Anti-tumor Lymphangiogenesis And Lymph Node Metastasis Drug Research On The Subcutaneous Transplanted Tumor Of Human Gastric Carcinoma In Nude Mice

Research background and Objective:Gastric carcinoma is one of the common Malignant tumor, ranking the forth in the Malignant tumor in the world, while the mortality rate in second place in the Malignant tumor. Lymph node metastasis is an important prognostic factor and the major cause of treatment failure and death of patients with gastric cancer. Approximately 60% of resectable gastric cancer in the time of diagnosis has occurred lymph node metastasis, resulting in advanced gastric cancer five-year overall survival rate is less than40%. There was an urgent need to explore effective method in inhibiting gastric lymph node metastasis. The purpose of this study is screening out of drugs which can effectively inhibit the lymphangiogenesis and lymph node metastasis of transplanted tumor which implanted under the skin of nude mice through animal experiments. It can provide a new approach that contain the gastric carcinoma and other tumors lymphangiogenesis, control lymphatic metastasis and improve the survival rate.Method:1. Cultured gastric cancer cell SGC-7901 and injected 0.2ml single cell suspension in nude mice. Twelve days later all the nude mice showed around 7mm diameter subcutaneous nodules. The subcutaneously transplanted tumor models in nude mice were established. (Three part different time experiments have the same step.)2. The three batches of subcutaneously transplanted tumor models of human gastric carcinoma in nude mice were randomly divided into four groups separately. Part I: saline group, 5-FU group, low-dose group of recombinant human vascular endothelial growth inhibitor and high-dose group of recombinant human vascular endothelial growth inhibitor.Part II: saline group, 5-FU group, low-dose arsenic trioxide group and high-dose arsenic trioxide group.Part III: saline group, 5-FU group, low-dose thalidomide group and high dose thalidomide group. And we started delivery on the same day. Drug delivery continued in the next 7 days.3. Executed nude mice and stripped tumor tissue. Calculated the inhibition rate of tumor weight.4.using the immunohistochemical method,investigated the tumor cell expression of VEGF-C and the new distribution of lymphatic vessels.5. The lymph node metastasis of transplanted tumor was detected by HE stainning.6. Using the TUNEL method detected tumor cell apoptosis in tumor tissue.Results:1. Tumor inhibition effect of Recombinant human vascular endothelial growth inhibitor (Endostar).(1) Tumor weight:Tumor weight of Saline group was 601±26 mg. Tumor weight of 5-FU group was 427±23 mg. Tumor weight of low concentration endostar group was 588±25 mg. Tumor weight of high concentration endostar group was 448±36 mg. 5-FU and high concentration endostar could Obviously inhibit the growth of transplanted tumor, in contrast to saline and low concentration endostar. Difference between 5-FU group and high concentration endostar group was no statistically significant. Difference between saline group and low concentration endostar group was no statistically significant.(2) Apoptosis rate:Apoptosis rate of Saline group was 8.8±2.0%. Apoptosis rate of 5-FU group was 30.5±7.4%. Apoptosis rate of low concentration endostar group was 12.5±3.9% .Apoptosis rate of high concentration endostar group was 28.3±5.8%. 5-FU and high concentration endostar could Obviously up-grade the rate of transplanted tumor apoptosis, in contrast to saline and low concentration endostar, the differences were significant. Difference between 5-FU group and high concentration endostar group was no statistically significant. Difference between saline group and low concentration endostar group was no statistically significant.(3) Optical density of the expression VEGF-C of Tumor cells:Optical density of Saline group was 0.88±0.11. Optical density of 5-FU group was 0.79±0.10. Optical density of low concentration endostar group was 0.78±0.15. Optical density of high concentration endostar group was 0.37±0.14. High concentration endostar could down-grade the expression VEGF-C of tumor cells, in contrast to saline,5-FU and low concentration endostar . The differences were statistically significant (p <0.05). Differences were not significant between saline group, 5-FU group and low concentration endostar group.