Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of primary lung cancer in the central China region.

BACKGROUND: To determine the imprinting status of the IGF2 in Chinese patients with primary lung cancer and to analyze the clinical significance of the loss of imprinting (LOI) of IGF2. MATERIALS AND METHODS: PCR- RFLP and RT-PCR-RFLP were carried out to select heterozygous cases for the ApaI polymorphism within exon 9 of the IGF2 gene and further analyze IGF2 LOI in 64 lung cancer patients, respectively. RESULTS: Of 64 lung cancer patients, 31 were heterozygous for IGF2. The positive rates of IGF2 LOI of lung cancer foci, matched paracancer tissues, and normal lung tissues were 77.4% (24/31), 61.3% (19/31), and 29.0% (9/31), respectively. The LOI differences for IGF2 among the three groups were statistically significant (χ2=15.267, p=0.000), and the LOI frequency of IGF2 in normal lung tissue was significantly lower than that in lung cancer foci and paracancer tissues (χ2=14.577, p=0.000; χ2=6.513, p=0.011). No statistical difference was observed between the lung tumor group and the matched paracancer group (χ2=1.897, p=0.168). The prevalence of advanced clinical stages (χ2=2.379; p=0.017) and lymph node metastasis (χ2=5.552; p=0.018) was significantly higher for LOI- positive paracancer tissues than for LOI-negative paracancer tissues. CONCLUSIONS: IGF2 LOI is highly frequent in Chinese primary lung cancer patients, especially those with increased risk of lymph node metastasis and advanced clinical stages. IGF2 LOI may be an early epigenetic event in human lung carcinogenesis.

[Expression of ASMase in alcoholic liver fibrosis in rats].

OBJECTIVE: To investigate the expression of the lysosomal enzyme acid sphingomyelinase (ASMase) in alcohol-induced hepatic fibrosis using a rat model. METHODS: The model of liver fibrosis was induced by administration of alcohol and high fat diet using 20 rats. Six rats given no alcohol and normal diet served as the control group. Real-time PCR, western blotting, and immunohistochemistry were used to evaluate fibrosis-related changes in the mRNA and protein expressions of ASMase. RESULTS: The fibrotic liver tissues of the model rats showed significantly higher expression levels of ASMase than the non-fibrotic liver tissues of the control rats (P less than 0.05). CONCLUSION: Expression of ASMase is increased in the fibrotic liver tissue of an alcohol-induced hepatic fibrosis rat model, suggesting that this lysosomal enzyme may contribute to development of this disease condition.

[The effects of sympathetic neurotransmitters and adrenergic receptors on liver fibrosis in murine schistosomiasis].

OBJECTIVE: To investigate the effects of sympathetic neurotransmitters and adrenergic receptors on liver fibrosis in murine schistosomiasis. METHODS: Mice were infestated with schistosoma by means of pasting cercariae on their abdomens. Thirty mice were randomly divided into a control group and a model group. Hematoxylin eosin and Van Gieson staining were used to view the histopathology of their livers. Immunofluorescence histochemistry and laser scanning confocal fluorescence microscopy were used to measure the a1A and beta2 adrenergic receptors in livers of the two groups of mice. High performance liquid chromatography-electrochemical detector (HPLC-ECD) was used to determine the concentration of norepinephrine (NE) and dopamine (DA) in the plasma of the mice. RESULTS: Immunofluorescence histochemistry showed that a1A and beta2 receptors were present in hepatocytes and hepatic sinusoids of the livers of the mice of the two groups, but there were many more in the livers of the schistosoma infected mice (t=-2.888; t=-6.648) (P<0.05). The results of HPLC-ECD showed that the levels of NE and DA in the model group were higher than those of the control group (t=-3.372; t=-4.428) (P<0.05). CONCLUSION: Sympathetic neurotransmitters and adrenergic receptors may participate in liver fibrogenesis in mice infected with schistosoma.

