BACKGROUND AND AIM: To examine the mutation of DNA repair genes of human bronchial epithelial cells malignant transformation induced by glycidyl methacrylate. MATERIALS AND METHODS: To evaluate the mutation of DNA repair genes XRCC1, hMSH2, XPD and XRCC3 by polymerase chain reaction-restriction fragments length polymorphism(PCR-RFLP), and the result was verified by the DNA sequencing. RESULTS: The mutation of hMSH2 IVS12-6 (T>C) was observed, while mutations in the other genes were not found.The results from PCR-RFLP and DNA sequencing were consistent. CONCLUSION:The mutation of DNA repair genes hMSH2 IVS12-6 (T>C)might be important and an initation step during the malignant transformation of human bronchial epithelial cells induced by glycidyl methacrylate.

BACKGROUND AND AIM: To study the biodistribution of the 125I labeled tyrosine-multiwalled carbon nanotubes via a single tail vein injection in ICR mice. MATERIALS AND METHODS: Tyrosine-multiwalled carbon nanotubes were prepared and labeled by 125I. Healthy adult female ICR mice received intravenous injection of 125I marked tyrosine-multiwalled carbon nanotubes. The mice were sacrificed at 10 min, 30 min, 1 h, 6 h, 12 h, 24 h and 48 h after injection. The tissues, including the blood, heart, liver, spleen, lung, kidney, brain, muscle, bone, stomach, intestine, skin and thymus gland, were immediately dissected. Each tissue was weighed and counted for 125I activity. The distribution in the tissues was represented by the percent of the injected dose per gram of tissue(％ID/g). RESULTS: 125I activity was mainly detected in the lung and without significant reduction at 48 h after injection (P>0.05). The second highest level was found in the kidney but rapidly declined within 48 h (P<0.05). 125I activity was found accumulated and remained constant in the liver and spleen. The 125I activity was very low in blood, heart, muscle, bone, stomach, intestine, skin and thymus gland and continuous decreased within 48 h. Lastly, it was rarely detected in brain. CONCLUSION: Intravenous tyrosine-multiwalled carbon nanotubes were mainly distributed in the lung of ICR mice and might be stored in the lung, liver and spleen.

BACKGROUND AND AIM: To investigate the expression and alterations of hypoxia inducible factor-1α (HIF-1α) during development of hepatocellular carcinoma(HCC). MATERIALS AND METHODS: Rat hepatoma model was established with 2-fluorenylacetamide (2-FAA) containing-feeds for 12 weeks in male Sprague-Dawley (SD) rats. Morphological changes in rat livers were examined by HE staining. The changes of HIF-1α in rat livers and sera were quantitatively analyzed by enzyme linked immunosorbent assay (ELISA). Simultaneously, the expression and cellular distribution of liver HIF-1α were assessed by immunohistochemistry. RESULTS: Rat hepatocytes showed granule-like degeneration to atypical hyperplasia and HCC development after ingestion of 2-FAA, with progressive increase of hepatic HIF-1α expression during the course. The levels of HIF-1α in hepatoma tissues and sera were significantly higher than those in normal and degenerative tissues. Immunohistochemistry evidence showed positive expression and hepatocyte distribution of HIF-1α in rat hepatoma. A positive relationship of HIF-1α level in hepatoma tissues and sera was found(r=0.474,P<0.05). CONCLUSION: Hepatic HIF-1α may participate in hepatocytic malignant transformation. Detection of HIF-1α expression during HCC development could be a useful molecular marker for early diagnosis and prognosis of HCC.

