组蛋白去乙酰化酶抑制剂诱导人胃癌细胞SGC-7901凋亡及其机制

目的研究去乙酰化酶转移酶抑制剂曲古抑菌素A(TSA)对人胃癌细胞SGC-7901的凋亡诱导作用及其机制。方法利用细胞计数、流式细胞仪及末端脱氧核苷酸转移酶生物素dUTP切口末端标记法(TUNEL)研究TSA对胃癌细胞SGC-7901的凋亡诱导作用。利用蛋白印迹法、基因芯片及实时定量PCR研究TSA对胃癌细胞凋亡相关基因表达的影响。结果TSA可诱导胃癌细胞SGC-7901发生凋亡;TSA可增加胃癌细胞SGC-7901p53,bax等基因的表达,降低BCL-2、生存素和半胱天冬酶的表达;TSA可使凋亡诱导因子抗侵袭因子(AIF)和核酸内切酶EndoG从线粒体释放并转移到细胞核内;TSA可通过调控多个凋亡相关基因的表达诱导胃癌细胞发生凋亡,且该凋亡是半胱天冬酶非依赖性的。结论TSA可通过调控多个凋亡相关基因来实现其诱导胃癌细胞凋亡的作用,这种凋亡诱导作用是通过半胱天冬酶非依赖途径进行的。

Implication of HPV16 Infection and P21 Gene Mutation in the Carcinogenesis and Prognosis of Gastric Cancer

Objective： To investigate whether there is a synergistic carcinogenesis between the infection of human papilloma virus （HPV） and the P21 gene mutation in gastric cancer tissue and their relationship with prognosis of the patients with gastric cancer. Methods： By using PCR technique, HPV16 infection in 46 gastric cancer tissue samples was measured and by using immunohistochemical S-P method, the P21 gene mutation in gastric cancer was detected. All patients were regularly followed up for 3 years by writing letter or clinics, to detect the infection of HPV16 by PCR and the p21 gene mutation by immunohistochemical method in 46 gastric cancer tissue specimens. Results： The positive rate of HPV16 was 41.3% and the gene mutation rate of p21 was 52.17% respectively. The recurrence or remote metastasis was observed in 21 of 46 patients. The recurrence rate was 73.68% in the patients positive for HPV16 and 66.6% in those positive for p21 gene mutation. In 8 cases positive for both HPV16 and P21, 6 had recurrence or remote metastasis. Conclusion： The HPV16 infection may be one factor causing gastric cancer and it has a synergistic carcinogenesis with the p21gene mutation. The latter may be one of the prognostic indices in gastric cancer.

Inhibitory effect of schisandrin B on gastric cancer cells in vitro

AIM： To investigate the inhibitory effect and possible mechanism of action of schisandrin B in SC-B on gastric cancer cells in vitro.METHODS： SC-B consisted of schisandrin B, aloeemodin, and Astragalus polysaccharides. Exponentially growing human gastric cancer SGO7901 cells were divided into six treatment groups： （1） control group （RPMI 1640 medium）; （2） negative control group （2% DMSO）; （3） positive control group （50 mg/L 5-Fluorouracil, 5-FU）; （4） low-dose group （LSC, final concentration of schisandrin B, 25 mg/L）, （5） moderate-dose group （MSC, final concentration of schisandrin B, 50 mg/L）; （6） highdose group （HSC, final concentration of schisandrin B, 100 mg/L）. Follow-up was done at 12-48 h. An MTT （Methylthiazolyldiphenyl-tetrazolium bromide） assay was used to examine the inhibitory effect of SOB on gastric cancer cells. The mitosis index was assessed using an inverted microscope. Flow cytometry was used to visualize the cell cycle. An RT-PCR （Reverse transcription-Polymerase chain reaction） -based assay was used to detect mRNA expression for cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase （GAPDH）.RESULTS： The MTT assay showed that the number of living cells in the LSC, MSC and HSC groups was significantly smaller than that in the DMSO-treated group （P 〈 0.05） at 12-48 h. The inhibitory rate （IR） of the LSC group was 41.15% ± 3.86%, 59.24% ± 5.34% and 69.93% ± 7.81% at 12, 24 and 48 h, respectively. The IR of the MSC group was 42.82% ± 4.94%, 62.68% ± 7.58% and 71.79% ± 8.12% at 12, 24 and 48 h, respectively. The IR of the HSC group was 37.50% ± 3.21%, 40.34% ± 2.98% and 61.99% ± 4.88% at 12, 24 and 48 h, respectively. These results suggested that a moderate dosage had the most obvious inhibitory efficacy at 48 h. Compared to the DMSO group, the mitosis index of the LSC, MSC, HSC groups was greatly decreased （P 〈 0.05） at all time points. Any dose of SC-B suppressed mitosis within 12-48 h. Compared to the DMSO group, the percentage of cells in the G0/G1 phase of the MSC group was greatly increased, and that of the S ＋ G2M phase was greatly decreased, while the percentage of cell inhibition （PCI） in the MSC group was greatly increased （P 〈 0.05）. This suggested that SC-B could exclusively arrest cells in the G0/G1 phase. Cyclin D1 mRNA expression was lower in the MSC group than that in the DMSO group （P 〈 0.05）.CONCLUSION： SC-B can inhibit the proliferation and aberrant mitosis of human gastric cancer SCG-7901 cells /n v/tro, This inhibitory effect may be due to the down- regulation of cyclin D1 mRNA expression, which causes cell cycle arrest of gastric cancer cells.

