Overexpressed SIRT6 ameliorates doxorubicin-induced cardiotoxicity and potentiates the therapeutic efficacy through metabolic remodeling

Figures

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  Figure 1. Decreased SIRT6 expression and increased lactate accumulation were observed in the plasma of anthracyclines-treated cancer patients. (A) Ten breast cancer patients received postoperative adjuvant therapy with anthracyclines-based chemotherapy regimens, and plasma was collected before and after chemotherapy from same patients. SIRT6 expression in the plasma of cancer patients was measured by ELISA (n = 10 patients). (B) Lactate concentration in the plasma of cancer patients was measured (n = 10 patients). The data are expressed as the mean ± standard deviation. Two-tailed paired t tests were performed to determine significant differences. ∗∗∗P < 0.001.

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  Figure 2. Ectopic SIRT6 overexpression ameliorates Dox-induced cytotoxicity in cardiomyocytes but exacerbates Dox-induced cytotoxicity in cancer cells. (A, B) After treatment with different concentrations of doxorubicin (Dox) for 24 h, the viability of primary cardiomyocytes and cardiac fibroblasts isolated from SIRT6 transgenic (TG) mice and wild-type (WT) littermates, mouse cardiomyocyte HL-1 cells, mouse Lewis lung carcinoma LLC cells, mouse breast cancer 4T1 cells, and mouse melanoma B16-F10 cells infected with lentivirus containing SIRT6 vector (Lenti-SIRT6) or control vector (Lenti-Ctrl) was assessed by the CCK-8 method (n = 3 different experiments). (C, D) The protein expression of SIRT6 in HL-1 cells, primary cardiomyocytes, cardiac fibroblasts, LLC cells, 4T1 cells, and B16-F10 cells was detected by Western blotting. β-Actin was used as a loading control. (E) Semiquantitative analyses of bands were performed using ImageJ software (n = 3 different experiments). The data are expressed as the mean ± standard deviation. Two-tailed unpaired t tests were performed to determine significant differences. ∗∗∗P < 0.001 compared to the control group.

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  Figure 3. SIRT6 overexpression ameliorates Dox-induced cardiotoxicity in mice. (A) The chronic low-dose treatment protocol and acute high-dose treatment protocol are shown. In the chronic treatment protocol, SIRT6 transgenic mice and wild-type littermates were injected with Dox (4 mg/kg/week, i.v.) or saline for 5 weeks, and the total cumulative dose was 20 mg/kg. In the acute treatment protocol, wild-type and SIRT6 transgenic mice were injected with Dox (20 mg/kg/week, i.v.) or saline for 2 weeks. (B) In the chronic treatment protocol, biochemical parameters associated with heart failure were measured in the plasma of the mice. The levels of CK-MB and LDH1 are shown in Fig. S1 (n = 6 or 10 animals per group). (C, D) In the chronic treatment protocol, cardiac function was measured using echocardiography and was assessed by ejection fraction, fractional shortening, diastolic left ventricular anterior wall thickness, and diastolic left ventricular internal diameter (n = 6 or 10 animals per group). (E) In the chronic treatment protocol, fibrosis, apoptosis and cell morphology in cardiac tissue were measured by Masson trichrome staining, TUNEL and H&E staining, respectively. Representative…

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  Figure 4. SIRT6 overexpression potentiates antitumor efficacy of Dox in mice. (A) The treatment protocol is shown. Wild-type C57BL/6 mice were inoculated subcutaneously with 1.5 × 106 LLC cells with lentivirus-mediated SIRT6 overexpression (Lenti-SIRT6) or control LLC cells (Lenti-Ctrl). After tumors became visible (approximately 7 days), the tumor-bearing mice were treated with Dox (10 mg/kg/week, i.v.) for 2 weeks, and the total cumulative dose was 20 mg/kg. (B, C) Tumor volume was visualized using the IVIS Imaging System before and after treatment, and representative bioluminescence images and quantitative analyses of the tumor-bearing mice are shown (n = 3 or 5 animals per group). (D, E) At treatment termination, tumors from each mouse were excised and weighed, and photographs of the tumors are shown (n = 6 or 9 animals per group). (F) Cell morphology, proliferative activity and apoptosis and in the tumors were measured by H&E, Ki67 immunofluorescence and TUNEL staining, respectively. Representative images are shown. Scale bar = 50 μm. (G) Quantitative analyses of the fluorescence of Ki67 (red-stained) and TUNEL-positive cells (brown-stained) in F were performed by ImageJ software (n =…

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  Figure 5. Dox impairs mitochondrial respiration and mtDNA integrity to induce cytotoxicity in cardiomyocytes and cancer cells. (A) RNA sequencing was performed on primary cardiomyocytes isolated from WT mice, after treated with Dox (0.5 μmol/L) or solvent for 24 h. Differentially expressed genes are shown in volcano plots. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes. (C, D) The oxygen consumption rate (OCR) was measured by a Seahorse XF96 extracellular-flux analyzer after cardiomyocytes and LLC cells were treated with Dox (0.5 μmol/L) for 12 h (n = 3 different experiments). (E, F) The extracellular acidification rate (ECAR) was measured by a Seahorse XF96 extracellular-flux analyzer after cardiomyocytes and LLC cells were treated with Dox (0.5 μmol/L) for 12 h (n = 3 different experiments). (G, H) Representative electron microscopic images of cardiomyocytes and LLC cells after Dox treatment (0.5 μmol/L) for 24 h are shown. Scale bar = 1 μm. The number of abnormal mitochondria was quantified blindly in 5 images from different fields. (I) Mitochondrial DNA (mtDNA) integrity was determined after cardiomyocytes and LLC cells were…

