Cell cycle arrest and apoptotic cell death in cultured human gastric carcinoma cells mediated by arsenic trioxide.
OBJECTIVE
To investigate the effect of arsenic trioxide on human gastric cancer cell line MKN45 with respect to both cytotoxicity and induction of apoptosis in vitro.


METHODS
MKN45 cells were treated with arsenic trioxide (As2O3) at the concentration of 1, 5, and 10 micromol/L, respectively, for three successive days. Cell growth and proliferation were observed by cell counting and trypan blue exclusion. Cytotoxicity of As2O3 was determined by MTT assay. Morphologic changes were studied with light microscopy. Flow cytometry was used to assay cell DNA distribution and apoptotic cells were confirmed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and DNA electrophoresis.


RESULTS
The growth of MKN45 cells was significantly inhibited by As2O3 which was confirmed by colony-forming assay. After 7 d of culture with various concentrations of As2O3, colony-forming capacity of MKN45 cells decreased with As2O3 increment in comparison with that of control group. The inhibitory rate of colony-formation was 38.5%, 99.1%, and 99.5% when the concentration of As2O3 was 1, 5, and 10 micromol/L in culture medium, respectively. The cell number of a single colony in drug treatment groups was less than that of control group. The cell-killing rate of As2O3 to MKN45 cells was both dose- and time-dependent with an IC50 of (11.05+/-0.25) micromol/L. After incubation in 10 micromol/L As2O3 for 24 h, the cell-killing rate was 27.1%, and it was close to 50% after 48 h. The results showed that As2O3 induced time- and dose-dependent apoptosis in MKN45 cells, blocked at G2/M phase. The apoptotic peak (sub-G1 phase) appeared and cell apoptotic rate in MKN45 cells was 18.3-32.5% after treatment by 10 micromol/L As2O3 for 48 h. The percentage of G2/M cell of the experimental groups was 2.0-5.0 times than that of the control group. Gel electrophoresis of DNA from cells treated with each concentration of As2O3 for 48 h revealed a "ladder" pattern, indicating preferential DNA degradation at the internucleosomal, linker DNA sections. TUNEL also demonstrated strand breaks in DNA of MKN45 cells treated with As2O3, while control cells showed negative labeling.


CONCLUSIONS
As2O3 can induce apoptosis of human gastric carcinoma cells MKN45, which is the basis of its effectiveness. It shows great potential in the treatment of gastric carcinoma.
Shao
Qin-Shu
QS
Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang Province, China.
Ye
Zai-Yuan
ZY
Ling
Zhi-Qiang
ZQ
Ke
Jin-Jing
JJ
D000230:Adenocarcinoma / Q000188:drug therapy* / Q000473:pathology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D006801:Humans; D010087:Oxides / Q000494:pharmacology*; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000473:pathology
D000970:Antineoplastic Agents; D001152:Arsenicals; D010087:Oxides; D000077237:Arsenic Trioxide