| Chemotherapy is one of the important developing strategies in cancer therapy. The major shortcoming of current cancer chemotherapies of many anti-cancer drugs is poor selectivity between tumor cells and healthy cells. Conjugating an anti-tumor agent with a polyamine motif elevates their affinity for tumor cells, and expresses excellent selectivity between tumor cells and normal cells. Naphthalimides and their derivates exhibit considerable potential as DNA intercalating anti-tumor compounds. Conjugating naphthalimides with a polyamine motif enhances their aqueous solubility and anti-tumor effects. Naphthalimide-polyamine conjugates are capable of specific entry into tumor cells. Then, developing naphthalimide- polyamine conjugates having the potential of transporting naphthalimide selectively into tumor cells is attractive in improving chemotherapeutic efficacy. However, the evaluation of their cytotoxic mechanism has not been comprehensive, and exact cytotoxic mechanism of these conjugates is not well known.This study focused on the effects of mononaphthalimide spermidine (MNISpd) conjugate on recognization by polyamine transport (PAT), selectivity between tumor cells and healthy cells, growth and survival, and the relationship between MNISpd-induced anti-tumor effects and reactive oxygen species (ROS) in HeLa cells. Our results determined that after 48 h, an IC50 of 4.71±0.21 and 16.68±0.18 was observed for HeLa cells and HELF cells respectively, and MNISpd exerted a higher cytotoxicity towards HeLa cells but with less cytotoxicity on HELF cells. An IC50 of 7.37±0.16 and 3.47±0.05 was observed for HeLa cells treated with MNISpd plus SPD and MNISpd plus DFMO for 48 h respectively, indicating a protection effects for SPD in MNISpd plus SPD treatment HeLa cells and a synergism effects for DFMO in MNISpd plus DFMO treatment HeLa cells. In characterizing the mechanism of MNISpd cytotoxicity in HeLa cells, inhibition of proliferation was observed when HeLa cells were treated with MNISpd < 6μM and MNISpd induced a cell cycle arrest in S phase correlated with enhanced p21 expression and decreased Cdc2 but not CDK2 expression in this range. There were evidences of apoptosis in MNISpd≥6μM treatments HeLa cells, and 9μM MNISpd was suitable to induce apoptosis during a 48-h period. Our findings further determined that MNISpd-induced apoptosis was correlated with decreased XIAP expression and a loss of mitochondrial membrane potential following cytochrome c release, elevation of caspase-3, -9 activity, apoptosis inducing factor (AIF) translocation and up-/down-regulation of Bax/Bcl-2 protein expression, but not caspase-8, and these effects were completely antagonized by pre-incubation with 10 mM NAC for 2 h. MNISpd induced a significant ROS accumulation following up-regulation in polyamine oxidase (PAO) activity and complex variations in glutathione levels. Based on all these evidences, it is proposed: (1) MNISpd could be recognized by PAT and transfer naphthalimide entry into HeLa cells via PAT, (2) MNISpd expressed a targeting effect to HeLa cells, (3) MNISpd-mediated cytotoxicity towards HeLa cells primarily results from apoptosis induction and proliferation inhibition dependent on variations: with MNISpd < 6μM treatment, effects on cell proliferation predominate, while MNISpd≥9μM, apoptosis develops, (4) MNISpd-induced apoptosis via both intrinsic caspase-dependent and AIF-mediated caspase independent apoptosis pathways, but not extrinsic caspase-dependent pathway in HeLa cells is related to oxidative stress, (5) MNISpd-induced ROS production results from GSH (reduced form of glutathione) pool depletion, and PAO is likely to be the source of ROS, (6) MNISpd- induced apoptosis susceptible to be inhibited with NAC treatment but not cell cycle arrest in HeLa cells.In addition, MNISpd-mediated cytotoxicity towards k562 cells was related to apoptosis induction and proliferation arrest.It is concluded that MNISpd could act as potential tumor targeting agent for further development.
|
PDF Full Text Request