BACKGROUND AND AIM: The homeostasis of histone acetylation can control gene expression and chromosome configuration. If the balance is disrupted the cells will be induced to form cancers.This study was designed to investigate the mechanisms of histone deacetylase inhibitors(sulfonamide anilide compound referred as the “Compound”) with antitumor activity against human bladder cancer cells. MATERIALS AND METHODS: Treated with different concentrations of the Compound and HCPT for different periods of time, human bladder cancer cell line BIU-87 growthwas measured by MTT assay. Apoptosis and changes in cell cycle were examined by means of flow cytometry (FCM). P21waf/cif1 mRNA expression was assessed by RT-PCR. RESULTS: The Compound and HCPT inhibited the proliferation of bladder cancer cells significantly at nanomolar concentrations in a time- and dose-dependent fasion,but the Compound was more effective. Both treatments caused cell arrest at G1 phase as shown by FCM and induced P21waf/cif1 mRNA expression. HCPT induced apoptosis but no apparent apoptosis peak. Real-time quantitative PCR analysis showed that the Compound could induce P21waf/cif1 mRNA expression in BIU-87.The above results clearly displayed volume-effect and time-effect relationships. CONCLUSION: Sulfonamide anilide compound could inhibit human bladder cancer cell growth in vitro, most likely through induction of P21waf/cif1 mRNA expression and subsequent arrest of cell cycling at G1 phase.

BACKGROUND AND AIM: In order to investigate the roles of TRAIL in vitamin E succinate (VES)-induced apoptosis in human gastric cancer MKN28 cells. MATERIALS AND METHODS: The MKN28 huamn gastric cancer cells were cultured with different concentrations of VES.The MKN28 cells were divided into four groups:5,10,20 μg/ml VES and control groups. Apoptosis was assessed by DAPI staining, and TRAIL-receptor(DR4,DR5,DcR1 and DcR2) protein expressions induced by VES at different doses and different time points were measured by western blot. RESULTS: The results showed that VES obviously induced cells to undergo apoptosis.The expression of DR4 and DR5 were increased by VES at 5,10,20 μg/ml for 12 h; however the expression of DcR1 and DcR2 were reduced .DR4 and DR5 were increased by the hour,then reached the top level at 12 h. CONCLUSION: The data implicated that TRAIL pathway might be invovled in VES-induced apoptosis.

BACKGROUND AND AIM: To explore the effect of hypoxia on cell growth and apoptosis of non-small cell lung cancer cell line and the change in expression of Survivin gene. MATERIALS AND METHODS: The hypoxia model was set up by using cobalt chloride (CoCl2). MTT assay was used to detect the growth inhibition of A549 in the presence of CoCl2 .Cell morphological transformation was examined with fluorescent microscope. The change of Survivin gene expression was measured by RT-PCR. RESULTS: The growth inhibition rate of A549 effected by CoCl2 was increased in a concentration and time-dependent manner. After exposure to CoCl2 at high concentrations, the Survivin mRNA expression in A549 was down-regulated. CONCLUSION: Hypoxia could inhibit the proliferation of A549 cell and induce apoptosis. The mechanism may be related to the down-regulation of Survivin gene expression.

BACKGROUND AND AIM: To investigate thel changes of genomic methylation during lung carcinogenesis in Wistar rats. MATERIALS AND METHODS: Squamous cell carcinoma of lung was induced with 3-methylcholanthrene (MCA) and diethyinitrosamine (DEN) in iodized oil by left intra-bronchial instillation in 80 Wistar rats. The status of genomic methylation was evaluated by immunohistochemistry with anti 5-mc antibody, and the mean optical density and integrated optical density were measured by image analysis system. RESULTS: A total of 154 specimens of various phases during carcinogenesis were obtained: 25 hyperplasia, 27 squamous metaplasia, 33 dysplasia, 30 carcinoma in situ and 25 infiltrating carcinoma. The immunohistochemical scores of 5-mc were significantly decreased in hyperplasia, squamous metaplasia, dysplasia, carcinoma in situ and infiltrating carcinoma (P<0.01). 5-mc immunostaining was significantly higher in the basal cells than in the luminal cells in normal and carcinogenesis phase (P<0.01). CONCLUSION: The decrease of genomic methylation level suggested that it may be an important epigenetic mechanism during carcinogenesis of lung squamous cell carcinoma in rats.