Short-term Celecoxib intervention is a safe and effective chemopreventive for gastric carcinogenesis based on a Mongolian gerbil model

AIM： To evaluate the optimal intervention point of a selective cyclooxygenase-2 （COX-2） inhibitor, Celecoxib, for inhibiting Helicobacter pylori （H pylori-associated gastric carcinogenesis in Mongolian gerbils （MGs）.METHODS： One hundred and twelve MGs were divided into six groups （A-F）. One hundred gerbils were inoculated with H pylor/（groups A-E）. Twelve gerbils were inoculated with vehicle broth only （group F）. After 4 wk, they were given N＇-methyl-N＇ -nitro-N-nitroso-guanidine （MNNG） （50 μg/mL） in the drinking water for 20 wk. in groups B-E, the animals were given the stock Celecoxib （10 mg/kg per day） diet from the 21st, 31st, 21st and 41st week respectively. The periods of administering Celecoxib were 30, 20, 20, and 15 wk respectively. On the 51st week, the animals were sacrificed for histological examination. Local PCNA expression was examined by the immunohistochemistry method. The expression of COX-2 protein was assessed by Western Blot. Analysis used the 2 test. The difference was regarded as significant when P value was less than 0.05. RESULTS： Seventeen percent （17/100） of H pyloriinfected MGs developed gastric cancer. All of these lesions were well-differentiated adenocarcinoma. The incidence rates of adenocarcinoma in groups A-F were 40%, 0%, 0%, 20%, 25%, and 0% respectively. The inflammatory scores were higher in group B than in other groups. There was no inflammatory response noted in group F. Celecoxib treatment resulted in a significant reduction in the proliferation of H pyloriinfected mucosal cells （groups B, C and D） （P 〈 0.01）. The expression of COX-2 protein was significantly attenuated in the groups which were Celecoxib-treated for more than 20 wk （groups B, C, D）. The groups treated with Celecoxib had a significantly lower rate of advanced gastric cancer （34% vs 75%, P 〈 0.001） There were no sudden deaths in any of the groups. CONCLUSION： Short-term treatment with Celecoxib has an anti-carcinogenic effect, and resulted in less severe inflammation and inhibited the invasive degree of gastric cancer.

