Etiological study of esophageal squamous cell carcinoma in an endemic region: a population-based case control study in Huaian, China

BACKGROUND: Continuous exposure to various environmental carcinogens and genetic polymorphisms of xenobiotic-metabolizing enzymes (XME) are associated with many types of human cancers, including esophageal squamous cell carcinoma (ESCC). Huaian, China, is one of the endemic regions of ESCC, but fewer studies have been done in characterizing the risk factors of ESCC in this area. The aims of this study is to evaluate the etiological roles of demographic parameters, environmental and food-borne carcinogens exposure, and XME polymorphisms in formation of ESCC, and to investigate possible gene-gene and gene-environment interactions associated with ESCC in Huaian, China. METHODS: A population based case-control study was conducted in 107 ESCC newly diagnosed cases and 107 residency- age-, and sex-matched controls in 5 townships of Huaian. In addition to regular epidemiological and food frequency questionnaire analyses, genetic polymorphisms of phase I enzymes CYP1A1, CYP1B1, CYP2A6, and CYP2E1, and phase II enzymes GSTM1, GSTT1, GSTP1, and microsomal epoxide hydrolase (EPHX) were assessed from genomic DNA using PCR based techniques. RESULTS: Consuming acrid food, fatty meat, moldy food, salted and pickled vegetables, eating fast, introverted personality, passive smoking, a family history of cancer, esophageal lesion, and infection with Helicobacter pylori were significant risk factors for ESCC (P < 0.05). Regular clean up of food storage utensils, green tea consumption, and alcohol abstinence were protective factors for ESCC (P < 0.01). The frequency of the GSTT1 null genotype was higher in cases (59.4%) compared to controls (47.2%) with an odds ratio (OR) of 1.68 and 95% confidence interval (CI) from 0.96 to 2.97 (P = 0.07), especially in males (OR = 2.78; 95% CI = 1.22–6.25; P = 0.01). No associations were found between polymorphisms of CYP1A1, CYP1B1, CYP2A6, CYP2E1, GSTM1, GSTP1, and EPHX and ESCC (P > 0.05). CONCLUSION: Our results demonstrated that dietary and environmental exposures, some demographic parameters and genetic polymorphism of GSTT1 may play important roles in the development of ESCC in Huaian area, China

Defective autophagic flux aggravates cadmium-induced Sertoli cell apoptosis

The male reproductive dysfunction accounts for 50% of infertile couples in the world. Cadmium (Cd) is one of the most harmful heavy metals to both the environment and inhabitants. Accumulating data suggest that Cd could cause male infertility. Sertoli cell (SC) is a somatic cell of testis and a key regulator of spermatogenesis by providing physical and nutritional support for developing sperm. Many studies showed that Cd induced dysfunction of SCs was directly related to male reproductive damage. However, the mechanism of SCs injury caused by Cd remains to be clarified. We found that Cd treatment caused a significant increase of apoptosis in SCs cells, accompanied by a marked increase in the production of ROS. These results were associated with the formation of mitochondria-containing autophagosomes and increased expression of LC3-II in vitro. Interestingly, our results showed that Cd did not promote but inhibited the fusion of mitochondria-containing autophagosomes with lysosomes by reducing the function of lysosomes. Together, this study provides insight into the negative effects of Cd, which interferes with autophagic flux and induces the apoptosis of SCs

Purification, structural identification, antioxidant activity, and inhibitory effects on digestive enzymes of α-glucan from Chuanminshen violaceum

Chuanminshen violaceum, as a food and folk medicine, is cultivated and grown only in China. Its polysaccharides play an important role in medicinal benefits. Herein, crude C. violaceum polysaccharides (CVPs) were purified to yield two polysaccharide fractions (CVPs-1-G and CVPs-2-G) using DEAE Cellulose-52 and Sephadex G-100 column chromatography. Structural analyses indicated that both CVPs-1-G and CVPs-2-G were glucans with average molecular weights of 8.1 kDa and 187.5 kDa, respectively. The main chain of the major component (CVPs-1-G) might be composed of (1 → 4)-linked α-Glcp, and the branched-chain might be an α-Glcp → 4)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 4)-α-D-Glcp, which connected to the main chain through the O-6 position of → 3,6)-α-D-Glcp-(1 →. In addition, CVPs-1-G displayed ABTS, DPPH, and hydroxyl radical scavenging activity, as well as competitive inhibitory activities of α-amylase and non-competitive inhibitory activities of lipase and pepsin. According to molecular docking, its inhibitory activity on digestive enzyme might be caused by hydrogen bond and hydrophobic interaction between them. Thus, CVPs exhibit strong potential for use in functional foods and pharmaceuticals

Promoter Hypomethylation of Maspin Inhibits Migration and Invasion of Extravillous Trophoblast Cells during Placentation.

