Arylsulfatase,β-galactosidase and lysozyme in gastric cancer cells and its relationship to invasion

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Enhancement of migration and invasion of hepatoma cells via a Rho GTPase signaling pathway

AIM:Intrahepatic extension is the main cause of liver failureand death in hepatocellular carcinoma patients.The smallGTPase Rho and one of its effector molecules ROCK regulatecytoskeleton and actomyosin contractility,and play a crucialrole in cell adhesion and motility.We investigated the roleof small GTPase Rho in biological behaviors of hepatocellularcarcinoma to demonstrate the importance of Rho in cancerinvasion and metastasis.METHODS:Using Western blotting,we quantitated Rhoprotein expression in SMMC-7721 cells induced byLysophosphatidic acid (LPA).Furthermore,we examined therole of Rho signaling in regulating the motile and invasiveproperties of tumor cells.RESULTS:Rho protein expression was stimulated by LPA.Using the Rhotekin binding assay to assess Rho activation,we observed that the level of GTP-bound Rho was elevatedtransiently alter the addition of LPA,and Y-27632 decreasedthe level of active Rho.LPA enhanced the motility of tumorcells and facilitated their invasion.Rho played an essentialrole in the migratory process,as evidenced by the inhibitionof migration and motility of cancer cells by a specific inhibitorof ROCK,Y-27632.CONCLUSION:The finding that invasiveness of hepatocellularcarcinoma is facilitated by the Rho/Rho-kinase pathway islikely to be relevant to tumor progression and Y-27632 maybe a new potential effective agent for the prevention ofintrahepatic extension of human liver cancer.     

Effect of staurosporine on cycle of human gastric cancer cells

AIM:TO study the effect of staurosporine (ST) on the cellcycle of human gastric cancer cell lines MGC803 andSGC7901.METHODS:Cell proliferation was evaluated by trypan bluedye exclusion method.Apoptotic morphology was observedunder a transmission electron microscope.Changes of cellcycle and apoptotic peaks of cells were determined by flowcytometry.Expression of p21~(WAFl)gene was examined usingimmunohistochemistry and RT-PCR.RESULTS:The growth of MGC803 and SGC7901 cells wasinhibited by ST.The inhibitory concentrations against 50?lls (IC_(50)) at 24 h and 48 h were 54 ng/ml and 23 ng/ml forMGC803,and 61 ng/ml and 37 ng/ml for SGC7901.Typicalapoptotic bodies and apoptotic peaks were observed 24 hafter cells were treated wth ST at a concentration of 200ng/ml.The percentage of cells at G_0/G_1 phase was decreasedand that of cells at G_2/M was increased significantly in thegroup treated wth ST at the concentrations of 40 ng/ml,60 ng/ml,100 ng/ml for 24 h,compared with the controlgroup (P<0.01).The expression levels of p21~(WAFl)gene inboth MGC803 and SGC7901 cells were markedly up-regulatedafter treatment with ST.CONCLUSION:ST can cause arrest of gastric cancer cellsat G_2/M phase,which may be one of the mechanisms thatinhibit cell proliferation and cause apoptosis in these cells.Effect of ST on cells at G_2/M phase may be attributed to theup-regulattion of p21~(WAFl) gene.     

Anti-cancer effects of COX-2 inhibitors and their correlation wit angiogenesis and invasion in gastric cancer

AIM:To observe the anti-cancer effects of COX-2 inhibitorsand investigate the relationship between COX-2 inhibitorsand angiogenesis,infiltration or metastasis in SGC7901cancer xenografts.METHODS:Thirty athymic mice xenograft models withhuman stomach cancer cell SGC7901 were established anddivided randomly into 3 groups of 10 each.Sulindac,onenon-specific COX inhibitor belonging to non-steroidal anti-inflammatory drugs (a series of COX inhibitors known asNSAIDs) and celecoxib,one selective COX-2 inhibitor (knownas SCIs) were orally administered to mice of treatmentgroups.Immunohistochemistry was used to examine theexpression of PCNA,CD44v6 and microvessel density (MVD).Apoptosis was detected by using TUNEL assay.RESULTS:Tumors in sulindac and celecoxib groups weresignificantly smaller than those in control group from thesecond week after drug administration (P<0.01).Intreatment group,the cell proliferation index was lower(P<0.05) and apoptosis index was higher (P<0.05) thanthose in control groups.Compared with the controls,microvessel density was reduced (P<0.01) and expressionof CD44v6 on tumor cells was weakened (P<0.05) intreatment groups.CONCLUSION:COX-2 inhibitors have anticancer effectson gastric cancer.They play important roles in angiogenesisand infiltration or metastasis of stomach carcinoma.Theanticancer effects of COX-2 inhibitors may include inducingapoptosis,suppressing proliferation,reducing angiogenesisand weakening invasiveness.     

