OBJECTIVE: To discuss the interaction between autophagy and endoplasmic reticulum stress in human gastric cancer SGC-7901 cells treated with vitamin E succinate (VES). METHODS:Human gastric cancer SGC-7901 cells were treated with VES of different doses (0, 5, 10, 15, 20 μg/mL), then after 24 h laser confocal microscope was used to study fluorescence intensity and distribution of the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3). Western blot was used to evaluate the level of endoplasmic reticulum stress markers glucose regulative protein 78(GRP78), glucose regulative protein 94(GRP94) and LC3, Beclin-1. SGC-7901 cells were pre-treated with autophagy inhibitors, 3-methyladenine(3-MA) and chloroquine (CQ), inhibitor of endoplasmic reticulum stress, 4-phenylbutyric acid(4-PBA), then human gastric cancer SGC-7901 cells were treated with 20 μg/mL VES for 24 h. Western blot evaluated the level of GRP78, GRP94, LC3 and Beclin-1. RESULTS:With increasing doses of VES, the LC3 in cells began to gather and intensity, GRP78 and GRP94 were decreased, then increased again and peaked at 24 h. The levels of GRP78 and GRP94 in VES+3-MA group and VES+CQ group were significately higher compared with VES alone (all P<0.05). The levels of LC3 and Beclin-1 in VES+4-PBA group were higher compared with VES alone(both of P<0.05). CONCLUSION:In VES-treated human gastric cancer cells, the autophagy and endoplasmic reticulum stress could interact and regulate each other.

OBJECTIVE: To analyze the expression and methylation of SOX30 gene in colorectal cancer cell lines and colorectal cancer tissues. METHODS:The mRNA expression and methylation of SOX30 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and methylation specific PCR (MSP). Demethylation experiment with 5-Aza-CdR was used to confirm the regulation of SOX30 expression by DNA hypermethylation. The methylation of SOX30 was further confirmed by sodium bisulfite DNA sequencing (BSP). RESULTS:Hypermethylation of SOX30 gene was found in all the colorectal cancer cell lines, with reduced mRNA expression. The mRNA expression of SOX30 increased in colorectal cancer cell lines treated with 5-Aza-CdR, indicating that SOX30 expression was regulated by DNA methylation. SOX30 hypermethylation was found in most of the colorectal cancer tissues (79.6%) , but not in tumor adjacent tissues (20%). After investigating possible associations between SOX30 methylation and clinicopathologic features, we found that SOX30 hypermethylation was associated with histological type, but not correlated to gender, age, smoking, clinical TNM stages and Ducks stages. CONCLUSION:Down-regulation of SOX30 by hypermethylation was always found in colorectal cancer cell lines, and its hypermethylation was obviously associated with histological type, suggesting SOX30 playing an important role in the carcinogenesis of colorectal cancer.

OBJECTIVE: To observe the effects of AMPK activation on intracellular endoplasmic reticulum stress (ER stress) in Hela cells. METHODS:Cultured HeLa cells were divided into groups:1‰ DMSO control, tunicamycin (2 μg/mL) or MG132 (500 nmol/L), tunicamycin or MG132+Compound C (0.5 mmol/L);tunicamycin or MG132+ AICAR(2 mmol/L) and tunicamycin or MG132+metformin (2 mmol/L). AMPK activation and markers of ER stress were determined by Western blot, and then intracellular Ca²⁺ concentration was measured in HeLa cells by Indo-1 ratiometric Ca²⁺ analysis. RESULTS:Our results showed that tunicamycin and MG132 could increase expression of ER stress. Activation of AMPK by AICAR and metformin could reduce the expression of above markers, and decrease the concentration of intracellular Ca²⁺. CONCLUSION:AMPK activation could relieve intracellular ER stress, maintain the homeostasis of intracellular Ca²⁺ concentration, and further, may contribute to cellular adaptation and resistance to stress stimuli.

OBJECTIVE: This study aimed to explore the effects of cyclophilin A (CyPA) over-expression in human lung adenocarcinoma cell line H1299 after treatments of high temperature, ultraviolet (UV) and cisplatin-induced apoptosis. METHODS:We set up H1299 CyPA over-expression cell lines via lentiviral transfection and used fluorescence microscope and Western blot analysis to analyze the expression of the target gene. H1299 and H1299 CyPA OE cell lines were cultured and exposed to treatments of high temperature, UV and cisplatin. High intension analysis platform was used to detect apoptosis of the two cell lines. RESULTS:Green fluorescent could be detected in H1299 CyPA OE cell line, and the protein CyPA expression was much higher than H1299 cell line, indicating that H1299 CyPA OE cell line was set up successfully. The apoptosis rate of H1299 CyPA OE cell line was much lower than H1299 cell line under different concentrations of cisplatin(P<0.05), but meanwhile the difference of apoptosis rates of H1299 CyPA OE cell line and H1299 cell lines showed no statistical significance(P>0.05). CONCLUSION:The over-expression of CyPA could confer resistance to apoptosis induced by cisplatin. CyPA may act as a potential gene target and provide new strategies of combined chemotherapy for lung cancer.

