Anticancer properties of novel aminoacetonitrile derivative monepantel (ADD 1566) in pre-clinical models of human ovarian cancer

Abstract

Monepantel (MPL) is a new anthelmintic agent approved for the treatment of nematode infections in farm animals. As a nematicide, it acts through a nematode-specific nicotinic receptor subtype which explains its exceptional safety in rodents and mammals. In the present study, we evaluated its potential as an anticancer agent. In vitro treatment of epithelial ovarian cancer cells with MPL resulted in reduced cell viability, inhibition of cell proliferation and suppression of colony formation. Proliferation of human ovarian surface epithelial cells and other non-malignant cells were however minimally affected. MPL-induced inhibition was found to be independent of the acetylcholine nicotinic receptor (nAChR) indicating that, its target in cancer cells is probably different from that in nematodes. Analysis of MPL treated cells by flow cytometry revealed G1 phase cell cycle arrest. Accordingly, MPL treated cells expressed reduced levels of cyclins D1 and A whereas cyclin E2 expression was enhanced. Consistent with a G1 phase arrest, cellular levels of cyclin dependent kinases (CDKs) 2 and 4 were lower, whereas expression of CDK inhibitor p27(kip) was increased. In cells expressing the wild-type p53, MPL treatment led to increased p53 expression. In line with these results, MPL suppressed cellular thymidine incorporation thus impairing DNA synthesis and inducing cleavage of poly (ADP-ribose) polymerase (PARP-1). Combined these pre-clinical findings reveal for the first time the anticancer potential of monepantel.

Keywords: Monepantel; PARP-1; cell cycle; cyclins; ovarian cancer.

Figure 1

Chemical structure of monepantel (MPL).

Figure 2

MPL inhibits growth of epithelial ovarian cancer cells. Human ovarian cancer cell lines OVCAR-3 and A2780 were grown in 6 well tissue culture plates under standard cell culture conditions in the presence of MPL (0, 5, 10, 25 μmol/L) for 72 h. Cells were then stained with Giemsa, washed and photographed (40 × magnification; using Leica DM IRB light microscope). Experiment repeated twice with the same result.

Figure 3

Effect of MPL on cell viability is cell specific. Effect of MPL on cell viability was determined using trypan blue dye exclusion assay. Treatment of ovarian cancer cells (top panel) with MPL (0, 5, 10, 25 μmol/L) for 72 h, reduced cell viability and induced cell-death in a concentration-dependent manner. Normal (non-malignant) cells exposed to the same concentrations of MPL over the same period of time were far less susceptible (bottom panel). Effect of MPL on the viability of human ovarian surface epithelial (HOSE) cells was minimal (p < 0.001 at all concentrations compared to OVCAR-3 cells). Each concentration was tested in replications of 8 and each experiment was repeated twice. Data represent mean ± SEM from two independent experiments combined. Population of live and dead cells at the end of treatment period are presented as percentage control (mean ± SEM).

Figure 4

MPL suppresses proliferation of ovarian cancer cells. The impact of MPL (0, 5, 10, 25, 50 and 100 μmol/L) on cell proliferation was assessed using the SRB assay. Control (vehicle treated) cells were taken to present 100% proliferation and values for the MPL treated groups are expressed as percentage of control (mean ± SEM). Each concentration was tested in replications of 8 and each experiment was repeated twice. Each drug concentration was tested in quadruplicate and each experiment was repeated at least twice. For statistical comparisons, each drug treated group was compared with the control group using Student’s t-test. Then, to confirm concentration dependency of the drug effect, ANOVA was used to compare the between group values.

Figure 5

MPL effects on the colony formation activity of ovarian cancer cells. Following incubation of OVCAR-3 and A2780 cells with MPL (0, 5, 10, 25 μmol/L) for 72 h, cells were washed and then transferred to agar plates, cultured with RPMI growth medium (drug free) and incubated under standard conditions for 14 days. Cells were then fixed with 100% methanol and stained with 0.1% crystal violet. Colonies (Cluster of cells greater than 50) were counted manually. Number of colonies counted for different experimental groups (MPL treated) is expressed as percentage of the control.* = p < 0.05; ** = p < 0.01 and ***= p < 0.001 as compared to control (vehicle treated) group using Student t-test.

Figure 6

MPL-antiproliferative effect is not mediated through acetylcholine nicotinic receptor. Pre-treatment (30 min) of OVCAR-3 cells with nicotinic agonist (nicotine, carbachol) or nicotinic antagonists (atropine, mecamylamine, tubocurarine or α-Bungarotoxin) at the indicated concentrations did not change the a ntiproliferative effects of MPL under the cell culture conditions. Proliferation was assessed using SRB assay. Each concentration was tested in replicates of 8 and each experiment was repeated twice. Data represent mean ± SEM from two independent experiments combined.

Figure 7

MPL effects on cell cycle progression of human ovarian cancer cells. OVCAR-3 and A2780 cells treated with either MPL or the vehicle (control group) for 48 h were harvested, washed, digested and stained with propidium iodide and analysed by flow cytometry. Distribution of cells in the various phases of the cell cycle (G1, S and G2/M) are presented as percentage, with values (mean ± SEM) representing mean of two independent experiments.

Figure 8

MPL modulates expression of the G1 cell cycle regulatory proteins. A. Western blot of lysates prepared from cells treated with MPL (0, 5, 10, 25 μmol/L for 48 h) were analysed for the expression of cyclin D1, CDK4, cyclin E2, cyclin A, CDK2, p27^kip and p53 proteins. The house-keeping gene (GAPDH) was used to confirm similar protein loading and blot transfer. B. Thymidine incorporation in MPL (0, 5, 10, 25, 50 and 100 μmol/L) treated cells. The y axis presents the actual counts per minute (CPM). These values are mean ± SEM) of two independent experiments. C. Immunoblot analysis for the detection of PARP-1 and cleaved PARP-1 in cells treated for the indicated period of time (24-72 h) with MPL (0, 5, 10, 25 μmol/L).