Effect Of Recombinant Adenovirus-p53 On Growth And Chemosensitivity Of Human Gastric Carcinoma Cell Lines

Gastric cancer is one of the most common malignant tumours in our country. Although the disease in the early stages is treatable by surgical resection, advanced gastric cancer does not generally respond to conventional chemotherapy. Further improving the effect of chemotherapy is an urgent problem to be settled.While drug tolerance of tumor against chemotherapy is the major reason for chemotherapy failure to gastric carcinoma.Point mutation of the p53 gene has been reported in more than 60% of gastric cancers, and can lead to the genetic instability and uncontrolled cell proliferation characteristic of human cancer. Studies have also shown that mutation and inactivation of p53 contributes to the drag tolerance of tumor against chemotherapy. Wild-type p53 is a primary mediator of cell cycle arrest, DNA repair and apoptosis, and involved intimately in tumor growth and response to chemotherapy . Chemical drags kill tumor cells by inducing cell cycle arrest and apoptosis. Wild-type p53 promotes cell cycle arrest and apoptosis of tumor cells following cytotoxicity, while mutated p53 abrogate this response. p53 gene mutation occurs in more than 50% of all human tumors, inducing tolerance to chemotherapy. Thus restoring Wild-type p53 gene into tumor cells which have p53 abnormality enhances chemosensitivity of tumor cells. Recombinant adenoviral p53 is a human type 5 adenovirus, in which the El region has been replaced with the cDNA of the wild-type p53. By transferring wild-type p53 could express its protein strongly in the malignant cells,and toxicity against hemopoietic system was observed minimal. In clinical trials and applications, we also observed that recombinant adenoviral p53 reduced the side effects caused by conventional chemotherapy and radiation therapy. It has been demonstrated by several clinical trials that wild-type p53 is effective against a variety of malignant tumors, including colon, head and neck, lung, breast, ovarian, gliomas et al. However, study on the combination of p53 with chemotherapy in gastric carcinoma is rarely published.Cisplatin and paclitaxel are critical component in most chemotherapy regimens for gastric cancer, but drug tolerance limits their clinical use. In the current study, wild-type p53 gene was transfered into the three gastric carcinoma cell lines with different p53 genetic status using recombinant adenoviral p53 to evaluate the effect of wild-type p53 on apoptosis and cheomsensitivity compared to that of cisplatin and paclitaxel alone. The trial were performed to observe the antineoplastic activity of rAd-p53 alone and combined with Cisplatin and paclitaxel currently used in chemotherapeutic protocols,and evaluate the effect of adenovirusmediated p53 gene on apoptosis and chemosensitivity of human gastric carcinoma cell lines and the value of gene therapy combined with chemotherapy. We expect that the results could provide the theoretical basis for clinical medication of gastric carcinoma.Methods:1. Three human gastric carcinoma cell lines with different p53 status, BGC-823-wtp53 cell (abbreviated W) containing wild-type p53; BGC-823-mutp53(abbreviated M) containing mutant-type p53; BGC-823-vect cell (abbreviated neo) without p53 gene, were used in this study and cultured with DMEM medium with 10% calf serum and antibiotics.2. Drugs included recombinant adenovirus-p53(rAd-p53), Cis-Dismine- dichloroplatinum(DDP), Paclitaxol(TAX).3. MTT assay was used to examine suppressive rate of cell growth dealt with various concentration of rAd-p53,TAX, DDP alone and in combination, and dose-response curves for proliferation inhibition at 0,24,48,72h were drew according MTT data.4. the development of cell cycle and apoptosis induced by different drugs alone and in combination was examined by flow cytometry at 48h.5. P53 protein expression was detected by immunohistochemistry assay and western blot assay.6. The data was