RhoA and RhoC -siRNA inhibit the proliferation and invasiveness activity of human gastric carcinoma by Rho/PI3K/Akt pathway

AIM: To evaluate the effects of adenovirus-mediated gene transfer of RhoA siRNA and RhoC siRNA on proliferation and invasion of SGC7901 cells by Rho/ PI3K/Akt pathway. METHODS: Plasmid of RhoA siRNA and RhoC siRNA were constructed and transfected into SGC7901 cells. siRNA and LY294002 (PI3K inhibitor) were designed as the control group. The mRNA and protein expressions of RhoA and RhoC were respectively detected with RT-PCR and western blotting. In order to fi nd out the changes of proliferation and invasion power of SGC7901 cell lines, we analyzed the data by MTT, Boyden chamber and evaluated apoptosis of cell with flow cytometry. We treated BALB /C nude mice with RhoA and RhoC-siRNA, and tumor control rate (%) in nude mice was calculated. RESULTS: RhoA and RhoC siRNA transfections specifically down-regulated the corresponding mRNA and protein levels in SGC7901 Cells.The experiment of permeated artificial basal membrane showed that the invasion power of SGC7901 cell lines are on the decline after treatment of Ad-RhoA and RhoC-siRNA (12.64 ± 3.27 vs 87.38 ± 17.38, P < 0.05). The values of 490 nm wavelength light absorption were different in the five groups. The number of alive cells in the group of RhoA and RhoC-siRNA was lower than others in the 6th d (0.71 ± 0.01 vs 3.82 ± 0,11 P < 0.05). The apoptosis rate of transfected RhoA and RhoC-siRNA group with FACS were 19.07% ± 1.78 and there were significant differences between treated and control groups (19.07 ± 1.78% vs 1.23 ± 0.11%, P < 0.01). The tumor transplantation experiment in BALB/C nude mice showed intratumoralinjection of RhoA or RhoC siRNA can inhibit tumor growth. CONCLUSION: RhoA and RhoC siRNA gene therapy mediated by adenovirus may be useful for inhibiting growth and invasion of SGC7901 through a PI3K/Akt pathway. These results provide a novel therapeutic target in preventing gastric cancer cell invasion and metastasis.

Inhibitory effects of apigenin on the growth of gastric carcinoma SGC-7901 cells

AIM： To explore the growth inhibition and apoptosisinducing effect of apigenin on human gastric carcinoma SGC-7901 cells. METHODS： The effects of apigenin on the growth, clone formation and proliferation of human gastric carcinoma SGC-7901 cells were observed by N-rr, done-forming assay, and morphological observation. Fluorescent staining and flow cytometry analysis were used to detect apoptosis of cells. RESULTS： Apigenin obviously inhibited the growth, clone formation and proliferation of SGC-7901 cells in a dosedependent manner. Inhibition of growth was observed on d 1 at the concentration of 80 μmol/L, while after 4 d, the inhibition rate （IR） was 90%. The growth IRs at the concentration of 20, 40, and 80 μmol/L were 38%, 71%, and 99% respedJvely on the 7^th d. After the cells were treated with apigenin for 48 h, the number of clone-forming in control, 20, 40, and 80 μmol/L groups was 217±16.9, 170±11.1 （P〈0.05）, 98±11.1 （P〈0.05）, and 25±3.5 （P〈0.05） respectively. Typical morphological changes of apoptosis was found by fluorescent staining. The cell nuclei had lost its smooth boundaries, chromatin was condensed, and cell nuclei were broken. Flow cytometry detected typical apoptosis peak. After the cells were treated with apigenin for 48 h, the apoptosis rates were 5.76%, 19.17%, and 29.30% respectively in 20, 40, and 80 μmol/L groups. CONCLUSION： Apigenin shows obvious inhibition on the growth and clone formation of SGC-7901 cells by inducing apoptosis.

Inhibitory effects of N-(4-hydrophenyl) retinamide on liver cancer and malignant melanoma cells