OBJECTIVE: APOBEC-3F (A3F) and -3G (A3G) inhibit viral replication of HBV and participate in hypermutation of HBV genome. HBcAg is considered as a potential interaction site. Therefore, we studied the interactions between A3F/A3G and HBcAg proteins. METHODS:The cDNAs of human A3F and A3G were cloned by RT-PCR from PHA-stimulated PBMC, and DNA of HBcAg was cloned by PCR from ayw subtype HBV. Then the yeast two hybrid expression plasmids pGADT7-A3F/-A3G and pGBKT7-HBcAg were constructed. To ascertain the direct interactions between HBcAg and A3F or A3G, pGBKT7-HBcAg was co-transformed into yeast AH109 with pGADT7-A3F or pGADT7-A3G, then the transformed yeast cells were cultured in synthetic double drop-out medium (DDO) and quadruple drop-out medium (QDO) containing X-α-gal, and Alpha galactose glucoside enzyme activity tests were carried out. The eukaryotic expression plasmids, containing HBcAg or A3F or A3G respectively, were constructed and transfected into HeLa cells, then co-immunoprecipiation (Co-IP) and western blot were performed to ensure the indirect interactions between HBcAg and A3F or A3G. RESULTS:The results of restriction endonuclease analysis and DNA sequencing showed that the plasmids involved were constructed successfully. Yeast two hybrid experiments with alpha galactosidase qualitative and quantitative assays displayed that A3F and A3G had no evident direct interaction with HBcAg. The results of Co-IP and western blot indicated A3F and A3G had interactions with HBcAg. CONCLUSION:We demonstrated the interactions between A3F/A3G and HBcAg, but the interactions might not be realized by direct bind of the proteins.

OBJECTIVE: To compare the genotoxic characteristics of micronucleus cytomic effect of the two differential fluorescence wave lengths CdSeS/ZnS-COOH alloyed quantum dots(QDs). METHODS:Using the cytokinesis block micronucleus cytomic assays as the major method, mouse lymphoma cells (L5178Y) were put under the test doses of 0.062 5, 0.125, 0.25, 0.5 and 0.1 mg/mL, the micronucleus cytomic effect, dose-effect and time-effect relationships were assessed. RESULTS:Compared with the negative control group, the two wavelengths of CdSeS/ZnS-COOH alloyed QDs at 0.062 5 mg/mL could induce the emergence of micronucleus(P<0.05). Both could induce types Ⅰ and Ⅱ micronucleus and nuclear buds effects, but 490 nm alloyed QDs could not induce nucleoplasmic bridge effect, while 540 nm alloyed QDs could. 490 nm alloyed QDs at the lowest dose could induce total micronucleus, typeⅠ micronucleus and nuclear buds, whilst 540 nm alloyed QDs at the lowest dose could induce typeⅠmicronucleus. With the increasing dose other effects emerged. The micronucleus cytomic effect of various groups had a clear dose-response relationship(P<0.05). 490 nm QDs could induce the emergence of nucleoplasmic bridges first after 9 h exposure, while 540 nm QDs induced the increase of total micronucleus and type Ⅱ micronucleus first. As time increased other effects began to develop, and reached their peaks at 27 h, then declined. CONCLUSION:Both QDs could induce micronuclus cytomic effect, but at the same dose their spectrum of effect, dose-effect and time-effect were different, which showed that they possessed different genotoxic characteristics.