performed by one-way analysis of variance and paired-samples T test using SPSS version10.0. Summary statistics were expressed as the means±the standard deviations. In all statistical analyses, a P value of≤0.05 was considered statistically significant, and all P values were two-sided.Results:1. By immunochemistry, cultures of the three human gastric carcinoma cell lines were infected with rAd-p53 at 5×10⁷vp/ml,48h later, the wild-type p53 gene was strongly expressed in each cell lines as shown by immunochemistry staining. In W cell lines infected with rAd-p53, p53 protein expression ratio of management groups were (50.20±4.84)% higher than that of control groups (14.15±3.44)%(P<0.001). In M cell line, p53 protein ratio of management groups were (63.74±4.21)% higher than control groups (30.80±5.32)% (P<0.001). In neo cell line p53 protein expression ratio of management groups were (48.87±5.88)%, compared with negative expression in control groups.2. By Western blotting, cultures of the three human gastric carcinoma cell lines were infected with rAd-p53 at 5×10⁷vp/ml,48h later the wild-type p53 gene was strongly expressed in each cell lines. Control group of neo cell didn't expressed.3. MTT assay showed that various concentration of rAd-p53,TAX, DDP single could effectively inhibit the growth of three human gastric carcinoma cell lines , compared all of the management groups with control groups (P<0.01). The results suggested that treatment of three human gastric carcinoma cell lines with various concentrations of rAd-p53, TAX, DDP alone resulted in a concentration -dependent increase in the suppressive rate of growth of cell lines, and demonstrated that rAd-p53 infection increased cellular proliferation inhibition in vitro independently on cellular intrinsic p53 status. TAX, DDP could more strongly inhibit the growth of W cell than M and neo cell (P<0.01), and the difference between M and neo cell was not significant (P>0.05). The value of IC50 of M and neo cell were higher than W cell.4. The project of rAd-p53+DDP, rAd-p53+TAX could also effectively inhibit the growth of three human gastric carcinoma cell lines in vitro. compared with all of the management groups with control groups(P<0.01). Same concentration of rAd-p53/DDP, rAd-p53/TAX could strongly inhibit the growth of M cell than W and neo cell (P<0.01), There is no difference between the W and neo cell (P>0.05) The results suggested that treatment of three human gastric carcinoma cell lines with various concentrations of rAd-p53+DDP, rAd-p53+TAX resulted in a concentration -dependent increase in the suppressive rate of growth of cell line. More obvious cellular proliferation inhibition induced by infection with rAd-p53 following with TAX, DDP treatment than either rAd-p53 or chemical drug alone (P<0.01). The value of IC50 of rAd-p53/DDP, rAd-p53/TAX was lower than TAX, DDP alone.5. Dose-response curves for proliferation inhibition show that various concentration of rAd-p53,TAX, DDP alone and rAd-p53+DDP, rAd-p53+TAX inhibit the growth of three human gastric carcinoma cell lines at 0,24,48,72h. The results suggested that treatment of three human gastric carcinoma cell lines with various concentrations of rAd-p53, alone and rAd-p53+DDP, rAd-p53+TAX resulted in a dose-dependent and time-dependent inhibition of cell proliferation.6. Cell cycle distribution and Sub-G₁ peak of three human gastric carcinoma cells infected with rAd-p53 alone and combination with TAX, DDP were assayed by flow cytometry:6.1 For W cell, infection of rAd-p53 induced G₂/M arrest and apoptotic Sub- peak. After exposed to DDP alone induced G₁ arrest and apoptotic Sub- peak. After exposed to TAX induced G₂/M arrest and apoptotic Sub- peak. Combined administration of rAd-p53 and DDP remarkably arrested in G₂/M,and cells in S/ G₁ phase significantly decreased. Meanwhile the apoptotic rate was 47.7%±1.66% in rAd-p53+DDP group,which was significantly higher than that in rAd-p53 group(9.33%±1.27%, P<0.001) and DDP group(30.8%±1.53%, P<0.001). Combined