AIM: To investigate the effect of N-(4-hydrophenyl) retinamide (4-HPR), the derivative of retinoic acid, on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatocellular carcinoma cells (HCCs) and malignant melanoma cells.METHODS: 4-HPR was chemically synthesized. Cellular migration and invasion were assayed by Borden chamber experiment. Cell growth was assayed by MTT chromometry.Apoptosis effect was measured using Hoechst 32258 staining and flow cytometry. Gene transfection was performed with lipofectamine.RESULTS: We observed that the migration of HCC and melanoma cells was significantly suppressed by 4-HPR and the migration cells were reduced to 58±5.03 (control 201±27.2, P＜0.05, n = 4) in SMMC 7721-k3 HCC, and to 254±25.04 (control 302±30.1, P＜0.05, n = 4) in melanoma cells after 6-h incubation with 4-HPR. The invasion through reconstituted basement membrane was also significantly reduced by 4-HPR treatment to 11.2±3.3 in SMMC 7721-k3 HCC (control 27±13.1), and to 24.3±3.2 in melanoma cells (control 67.5±10.1, P＜0.05, n = 3). Cell growth, especially in melanoma cells, was also significantly inhibited.Furthermore, 3 μmol/L of 4-HPR induced apoptosis in B16 melanoma cells (37.11±0.94%) more significantly than all-trans retinoic acid (P＜0.05), but it failed to induce apoptosis in SMMC 7721-k3 HCC. The mechanism for 4-HPR-induced apoptosis was not clear, but we observed that 4-HPR could regulate p27kip1, and overexpression of cerebroside sulfotransferase (CST) diminished the apoptosis induced by 4-HPR in melanoma cells.CONCLUSION: 4-HPR is a potent inhibitor of HCC migration and inducer of melanoma cell apoptosis. CST and p27kip1 expression might be associated with 4-HPR-induced apoptosis.

Influence of a microscopic positive proximal margin in the treatment of gastric adenocarcinoma of the cardia

AIM： To investigate the influence of a positive proximal margin in total gastrectorny patients with gastric adenocarcinorna of the cardia. METHODS： Medical records of 191 patients with total gastrectornies for adenocarcinorna of the cardia between 1995 and 2000 were reviewed. The clinicopathologic features associated with a positive margin were determined, and the predictors for survival were analyzed. RESULTS： The incidence of positive proximal margin was 8.4% （16/191）. The positive margins were associated with advanced diseases. The tumor size and the depth of tumor invasion were independent risk factors for a positive margin. The mean survival in the positive margin group was 33.9 mo as compared.with 62.4 mo in the negative group （P 〈 0.001）. However, the difference in survival lost significance in subgroup analysis according to stage. Multivariate analysis identified that a positive margin was not an independent prognostic factor for survival. CONCLUSION： A positive margin is more of an indication of advanced disease in patients with gastric adenocarcinoma of the cardia rather than an independent prognostic factor for survival.

Down-regulation of Bcl-X_L by RNA interference suppresses cell growth and induces apoptosis in human esophageal cancer cells

AIM： To determine the inhibitory effect of the vectorgenerated small interfering RNAs （siRNAs） on the expression of the BcI-XL gene in established human esophageal cancer cells, and to investigate the effect of the BcI-XL siRNAs on cell growth and apoptosis in esophageal cancer cells. METHODS： Three siRNA-expressing vectors targeting different sites of the Bcl-XL gene were constructed from pTZ-U6＋I vector. Cultured esophageal cancer cells were transfected with the siRNA-expressing vector （or the control vector） using lipofectamine 2000. BcI-XL gene expression was determined with semiquantitative RT- PCR assay and Western blotting. Among the three siRNA- expressing vectors, the most highly functional vector and its effect on cell growth and apoptosis in esophageal cancer ceils was further analyzed. RESULTS： Of the three siRNA-expressing vectors, siRNA- expressing vector No.1 was the most potent one which suppressed Bcl-XL mRNA production to 32.5% of that in the untreated esophageal cancer cells. Western blotting analysis showed that siRNA-expressing vector No.1 markedly down-regulated the expression of Bcl-XL in human esophageal cancer cells. Treatment of esophageal cancer cells with siRNA-expressing vector No.1 resulted in inhibition of cell growth and induction of apoptosis. CONCLUSION： Down-regulation of BcI-XL by vectorgenerated small interfering RNAs can suppress cell growth and induce apoptosis in human esophageal cancer cells.