OBJECTIVE: Insulin resistance (IR) is a major characteristic of type 2 diabetes millitus which is difficult to be prevented and treated, and its pathogenesis is still uncertain. The aim of this study was to investigate the change of oxidative stress and nuclear-mitochondria axis in hepatic tissue during the process of insulin resistance induced by high-fat-diet in mice. METHODS:Male C57 mice were randomly divided into 2 groups, the mice in control group were fed with normal chow while those in high fat diet group were fed with 10% lard oil containing diet to induce insulin resistance. The hepatic reactive oxygen species level, malondialdehyde (MDA) content and lipid accumulation were tested. P-Akt, Nrf2, NRF-1 and mtTFA expression in liver tissue were determined by Western blot. RESULTS:Compared with controls, mice fed with a high fat diet could induce insulin resistance. During this process, hepatic fat and ROS accumulation were both increased, the MDA content was also elevated by about 30%. The expression of insulin signal transduction protein P-Akt was attenuated by 45% whilst the expressions of Nrf2, NRF-1 and mtTFA were decreased by 20%-30%. CONCLUSION:Oxidative stress and dysfunction of Nrf2/NRF-1/mtTFA axis may play important roles in the development of insulin resistance induced by a high fat diet.

OBJECTIVE: To explore the effects of folate and choline on human DNA mismatch repair genes hMLH1, hMSH2 expressions in human colonic adenocarcinoma cells. METHODS:Modified RPMI-1640 with the combinations of 12 μmol/L choline chlorine (CC) and 15, 30, 120 nmol/L of folic acid (FA) or 5-methyltetrahydrofolate (5-MeTHF), FA 120 nmol/L and CC (1.5, 3 μmol/L) were set as the intervening culture medium. COLO205 cells were cultured for 20 days. The transcription levels of hMLH1 and hMSH2 were analyzed by real-time fluorescence quantitative PCR. RESULTS:Transcription levels of hMLH1 and hMSH2 were significantly negatively correlated significantly with the concentrations of FA, 5-MeTHF or CC in tested cells (P<0.01). 5-MeTHF was more effective on hMLH1 and hMSH2 transcription regulation than same concentration of FA at 30 and 120 nmol/L (P<0.01 or 0.05). CONCLUSION:We concluded that FA, 5-MeTHF and CC deficiency up-regulated the expressions of hMLH1 and hMSH2. This may be the cellular response to DNA damage or abnormal methylation induced by folate and choline deficiency.

OBJECTIVE: It has been demonstrated that HBV is able not only to pass through the blood-testis barrier and integrate into sperm genome, leading to increase of the instability of sperm chromosomes, resulting in a various types of chromosomal aberrations, but also to destroy mitochondrial functions and induce loss of mitochondrial membrane potential (MMP), causing low sperm motility and reducing fertilization rate and fertilizing index. However, the exact pathogenic mechanism of such events is largely unknown so far. The purpose of this study is to explore the relationship between hepatitis B virus S protein (HBs) and oxidative stress in human sperm cells. METHODS:After the co-incubation of sperm cells with (0, 25, 50, 100 μg/mL) HBs, the reactive oxygen species (ROS) production was assessed by flow cytometric analyses;the total antioxidant capacity (TAC) level and the Aldetect (MDA-specific) lipid peroxidation (LP) level of the human sperm were measured by microplatereader. RESULTS:After incubation with 25 μg/mL of HBs for 3 h, the average rates of ROS-positive cells was significantly increased in the test groups as compared to those in the control groups, while TAC level was decreased when compared with the control. There was significantly higher level of MDA in the sperm cells exposed to 50 μg/mL of HBs for 3 h than that in the controls (P<0.05 or 0.01). HBs increased the MDA levels and the numbers of ROS-positive cells in a dose-dependent manner. HBs decreased the TAC levels in sperm cells in a dose-dependent manner. CONCLUSION:HBs exposure could lead to ROS generation, lipid peroxidation, TAC reduction, resulting in loss of sperm membrane integrity and causing sperm dysfunctions.

OBJECTIVE: To evaluate the DNA damage of different sub-groups of workers in the electronics industry, and to set up major preventive measures. METHODS:The 563 workers of an electronics factory in the Pearl River Delta Region were studied, according to their jobs we divided them into 5 groups, including lead exposure group (n167), isopropyl alcohol group (n287), noise group (n39), lead+high temperature group (n34), and isopro-panol+high temperature group (n36), with 90 pre-employment workers as controls. DNA damage was detected using micro-whole blood specimen by the comet assay method. RESULTS:Nuclear DNA content of lead+high temperature group was lower than the control group (P<0.05), and its tail DNA content, tail length, and tail moment were higher than the control group (P<0.05). The nuclear DNA content of isopropyl alcohol+high temperature group was lower than the control group (P<0.05), the comet tail DNA content, tail length, comet length, olive tail moment and tail moment were higher than the control group (P<0.05). CONCLUSION:Workers of different types in electronics industry chosen in this study had obvious DNA damage, especially isopropanol+high temperature group, followed by lead+high temperature group, to which special attention should be paid to these.

