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.

Study question Do human spermatozoa express insulin and the enzymes responsible for the cleavage of proinsulin? Is the secretion of insulin by spermatozoa responsive to glucose stimuli? Summary answer Human spermatozoa express insulin and PC1/3 and PC2, enzymes responsible for the cleavage of proinsulin. Spermatozoa produce and release insulin in a glucose concentration-dependent manner. What is known already Spermatogenesis is dependent on a finely regulated hormonal and metabolic control. Insulin is considered one of the most important regulators in the metabolic regulation of spermatogenesis, acting directly on the differentiation of spermatogonia into primary spermatocytes or indirectly through the metabolic modulation of both Sertoli and Leydig cells. In addition, insulin is hypothesized to play a major role in human spermatozoa capacitation, although the mechanisms that regulate its production and secretion remain to be disclosed. Study design, size, duration The main objective of this study was to estimate the capacity of insulin production by human spermatozoa and if they express PC1/3 and PC2, which are enzymes responsible for the cleavage of proinsulin. In addition, we hypothesized that insulin production could respond to glucose stimuli. Seminal samples of normozoospermic men (n = 15) seeking fertility treatment were collected for these experiments. Participants/materials, setting, methods Spermatozoa were submitted to a density gradient centrifugation and two fractions were collected according to their motility (high vs low motility). The expression of insulin, PC1/3 and PC2 mRNA was evaluated by RT-qPCR. The protein expression of insulin, PC1/3 and PC2 was evaluated by immunofluorescence. High motility spermatozoa were incubated in BWW medium with increasing glucose concentrations (0, 5.5, 11, and 22 mM). After 6 h, the concentration of secreted insulin was quantified by ELISA. Main results and the role of chance We were able to identify the expression of insulin, PC1/3, and PC2 mRNA, as well as the respective proteins, in human spermatozoa. Through immunofluorescence, we observed that insulin and both enzymes are mainly expressed in the midpiece. Human spermatozoa also express insulin receptor, mainly in the midpiece and tail. The mRNA expression of insulin and PC1/3 was found to be higher in the highly motile spermatozoa. Human spermatozoa produce very small quantities of insulin (pg/mL range), which suggests an autocrine or paracrine action. The release of insulin to the medium was found to occur in a glucose concentration-dependent manner. Limitations, reasons for caution This is an in vitro study with relatively small sample size. Future studies are required to further evaluate the role of insulin and its signalling pathway on human spermatozoa capacitation and fertilization capacity. Wider implications of the findings This study shows that human spermatozoa express PC1/3 and PC2, enzymes responsible for the cleavage of proinsulin. In addition, human spermatozoa release insulin in a glucose concentration-dependent manner. The study of insulin signalling opens the path for a deeper understanding of human spermatozoa bioenergetics and the development of novel therapies. Trial registration number Not applicable