Angiostatin inhibits pancreatic cancer cell proliferation and growth in nude mice

AIM: To observe the biologic behavior of pancreatic cancer cells in vitro and in vivo, and to explore the potential value of angiostatin gene therapy for pancreatic cancer.METHODS: The recombinant vector pcDNA3.1(+)-angiostatin was transfected into human pancreatic cancer cells PC-3 with Lipofectamine 2000, and paralleled with the vector and mock control. Angiostatin transcription and protein expression were determined by immunofluorescence and Western blot. The stable cell line was selected by G418. The supernatant was collected to treat endothelial cells. Cell proliferation and growth in vitro were observed under microscope. Cell growth curves were plotted.The troms-fected or untroms-fected cells overexpressing angiostatin vector were implanted subcutaneously into nude mice. The size of tumors was measured, and microvessel density count (MVD) in tumor tissues was assessed by immunohistochemistry with primary anti-CD34antibody.RESULTS: After transfected into PC-3 with Lipofectamine 2000 and selected by G418, macroscopic resistant cell clones were formed in the experimental group transfected with pcDNA 3.1(+)-angiostatin and vector control. But untreated cells died in the mock control. Angiostatin protein expression was detected in the experimental group by immunofluorescence and Western-blot. Cell proliferation and growth in vitro in the three groups were observed respectively under microscope. After treatment with supernatant, significant differences were observed in endothelial cell (ECV-304) growth in vitro. The cell proliferation and growth were inhibited. In nude mice model, markedly inhibited tumorigenesis and slowed tumor expansion were observed in the experimental group as compared to controls, which was parallel to the decreased microvessel density in and around tumor tissue.CONCLUSION: Angiostatin does not directly inhibit human pancreatic cancer cell proliferation and growth in vitro,but it inhibits endothelial cell growthin vitro. It exerts the anti-tumor functions through antiangiogenesis in a paracrine way in vivo.

Indomethacin suppresses growth of colon cancer via inhibition of angiogenesis in vivo

AIM: It has been reported that regular consumption of nonsteroidal anti-inflammatory drugs like indomethacin decreases the incidence and mortality rate of a number of gastrointestinal cancers. We aimed to explore the efficacy and possible mechanisms of indomethacin on tumor growth and tumor angiogenesis of human colon cancer xenografts in nude mice. METHODS: MTT (thiazolyl blue) assay was used to assess the effect of indomethacin on cultured human colorectal cancer cell line HCT116. HCT116 cells were inoculated subcutaneously into BALB/c-nu/nu mice. After oral administration of indomethacin, 3 mg/kg·d for 4 wk, animals were sacrificed by cervical dislocation. Immunohistochemical staining was employed to determine the microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression in tumor tissues. RESULTS: Indomethacin, a non-selective COX inhibitor, significantly decreased the viability of HCT116 cells in a dose-dependent manner (P<0.05) with 50% inhibition at approximately 318.2±12.7 μmol/L Growth of HCT116 cell tumor was significantly suppressed by indomethacin. The tumor volume was significantly decreased in the treated group (458.89±32.07 mm3) compared to the control group (828.21±31.59 mm3) (P<0.05). The MVD of the treated group (19.50±5.32) was markedly decreased compared to the control group (37.40±4.93) (P<0.001). The VEGF expression of the treated group (1.19±0.17) was obviously reduced as compared to the control group (1.90±0.48) (P<0.01). The decrease in MVD was positively correlated with the decrease of VEGF expression (rs = 0.714, P<0.05). We did not see gastrointestinal complications in the treated group and no differences were noted in the body weight of the mice between the two groups throughout the study CONCLUSION: Indomethacin can significantly decrease the viability of cultured HCT116 cells and retard human colorectal HCT116 cell tumor growth via inhibiting tumor angiogenesis, which might be through reduction of VEGF expression.

Tumor suppressor and hepatocellular carcinoma

A few signaling pathways are driving the growth of hepatocellular carcinoma.Each of these pathways possesses negative regulators.These enzymes,which normally suppress unchecked cell proliferation,are circumvented in the oncogenic process,either the overactivity of oncogenes is sufficient to annihilate the activity of tumor suppressors or tumor suppressors have been rendered ineffective.The loss of several key tumor suppressors has been described in hepatocellular carcinoma.Here,we systematically review the evidence implicating tumor suppressors in the development of hepatocellular carcinoma.

