The Mechanism Of Radiosensitivity In Esophageal Cancer By Thalidomide

Objective:Molecular biology, radiobiology and pathology techniques were adopted to clarify the effect of thalidomide on tumor radiotherapy at tissue, serum, cell ,protein and molecular level, in an attempt to search for the key factor in the signal pathway, screen out effective bio-molecular Markers for evaluation of radiosensitivity and efficacy for esophageal cancer, besides, short-term effect and security in thalidomide treated esophageal cancer was observed.Methods:1.The effect of thalidomide on proliferative activity of esophageal carcinoma cell was analyzed by MTT assay.2.The effect of thalidomide on cells attaching was evaluated by cells attachment assay.3. The effect of thalidomide on invasive and metastasis ability of esophageal carcinoma cell was analyzed by cell scratch assay.4. The effect of thalidomide on VEGF protein expression of esophageal carcinoma cell was detected by immunohistochemical staining.5. The effect of thalidomide on cell cycle distribution of esophageal carcinoma cell was assayed by flow cytometry.6. The effect of thalidomide combined with X-ray on DNA synthesis of esophageal carcinoma was investigated by ³H- TdR incorporation assay.7. The effect of thalidomide on radiosensitivity of esophageal carcinoma cell was investigated by clone formation experiment and changes of radiosensitivity parameters such as D₀, D_q, SF₂ and etc. were analyzed simultaneously.8. The effect of thalidomide combined with X-ray on VEGF mRNA expression of esophageal carcinoma cell was determined by RT-PCR. 9. The effect of thalidomide combined with X-ray on VEGF protein expression of esophageal carcinoma cell was assayed by Western blot.10. Nude mice transplantation tumor models were established to observe the influence of thalidomide combined with irradiation on the growth of xenograft in subcutaneous of nude mice.11. MVD、VEGF、VEGFR、HIF-1αexpression in staining tumor tissues of tumor-bearing mice was detected by immunohistochemistry.12. VEGF level expression in serum from esophageal carcinoma patients were assayed by ABC-ELISA.Results:1. MTT assay showed that a decrease of TE1 cell growth was observed with an increase of thalidomide concentration and prolonging of time in vitro, in a time and concentration dependant fashion.2. Cells attachment assay results showed that cell attachment ability decreased with the increasing of thalidomide concentration in vitro.3. Cell scratch assay showed that cell invasion and migration ability decreased with the increasing of thalidomide concentration in vitro.4. Immunohistochemical staining showed that VEGF protein expression decreased with an increase of thalidomide concentration in vitro.5. 3H-TdR incorporation assay result showed that either thalidomide or X-ray alone in vitro had inhibition effect on DNA synthesis of TE1 cell, and DNA synthesis rate decrease gradually with increasing of the thalidomide concentration or irradiation dose. DNA synthesis inhibition rate of thalidomide combined with X-ray group was higher than that of thalidomide or X-ray alone group.6. Cell clone test result showed that TE1 colony numbers decreased with the increasing of thalidomide concentration at the same doses of radiation and likewise TE1 colony numbers decreased with the increasing of radiation doses at the same concentration of thalidomide. D0、Dq、SF2 value for TE1 cell decreased with the increasing of thalidomide concentration, which present as concentration dependant.7. Flow cytometry assay result showed that thalidomide had no obvious effect on the cell cycle distribution of TE1 cell in vitro.8. RT-PCR result showed that relative expression of TE1 cell VEGF mRNA decrease gradually with the increasing of drug concentration in thalidomide alone group, while no significant change was found in X-ray alone group. This expression in thalidomide combined with X-ray group is lower than thalidomide group.9. Western blot result showed that compared with control group, expression of VEGF protein decreased in thalidomide alone group, while no significant change was found in X-ray alone group. And VEGF protein expression in thalidomide combined with X-ray group is lower than thalidomide group.10. Nude mice transplantation tumor models study results showed that with the days passed by, the tumor volume increased continuously, and the change rate was different between each group: tumor volume for control group was significant larger than every other group, the average tumor volume of thalidomide combined with irradiation （20Gy） group reduced 8 days later and it was significant lower than irradiation （20Gy） alone group. Volume weight of 10Gy, 10Gy+thalidomide, 20Gy, 20Gy+thalidomide were all lower than control group, and the tumor inhibition rate of 20Gy+thalidomide group reached to 60.61%.11. Immunohistochemistry assay results showed that the HIF-1αprotein of esophageal carcinoma tissue mainly expressed in nucleus, a little in cytoplasm, the VEGF protein mainly located at cytoplasm, and VEGFR2 protein mainly expressed in cytoplasm and the membrane of the vascular endothelial cells of tumor interstitial vessel. Expression level of HIF-1α, VEGF and VEGFR2 protein for irradiation alone group as all higher than control group, and was scored as strong positive（+++）. Thalidomide alone group was scored as weak positive to positive（+～++） and showed no significant difference compared with control group. And thalidomide combined with irradiation group was scored as weak positive （+）, which was lower than control group and X-ray alone group. MVD for thalidomide alone group （13.33±3.39） was lower than control group（5.73±1.75）, and combined group （8.13±2.59） was lower than irradiation alone group（13.80±2.65）and control group.12. Serum VEGF detection results showed that VEGF level in serum esophageal carcinoma patients group was higher than that in healthy control group, which was related to primary tumor size, lymph node metastasis, pathologic type and clinical stage, but not to lesion site, distant metastasis, X-ray pathologic type, gender or age. Thalidomide was given for those serum VEGF level increasing during radiotherapy till the end of radiation treatment, and VEGF level in serum at the end of radiotherapy was found decreased compared with that during radiotherapy.Conclusion:1. In vitro, thalidomide had inhibition ability on the cell proliferation, adhesion, migration and DNA synthesis of TE1 cells in vitro.2. X-ray combined with thalidomide could enhance the inhibition effect on the growth and DNA synthesis ability of TE1 cells in vitro which indicated that thalidomide has a certain radiosensitizing effecton TE1 cells.3. Thalidomide can inhibit the growth of xenograft in subcutaneous of nude mice, and this inhibition effect can be enhanced by combination of thalidomide with irradiation , and the related mechanism may involve: （1） Direct tumor inhibition effect; （2） Indirect tumor inhibition effect through down-regulating of VEGF expression; （3） Reducation of the hypoxic ratio in carcinoma, which causees hypoxia reoxygenation, weaks its damage repair ability, finally reduces radiation radioresistance.4. The level of VEGF expression in serum from esophageal carcinoma patients was related to primary tumor size, lymph node metastasis, pathologic type and clinical stage, but not to lesion site, distant metastasis, X-ray pathologic type, gender and age.5. The level of VEGF expression in serum from esophageal carcinoma patients was related with individual radiation resistance and the radio therapy effect was poor for those patients whose VEGF level increased during radiation treatment. When given thalidomide, serum VEGF level decreased, and radiotherapy increased correspondingly, which suggested that thalidomide had a certain radiosensitivity effect for those esophageal patients with high expression of serum VEGF. From these results, serum VEGF level has important reference value for predicting radiosensitivity and judging prognosis for esophageal carcinoma. Thalidomide combined with X-ray provides a new strategy for increasing radio therapy effect on esophageal carcinoma.