1 Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, Institute of Pharmacology & Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China
2 Department of Clinical Pharmacology, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
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Accumulating clinical evidence indicates that diabetic liver cancer patients are less sensitive to intra-arterial chemotherapy than non-diabetic cancer patients. However, the underlying mechanism remains largely uncharacterized. Here, we report that hyperglycemia inhibits AMPK pathway and subsequently reduces adriamycin (ADR)-induced DNA damage, resulting in decreased chemotherapeutic sensitivity of ADR. HepG2 and Bel-7402 cells were treated with ADR in various glucose conditions and then subjected to cell proliferation assay and apoptosis. The IC50 of ADR greatly increased with the increasing concentration of glucose (15 ± 4 nM to 93 ± 39 nM in HepG2, 78 ± 8 nM to 1310 ± 155 nM in Bel-7402). Both FACs and Western blot analysis indicated that high concentration of glucose protected cells from ADR-induced apoptosis. Mouse hepatoma H22 xenografts were established both in db/db diabetic mice and STZ-induced diabetic mice. The inhibitory effect in tumor growth of ADR was significantly reduced in diabetic mice, which could be recovered by insulin therapy. Hyperglycemia greatly ameliorated AMPK activation and H2AX expression caused by ADR treatment. Pretreatment with compound C or AMPK silencing eliminated hyperglycemia reduced cytotoxicity of ADR. However, the impaired cytotoxicity in hyperglycemia was recovered by treatment with AMPK activator AICAR. This study indicates that hyperglycemia impairs the chemotherapeutic sensitivity of ADR by downregulating AMPK pathway and reducing ADR-induced DNA damage.
Figure S1. The different concentrations of mannitol did not affect the cytotoxicity of ADR in HepG2 cells and Bel-7402 cells. (A,B) Cells were treated with ADR as indicated in 5.5 mM,11 mM and 25mM mannintol respectively for 24 h(A) and 48 h(B), and cell survival was examined by SRB. HepG2(C) cells and Bel-7402(D) cells were exposed to ADR in 5.5 mM, 11mM and 25mM glucose conditions respectively for 24h, and cell survival was analyzed by SRB. Data are showed as the mean±SD, n=3.
Figure S2. The body weight of two animal models. (A) The body weight of the db/db or C57BL/C mice which were inoculated with the H22 cells and treated with the ADR during the course. (B) The body weight of the ICR mice and STZ-induced diabetic mice (DM mice) or normal mice which were inoculated with the H22 cells and treated with the ADR.
Figure S3. The effects on ADR cytotoxicity by modulation the GLUT2 expression. (A,C) Bel-7402 cells and HepG2 cells were transfected with siRNAs targeting GLUT2 or control siRNAs and 12 h later the cells were exposed to ADR in 5.5 mM,11 mM and 25Mm glucose conditions respectively for 24 h. (B,D)Bel-7402 cells and HepG2 cells were transfected with GLUT2 plasmid for 24 h, then they were exposed to ADR in various glucose conditions for 24 h, and cell-survival fraction were examined by SRB. Data are expressed as the mean±SD, n=3.