Trimethory-Benzaldehyde Ciprofloxacin Schiff Base Induces Apoptosis Of Human Hepatocarcinoma Cells

ObjectiveTo screened12novel antibacterial fluoroquinolone derivative,select a compounds which have highinhibiting activity to proliferation against human hepatocarcinoma SMMC-7721cells, hrough theidentification of the induced tumor cell apoptosis effect and studied the mechanism of the compoundsinduction of cell apoptosis in order to provide references for the development of new anticancer drugs.MethodsWe used MTT assay to detect the the inhibition effect on the proliferation of the humanhepatocarcinoma cells （SMMC-7721） with Quinolone derivatives and calculate50%inhibitoryconcentration. then choice M27of proliferation inhibition of significant compound for hepatocarcinomacell, detection the inhibition effect of M27on human colon adenocarcinoma cells（HCT–16）and leukemiacell line JURKET，and ciprofloxacin its precursor compound on SMMC-7721cell. Then protracted theproliferation curve with drug concentrations changes; Apoptosis morphological changes and the apoptosisrate of cell were determined using Hoechst33258fluorescence staining and TUNEL assay. Mitochondrialmembrane potenitial（△ψ m）was measured using a high content screening imaging system. Change ofProteins expression correlated apoptosis,such as p53、Caspase-9、Caspase-3、Caspase-8、Bcl-2、Bax andcytochrome c was detedted with western blotting analysis.ResultsThe results of drug screening indicated that12quinolone derivatives have varying degrees ofinhibiting activity on the proliferation cells, such as M25、M26、M27、M28、M32、M36have highinhibiting activity at20μmol·L^-160μmol·L^-1concentration on the proliferation cells，and the cellpro-liferation was potently inhibited by M27,{trimethory-benzaldehyde ciprofloxacin schiffbase,1-cyclopropyl-6-fluoro-7-（4-H-piperazin-1-yl）-3-[S-（3,4,5-3-benzyl-sulfanyl）-4-（3,4,5-trimethoxy-benzylidene-amino）-（1,2,4-triazol-3yl）]-1H-quinolin-4-ketone}at10μmol·L^-160μmol·L^-1in dose and time dependentmanners. The approximate the concentration of50%growth inhibition (IC₅₀) values of M27after treatment for24h、48h、72h were39.97μmol·L^-1、26.67μmol·L^-1、23.04μmol·L^-1, respectively;Treatment with M27inhibited proliferation of the HCT-116and JURKET cells in dose and time dependent manners. ForHCT-116cells,the IC₅₀values after treatment for24h、48、72h were46.52μmol·L^-1、39.57μmol·L^-1、16.13μmol·L^-1, respectively. For JURKET cells,the IC₅₀values after treatment for24h、48h、72h were44.33μmol·L^-1、30.89μmol·L^-1、21.91μmol·L^-1,respectively. Ciprofloxacin showed weak cytotoxicityagainst SMMC-7721cell. The IC₅₀values at24h、48h、72h were180.23μmol·L^-1、152.34μmol·L^-1、140.90μmol·L^-1. SMMC-7721cells Treament with M27(0，21.68，37.97，47.73μmol·L^-1) for24h,typicalty morphological features of apoptosis, such as chromatin condensation and nucleic fragmentationwere displayed following Hoechst33258staining; The TUNEL result shown that M27dose-dependentlyincrease the percentage of apoptotic cells, percentage of apoptosic significantly higher than control group（P<0.05）. Cell mitochondrial membrane potential（△ψ m）was decreased（11.12±8.80）%,（46.52±18.74）%,（63.05±22.84）%, respectively.The difference compared to the control were statistically significant（P<0.05）. The result of Western-blot test indicated that the protein expression of p53、Caspase-8、Caspase-9and Caspase-3was increased in M27treated groups compared to the controls, and cleave activefragment of Caspase-9、 Caspase-8and Caspase-3was also increased in SMMC-7721cells; theanti-apoptotic protein Bcl-2expression was decreased, while the pro-apoptotic peotein Bax wasincreased.M27significantly increase cytochrome c contents in the cytosolic, and a decrease cytochrome cin the mitochondrial compartment.Conclusions1. M27dispaly high inhibitory effec of human hepatocarcinoma SMMC-7721proliferation, indose and time dependent manners.2. M27can induce apoptosis of SMMC-7721cells significantly.3. M27induce apoptosis of SMMC-7721cells predominantly through mitochondrial-depe-ndent pathways, however, Death receptor pathway can not rule out.