Prevention of VP-16 Resistance by a Disiloxane, SILA409: Effects of SILA409 on the Expression of GRP78 in NCI-H446 Human Small Cell Lung Cancer Cells

Encheng      Li; Jinhui      Zhang; Jiarui      Wang; Fei      Gao; Zhonghai      Yang; Qiang      Meng; Qianqian      Zhang; Na      Li; Ming      Huang; Gabriella      Spengler; Joseph      Molnar; Qi      Wang

Abstract

Aim: To investigate the effects of an MDR inhibitor and Ca2+ antagonist, the SILA409, on the expression of glucose-regulated protein 78 (GRP78) and the resistance to VP-16 in the NCI-H446 cell line. Methods: Conventional RTPCR, western blotting and immunofluorescence were used to detect the expression of GRP78 at the mRNA and protein levels in the NCI-H446 cell line treated with varying amounts of SILA409. The viability 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. Results: The expression of GRP78 at the mRNA and protein levels in the A23187 Ca2+ ionophore-induced and SILA409-post-treated cells was significantly lower than that in the positive control in the A23187-treated group (p < 0.05), whereas no difference was found between the SILA409-treated group and the untreated negative control group. The IC50 values for VP-16 in the SILA409- treated and A23187-pretreated and SILA409-post-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). The immunofluorescence assay confirmed the western blotting results. DAPI staining indicated that the apoptosis of the cells was significantly higher in the SILA409-treated group and the A23187-pretreated and SILA409-post-treated group relative to the A23187- treated group (p < 0.05). The survival of NCI-H446 cells exposed to VP-16 in the presence of the Ca2+ ionophore followed by SILA409 treatment was significantly decreased as compared with that of the Ca2+ ionophore-treated cells. The apoptosis induced by SILA409 treatment, in Ca2+ ionophore-pretreated and SILA409-post-treated cells was significantly increased relative to Ca2+ ionophore-treated cells. An elevated glucose concentration itself does not affect the MDRreversing effect of SILA409. Conclusion: SILA409 can significantly decrease the overexpression of GRP78 induced by A23187 and increase the sensitivity to VP-16 in the NCI-H446 cell line.

Keywords: Disiloxane, GRP78, multidrug resistance, SCLC, SILA409, VP-16, MDR, Ca 2+ ionophore-induced, DAPI staining, apoptosis, immunofluorescence, chemoresistance, glucose-regulated proteins (GRPs), MCF7, small cell carcinoma, Downregulation