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  Figure 6. SIRT6 overexpression differentially regulates ATP production in cardiomyocytes and cancer cells during Dox treatment by enhancing mitochondrial respiration and inhibiting glycolysis. (A, B) Primary cardiomyocytes isolated from SIRT6 TG and WT littermates, LLC cells with lentivirus-mediated SIRT6 overexpression (Lenti-SIRT6) and control LLC cells (Lenti-Ctrl) were treated with Dox (0.5 μmol/L) for 12 h, and the oxygen consumption rate (OCR) was measured by a Seahorse XF96 extracellular-flux analyzer, (C, D) the lactate concentration was measured, (E–H) and the real-time ATP production rate assay was performed by a Seahorse XF96 extracellular-flux analyzer (n = 3 different experiments). The data are expressed as the mean ± standard deviation. One-way ANOVA followed by Bonferroni multiple comparisons were performed to determine significant differences. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 compared to the control group. #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to the Dox-treated group.

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  Figure 7. SIRT6 deacetylates and inhibits Sgk1 to regulate mitochondrial biogenesis and mitophagy. (A) RNA sequencing was performed on primary cardiomyocytes isolated from SIRT6 TG and WT littermates. The differentially expressed genes were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. (B) The heatmap that was created from the clustering of 14 enriched genes in the PI3K/Akt signaling pathway, which as the most highly enriched pathway identified by KEGG analysis. (C, D) Western blotting analysis of SGK1 in (C) the cardiac tissue of SIRT6 TG mice and WT littermates, as well as SIRT6-knockout (KO) mice and WT littermates, (D) LLC cells with lentivirus-mediated SIRT6 overexpression (Lenti-SIRT6) and control LLC cells (Lenti-Ctrl), and LLC cells transfected with SIRT6 siRNA or control siRNA. β-Actin was used as a loading control. Semiquantitative analyses of bands were performed using ImageJ software (n = 3 different experiments). (E, F) Mitochondrial mass as assessed by mitochondrial DNA (mtDNA) copy number was measured in (E) cells infected with lentivirus containing the SGK1 vector or control vector and (F) cells transfected with SGK1 siRNA or control siRNA (n…

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  Figure 8. Ellagic acid-induced SIRT6 overexpression ameliorates Dox-induced cytotoxicity in cardiomyocytes but exacerbates Dox-induced cytotoxicity in cancer cells. (A) The 2D structure of ellagic acid. (B) The 3D binding model of ellagic acid and SIRT6. The ellagic acid is colored in cyan. The surrounding residues in the binding pocket are colored in yellow. The backbone of the receptor is depicted as gray cartoon. The hydrogen bond and Pi–Pi stacking interaction are depicted as green dashed lines. (C, D) After treatment with different concentrations of ellagic acid for 24 h, the expression of SIRT6, SGK1, H3K9 and H3K56 in primary cardiomyocytes isolated from WT mice and LLC cells was examined by Western blotting. β-Actin was used as a loading control. Semiquantitative analyses of bands were performed using ImageJ software (n = 3 different experiments). (E, F) Primary cardiomyocytes isolated from WT mice and LLC cells were transfected with SIRT6 siRNA or control siRNA. After pre-treated with ellagic acid (10 μmol/L) for 24 h, Dox (0.5 μmol/L) was added to culture medium for subsequent 24 h, and apoptosis was analyzed by TUNEL assays. Representative images are shown (scale bar = 100 μm).…

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  Figure 9. Ellagic acid alleviates Dox-induced cardiotoxicity and potentiates Dox-mediated tumor regression in mice. (A) The treatment protocol is shown. Wild-type C57BL/6 mice were inoculated subcutaneously with 1.5 × 106 LLC-Luc cells (LLC cells stably expressing the luciferase gene). After tumors became visible (approximately 7 days), the tumor-bearing mice were treated with Dox (10 mg/kg/week, i.v.) or/and ellagic acid (100 mg/kg/2 days, i.g.) for 2 weeks. (B) Cardiac function was measured using echocardiography as assessed by ejection fraction and fractional shortening in the mice. The diastolic left ventricular anterior wall thickness and diastolic left ventricular internal diameter are shown in Supporting Information Fig. S8 (n = 6 or 10 animals per group). (C, D) Tumor volume was visualized using the IVIS Imaging System before and after treatment, and representative bioluminescence images and quantitative analyses of the tumor-bearing mice are shown (n = 3 or 5 animals per group). (E) At treatment termination, tumors from each mouse were excised and weighed, and photographs of the tumors are shown in Fig. S8 (n = 6 or 10 animals per group). (F) Cell morphology, fibrosis and…

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  Figure 10. Schematic framework of the SIRT6-mediated differential responses to Dox between cardiomyocytes and cancer cells. Dox-impaired mitochondrial DNA led to metabolic remodeling of decreased mitochondrial respiration and increased glycolysis, thus inhibiting cellular ATP production, which were responsible for Dox-induced cytotoxicity. SIRT6 has been reported to function as a histone H3K9 deacetylase and inhibits the transcription of multiple glycolytic genes. Meanwhile, SIRT6 regulated mitochondrial biogenesis and mitophagy to maintain mitochondrial homeostasis by deacetylating and inhibiting SGK1, thus improving mitochondrial respiration when mitochondrial function is impaired by Dox. Thus, the differences in metabolic patterns between cardiomyocytes and cancer cells are responsible for the differential regulation of SIRT6 on energy production. In cardiomyocytes that depend on mitochondrial respiration for energy production, SIRT6 overexpression reversed the Dox-induced reduction in ATP production and improved cellular energy supply by increasing mitochondrial respiration. In cancer cells that depend on glycolysis for energy production, SIRT6 overexpression enhanced the Dox-induced…