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. 2020 Nov 25;23(1):80–98. doi: 10.1016/j.neo.2020.11.004

Figure 4.

Figure 4:

Regulation of DNA damage repair related genes by bortezomib-combined chemotherapy in vitro: BON cells were either treated with 25 nM bortezomib, 10 µM cisplatin, combination of both or control for 24 h in vitro and analyzed by nCounter mRNA expression array. (A) Cisplatin induced the upregulation of several DNA repair genes whereas (B) Addition of bortezomib reversed the effect and triggered the downregulation of identical genes and genes associated with the same repair pathways, respectively. Arrows exemplarily mark DDB2, RAD51, FEN1, and BRCA1. (C) BON cells were treated with 25 nM bortezomib, 10 µM cisplatin, combination of both or control for 24 h and analyzed by western blot. Crucial DNA damage repair proteins were downregulated after bortezomib and combined treatment. These include proteins of the HR (RPA, SKP2), FA (FANCA), MMR (RPA, RFC), BER (FEN1) and primarily of the NER (RPA, DDB2, XPA, XPD, ERCC1) pathways of DNA repair. These effects strongly induced DNA damage stress, indicated by histone H2A.X phosphorylation. Representative data of three independent experiments are shown. Corresponding data obtained from KRJ-I and LCC-18 cells are presented in Supplementary Figure 2. (D) Bortezomib affected important molecules of the human response to DNA damage: Alkylating agents, ionizing radiation (IR), ultra violet (UV) radiation, crosslinking agents (e.g., platin-based chemotherapeutics), inhibitors of topoisomerases (TOPOi), PRRT or nucleoside analogs induce different DNA lesions (list is not exhaustive). Major pathways of DNA repair, which depend on the type of damage and the cell cycle phase, are direct repair (e.g., by methyltransferases), base excision repair (BER), nucleotide excision repair (NER), interstrand crosslink repair (ICLR), nonhomologous end joining (NHEJ), homologous recombination (HR) and mismatch repair (MMR). Bolded proteins have been shown to be downregulated by the proteasome inhibitor bortezomib. BER, Base Excision Repair; FA, Fanconi Anemia; HR, Homologous Recombination; MMR, Mismatch Repair; NER, Nucleotide Excision Repair.