ABSTRACT
Autophagy is the multistep mechanism for the elimination of damaged organelles and misfolded proteins. This mechanism is preceded and may induce other program cell deaths such as apoptosis. This study unraveled the potential pharmacological effect of 24MD in inducing the autophagy of lung cancer cells. Results showed that 24MD was concomitant with autophagy induction, indicating by autophagosome staining and the induction of ATG5, ATG7 and ubiquitinated protein, p62 expression after 12-h treatment. LC3-I was strongly conversed to LC3-II, and p62 was downregulated after 24-h treatment. The apoptosis-inducing activity was found after 48-h treatment as indicated by annexin V-FITC/propidium iodide staining and the activation of caspase-3. From a mechanistic perspective, 24-h treatment of 24MD at 60 µM substantially downregulated p-mTOR. Meanwhile, p-PI3K and p-Akt were also suppressed by 24MD at concentrations of 80 and 100 µM, respectively. We further confirmed m-TOR-mediated autophagic activity by comparing the effect of 24MD with rapamycin, a potent standard mTOR1 inhibitor through Western blot and immunofluorescence assays. Although 24MD could not suppress p-mTOR as much as rapamycin, the combination of rapamycin and 24MD could increase the mTOR suppressive activity and LC3 activation. Changing the substituent groups (R groups) from dimethylphenol to ethylphenol in EMD or changing methylazanedyl to cyclohexylazanedyl in 24CD could only induce apoptosis activity but not autophagic inducing activity. We identified 24MD as a novel compound targeting autophagic cell death by affecting mTOR-mediated autophagy.
Subject(s)
Autophagic Cell Death , Lung Neoplasms , Apoptosis , Autophagy , Caspase 3/metabolism , Cell Line, Tumor , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Propidium/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/metabolism , Ubiquitinated Proteins/pharmacology , Ubiquitinated Proteins/therapeutic use , XylenesABSTRACT
A simple and effective strategy was developed to enrich ubiquitinated proteins (UPs) from cancer cell lysate using the α-Al2O3 nanoparticles covalently linked with ubiquitin binding protein (Vx3) (denoted as α-Al2O3-Vx3) via a chemical linker. The functionalized α-Al2O3-Vx3 showed long-term stability and high efficiency for the enrichment of UPs from cancer cell lysates. Flow cytometry analysis results indicated dendritic cells (DCs) could more effectively phagocytize the covalently linked α-Al2O3-Vx3-UPs than the physical mixture of α-Al2O3 and Vx3-UPs (α-Al2O3/Vx3-UPs). Laser confocal microscopy images revealed that α-Al2O3-Vx3-UPs localized within the autophagosome of DCs, which then cross-presented α-Al2O3-Vx3-UPs to CD8+ T cells in an autophagosome-related cross-presentation pathway. Furthermore, α-Al2O3-Vx3-UPs enhanced more potent antitumor immune response and antitumor efficacy than α-Al2O3/cell lysate or α-Al2O3/Vx3-UPs. This work highlights the potential of using the Vx3 covalently linked α-Al2O3 as a simple and effective platform to enrich UPs from cancer cells for the development of highly efficient therapeutic cancer vaccines.
