Acetyl-macrocalin B, an ent-kaurane diterpenoid, initiates apoptosis through the ROS-p38-caspase 9-dependent pathway and induces G2/M phase arrest via the Chk1/2-Cdc25C-Cdc2/cyclin B axis in non-small cell lung cancer

ABSTRACT

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, and novel effective drugs against NSCLC are urgently needed. Isodon species are rich in ent-kaurane diterpenoids that have been reported to have antitumor bioactivity. Acetyl-macrocalin B (A-macB) is a novel ent-kaurane diterpenoid isolated from Isodon silvatica, and its antitumor efficacy against NSCLC and the underlying mechanisms were scrutinized in depth. The viability of cells treated with A-macB was detected by CCK-8 and colony formation assays. Apoptosis and cell cycle distribution were analyzed by flow cytometry. The mechanisms were investigated by detecting ROS and performing western blotting and verification experiments with specific inhibitors. The in vivo effect of A-macB was explored in a nude mouse xenograft model. A-macB effectively inhibited H1299 and A549 cell viability, triggered apoptosis and delayed cells in the G2/M phase. A-macB induced cellular ROS production and then activated the p38 MAPK-mediated, caspase 9-dependent apoptotic pathway. Both the ROS scavenger NAC and the specific p38 inhibitor SB203580 inactivated the function of p38 induced by A-macB, thus preventing cells from apoptosis. A-macB activated the Chk1/2-Cdc25C-Cdc2/cyclin B1 axis to induce G2/M phase arrest. AZD7762 abrogated the function of Chk1/2, abolished the G2/M delay and enhanced the cytotoxicity of A-macB. Moreover, A-macB efficiently suppressed tumor growth in a mouse xenograft model without noticeable toxicity to normal tissues. Having both efficacy and relative safety, A-macB is a potential lead compound that is worthy of further exploration for development as an anticancer agent.

KEYWORDS:

• Acetyl-macrocalin B
• reactive oxygen species
• p38 MAPK
• apoptosis
• Chk1/Chk2
• G2/M phase arrest
• NSCLC

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Ethical approval

The in vivo animal experiments were approved by the Cancer Institute and Hospital of the Chinese Academy of Medical Sciences Institutional Animal Care and Use Committee (IACUC; permission number: NCC2015A095). All applicable institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

Acknowledgments

The authors thank X. Zeng (WuXi AppTec., Shanghai) for his help and support in the animal experiments.

Additional information

Funding

This work was supported by the National Key Research Development Plan (2016YFC0905400), the National Key Basic Research Development Plan (2014CB542006), the International Science and the Technology Corporation and Exchange Project (2015DFA31090), the CAMS Innovation Fund for Medical Sciences (2016-I2M-1-001), the Research Special Fund for Public Welfare Industry of Health (201402003), the National Natural Science Foundation of China (81673329) and the NSFC-Joint Foundation of Yunnan Province (Grant U1302223).