT trail did not show apparent difference between the treatment and placebo group in alleviating the esophageal precancerous lesions and abnormal cell proliferation. For the calcium intervention study, after 11 years' follow-up, 10 subjects had developed into cancers in the calcium group (10%, 8 EC and 2 GCA), and 8 subjects developed into EC in the placebo group (8%). All these patients were diagnosed at very early stage of cancer (symptom-free). Of the 578 subjects, 25 (18 males and 7 females) had developed into EC (n = 23, 4.3%) and gastric cardia cancer (GCA, n = 2, 0.3%), during the 11 years' follow-up. The mean time of cancer development (from entry of the follow-up study to the cancer detection) was 5.0 +/- 2.9 years (males) and 4.7 +/- 3.2 years (females). Of the 25 patients with EC and GCA, 11 were from the 387 followed subjects with "normal" histomorphology of biopsy at the entry of the follow-up study (3%, 11/387), 2 were from the subjects with basal cell hyperplasia, grade I (BCH I, 2%, 2/94), 7 from the subjects with BCH grade II (BCH II, 10%, 7/72), and 5 from BCH III and dysplasia (20%, 5/25). CONCLUSIONS: DGT trail was not shown to have beneficial effects in alleviating esophageal precancerous lesions and abnormal cell proliferation patterns. Calcium supplementation did not produce apparent long-term effects on EC. BCH II could be considered as precancerous lesions of EC. The quantitative histopathological analysis in terms of number of proliferating basal cell layers is of importance in determining the high-risk subjects for EC and evaluating the intervention results. Follow-up studies with repeated endoscopic biopsies are the powerful strategy for early detection and mortality control of EC and GAC in the high incidence area.

Genome-wide association study of esophageal squamous cell carcinoma in Chinese subjects identifies susceptibility loci at PLCE1 and C20orf54.

We performed a genome-wide association study of esophageal squamous cell carcinoma (ESCC) by genotyping 1,077 individuals with ESCC and 1,733 control subjects of Chinese Han descent. We selected 18 promising SNPs for replication in an additional 7,673 cases of ESCC and 11,013 control subjects of Chinese Han descent and 303 cases of ESCC and 537 control subjects of Chinese Uygur-Kazakh descent. We identified two previously unknown susceptibility loci for ESCC: PLCE1 at 10q23 (P(Han combined for ESCC) = 7.46 x 10(-56), odds ratio (OR) = 1.43; P(Uygur-Kazakh for ESCC) = 5.70 x 10(-4), OR = 1.53) and C20orf54 at 20p13 (P(Han combined for ESCC) = 1.21 x 10(-11), OR = 0.86; P(Uygur-Kazakh for ESCC) = 7.88 x 10(-3), OR = 0.66). We also confirmed association in 2,766 cases of gastric cardia adenocarcinoma cases and the same 11,013 control subjects (PLCE1, P(Han for GCA) = 1.74 x 10(-39), OR = 1.55 and C20orf54, P(Han for GCA) = 3.02 x 10(-3), OR = 0.91). PLCE1 and C20orf54 have important biological implications for both ESCC and GCA. PLCE1 might regulate cell growth, differentiation, apoptosis and angiogenesis. C20orf54 is responsible for transporting riboflavin, and deficiency of riboflavin has been documented as a risk factor for ESCC and GCA.

[Serum proteomic profiles of the subjects with esophageal precancerous and cancerous lesions from Linzhou, an area with high incidence of esophageal cancer in Henan Province, Northern China].

BACKGROUND & OBJECTIVE: Some molecular changes occurred in esophageal precancerous and cancerous lesions could be reflected in the serum, but the clinical application is limited because of their low sensitivity and specificity. Serum proteomic profiling is much desirable in identifying the proteins closely related to esophageal carcinogenesis. This study was to characterize the serum protein profiles of the subjects with normal esophagus, esophageal precancerous and cancerous lesions from Linzhou, the area with the highest incidence of esophageal carcinoma (EC) in Henan Province, Northern China. METHODS: Proteomic spectra were generated with surface-enhanced laser desorption/inionation-time of flight-mass spectra (SELDI-TOF-MS) and weak cation exchange (WCX2) protein chip system, and analyzed by bioinformatics like decision tree classification algorithm on a set of serum from 130 symptom-free subjects [including 63 cases with normal esophageal epithelia, 40 with basal cell hyperplasia (BCH), and 27 with dysplasia (DYS)] and 30 EC patients from Linzhou. RESULTS: One protein in BCH group with a ratio of mass to charge (M/Z) of M9 306.61 u, 1 in DYS group with a M/Z ratio of M13 765.9 u, and 2 in EC group with M/Z ratios of M2 942.15 u and M15 953.4 u were selected to build 3 decision tree classification models to identify the subjects with BCH, DYS, and EC, respectively. With these classification models, the sensitivities of identifying BCH, DYS and EC were 57.5% (23/40), 88.8% (24/27) and 96.6% (29/30), respectively, in the training sets, and 57.5% (23/40), 66.6% (18/27) and 60.0% (18/30), respectively, in the test sets; the specificities of identifying BCH, DYS and EC were 96.8% (61/63), 63.4% (40/63) and 92.0% (58/63), respectively, in the training sets, and 95.2% (60/63), 71.4% (45/63) and 84.1% (53/63), respectively, in the test sets. CONCLUSION: The protein sets with M/Z ratios of M9 306.61 u, M13 765.9 u, M2 942.15 u, and M15 953.4 u may contain promising serum biomarkers for screening the subjects with high-risk of EC.