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Aim To investigate the effects of diallyl di-sulfide( DADS) on G2/M arrest in Chk1/MGC803 and Chk2/MGC803 cells so as to establish stable human gastric cancer MGC803 cells with overexpression of Chk1/2 gene. Methods The colony formation, flow cytometry, RT-PCR and Western blot were used to de-tect the proliferation, cell cycle, and expression of Chk1/2 mRNA and protein, p-Chk1/2, CDC25C and cyclinB1, respectively. Results The colony formation showed that the colony forming efficiency in Chk1/MGC803 and Chk2/MGC803 cells treated by 30 mg· L-1 DADS was lower than in control group and vector group ( P <0. 05 ) . Flow cytometry demonstrated that 41. 3%, 57. 4%, 68. 9% and 42. 9% of G2/M cells in Chk1/MGC803 were increased than in MGC803 and Chk2/MGC803 , respectively after treated by DADS in 12,24, 36 and 48 h(P <0. 05). At the same time, RT-PCR disclosed that expression of Chk1 and Chk2 mRNA had no marked change. Western blot showed that total proteins of Chk1 and Chk2 and p-Chk2 had invisible change, but expression of p-Chk1 was up-reg-ulated, and CDC25C and cyclinB1 were down-regula-ted time-dependently in Chk1/MGC803 cells ( P <0. 05 ) . Conclusion DADS arrests MGC803 cells at G2/M by increasing p-Chk1 expression to cause down-regulation of CDC25C and cyclinB1 simultaneously.
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Objective:This work aims to investigate diallyl disulfide (DADS) inhibition of cell migration and invasion in human colon cancer SW480 cells through the Rac1-ADF/cofilin1 pathway. Methods:The potential of cell migration and invasion was examined by scratch healing assay and transwell membrane assay. The expression of Rac1-ADF/cofilin1 pathway was detected by RT-PCR and Western blot. Results:After the SW480 cells were treated with 40 and 50 mg·L-1 of DADS for 24 h, the number of transmembrane cells through the Matrigel obviously decreased by 57.12%and 64.59%, respectively (P0.05). After the treatment with 45 mg·L-1 of DADS for 6, 12, 24, and 48 h, the expression of Rac1, Rock1, PAK1, LIMK1, and Destrin proteins respectively decreased in a time-dependent manner compared with the control group (P0.05). Moreover, the expression of p-LIMK1 and p-cofilin1 notably decreased in a time-dependent manner (P<0.05). Conclusion:DADS inhibits cell migration and invasion, which is related to the down-regulation of Rac1, Rock1, PAK1, LIMK1, p-LIMK1, p-cofilin1, and destrin through the Rac1-ADF/cofilin1 pathway.
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Aim To investigate whether DATS induce MGC803 cell apoptosis and the relationship betweenapoptosis and Ca~(2+) disruption. Methods MGC-803 cell growth inhibition was measured by MTT assay. Tunnel and flow cytometry methods were used to determine the induction of apoptosis and Ca2+ homeostasis disruption. Result MTT assay showed that the inhibitory rates on MGC-803 cell growth of different concentrations of DATS 4,8,12,16 and 24 mg?L-1 were 0.231?0.037,0.305?0.036,0.455?0.029,0.607?0.058,0.751?0.019 respectively. Flow cytometry analysis showed that treating MGC803 cell with DATS significantly increased the percentage of apoptosis cells and intracellular Ca2+. Treatment of cells with 1,2-bis(2-aminophenoxye-thane)-N,N,N-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), cellular Ca2+ chelator, resulted in abolishment of the elevation of intracellular Ca~(2+) and blockage of DATS induced apoptotic of MGC-803. Conclusoin DATS could induce apoptosis of MGC-803 cells through the mechanism of Ca~(2+) homeostasis disruption.
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Aim To construct a subtracted cDNA library o f differentially expressed genes in human gastric carcinoma induced by diallyl dis ulfide(DADS). Methods Differentially expressed cDNA species induc ed by DADS in MGC 803 human gastric carcinoma cell line was determined by using suppression subtractive hybridization (SSH). Then these cDNA species were direct ly inserted into T/A cloning vector to set up the subtractive library. Amplification of th e library was carried out with transformation of E.coli by high voltage electrop erforation. One hundred positive bacteria clones were randomly picked and identi fied using PCR method. Results The amplified library contained more than 1,000 positive bacteria clones. Random analysis of 100 clones with PCR m ethod showed that all clones contained 100～600 bp inserts.Conclusions A subtracted cDNA library of differentially expressed genes in MGC 803 hum an gastric carcinoma cell line induced by DADS is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundati on for screening and cloning new and specific tumor correlative genes of human g astric carcinoma, and provides a new idea for further exploring the mechanism of DADS effects on carcinoma cells.
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Aim To observe effect of DADS on the differentiation of gastric adenocarcinoma cells and acetylation of human gastric cancer tumor cell transplantable histone.Methods Human gastric adenocarcinoma heterotransplantable tumor model was constructed through subcutaneously injecting MGC803 cells to nude mice.Morphologic changes of xenograft tumor cells were observed with optical microscope,the influence of DADS on xenograft tumor cells generationcycle distribution,the expression of p21~(WAF1) protein,histone H3 and and H4 acetylion were analyzed with flow cytometry and Western blot.Results There was obvious growth inhibitory effect of xenograft tumor while abdominal injection dose were 100 and 200 mg?kg~(-1)DADS;cells density and heteromorphism decreased after treated with DADS.Flow cytometry analysis revealed that treating xenograft tumor cells with increasing quantities of DADS increased the percentage of cells in the G_2/M phase.The proportion of xenograft tumor cells in the G_2/M phase after treatment with 100 mg?kg~(-1) and 200 mg?kg~(-1) DADS was 2.22 and 3.37 times of that in NS group.Western blot analysis showed H3 acetylion increase along with G_2/M arrest of xenograft tumor cells by DADS.DADS didn′t influence the expression level of H4 acetylion;the expression of p21~(WAF1) protein in xenograft tumor increased along with the increases in the concentration of DADS.Conclusion DADS cansignificantly inhibit the growth of human gastric carcinoma xenograft in BALB/C nucle mice and induce cell differentiation,which might be related with up-regulation of histone a cetylization and p21~(WAF1) protein level.