Increased sensitivity to cisplatin in non-small cell lung cancer cell lines after FHIT gene transfer

Abstract

To evaluate the relevance of fragile histidine triad (FHIT) status in relation to drug treatment, we analyzed the sensitivity of the Fhit-negative non-small cell lung cancer (NSCLC) cell line NCI-H460 to different drugs, after treatment with an adenoviral vector expressing the FHIT transgene. Expression of Fhit resulted in reduced sensitivity to etoposide, doxorubicin, and topotecan. This feature was associated with Fhit-induced downregulation of DNA topoisomerases I and II. In contrast, expression of Fhit did not modulate sensitivity to Taxol, but produced a slight increase in sensitivity to cisplatin, as shown by colony-forming assays. Analysis of apoptosis revealed that, after cisplatin exposure, the number of apoptotic cells was two-fold higher in Fhit-expressing H460 cells. Moreover, it appeared that wildtype p53 was required for sensitization to cisplatin because the effect was marginal in A549 and Calu-1 cells, where the p53 pathway is altered and simultaneous restoration of p53 and Fhit in Calu-1 cells increased cisplatin sensitivity. Fhit could also partially restore sensitivity to cisplatin in Bcl-2- and Bcl-x(L)-overexpressing H460 cells that are normally resistant to this drug. Our results support the possible relevance of FHIT in cisplatin-based chemotherapy as well as in the reversal of drug resistance in NSCLC.

Figure 1

Western blot analysis of Fhit expression in different cell systems. (A) Analysis of Fhit expression in Ad-Fhit-transduced NSCLC cells. H460 cells were infected at an MOI = 5 for Ad-Fhit, and Fhit expression was analyzed at different time points. The Fhit protein is already detectable at 24 hours, increases at 36 hours, and then remains stable. (B) Levels of expression of the Fhit protein in H460 clones stably transfected with the antiapoptotic proteins Bcl-2 and Bcl-x_L.

Figure 2

Effect of Fhit expression on the sensitivity of H460 cell line to different antitumor drugs. Cell sensitivity was assessed by SRB assay after a 48-hour drug exposure. H460 cells were infected with Ad-Fhit and Ad-LacZ as controls at an MOI = 5 and were harvested 48 hours later. Cells were then seeded and exposed to drugs at indicated doses for 2 days. IC₅₀ = concentration of a drug required to reduce absorbance by 50% at 550 nm.

Figure 3

Western blot analysis of topoisomerase I and IIα expression. H460 cells were infected with an MOI = 5 of Ad-Fhit and Ad-LacZ, or treated with medium. The expression of Fhit was associated with a decrease in topoisomerase protein expression levels, which was more evident in topoisomerase IIα. Vinculin was used as a control.

Figure 4

Antiproliferative effect of Ad-Fhit and cisplatin treatment. (A) Clonogenic survival of cisplatin-treated H460 cell line. H460 cells were infected with Ad-LacZ or Ad-Fhit for 24 hours and then treated with cisplatin for 1 hour before performing colony-forming assay. (B and C) Modulation of p21^Waf1 expression. H460 cells were infected with Ad-Fhit and Ad-LacZ for 24 hours and treated with cisplatin. One hour later, the drug was removed; after 6 and 20 hours, p21^Waf1 mRNA levels were assayed by real-time PCR (B). p21^Waf1 protein expression was analyzed by Western blot analysis (C).

Figure 5

Apoptosis analysis. H460 cells were transduced with Ad-Fhit and Ad-LacZ at an MOI = 5, or treated with medium for 24 hours and then exposed to 10 µM cisplatin for 24 and 48 hours. Apoptosis was determined by TUNEL assay. The experiment is representative of assays, performed at least three times, that gave comparable results.

Figure 6

Effect of combination treatment with cisplatin and Ad-Fhit on Bcl-2-and Bcl-x_L-overexpressing clones. (A) Clonogenic survival. Cells were infected with Ad-Fhit and Ad-LacZ for 24 hours at an MOI = 5 and were then exposed to cisplatin for 1 hour before clonogenic survival analysis. (B) Apoptosis analysis. Cells were transduced with Ad-Fhit and Ad-LacZ for 24 hours at an MOI = 5 and then treated with 16 µM cisplatin for 24 hours. The presence of apoptotic cells was assessed by TUNEL assay, and the experiment is representative of assays, performed at least three times, that gave comparable results.

Figure 7

Sensitivity of A549 and Calu-1 cell lines to a combination treatment of cisplatin and Ad-Fhit. (A) Clonogenic survival in A549 cell line. A549 cells were infected with Ad-Fhit and Ad-LacZ at an MOI = 5 for 24 hours and then treated with different doses of cisplatin for 24 hours. (B) Apoptosis analysis in A549 and Calu-1 cell lines. Cells were infected with Ad-Fhit and Ad-LacZ or treated with medium as control for 24 hours and then treated with 32 and 64 µM cisplatin, respectively, for 24 hours. Results are expressed as percentages of apoptotic cells relative to medium-treated cells and represent the mean of three different independent analyses. (C) Calu-1 cells expressing inducible Fhit, p53, or both were incubated in the absence or in the presence of Pon A to induce transgene expression. After 24 hours, the cells were treated with 64 µM cisplatin for 24 hours, and the presence of apoptotic cells was assessed by TUNEL assay. The results are expressed as percentages of apoptotic cells relative to controls, represented by uninduced cells.