| Objectives To evaluate the mechanisms of heparanase (HpA) antisense oligodcoxy (AS-ODN) inhibited and induced apoptosis of gastric cancer cells by detecting expression of HPA-raRNA, p53 , bcl-2 , Survivin , CD54 , VEGF , MMP-2and TIMP-2 in human gastric adenocarcinoma cell line SGC-7901 before and after AS-ODN tranfection in vitro.Methods The AS-ODN complementary to the start codon region of heparanase mRNA and its control, scrambled nonsense oligodeoxyuncleotide (NS-ODN) were desiged and synthesized and phosphorthioated. The ODNs were embedded in cationic liposome Lipofection and transfected into SGC-7901 cells and cultured in 37 ℃ ,5%CO2 culture box for 24h, 48h and 72h. The expression of HPA-mRNA, p53 and bcl-2 were detected by reverse transcription-polymerase chain reaction analysis (RT-PCR) before and after transfection.The morpologic test, TUNEL staining and flow cytometery were used to measure apoptosis of gastric cancer cells. The expression of CD54 and bcl-2 were detected by flow cytomety before and after transfection. The cells proliferation were detected by MTT method. The expression of Survivin, VEGF, MMP-2 and TIMP-2 were detected byimmunohistochemical staining method. :Results The results of RT-PCR slowed that HPA-mRNA, p53 and bcl-2 of SGC-7901 treated with AS-ODN of different final concentration were significanthy changed (HPA-mRNA and bcl-2 were decreased, p53 was increased)compared with that of the controls. AS-ODN has a significant inhibitory effect on the SGC-7901 and influence on the expression of p53 and bcl-2 in a dose-dependent manner.After AS-ODN transfection, SGC-7901 cell present some typical morphologic features of apoptosis, including nuclear shrinkage chromatin condensation. DNA fragmentation and formation of apoptotic bodies.The results by TUNEL showed that AS-ODN induced SGC-7901cells apoptosis and the apoptosis cells increased signifantly with the incroasily of concentration of AS-ODN. After 48h danstected with AS-ODN, The number of cells increased from 3.88 + 1.06% before transfection to 10.31 + 1.63% after transfeotion in 0.4umol/L group; Cell numbers increased after transfection in 0.8umol/L group. There was significant difference (P<0.01).The results by flow cytometry showed typical sub-diploid "apoptotic peak" in NDA histogram.24 hours aften the transfection with AS-ODN at the concentration of 0.2, 0.4 and 0.8umol/L, the apoptotic rates of SGC-7901 cells were 5.97, 9.13, 16% respectively; 48 hours after, the rates were 11.09, 19.63 and 30.77%, respectively; 72 hours after, the rates were 13.32, 17.2 and 19%,respectively. In a multivariate analysis, there were signifcant difference in different groups with concentration (P<0.01) and no significanl difference with time (P>0.05).The results by flow cytometry also shoned that AS-ODN couldinhibit the expression of SGC-7901, CD54, bcl-2.24 hours after the transfection with AS-ODN at the concentration of 0.2, 0.4 and 0.8umol/L, the equal fluorescent intensity of CD54 showed 155.6, 110.62 and 94.00, respectively; 48 hours after the intensity showed 52.68, 50.90 and 30.94, respecticely; 72 hours after, the intensity showed 36.95, 14.37 and 7.11, respectively.In a multivariate anslysis, the mean of equal fluorescent intensity showed statistical significance in different groups with time and concentration.And the less expression of CD54 with the increasing of time and concentration. The inhibition on CD54 expression was in a time-dependent and concentration-dependent manner(P<0.01).24 hours after the transfection with AS-ODN at the concentration of 0.2, 0.4 and 0.8nmol/L, the equal fluorescent intensity of bcl-2 showed 94.20, 77.29 and 60.08, respectively; 48 hours after, the intensity showed 24.23, 21.81 and 21.75, respectively; 72 hours after, the intensity showed 12, 7.99 and 6.10, respectively.In a multivariate analysis, the mean of equal fluorescent intensity showed statistical significance in different group with time and concentration. And the bcl-2 expression cut down gradually with the increasing of time and concentration. The inhibition on expression of bcl-2 was in a time-dependent and concentration-dependent manner (P<0.05).24h