OBJECTIVE: Trichloroethylene (TCE), a common industrial contaminant in the environment, could induce hepatocellular carcinoma but not kidney cancer in mice. This study aimed to understand the molecular mechanisms of TCE hepatocarcinogenicity by examining the effect of TCE on proliferation-related mRNA expression and DNA methylation in mouse liver and kidney. METHODS:B6C3F1 male mice aged six weeks were randomly divided into 3 groups, with 4 mice in each group. Mice were fed with TCE at 0, 500 and 1 000 mg/kg for 5 days. Using qPCR, we examined the mRNA expression of genes involved in cell proliferation and in the regulation of DNA methylation in TCE-exposed mouse liver and kidney. The promoter methylation status of Cdkn1a and the DNA methylation level of repetitive sequences were detected by combined bisulfite restriction analysis. RESULTS:Compared with control, TCE increased the mRNA expression of Cdkn1a, Jun and Mki67 in a dose-dependent manner in mouse liver. Moreover, in mouse treated with TCE at 1 000 mg/kg, the mRNA expression of a number of key DNA methylation regulation genes including Dnmt3a, Dnmt3b and Tet2 were down-regulated, while the mRNA expression level of Uhrf1 was up-regulated (all P<0.05). TCE also induced hypomethylation of the promoter region of Cdkn1a in mouse hepatocytes. However, TCE should no detectable effects on mRNA expression and DNA methylation in mouse kidney. CONCLUSION:Alterations in the mRNA expression of proliferation-related genes and DNA methylation changes might be crucial to TCE-induced hepatocarcinogenesis in mice.

OBJECTIVE: To investigate the effects of soybean isoflavone and di-(2-ethylhexyl) phthalate(DEHP) on the hormone level in female rats. METHODS:Thirty-two female Sprague-Dawley rats were randomly divided into 4 groups:86 mg/kg soybean isoflavone group, 68 mg/kg DEHP group, isoflavone(86 mg/kg)+DEHP(68 mg/kg) group and control group, 8 rats in each group. Food intake and body weight were recorded weekly, at the end of the fourth week, rats were sacrificed, blood collected and viscera weighed. High performance liquid chromatography (HPLC) was used to measure serum DEHP content and radioimmunoassay method was used to assess serum estrogen and testosterone levels. The expression of ERα in endometrium was tested by immunohistochemistry. RESULTS:When compared to the control group, no significant difference was found in all of the treated groups with respect to weight gain (P value were all >0.05). DEHP significantly increased the organ/body ratio of spleen and liver when compared to the control group (P<0.05). The levels of serum DEHP in the DEHP group and isoflavone+DEHP group were significantly higher than that in the control group (P<0.05) and the isoflavone+DEHP group had significant lower DEHP level than the DEHP group (P<0.05). Furthermore, testosterone level was not significantly different among different groups, however, serum estrogen in isoflavone+DEHP and DEHP group was significantly lower than that in the control group (P<0.05). Immunohistochemistry suggested although 86 mg/kg soybean isoflavone group, 68 mg/kg DEHP group and isoflavone+DEHP group had higher expresson level of ERα in rats'endometrium tissue, no significant difference was found among them(P>0.05). CONCLUSION:DEHP could significantly increase the organ/body ratio of the liver and spleen and it also could reduce serum estrogen level in female rats. Soybean isoflavone could reduce the residual serum DEHP to some extent.

OBJECTIVE: To study the effects of 2, 4-dichlorophenoxyacetic acid (2, 4-D) on early neuro-behavior development and oxidative stress in offsprings. METHODS:Pregnant Wistar rats were treated with 2, 4-D by gavage, in doses of 0, 25, 50, 100 mg/kg once a day from gestation day 2 until postnatal day 21. Then the early physiological and neurobehavioral indexes in the offsprings were measured. The levels of MDA, GSH and the GSH-Px activities in brain tissues of newborn rat were determined. RESULTS:The weights of offspring in the 100 mg/kg 2, 4-D group were lower than in control after postnatal day 14(P<0.05) and the weights of offspring in the 50 mg/kg 2, 4-D were lower than in control after postnatal day 21(P<0.05). The physiologic markers including eye opening, pinna detachment, hair growth, tooth growth etc in all treated groups were not affected in contrast to the control group, cliff avoidance, mid-air righting and acoustic startle in the 100 mg/kg 2, 4-D group were delayed(P<0.05). The level of GSH and GSH-Px activities decreased in the 100 mg/kg 2, 4-D group, while the level of MDA was elevated in the 50 and 100 mg/kg 2, 4-D groups. CONCLUSION:Early neurobehavioral development of offspring rats could be impaired by 2, 4-D due to its disturbance to the homeostasis of oxidation and anti-oxidation system.

