Abstract
Most chemotherapeutic agents for leukemia are DNA damaging agents. However, DNA lesions can be repaired by activities of DNA repair systems. Increasing evidence have shown that enhanced DNA damage repair capacity contributes to chemotherapy resistance in leukemia cells. Thus, targeting DNA repair mechanisms is a promising strategy for novel leukemia treatment. SIRT1 expressions were downregulated by lentivirus-delivered SIRT1 shRNA in myeloid leukemia cells. SIRT1 mRNA and protein levels were analyzed by real-time PCR and Western blot, respectively. Flow cytometry was carried out to analyze cell cycle progression, apoptosis and DNA damage repair efficiency. DNA damage levels were assessed by alkaline comet assay, and H2AX phosphorylation was analyzed by immunoblotting and immunofluorescence. A mouse leukemia model was established by transplanting lentivirus-infected K562 cells containing SIRT1 shRNA into sublethally irradiated NOD/SCID mice, and tumorigenesis was evaluated by detecting tumor weights and mice survival. SIRT1 expressions were upregulated in myeloid leukemic patients. Downregulation of SIRT1 by RNAi promoted etoposide-induced DNA damage in myeloid leukemia cells accompanied by reduced NHEJ activity, and increased Ku70 acetylation. Furthermore, SIRT1 knockdown resulted in cell cycle arrest, induction of apoptosis and reduction of K562 cell proliferation accompanied by enhanced p53 and FOXO1 acetylation in K562 cells after etoposide treatment. Importantly, SIRT1 downregulation reduced the tumorigenesis ability of K562 cells in mouse xenografts following chemotherapy treatment. These results revealed that SIRT1 promotes the NHEJ repair pathway by deacetylating Ku70 in K562 cells, suggesting that SIRT1 is a novel therapeutic target for treating myeloid leukemia.
Highlights
RESULTSDNA damage-based chemotherapy is currently the first choice for treating leukemia
Different types of DNA damage are repaired by distinct repair systems such as base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR) and nonhomologous end joining (NHEJ) [2]
SIRT1 knockdown resulted in cell cycle arrest, induction of apoptosis, and reduction of cell proliferation in K562 cells accompanied by enhanced p53 and FOXO1 acetylation in myeloid leukemia cells after etoposide treatment
Summary
DNA damage-based chemotherapy is currently the first choice for treating leukemia. DNA damage leads to cell cycle arrest and cell death. Further immunofluorescence staining demonstrated an increased number of γ-H2AX foci in K562 cells infected with shSIRT1-KD, compared with that of cells infected with shRNA-NC following etoposide treatment (P < 0.05, Figure 2C) These results clearly demonstrate that the silencing of SIRT1 lead to enhanced DNA damage in response to etoposide treatment in K562 www.impactjournals.com/oncotarget. Following treatment with 20 μM of etoposide for four hours, a significant increase in apoptotic rate was observed in the shSIRT1-KD group compared with the shRNA-NC group after 48 hours (P < 0.05); demonstrating that SIRT1 knockdown enhanced cell apoptosis in response to etoposide treatment in K562 cells. The group engrafted with shSIRT1-KD K562 cells had a longer life-span than the shRNA-NC group after etopside treatment(Figure 5D) These results revealed that SIRT1 inhibition effectively reduced the tumorigenesis ability of K562 cells in vivo. SIRT1 knockdown increased FOXO1 acetylation without affecting the total FOXO1 protein expression (Figure 6E)
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