BACKGROUND AND AIM: This study was to evaluate leptin and OB-Rb expression in human lung cancer and adjacent normal lung tissuee,and xplore the role of extracellular singnal-regulated kinase 1/2(ERK1/2) in leptin-induced lung cancer A549 cell proliferation . MATERIALS AND METHODS: Immunohistochemical staining was used to evaluate the protein expression of leptin and OB-Rb in tumor tissues and samples of corresponding adjacent lung tissue,the expression of OB-Rb in A549 cells was evaluated by fluoroimmunoassay and reverse transcription polymerase chain reaction(RT-PCR). The action of leptin on A549 was examined by MTT assay to determine cell proliferation,and activation of extracellular singnal-regulated kinase 1/2(ERK1/2) was assessed by Western blot. RESULTS: The positive protein expression of leptin and OB-Rb were detected in a significantly greater proportion of lung cancer(83.37％ and 66.7％,respectively)than adjacent normal lung tissue(33.3％ and 29.2％,respectively),P<0.01. Fluoroimmunoassay and RT-PCR showed the presence of OB-Rb in A549 cells. Leptin could stimulate the proliferation of A549 cells,and this effect was maximal at 100 ng/ml after 24-hour treatment. Blocking ERK1/2 phosphorylation by PD98059 significantly reduced the proliferation of A549 cells stimulated by leptin. CONCLUSION: Enhanced expression of leptin and OB-Rb in lung cancer and leptin could promote the proliferation of A549 cells by activating ERK1/2 signaling pathway suggested that continued activation of ERK1/2 pathways by leptin might promote lung cancer growth and development.

BACKGROUND AND AIM: To construct the expression vector of TrKA small interfering RNA,and to observe its effect on cell proliferation induced by nerve growth factor(NGF) and cell cycle. MATERIALS AND METHODS: Using the mRNA complete sequence of TrKA gene provided by Genbank, DNA sequence which could transcribe short hairpin RNAs was selected and designed by software,and was connected with the vector of PsilencerTM 4.1-CMV neo .Then it was transfected into MCF-7 cells after confirmed by sequencing. The stable cell line expressing TrKA small interfering RNA were selected by G418. The mRNA and protein levels of TrKA were tested by real-time PCR, Western blot, and immunohistochemistry. The alteration of cell proliferation induced by NGF was assessed by MTT assay after TrKA interference and cell cycle was measured by flow cytometry. RESULTS: The expression vector of TrKA siRNA was successfully constructed . The level of TrKA mRNA and protein was decreased by 74.7％ (P<0.01) and 80.5％,respectively. Immunohistochemistry also showed that TrKA was down-regulated.The expression of GAPDH gene was decreased by 85.0％ (P<0.05). The density value of NGF group was higher than the group of NGF＋siRNA,siRNA and control each time(P<0.05),but the density value of NGF＋siRNA group was lower than other group(P<0.05) . The result indicated that TrKA could effectively inhibit the proliferation of breast cancer cells MCF-7 induced by NGF.The cell cycle showed that compared with control, G0/ G1 period of NGF＋siRNA was higher and the S cell of siRNA was lower (P<0.05), cell cycle was arrested by G0/G1. CONCLUSION: The expression vector of TrKA siRNA could decrease the expression of TrKA in MCF-7 cells effectively,and inhibit the proliferation of MCF-7 induced by NGF.

BACKGROUND AND AIM: To examine the pathway and mechanisms of benzo[a]pyrene(BaP) effects on cell cycle. MATERIALS AND METHODS: We administratered BaP to human embryonic lung fibroblasts(HELF) and monitored the changes in cell growth by cell counting, the cell size and cell cycle distribution by FACScan flow cytometer, and the mRNA and protein expression levels of cell cycle genes by RT-PCR and Western blotting. RESULTS: BaP promoted HELF cell growth, enlarged cell size, enhanced the transition of G1 phase to S and G2/M phase. The mRNA and protein expression levels of p53-related genes were detected indicating that BaP up-regulated the p53 mRNA and protein expression, and increased the expressions of cyclin D1, CDK2, CDK4 and p21, while decreasing the expresson of cyclin E. CONCLUSION: BaP affected cell cycle progression via the expressions of cyclin D1, CDK2 and CDK4, escaping from the p53-p21 mediated G1 checkpoint and was cyclinA-p27 independent.

