Abstract
Purpose
We evaluated the expression of proteasome subunits to assess whether the proteasome could be a therapeutic target in cisplatin-resistant lung cancer cells.
Methods
Cisplatin-resistant (CR) variants were established from three non-small cell lung cancer (NSCLC) cell lines (A549, H1299, and H1975) and two small cell lung cancer (SCLC) cell lines (SBC3 and SBC5). The expression of proteasome subunits, the sensitivity to immunoproteasome inhibitors, and 20S proteasomal proteolytic activity were examined in the CR variants of the lung cancer cell lines.
Results
All five CR cell lines highly expressed one or both of the immunoproteasome subunit genes, PSMB8 and PSMB9, while no clear trend was observed in the expression of constitutive proteasome subunits. The CR cells expressed significantly higher levels of PSMB8 and PSMB9 proteins, as well. The CR variants of the H1299 and SBC3 cell lines were more sensitive to immunoproteasome inhibitors, and had significantly more proteasomal proteolytic activity than their parental counterparts.
Conclusions
The immunoproteasome may be an effective therapeutic target in a subset of CR lung cancers. Proteasomal proteolytic activity may be a predictive marker for the efficacy of immunoproteasome inhibitors in cisplatin-resistant SCLC and NSCLC.
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280_2020_4061_MOESM1_ESM.tif
Supplementary Figure S1. Quantitative reverse transcription PCR analysis showing mRNA levels of 20S proteasome subunits in cisplatin-resistant variants from A549, H1299, H1975, SBC3, and SBC5 cells. (TIF 550 kb)
280_2020_4061_MOESM2_ESM.tif
Supplementary Figure S2. The effect of the immunoproteasome inhibitor PR957 on cisplatin-resistant (CR) variants derived from (a) A549, (b) H1299, (c) H1975, (d) SBC3, and (e) SBC5 lung cancer cell lines by MTT assay. (TIF 462 kb)
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Supplementary Figure S3. (a) Western blot analysis showing the accumulation of ubiquitinated proteins after carfilzomib (CFZ) treatment. (b) Quantification of western blot analysis shown in (a) and normalized to actin. (TIF 803 kb)
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Supplementary Figure S4. Simultaneous knockdown of PSMB5, PSMB8, and PSMB9 by small interfering RNA (siRNA) in the cisplatin-resistant variants of A549 and H1299. (a) Knockdown efficiency of PSMB5, PSMB8, and PSMB9 was confirmed by western blot analysis. Simultaneous knockdown of PSMB5, PSMB8, and PSMB9 suppressed 20S proteasome chymotrypsin-like activity (b) but did not impair cell viability (c). The effects of the triple knockdown on the sensitivity to cisplatin (d) and carfilzomib (CFZ) (e) were analyzed by MTT cell proliferation assay. NT, non-target. **P<0.01, Welch t test. (TIF 831 kb)
280_2020_4061_MOESM5_ESM.tif
Supplementary Figure S5. Combination of antioxidant agent and carfilzomib (CFZ) in the cisplatin-resistant (CR) variants of A549 and H1299. (a) 1000 mmol/L of glutathione (GSH) or 100 mmol/L of N-acetylcysteine (NAC) reduced intracellular reactive oxygen species levels. The effect of antioxidant agent on the sensitivity of CFZ was analyzed by MTT cell proliferation assay in the CR variants of A549 (b) and H1299 (c). **P<0.01, ***P<0.001, Welch t test. (TIF 481 kb)
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Supplementary Figure S6. Western blot analysis showing the expression of proteins involved in controlling G2/M cell cycle progression in the indicated cells after carfilzomib (CFZ) treatment. (TIF 1062 kb)
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Supplementary Figure S7. Western blot analysis showing the expression of proteins involved in endoplasmic reticulum stress in the indicated cells after carfilzomib (CFZ) treatment. (TIF 924 kb)
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Shoji, T., Kikuchi, E., Kikuchi, J. et al. Evaluating the immunoproteasome as a potential therapeutic target in cisplatin-resistant small cell and non-small cell lung cancer. Cancer Chemother Pharmacol 85, 843–853 (2020). https://doi.org/10.1007/s00280-020-04061-9
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DOI: https://doi.org/10.1007/s00280-020-04061-9