Arenobufagin intercalates with DNA leading to G2 cell cycle arrest via ATM/ATR pathway.
Arenobufagin, a representative bufadienolide, is the major active component in the traditional Chinese medicine Chan'su. It possesses significant antineoplastic activity in vitro. Although bufadienolide has been found to disrupt the cell cycle, the underlying mechanisms of this disruption are not defined. Here, we reported that arenobufagin blocked the transition from G2 to M phase of cell cycle through inhibiting the activation of CDK1-Cyclin B1 complex; The tumor suppressor p53 contributed to sustaining arrest at the G2 phase of the cell cycle in hepatocellular carcinoma (HCC) cells. Moreover, arenobufagin caused double-strand DNA breaks (DSBs) and triggered the DNA damage response (DDR), partly via the ATM/ATR-Chk1/Chk2-Cdc25C signaling pathway. Importantly, we used a synthetic biotinylated arenobufagin-conjugated chemical probe in live cells to show that arenobufagin accumulated mainly in the nucleus. The microscopic thermodynamic parameters measured using isothermal titration calorimetry (ITC) also demonstrated that arenobufagin directly bound to DNA in vitro. The hypochromicity in the UV-visible absorption spectrum, the significant changes in the circular dichroism (CD) spectrum of DNA, and the distinct quenching in the fluorescence intensity of the ethidium bromide (EB)-DNA system before and after arenobufagin treatment indicated that arenobufagin bound to DNA in vitro by intercalation. Molecular modeling suggested arenobufagin intercalated with DNA via hydrogen bonds between arenobufagin and GT base pairs. Collectively, these data provide novel insights into arenobufagin-induced cell cycle disruption that are valuable for the further discussion and investigation of the use of arenobufagin in clinical anticancer chemotherapy.
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Li-Juan
LJ
Deng
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Qun-Long
QL
Peng
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Long-Hai
LH
Wang
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Jun
J
Xu
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510632, P.R. China.
Jun-Shan
JS
Liu
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Ying-Jie
YJ
Li
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Zhen-Jian
ZJ
Zhuo
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Liang-Liang
LL
Bai
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Li-Ping
LP
Hu
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Wei-Min
WM
Chen
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Wen-Cai
WC
Ye
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China.
Dong-Mei
DM
Zhang
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D015153:Blotting, Western; D002018:Bufanolides / Q000494:pharmacology*; D002151:Calorimetry; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D020552:Comet Assay; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D006801:Humans; D047468:Immunoprecipitation; D007364:Intercalating Agents / Q000494:pharmacology; D008113:Liver Neoplasms / Q000473:pathology*; D008958:Models, Molecular; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
D000970:Antineoplastic Agents; D002018:Bufanolides; D007364:Intercalating Agents; D034741:RNA, Small Interfering; C055393:arenobufagin; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins
DNA damage response; DNA intercalator; G2 cell cycle arrest; arenobufagin