BACKGROUND AND AIM: To investigate the effects of overexpression of the 24-amino-acid N-terminal end of p55γ regulatory subunit of phosphoinositide-3 kinase on proliferation and migration mediated by adenovirus in MGC803 gastric carcinoma cells. MATERIALS AND METHODS: The recombinant adenovirus-Ad-N24-GFP and control virus Ad-GFP were amplified in HEK293 cells. The virus infection rate of tumor cells was determined by fluorescence microscope. The effects of Ad-N24-GFP on cell proliferation and cell cycle were detected by cell growth curve and flow cytometry. The effect of Ad-N24-GFP on cell migration was detected by Transwell migration assay. RESULTS: The infection rate of recombinant adenovirus of MGC803 cells was highest when MOI=100. Compared with control cells, the growth of MGC803 cells after Ad-N24-GFP infection was suppressed and cell doubling time was prolonged. The percentage of Ad-N24-GFP cells in G0/G1 phase was (84.2±5.4)％, significantly higher than that of control cells in G0/G1 phase (68.7±5)％ (P<0.05). Overexpression of the Ad-N24-GFP fusion protein markedly resulted in decrease of MGC803 cell migration compared with the control cells (P<0.05). CONCLUSION: Overexpression of the p55 γ gene N-terminal 24 peptide mediated by adenovirus inhibited cell proliferation and migration in gastric carcinoma MGC803 cells. It could have potential application in gastric carcinoma gene therapy.

BACKGROUND AND AIM: To study the ability of(RGD)3 -tTF thrombosis targeted protein binding to tumor vascular markers such as integrin αv β3, and to investigate the relationship between the targeting ability to integrin αv β3 and the chemical structure of RGD polypeptide of the fusion protein. MATERIALS AND METHODS: The (RGD)3 -tTF fusion gene was synthesized by the truncated tissue factor (tTF) and three RGD polypeptides in series and expressed in Escherichia coli(E.coli)BL21 (DE3). The fusion protein was then purified by Nickel affinity chromatography column. The activity of the tTF fusion protein was evaluated by clotting assay. The binding ability to intergin αv β3 specially was analyzed by indirect enzyme linked immunosorbent assay (ELISA), and compared with the activity of RGD-tTF fusion protein. RESULTS: The coagulation activity of (RGD)3 -tTF and RGD-tTF purified fusion protein was alike (F=0.019，P>0.05). However, the ability of the (RGD)3 -tTF fusion protein binding to intergrin αv β3 specifically was elevated significantly (F=140.17，P<0.01). When the concentration was 0.24 μmol/L, the OD405nm of the (RGD)3 -tTF fusion protein was 1.3 times more than that of RGD-tTF fusion protein(1.25/0.95). CONCLUSION: Because the (RGD)3 -tTF fusion protein has two disufides and three RGD polypeptides, the coagulation activity of tissue factor was retained and the ability binding to integrin αv β3 was increased significantly.It may provide a foundation for the further study of thrombosis- targeted therapy in animal tumor models.

BACKGROUND AND AIM: Alkaline sphingomyelinase(alk-SMase) is the key enzyme of sphingomyelin(SM) metabolism, and an inhibitor of colon cancer. Through vitro experiments, we examined the alk-SMase expression in human colon cancer HT-29 cells, which produced the hydrolysis products of SM, ceramide(Cer) and sphingosine(Sph). MATERIALS AND METHODS: After treatment with 12.5,25 and 50 μmol/L of C2-Cer and Sph for 12,24 and 48 hours, we tested the alk-SMase mRNA and protein expressions in HT-29 cells. DMSO was the control. RESULTS: Compared with the control group, the alk-SMase expression of protein and mRNA level was reduced by C2-Cer and Sph in HT-29 cells, With a clear dose-response relationship. CONCLUSION: Cer and Sph exerted negative feedback on alk-SMase. Cer and Sph might have induced apoptosis directly in colon cancer cells, rather than through hydrolysis of SM.

BACKGROUND AND AIM: To investigate the effects of microcystin LR on the mRNA expression of protein phosphotase 2A subunits of human amniotic (FL) cells and human hepatocyte HL7702 cells. MATERIALS AND METHODS: After treated with 100 nmol/L MCLR for 24 h, the mRNA expression of protein phosphotase 2A subunits of human amniotic cells and human hepatocyte cells was measured by real time PCR. RESULTS: The changes of subunits expression were diverse in human amniotic cells but increased significantly in human hepatocyte cells. CONCLUSION: The special effects of MCLR on HL7702 cells could be account for its hepatotoxicity.