(4) Lymphatic number:Lymphatic number of Saline group was 8.5±1.5. Lymphatic number of 5-FU group was 7.4±1.7. Lymphatic number of low concentration endostar group was 7.1±1.6. Lymphatic number of high concentration endostar group was 3.7±1.1. High concentration endostar could depress the number of tumor Lymphatic, in contrast to saline, 5-FU and low concentration endostar. Differences were statistically significant (p<0.05). Differences were no significant between saline group, 5-FU group and low concentration endostar group.(5) Lymph node metastasis rate:Lymph node metastasis rate of Saline group was 3.5±0.9. Lymph node metastasis rate of 5-FU group was 1.3±0.5. Lymph node metastasis rate of low concentration endostar group was 3.4±0.5. Lymph node metastasis rate of high concentration endostar group was 1.1±0.3. High concentration endostar and 5-FU could depress Lymph node metastasis of Tumor, in contrast to saline and low concentration endostar, differences were statistically significant (p <0.05). Difference between 5-FU group and high concentration endostar group was no statistically significant. Difference between saline group and low concentration endostar group was no statistically significant.2. Tumor inhibition effect of arsenic trioxide.(1) Tumor weight:Tumor weight of Saline group was 604±29 mg. Tumor weight of 5-FU group was 418±10 mg. Tumor weight of low concentration arsenic trioxide group was 438±25 mg. Tumor weight of high concentration arsenic trioxide group was 430±21 mg. 5-FU, low concentration As2O3 and high concentration As2O3 could Obviously inhibit the growth of transplanted tumor, in contrast to saline. The differences were significant (P <0.05). Tumor weight differences between 5-FU group, low concentration arsenic trioxide group and high concentration arsenic trioxide group were no statistically significant. Arsenic trioxide can significantly depress tumor growth.(2) Apoptosis rate:Apoptosis rate of Saline group was 8.5±2.0%. Apoptosis rate of 5-FU group was 27.3±2.8%. Apoptosis rate of low concentration arsenic trioxide group was 27.8±3.9%. Apoptosis rate of high concentration arsenic trioxide group was 28.0±4.5%. Transplanted tumor apoptosis rates of low concentration arsenic trioxide group , high concentration arsenic trioxide group and 5-FU group were significantly higher than normal saline group, and the differences were significant (P <0.05). Tumor apoptosis rate differences between 5-FU group, low concentration arsenic trioxide and high concentration arsenic trioxide were no statistically significant. These suggested the significant roles of arsenic trioxide and 5-FU in promoting tumor cell apoptosis.(3) Optical density of the expression VEGF-C of Tumor cells:Optical density of Saline group was 0.85±0.19. Optical density of 5-FU group was 0.82±0.14. Optical density of arsenic trioxide low concentration group was 0.39±0.09. Optical density of arsenic trioxide high concentration group was 0.41±0.11. Low concentration arsenic trioxide and high concentration arsenic trioxide could down-grade the expression VEGF-C of Tumor cells in contrast to saline and 5-FU. Difference between Low concentration arsenic trioxide and high concentration arsenic trioxide group was no statistically significant. Difference between saline group and 5-FU group was no statistically significant.(4) Lymphatic number:Lymphatic number of Saline group was 8.0±1.7. Lymphatic number of 5-FU group was 7.0±1.3. Lymphatic number of arsenic trioxide low concentration group was 3.5±0.9. Lymphatic number of arsenic trioxide high concentration group was 3.6±1.0. Compared with normal saline and 5-FU, low concentration arsenic trioxide and high concentration arsenic trioxide inhibited the growth of lymphatic vessels and the differences were statistically significant (P <0.05). No significant difference between 5-FU group and saline group. No significant difference between low concentration arsenic trioxide and high concentration arsenic trioxide.