Interleukin 1-β gene polymorphisms and risk of gastric cancer in Sweden

Objective. Helicobacter pylori (H. pylori) infection stimulates the production of interleukin (IL)-1β, a pro-inflammatory cytokine and suppressor of gastric acid secretion. As both inflammation and hypochlorhydria, which might facilitate proximal colonization of H. pylori and other bacterial species alike, have been implicated in gastric carcinogenesis, much attention has been directed to functional genetic polymorphisms that affect the production of IL-1β. The purpose of this study was to clarify the role of these polymorphisms. Material and methods. We analysed a population-based, case-control study in 5 Swedish counties and a hospital-based, case-control study conducted in 8 Swedish hospitals, with a total of 351 gastric cancer cases and 539 controls. The IL1B-31, IL1B-511 and IL1B+3954 biallelic polymorphisms were genotyped using pyrosequencing. The variable number of tandem repeats (VNTR) polymorphism of IL1-RN was analysed using polymerase chain reaction (PCR) followed by gel electrophoresis. Relative risks were estimated by odds ratios with 95% confidence intervals, derived from unconditional logistic regression. Results. The risk of gastric cancer was unrelated to genotype in all of the studied polymorphic loci, and the absence of any association was confirmed in both the population-based and hospital-based case-control studies. Analyses confined to histological subtypes (intestinal or diffuse) and site-specific tumours (cardia or distal stomach), as well as analyses stratified by H. pylori infection status and family history of gastric cancer, did not reveal any significant increases or decreases in risk. Conclusion. Our results do not lend support to the hypothesis that human genetic polymorphisms related to the production of IL-1β are associated with the risk of gastric cancer.     

Survivin antisense oligodeoxynucleotide inhibits growth of gastric cancer cells

AIM:To investigate the effect of transfected survivin antisenseoligonucleotide (ASODN) on proliferation and apoptosis ofgastric cancer ceils.METHODS:The authors designed ASODNs targetingdifferent regions of survMn mRNA,including survivingASODN1,ASODN2 and ASODN3.ASODNs were transfectedinto gastric cancer cell line SGC 7901,cell growth was detectedby MTT assay.Cells exposed to the potent oligonucleotidewere also examined for apoptosis induction by FCM andfluorescence microscopy.Semiquantitive RT-PCR andWestern blot examinations were carried for expression ofsurvivin mRNA and protein.RESULTS:ASODN3 caused a statistically significantreduction of cell viability to 60.6% (±2.9%) (P<0.01),whileASODN1 and ASODN2 had no such changes (P>0.05).Thecell growth was also significantly inhibited by ASODN3,compared with reversal and scrambled sequence.A significantloss of survivin mRNA was presented in ASODN3 treatedceils and this was not seen in treatment with sense ODNor scramble ODN.Protein level was significantly decreased48 h after survivin ASODN trasfected by approximately 2-folddecrease comparedwith untreated controls.However.ASODN3 did not induce significant apoptosis response until48 h after transfection (P>0.05).CONCLUSION:ASODN3,which targets translation initiationpart,can be identified as a most potent antisense compound.Srvivin ASODN3 may provide a novel approach to therapyof gastric cancer.     