after the transfection with AS-ODN at the concentration of 0.1, 0.2, 0.4 and 0.8umol/L, the proliferative rates of cells were 90.41, 87.48, 78.29 and 75.40, respectively; 48h after, the rates were 85.42, 76.58, 52.95 and 42.38, respectiuely; 72h after, the rates were 80.17, 60.92, 42.33 and 25.30, respectively. The results by MTT showed that AS-ODN could inhibit the proliferation of SGC-7901 cells. In a multivariate anslysis, the mean of proliferative rates of cells showed statistical significance in different group with time and concentration.And the inhibition enhanced gradually with the extension of time and the increasing of concentration. The inhibitory effects on SGC-7901 were in a dose-dependent and time-dependent manner(P<0.01).The results by immunohistochemical staining showed that AS-ODN could affect the expression of Survivin, VEGF, MMP-2 and TIMP-2 in varying degrees. Cultivated for 48 hours, the expression of Survivin was 0.3266 + 0.131 8 befor the transfection with AS-ODN, and 0.1999 + 0.0503, 0.1961 ±0.0552 after the transfection with AS-ODN at the concentration of 0.4 and 0.8umol/L, respectively. The expression showed a tendency to drop. However in one-way analysis of varicance, the mean of light density showed no statistical significance in different group with concentration (P>0.05).The light density of VEGF was 0.3591 ± 0.1943 before the transfection, and 0.2990 ± 0.1165, 0.2969 + 0.0542 after the transfection at the concentration of 0.4 and 0.8 umol/L, respectively. The expression showed a tendency to drop, too. But in one-way analysis of varicance, the mean of light density showed no statistical significance in different group with concentration(P>0.05).The light density of MMP-2 was 0.2695 ± 0.069 before the transfection with AS-ODN, and 0.1484 + 0.0312, 0.444 + 0.0209 after the transfection at the concentration of 0.4 and 0.8 umol/L, respectively. In one-way analysis of variance, the mean of light density showed statistical significance in different group with concentration(P<0.01). The inhibitory effects on MMP-2 were in a concentration-dependent manner.The light density of TIMP-2 was 0.2797 + 0.1087 and 0.2923 + 0.0001 before the transfection with AS-ODN at the concentration of 0.4 and 0.8umol/L, respectiuely. The expression showed a tendency to improve. But in one-way analysis of varicance, the mean of lightdensity showed no statistical significance in different group with concentration(P>0.05).Conclusions: 1. The concentration of HPA-mRNA after with AS-ODN declined,which indicated heparanase AS-ODN could downregulate the expressin of HPA by closiny genes of HPA.2. SGC-7901 cells transfected with AS-ODN changed typically in features. Increased significantlly in index of apoptosis and showed typical subdiploid "apoptotic peak"in histogram, where apoptotic rates of cell increased and cell proliferation-inhibited. Which indicated the apoptosis induced was one of the mechnisms how the Heparancise AS-ODN inhibited gastric cancer cells. 3. Apoptosis inducing gene p53 transfected with AS-ODN upregulated, apoptosis inhibiting gene bcl-2 downregulated, but apoptosis inhibiting gene Survivin unchanged.Which indicated the apoptosis induced by Heparanase AS-ODN were correlated with the expression of p53 and bcl-2, and no correlation with Survivin.4. The expression of CD54 and MMP-2 transfected by AS-ODN declined obviously, whereas the expression of VEGF and TIMP-2 unchangable, implying the downregulation of CD54 an MMP-2 was related to the less adhesin to tumor cells and the less desradation of excracelluan matrix and basemembrance, respectively, wheareas no correlation with the expression of VEGF and TIMP-2.Signifance Heparanase AS-ODN could downregulate the expression of HPA-mRA. The apoptosis on gastric caner cells were related to the expression of p53 and bcl-2, The less expression of CD54 and MMP-2 might affect the adhesion on caner cells and the degradation of ECM and basemembrance.In short, it was concluded that HPA AS-ODN might play an important role in the inhibition of proliferation, invasion and metastasis of gastric cancer cells. It showld be considered in futrue... |
PDF Full Text Request