Abstract
Autophagy has been recognized as a stress tolerance mechanism that maintains cell viability, which contributes to tumor progression, dormancy, and treatment resistance. The inhibition of autophagy in cancer has the potential to improve the therapeutic efficacy. It is therefore of great significance to search for new autophagy inhibitors. In the present study, after screening a series of curcumin derivatives synthesized in our laboratory, (E)-3-((E)-4-chlorobenzylidene)-5-((5-methoxy-1H-indol-3-yl)methylene)-1-methylpiperidin-4-one (CB-2) was selected as a candidate for further study. We found that CB-2 increased the LC3B-II and SQSTM1 levels associated with the accumulation of autophagosomes in non-small cell lung cancer (NSCLC) A549 cells. The increased level of LC3B-II induced by CB-2 was neither eliminated when autophagy initiation was suppressed by wortmannin nor further increased when autophagosome degradation was inhibited by chloroquine (CQ). CB-2 enhanced the accumulation of LC3B-II under starvation conditions. Further studies revealed that CB-2 did not affect the levels of the key proteins involved in autophagy induction but significantly blocked the fusion of autophagosomes with lysosomes. High-dose CB-2 induced the apoptosis and necrosis of A549 cells, while a lower dose of CB-2 mainly impaired the migrative capacity of A549 cells, which only slightly induced cell apoptosis. CB-2 increased the levels of mitochondrial-derived reactive oxygen species (ROS) while decreasing the mitochondrial membrane potential (MMP). Scavenging ROS via N-acetylcysteine (NAC) reversed CB-2-induced autophagy inhibition and its inhibitory effect against A549 cells. In conclusion, CB-2 serves as a new late-stage autophagy inhibitor, which has a strong inhibitory potency against A549 cells.
Highlights
A series of derivatives of curcumin were screened for discovering a novel autophagy inhibitor using GFP-LC3B stably transfected U87 cells
After AO staining, bright red fluorescent dots appeared in A549 cells treated with CB-2 for 24 h, indicating that this compound induced the accumulation of acidic vesicular organelles in the cytoplasm (Figure S1)
We investigated whether the overproduction of reactive oxygen species (ROS) contributes to CB-2We investigated whether the overproduction of ROS contributes to CB-2-induced induced autophagy and its inhibitory effects on A549 cells. to According to the autophagy inhibitioninhibition and its inhibitory effects on A549 cells
Summary
In addition to its essential role in maintaining the balance of the intracellular environment in normal cells, autophagy is involved in various pathological processes, especially cancer [2]. Most cancer cells are exposed to hypoxic conditions within tumor regions, and they have elevated levels of basal autophagy to resist environmental stress [3]. Most chemo/radiotherapy inevitably leads to cellular stress. In this case, autophagy is often activated, enabling cancer cells to resist anticancer treatments and facilitating tumor dormancy [5]. The blockade of autophagy forces the proliferation of dormant tumor cells, which makes them more vulnerable to be killed by chemo/radiotherapy [5]. The inhibition of autophagy emerges as the efficient antitumor treatment
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