(5) Lymph node metastasis rate:Lymph node metastasis rate of Saline group was 3.6±1.0. Lymph node metastasis rate of 5-FU group was 1.6±0.7. Lymph node metastasis rate of low concentration arsenic trioxide group was 1.5±0.6. Lymph node metastasis rate of high concentration arsenic trioxide group was 1.4±0.7. Low concentration arsenic trioxide, high concentration arsenic trioxide and 5-FU inhibited the Lymph node metastasis, compared with normal saline, and the differences were statistically significant (P <0.05). The differences were no statistically significant between Low concentration arsenic trioxide group , high concentration arsenic trioxide group and 5-FU group.3. Tumor inhibition effect of Thalidomide.(1) Tumor weight:Tumor weight of Saline group was 604±30 mg. Tumor weight of 5-FU group was 427±20 mg. Tumor weight of low concentrations thalidomide group was 588±31 mg. Tumor weight of high concentrations thalidomide group was 579±28 mg. Transplanted tumor weights of 5-FU group significantly lower than low concentration thalidomide group, high concentration thalidomide group and saline group, the differences were significant (P <0.05). Tumor weight differences between saline group, Low concentration thalidomide group and high concentration thalidomide group were no significant.(2) Apoptosis rate:Apoptosis rate of Saline group was 9.1±2.8%. Apoptosis rate of 5-FU group was 28.5±3.4%. Apoptosis rate of low concentration thalidomide group was 11.5±1.5% .Apoptosis rate of high concentration thalidomide group was 29.0±4.6%. Transplanted tumor apoptosis rates of 5-FU group and high concentration thalidomide group were significantly higher than normal saline group and low concentration thalidomide group. The differences were significant (P <0.05). Difference between 5-FU group and high concentration thalidomide group was no significant. Difference between saline group and low concentration thalidomide group was no significant.(3) Optical density of the expression VEGF-C of Tumor cells:Optical density of Saline group was 0.87±0.16. Optical density of 5-FU group was 0.80±0.15. Optical density of thalidomide low concentration group was 0.85±0.16. Optical density of thalidomide high concentration group was 0.33±0.10. High concentration thalidomide could down-grade the expression VEGF-C of Tumor cells, in contrast to saline, 5-FU and low concentration thalidomide. The differences were significant (P <0.05). Differences were no significant between saline group, 5-FU group and low concentration thalidomide group.(4) Lymphatic number:Lymphatic number of Saline group was 8.2±1.6. Lymphatic number of 5-FU group was 7.5±1.5. Lymphatic number of low concentration thalidomide group was 7.3±1.7. Lymphatic number of high concentration thalidomide group was 3.4±1.0. High concentration thalidomide could depress the number of lymphatic, in contrast to saline, 5-FU and low concentration thalidomide. The differences were significant (P <0.05). Differences were no significant between saline group, 5-FU group and low concentration thalidomide group.(5) Lymph node metastasis rate:Lymph node metastasis rate of Saline group was 3.7±0.9. Lymph node metastasis rate of 5-FU group was 1.4±0.6. Lymph node metastasis rate of low concentration thalidomide group was 3.2±0.9. Lymph node metastasis rate of high concentration thalidomide group was 1.3±0.5. High concentration thalidomide and 5-FU could depress Lymph node metastasis, in contrast to saline and low concentration thalidomide, the differences were significant (p <0.05). Difference between 5-FU group and high concentration thalidomide group was no significant. Difference between saline group and low concentration thalidomide group was no significant.Conclusion:1. High concentration recombinant human vascular endothelial growth inhibitor could promote tumor cell apoptosis and inhibit tumor growth, tumor cell VEGF-C expression, tumor lymphangiogenesis and Lymph node metastasis. Low concentration recombinant human vascular endothelial growth inhibitor did not show the above role.2. Low concentration arsenic trioxide and high concentration arsenic trioxide could promote tumor cell apoptosis and inhibit tumor growth, tumor cell VEGF-C expression, tumor lymphangiogenesis and Lymph node metastasis.3. High concentration thalidomide could promote tumor cell apoptosis and inhibit tumor cell VEGF-C expression, tumor lymphangiogenesis and lymph node metastasis. But low concentration thalidomide did not show the above-mentioned role. 4. 5-FU could promote tumor cell apoptosis and inhibit tumor growth and lymph node metastasis but it has serious toxicity effect.