[논문]Long-term depletion of cereblon induces mitochondrial dysfunction in cancer cells

Park, Seulki; Kim, Kidae; Haam, Keeok; Ban, Hyun Seung; Kim, Jung-Ae; Park, Byoung Chul; Park, Sung Goo; Kim, Sunhong; Kim, Jeong-Hoon

doi:10.5483/bmbrep.2021.54.6.218

Long-term depletion of cereblon induces mitochondrial dysfunction in cancer cells 원문보기

BMB reports, v.54 no.6, 2021년, pp.305 - 310

Park, Seulki (Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) , Kim, Kidae (Division of Biomedical Informatics, Center for Genome Science, National Institute of Health, KCDC) , Haam, Keeok (Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology) , Ban, Hyun Seung (Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology) , Kim, Jung-Ae (Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology) , Park, Byoung Chul (Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) , Park, Sung Goo (Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) , Kim, Sunhong (Drug Discovery Center, LG Chem) , Kim, Jeong-Hoon (Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))

Abstract ▼ AI-Helper

Cereblon (CRBN) is a multi-functional protein that acts as a substrate receptor of the E3 ligase complex and a molecular chaperone. While CRBN is proposed to function in mitochondria, its specific roles are yet to be established. Here, we showed that knockdown of CRBN triggers oxidative stress and calcium overload in mitochondria, leading to disruption of mitochondrial membrane potential. Notably, long-term CRBN depletion using PROteolysis TArgeting Chimera (PROTAC) induced irreversible mitochondrial dysfunction, resulting in cell death. Our collective findings indicate that CRBN is required for mitochondrial homeostasis in cells.

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표/그림 (4)

그림 Fig. 1. CRBN knockdown leads to excessive ROS production and mitochondrial dysfunction. (A) Levels of CRBN mRNA (top) and protein (bottom) were examined via qRT-PCR and western blot, respectively, in shControl (shCon) and stable CRBN-depleted (shCRBN) HepG2 cells. (B, C) Fluorescence staining of each ROS species. shControl and shCRBN HepG2 cells were stained with (B) DHR123, an indicator of intracellular hydrogen peroxide, or (C) MitoSOX Red, an indicator of mitochondrion-derived superoxide. (D) To monitor mitochondrial membrane potential, shControl HepG2 cells treated with or without H₂O₂ 100 μM and shCRBN HepG2 cells were stained using JC-1 fluorescent dye and images visualized under a fluorescence microscope. Representative images are shown. Original magnification (× 200); Scale bar: 50 μm. (E) Quantitative measurement of ATP production using the Seahorse Extracellular Flux Analyzer. (F) Basal respiration was calculated as the mean value of the first three baseline readings. (G) ATP-linked respiration OCR was measured based on the subtracted value of the mean of three readings following oligomycin (1.5 μM) treatment from basal respiration. All results are presented as mean ± S.D. of at least three independent experiments. P-values were calculated using unpaired Student’s t-test. *P < 0.05 and **P < 0.01 vs control.
그림 Fig. 2. CRBN knockdown induces mitochondrial calcium overload. (A) Analysis of calcium flux in mitochondria using Rhod2-AM fluorescent dye. MitoTracker green and Hoechst were employed as mitochondrial and nuclear indicators, respectively. All cells were stained with indicators and imaged under a confocal microscope for 240 seconds following UTP treatment (100 μM). Original magnification (X630) Scale bar: 50 μm. (B, C) Quantitative analysis of mitochondrial calcium flux in (B) shControl and shCRBN HepG2 cells and (C) shControl and shCRBN HeLa cells. The Y axis represents fluorescence intensity of Rhod2-AM analyzed using Zen2 (blue edition) program. P-values were calculated using unpaired Student's t-test. *P < 0.05 and **P < 0.01 vs control.
그림 Fig. 3. Long-term depletion of CRBN promotes mitochondrial ROS accumulation and irreversible mitochondrial damage. (A) Exogenous CRBN expression in shControl and shCRBN cells determined via western blot. (B) Quantitative analysis of mitochondrial superoxide production using MitoSOX Red fluorescent dye. (C) Quantitative analysis of mitochondrial membrane potential via flow cytometry in CRBN-overexpressing cells. (D) Western blot analysis of CRBN levels in TD-165-treated HepG2 cells. (E) Quantitation of intracellular hydrogen peroxide production after TD-165 treatment (1 μM) for 3 and 12 days. (F) Quantitation of mitochondrion-derived superoxide production after TD-165 treatment (1 μM) for 3 and 12 days. (G) Quantitative analysis of mitochondrial membrane potential via flow cytometry. (H) Immunofluorescence staining of shControl and shCRBN HeLa cells using TOM20 antibody. Images were visualized via confocal microscopy. Arrows indicate changes in the shape of mitochondria. Original magnification (X630) Scale bar: 50 μm. (I) Western blot analysis of CRBN and mitochondria-related protein expression after isolation of mitochondrial and cytosol extracts from CRBN-depleted HepG2 cells. TOM20 and alpha-tubulin were used as the controls for mitochondria and cytosol, respectively. Results are presented as mean ± S.D. of at least three independent experiments. P-values were calculated using unpaired student's t-test. *P < 0.05 and **P < 0.01 vs control.
그림 Fig. 4. Long-term depletion of CRBN promotes cancer cell death. (A, B) Effects of CRBN knockdown on viability of HepG2 and HeLa cells were determined with the Cell titer-Glo assay. (C) Flow cytometry analysis of cell cycle changes in shControl and shCRBN HepG2 cells. (D) Quantification of caspase 3/7 activity in shControl and shCRBN cells. (E) Western blot analysis of proteins related to apoptosis (cleaved caspase-7, cleaved PARP, p53 and p21). (F-G) Quantitative analysis of viability following TD-165 treatment for 12 days in (F) HepG2 and (G) HeLa cells. All results are presented as mean ± S.D. of at least three independent experiments. P-values were calculated using unpaired student's t-test. *P < 0.05 and **P < 0.01 vs control.

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