BACKGROUND AND AIM: To investigate effects of NADH on mutations of p53 gene and c-erbB2 gene in L02 cells induced by diethylnitrosamine(DENA). MATERIALS AND METHODS: The anti-mutagenesis effects of NADH on DENA-induced mutations of p53 gene in L02 cells were investigated by PCR-SSCP and PCR-RFLP analysis. The effect of NADH on c-erbB2 gene amplification was assessed by Southern blotting analysis. RESULTS: PCR-SSCP analysis showed that the mutation rate of p53 exon7 in DL02-Ⅲ and DL02-B cells in NADH protection groups decreased to 33.3％(4/12)and 50％(6/12), respectively. The difference was significant(P<0.01)compared with DENA mutagenesis group. PCR-RFLP analysis showed that NADH decreased the mutation frequency of the 249th codon of p53 in L02 cells induced by DENA. Southern blotting showed that NADH down-regulated the amplification of c-erbB2 gene in L02 cells induced by DENA. CONCLUSION: Reduced coenzyme NADH had anti-mutagenesis effects and could decrease mutation of p53 and inhibit the expression of c-erbB2 in L02 cells induced by DENA.

BACKGROUND AND AIM: To investigate the effect of nerve growth factor(β-NGF) on the proliferative ability and cell cycle of human pancreatic carcinoma cells MIA PaCa-2. MATERIALS AND METHODS: Colony forming efficiency of MIA PaCa-2 cells treated with β-NGF was higher than normal group(P<0.05),especially with the 100 ng/ml β-NGF group.Absorbence gradually increased,along with β-NGF concentration enhancement and culture time prolongation.Proliferation rate reached 48.68％ when cultured 72 hours in 100 ng/ml β-NGF group.However, absorbence gradually decreased in K252a group at 24,48,72 h and 96 h.Inhibition rate was reduced to 18.05％ in 100 ng/ml K252a group.Effect of different concentrations of β-NGF and K252a on MIA PaCa-2 cells proliferation was evaluated by MTT and Flow Cytometry. RESULTS: β-NGF promoted MIA PaCa-2 proliferation and growth which were markedly inhibited by K252a(P<0.05). CONCLUSION: β-NGF could enhance the proliferation of MIA PaCa-2 pancreatic carcinoma cells.

BACKGROUND AND AIM: To investigate the effects of RNAi targeted to STAT3 on apoptosis induction and on STAT3 signaling in human hepatocellular carcinoma cell line SMMC7721. The research provided theoretical and experimental basis for further development and clinical application of targeting STAT3 signaling pathway therapy for cancer. MATERIALS AND METHODS: Applying RNAi technique to specifically silence STAT3 gene in SMMC7721 cells, and expression of STAT3 protein in cells was determined by Western blot. Effect of RNAi on SMMC7721 apoptosis was determined by flow cytometry. Expressions of apoptosis-associated genes survivin and bcl-2 were determined by semi-quantitative RT-PCR. RESULTS: RNAi technology could effectively and specifically silence the expression of STAT3. Compared to the control group, the apoptosis rate was significantly increased after RNAi silencing STAT3 (P<0.01). Expressions of survivin and bcl-2 mRNA were significantly inhibited compared to the control group (P<0.01). CONCLUSION: RNAi technology targeted to STAT3 could effectively silence STAT3 expression in SMMC7721. RNAi targeted to STAT3 induced apoptosis by down-regulating the expressions of bcl-2 and survivin mRNA.

BACKGROUND AND AIM: This study was to explore the inhibitory effect of periplocin extracted from cortex periplocae (CPP) on tumor formation activity of human colorectal cancer cell line SW480 in nude mice, and to investigate its antitumor mechanisms. MATERIALS AND METHODS: SW480 cells were injected subcutaneously into nude mice to construct Balb/c-nu/nu mice model of SW480 cells. After treated with CPP, the transplanted tumor volume and weight of nude mice were measured. Morphology of SW480 cells in transplanted tumors were observed under light microscope after HE staining. Expression of β-catenin, Survivin and C-myc involved in Wnt/β-catenin signaling pathway were detected by immunohistochemistry. RESULTS: The growth of transplanted tumor in nude mice was inhibited markedly by CPP, the volume and weight of tumors were significantly less, the inhibition rate was 61.41％ (P<0.01). There were inflamatory cells and noticeable necrosis in transplanted tumor tissue of CPP-treated mice. Expression levels of β-catenin, Survivin and C-myc in transplanted tumor were significantly lower in CPP-treated group than in control group. CONCLUSION: CPP exerted significant inhibitory effect on the proliferation of human colon cancer SW480 cells in nude mice. The mechanisms may be associated with down-regulating the Wnt/β-catenin signaling pathway.

