| Objective:This experiment aimed to evaluate the effect of thalidomide on proliferation and and the expression of VEGF (vascular endothelial growth factor), HIF-1α(hypoxia-inducible factor-1α), NF-κB(nuclear factor-κB), MMP-1(matrix metallo- proteinase -1), TIMP-3 (tissue inhibitor of metalloproteinase -3)in small cell lung cancer cell line in vitro, to further investigate possible mechanism of its role as angiogenesis inhibitor and activity of anti-tumor, meanwhile, investigate the effects of thalidomide combined with anti-cancer agents VP-16 on human small cell lung cancer cell line NCI-H446 in vitro.Methods: 1 Human small cell lung cancer cell line NCI-H446 was incubated in culture medium in vitro, using MTT assay to detect the growth rate among different thalidomide concentration groups (25,50,100μg/ml) and different time groups (24,48,72h). Apoptosis and distribution of cell cycle were examined with flow cytometry. 2 According to the result of MTT, establishing control and experimental group, The expression of VEGF,HIF-1α,NF-κB protein was qualitationly studied by immunocytochemistry staining in NCI-H446 cells treated before and after with thalidomide. VEGF,HIF-1α,MMP-1,TIMP-3 was semi-quantitately examined by flow cytometry in NCI-H446 cells treated before and after with thalidomide. 3 MTT colorimetric methods was performed to evaluate the potential cytostatic effect of combining thalidomide with VP-16 on human small cell lung cancer cell line NCI-H446 and the OD values were computed. To determine whether the combination of thalidomide with VP-16 results in a synergistic cytostatic effect, Isobolanalysis formula was performed. In the analysis, there is synergy when the interaction index is less than 1; additivity when the interaction index is equal to 1; and antagonism when the interaction index is more than 1. 4 MTT colorimetric method was performed to evaluate the different potential cytostatic effect of combining thalidomide with VP-16 in different order on human small cell lung cancer cell line NCI-H446.Results: 1 MTT assay results:Within the concentration of (25~100)μg/ml, thalidomide can obviously inhibit proliferation of NCI-H446 in vitro. The inhibition ratio was stepping up as the concentration and time increasing, and the highest inhibition ratio was in the group with the concentration of 100g/ml and 72h. There was significantly statistical significance of inhibition ratio in different concentration groups of NCI-H446 cells after being treated thalidomide (P<0.01). Treatment with thalidomide for 48h,72h, the IC50 was 75.42μg/ml,40.62μg/ml, separately. When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the results were: After treatment with 25,50,100μg/ml thalidomide for 48h, the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase and G2/M phase decreased grudually. That was, thalidomide could induce an arrest of cell cycle in G1 phase by a dose-dependent manner. In addition, after being treated with 0,25,50,100μg/ml thalidomide for 48h, the typical apoptotic peak which enhanced gradually with the increasing concentration of thalidomide could be observed ,and the apoptotic percentage was 4.56%,13.47%,19.12%,31.10%, seperately,and there was statistically significant difference between control group and every treatment group(P<0.01). 2 Immunocytochemistry staining results:In the control group, the staining of VEGF,HIF-1α,NF-κB was positive in NCI-H446 cells and the buffy grains could be seen in endochylema, and the buffy grains was very thick;thalidomide weakened the stain degree of VEGF,HIF-1α,NF-κB in the NCI-H446 cells,the stain and the buffy grains became thinner and the masculine cell population was less than control group. and there was statistically significant difference between control group and every treatment group(P<0.05). VEGF and HIF-1αare positive correlate(P<0.05), VEGF and NF-κB are positive correlate (P<0.05),HIF-1αand NF-κB are positive correlate (P<0.05). FCM assay results:The expression of VEGF,HIF-1α,MMP,TIMP protein had been examined in NCI-H446 cells being treated with 25,50,100μg/ml thalidomide for 48h, the FI-value of VEGF in NCI-H446 was 0.842,0.718,0.558, respectively. the FI-value of HIF-1αin NCI-H446 was 0.903,0.838,0.709, respectively. the FI-value of MMP-1 in NCI-H446 was 0.849,0.733,0.599, respectively. the FI-value of TIMP-3 in NCI-H446 was 1.663,2.110,2.677, respectively. The difference was statistically significant (P<0.05). VEGF and MMP-1 are positive correlate(P<0.05). VEGF and TIMP-3 are negative correlate(P<0.05), MMP-1 and TIMP-3 are negative correlate(P<0.05). 3 MTT colorimetric methods were performed to evaluate the potential cytostatic effect of combining thalidomide with anti-cancer agents etoposide in NCI-H446 cells. The results showed: thalidomide excerted a synergistic cytostatic effect in NCI-H446 cells when combined with etoposide. In the range of concentration from 25μg/ml to 100μg/ml, thalidomide had the enhancing synergistic effect combined with etoposide with the increasing concentration. The IC50 of etoposide in NCI-H446 cells alone was 1.66μg/ml. When combined with 25,50μg/ml thalidomide, the IC50 decreased to 1.16,0.68μg/ml;Thus, there was synergistic interaction between thalidomide and etoposide in NCI-H446 cells. 4 The analysis of isobolanalysis showed that the interaction index for thalidomide combined with etoposide used in NCI-H446 cells was 0.55 as the inhibition ratio was 50% respectively. It proved that thalidomide indeed had a synergistic effect on inhibiting the proliferation of NCI-H446 cells combined with etoposide. 5 MTT colorimetric methods was performed to evaluate the different potential cytostatic effect of combining thalidomide with etoposide in different order on human small cell lung cancer cell line NCI-H446. The results showed: the synergistic cytostatic effect of the thalidomide after etoposide groups is stronger than the adverse groups, the difference was statistically significant (P<0.05).Conclusions: 1 We comfirmed that thalidomide had the capability of inhibiting the proliferation of NCI-H446 cells in vitro by a dose-and time-dependent manner. 2 We confirmed that thalidomide induced an arrest of cell cycle in G1 phase as well as apoptosis in NCI-H446 cells, expressing the inducement apoptotic is one of the mechanisms of its anti-tumor. 3 Within a certain drug concentration, thalidomide can inhibit the expression of VEGF,HIF-1αand NF-κB,MMP-1, and increase TIMP-3 in NCI-H446 cells. VEGF and HIF-1αare positive correlate, VEGF and NF-κB are positive correlate,HIF-1αand NF-κB are positive correlate. VEGF and MMP-1 are positive correlate.VEGF and TIMP-3 are negative correlate, MMP-1 and TIMP-3 are negative correlate. 4 The thalidomide combined with etoposide had a synergistic effect on inhibiting the proliferation of NCI-H446 cells. 5 This research proved that the different order of the medication has different synergistic effect on inhibiting the proliferation of NCI-H446 cells, the synergistic cytostatic effect of the thalidomide after etoposide is stronger than the adverse. This provides a new theoretic basic for the order of conjoined mediation. 6 This research proved that thalidomide has the effect of anti-pulmonary carcinoma and inriched the treatment of pulmonary carcinoma through combining thalidomide with etoposide. This provides a new theoretic way for pulmonary carcinoma integrational therapy.
|
PDF Full Text Request