Anti-tumor Effects And Its Mechanisms Of ANTMHspd, A Novel Homospermidine Conjugate

The natural polyamines, putrescine (PUT), spermidine (SPD) and spermine (SPM), play an essential role in cellular growth and differentiation. The elevated requirement of tumor cells for polyamines, which can be realized by internal biosynthesis and/or external uptake, makes the polyamine pathway attractive for tumor targeted chemotherapy. Previous efforts revealed that the non-native triamine, homospermidine, showed a better polyamine transporter (PAT) recognization than natural polyamines. A leading conjugate, anthracenylmethyl homospermidine (ANTMHspd), was found to display an excellent PAT selectivity, exert cytotoxicity on several tumor cell lines. In this study, the anti-tumor potency of ANTMHspd was investigated in many tumor cell lines. We also investigated the effects of ANTMHspd on the redox status, differentiation and the possible apoptotic mechanism in tumor cells. Moreover, we also study the anti-tumor effects of ANTMHspd in graft-tumor model in C57BL/6 and KM mice.The present data demonstrated that ANTMHspd inhibited growth effectively in B16 cells, BEL-7402 cells, SMMC-7721 cells, HepG2 cells, K562 cells, HL 60cells, U937 cells and Hela cells in a dose- and time-dependent manner. The IC₅₀(48 h) is 0.95±0.13μM, 0.56±0.08μM, 1.76±0.09μM, 1.69±0.12μM, 0.95±0.13μM, 0.97±0.05μM, 1.61±0.15μM and 1.54±0.04μM, respectively. Moreover, DFMO could enhance the growth inhibition activity in HL60 cells.ANTMHspd induced lipid peroxidation of tumor cells significantly, shown by an increase in the level of malondialdehyde (MDA) and PAO activity. ANTMHspd also induced a significant decrease in the content of intracellular glutathione (GSH), concomitantly an obvious increase in the production of intracellular reactive oxygen species (ROS) in B16 cells, BEL-7402 cell and K562 cells. ANTMHspd also induced decreases in the activities of intracellular antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). These results indicated that ANTMHspd could induce significant intracellular oxidative stress. Hoechst 33258 staining and the flow cytometry assay showed that ANTMHspd induced cell apoptosis and cell cycle perturbation. Furthermore, ANTMHspd could induce mitochondrial membrane potential loss and cytochrome c release, and enhance cleaved caspase-3, cleaved caspase-9 and Bax protein expression without caspase-8 activation. ANTMHspd could also decrease the expression of Bcl-2 and cytochrome c in mitochondria. In addition, the specific inhibitors of caspase-9 and caspase-3 almost abolished the ANTMHspd-induced caspase-9 and caspase-3 activation, respectively. In conclusion, ANTMHspd could induce many tumor cells apoptosis via the mitochondrial/caspase-dependent pathway and the Bcl-2 family was involved in the control of apoptosis.Our studies also demonstrated that ANTMHspd could induce B16 cells, K562 cells and HL60 cells differentiation. ANTMHspd induced B16 cells differentiation via enhancement of melanogenesis and increase the activity of tyrosinase at lower doses (<0.1μM) . Moreover, ANTMHspd also induced K562 cells and HL60 cells toward erythroid and neutrophils differentiation, respectively.We found that ANTMHspd was highly anti-tumor active against i.p. and sc inoculated sarcoma 180 tumors in mice when administered by the i.p for consecutive 8 days in vivo. The results demonstrated that ANTMHspd inhibited S180 tumor growth and enhanced survival time of the mice which inoculated sarcoma 180 ascitic tumor. Furthermore, ANTMHspd also inhibited B16 and Lewis tumor growth, and interrupted lung invasion and metastasis of B16 and Lewis tumor in C57BL/6 mice.In conclusion, ANTMHspd showed significant anti-tumor activity in vitro and in vivo.