Cytotoxic diarylheptanoid induces cell cycle arrest and apoptosis via increasing ATF3 and stabilizing p53 in SH-SY5Y cells.
OBJECTIVE
The aim of the study is to dissect the cytotoxic mechanisms of 1-(4-hydroxy-3-methoxyphenyl)-7-(3,4-dihydroxyphenyl)-4E-en-3-heptanone (compound 1) in SH-SY5Y cells and therefore to provide new insight into neuroblastoma chemotherapy.


METHODS
Nine diarylheptanoids were isolated from Alpinia officinarum by chromatography and their cytotoxicity was evaluated by an MTS assay. Flow cytometry, BrdU incorporation assay and fluorescence staining were employed to investigate cytostatic and apoptotic effects induced by the compound 1. In addition, Western blot, qPCR and siRNA techniques were used to elucidate the molecular mechanisms of the cytotoxicity.


RESULTS
The study to elucidate the cytotoxic mechanisms of compound 1, the most potent diarylheptanoid showed that cell cycle-related proteins, cyclins, CDKs and CDKIs, as well as two main apoptotic related families, caspase and Bcl 2 were involved in S phase arrest and apoptosis in neuroblastoma cell line SH-SY5Y. Furthermore, following the drug treatment, the protein expression of p53, phospho-p53 (Ser20) as well as the p53 transcriptional activated genes ATF3, puma and Apaf-1 were increased dramatically; MDM2 and Aurora A, the two p53 negative regulators were decreased; the p53 protein stability was enhanced, whereas the p53 mRNA expression level slightly decreased and ATF3 mRNA expression apparently increased. In addition, the knockdown of ATF3 gene by siRNA partially suppressed p53, caspase 3, S phase arrest and apoptosis triggered by compound 1.


CONCLUSIONS
These results suggest that compound 1 induces S phase arrest and apoptosis via up regulation of ATF3 and stabilization of p53 in SH-SY5Y cell line. Therefore, compound 1 might be a promising lead structure for neuroblastoma therapy.
Tian
Ze
Z
Informatics Program, MIT Division of Health Sciences and Technology, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA.
An
Ning
N
Zhou
Bin
B
Xiao
Peigen
P
Kohane
Isaac S
IS
Wu
Erxi
E
D051700:Activating Transcription Factor 3 / Q000235:genetics* / Q000502:physiology; D032429:Alpinia / Q000737:chemistry; D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D036381:Diarylheptanoids / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D015394:Molecular Structure; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D055550:Protein Stability; D034741:RNA, Small Interfering / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*
C539887:1-(4-hydroxy-3-methoxyphenyl)-7-(3,4-dihydroxyphenyl)-4E-en-3-heptanone; C490449:ATF3 protein, human; D051700:Activating Transcription Factor 3; D000972:Antineoplastic Agents, Phytogenic; D036381:Diarylheptanoids; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53