[Expression and significance of E-cadherin, CD44v6, and proliferating cell nuclear antigen in non-small cell lung cancer].

BACKGROUND & OBJECTIVE: E-cadherin (E-cad), CD44v6 and proliferating cell nuclear antigen (PCNA) play important roles in invasion and metastasis of cancers. This study was to investigate the correlations of the expression of E-cad, CD44v6, and PCNA to the invasion, metastasis, and prognosis of non-small cell lung cancer (NSCLC). METHODS: The expression of E-cad, CD44v6 and PCNA in 86 specimens of NSCLC and 40 specimens of adjacent normal tissues were detected by EnVision immunohistochemistry. RESULTS: The high expression rate of E-cad was significantly lower in NSCLC than in adjacent normal tissues (53.5% vs. 80.0%, P<0.05). E-cad staining in NSCLC tissues was correlated to differentiation, lymph node metastasis and TNM stage (P<0.05). The high expression rate of CD44v6 was 44.2% in NSCLC, and 0 in adjacent normal tissues. CD44v6 staining in NSCLC tissues was correlated to classification, lymph node metastasis and TNM stage (P<0.05). The high expression rate of PCNA was 48.8% in NSCLC, and 0 in adjacent normal tissues. PCNA staining was correlated to lymph node metastasis (P<0.05). PCNA expression was negatively correlated to E-cad expression (r=-0.554, P<0.05), and positively correlated to CD44v6 expression (r=0.688, P<0.05). Univariate analysis indicated that E-cad, CD44v6, and PCNA were prognostic factors of NSCLC. Multivariate analysis showed that E-cad and TNM stage were independent prognostic indicators (P<0.05). CONCLUSIONS: E-cad, CD44v6 and PCNA play important roles in invasion and metastasis of NSCLC. The expression of E-cad, CD44v6 and PCNA may be of prognostic value in patients with NSCLC.

Growth inhibition and apoptosis inducing mechanisms of curcumin on human ovarian cancer cell line A2780.

OBJECTIVE: To explore the growth inhibition effects and apoptosis inducing mechanisms of curcumin on human ovarian cancer cell line A2780. METHODS: After treatment with 10 - 50 micromol/L curcumin for 6 - 24 h, the growth activity of A2780 cancer cells were studied by [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) colorimetry. Cellular apoptosis was inspected by flow cytometery and acridine orange-ethidium bromide fluorescent staining methods. The fragmentation of cellular chromosome DNA was detected by DNA ladder, the ultrastructural change was observed under a transmission electron microscope, and the protein levels of nuclear factor-kappa B (NF-kappaB, P65) and cysteinyl aspartate specific protease-3 (Caspase-3) in ovarian cancer cells were measured by immunohistochemistry. RESULTS: After treatment with various concentrations of curcumin, the growth inhibition rates of cancer cells reached 62.05% - 89.24%, with sub-G(1) peaks appearing on histogram. Part of the cancer cells showed characteristic morphological changes of apoptosis under fluorescence and electron microscopes, and the rate of apoptosis was 21.5% - 33.5%. The protein expression of NF-kappaB was decreased, while that of Caspase-3 was increased in a time-dependent manner. CONCLUSION: Curcumin could significantly inhibit the growth of human ovarian cancer cells; inducing apoptosis through up-regulating Caspase-3 and down-regulating gene expression of NF-kappaB is probably one of its molecular mechanisms.

[Expression and significance of hypoxia inducible factor-1 alpha in hepatocellular carcinoma tissues].