Extravillous trophoblast (EVT) cells invade the endometrium and the maternal spiral arterioles during the first trimester. Mammary Serine Protease Inhibitor (Maspin, SERPINB5) plays a putative role in regulating the invasive activity of cytotrophoblasts. The maspin gene is silenced in various cancers by an epigenetic mechanism that involves aberrant cytosine methylation. We investigated the effect of the methylation status of the maspin promoter on the maspin expression and the aggressiveness of EVT cells.Western blotting was used to detect the maspin protein expression in EVT cells upon hypoxia. The proliferative ability, the apoptosis rate and the migration and invasiveness were measured with Cell Counting Kit-8 assay, Flow Cytometry technology and Transwell methods. Subsequently, we treated cells with recombinant maspin protein. The methylation degree of maspin promoter region upon hypoxia/ decitabine was detected by bisulfite sequencing PCR and methylation-specific PCR. Finally, we explored the effects of decitabine on maspin protein expression and the aggressiveness of EVT cells.Hypoxia effectively increased maspin protein expression in EVT cells and significantly inhibited their aggressiveness. The addition of recombinant maspin protein inhibited this aggressiveness. Decitabine reduced the methylation in the maspin promoter region and effectively increased the maspin protein expression, which significantly weakened the migration and invasiveness of EVT cells.The methylation status of the maspin promoter is an important factor that affects the migration and invasion of EVT cells during early pregnancy. A decrease in the methylation status can inhibit the migration and invasion of EVT cells to affect placentation and can result in the ischemia and hypoxia of placenta

Status of methylation at the CpG site in the maspin promoter region of TEV-1 cells upon hypoxia and DI A-D: Bisulfite sequencing revealed a methylated CpG island in the maspin promoter region of TEV-1 cells in four groups.

<p>Each square represents an individual CpG site, and the sizes of the yellow parts indicate fractional methylation. The yellow parts represent methylation-positive results, and the blue parts represent methylation-negative results. E: The extent of methylation at the CpG site was calculated using the following formula: [extent of methylation/(extent of methylation + extent of unmethylation)]. The methylation rates of the maspin promoter in the HOX group were significantly decreased compared to the NOX group at 24h and 48h. (*P < 0.05). F: DI significantly reduced the methylation rates of the maspin promoter in the NOX group, but it had no significant effect on the methylation rates of the maspin promoter in the HOX group. (*P < 0.05). G: MSP analysis of maspin promoter in TEV-1 cells of four groups. All the 4 groups show the presence of both unmethylated and methylated DNA in the placental tissues, respectively. The bands detected by the methylated primer represent methylated maspin (M), and the bands detected by the unmethylated primer represent unmethylated maspin (U).</p

Effect of recombinant maspin protein on proliferative, apoptotic, migrative and invasive ability in TEV-1 A: The number of cells in NOX, HOX, r.M and HOX+r.M groups was measured by ELISA.

<p>Recombinant maspin protein significantly weakened the proliferative ability of TEV-1 cells in NOX group at 24h, but overall it had no significant effect on the proliferative ability of TEV-1 cells in either NOX or HOX group. B: The apoptosis rate of cells in NOX, HOX, r.M and HOX+r.M groups was detected by FCM. Recombinant maspin protein had no significant effect on the apoptosis rate of TEV-1 cells in either NOX or HOX group. C: The migrated/invaded cells were counted to estimate the effect of recombinant maspin protein on normoxic EVT cells. Recombinant maspin significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the normoxic EVT cells. (*P < 0.05) D: The migrated/invaded cells were counted to estimate the effect of recombinant maspin protein on hypoxic EVT cells. Recombinant maspin significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the hypoxic EVT cells. (*P < 0.05).</p

Effect of DI on maspin expression, aggressive ability of TEV-1 cells.

<p>A: Representative Western blot analysis of maspin protein expression. DI increased the protein expression of maspin in TEV-1 cells in the NOX group at 24h and 48h. DI increased the protein expression of maspin in TEV-1 cells in the HOX group at 24h, but had no significant effect on the protein expression of maspin in TEV-1 cells in the HOX group at 48h. (*P < 0.05). B: We investigated the effect of decitabine on the apoptosis rate of the normoxic/hypoxic EVT cells. DI had no significant effect on the apoptosis rate of TEV-1 cells in the NOX and HOX group. C: Representative Western blot analysis of maspin protein expression in the NOX and HOX groups upon DI at two different culture time points (24h, 48h). (+: with DI;-: without DI). D: The migrated/invaded cells were counted to estimate the effect of decitabine on normoxic EVT cells. Decitabine significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the normoxic EVT cells. (The control was the NOX group. *P < 0.05) E: The migrated/invaded cells were counted to estimate the effect of decitabine on hypoxic EVT cells. Decitabine significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the hypoxic EVT cells. (The control was the NOX group. *P < 0.05) F: Photo of migrated/invaded cells in the NOX, DI, HOX and HOX+DI group (200X).</p

Effect of hypoxia on protein expression of maspin, apoptotic and invasive ability in TEV-1 A: The maspin protein in the normoxic and hypoxic groups expressed as a relative measure compared to GAPDH and normalized to a control sample run on each gel at two different culture time points (24h, 48h).

<p>The protein expression of maspin in the HOX group was significantly higher compared to the NOX group at both 24h and 48h, the protein expression of maspin at 48h was significantly higher compared to 24h; (*P < 0.05). B: Representative Western blot analysis of maspin protein expression in the NOX and HOX groups; C: Apoptosis rate of cells was measured by flow cytometry. The apoptosis rate in the HOX group was significantly higher compared to the NOX group at 12h, 24h and 48h. (*P < 0.05) D: The invasive ability of cells were tested by transwell method. The invaded cells in the HOX group was significantly fewer compared to the NOX group at 48h. (*P < 0.05) E: Photo of invaded cells in the NOX and HOX group (200X).</p