BACKGROUND AND AIM: To investigate proliferation-inhibiting effects and mechanisms of shikonin on human choriocarcinoma JEG-3 cells. MATERIALS AND METHODS: 3-(4,5- dimethylthiazol-2-yl)-2,5- diphemyltetra-zolium Bromide (MTT) assay was used to determine the inhibitory rate of shikonin on the proliferation of JEG-3 cells. Apoptosis induced by shikonin was detected with Hoechst 33258 dye , flow cytometry (FCM) and Annexin V/PI assay. Western blot was used to evaluate the changes of pro-caspase-3, cleaved PARP, active MAPK in protein levels in JEG-3 cells. RESULTS: Shikonin induced JEG-3 apoptosis in a time- and dose-dependent manner. The IC50 of a 24 h time course for JEG-3 cells was 6.3±0.6 μmol/L. Typical morphological changes of apoptosis were observed in JEG-3 cells with Hoechst staining after induced by shikonin. The shikonin-treated JEG-3 cells with condensed and fragmented chromatin, apoptosis peak and hypo-diploid cells were revealed by proidum iodide (PI) staining. Furthermore, Annexin-V/PI staining showed the early apoptotic cells. To elucidate the apoptotic pathways induced by shikonin, we assessed the expression of active caspase-3, cleavages of poly(ADP-ribose) polymerase (PARP) and MAPK. Caspase-3 in JEG-3 cells was activated after 12 h treatment and PARP was cleaved subsequently. Phosphorylation of ERK and JNK was respectively blocked after 8 h and 12 h treatment. CONCLUSION: Shikonin could significantly inhibit the proliferation of JEG-3 cell and induce apoptosis. Activation and inhibition of MAPK pathway may affect apoptosis.

BACKGROUND AND AIM: To examine the significance of P504S, p63, 34βE12 cocktail in the differential diagnosis of prostate lesion in needle biopsy specimen. MATERIALS AND METHODS: The expressions of P504S,P63 and 34βE12 were examined in the biopsy specimens of 32 prostate cancer(PCa),16 prostatic atypical adenomatous hyperplasia (AAH)and 44 benign prostatic hyperplasia (BPH) using immunohistochemical method with triple-antibody cocktail (P504S, P63, 34βE12)staining and double-color chromogens in single paraffin sections. RESULTS: PCa stained positive for P504S and negative for P63 and 34βE12, and the positivity rate of P504S was 100％. The positive rates in AAH were all 75％ for the three proteins. BPH strongly expressed P63 and 34βE12, but not P540S. The expressions of P504S, P63 and 34βE12 were significantly different among PCa, AAH and BPH (P<0.05). CONCLUSION: Cocktail staining may be an effective approach in the diagnosis and differential diagnosis of prostate lesion.P504S was a sensitive and specific marker for PCa. P63 and 34βE12 cocktail staining could increase the sensitivity and specificity for the basal cell layer. The triple-antibody cocktail staining could improve diagnostic accuracy of prostate lesion in needle biopsy specimen, and important for the early diagnosis of prostate cancer.

BACKGROUND AND AIM：To investigate the association between the MYH(MutY homologue)genovariation and colorectal cancer risk. MATERIALS AND METHODS： Denaturing high performance liquid chromatography (DHPLC)and DNA sequencing were used to delineate the variants in 7 of 16 exon-districts in the 140 colorectal cancer patients and 280 controls. All data was analyzed by SPSS software. RESULTS：Four genovariation sites, Exon1-316 G>A, Exon1-292 G>A and Intron1＋11 C>T, in exon 1 district were detected in all variants of cases and controls simultaneouly. The variation frequency in cases was significantly higher than that in controls, genovariation risk in cases was 8.16-fold more than controls(P=0.04；OR=8.16，95％ CI=1.01～203.70). A missense variation, Exon14＋74 T>A, p.V463E, was found in exon 14. A deletion variation, nt 1678-80 del GTT, was found in exon 16. In cases, the rectal cancer genovariation frequency was higher than colon cancer, variation risk in the former was 7.18-fold more than the latter(P=0.04；OR=7.18，95％ CI=1.102～165). CONCLUSION： MYH variation may function as a risk factor for colorectal cancer development. Most patients had rectal cancer. Similar to other Asian races, the MYH variant frequency in Chinese is lower than Caucasian.No variant is found in and around Exon 7 which is one of the most frequently mutated exons in Caucasians. This indicates that the differences of MYH variants may be related to racial differences.

