| Objective In order to investigate the effects of Act D on the growth and proliferation of gastric adenocarcinoma cells and apoptosis of gastric adenocarcinoma cells induced by ActD. To study the effect of ActD on Survivin,Cox-2 and HSP70 mRNA expression of gastric adenocarcinoma cells (BGC823), in vitro. To investigate the prevalence and their clinical significance of Survivin,Cox-2 and HSP70 in gastric carcinoma and screen the prognostic factors of gastric carcinoma.Methods MTT assay was used to observe the viability and growth inhibition of human gastric adenocarcinoma cells (BGC823) after different time treatment with various concentrations of ActD and ActD analogs. The percentage of apoptotic cells and the cell-cycle distribution of BGC823 cells treated with ActD were measured by flow cytometry (FCM). Using the transmission electron microscopy (TEM), the change of ultrastructural organization of BGC823 cells was observed. Expression of Survivin,Cox-2 and HSP70 mRNA was detected using RT-PCR in gastric cancer cells (BGC823).To identify and analysis the differentially expressed genes profile on gastric cancer cells (BGC823) by cDNA microarray.The gastric cancer tissue microarrays (TMA) containing 99 gastric cancer specimens from different clinical stages were used to determine Survivin,Cox-2 and HSP70 expression by immunohistochemistry.Results 1.When the concentrations of ActD were higher than 5.0×10-9mol/l, the cell growth of BGC823 was suppressed statistically in dose- and time- dependent manner (P<0.05). ActD analogs [D-Phe2, D-Me-Phe5]2 AMD and [D-Phe2, D-Me-Val5] AMD2 were similar with ActD suppressed the cell growth of BGC823.The inhibition ratio of ActD analogs were lower than that ActD.2.After 48h and 72h treatment with the higher concentrations of ActD (1.0×10-7, 5.0×10-8mol/l), the cell growth of BGC823 was suppressed remarkably and necrotic peak was observed in all specimens.The percentage of apoptotic cells increased to 8.0%, 28.0% and 7.8% after 72h treatment with 1.0×10-8, 5.0×10-9 and 1.0×10-9 mol/l of ActD, respectively, and increased to 6.0% after 48h treatment with 5.0×10-9mol/l of ActD, while the percentage of apoptotic cells was unchanged after less than 72h incubation with 1.0×10-8 and 1.0×10-9mol/l of ActD.After 24h treatment with 1.0×10-8, 5.0×10-9 and 1.0×10-9mol/l of ActD, the G1 phase content of BGC823 cells decreased and the G2 phase content of BGC823 cells increased as compared with control group. However, the G1 phase content of BGC823 cells increased and the G2, S phase content of BGC823 cells decreased as compared with control group after 48h and 72h treatment.3.After 48h and 72h incubation with the higher concentrations of ActD (1.0×10-7, 5.0×10-8mol/l,1.0×l0-9mol/l), nuclear condensation, nuclear fragmentation and apoptotic bodies were observed.4. After 72h treatment with 1.0×10-9 and 5.0×10-9mol/l of ActD, the relative expression level of survivin mRNA was down in gastric cancer cells.The relative expression level of Cox-2 and HSP70 mRNA treatmented with 5.0×10-9mol/l was lower obviously it treatmented with 1.0×10-9 mol/l.5. After the gastric cancer cells was treatment with of ActD, 16 genes were up-regulated and 3 genes were down-regulated.The profile of these differentially expressed mainly focus on DNA repair genes,immunologic genes,apoptosis genes,cell proliferation genes,signal transduction genes,etc.6.The expression rates of Survivin,Cox-2 and HSP70 were 58.6% (58/99),60.6% (60/99) and 61.6%(61/99), respectively. There were a significant correlation between Survivin,Cox-2 and HSP70 expression and the depth of invasion(P<0.05).Cox-2 and HSP70 were found to be statistically associated with lymph node metastasis of gastric cancers (P<0.05). Coexpression rates of survivin and Cox-2 and HSP70 were observed in 37.4%,35/99,41/99,respectively. But no statistically significant correlation was found (P>0.05). Kaplan-Meier survival plots for 89 patients showed no significant relationship(P>0.05) between Survivin,Cox-2 and HSP70 expression and the 5-year survival rates of patients with gastric carcinoma.In the initial univariate analysis of 89 gastric cancer cases followed up, the depth of invasion, sex, lymph node metastasis,Cox-2 expression were all significant(P<0.05). Multiple Cox regression analyses demonstrated that the depth of invasion,sex and lymph node metastasis were independent prognosticators(P<0.05). According to regression coefficients of the depth of invasion,sex and lymph node metastasis in the final Cox regression analysis, a risk function of postoperative patients with gastric cancers was established as follows:h(t)=[h0(t)]e0.411X1+0.635 X3+0.630X6 Conclusions 1. The higher concentrations of ActD cause cell death directly by cytotoxic role and suppress the cell growth of BGC823 notablely in dose- and time- dependent manner. After longer time treatment with the lower concentrations of ActD, the growth and proliferation of gastric adenocarcinoma cells are suppressed significantly due to apoptosis of cells.The lower concentrations of ActD influence the growth of gastric cancer cells by blocking cell cycle. BGC823 cells can be arrested in G2 phase after treatment with the lower concentrations of ActD for shorter time. For longer time, ActD mainly affects G1 phase and arrests cells in G1 phase, i.e, transition from G1 to S phase of gastric cancer cells can be blocked by ActD.The lower concentrations of ActD can cause down-regulated expression of Survivin,Cox-2 and HSP70 mRNA in gastric cancer cells. According to the results of flow cytometry (FCM), transmission electron microscopy (TEM), laser confocal scanning microscopy (LCSM) and RT-PCR, down-regulation of Survivin,Cox-2 and HSP70 mRNA possibly participates in apoptosis of gastric cancer cells induced by ActD.2. The inhibition ratio of ActD analogs were lower than that ActD.It is necessary that the reforming frame of ActD are studied further.3. ActD showed significant effecton the differential expression of cells apoptosis genes and other related genes in human gastric carcinoma cells (BGC823).4.Overexpression of Survivin,Cox-2 and HSP70 are observed in gastric cancers. The upregulated expression of Survivin in gastric carcinoma plays positive role in early stage of human gastric carcinogenesis. Cox-2 and HSP70 may contribute to the growth, the invasion and the lymph node metastasis in gastric carcinoma and display the poor clinical prognosis of patients with gastric carcinoma as an independent prognosticator. The depth of invasion, sex, lymph node metastasis and C0x-2 expression are significant prognostic factors. However, only the depth of invasion, sex and lymph node metastasis can indicate prognosis of patients with gastric carcinoma independently.
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