Antitumor profiles and cardiac electrophysiological effects of aurora kinase inhibitor ZM447439

Abstract

Aurora kinases inhibitors, including ZM447439 (ZM), which suppress cell division, have attracted a great deal of attention as potential novel anti-cancer drugs. Several recent studies have confirmed the anti-cancer effects of ZM in various cancer cell lines. However, there have been no studies regarding the cardiac safety of this agent. We performed several cytotoxicity, invasion and migration assays to examine the anti-cancer effects of ZM. To evaluate the potential effects of ZM on cardiac repolarisation, whole-cell patch-clamp experiments were performed with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cells with heterogeneous cardiac ion channel expression. We also conducted a contractility assay with rat ventricular myocytes to determine the effects of ZM on myocardial contraction and/or relaxation. In tests to determine in vitro efficacy, ZM inhibited the proliferation of A549, H1299 (lung cancer), MCF-7 (breast cancer) and HepG2 (hepatoma) cell lines with IC₅₀ in the submicromolar range, and attenuated the invasive and metastatic capacity of A549 cells. In cardiac toxicity testing, ZM did not significantly affect I _Na, I _Ks or I _K1, but decreased I _hERG in a dose-dependent manner (IC₅₀: 6.53 µM). In action potential (AP) assay using hiPSC-CMs, ZM did not induce any changes in AP parameters up to 3 µM, but it at 10 µM induced prolongation of AP duration. In summary, ZM showed potent broad-spectrum anti-tumor activity, but relatively low levels of cardiac side effects compared to the effective doses to tumor. Therefore, ZM has a potential to be a candidate as an anti-cancer with low cardiac toxicity.

Keywords: Anticancer agents; Aurora kinases inhibitor; Cardiotoxicity; Efficacy testing; ZM447439.

Fig. 1. Cytotoxicity of ZM447439 (ZM) on human cancer cell lines and normal lung fibroblast.

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays were performed with various cancer cell lines—(A) A549 (non-small cell lung cancer), (B) MCF-7 (breast cancer), (C) NCI-H1299 (non-small cell lung cancer), (D) HepG2 (human hepatocellular carcinoma) and (E) HLF (human lung fibroblast) cells—after treatment with ZM at several concentrations (0.01, 0.03, 0.1, 1, 3, and 10 µM). After 24, 48 or 72 h of incubation, cell viability was examined by MTS assay. Data are presented as mean ± standard deviation (n = 3).

Fig. 2. Invasion assay on ZM447439 (ZM) treated A549 cells.

(A) Representative images of the invasiveness of A549 cells that migrated through transwell membranes. Cells were treated with 0.1, 1, and 10 µM ZM for 24, 48, and 72 h. (B) Quantification of the transwell assay by counting the number of invasive cells through the transwell membranes. Data are presented as mean ± standard deviation and compared by ANOVA followed by Dunnett's test (each n = 3). ^**p < 0.01; ^***p < 0.001, compared with vehicle control (VC, 0.1% dimethyl sulfoxide [DMSO] treated cells).

Fig. 3. Wound healing assay on ZM447439 (ZM) treated A549 cells

Wound healing assay was performed to assess the effect of ZM on the migration of A549 cells. (A) The representative images for the cells treated with 0.1% dimethyl sulfoxide (vehicle control, VC) or 10 µM ZM at 0 and 24 hours post-scratch are shown. (B) Quantification of the wound healing assay by migration area and % wound healing of the initial scratch area was compared. Data are presented as mean ± standard deviation and compared by t-test (each n = 3). ^***p < 0.001 compared with VC.

Fig. 4. Effects of ZM447439 (ZM) on action potential (AP) parameters of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

(A) Representative action potential traces of hiPSC-CMs in the absence (control) and presence of 0.3, 1, 3, and 10 µM ZM. (B) Normalized AP parameters of hiPSC-CMs in control (0 µM) and in the presence of 0.3, 1, 3, and 10 µM ZM. Data are expressed as mean ± standard error of the mean and compared by ANOVA followed by Dunnett's test. MDP, maximal diastolic potential; APA, action potential amplitude; dV/dt_max, maximum upstroke velocity; APD₅₀ or APD₉₀, action potential duration at 50% or 90% repolarization. ^**p < 0.01 compared to control (n = 3).

Fig. 5. Effects of ZM447439 (ZM) on cardiac ion channel currents.

(A) Representative traces demonstrating the effect of ZM on I_hERG at doses of 0.3, 1, 3, and 10 µM, respectively (left). Dose-response relationship of ZM showing an IC₅₀ value for I_hERG (mean ± standard error of the mean [SEM], n = 4) (right). (B) The representative I–V traces of I_hERG in control (left) and in the presence of 10 µM ZM (bottom). I–V relationships of I_hERG in the control and 10 µM ZM (mean ± SEM, n = 3) (right). (C–F) Representative traces demonstrating the effect of ZM on I_Ks (C), I_K1 (D), I_Na (E) and I_Ca (F) at doses of 10, 30, and 100 µM, respectively (each left) and dose-response relationship of ZM for each ion currents (mean ± SEM, n = 3) (each right).

Fig. 6. Effects of ZM447439 (ZM) on contraction of rat ventricular myocyte.

(A) Representative raw traces of sarcomere shortening and lengthening in the absence (control) and presence of 10 nM, 100 nM, and 1 µM nifedipine. (B) Typical raw traces of sarcomere shortening/lengthening in control and the presence of ZM at concentrations of 100 nM, 1 µM, and 10 µM.