Resistance to platinum-based chemotherapy in lung cancer cell lines

Abstract

Purpose: A series of six lung cancer cell lines of different cell origin (including small cell and mesothelioma) were characterized immunohistochemically and the role of a series of protein candidates previously implicated in drug resistance were investigated.

Methods: These include colony-forming and cell growth assays, immunohistochemistry, siRNA knockouts, real-time PCR and western blots.

Results: No correlation was found with AKT, HO-1, HO-2, GRP78, 14-3-3zeta and ERCC1 levels and cisplatin nor oxaliplatin cytotoxicity, but an association was observed with levels of the enzyme, dihydrodiol dehydrogenase (DDH); an enzyme previously implicated in the development of platinum resistance. The relationship appeared to hold true for those cell lines derived from lung epithelial primary tumors but not for the neuroendocrine/small-cell and mesothelioma cell lines. siRNA knockouts to DDH-1 and DDH-2 were prepared with the cell line exhibiting the greatest resistance to cisplatin (A549) resulting in marked decreases in the DDH isoforms as assessed by real-time PCR, western blot and enzymatic activity. The DDH-1 knockout was far more sensitive to cisplatin than the DDH-2 knockout.

Conclusion: Thus, sensitivity to cisplatin appeared to be associated with DDH levels in epithelial lung cancer cell lines with the DDH-1 isoform producing the greatest effect. Results in keeping with transfection experiments with ovarian and other cell lines.

Fig. 1

Protein expression of AKT, HO-1, HO-2, 14-3-3zeta and GRP78 in six lung cancer cell lines; Lane 1 (A549), 2 (H460), 3 (H520), 4 (H23), 5 (H226) and 6 (DMS114), as analyzed by Western blot analysis. The protein extraction and separation was performed as described in the Material and Methods Section. Actin was used as an internal control to ensure equal loading of each lane

Fig. 2

(a) - Protein expression of DDH-1, DDH2, DDH-3 in six lung cancer cell lines; Lane 1 (A549), 2 (H460), 3 (H520), 4 (H23), 5 (H226) and 6 (DMS114) as visualized following western blot analysis. The origin of the antibodies, condition for protein separation and visualization of the bands was performed as described in the Material and Methods Section. Actin was used as an internal control to ensure equal loading of each lane (b) Densitometry of the western blots (2 gels in duplicate) of total DDH (■) and DDH-1 (□) in six lung cancer cell lines

Fig. 3

(a) - Semiquantitative RT-PCR of DDH-1, DDH-2, DDH-3 mRNA in the A549 cells (lane 1), the DDH-1 siRNA transfected cells A8n3 (lane 2) and the DDH-2 siRNA transfected cells A7nl (lane 3). GAPDH mRNA was run as an internal control (b) Protein expression of DDH-1, DDH-2 and DDH-3 as visualized by western blot analysis; A549 (lane 1), A8n3 (lane 2), and A7nl (lane 3). Actin is used as an internal control to assure equal loading in each lane (c) Densitometry of the western blots (2 gels in duplicate) of total DDH (■) and DDH-1 (□) in the A549, A8n3 and A7nl cells