Hyperglycemia decreases anti-cancer efficiency of adriamycin via AMPK pathway

in Endocrine-Related Cancer
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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.

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  • 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.

 

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    High glucose attenuates the cytotoxity of ADR in HCC cell lines. (A) Human hepatocellular carcinoma HepG2 and Bel-7402 cells were exposed to ADR as indicated in 5.5, 11 and 25 mM glucose respectively for 48 h. Cell survival was evaluated by SRB assay and the IC50 values were calculated by Calcusyn software. Data are expressed as the mean ± s.d., n = 3. (B) Cell apoptosis was evaluated by Annexin V/PI staining. (C) Whole cell lysates were harvested and determined by Western blotting with antibodies against c-PARP, cleaved caspase-3 and β-actin.

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    The antitumor effect of ADR was decreased in H22 tumor-bearing db/db mice accompanied with hyperglycemia and hyperinsulinemia. (A) H22 mouse hepatocellular carcinoma model was established by subcutaneously inoculating 1 × 106 cells into db/db or C57BL/C mice. After inoculation, db/db and C57BL/C mice were randomized into control and ADR treatment groups respectively according to blood glucose levels. The mice in control groups received NS (i.p.) while mice in the treatment groups received ADR (2 mg/kg, i.p.) every 2 days for 11 days. (B) Non-fasting blood glucose levels were studied on Days 1, 5 and 11 respectively during ADR treatment. (C) On Day 11, mice were killed and tumor weight was measured inhibition ratio was calculated. (D) Apoptosis cells in tumor tissues were detected by TUNEL assay. (E) Blood glucose levels after administration within 24 h were measured and areas under the blood glucose curve (AUC) were calculated using the trapezoidal method. The correlations between blood glucose levels and the anti-cancer activity of ADR were analyzed. (F) Plasma insulin levels on Day 11 were measured by ELISA kit. Data are expressed as the mean ± s.e., n = 6. *P < 0.05, **P < 0.01, ***P < 0.001 vs C57BL/C control. #P < 0.05, ###P < 0.001 vs C57BL/C+ADR 2 mg/kg.

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    Hyperglycemia attenuates the antitumor effect of ADR on mouse hepatoma H22 xenograft in STZ-induced diabetic mice. (A) ICR mice were injected with streptozotocin (i.p.) after fasted overnight to establish a hyperglycemia model. H22 xenografts were established by subcutaneously inoculating cells into STZ-induced diabetic mice (DM mice) or normal mice. After inoculation, normal mice and DM mice were randomized into control and treatment groups to receive the following treatments: NS (i.p.), ADR (2 mg/kg, i.p.), insulin (12.5 IU/kg, s.c.), combination therapy (12.5 IU/kg insulin + 2 mg/kg ADR). (B and C) Non-fasting blood glucose levels and blood glucose levels during 24 h after administration were analyzed during the course. (D) At the end of the treatment on Day 10, mice were killed and the tumors were excised and weighted. (E) Apoptosis cells in tumor tissue were visualized by TUNEL assay. (F) On Day 10, blood was collected and the plasma insulin levels were measured by ELISA kit. Data are expressed as the mean ± s.e., n = 6. **P < 0.01, ***P < 0.001 vs control.

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    AMPK activation modulates ADR-induced DNA damage in hepatoma cells. (A and B) Human hepatocellular carcinoma HepG2 and Bel-7402 cells were seeded in six-well plates and exposed to DMSO or 0.5 μM ADR in different glucose conditions for 2 h. Whole cell lysates were harvested and determined by Western blotting with antibodies against p-AMPK, AMPK, p-ACC, ACC, γ-H2AX and β-actin. (C) HepG2 and Bel-7402 cells were seeded in six-well plates and exposed to 0.5 μM ADR, 1 mM AICAR or the combination of both compounds for 2 h. (D) HepG2 and Bel-7402 cells were seeded in six-well plates and exposed to 0.5 mM ADR, 5 μM compound C or the combination of both compounds for 2 h. After incubation, whole cell lysates were harvested and determined by immunoblotting with antibodies against p-AMPK, γ-H2AX and β-actin. Western blotting bands were quantified by IPP plus 6.0. Data are expressed as the mean ± s.d., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 vs control. #P < 0.05, ##P < 0.01, ###P < 0.001 vs ADR in 5.5 mM glucose conditions.

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    High glucose decreases the cytotoxicity of ADR by downregulating AMPK pathway. (A and B) HepG2 and Bel-7402 cells were pretreated with 5 μM compound C for 2 h. (C and D) HepG2 and Bel-7402 cells were transfected with siRNAs specifically targeting AMPK or control siRNAs. (E and F) HepG2 and Bel-7402 cells were pretreated with 0.5 mM AICAR for 2 h. Then, cells were exposed to ADR as indicated in different glucose conditions for 48 h. Cell-proliferation inhibitory activities were determined by SRB assay. Protein expressions were detected by Western blot analysis.

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