Pixantrone, a new anticancer drug with the same old cardiac problems? An in vitro study with differentiated and non-differentiated H9c2 cells

Abstract

Pixantrone (PIX) is an anticancer drug approved for the treatment of multiple relapsed or refractory aggressive B-cell non-Hodgkin's lymphoma. It is an aza-anthracenedione synthesized to have the same anticancer activity as its predecessors, anthracyclines (e.g. doxorubicin) and anthracenediones (e.g. mitoxantrone), with lower cardiotoxicity. However, published data regarding its possible cardiotoxicity are scarce. Therefore, this work aimed to assess the potential cytotoxicity of PIX, at clinically relevant concentrations (0.1; 1; and 10 μM) in both non-differentiated and 7-day differentiated H9c2 cells. Cells were exposed to PIX for 48 h and cytotoxicity was evaluated through phase contrast microscopy, Hoescht staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and neutral red (NR) uptake assays. Cytotoxicity was observed in differentiated and non-differentiated H9c2 cells, with detached cells and round cells evidenced by phase contrast microscopy, mainly at the highest concentration tested (10 μM). In the Hoechst staining, PIX 10 μM showed a marked decrease in the number of cells when compared to control but with no signs of nuclear condensation. Furthermore, significant concentration-dependent mitochondrial dysfunction was observed through the MTT reduction assay. The NR assay showed similar results to those obtained in the MTT reduction assay in both differentiated and non-differentiated H9c2 cells. The differentiation state of the cells was not crucial to PIX effects, although PIX toxicity was slightly higher in differentiated H9c2 cells. To the best of our knowledge, this was the first in vitro study performed with PIX in H9c2 cells and it discloses worrying cytotoxicity at clinically relevant concentrations.

Keywords: H9c2 cells; Pixantrone; cardiotoxicity.

Figure 1

Chemical structure of DOX, MTX and PIX. DOX is composed by a tetracyclic quinone-hydroquinone chromophore, a carbonyl-containing side chain, and an aminosugar (daunosamine). MTX is a three-ring quinone-hydroquinone anthracenedione; its side chains lack carbonyl groups and MTX also lacks daunosamine. PIX differs from MTX in its lack of the hydroquinone, insertion of a nitrogen heteroatom in the same ring (black arrow), and substitution of (ethylamino)-diethylamino for (hydroxyethylamino)-ethylamino side chains (grey circles).

Figure 2

PIX causes cytotoxicity in non-differentiated H9c2 without signs of condensed nuclei. Phase contrast microscopy (A, B, C, D) and fluorescence microscopy (Hoechst 33258 staining) (E, F, G, H) images of non-differentiated H9c2 cells after a 48-h exposure to PBS (A and E), 0.1 μM PIX (B and F), 1 μM PIX (C and G) or 10 μM PIX (D and H). Images are representative of two independent experiments (scale bar 100 μm).

Figure 3

PIX leads to lysosomal and mitochondrial damage in non-differentiated H9c2. NR uptake (A) and MTT reduction (B) assays in non-differentiated H9c2 cells exposed to 0.1, 1 or 10 μM PIX for 48 h. Values are expressed as percentage of control and are shown as mean ± standard deviation. The results were obtained from 4–5 independent experiments using 20–24 wells. The statistical analyses were made using the Kruskal-Wallis test, followed by the Dunn’s post-hoc test (*p<0.05 and ****p<0.0001 versus control; ^####p<0.0001 versus PIX 0.1 μM; ⁺⁺p<0.01, ⁺⁺⁺p<0.001 versus PIX 1 μM).

Figure 4

PIX evokes cellular damage in a concentration dependent manner in differentiated H9c2 cells. Phase contrast microscopy (A, B, C, D) and fluorescence microscopy (Hoechst 33258 staining) (E, F, G, H) images of 7-day differentiated H9c2 cells after a 48-h exposure to PBS (A and E), 0.1 μM PIX (B and F), 1 μM PIX (C and G) or 10 μM PIX (D and H). Images are representative of two independent experiments (scale bar 100 μm).

Figure 5

PIX induces lysosomal and mitochondrial disruption in differentiated H9c2 cells. NR uptake (A) and MTT reduction (B) assays in differentiated H9c2 cells exposed to 0.1, 1 or 10 μM PIX for 48 h. Values are expressed as percentage of control and are shown as mean ± standard deviation. The results were obtained from 4 independent experiments using 20 wells. The statistical analyses were made using the Kruskal-Wallis test, followed by Dunn’s post-hoc test (A) and the ANOVA test, followed by the Tukey post hoc test (B) (*p<0.05 and ****p<0.0001 versus control; ^####p<0.0001 versus PIX 0.1 μM; ⁺p<0.05, ⁺⁺⁺⁺p<0.0001 versus PIX 1 μM).