BACKGROUND AND AIM：To explore the mechanisms of Reinforcing Qi and Nourishing Yin in inhibiting tumor metastasis. MATERIALS AND METHODS：By duplicating spontaneously metastazing tumor model, the changes of the tumor cell phenotype were studied. Total RNA of tumor tissues was extracted, and fluorescent cDNA probes were prepared through reverse transcription of the isolated mRNAs. The RNA samples were labeled with Cy3-dUTP or Cy5-dUTP, then hybridized with microarrays and the chips were scanned with a ScanArray. The acquired images were analyzed using GenePixPro 3.0 software. RESULTS：Reinforcing Qi and Nourishing Yin could inhibit spontaneous tumor metastasis.Gene expression spectrum revealed that it could regulate multiple target genes such as tumor filtration and metastasis，signal transduction，cell proliferation，apoptosis and metabolism-related genes. CONCLUSION：Reinforcing Qi and Nourishing Yin had effects on anti-tumor metastasis. cDNA microarrays technology may be powerful tools in discovering the possible mechanisms of drug at the gene level, and provides new information for further research.

BACKGROUND AND AIM: Effects of quercetin on expressions of mdr1 gene and P-glycoprotein in B-MD-C1(ADR＋/＋) cell line were studied in vitro. MATERIALS AND METHODS: After treatment with or without various concentrations of quercetin (10, 20, 40 μmol/L) for 48 h, the expression in mdr1 gene in B-MD-C1(ADR＋/＋) cell was detected by RT- PCR and expression of P-gp protein with IHC. The chemosensibility of B-MD-C1(ADR＋/＋) cells was evaluated by means of MTT. Changes of subcellular distribution of ADM in B-MD-C1(ADR＋/＋) cell was examined by confocal scanning laser microscopy (CSLM). RESULTS: After treatment with different concentrations of quercetin (10, 20, 40 μmol/L) for 48 h, the expression levels of mdr1 gene and P-glycoprotein in B-MD-C1(ADR＋/＋) cells were decreased significantly(P<0.05). The abnormal distribution of ADM in B-MD-C1(ADR＋/＋) cells was restored. CONCLUSION: Quercetin could reverse the multidrug resistance by down-regulating the expression of mdr1 gene and P-glycoprotein and restoring the distribution of ADM in B-MD-C1 (ADR＋/＋) cells.

BACKGROUND AND AIM: To identify the TPA responsiveness of ezrin gene promoter and the mitogen-activated protein kinase(MAPK) signal pathway of TPA-induced ezrin transcription in esophageal carcinoma cells. MATERIALS AND METHODS: The potential transcription factors and TPA-responsive elements(TRE) were analyzed and predicted using transcription factor database. Using dual-luciferase reporter assay system, we determined the promoter activity and TPA responsiveness of ezrin gene －87/＋134 sequence, the transactivation of TRE binding proteins Sp1 and AP-1 on ezrin, and the inhibition of MAPK inhibitors on TPA-induced ezrin transactivation. RESULTS: Ezrin gene －87/＋134 sequence had potential TPA-responsive elements and exhibited promoter activity. 5 ng/ml TPA increased ezrin promoter activity significantly(P<0.01). Sp1 and AP-1 transactivated ezrin gene. MEK1/2 specific inhibitors U0126 and PD98059 decreased the TPA-induced ezrin transactivation. CONCLUSION: Ezrin gene promoter demonstrated TPA responsiveness in esophageal carcinoma cells. TPA may regulate ezrin transcription via MEK/ERK1/2 phosphoryla- ting Sp1 and AP-1 pathways.