BACKGROUND AND AIM: To investigate the relationships of Survivin, PTEN and Connexin43(Cx43)with development of skin basal cell carcinoma(BCC).MATERIALS AND METHODS: The expressions of Survivin, PTEN and Cx43 proteins were measured by immunohistochemical S-P method in 35 patients with BCC and 10 specimens of normal skin. The expressions of Cx43 mRNA was evaluated by in situ hybridization.All data in normal epidermis and BCC tissues were analyzed by computerized image-analysis. RESULTS: Cx43 protein and its mRNA showed strongly positive expression in normal epidermis，and positive or weakly positive expression in BCC group. Compared with normal skin group and SCC group，the expression(positive area,optical density)of Cx43 protein and its mRNA in BCC group were statistically significant(P<0.01). Survivin protein was mostly negative in normal epidermis, and mostly weakly positive in BCC group.The expression of Survivin protein was statistically different between BCC and normal skin(P<0.05). PTEN protein showed strongly positive expression in normal epidermis,and positive or weakly positive expression in BCC group. Compared with normal skin group，the expression of PTEN protein in BCC group was statistically significant(P<0.05). There was positive correlation between the expressions of PTEN and Cx43 protein(r=0.519，P<0.01)， and between the expressions of Cx43 protein and its mRNA(r=0.732，P<0.01) in BCC tissues. CONCLUSION: The lower expression of Cx43, PTEN proteins and Cx43 mRNA and the higher expression of Survivin protein in BCC may play important roles in its development.

BACKGROUND AND AIM: To explore the possible mechanism that two administrations of cyclophosphamide (CP) could potentiate immunity in normal SD rats. MATERIALS AND METHODS: Peripheral blood was analyzed by ADVIA 2120 blood-counter system and flow cytometry after two treatments with 60 mg/kg of CP in vivo. Spleen lymphocyte proliferation was measured with Mono-nuclear cell direct cytotoxicity assay (MTT), the anti-OVA antibody response were assessed by ELISA. RESULTS: Cells in peripheral blood were decreased six days after the first CP administration, but increased nine days after second CP injection. A significant elevation was also observed in splenic proliferation. Consistently, the ratio of CD4＋ T lymphocytes and CD4＋/CD8＋ was markedly increased (P<0.05). However,anti-OVA antibody response was suppressed during these processes. CONCLUSION:These changes might be associated with the immune potentiating effect of CP, treated with two CP (60 mg/kg) injections in 6 days might enhance the immunological function of SD rats.

BACKGROUND AND AIM: To study the effect of trace element copper on lipid peroxidation by analyze relations among copper concentration, copper-zinc ratio (Cu/Zn) and the biomarkers of lipid peroxidation in copper deficiency in rats. MATERIALS AND METHODS: Determined the content of copper in blood, the activities of Superoxide Dismutase (SOD), Glutathione Peroxidase (GSH-pX) and Catalase (CAT) and the content of Malondialdehyde (MDA) in copper deficiency. RESULTS: Copper-defficient rats treated with copper gluconate (Cu-G) in different doses showed fluctuating copper concentrations in the blood, while copper-zinc ratio (Cu/Zn) showed an ascending trend following the increase of copper intake and statistical significant results (P<0.01). In copper deficiency state, the SOD activity was lower than the normal level, and then followed an upward trend with increased copper intake, equivalent to the copper-zinc (Cu/Zn) ratio. The content of MDA was higher than the normal level, and its content decreased with increasing copper intake and the Cu/Zn ratio, and remained at a relatively low level. The decreased Cu/Zn ratio could induce the increase of the CAT activity, but with no linear correlation between them. No significant effect on the GSH-pX activity in rat blood was found during inadequate copper intake. CONCLUSION: Cu-Zn ratio has more practical significance in the research of physiological and biochemical roles of the trace element copper. In copper deficiency, theSOD activity in the rat blood was lower than the normal level and the content of MDA higher. With the increase of Cu/Zn ratio, the SOD activity increased gradually, while the content of MDA was reduced. Insufficient copper intake could enhance the CAT activity in blood with no effect on GSH-pX.