Methylation in esophageal carcinogenesis

Genetic abnormalities of proto-oncogenes and tumor suppressor genes have been demonstrated to be changes that are frequently involved in esophageal cancer pathogenesis. However, hypermethylation of CpG islands, an epigenetic event, is coming more and more into focus in carcinogenesis of the esophagus. Recent studies have proved that promoter hypermethylation of tumor suppressor genes is frequently observed in esophageal carcinomas and seems to play an important role in the pathogenesis of this tumor type. In this review, we will discuss current research on genes that are hypermethylated in human esophageal cancer and precancerous lesions of the esophagus. We will also discuss the potential use of hypermethylated genes as targets for detection, prognosis and treatment of esophageal cancer.

Inhibitory effect of a new gossypol derivative apogossypolone (ApoG2) on xenograft of human prostate cancer cell line PC-3

Objective: To investigate the inhibitory effect of apogossypolone (ApoG2) on prostate cancer cell line PC-3 in vivo, and explore its mechanism. Methods: The models of transplantation tumors in Balb/c nu/nu mice were established via subcutaneous injection of PC-3 cells and the tumor-transplanted mice were divided into 4 groups: control group and three ApoG2 treatment groups, with 10 mice in each group. Volumes of the tumor were estimated every 2 d and the morphology of tumor tissues was observed. Immunohistochemistry was employed to observe the expression of Bcl-2, PCNA, CD31, caspase-3 and caspase-8 in tumor tissues. Results: ApoG2 (2.5 mg/kg-10 mg/kg) given intraperitoneally once a day can obviously inhibit the growth of subcutaneous prostatic carcinoma implant. The tumor volume decreased obviously when the treatment dosage was bigger than 5.0 mg/kg (P<0.01). Meanwhile, ApoG2 decreased the expression of PCNA and CD31, and enhanced the expression of caspases-3, caspase-8 in tumor tissues. Conclusion: ApoG2 exert an inhibitory effect on prostatic carcinoma possibly by inducing apoptosis and inhibiting tumor angiogenesis.

Inhibitory effect of mycophenolate mofetil on human hepatocellular carcinoma cell line HepG-2

To investigate the inhibitory effect of mycophenolate mofetil on human hepatocellular carcinoma cell line HepG-2. Methods： HepG-2 cells were cultured in the presence of the different concentrations of mycophenolate mofetil in vitro. MTT assay was used to analyze the inhibition of cell viability conferred by mycophenolate mofetil. Cell apoptosis was observed using Hoechst33258 staining, and the percentage of HepG-2 cells at different cell cycles was determined through flow cytometry. The ability of cell adhesion was evaluated by in vitro adhesion assay. Gene expressions of factors （ICAM-1 and VCAM-1） were detected by RT-PCR. Results： Mycophenolate mofetil significantly inhibited the growth of HepG-2 cells by inducing the apoptosis of cells and this drug also inhibited the adhesion of HepG-2 cells in a dose-dependent manner. Marked morphological changes characterized in cell apoptosis were demonstrated through Hoechst33258 staining. In addition, mycophenolate mofetil decreased the proportion of S phase cells and increased that of G0/G1 phase cells. [^3H]-Thymidine uptake assay indicated that the application of mycophenolate mofetil at different concentrations significantly inhibited the cell proliferation. RT-PCR identified the expression levels of ICAM-1 and VCAM-1 genes in liver cancer cells after cultured for 72 h with different concentrations of drug. An inverse relationship was found between the expressions of ICAM-1 and VCAM-1 and drug concentrations. Conclusion： Mycophenolate mofetil has remarkable inhibitory effect on hepatocarcinoma HepG-2 cells.