BACKGROUND AND AIM: To study the relationship between malignant transformation of skin scar and connexin 43(Cx43) protein and Cx43 mRNA. MATERIALS AND METHODS: We compared skin scar and skin scar carcinoma with normal skin tissues. The expressions of Cx43 protein was evaluated by immunohistochemical S-P method, and the expression of Cx43 mRNA was assessed by in situ hybridization. RESULTS: The expressions of Cx43 and Cx43 mRNA in normal epidermis, skin scar epithelium and skin scar carcinoma were strongly positive, positive and weakly positive, respectively. The expressions of Cx43 protein and Cx43 mRNA in skin scar epithelium and scar carcinoma were statistically different to that in normal skin epidermis. The expression levels of Cx43 protein and Cx43 mRNA showed positive correlation in skin carcinoma tissues. CONCLUSION: The low expressions of Cx43 proteins and Cx43 mRNA may play important roles in the development or progression of skin scar carcinoma.

BACKGROUND AND AIM: To investigate the effects of oxidative stress induced by smoking by quantifying various biomarkers of oxidative DNA damage, lipid peroxidation and antioxidative defense mechanisms. MATERIALS AND METHODS: Sixty male smokers and thirty male nonsmokers aged 18-25 were recruited and divided into two groups.8-OHdG in 24 h urine was analysed by HPLC-ECD; peripheral lymphocyte DNA strand breaks by comet assay; MDA, ACON and PP in 24 h urine were analysed by HPLC-UV and plasma antioxidant enzymes were assessed. RESULTS: 8-OHdG and DNA strand breaks were significantly greater by 185％ and 97％,respectively, in 60 smokers than in 30 nonsmokers. MDA, ACON and PP in urine were significantly higher than nonsmokers(P<0.01). Superoxide dismutase(SOD), glutathione peroxidase (GPX), and catalase were significantly lower by 15％,10％,and 9％, respectively, in smokers than nonsmokers(P≤0.01). CONCLUSION: Oxidative stress imposed by cigarette smoking had an impact upon certain pathways involved in DNA damage, lipid peroxidation and antioxidative defense system.

BACKGROUND AND AIM: To explore the effects on the variation of nuclear factor kappa B (NF-κB), C-IAP2 and Caspase-3 during apoptosis of human melanoma A375 cells induced by arsenic trioxide. MATERIALS AND METHODS: A375 cells were treated with arsenic trioxide alone or together with Ac-devd-cho (N-Acetyl-Asp-Glu-Val-Asp-CHO). The expression of NF-κB p65 nuclear protein and the activity of Caspase-3 were examined by Western blot, the cleaved Caspase-3 was detected by immunohistochemistry, the expression of C-IAP2 mRNA was examined by semi-quantitative RT-PCR. RESULTS: Treated with 5.0 μmol/L As2O3 alone for 12 and 24 hours; co-treatment with 5.0 μmol/L As2O3 and 20 μmol/L Ac-devd-cho for 24 hours; with increasing concentration of As2O3 , Caspase-3 was activated dramatically, NF-κB p65 and C-IAP2 mRNA expressions were decreased dramatically. Immunohistochemistry demonstrated that the cleaved Caspase-3 protein advanced in parallel with As2O3 concentration increase and response time extension, but could be partly blocked by Ac-devd-cho. CONCLUSION: As2O3 could induce the apoptosis of A375 cells and could be partially blocked by Ac-devd-cho. With the concentration increased and response time extension, the Caspase-3 protein was activated markedly,but blocked by Ac-devd-cho. NF-κB and C-IAP2 were suppressed, and could not be blocked by Ac-devd-cho. The signal transduction pathways of NF-κB, C-IAP2 and Caspase-3 might be associated with the apoptosis of A375 cells induced by As2O3.

