[Apoptosis of human leukemia HL-60 cells induced by rhabdastrellic acid-A and its mechanisms].
OBJECTIVE
Rhabdastrellic acid-A is an isomalabaricane triterpenoid isolated from the sponge Rhabdastrella globostellata from South China Sea. Our previous study indicated that rhabdastrellic acid-A can inhibit the proliferation of many types of tumor cells with minor toxicity. This study was to investigate the apoptosis of human leukemia HL-60 cells induced by rhabdastrellic acid-A and its possible mechanisms.


METHODS
Inhibitory effect of rhabdastrellic acid-A on the proliferation of HL-60 cells was evaluated by MTT assay. DNA fragmentation was analyzed by agarose electrophoresis. Cell morphology was observed under fluorescent microscope. The protein levels of Caspase-3, poly(ADP-ribose) polymerase (PARP), P73, Bcl-2 and Bax were analyzed by Western blot. The expression profile of apoptosis-related genes was analyzed by gene microarray. Reverse transcription-polymerase chain reaction (RT-PCR) was conducted to confirm some altered genes identified by gene microarray.


RESULTS
Rhabdastrellic acid-A inhibited the proliferation of HL-60 cells and the 50% inhibition concentration (IC50) was (0.64+/-0.21) microg/ml. When treated with 1 microg/ml rhabdastrellic acid-A for 36 h, condensation of nuclear chromatin of HL-60 cells was observed under fluorescent microscope and DNA fragmentation was observed by agarose electrophoresis. Also, rhabdastrellic acid-A induced cleavage of PARP and Caspase-3. The mRNA levels of 44 genes, including p73, JunD, TNFAIP3 and GADD45A, were up-regulated and the mRNA levels of 16 genes, including MAP2K5 and IGF2R, were down-regulated. The results were further confirmed by RT-PCR. The protein level of P73 was up-regulated after rhabdastrellic acid-A treatment.


CONCLUSIONS
Rhabdastrellic acid-A could induce the apoptosis of HL-60 cells which may be related to the up-regulation of apoptosis-related genes such as p73 and JunD, and the down-regulation of MAP2K5 and IGF2R.
Guo
Jing-Feng
JF
State Key Laboratory of Oncology in South China, Guangzhou, Guangdong, 510060, PR China.
Zhou
Jun-Min
JM
Feng
Gong-Kan
GK
Liu
Zong-Chao
ZC
Xiao
Ding-Jun
DJ
Deng
Song-Zhi
SZ
Deng
Rong
R
Zhu
Xiao-Feng
XF
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D053938:DNA Fragmentation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D048671:MAP Kinase Kinase 5 / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D014315:Triterpenes / Q000494:pharmacology*; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016755:Proto-Oncogene Proteins c-jun; D017527:Receptor, IGF Type 2; C000606460:TP73 protein, human; D014315:Triterpenes; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins; D051028:bcl-2-Associated X Protein; C529429:rhabdastrellic acid A; D011065:Poly(ADP-ribose) Polymerases; D048671:MAP Kinase Kinase 5; C482563:MAP2K5 protein, human; D053148:Caspase 3