Levofluoroquinolone-Chalconederivative Induces Apoptosis And Autophagy Of Human Hepatocarcinoma SMMC-7721cells

Objective The study aims to observe the process of cell apoptosis and autophagy and to investigate the relation and mechanism of them two by utilizing SMMC-7721 cells treated with different concentrations of Levofloxacin aldehyde condensation of methoxyl acetophenone.Methods With the different concentrations of the HGQ5 treated SMMC-7721 cells, The inhibitory effect of HGQ5 on the proliferation of SMMC-7721 cells was analyzed with the method of MTT assay.Test the effects of chloroquine(CQ) inhibition on the growth of SMMC-7721 cells at the time of 12 h and48 h. Also Tests the effects of HGQ5 and HGQ5 unite chloroquine(CQ) on cells SMMC-7721 cells at 48h;Cell apoptosis was observed by DAPI staining and FCM(flow cytometry);The apoptosis rate of SMMC-7721 cell induced by HGQ5 and HGQ5 unite chloroquine were calculated by TUNEL(terminal dexynucleotidyl transferase(Td T)-mediated d UTP nick end labeling) assay;Autophagy effector protein LC3 and autophagy substrate p62 were immunostained. The ultrastructure of autophagosome was observed under the transmission electron microscope. Expressions of LC3 and p62 were assayed respectively with Western blotting.With a combined autophagy inhibitor CQ and HGQ5 to explores the expression of autophagy related protein p62, LC3 ?/LC3 ? expression of the ratio of volume and change.Results Comapred with the control after 24 h,HGQ5 at the concentration of 1.25?mol/L,2.5?mol/L,5.0?mol/L and 10?mol/L respectively reduced the proliferation of SMMC-7721 cells to 30.06%(P<0.05),39.12%(P<0.01),51.71%(P<0.05)and 65.22%(P<0.01),The IC50 values of HGQ5 for 24 h was 5.0?mol/L dependent manners, with an IC50 value of 4.5 ?mol/L after 48 hours,with an IC50 value of4.48?mol/L after 72 hours.That HGQ5 significantly inhibited the proliferation of cells in dose-dependent manner was revealed by MTT assay. There was no statistically significant of chloroquine treated cells 12 hand 48h in the concentration of 6.25?mol/L and 0?mol/L(P<0.05). HGQ5 unite chloroquine treatment SMMC-7721 cells after 48 h,at alow doses(0.3125 to 2.5 ?mol/L) the cell growth inhibition rate is higher than single HGQ5 group, The situation is the opposite at high doses(5.0-10 ?mol/L).DAPI fluorescent staining was used to observe the morphological changes of the nucleus in the apoptosis cell. The results show that the apoptosis cells were significantly increased in the treatment group. The characteristic changes of apoptosis were observed such as the side accumulation of the cell nucleus, chromatin condensing of the cells and the cell nucleus cracking.The TUNEL detection result shown that increased with the concentration of HGQ5,the ratio of apoptosic cells also increased obviously.which were in dose-dependent manner,The difference between controls and HGQ5 treated groups were statistically significant(p<0.05); The TUNEL result shown that the ratio of apoptosic cells of HGQ5 unite chloroquine was the same as MTT.This situation explains that HGQ5 induces apoptosis was associated with autophagy.Immunostaining has indicated that LC3 was expression in SMMC-7721 cellular cytosol. The expressions of LC3 and p62 were analyzed with Western blotting assay. Compared with the group in control, the expressions of LC3-II and p62 respectively reached the peak at 6h,12 h,24h and 3h,6h,12 h after the SMMC-7721 cells were treated with 5.0?mol/L at the different time, statistically significant compared with control group. There was a dose-dependent manner that HGQ5 treat the SMMC-7721 cells in different time and concentrations. When HGQ5 unite chloroquine treatment SMMC-7721 cells 12 h and48 h,the expression of p62 protein at 48 h is higher than 12 h(p<0.05).Conclusion HGQ5 could dose-dependently inhibit cell proliferation and autophagy is involved in this HGQ5-inuced injury on SMMC-7721 cells.HGQ5 induces apoptosis was associated with autophagy.