Digitoxin and its synthetic analog MonoD have potent antiproliferative effects on lung cancer cells and potentiate the effects of hydroxyurea and paclitaxel

Abstract

Despite significant advances in the understanding of lung cancer biology, the prognosis of cancer patients remains poor. Part of the failure of anticancer therapy is due to intratumoral heterogeneity in these patients that limits the efficacy of single agents. Therefore, there is an urgent need for new anticancer drugs or drug combination regimens that possess increased activity against all cellular subtypes found within the tumor. In this study, we evaluated the in vitro antiproliferative activity of the cardiac glycosides (CGs) digitoxin and its synthetic analog MonoD on H460 lung cancer cells grown under different culture conditions. The CGs were tested alone in H460 cells under routine culture as well as in cells growing under short (24-72 h) and prolonged serum starvation (7 days) in order to evaluate the activity of drugs on cancer cells under varied degrees of proliferation. Our results showed that both CGs, and MonoD in particular, have potent antiproliferative activity at clinically relevant concentrations against cells in all the tested culture conditions. In contrast, paclitaxel, hydroxyurea and colchicine were only active in cells growing in routine culture conditions, and relatively inactive in serum-starved conditions. Importantly, both CGs were able to potentiate the effect of clinically relevant concentrations of hydroxyurea or paclitaxel in serum-starved conditions. When paclitaxel was used in combination with CGs, the highest antiproliferative effect was obtained when paclitaxel was administered first, followed by either digitoxin or MonoD. Our results indicate that CGs have potential clinical applications in translational oncology especially in combination with other drugs, and warrants further investigation of CGs in more advanced preclinical models of lung cancer.

Figure 1

Serum potentiates the antiproliferative effect of digitoxin and MonoD. H460 cells were incubated in serum-free media or media + 5% FbS with 0, 1, 5, 10, 25, 50, 100 or 200 nM digitoxin or MonoD for 24, 48 for 72 h. Cell viability was assessed by the MTT assay after drug exposure for 24, 48 or 72 h. The table show the IC₅₀ ± ES for each drug. Results are representative of two experiments performed by quadruplicates.

Figure 2

Short periods of serum starvation slightly attenuates the antiproliferative effect of digitoxin and MonoD. H460 cells were serum starved overnight and then incubated in serum-free media or media + 5% FbS with 0, 1, 5, 10, 25, 50, 100 or 200 nM Digitoxin or MonoD for 24, 48 for 72 h. Cell viability was assessed by the MTT assay after drug exposure for 24, 48 or 72 h. The table shows the IC₅₀ ± ES for each drug. Results are representative of two experiments performed by quadruplicates.

Figure 3

Effect of digitoxin and MonoD in combination with hydroxyurea. (A) Concomitant treatment with Dig 20 nM or MonoD 20 nM increases the antiproliferative effect of HU. (B) Dig and MonoD exerts antiproliferative effect on cells that survived treatment with HU 2 mM for 24 h with similar potency compared to HU-untreated cells.

Figure 4

Prolonged periods of serum starvation slightly attenuates the antiproliferative effect of digitoxin and MonoD but markedly affect paclitaxel, colchicine and hydroxyurea sensitivity. (A) effect of PX and colchicine on H460 cells growing in CM. Cells were treated with drugs in CM. (B) effect of PX, colchicine, HU, Dig and MonoD on seven-day serum-starved H460 cells. Cells were treated with drugs in SFM. PX, paclitaxel; HU, hydroxyurea; Dig, digitoxin; SFM, serum-free media.

Figure 5

Effect of digitoxin and MonoD in combination with paclitaxel on H460 cancer cells. (A) Concentration-dependent effect of PX alone or in the presence of Dig (20 nM) or MonoD (20 nM). Cell viability was measured by the MTT assay after treatment for 48 or 72 h. (B) Left panel, representative digital images showing colonies produced by H460 cells following plating of 200 cells. Cells were treated with Dig (20 nM), MonoD (20 nM) or PX (10 nM) alone or in combination (PX 10 nM) + Dig (20 nM) or MonoD (20 nM) for 72 h in and then allowed to form colonies for 10 days. C, Control cells were treated with DMSO alone. Right, quantification of the colony forming assay. ^**P<0.05, significantly different between PX 10 nM vs. the combined treatments. PX, paclitaxel; Dig, digitoxin.

Figure 6

Sequential treatment: single drug → co-treatment (A and b) or single drug → single drug (C and D). Cells were incubated for 24 h with drugs alone at the indicated concentration for 24 h followed by incubation with the indicated combination or drug alone for 48 h. The symbol '–>' indicates the sequence in which the drugs were added after 24 h. Control cells (DMSO) were incubated with DMSO that was also included at equivalent concentrations for all treatments. *P<0.001 and ^**P<0.05.