BACKGROUND AND AIM: To study testis index and mtATPase6 and D-Loop point mutations of mice testicle after exposure to cadmium. MATERIALS AND METHODS: 40 male mice were divided into 4 groups with 10 animals per group (weight:19.5±2.5 g).3 groups were treated with intraperitoneal cadmium chloride of 1,5,10 μmol/kg,once every 2 days,10 times in total,and one normal saline control group. After exposure to cadmium chloride for 20 days, we determined the testis index. The mtATPase6 and D-Loop genes were amplified by PCR from the genomic DNA of mice testicle tissue, with their mutant sites sequenced. RESULTS: The testis index of mice in the 10 μmol/kg cadmium chloride group were lower than that of the control group(P<0.01). No mtATPase6 or D-Loop mutation was found in the murine spermatogenic cells. CONCLUSION: Mice testis index was lower by 10 μmol/kg cadmium chloride. But cadmium chloride couldn't induce mtATPase6 and D-Loop mutations in the mice testicle. Testis index and mtATPase6 and D-Loop point mutations did not seem to be related.

BACKGROUND AND AIM: To explore the combined toxic effects of benzene and formaldehyde on mouse embryos in vitro. MATERIALS AND METHODS： Whole embryo culture system was adopted. According to 3×4 factorial design, 12 groups were set up. The 8.5-day isolated mouse embryos were cocultured for 48 hours in freshly centrifuged serum with different concentrations of benzene and formaldehyde. The growth, development and morphodifferentiation of tissues and organs of mouse embryos were studied. The type of combined toxic effects of benzene and formaldehyde was analyzed by ANOVA and direct-viewing analysis. RESULTS:There were no statistically significant differences in embryonic growth and development among control groups in vitro and in vivo group (P>0.05). Benzene and formaldehyde had obvious toxic effect on growth and development in embryos in a dose-dependent manner every index of growth and development, morphodifferentiantion of tissues and organs demonstrated significant differences compared with the solvent control (P<0.05). With the increasing concentrations of benzene and formaldehyde,the incidence of malformation and death of embryo increased. Structure, arrangement and cell polarity of VYS endodermis cells were influenced. When benzene was combined with formaldehyde, the severity of the toxic effects on the embryo was stronger than the addition of the two agents alone. CONCLUSION:Benzene and formaldehyde could cause synergistic developmental toxicity and teratogenicity to embryo in vitro.

BACKGROUND AND AIM: To explore the mechanism of xiongshao capsule on inducing angiogenesis by measuring its effects on nitrogen monoxide(NO), inducible nitric oxide synthase(iNOS) and endothelial nitric oxide synthase(eNOS) in human umbilical vein endothelial cells. MATERIALS AND METHODS: Serologic pharmacology method and reversed transcriptive polymerase chain reaction(RT-PCR)assay were used to measure the effects of serum containing different concetrations of xiaongshao capsule(4.17, 8.33 and 16.66 mg/ml, respectively) on NO and iNOS secretion and expressions of iNOS and eNOS mRNA. RESULTS: Serum containing 4.17 mg/ml xiaongshao capsule could promote the secretion of NO(P<0.01), whereas the concentrations of 4.17 and 8.33 mg/ml could promote the secretion of iNOS(P<0.01). RT-PCR showed serum containing 4.17 mg/ml xiaongshao capsule up-regulated the mRNA expressions of eNOS and iNOS significantly(P<0.01),and the concentraions of 8.33 and 16.66 mg/ml down-regulated the mRNA expressions of eNOS and iNOS significantly(P<0.01). CONCLUSION: The angiogenetic effect of serum containing 4.17 mg/ml xiongshao capsule may be attributed up-regulating the expressions of iNOS and eNOS mRNA and promoting nitrogen monoxid synthesis and secretion.