OBJECTIVE: To explore the effects of di-(2-ethylhexyl) phthalate (DEHP) on protein expression of hormonal-related genes including 3β-hydroxysteroid dehydrogenase (3β-HSD), gonadotropin-releasing hormone receptor (GnRHR), follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in rats. METHODS:A total of 80 5-week-old male and female Sprague-Dawley rats were randomly divided into four groups with 20 rats in each group. The DEHP doses were 0 mg/kg in control group (corn oil), 100 mg/kg in low dose group, 500 mg/kg in medium dose group and 1 500 mg/kg in high dose group. Rats were exposed to DEHP via gavage administration for consecutive six weeks, DEHP administration was once a day and five times a week. After the exposure, rats were anesthetized through ether, then sacrificed for the further studies. The protein expressions of 3β-HSD, GnRHR and FSHR in testis and ovary was analyzed by Western blot. RESULTS:Compared with control group, the expression of 3β-HSD protein was significantly increased in DEHP 500 and 1 500 mg/kg groups ( P<0.01), GnRHR was decreased in DEHP 1 500 mg/kg group (P<0.01), FSHR was decreased in DEHP 500 and 1 500 mg/kg groups (P<0.05 or P<0.01, respectively). The protein expressions of LHR in DEHP 500 and 1 500 mg/kg groups were decreased by 25% to 35% when compared with control, whilst GnRHR protein expressions were decreased by 60% to 80% in DEHP 500 and 1 500 mg/kg groups (P<0.05 or P<0.01). CONCLUSION:DEHP could cause endocrine disorder and interfere with the synthesis of sex hormones in rats, ultimately leading to male and female reproductive dysfunction.

OBJECTIVE: To investigate the expression of semaphorin 4C(Sema4C) in laryngeal squamous carcinoma and its role in tumor proliferation and lymphatic metastasis. METHODS:We investigated the expression of Sema4C and MCM2 in 82 cases of laryngeal squamous carcinoma and normal laryngeal tissues by immunohistochemical technique.Furthermore, we used D2-40 to label the lymphatic microvessel and measure the lymphatic microvessel density(LMVD) to analyze their roles in laryngeal squamous carcinoma. RESULTS:The expressions of Sema4C and MCM2 in laryngeal squamous carcinoma were significantly higher than that in the normal laryngeal tissues (P<0.01), but the difference of LVMD was not significant (P0.077). The aberrant expression rates of Sema4C, MCM2 and LVMD in laryngeal squamous carcinoma were closely related to lymph node metastasis but did not correlate with age and T stage. LMVD in laryngeal squamous carcinoma should positive correlation with the expressions of Sema4C (P<0.05) and MCM2 (P<0.01)． Moreover, there was positive correlation between Sema4C and MCM2 (P<0.05)．CONCLUSION:Sema4C may play a role in the development of laryngeal squamous carcinoma. It may be possible to follow the progression of the disease and provide prognosis by detecting the expression of Sema4c.

OBJECTIVE: This study aimed to investigate the expression of integrin αvβ6 and JunB in oral squamous cell carcinoma (OSCC) and the correlation with clinicopathologic characteristics and prognosis. METHODS:The expression of integrin αvβ6 and JunB protein was detected with immunohistochemical staining assay in 120 cases of OSCC and 15 samples of normal oral mucosa. The correlation with clinicopathologic characteristics and prognosis of the patients were evaluated. RESULTS:Integrin αvβ6 was detected in both cytoplasm and cellular membrane of OSCC, but JunB protein was localized in the nuclei of both OSCC and normal oral mucous tissues. The expressions of integrin αvβ6 and JunB protein in OSCC were significantly higher than that in normal oral mucosa (P<0.01). The expression of integrin αvβ6 was positively related to depth of invasion(T stage), tumor differentiation degree and lymph node metastasis(N stage) (P<0.05). The expression of JunB was positively related to depth of invasion and lymph node metastasis (P<0.05). Survival as a single factor analysis revealed that patients with over-expression of integrin αvβ6 had a shorter overall survival(OC) (P=0.021). Through Cox multivariate regression analysis, lymph node metastasis (N stage) was an independent risk factor that affected the prognosis. CONCLUSION: The over-expressions of integrin αvβ6 and JunB were common in OSCC and closely correlated with tumor invasion and metastasis. Integrin αvβ6 may be used as a prognostic marker for therapy.