OBJECTIVES: To investigate the expression and significance of HIF1 alpha in hepatocellular carcinoma (HCC) tissues and in hepatoma carcinoma cell line HepG2. METHODS: The expression of the HIF1 alpha mRNA and protein were detected with immunohistochemistry (IHC), Western blot and RT-PCR techniques in HCC, normal liver tissues and HepG2. Their relationship with the pathological characteristics of the HCC was also analyzed. RESULTS: HIF1 alpha protein was obviously expressed in HCC. The positive rate of HIF1 alpha protein in HCC tissues was 76.4% and was higher than that in normal hepatic tissues. The expression of HIF1 alpha had a correlation to the differentiation degree of HCC tissues and intrahepatic and extrahepatic metastases (P<0.05), but there was no correlation to the existence of portal vein tumor emboli, the status of HBsAg and the prognosis (P<0.05). The results of Western blot and RT-PCR were similar to the results of IHC. The positive rate of HIF1 alpha in HepG2 was 93.6%. The levels of HIF1 alpha protein and mRNA began to increase after being treated two hours with hypoxia or with CoCl(2) (150 micromol/L). CONCLUSIONS: HIF1 alpha protein is obviously expressed in HCC and it is mainly affected by hypoxia. The expression of HIF1 alpha is related to the differentiation of the HCC and its intrahepatic and extrahepatic metastases but has no correlation to the existence of portal vein tumor emboli, the status of HBsAg and the prognosis.

[Inhibition of osteosarcoma cell proliferation by a short hairpin RNA targeting proliferation cell nuclear antigen].

OBJECTIVE: To construct a plasmid vector expressing the short hairpin RNA (shRNA) targeting proliferation cell nuclear antigen (PCNA), and to investigate its effect in vitro on the expression of PCNA, proliferation and apoptosis of human osteosarcoma cells. METHODS: A plasmid vector expressing the short hairpin RNA targeting at PCNA was constructed and transfected into human osteosarcoma cell line MG-63 by dosper liposomal method. PCNA mRNA and protein expressions were examined using RT-PCR and immunohistochemical staining, respectively. Inhibition of the cell proliferation was studied by MTT method and colony forming assay. DNA synthesis was analyzed by (3)H-TdR incorporation and the cell cycle was determined by flow cytometry. The apoptotic cells were stained with acridine orange. RESULTS: Expression of PCNA mRNA after the transfection was markedly inhibited by 80.51% with a PI value of 25.68% of that of the control group. PCNA shRNA inhibited MG-63 cell growth detected by using MTT method, with an inhibition rate of 61.78% at 48 h. DNA synthesis rate also decreased in the (3)H-TdR incorporation test. Flow cytometry analysis showed an increase of the percentage of G(0)/G(1) phase cells, along with a decrease of cell population in the S phase. The apoptosis rate of cells transfected with the plasmid vector was 16.54%. CONCLUSIONS: PCNA shRNA significantly suppresses the expression of PCNA at both mRNA and protein levels, corresponding to an inhibition of the proliferation of MG-63 cell and an increase of the cellular apoptosis.

[Regulatory effects on apoptotic activities of gastric cancer cell line by over-expression of Smac gene].

OBJECTIVE: To investigate the effects of overexpression of second mitochondria-derived activator of caspases (Smac) gene on apoptosis of gastric cancer cells. METHODS: Under the induction of liposome, MKN-45 cells were transfected by Smac gene and incubated with G418 for subclone selection. Reverse transcriptase-polymerase chain reaction and Western blot were used to determine cellular Smac gene expression. After induction of apoptosis by mitomycin (MMC), cell viabilities were analyzed using trypan blue stain. Apoptosis was measured by electronic microscopy, acridine orange-ethidium bromide fluorescent staining and in situ terminally labelled transferase technique (TUNEL). Western blot and colorimetry were used to assess cellular caspase-3 expression and its activity. RESULTS: The Smac mRNA and protein levels in MKN-45/Smac subclone cells (subclone consistently expressing Smac gene) were significantly higher than those in MKN-45 (P < 0.01). When compared with those in MKN-45, cell viabilities of MKN-45/Smac were reduced by 10.0% to 30.8% (P < 0.01), after treatment with 10 microg/ml MMC for 6 to 24 hours. Some of the MKN-45/Smac cells showed characteristic morphologic changes of apoptosis, their apoptotic rate being increased by 21.2% (P < 0.01). After treatment with MMC, caspase-3 expression and its activity in MKN-45/Smac cells were significantly higher than those in MKN-45 (P < 0.01). CONCLUSIONS: Overexpression of Smac in gastric cancer cell line significantly improves expression and activity levels of caspase-3 after induction by MMC. Such apoptosis-inducing effect establishes a novel strategy for regulating the apoptosis activity of gastric cancer.