A p53 genetic polymorphism of gastric cancer： Difference between early gastric cancer and advanced gastric cancer

INTRODUCTIONGastric cancer is one of the most common malignancies worldwide,although the overall incidence of gastric cancer has been decreasing over the past few decades.Chronic H pylori infection and dietary factors,such as those high in salt or nitrate…     

Hypermethylation of TGF-β1 gene promoter in gastric cancer

AIM:To examine transforming growth factor-β1(TGF-β1)promoter methylation in gastric cancer and to determine if Helicobacter pylori(H.pylori)or interleukin(IL)-1β could induce TGF-β1 hypermethylation in vitro.METHODS:We examined the frequency and extent of TGF-β1 promoter methylation using methylationspecific PCR in the gastric tissues from 47 gastric cancer patients and 39 non-gastric cancer subjects.H.pylori infection was confirmed by a positive result from either a serological test,histological analysis or C13urea breath test.GES-1 and MKN-45 cells co-cultured with H.pylori or treated with IL-1β for 12,24 and 48 h in vitro tested the effects of H.pylori or IL-1β on TGF-1β.RESULTS:Twenty-four/forty-seven(51%)cases of gastric cancer(GC)tissues showed TGF-β1 promoter methylation,15/47(31.9%)cases of matched noncancerous gastric mucosa tissues from the GC patients,and 11/39(28%)case of the normal gastric mucosa tissues from non-GC subjects showed TGF-β1 promoter methylation(51%vs 28%,P<0.05).Significantly higher levels of methylation of TGF-β1 were found in the tumor tissues than in non-tumor tissues from GC patients(0.24±0.06 vs 0.17±0.04,P<0.05)and normal gastric tissues from non-GC subjects(0.24±0.06 vs 0.15±0.03,P<0.05).TGF-β1 methylation was found in 48.3% of H.pylori-positive gastric mucosal tissues whereas only 23.1% of H.pylori-negative gastric mucosal tissues showed TGF-β1 methylation(48.3%vs 23.1%,P<0.05).IL-1β appeared to induce a dose-dependent methylation of TGF-β1 and the strongest methylation was observed in GES-1 cells treated with 2.5 ng/mL of IL-1β for 48 h.Further studies showed that pre-treatment of GES-1 cells with 20ng/mL IL-1RA for 1 h could partially abolish the effect of IL-1β on TGF-β1 methylation.Infection of GES-1cells by H.pylori was not found to induce significant TGF-β1 promoter methylation.CONCLUSION:Our data revealed that TGF-1 promoter is methylated in GC patients.IL-1β may be an important mediator for H.pylori induced gene methylation during GC     

Evaluation of VEGF and VEGF-C expression in gastric cancer cells producing α-fetoprotein

Background. Gastric cancers producing α-fetoprotein (AFP) are known to have a poor prognosis and to show a high incidence of liver metastasis. Vascular endothelial growth factor (VEGF) and its isoform VEGF-C are reported to be associated with tumor progression through an angiogenic or lymphangiogenic function. In the present study, to clarify the cellular characteristics of AFP-producing gastric cancers, the expression of VEGF and that of VEGF-C in AFP-producing gastric cancer was compared with their expression in gastric cancers that do not produce AFP. Methods. Twenty-six patients with AFP-producing gastric cancers [AFP(+)] and 26 patients with stage-matched gastric cancers that did not produce AFP [AFP(−)] were evaluated for VEGF and VEGF-C expression and vessel density, using immunohistochemical analysis. Results. The survival rate of the AFP(+) group was significantly worse than that of the stage-matched AFP(−) group (P < 0.05). The frequency of VEGF-C expression was significantly higher in the AFP(+) group than in the AFP(−) group (P < 0.01). There was no significant difference in VEGF expression between the AFP(+) and AFP(+) groups. The microvessel density was higher in the AFP(+) group than in the AFP(−) group (P < 0.05). Conclusions. A higher expression of VEGF-C might be one explanation for the poorer prognosis of AFP-producing gastric cancers. Received: June 7, 2002 / Accepted: October 25, 2002 RID="*" ID="*" Reprint requests to: K. Kono     

