Mechanisms Of Action Of Lidamycin Influenced By Cell Prosurvival Molecules

Lidamycin(LDM) as a member of the enediyne antibiotics family is developed independently by our institute,and it has been in phaseâ…¡clinical trial.Previous studies showed that high concentrations of LDM induced tumor cells to non-caspase dependent apoptosis,however,LDM at lower concentrations induced mitotic death with senescence-like phenotype(SLP).The mechanism of different responses to tumor cells for LDM is still unknown.Using different sensitive and drug-resistant tumor cells,we examine the effect mechanisms from cell survival molecules profile.Firstly,the results of Western blot,FACS,SA-β-gal staining and chromatin condensation experiment indicated that different concentrations of LDM could induce two modes of death characteristic in human hepatoma BEL-7402 cells.The cells treated with 10 nM LDM for 24h appeared classic apoptotic characteristics,such as obvious increase of p53 protein,cleavaged fragments of PARP and SIRT1 as well as sub-G₁ peak.However,the cells treated with 0.5 nM LDM could induce mitotic death with SLP. The cells were arrested in G₂/M phase firstly,then the percentage of the cells with senescence-associatedβ-galactosidase activities(72h) was increased obviously.At the same time,the expression of p53 protein was no obvious change,and the survival molecule SIRT1 also sustained its expression.In addition,we also detected the expression changes of other survival molecules by Western blot.The cells were treated with 10 nM LDM for 24h and collected at indicated time points.The results showed that the phosphorylation level of p38 protein was obviously increased,and the expression of FOXO3a,Bim was declined after LDM treatment.The results also indicated that the expression levels of MnSOD and Cu/ZnSOD was not increased.At 0.5 nM LDM treatment group,the expressions of FOXO3a and Bim were gradually increased from 4h to 72h after treatment.MnSOD and Cu/ZnSOD were transient increased within 4～8h and Akt was activated obviously until 48h.These studies indicated that survival molecules influenced the action of LDM and resulted in different cell death modes ultimately.The genome DNA cleavage after 1μM LDM treatment for 1h was observed in MCF-7 cells by chromatin condensation and DNA electrophoresis,but did not observed in doxorubicin-resistant MCF-7 cells.This phenomenon was related with rapidly decrease of PARP and P53 in MCF-7 cells,but not in MCF-7/DOX cells detected by Western blot.Moreover,it was not related with P-glucoprotein,glutathione and prosurvival molecules such as SIRT1,Akt.Furthermore,the caspase inhibitor did not inhibit this effect of LDM.The inhibition of proliferation in human p53 wild-type and p53 knocked out colorectal HCT116 cells treated with 10 nM LDM for 24h assayed by MTT showed that p53 wt HCT116 cells was more sensitive to LDM induced toxicity than p53 ko HCT116 cells.The mechanism within it might be related with SIRT1 and Akt.Furthermore, LDM could induce the cells appearing SLP,G₂/M arrest and chromatin condensation.The result of MTT between human liver normal L-02 cells and human hepatoma BEL-7402 cells treated with LDM showed that the toxicity to L-02 cells was lower than to BEL-7402 cells.The result of Western blot indicated that the expression level of PARP was no obvious difference between untreated L-02 cells and BEL-7402 cells.But, compared with L-02 cells,the expression levels of SiRT1 and P53 were higher and the level of Akt was lower in BEL-7402 cells.This study demonstrated initially that prosurvival molecules influenced the action of LDM in tumor cells and resulted in different modes of cell death by using various types of cells.And it is helpful to explain the mechanisms of LDM.