| Background and aims:Gastric cancer (GC) is one of the most common malignant tumors in the world, and China alone accounts for 42% of all gastric cancers in the world, and a survey in 2004-2005 showed that GC is the third leading cause of cancer mortality in China. The disease in early stages is treatable by radical resection. However, 15-50% or more patients have developed a peritoneal carcinomatosis at the surgical exploration, thus curative treatment is no longer available. To make the matter worse, it has long been recognized that GC does not generally respond to conventional chemotherapy. With the development of cancer genetics, gene therapy has stood out as a promising multidisciplinary treatment approach against GC.The tumor suppressor p53 gene, located on chromosome 17p13, is the most frequently mutated gene in human cancers. Normal p53 gene (wild-type) regulates the cell cycle, DNA reparation, and cell apoptosis. Mutated p53 gene defect these functions and appeared to be carcinogenic. It is reported that 60% of human GC had a p53 gene mutation.Gendicine (recombinant human Ad-p53 injection, rAdp53, Shenzhen SiBiono GeneTech Co., Ltd., Shenzhen, China) was the first commercial gene therapy product in the world, approved by the China State Food and Drug Administration in 2003, first for the treatment of head and neck cancers and later for other malignancies. It was based on a modified serotype 5 adenovirus vector engineered to express p53 gene to initiate the apoptosis pathway in the cell nucleus and the cytoplasm. Gendicine can also promote drug sensitivity through a by-stander effect. It is reported that head and neck squamous cell carcinoma (HNSCC), lung cancer, breast cancer, and liver cancers have been treated with Gendicine and appeared to be effective and well tolerated. Although the initial results were encouraging, it has also been recognized that gene therapy alone has not met the high expectations, and combination of gene therapy with other therapeutic approaches such as chemotherapy might provide more treatment benefit. Therefore, this study was designed to explore the potential of recombinant human Ad-p53 in gastric caner treatment, either alone or in combination with chemotherapy, by in vitro and in vivo test. Methods:(1) Human gastric adenocarcinoma cell line SGC-7901 were cultured in RPMI-1640 medium supplemented with 10% standard newborn bovine serum in the 5% CO2, saturated humidity,37℃incubator. SGC-7901 cells were plated in 24-well culture plates, at a density of 3.6×104/well. Four groups were treated with 109 vp of rAdp53,1μg of EPI, both of the two at the same dose, and a blank control with culture medium, respectively. Cells of each group were harvested and counted daily with counting plate from day 1 (after 24 h) to day 6. The cell growth curve was drawn for each treatment group. (2) The effect of the rAd-p53 and EPI on cell cycle progression was assessed by flow cytometry. (3) SGC-7901 cells were plated in 6-well culture plates with cover slips for 48 h as above. The slips were stained with Wright's stain for 15 min at room temperature after being air dried. Cover slips were washed completely and cellular morphology was observed under a microscope. (4) SGC-7901 cells (5x106) were subcutaneously inoculated into the back of one nude mouse as a donor tumor, which was resected when it reaches 8-10 mm in diameter after 20 days and cut into cubes～2 mm in diameter, then transplanted under the left dorsal skin of 4-5-week old female nude mice surgically under sterile conditions. The tumor-bearing nude mice were allocated to three groups, the rAd-p53 therapy group (n=8), EPI therapy group (n=8), and the control group (n=6) were given 10μl of 1012 vp/ml of rAd-p53,1.25 mg/kg body weight of EPI, and 10μl of 0.9% saline, respectively. Each mouse received intra-tumor injection of the study drugs every three days over a period of three weeks. The body weights and tumor volumes were measured before every injection. Two days after the last injection, all nude mice were euthanized. The blood, tumor, major organs such as the heart, the kidneys, the lungs and the liver were collected for biochemical and histological studies. (5) Formalin-fixed, paraffin-embedded tumor tissues were cut at 5-μm thickness. Tumor feature were observed under light microscope after henatoxylin and eosin (H&E) staining. (6) Tumors of each group were lysed, and the p53 protein concentration of each sample was determined by the Western blot assay. (7) The serum of each nude mouse was diluted at a 1:10 ratio with 0.9% saline. The liver, renal and heart functions were studied in serum of each nude mouse using Biochemical Analyzer. Histopathological study of the liver and heart were also performed on paraffin-embedded specimens with H&E staining.Results:(1) The inhibition rate of rAd-p53, EPI, combined therapy on SGC-7901 were 32.26,35.48,43.44% on