Hsp90 inhibitor BIIB021 enhances triptolide-induced apoptosis of human T-cell acute lymphoblastic leukemia cells in vitro mainly by disrupting p53-MDM2 balance.
OBJECTIVE
To investigate the effects of BIIB021, an inhibitor of heat shock protein 90 (Hsp90) alone or in combination with triptolide (TPL) on T-cell acute lymphoblastic leukemia (T-ALL) and the mechanisms of action.


METHODS
Human T-ALL cells line Molt-4 was examined. The cell viability was measured using MTT assay. Apoptotic cells were studied with Hoechst 33258 staining. Cell apoptosis and cell cycle were analyzed using flow cytometry with Annexin V/PI staining and PI staining, respectively. The levels of multiple proteins, including Akt, p65, CDK4/6, p18, Bcl-2 family proteins, MDM2, and p53, were examined with Western blotting. The level of MDM2 mRNA was determined using RT-PCR.


RESULTS
Treatment of Molt-4 cells with BIIB021 (50-800 nmol/L) inhibited the cell growth in a dose-dependent manner (the IC50 value was 384.6 and 301.8 nmol/L, respectively, at 48 and 72 h). BIIB021 dose-dependently induced G0/G1 phase arrest, followed by apoptosis of Molt-4 cells. Furthermore, BIIB021 increased the expression of p18, decreased the expression of CDK4/6, and activated the caspase pathway in Molt-4 cells. Moreover, BIIB021 (50-400 nmol/L) dose-dependently decreased the phospho-MDM2 and total MDM2 protein levels, but slightly increased the phospho-p53 and total p53 protein levels, whereas TPL (5-40 nmol/L) dose-dependently enhanced p53 activation without affecting MDM2 levels. Co-treatment with BIIB021 and TPL showed synergic inhibition on Molt-4 cell growth. The co-treatment disrupted p53-MDM2 balance, thus markedly enhanced p53 activation. In addition, the co-treatment increased the expression of Bak and Bim, followed by increased activation of caspase-9.


CONCLUSIONS
The combination of BIIB021 and TPL may provide a novel strategy for treating T-ALL by overcoming multiple mechanisms of apoptosis resistance.
Institute of Hematology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Min
M
Li
Xiang
X
Zhang
Wen-jing
WJ
Zhou
Yue-hua
YH
Chen
Hui
H
Liu
Lin
L
Liu
Chun-mei
CM
Yang
Wen-bin
WB
Qan
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D004224:Diterpenes / Q000494:pharmacology*; D004852:Epoxy Compounds / Q000494:pharmacology; D005434:Flow Cytometry; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D010616:Phenanthrenes / Q000494:pharmacology*; D054218:Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / Q000378:metabolism / Q000473:pathology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
C521963:6-chloro-9-(4-methoxy-3,5-dimethylpyridin-2-ylmethyl)-9H-purin-2-ylamine; D017931:DNA Primers; D004224:Diterpenes; D004852:Epoxy Compounds; D018841:HSP90 Heat-Shock Proteins; D010616:Phenanthrenes; D011725:Pyridines; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C001899:triptolide; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D000225:Adenine