Abstract
BackgroundAutophagy is an indispensable lysosomal self-digestion process involved in the degradation of aggregated proteins and damaged organelles. Autophagy is associated with the several pathological processes, including cancer. Cancer stem cells (CSCs) play significant roles in cancer initiation, progression and drug resistance. Recent studies have demonstrated the antitumor activities of plant-derived chemopreventive agent rottlerin (Rott). However, the molecular mechanism by which Rott induces autophagy in breast CSCs has not been investigated.ResultsThe objectives of this study were to examine the molecular mechanism by which Rott induces autophagy which leads to apoptosis in breast CSCs. Treatment of breast CSCs with Rott for 24 h resulted in a concentration dependent induction of autophagy, followed by apoptosis as measured by flow cytometry. Electron microscopy confirmed the presence of autophagosomes in Rott treated breast CSCs. Western blot analysis showed that Rott treatment increased the expression of LC3, Beclin-1 and Atg12 that are accumulated during autophagy. Prolonged exposure of breast CSCs to Rott caused apoptosis which was associated with the suppression of phosphorylated Akt and mTOR, upregulation of phosphorylated AMPK, and downregulation of anti-apoptosis Bcl-2, Bcl-XL, XIAP and cIAP-1. Knock-down of Atg7 or Beclin-1 by shRNA inhibited Rott-induced autophagy at 24 h. Our study also demonstrates that pre-treatment of breast CSCs with autophagosome inhibitors 3-methyladenine and Bafilomycin, as well as protein synthesis inhibitor cycloheximide inhibited Rott-induced autophagy and apoptosis. Rott induces autophagy via extensive cytoplasmic vacuolization in breast CSCs. Molecular docking results between C2-domain of protein kinase C-delta and Rott indicated that both hydrogen bonding and hydrophobic interactions contributed significantly for ligand binding with minimum binding affinity of ≈ 7.5 Kcal/mol. Although, autophagy inhibitors suppress the formation of cytoplasmic vacuolization and autophagy in breast CSCs, the potency of Rott to induce autophagy and apoptosis might be based on its capability to activate several pathways such as AMPK and proteasome inhibition.ConclusionsA better understanding of the relationship between autophagy and apoptosis would eventually allow us to discover novel drugs for the treatment of breast cancer by eliminating CSCs.
Highlights
Autophagy is an indispensable lysosomal self-digestion process involved in the degradation of aggregated proteins and damaged organelles
Rottlerin induced cytoplasmic vacuolation and cell death in breast Cancer stem cells (CSCs) To examine whether Rott induces cytotoxic effect on breast CSCs, we treated breast CSCs with different concentrations of Rott (0, 0.5, 1 and 2 μM) for various time points
In this study we demonstrated that Rott induces early autophagy as a survival strategy against late apoptosis through Adenosine monophosphate-activated protein kinase (AMPK) and Akt/mTOR cascade dependent pathways in human breast CSCs
Summary
Autophagy is an indispensable lysosomal self-digestion process involved in the degradation of aggregated proteins and damaged organelles. Autophagy is a highly conserved cellular process that is involved in several catabolic processes, cellular development [1], autoimmunity [2], degradation of long-lived proteins and organelles [3], and cell death [4] It has been involved in several other cellular mechanism which are directly or indirectly related to diseases like neurodegeneration, cardiovascular, aging and cancer [5]. It has been reported that the chemotherapeutic agents [13,14] induced the early stage of autophagy in cancer stem cells (CSCs) [15,16], and it is regulated by several ‘Atg’ (Autophagy-related) genes [17] and proteins which have been implicated in autophagosome formation [18]. An important autophagy-regulatory gene such as Beclin-1 functions as a haplo-insufficient tumor suppressor gene [23], further emphasizing the clinical importance of autophagic cell death and apoptosis
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