The Effects And Mechanisms Of Naphthalimide-polyamine Conjugates 11e And 6k On Hepatoma Carcinoma Cells

Polyamines(PA)are organic compounds,containing three or more than three free nitrogen atoms.Natural polyamines,small molecular aliphatic polyvalent cations,are amines widely existing in the cells,including putrescine(Put),spermidine(Spd)and spermine(Spm).Polyamines maintain normal cell metabolism by maintaining the structure of chromosomes,regulating the associated ion channels,maintaining the stability of the cell membrane and scavenging free radicals.The majority of tumor cells have a higher concentration of polymines than normal cells.Polyamines are involved in the regulation of growth signals,have the impact on ion channels of cell membrane,receptors,transcription and expression of nuclear genes in the nucleus,result in the proliferation and differentiation of abnormal cells,and promote tumorigenesis.So polyamine pathway has gradually become a hot spot of anti-tumor research in recent years.Naphthalimides have been developed rapidly in recent years as antitumor drugs,such as Amonafide(Amo).But they also exist many deficiencies,like the disadvantages of neurotoxicity and the poor tumor selectivity.Naphthalimides have cytotoxicity.The naphthalimide-polyamine conjugates are obtained through connecting the polyamines and naphthalimide compounds.The conjugates are identified and uptook into cells by Polyamine transporter(PAT).They reduce intracellular natural polyamines,and improve the targeted antitumor effects of naphthalimide.In this paper,based on the use of the reactive group of naphthalimide modified with polyamines,we designed the new naphthalimide-polyamine conjugates 11e and 6k.Through cellular models and tumor bearing animal models,we evaluated the antitumor effects and molecular mechanisms of 11e and 6k in vivo and in vitro.We hope to find more effective antitumor compounds.The present in vitro data of 11e demonstrated that 11e inhibited the growth of human hepatoma HepG2 and SMMC-7721 cells effectively in dose-dependent manners.The IC50(48 h)was 6.33±0.52 ?M and 11.12±0.83 ?M respectively.11e induced the tumor cells'apoptosis,changed the morphology of the cell,decreased mitochondrial membrane potential,increased ROS levels,and caused autophagy.In the study of apoptosis-related proteins,11e increased the expression of Caspase-3,Caspase-9,Cytochrome C,Bax and Bad,and decreased the expression of Bcl-2,IKKa and NF-?B.When being combined with DFMO(polyamine synthetase inhibitor)or Aspirin(Asp,polyamine metabolizing enzyme activator)individually,the tumor cells' growth inhibition,apoptosis induction and autophagy by 11e were enhanced.11e significantly inhibited the growth of solid tumors,pulmonary metastasis and improved the life prolonging ratio of H22 cells in vivo.But when being combined with DFMO or Asp respectively,these in vivo effects were reduced,which was conflicted with the in vitro results.These results may not be consistent with in vivo or in vitro tests,or may be less dependent on polyamines about the mechanisms of action.The present in vitro data of 6k demonstrated that 6k inhibited the growth of human hepatoma HepG2 and SMMC-7721 cells effectively in dose-dependent manners.The IC50(48 h)was 20.12± 1.37 ?M and 18.71 ± 1.05 ?M respectively.6k induced the tumor cells'apoptosis,changed the morphology of the cell,decreased mitochondrial membrane potential,increased ROS levels,and caused autophagy.In the study of apoptosis-related proteins,6k increased the expression of Caspase-3,Caspase-9,Cytochrome C,Bax and Bad,and decreased the expression of Bcl-2,IKKa and NF-?B.6k inhibited the proliferation and migration of SMMC-7721 cells,increased the expression of E-cadherin and ?-catenin,and decreased the expression of VEGF,Integrin a6,Cathepsin D.So 6k decreased the activity of SMMC-7721 cells.When being combined with DFMO or Asp individually,the tumor cells'growth inhibition,apoptosis induction and autophagy by 6k were enhanced.6k significantly inhibited the growth of solid tumors,pulmonary metastasis and improved the life prolonging ratio of H22 cells in vivo.When being combined with DFMO or Asp respectively,these effects were increased.The in vivo and in vitro test results of 6k were consistent.The roles of 6k are highly correlated with poly amine dependence.The results showed that the two compounds 11e and 6k both showed significant in vivo and in vitro antitumor activity.Combined with DFMO or Asp individually,the in vivo antitumor activity of 6k was enhanced compared with the single use of 6k.However,the in vivo antitumor activity of 11e was lower than the single use of 11e.These suggested that the mechanisms between the two may be different.The antitumor effect of 6k is polyamine dependent,whereas 11e is non-polyamine dependent.To sum up,11e and 6k induced apoptosis and autophagy of HepG2 cells and SMMC-7721 cells in vitro.Moreover,11e and 6k can effectively inhibit the proliferation and metastasis of H22 tumor cells and prolong the life span of mice in vivo.In conclusion,naphthalimide-polyamine conjugates have good prospects as antitumor drugs.