Inhibitory effect of Fuzheng Yiliuyin in combination with chemotherapeutics on human gastric carcinoma cell strain

AIM： To study the inhibitory effects of Fuzheng Yiliuyin （Decoction for Suppressing Tumors by Strengthening the Body Resistance） in combination with chemotherapeutics on human gastric carcinoma cell strain. METHODS： Fuzheng Yiliuyin （ZY） combined with various kinds of chemotherapeutics was put into two kinds of cultivated human gastric carcinoma cell strains, then its inhibitory effects on human gastric carcinoma cell strains were determind by the MTT method. Flow cytorneter was used to assay the apoptosis rate, and the ultrastructure of gastric carcinoma cells was observed under transmission electron microscope. RESULTS： Obvious apoptosis was seen in gastric carcinoma cells after treatment with ZY for 72 h. ZY and chemical drugs had synergistic inhibition effects on the cultivated gastric carcinoma cells, but the effects were different on various cell strains. The inhibitory effects of ZY could be strengthened by cytotoxic action and apoptosis. ZY combined with tluorouracil, etoposide and cisplatin （EFP） chemotherapeutics had better inhibitory effects on SGC-7901, while ZY combined with EFP or with DDP chemotherapeutics had better inhibitory effects than other drugs on MGC-803. CONCLUSION： ZY induces apoptosis and inhibits the growth of gastric carcinoma cells. ZY has the synergistic function of chemotherapeutics.

Downregulation of survivin by RNAi inhibits growth of human gastric carcinoma cells

AIM: To investigate the inhibitory effect of a specific small survivin interfering RNA (siRNA) on cell proliferation and the expression of survivin in human gastric carcinoma cell line SGC-7901. METHODS: To knockdown survivin expression, a small interfering RNA targeting against survivin was synthesized and transfected into SGC-7901 cells with lipofectamineTM2000. The downregulation of survivin expression at both mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Cell proliferation inhibition rates were determined by methyl thiazolyl tetrazolium (MTT) assay. The effect of survivin siRNA on cell cycle distribution and cell apoptosis was determined by flow cytometry (FCM). RESULTS: RNA interference could efficiently suppress the survivin expression in SGC-7901 cells. At 48 h after transfection, the expression inhibition rate was 44.52% at mRNA level detected by RT-PCR and 40.17% at protein level by Western blot analysis. Downregulation of survivin resulted in significant inhibition of tumor cell growth in vitro. The cell proliferation inhibition rates at 24, 48 and 72 h after survivin siRNA and non-siliencing siRNA transfection, were 34.06%, 47.61% and 40.36%, respectively. The apoptosis rate was 3.56% and the number of cells was increased in G0/G1 phase from 38.2% to 88.6%, and decreased in S and G2/M phase at 48 h after transfection. CONCLUSION: Downregulation of survivin results in significant inhibition of tumor growth in vitro. The inhibition of survivin expression can induce apoptosis of SGC-7901 cells. The use of survivin siRNA deserves further investigation as a novel approach to cancer therapy.

Science Letters:IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis with its expression associated with DNA hypomethylation of exon 1

Insulin-like growth factor binding-protein-7 （IGFBP7） was obtained from our previous colonic adenocarcinoma （CRC） and normal mucosa suppression subtraction hybridization （SSH） cDNA libraries. By RT-PCR and immunohistochemistry, we found that IGFBP7 was overexpressed in CRC tissue compared to normal tissue. However, our in vitro experiments performed in 10 CRC cell lines showed that IGFBP7 expressed only in SW480 and Caco2 cell lines, which implied an underlying reversible regulatory mechanism. Using methylation-specific PCR （MSP） and bisulfite sodium PCR （BSP）, we found that its expression was associated with DNA hypomethylation of exonl. This was further supported by the in vitro study which showed restored IGFBP7 expression after demethylation agent 5-aza-2＇-deoxycytidine treatment. Correlation analysis between IGFBP7 expression and prognosis indicated that overexpression of IGFBP7 in CRC tissue correlated with favourable survival. Investigation of the functional role of IGFBP7 through transfection studies showed that IGFBP7 protein could inhibit growth rate, decrease colony formation activity, and induce apoptosis in RKO and SW620 cells, suggesting it a potential tumor suppressor protein in colorectal carcinogenesis. In conclusion, our study clearly demonstrated that IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis and its expression is associated with DNA hypomethylation of exon 1.

