Bufalin induces apoptosis in human osteosarcoma U-2OS and U-2OS methotrexate300-resistant cell lines.
OBJECTIVE
To investigate the antiproliferative activity and apoptosis-inducing effects of bufalin on human osteosarcoma cell lines.


METHODS
U-2OS and U-2OS methotrexate (MTX) 300-resistant cell lines were treated with bufalin. Cell viability was assessed using the MTT assay. Cell-cycle status, apoptosis-inducing effects, and the expression of apoptosis-related proteins were evaluated by flow cytometry, fluorescent staining, DNA fragmentation assays, and Western blotting. The effect of bufalin on dihydrofolate reductase (DHFR) expression was studied by RTPCR and Western blotting.


RESULTS
Bufalin inhibited cell growth in both U-2OS and U-2OS MTX300 cells. The induction of G2/M cell-cycle arrest was also seen in the cells treated with bufalin. The induction of apoptosis by bufalin was confirmed by increased expression of the tumor suppressor protein p53 and the increased ratio of the Bax/Bcl-2 proteins. Bufalin induced apoptosis to the same extent in both cell lines without regard to DHFR levels in the cells.


CONCLUSIONS
Bufalin inhibited the growth of and induced apoptosis in both MTX-sensitive and MTX-resistant human osteosarcoma U-2OS cells. The apoptosis-inducing effect of bufalin was not influenced by the presence of high levels of the DHFR protein.
Yin
Jun-Qiang
JQ
Department of Orthopaedic Oncology, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China.
Shen
Jing-Nan
JN
Su
Wei-Wei
WW
Wang
Jin
J
Huang
Gang
G
Jin
Song
S
Guo
Qian-Chen
QC
Zou
Chang-Ye
CY
Li
Hao-Miao
HM
Li
Fo-Bao
FB
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002018:Bufanolides / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D012516:Osteosarcoma / Q000378:metabolism / Q000503:physiopathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000970:Antineoplastic Agents; D002018:Bufanolides; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D013762:Tetrahydrofolate Dehydrogenase; C022777:bufalin; D008727:Methotrexate