Roles of PLC-γ2 and PKCα in TPA-induced apoptosis of gastric cancer cells

AIM: To investigate the roles of PLCγ2 and PKCα in TPA-induced apoptosis of gastric cancer cells. METHODS: Human gastric cancer cell line MGCS0-3 was used. Protein expression levels of PLCγ2 and PKCα were detected by Western blot. Protein localization of PLCγ2 and PKCα was shown by immunofluoscence analysis under laser-scanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among I 000 cells randomly. RESULTS: Treatment of gastric cancer cells MGCS0-3 with TPA not only up-regulated expression of PLC-γ2 protein, but also induced PLC-γ2 translocation from the cytoplasm to the nucleus. However, this process was not directly associated with apoptosis induction. Further investigation showed that PKCa translocation from the cytoplasm to the nucleus was correlated with initiation of apoptosis. To explore the inevitable linkage between PLC-γ2 and PKCα during apoptosis induction, PLC inhibitor U73122 was used to block PLC-γ2 translocation,in which neither stimulating PKCα translocation nor inducing apoptosis occurred in MGC80-3 cells. However, when U73122-treated cells were exposed to TPA, not only PLC-γ2, but also PKCα was redistributed. On the other hand, when cells were treated with PKC inhibitor alone, PLC-γ2 protein was still located in the cytoplasm. However, redistribution of PLC-γ2 protein occurred in the presence of TPA, no matter whether PKC inhibitor existed or not. CONCLUSION: PLC-γ2 translocation is critical in transmittingTPA signal to its downstream molecule PKCα. As an effector, PKCα directly promotes apoptosis of MGC80-3 cells. Therefore, protein translocation of PLCγ2 and PKCα is critical event in the process of apoptosis induction.     

Quantitation of CD 44 in tumor cells and peripheral blood of patients with gastric cancer

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Effect of pseudolaric acid B on gastric cancer cells： Inhibition of proliferation and induction of apoptosis

AIM: To examine the effect of pseudolaric acid B on the growth of human gastric cancer cell line, AGS, and its possible mechanism of action. METHODS: Growth inhibition by pseudolaric acid B was analyzed using MTT assay. Apoptotic cells were detected using Hoechst 33258 staining, and confirmed by DNA fragmentation analysis. Western blot was used to detect the expression of apoptosis-regulated gene Bcl-2, caspase 3, and cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). RESULTS: Pseudolaric acid B inhibited the growth of AGS cells in a time- and dose-dependent manner by arresting the cells at G2/M phase, which was accompanied with a decrease in the levels of cdc2. AGS cells treated with pseudolaric acid B showed typical characteristics of apoptosis including chromatin condensation and DNA fragmentation. Moreover, treatment of AGS cells with pseudolaric acid B was also associated with decreased levels of the anti-apoptotic protein Bcl-2, activation of caspase-3, and proteolytic cleavage of PARP-1. CONCLUSION: Pseudolaric acid B can dramatically suppress the AGS cell growth by inducing apoptosis after G2/M phase arrest. These findings are consistent with the possibility that G2/M phase arrest is mediated by the down-regulation of cdc2 levels. The data also suggest that pseudolaric acid B can trigger apoptosis by decreasing Bcl-2 levels and activating caspase-3 protease.     

Ecto-5′-nucleotidase promotes invasion, migration and adhesion of human breast cancer cells

Purpose

Associated with many molecules, metastasis includes cell adhesion to extracellular matrix, migration towards specific direction and invasion into local vessel of distant organs. The purpose of the present study was to evaluate the role of ecto-5′-nucleotidase (eN, ecto-5-NT, CD73) generated extracellular adenosine in biologically malignant behaviors of human breast cancer cell lines.

Materials and methods

Two human breast cancer cell lines, T-47D with lower expression of CD73 and MB-MDA-231 with higher expression of CD73, were used to investigate the functions of CD73. The effects of CD73 over-expression on invasion, migration and adhesion were observed in T-47D transfected with pcDNA-NT5E plasmid. The effects of specific CD73 inhibitor, α, ß-methylene ADP (APCP), were observed in MB-MDA-231 cells.

Results

The results showed CD-73 overexpression increased invasion, migration and adhesion to ECM of the pcDNA-NT5E transfected T-47D cells compared to the saline and mock vector controls. The increased cell mobility of CD-73-overexpressed T-47D cells was blocked by APCP. Adenosine increased the mobility of wild type T-47D cells. APCP inhibited the mobility of the MB-MDA-231 cells.

Conclusion

Taken together, our results indicated that CD73 may facilitate the adhesion, migration and invasion of human breast cancer cells through its enzyme activity of generating adenosine. This study provided a possibly molecular mechanism of metastasis of breast carcinoma.