BACKGROUND AND AIM: To examine the expressions and their significance of oncogene protein MDM2, anti-oncogene protein P53 and cell cycle protein P27 in esophageal squamous cell carcinom and adjacent mucosa. MATERIALS AND METHODS: Immunohistochemistry was used to detect the expressions of MDM2, P53 and P27 proteins in eighty-five cases of esophageal squamous cell carcinoma and their corresponding adjacent mucosa. Flow cytometry(FCM) was applied to measure the quantities of these protein expressions in 48 fresh esophageal squamous cell cancers and adjacent tissue and 12 relatively normal mucosa at the edge of the excision. RESULTS: The changes of esophageal epithelia from simple hyperplasia to mild to moderate to severe dysplasia to carcinoma in situ and infiltrating carcinoma，revealed that the P53 protein was not expressed in normal mucosa but appeared in the early stages of esophagus carcinogenesis. The MDM2 and P27 proteins had different degrees of expression in normal esophageal mucosa,with MDM2 protein markedly increased in the advanced stages of esophageal cancer. CONCLUSION: P53 and P27 proteins appeared in the early stages of esophagus oncogenesis, however the changes of MDM2 expression occurred in the advanced stage of esophageal carcinogenesis. Flow cytometry quantitative and immunohistochemistry qualitative analyses of gene proteins, could explore the expressions of oncoproteins and their clinical significances.

BACKGROUND AND AIM: To assess the interactions of MutT homologue (MTH1) and 8-oxoguanine-DNA glycosylase1 (OGG1), two important genes of base excision repair. We examined 8-hydroxyguanine(8-oxo-dG) formation and 8-oxo-dG repair enzyme activity in pulmonary type-II-like epithelial cells to determine the association of hOGG1 and hMTH1 under oxidative stress induced by H2O2 . MATERIALS AND METHODS: A549 cells were incubated with H2O2 at concentrations of 0，50,100,200 and 300 μmol/L for 24 h. We then evaluated 8-oxo-dG formation, 8-oxo-dG repair enzyme activity and expressions of hOGG1 and hMTH1 genes. This was done using a high-performance liquid chromatography system equipped with an electrochemical detector, endonuclease nicking assay and reverse transcription polymerase chain reaction, respectively. RESULTS: H2O2 induced the formation of 8-oxo-dG in DNA at concentrations of 100 and 200 μmol/L. 8-oxo-dG levels peaked at 12 h and had declined to near baseline at 24 h, whereas 8-oxo-dG repair enzyme activity peaked at 18 h post-H2O2 exposure. hOGG1 and hMTH1 mRNA levels were also increased by H2O2 exposure, with alternating peaks. CONCLUSION: These data suggested that hOGG1 and hMTH1 displayed alternating effects in the process of oxidized DNA repair.

BACKGROUND AND AIM: To investigate oxidative damage of mitochondria induced by olaquindox in human hepatoma G2 (HepG2) cells. MATERIALS AND METHODS: HepG2 cells were treated with 0，200，400 and 800 μg/ml olaquindox for 24 h. The value of IC50 of olaquindox in HepG2 cells was determined by MTT assay. The levels of reactive oxygen species (ROS) were detected by 2, 7-dichlorodihydrofluorescein diacetate (DCFDA) and Dihydroethidium (DHE). The level of mitochondrial membrane potential(△Ψm) and calcium concentration were measured by Rhodamine123(Rh-123) and Fluo-3AM, respectively. Finally, the damage ratio of mtDNA and nDNA was assessed by quantitative PCR. RESULTS: MTT assay revealed that the HepG2 cells viabilities were significantly inhibited by olaquindox in dose- and time- dependent manners. Olaquindox induced increased levels of ROS and calcium in HepG2 cells, and reduced the level of △Ψm . Quantitative PCR assay showed that olaquindox led to a dose-dependent decrease in the amplification of both mtDNA and nDNA. These data also suggested that the olaquindox-induced damage to mtDNA was more extensive than its damage to nDNA. CONCLUSION: Olaquindox could cause oxidative damage of mitochondria in HepG2 cells.

BACKGROUND AND AIM: To study the effect of oxidative damage in endothelial dysfunction induced by homocysteine and to explore the protective effects of Genistein. MATERIALS AND METHODS: MTT assay was used to detect cell viability, and intracellular reactive oxygen species(ROS) level was measured by flow cytometry(FCM). RESULTS: The cellular viability of 5.0 mmol/ L HCY(41.27％±5.09％) were lower than the others. After co-treatment with 5.0 mmol/L HCY and different concentrations GEN(10,50,100 μmol/L), the cellular viability of each group was elevated. The level of ROS after 5.0 mmol/L HCY treatment increased obviously compared with control group(P<0.01). Co-treatment with 5.0 mmol/L HCY and different concentrations of GEN, the level of ROS was decreased. CONCLUSION: HCY could lead to endothelial dysfunction in ECV-304 through oxidative damage. GEN could protect cell viability and decreased intracellular reactive oxygen species.