BACKGROUND AND AIM: In this study we tried to evaluate the possible effects on chemosensitization of bladder cancer cell line BIU-87. MATERIALS AND METHODS: The expression of estrogen receptor(ER) was evaluated by immunohistochemical S-P method.In vitro chemosensitivity tests were done with bladder cancer cell line BIU-87 and Doxorubicin(0.1,1,10 μmol/L) in the presence or absence of graded concentrations of Tamoxifen(1,5,10 μmol/L). The growth and the sensitivity to Tamoxifen of cells were investigated by MTT assay. The level of bcl-2 gene mRNA expression was determined by in situ hybridization. RESULTS: Immunohistochemistry showed positive ER in BIU-87 cells. Tamoxifen stimulated a concentration- dependent and time-dependent decrease in bcl-2 mRNA.Tamoxifen at concentrations of 5 and 10 μmol/L significantly enhanced the cytotoxicity of the chemotherapeutic agent to the cell lines. Tamoxifen alone caused significant toxic effects to BIU-87 at 10 μmol/L. Tamoxifen at 5 and 10 μmol/L down-regulated the secretion of bcl-2 in a concentration-dependent manner. The effect of chemosensitization was evident in cells treated with 1,5 and 10 μmol/L Tamoxifen plus Doxonubicin in which 1.77 to 6.96-fold IC50 reduction was observed. CONCLUSION: Tamoxifen increased the chemosensitivity of Tamoxifen in human bladder cancer BIU-87 cell line in vitro,inhibited cell proliferation and promoted apoptosis.

BACKGROUND AND AIM: To observe the acute toxicity and the genotoxicity of alfalfa total saponins(ATS). MATERIALS AND METHODS: Kunming mice were used to test the LD50 of ATS. The mouse bone marrow micronucleus test and the mouse sperm morphology test were used to evaluate genotoxicity of ATS. RESULTS: The LD50 of ATS was 13.01 g/kg, 95％ confidence interval was 12.46～13.58 g/kg. The micronucleus rates and sperm abnormality rates were negative. CONCLUSION: The ATS was considered a substance with no toxicity and genotoxicity in this study.

BACKGROUND AND AIM: To investigate the teratogenicity of 1-chloromethylsilatrane in rats. MATERIALS AND METHODS: SD rats divided into 5 groups. On day 6-15 of gestation, SD rats received the test material at three doses of 10.29, 32.54 and 102.89 mg/kg by gavage. And at the same time, 2 similar group were dosed with 3％ starch paste (solvent control) and N,N'-methylene-bis-(α-amino-1,3,4-thiadiazole) (MATDA) (positive control). On day 20 of gestation, all rats were sacrificed. Body weight and observation data were collected during the study. RESULTS: The fetal skeletal abnormalities increased in groups treated with 32.54 and 102.89 mg/kg, showing sternum abnormalities with dysostosis, absent and abnormal ossification. The minimum teratogenic dose was 32.54 mg/kg. The body weight and fetal toxicity of pregnant rats were not different in treated groups compared with the control group. No abnormality was observed in growth effect, morphology and internal organs in rat fetuses in any group. CONCLUSION: Under this experimental condition, 1-chloromethylsilatrane had teratogenic toxicity on SD rats when given at certain dose.

BACKGROUND AND AIM: To study the protective effects of ursolic acid extract from Ledum Pulastre L. against genetic damage. MATERIALS AND METHODS: We used mice with genetic damage caused by cyclophosphamide,(CP), as a model of injury. Through mouse cells of bone marrow PCE micronuclear test, the protective effects of ursolic acid extract on genetic damage was measured. RESULTS: The bone marrow cells-nuclear PCE rates of ursolic acid extract at 1.16,2.30,4.60 g/kg dose were 2.20‰ ±0.37‰,2.00‰ ±0.45‰,1.80‰±0.37‰, respectively. There was no significant difference with the normal group (2.00‰±0.45‰) (P>0.05), but showed a significant difference with positive group (24.00‰±0.71‰)(P<0.01). The bone marrow cells-nuclear PCE rates of ursolic acid at 1.16,2.30,4.60 g/kg dose with added CP were 17.20‰±1.24‰,16.40‰±1.53‰,16.00‰±0.51‰,respectively,significantly different from the positive group(24.00‰±0.71‰)(P<0.05). CONCLUSION: Ursolic acid extract from Ledum Pulastre L. demonstrated protective effects against genetic damage.