administration of rAd-p53 and TAX remarkably arrested in G₂/M,and cells in S , G₁ phase significantly decreased. Meanwhile the apoptotic rate was 52.82%±2.35% in rAd-p53+TAX group,which was significantly higher than that in rAd-p53 group(9.33%±1.27%, P<0.001) and TAX group(33.5%±2.05%, P<0.001). Chemo-enhancement ratios of rAd-p53 were 1.55 and 1.58, for DDP and TAX.6.2 For M cell, infection of rAd-p53 induced G₂/M arrest and apoptotic Sub- peak. After exposed to DDP alone induced G₁ arrest and apoptotic Sub- peak. After exposed to TAX induced G₂/M arrest and apoptotic Sub- peak. Combined administration of rAd-p53 and DDP remarkably arrested in G₂/M,and cells in S , G₁ phase significantly decreased. Meanwhile the apoptotic rate was 51.88%±2.03% in rAd-p53+DDP group,which was significantly higher than that in rAd-p53 group(11.76%±2.33%, P<0.001) and DDP group(24.7%±2.42%, P<0.001). Combined administration of rAd-p53 and TAX remarkably arrested in G₂/M,and cells in S , G₁ phase significantly decreased. Meanwhile the apoptotic rate was 60.2%±2.33% in rAd-p53+TAX group,which was significantly higher than that in rAd-p53 group(11.76%±2.33%, P<0.001) and TAX group(29.89%±1.9%, P<0.001). Chemo-enhancement ratios of rAd-p53 for DDP was 2.1, and 2.01 for TAX, respectively.6.3 For neo cell, infection of rAd-p53 induced G₂/M arrest and apoptotic Sub- peak. After exposed to DDP alone induced G₁ arrest and apoptotic Sub- peak. After exposed to TAX induced G₂/M arrest and apoptotic Sub- peak. Combined administration of rAd-p53 and DDP remarkably arrested in G₂/M,and cells in S , G₁ phase significantly decreased. Meanwhile the apoptotic rate was 44.3%±2.26% in rAd-p53+DDP group,which was significantly higher than that in rAd-p53 group(10.97%±1.5%, P<0.001) and DDP group(23.2%±1.58%, P<0.001). Combined administration of rAd-p53 and TAX remarkably arrested in G₂/M,and cells in S , G₁ phase significantly decreased. Meanwhile the apoptotic rate was 50.53%±2.28% in rAd-p53+TAX group,which was significantly higher than that in rAd-p53 group(10.97%±1.5%, P<0.001) and TAX group(26.8%±1.47%, P<0.001). Chemo-enhancement ratios of rAd-p53 for DDP was1.91, and 1.89 for TAX, respectively.7. 24,48h after infection with rAd-p53, protein caspase-3 expression in each cell lines was induced as demonstrated by flow cytometry. Fluorescence index(FI) demonstrated relative contents of protein caspase-3. FI>1.0 was positive expression, FI≦ 1.0 was negative expression, it showed that the expression rate of protein caspase-3 was increased obviously in the W, M, neo cell of rAd-p53-added.Control group of neo cell was negative expression, the expression rate of protein caspase-3 was associated with time-dependent. There were significant difference compared with control group, (P<0.01).Conclusion1. rAd-p53 can inhibit the growth of three human gastric carcinoma cell lines irrespective of the status of endogenous p53 gene. the suppressive rate of growth of cell lines is dose-dependent and time-dependent.Infection of rAd-p53 induced G₂/M arrest and apoptotic Sub-G₁ peak in each cell line.2. Western blotting and immunohistochemistry show that the p53 protein in the three cell lines was strongly expressed 48h after infection with rAd-p53. rAd-p53 could up-regulated the express rate of protein caspase-3 after exposed to rAd-p53 for 24,48 hours.3. rAd-p53 combined with TAX, DDP, respectively , could significantly inhibit the growth and proliferation of three human gastric carcinoma cell lines in vitro. The dose of combined chemotherapy to obtain the same effect is less than single chemical drug. The apoptosis rate of rAd-p53 combined with TAX, DDP, respectively, was higher than single. 4. The cell lines containing mutant-type p53 and without p53 gene induced tolerance to TAX, DDP. Infection of rAd-p53 can transfer and restore wild-type p53 gene into the three gastric carcinoma cell lines with different p53 genetic status and enhance chemosensitivity of gastric carcinoma cells to TAX, DDP.