Piperonal ciprofloxacin hydrazone induces growth arrest and apoptosis of human hepatocarcinoma SMMC-7721 cells.
OBJECTIVE
To investigate the cytotoxic effects of piperonal ciprofloxacin hydrazone (QNT4), a novel antibacterial fluoroquinolone derivative, against human hepatocarcinoma SMMC-7721 cells.


METHODS
Human hepatocarcinoma cells (SMMC-7721), human breast adenocarcinoma cells (MCF-7) and human colon adenocarcinoma cells (HCT-8) were tested. The effects of QNT4 on cell proliferation were examined using MTT assay. Cell apoptosis was determined using Hoechst 33258 fluorescence staining, TUNEL assay and agarose gel electrophoresis. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. Mitochondrial membrane potential (Δψm) was measured using a high content screening imaging system. Protein expression of caspase-9, caspase-8, caspase-3, p53, Bcl-2, Bax, and cytochrome c was detected with Western blot analysis.


RESULTS
Treatment with QNT4 (0.625-10 μmol/L) potently inhibited the proliferation of the cancer cells in time- and dose-dependent manners (the IC(50) value at 24 h in SMMC-7721 cells, MCF-7 cells and HCT-8 cells was 2.956±0.024, 3.710±0.027, and 3.694±0.030 μmol/L, respectively). Treatment of SMMC-7721 cells with QNT4 (0.2146, 2.964, and 4.600 μmol/L) for 24 h dose-dependently increased the percentage of apoptotic cells, elicited characteristic DNA "ladder" bands, and decreased the mitochondrial membrane potential. QNT4 dose-dependently increased topoisomerase II-mediated DNA breaks while inhibiting DNA relegation, thus keeping the DNA in fragments. Treatment of SMMC-7721 cells with QNT4 significantly increased cytochrome c in the cytosol, and decreased cytochrome c in the mitochondrial compartment. QNT4 (3-7.39 μmol/L) significantly increased the protein expression of p53, Bax, caspase-9, caspase-3, and the cleaved activated forms of caspase-9 and caspase-3 in SMMC-7721 cells. In contrast, the expression of Bcl-2 was decreased, while caspase-8 had no significant change.


CONCLUSIONS
QNT4 induced the apoptosis of SMMC-7721 cells via inhibiting topoisomerase II activity and modulating mitochondrial-dependent pathways.
Institute of Neurobiology, College of Nursing, He-nan University, Kaifeng, China.
Zhen-yu
ZY
Shi
Yong-qiang
YQ
Li
Yu-hua
YH
Kang
Guo-qiang
GQ
Hu
Chao-shen
CS
Huang-fu
Jin-Bo
JB
Deng
Bin
B
Liu
D000230:Adenocarcinoma / Q000188:drug therapy / Q000378:metabolism; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002939:Ciprofloxacin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D005260:Female; D006801:Humans; D006835:Hydrazones / Q000737:chemistry / Q000494:pharmacology; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects
D000970:Antineoplastic Agents; D006835:Hydrazones; D002939:Ciprofloxacin; D004250:DNA Topoisomerases, Type II