Effects Of SILA409 On The Expression Of GRP78 In Nci-h446 Human Small Cell Lung Cancer Cells And Its Correlation With Chemotherapy Resistance To VP-16

Objective: To investigate the effects of a new compound named SILA409 on the expression of glucose-regulated protein 78 (GRP78) and analyze their association with chemoresistance to VP-16 in the NCI-H446 cell line.Methods: Cultured cells which were in good condition were divided into two groups: experimental group and control group, however the experimental group was divided into A23187 treated group, A23187â†'SILA409 treated group and SILA409 treated group. Conventional RT-PCR was used to detect the expression of GRP78 at the mRNA level in the control group and experimental group. Western blotting and immunofluorescence were used to exam the the expression of GRP78 at the protein level in the control group and experimental group. The viability of the two group of cells treated with VP-16 was examined by means of the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and apoptosis was studied via DAPI staining. The Ca2+ ionophore A23187 was used as an upregulator of GRP78 in the experiments. SPSS 11.5 windows software was used to analyze the data by ANOVA. Here values of p<0.05 were considered statisitically significant. All experiments were repeated at least three times.Results: 1.RT-PCR results showed that the expression of GRP78 at the mRNA level in the A23187 treated group cells was significantly higher than that of the control cells (p<0.05), while the GRP78 mRNA level in the A23187â†'SILA409 treated group cells was significantly lower than that of A23187 treated cells (p<0.05), showing non-dose dependent manner on SILA409 Interestingly, there is no difference in GRP78 mRNA level between the SILA409 treated group cells and the untreated negative control group cells. 2. Western blot results indicated that the protein expression of GRP78 in the A23187 treated group was higher compared with the control group (p<0.05).While compared with the A23187 treated group cells, GRP78 protein level was lower in the A23187â†'SILA409 treated group, similarly, showing non-dose dependent feasure on SILA409. The GRP78 protein expression in SILA409 treated group cells didn't show obvious alteration compared with the control cells 3. In according to Western blot results, the immunofluorescence assay showed the GRP78 protein lay round the nucleus, and the intensity of the fluorescence for the A23187-pretreated cells was abviously stronger than that of the control cells,and the density of the A23187â†'SILA409 treated group was lower than that of the A23187 treated cells. 4. Survival curves by MTT asssays showed that A23187 induction caused a significant prolonged survival for the cells subjected to VP-16 compared with the control cells. (p<0.05) cell survivals in the A23187 treated group were more than control group and A23187â†'SILA409 treated group. The IC₅₀ values for VP-16 in the SILA409 treated and A23187â†'SILA409 treated groups were obviously decreased as compared with the A23187-treated group (33.16±4.91 vs 47.21±8.34, and 33.05±2.97 vs 47.21±8.34, respectively, p<0.05). 5. DAPI staining showed the condensed status or fragmentation of cell nucleus and indicated that the apoptosis of the cells was significantly higher in the SILA409 treated group and the A23187â†'SILA409 treated group relative to the A23187 treated group.Conclusion: We can draw a conclusion in the following. 1. SILA409 has no effect on the expression of GRP78 mRNA and protein and cell viability of NCI-H446 cells. 2. SILA409 can significantly decrease the overexpression of GRP78 induced by A23187. 3. SILA409 can increase the sensitivity to VP-16 in the NCI-H446 cell line. This study may therefore be a favorable approach to the reversal of chemoresistance in small cell lung cancer therapy.