Experimental study of effect of Ganyanping on fibrosis in rat livers.

AIM: To observe the effects of Ganyanping on CCl(4)-induced hepatic fibrosis in rats. METHODS: The rats were separated randomly into five groups. Groups A to group D, each consisting of 15 rats, were for different tests, while 8 rats were used as normal controls (N). For group D, CCl(4) was injected subcutaneously, at a dosage of 3 ml/kg for 9 weeks. For group A, Ganyanping was administered via gastric tube at a dosage of 10 ml/kg. For group B, the treatment with Ganyanping was started 4 weeks after CCl(4) administration. In group C, Ganyanping was administered 8 weeks after the intoxication, and treatment lasted for 4 weeks. Liver tissues were fixed in 10 % formalin and embedded in paraffin. Pathologic changes, particularly fibrosis, were evaluated on the HE and V-G-stained sections. Ten middle-power fields were randomly selected for assessment of collagen deposition. RESULTS: Loss of normal hepatic architecture, some with pseudo-lobule formation, was observed in group D, while hepatocytes steatosis and fibrosis were less pronounced in the animals treated with Ganyanping. Pseudo-lobule formation was not evident in the latter groups. The total collagen area and ratio were 840.23+/-81.65 and 7.0+/-0.9, respectively in group D, the ratio being reduced greatly in the Ganyanping-treated groups (148.73+/-45.89 and 1.16+/-0.33, respectively). The activities of MAO and ACP were elevated and that of SDH in group D decreased in the hepatic tissue as compared to the control group. The treatment with Ganyanping abrogated these enzymatic changes. CONCLUSION: Our data approved that Ganyanping could improve the microcirculation in the liver, reduce oxygen-derived free radicals, and enhance the cellular metabolism and immune function, all resulting in an anti-fibrotic effect. Hence, Ganyanping can protect the liver from fibrosis. It may be a safe and effective preparation for patient with fibrosis.

[Inhibitory effects of curcumin on apoptosis of human ovary cancer cell line A2780 and its molecular mechanism].

BACKGROUND & OBJECTIVE: Curcumin is the major effective component of curcuma, which is a kind of traditional Chinese medicine. It has been paid more attention to curcumin recently for its specific proliferation inhibition and apoptosis inducing effects on tumor cells; however, the involved mechanisms were not clear. This study was designed to explore the apoptosis inducing effects of curcumin on human ovary A2780 cell line and its related molecular mechanisms. METHODS: A2780 cancer cells were treated with 10-50 mumol/L curcumin for 6-24 h and the growth inhibition rates of A2780 cancer cells were measured by MTT method. Cell apoptosis was inspected by flow cytometry (FCM) and acridine orange-ethidium bromide fluorescent staining method. The protein levels of NF-kappa B (P65) and Caspase-3 in cancer cells were observed by SP immunohistochemistry. RESULTS: The growth inhibition rates of the cancer cells reached 62.05%-89.24%, with the peak of sub G1 appeared on DNA histogram in FCM. Partial cells presented the characteristic morphological changes of apoptosis under the fluorescent microscope; the apoptosis rates were 21.5%-33.5%. The NF-kappa B (p65) expression was decreased while Caspase-3 expression was increased, which depended on the action time. CONCLUSIONS: Curcumin could significantly inhibit the growth of ovary cancer cells; inducing apoptosis through up-regulating Caspase-3 and down-regulating expression of NF-kappa B was probably one of its molecular mechanisms.