Protective effect of ascorbic acid in experimental gastric cancer: reduction of oxidative stress

AIM;Oxidative stress participates in the cell carcinogenesis by inducing DNA mutations.Our aim was to assess whether ascorbic acid,an antioxidant,could have a role in preventing ROS(Reactive Oxygen Species)generation in experimental gastric carcinoma in a rat model. METHODS:Experimental gastric cancer was induced in twelve Wistar male rats(weighting 250-350 g)by profound duodeno-gastric reflux throught split gastrojenunostomy.The rats were allocated to the following groups:Group Ⅰ(n=6) was the control;Group Ⅱ(n=6)which was mantained with daily intake of tape water with Vitamin C(30 mg/Kg).After 6 or 12 months,samples of gastric tumor or non tumor mucosa were taken from the anastomosis of both groups. Oxidative stress was measured by superoxide quantification through lucigenin-amplified chemiluminescence base and by staining with Nitrobluetetrazolium.The histopathologic confirmation of adenocarcinoma was made by eosin- hemathoxilin method. RESULTS:The intestinal type of gastric adenocarcinoma was microscopically identified in all animals of group Ⅰ whereas only 3 rats of group Ⅱ showed an adenocarcinoma without macroscopic evidence of them.The cancers were located in the anastomosis in all cases.Basal luminescence from tumor gastric tissue generated 38.4±6.8 count per minute/mg/×10~6(mean±SD)and 14.9±4.0 count per minute/mg/×10~6,respectively,in group Ⅰ and Ⅱ animals(P <0.05).The Nitrobluetetrazolium method showed intense staining in tumor tissues but not in non neoplasic mucosa. CONCLUSION:Experimental gastric tumors seem to produce more reactive oxygen species than non neoplasic gastric tissue.The reduction of oxidative stress and gastric tumor incidence in rats were induced by the intake of ascorbic acid.Therefore,it may have a role in the prevention of gastric carcinoma. Oliveira CPMS Kassab P Lopasso FP Souza HP Janiszewski M Laurindo FRM Iriya K Laudanna AA.Protective effect of ascorbic acid in experimental gastric cancer:reduction of oxidative stress.World J Gastroenterol 2003;9(3):446-448 http://www.wjgnet.com/1007-9327/9/446.htm

Effect of superoxide dismutase and malondialdehyde metabolic changes on carcinogenesis of gastric carcinoma

AIM： To investigate the relationship between the superoxide dismutase （SOD）, malondialdehyde （MDA） metabolic changes and the gastric carcinogenesis.METHODS： The SOD activity and MDA content were measured in the gastric tissues from the focus center, peripheral and far-end areas of gastric carcinoma （n = 52） arid gastric ulcer （n = 10）. All the tissues were subjected to routine histological examinations and classifications.RESULTS： The SOD activity was greatly reduced but the MDA content was markedly increased in the center areas of the non-mucous gastric carcinoma （non-MGC）; and the poorly differentiated gastric carcinoma varied. The SOD activity was gradually decreased and the MDA content was gradually increased in the tissues from the focus far-end, peripheral to center areas of non-MGC. Both of the SOD activity and the MDA content were significantly declined and were respectively at same low level in the tissues from the focus center, peripheral, and far-end area with the mucous gastric carcinoma （MGC）. In contrast to the gastric ulcer and grade I or II of non-MGC, the same level of the SOD activity and the MDA content were found in the focus center areas. Between non-MGC （groups A-D） and gastric ulcer （group F）, the differences of SOD activity and MDA content were very noticeable in the gastric tissues from the focus peripheral and far-end areas, in which the SOD activity showed noticeable increase and the MDA content showed noticeable decreasein the gastric ulcer.CONCLUSION： The active free radical reaction in the gastric tissues can induce the carcinogenesis of non-MGC. The utmost low ability of antioxidation in the gastric tissues can induce the carcinogenesis of MGC. The metabolic change of the free radicals centralized mostly in the center of ulcerated lesions only, which suggested the ability of antioxidation was declined only in these lesions. However, the metabolism of free radicals varied significantly and the ability of antioxidation declined not only in the local focus area but also in the abroad gastric tissues with gastric carcinoma.