Effects of the Combination of Gliotoxin and Adriamycin on the Adriamycin-Resistant Non-Small-Cell Lung Cancer A549 Cell Line

Abstract

Acquired drug resistance constitutes an enormous hurdle in cancer treatment, and the search for effective compounds against resistant cancer is still advancing. Marine organisms are a promising natural resource for the discovery and development of anticancer agents. In this study, we examined whether gliotoxin (GTX), a secondary metabolite isolated from marine-derived Aspergillus fumigatus, inhibits the growth of adriamycin (ADR)-resistant non-small-cell lung cancer (NSCLC) cell lines A549/ADR. We investigated the effects of GTX on A549/ADR cell viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the induction of apoptosis in A549/ADR cells treated with GTX via fluorescence-activated cell sorting analysis, Hoechst staining, annexin V/propidium iodide staining, tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, and western blotting. We found that GTX induced apoptosis in A549/ADR cells through the mitochondria-dependent pathway by disrupting mitochondrial membrane potential and activating p53, thereby increasing the expression levels of p21, p53 upregulated modulator of apoptosis (PUMA), Bax, cleaved poly (ADP-ribose) polymerase (PARP), and cleaved caspase-9. More importantly, we discovered that GTX works in conjunction with ADR to exert combinational effects on A549/ADR cells. In conclusion, our results suggest that GTX may have promising effects on ADR-resistant NSCLC cells by inducing mitochondria-dependent apoptosis and through the combined effects of sequential treatment with ADR.

Keywords: NSCLC; adriamycin resistance; apoptosis; gliotoxin.

Figure 1

Characterization of the adriamycin (ADR)-resistant A549 cell line. (a) The morphologies of A549 and A549/ADR cells were observed under a microscope; (b) Effects of ADR on A549 and A549/ADR cells for 48 h. Cell viability was determined by the MTT assay. The results of independent experiments were averaged and are presented as percentage cell viability. Values represent means ± standard deviation (SD) (n = 3) (* p < 0.05).

Figure 2

Gliotoxin (GTX) treatment reduces A549/ADR cell viability. (a) Chemical structure of GTX; (b) Effects of GTX on A549 and A549/ADR cells for 48 h. Cell viability was determined by the MTT assay. Results of independent experiments were averaged and are presented as percentage cell viability. Values represent means ± standard deviation (SD) (n = 3) (* p < 0.05).

Figure 3

GTX treatment induces apoptosis in A549/ADR cells. (a) Cell cycle analysis of A549/ADR cells treated with GTX. Cells were seeded in 60-mm dishes and treated with different concentrations of GTX (0, 0.0625, 0.125, 0.25, and 0.5 µM) for 24 h. Cells were then stained with propidium iodide (PI) solution and analyzed by flow cytometry; (b) Cells were treated with increasing doses of GTX. After 24 h, apoptotic cells were detected by staining with Hoechst 33342 and observed under a fluorescence microscope; (c) Annexin V/PI staining analysis by flow cytometry. After cells were treated with 0, 0.125, 0.25, and 0.5 µM GTX for 24 h, they were stained with PI and annexin V-fluorescein isothiocyanate (FITC) together with binding buffer for 15 min before analysis. Values represent means ± standard deviation (SD) (n = 3) (* p < 0.05).

Figure 4

GTX attenuates mitochondrial membrane potential in A549/ADR cells. Cells were treated with three concentrations of GTX for 24 h, stained with tetraethylbenzimidazolylcarbocyanine iodide (JC-1)-FITC together with binding buffer, and analyzed by flow cytometry. Values represent means ± SD (n = 3) (* p < 0.05).

Figure 5

GTX induces A549/ADR cell death via the mitochondria-dependent pathway. Cells were treated with 0, 0.125, 0.25, and 0.5 µM GTX for 24 h and cell lysates were subjected to western blotting for various apoptosis-related markers including p53, phosphorylated p53 (p-p53), p21, PUMA, Bax, PARP, cleaved PARP (c-PARP), cleaved caspase-9 (c-caspase-9), cleaved caspase-8 and Bid. β-actin was used as a loading control. The graphs are the densitometric quantification of the western blotting result (* p < 0.05).

Figure 6

Pre-treatment with GTX for 24 h potentiates the effects of ADR in A549/ADR cells. A549/ADR cells were pretreated with GTX for 24 h and subsequently treated with ADR. The effects of the combination treatment were investigated by MTT assay (a); cell cycle analysis (b); and western blotting (c), the bar graphs represent the densitometric quantification of the western blotting result (* p < 0.05). Columns with different letters are significantly different between samples of the same ADR dose treatment (p < 0.05).