Abstract
Cancers illustrating resistance towards apoptosis is one of the main factors causing clinical failure of conventional chemotherapy. Innovative therapeutic methods which can overcome the non-apoptotic phenotype are needed. The AMP-activated protein kinase (AMPK) is the central regulator of cellular energy homeostasis, metabolism, and autophagy. Our previous study showed that the identified natural AMPK activator is able to overcome apoptosis-resistant cancer via autophagic cell death. Therefore, AMPK is an ideal pharmaceutical target for chemoresistant cancers. Here, we unravelled that the bisbenzylisoquinoline alkaloid thalidezine is a novel direct AMPK activator by using biolayer interferometry analysis and AMPK kinase assays. The quantification of autophagic EGFP-LC3 puncta demonstrated that thalidezine increased autophagic flux in HeLa cancer cells. In addition, metabolic stress assay confirmed that thalidezine altered the energy status of our cellular model. Remarkably, thalidezine-induced autophagic cell death in HeLa or apoptosis-resistant DLD-1 BAX-BAK DKO cancer cells was abolished by addition of autophagy inhibitor (3-MA) and AMPK inhibitor (compound C). The mechanistic role of autophagic cell death in resistant cancer cells was further supported through the genetic removal of autophagic gene7 (Atg7). Overall, thalidezine is a novel AMPK activator which has great potential to be further developed into a safe and effective intervention for apoptosis- or multidrug-resistant cancers.
Highlights
The AMP-activated protein kinase (AMPK), a serine/threonine kinase, is a heterotrimeric complex containing one catalytic α subunit and regulatory β and γ subunits
AMPK directly phosphorylates a number of downstream targets including ACC, ULK, and Mammalian target of rapamycin complex 1 (mTORC1), which act as mediator/effectors to metabolism, cell growth, and autophagy [1, 4,5,6]
Immunoblot results indicated an increase in AMPK phosphorylation accompanied by a reduction in phosphorylated p70S6K, a downstream target of mTOR, in response to thalidezine after eight hours of treatment (Figure 1D)
Summary
The AMP-activated protein kinase (AMPK), a serine/threonine kinase, is a heterotrimeric complex containing one catalytic α subunit and regulatory β and γ subunits. AMP or ADP can directly bind to the regulatory γ subunits of AMPK, leading to a conformational change that promotes its activation [1]. Full AMPK activation requires specific phosphorylation of the α subunit at Thr172 by upstream kinases [2, 3]. AMPK restores cellular energy levels by promoting catabolic and inhibiting anabolic processes. AMPK directly phosphorylates a number of downstream targets including ACC, ULK (protein kinases that initiate autophagy), and mTORC1 (mammalian target of rapamycin complex 1), which act as mediator/effectors to metabolism, cell growth, and autophagy [1, 4,5,6]. The process engulfs regions of cytoplasm, protein aggregates, and damaged organelles into doublemembrane autophagosomes, which fuse with lysosomes for further degradation and cellular recycling [5,6,7]
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