BACKGROUND AND AIM： To investigate the expressions of heat shock protein 27(HSP27) and trefoil factors2 (TFF2) in gastric cancer and precancerous condition and its correlation with helicobacter pylori(Hp) infection. MATERIALS AND METHODS： The expressions of HSP27 and TFF2 and the infection of Hp were studied by immunohistochemical methods in 140 gastric specimens including 25 chronic superficial gastritis(CSG), 30 gastric ulcer (GU), 35 chronic atrophic gastritis (CAG) and 50 gastric cancers (GC). RESULTS： In CSG,GU,CAG and GC, the expressions of HSP27,TFF2 and Hp were increased, but the expression of TFF2 was decreased in gastric cancer. The expression of HSP27 in Hp positive group was significantly higher than that in negative group. The expression of HSP27 was positively correlated with the infection of Hp (r=0.235,P<0.05).The expression of TFF2 in Hp positive group was significantly decreased compared with that in negative. The expression of TFF2 was negatively correlated with Hp infection (r=－0.259,P<0.05). CONCLUSION： HSP27,TFF2 and Hp may play important roles in carcinogenesis and progression of gastric carcinoma. The evaluation of HSP27 and TFF2 and Hp could provide theoretical basis for diagnosing, evaluating prognosis and guiding treatment of gastric cancer.

BACKGROUND AND AIM: To investigate the correlation between infection of helicobacter pylori L-form (Hp-L) and invasion and metastasis of gastric carcinoma. MATERIALS AND METHODS: Infection of Hp-L was examined on 130 patients with gastric carcinoma and control group by Gram staining, immunohistochemical stain (SP method) and transmission electron microscopy. Immunohistochemical stain was used for measuring the protein expressions of VEGF and MMP-2 in 180 cases of gastric carcinoma and control group. RESULTS： The rate of Hp-L infection in gastric carcinoma was 67.69％(P<0.05).Compared with the control group, the positive rate of Hp-L form in gastric carcinoma group was notably high. The difference in the positive rates of VEGF and MMP-2 expressions between gastric carcinoma and control group was obvious (P<0.01).There was a definite correlation between infection of Hp-L and the expression of VEGF and MMP-2 (P<0.05). The positive rate of Hp-L in the gastric carcinoma group correlated with depth of invasion, metastasis to the paragastric and distant lymph nodes (P<0.01)but not tumor size. CONCLUSION： Hp-L infection correlated with the occurrence and progression of gastric carcinoma, and may be an important promoting factor in gastric carcinoma growth, invasion and metastasis.

BACKGROUND AND AIM: KLF4 (Krüppel-like factor 4) is a zinc-finger transcriptional factor expressed in a variety of tissues in humans, and plays an important role in different physiological functions. Studies suggested that KLF4 is associated with the development and progression of various tumors. The study was to investigate the expression of KLF4 in gastric cancer and its correlation with clinicopathology. MATERIALS AND METHODS: The expression of KLF4 was detected in cancer and adjacent tissues of 102 surgical specimens by immunohistochemistry. The normal gastric tissues collected simultaneously was also examined. RESULTS: KLF4 was expressed in cell nuclei and cytoplasm. Strong expression was detected in adjacent tissue and normal gastric mucosa. Weak expression was detected in intraepithelial neoplasia and negative expression in gastric cancer. There is statistical significance among groups(P<0.05). The KLF4 expression in gastric cancer decreased with the progression from stage I to stage IV(P<0.05). The expressions of KLF4 in groups with and without lymph node metastasis were 63.3％ and 78.6％, respectively. There was statistical significance between the two groups(P<0.05). CONCLUSION: The expression of KLF4 decreased in gastric cancer and showed correlation with gastric carcinogenesis, staging and lymph node metastasis. KLF4 might be the prognostic marker and potential therapeutic target for gastric cancer.

