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Identification of Compound CB-2 as a Novel Late-Stage Autophagy Inhibitor Exhibits Inhibitory Potency against A549
Zhihui Liu1, Lu Zhang1, Yachao Liu1
1College of Bioengineering, Henan University of Technology, Lianhua Street, Zhengzhou 450001, China.
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.
Insights
A novel compound, CB-2, inhibits late-stage autophagy by blocking autophagosome-lysosome fusion in non-small cell lung cancer cells. This autophagy inhibition leads to cell death and reduced migration, suggesting potential as a cancer therapeutic.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Autophagy is crucial for cancer cell survival, progression, dormancy, and treatment resistance.
- Inhibiting autophagy presents a promising strategy to enhance cancer therapy efficacy.
- There is a significant need for novel autophagy inhibitors in cancer treatment.
Purpose of the Study:
- To identify and characterize novel autophagy inhibitors from synthesized curcumin derivatives.
- To investigate the mechanism of action of the selected compound, CB-2, in non-small cell lung cancer (NSCLC) cells.
- To evaluate the anti-cancer potential of CB-2 against A549 NSCLC cells.
Main Methods:
- Screening of synthesized curcumin derivatives to identify autophagy inhibitors.
- Treatment of A549 NSCLC cells with CB-2 and assessment of autophagy markers (LC3B-II, SQSTM1).
- Investigation of CB-2's effect on autophagy initiation, degradation, and autophagosome-lysosome fusion using wortmannin and chloroquine.
- Analysis of CB-2's impact on apoptosis, necrosis, cell migration, mitochondrial reactive oxygen species (ROS), and mitochondrial membrane potential (MMP).
- Evaluation of ROS scavenging effects using N-acetylcysteine (NAC).
Main Results:
- CB-2 treatment led to the accumulation of autophagosomes by inhibiting autophagosome-lysosome fusion, classifying it as a late-stage autophagy inhibitor.
- CB-2 induced apoptosis and necrosis at high doses, and impaired migratory capacity at lower doses in A549 cells.
- CB-2 increased mitochondrial ROS production and decreased mitochondrial membrane potential, effects reversed by NAC.
- CB-2 did not affect autophagy initiation proteins but significantly blocked autophagosome-lysosome fusion.
Conclusions:
- CB-2 is identified as a novel late-stage autophagy inhibitor with potent inhibitory effects against A549 NSCLC cells.
- CB-2 exerts its anti-cancer effects by disrupting autophagosome-lysosome fusion, inducing oxidative stress, and impairing cell migration.
- CB-2 demonstrates significant potential as a therapeutic agent for non-small cell lung cancer.