BACKGROUND AND AIM: In view of the follicles proliferation is sensitive to the physical and chemical stimuli, to establish the follicles culture in vitro growth system, as an efficient biological model of reproductive toxicity. MATERIALS AND METHODS: 140-160 μm preantral follicles were selected to culture in vitro for ten days, the diameter of follicles was measured each other day. The ovulation of survival follicles was induced by HCG hormone. About 16-24 hours later, the matured oocytes, germinal vesicle breakdown (GVBD) and germinal vesicle (GV) oocytes were calculated under the stereomicroscope. Chromosomal abnormalities of matured oocytes including numerical and structural aberrations were investigated and recorded. RESULTS: Total of 332 follicles were separated from ovaries of 4 female mice, the mean diameter of follicles was increased at the early culture time in vitro, then follicles grow as ‘Frangipani’, some of them formed the antral-like cavity. On the 10 day of culture, the survival rate was 95.78％ (318/332)，the rate of ovulation induced by hormone in vitro was 72.01％ (229/318)，including 11.80％ oocytes arrested at germinal vesicles stage, 39.30％ oocytes showed germinal vesicle breakdown, 47.16％ oocytes emitted the first polar body and 1.74％oocytes were parthenogenesis. The chromosomal abnormalities of matured oocytes have no significant difference comparing with that of oocytes in vitro maturation (IVM) and oocytes superovulation (IVC). CONCLUSION: The oocytes matured from preantral follicles cultured in vitro were similar to those matured from in vivo growth. Therefore, the preantralfollicles cultured in vitro growth system maybe a good testing model of reproductive toxicology or follicular developmental mechanism in vitro.

BACKGROUND AND AIM： To develop a chemically-induced, easily established short limb malformation model with high incidence rate and discrete abnormal phenotype. MATERIALS AND METHODS: After the male and female ICR mice copulated, the presence of vaginal plug the following morning was considered as gestational day 0 (GD0). At GD9, GD10, GD11 and GD12, the pregnant mice in the treatment group were treated with 80 mg/kg all-trans-retinoic acid (atRA), and those of the control group received the same volume of soybean oil. At GD18, pregnant mice were killed and the embryos were harvested. The abnormalities of the embryos in the treatment group were studied. RESULTS: In the group pregnant mice were treated with atRA at GD10, the forelimbs of embryos were short and small, and the humerus, radius and ulna were shorter than those of the control group. In the group treated with atRA at GD11, the forelimbs and the hindlimbs of embryos were short and small, and the humerus, radius, ulna, femur and tibia were shorter than those of the control group, and most of the embryos had no fibula. In the group treated with atRA at GD12, the ulna of the embryos were shorter than those of the control group. CONLUSION: We developed a model of short limbs induced by atRA during ICR mouse embryogenesis, providing a basis to investigate the molecular mechanism of limb deformity.

BACKGOUND AND AIM: To investigate genetic toxicity and carcinogenetic potential of anticancer drugs on human. MATERIALS AND METHODS: To evaluate genetic toxicity and carcinogenetic potential of anticancer drugs with chromosomal aberration test in human peripheral blood lymphocytes in vitro. The agent was administrated at three different doses (0.01,0.02,0.04 μg/ml), then the type of aberrations and the rates of chromosomal aberrations and frequencies of sister chromatid exchange (SCE) were examined. RESULTS: Ifosfamide,Adriamycin, Vinblastine and Harringtonine, even at dose of 0.01 μg/ml, could significantly increase frequencies of SCE and rates of chromosomal aberration as compared with negative control group(P<0.05). Busulfan could significantly increase frequencies of SCE and rates of chromosomal aberration in high dose group, but there was no statistical significance in low dose group (P>0.05). SCE frequencies and rates of chromosomal aberrations were not significantly different between Mithramycin group and control group (P>0.05). CONCLUSION: Antineoplastic drugs including Ifosfamide,Adriamycin,Vinblastine,Harringtonine and Busulfan demonstrated genetic toxicity and carcinogenetic potential. However, Mithramycin showed negative results. Antineoplastic drugs should be handled carefully by clinical personnel.

BACKGROUND AND AIM: To evaluate the genotoxicity of bezoar antidotal tablets. MATERIALS AND METHODS: The Ames test,the mouse micronucleus test and the mouse sperm abnormality test were used to evaluate the genotoxicities of bezoar antidotal tablet. RESULTS: In the Ames test, the number of revertants in all dose groups was less than twice that of solvent control group.In the mouse micronucleus test and sperm abnormality test,no significant differences were observed in the three dose groups compared with that of control group. CONCLUSION: Under this experimental condition, no genotoxic effects had been found in bezoar antidotal tablets.