day 1 and 70.62,78.82,87.15% on day 2, respectively. rAd-p53 and EPI inhibited the growth of SGC-7901 cells effectively compared to the blank control (p<0.01), and rAd-p53 combined with EPI clearly improved the inhibition (p<0.05). After treating SGC-7901 cells for 5 days, the inhibition rate reached 93.75% for rAd-p53 and 98.96% for EPI. The combination of them resulted in-100% inhibition of growth (p<0.01). (2) The effect of rAd-p53 and/or EPI on the cell cycle is shown that as compared to the controlled group, rAd-p53 and/or EPI affected the SGC-7901 cell cycle. The percentage of cells in phase GO/G1 was decreased and phase S was increased compared to the control (p<0.05). An obvious sub-G1 peak in the rAd-p53 treated group and the combined group were observed, indicating apoptotic cells. (3) After Wright staining the control SGC-7901 cells were fusiform, grown to confluence, the cell structures were complete and clear. The confluent cells in EPI treated group seemed less than the control, and dispersed cells appeared. The junction between cells could not been seen after treating with rAd-p53 for 48 h. The number of SGC-7901 cells were obviously decreased, the cells were dispersed, single, changed from normal shape. Cytoplasm was stained deeper and nucleolus was disrupted into small pieces, several apoptotic cells were seen under the microscope. Cells were rare in the combined treatment group, several cell fragments could be seen, and there were few complete cells. (4) We implanted the SGC-7901 cells into nude mice and assessed the efficacy of rAd-p53 in vivo. The three different therapies were performed to compare the tumor growth of the three groups, which show that the tumor volume of rAd-p53 group was significantly smaller than the control group from the sixth injection to the end point (p<0.01). The difference in rAd-p53 and EPI therapy group was not statistically significant. The weight of the three groups over the period of therapy was not different (p>0.05). (5) The H&E stained tumor slices were observed under light microscope. In the control group, there was no necrosis found, and tumor shape and structure were well stained. Necrosis in the tumor tissue of nude mice in the EPI treated group was not obvious. Remarkable necrosis was observed in the tumor tissue of nude mice treated with rAd-p53, and there was some leukomonocytes infiltrating the tumor. (6) Western blotting of the p53 protein among different groups is shown that the gray scale ratios of rAd-p53, EPI and control groups were 0.234,1.012 and 1.309, respectively. (7) The serum biochemical parameters were tested to analyze the toxicity of the therapy agents. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) in control group was 92.5±8.86 U/l,198.75±50.83 U/l,1227.5±314.22 U/l, respectively. Two nude mice in the rAd-p53 group had a remarkable increase of the above three parameters. The results were validated by liver H&E staining, which showed the two livers had serious hepatitis. The CK and CK-MB in control group were 743.75±214.339 U/l,773.75±227.718 U/l, respectively. One case in the EPI group had a remarkable increase of the two enzymes, and the result was validated by histopathology. The index of renal function (BUN) of the EPI treatment group was significantly higher than the other two groups (p<0.05). Other enzymes in nude mice in therapy groups did not show any abnormal signs.Conclusions:The in vivo study confirmed that rAd-p53 had similar tumor inhibition effect to EPI (p>0.05), but significantly greater effect than the control group (p<0.01). Histological study revealed conspicuous tumor necrosis and apoptosis in the rAd-p53 treatment group. We performed Western blotting to measure p53 protein expression and found p53 protein expression level was lower in rAd-p53 and EPI groups than control group. In our study,2 of 8 nude mice in the rAd-p53 group developed liver toxicity, characterized by the significantly increased serum ALT, AST and LDH levels, diffused hepatocyte necrosis with lymphocyte infiltration. No special treatments were delivered to these animals. Nevertheless, there were no deaths in these animals during the study period. Such liver toxicity did not occur in the EPI group or the control group. In the EPI treatment group,1 of 8 nude mice developed cardiac toxicity, characterized by the significantly increased serum CK and CK-MB levels, and scattered piecemeal myocardium necrosis. Again, no unexpected animal deaths occurred in this group during the study period, suggesting that the rAd-p53 and EPI used at the study dosage was safe. In summary, this study demonstrated that rAd-p53 significantly inhibited gastric cancer SGC-7901 cells both in vitro and in vivo, and rAd-p53 had synergetic effect with EPI. This opens a new possibility of chemogene-therapy for gastric cancer.
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