BACKGROUND AND AIM: To observe the toxic effect of seal oil emulsion on the ability of maturity, mating, fertility and early fetal growth and development. MATERIALS AND METHODS: Wistar rats were randomly allocated into 0,250,500 and 1 000 mg/kg dose groups，and given seal oil emulsion orally for 5 weeks in male rats, 2 weeks in female rats respectively before mated, and then mated for 10 days. When the female rats were confirmed pregnant, the male rats were executed and their testicle, epididymis and sperm were examined.The seal oil emulsion treatment continued until 14th day of pregnancy and the ovary, uterus and embryo of the female rats were studied. RESULTS: The treatment groups were normal in spirit, behavior, activity and hair quality but the body weight gain was slow (average most value 50 g,P<0.05). The genitalia development, sexual function, the quantity and quality of sperm were normal. The number of live embryo (litter average 2.0) , the fetal body weight (average 0.03 g), and the mating ratio in 1 000 mg/kg group was 65％(P<0.05).There were no significant differences in the pregnancy rate, the number of corpus luteum, implantation, absorbed embryo and dead embryo, the rate of lost oosperm and fetus abnormality between the treatment and the control groups (P>0.05). CONCLUSION: Within the range of experiment doses, seal oil emulsion obviously decreased body weight gain of male rats, impaired embryonic and fetal development but did not effect the development of genitalia, mating behavior, sperm and ovum maturation and live fetus.

BACKGROUND AND AIM: To explore the effects of folic acid (FA) on in vitro maturation of mouse oocytes. MATERIALS AND METHODS: Naked mouse oocytes were matured in vitro either spontaneously(M16) or in the presence of hypoxanthine (Hx) or Hx plus FA for 24 h. At the end of the culture period, the nuclear status of mouse oocytes was assessed under inverted microscope. Immunoflurescence staining was used to evaluate the chromosome and spindle configuration of oocytes. RESULTS: The rates of germinal vesicle breakdown (GVBD) were 98％,22.2％,40.1％and the rates of the first polar body (PB1) extrusion were 77.7％,15.5％,27.6％ for M16,Hx and FA groups,respectively. The rates of GVBD and PB1 were all increased with the addition of FA compared with the Hx groups and comparison was significant (P<0.05). Hx and FA did not influence chromosome, but FA reversed the size of metaphase spindle elongated by Hx. CONCLUSION: FA appeared to promote the resumption of meiosis and subsequent maturation of mouse oocytes in vitro.

BACKGROUND AND AIM: To investigate the skin phototoxicity of di-sulfo-di-phthalimidomethyl phthalolcyanine zinc (ZnPcS2 P2)-based-photodynamic therapy. MATERIALS AND METHODS: Kunming mice were assigned to five dose groups,receiving intravenous administration of 0.9％NS at 0.2 ml/10 g body weight,the solvent at 0.2 ml/10 g body weight,hemoporphyrin injection at dosage of 15 mg/kg body weight, ZnPcS2 P2 at dosages of 2 or 5 mg/kg body weight. Mice were exposed to simulated sunlight (10,20,30,40 mW/cm2) 1,3,24,48,72,96 h and 7 d after a single intravenous injection.Skin phototoxicity was investigated by the weight of the slices taken from earflaps by perforex(diameter=6 mm). RESULTS: When mice were exposed to light(40 mW/cm2) 1,3,24 h after dosing, there were significant differences of the weight of ears between ZnPcS2 P2(2 or 5 mg/kg) and control groups(P<0.05). There were no significant findings in ZnPcS2 P2(2 mg/kg)ZnPcS2 P2 group 48 h after dosing,and in ZnPcS2 P2(5 mg/kg) group 7 d after dosing.When ZnPcS2 P2(2 mg/kg) group was exposed to light(10,20,30,40 mW/cm2) 1 or 3 hr after administration, there were significant differences of the weight of ears between 10 mW/cm2 and 20，30，40 mW/cm2 groups(P<0.05). CONCLUSION: In Kunming mice ZnPcS2 P2(2 mg/kg) caused phototoxicity 1,3，24 h after administration, the potential for phototoxicity was essentially gone after 48 h.Phototoxicity was gone in the skin of mice exposed to simulated sunlight 7 d after injection of ZnPcS2 P2(5 mg/kg). ZnPcS2 P2(2 mg/kg) caused skin phototoxicity,the saturated irradiance of the light was 20 mW/cm2.