Fig 2: Vandetanib induced hepatocyte apoptosis by upregulating CTSB. (A) GO enrichment analysis of alterable protein expression after vandetanib's treatment. The red font highlighted parts mainly divided into apoptosis and lysosome pathways. (B) GSEA plots for apoptosis and lysosome KEGG pathways significantly enriched after vandetanib's treatment. (C) The mRNA levels of Ctsb and Ctsl in liver tissues were measured by qRT-PCR. (n = 5). (D) HL-7702 cells were treated with 0, 10, 20 and 30 μM vandetanib for 24 h. The mRNA levels of CTSB and CTSL were measured by qRT-PCR. (n = 3). (E) The protein levels of CTSB and CTSL in liver lysates were measured by western blot. (n = 6). (F) Human primary hepatocytes (HPH), murine primary hepatocytes (MPH), and AML12 cells were treated with 20 μM vandetanib for 0, 12, 24, 36 h or 0, 10, 20, 30 μM vandetanib for 36 h. The expression levels of c-PARP and CTSB were measured by western blot. (G-I) HL-7702 cells were transfected with vector, 0.5, 1 or 1.5 μg pcDNA3.0-CTSB-Flag plasmid for 36 h. (G) The survival rates were measured by SRB staining. (n = 3). (H) The apoptosis rates were detected by flow cytometry of Annexin V/PI staining. (n = 3). (I) The expression levels of c-PARP in total cell lysates were detected by western blot. (J-K) HL-7702 cells were transfected with negative control or siRNA targeting CTSB, and then treated with or without 20 μM vandetanib for 36 h. (J) The expression levels of c-PARP and CTSB in HL-7702 cells were measured by western blot. (K) The apoptosis rates were detected by flow cytometry of Annexin V/PI staining. Data are represented as the mean ± SD. ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired two-sided Student's t test for (C) and (E). One way ANOVA followed by Dunnett T3 post hoc test for (D), (G) and (H). One way ANOVA followed by Tukey post hoc test for (K).

Fig 3: Vandetanib induced lysosomal damage via CTSB-mediated cleavage of MCOLN1. (A) The expression levels of CTSB and c-MCOLN1 in liver tissues of mice. (n = 6). (B) The expression levels of p-AMPK Thr172/183 in liver tissues of mice. (n = 6). (C) The expression levels of Galectin-3 and LAMP1 in HL-7702 cells treated with 20 μM vandetanib for 36 h were measured by immunofluorescence. Scale bar, 20 μm. (D-E) HL-7702 cells were transfected with negative control or siRNA targeting CTSB, and then treated with 20 μM vandetanib for 36 h. (D) Representative images of Lyso-Tracker staining in HL-7702. Scale bar, 20 μm. (E) The expression levels of Galectin-3 and LAMP1 in HL-7702 cells were measured by immunofluorescence. Scale bar, 20 μm. (F) HL-7702 cells were treated with 20 μM vandetanib for 36 h. The expression levels of LC3-II in HL-7702 cells were measured by immunofluorescence. Scale bar, 25 μm. (G) The expression levels of SQSTM1 and LC3-I/II in HL-7702 cells treated with 20 μM vandetanib for 0, 10, 20, 30 μM vandetanib for 36 h were detected by western blot. (H) The expression levels of LC3-II and SQSTM1 in liver tissues of mice were detected by immunohistochemical analysis. Scale bar, 100 μm. (I) Representative confocal fluorescence micrographs of HL-7702 cells transfected with Ad-mCherry-GFP-LC3B and treated with 0, 10, 20, 30 μM vandetanib for 24 h. Scale bar, 20 μm. (J) The expression levels of c-PARP and LC3-I/II in HL-7702 cells treated with 20 μM vandetanib for 0, 3, 6, 9, 12 and 24 h were detected by western blot. Data are represented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired two-sided Student's t test for (A), (B) and (H).

Fig 4: Knockdown of CTSB ameliorated vandetanib-induced hepatotoxicity in vivo. (A-D) C57BL/6 mice were randomly divided into 4 groups. After injection of AAV9-shCtsb adeno virus by tail vein for 3 weeks, mice were treated with 0.5% CMC-Na or 100 mg/kg/day vandetanib by gavage for another 4 weeks. (A) (Left panel) Representative photographs of mice liver. (Right panel) Representative images of H&E staining in liver tissues. Scale bar, 100 μm. (B) The levels of serum ALT and AST. (n = 8). (C) The expression levels of cleaved Caspase 3, p-AMPK Thr172/183, CTSB and LC3-II in liver tissues were detected by immunohistochemical analysis. Scale bar, 100 μm. (D) The expression levels of c-PARP, p-AMPK Thr172/183, AMPK, CTSB and LC3-I/II in liver tissues were measured by western blot. (n = 4). (E) Representative images of TUNEL staining in liver tissues. Scale bar, 100 μm. Data are represented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. One way ANOVA followed by Tukey post hoc test for (B), (C), (D) and (E).

Fig 5: Targeting CTSB alleviated vandetanib-induced cardiac injury. (A-E) C57BL/6 mice were randomly divided into 4 groups (n = 8 per group). After injection of AAV9-shCtsb adeno virus by tail vein for 3 weeks, mice were treated with 0.5% CMC-Na or 100 mg/kg/day vandetanib by gavage for another 4 weeks. (A) Representative M-mode echocardiogram images. (B) Quantifications of Ejection fraction and Fractional shortening ratios. (C) (Upper photos) Representative photographs of heart tissues. Scale bar, 500 μm. (Lower photos) H&E staining of heart tissues. Scale bar, 50 μm. (D) Representative images of immunohistochemistry for cleaved Caspase 3 staining in heart tissues. Scale bar: 50 μm. (E) Total RNA was extracted from mice hearts and the expression levels of Myh6, Myh7, Nppa and Nppb were measured by qRT-PCR. (F-J) C57BL/6 mice were randomly divided into 4 groups (n = 6 per group). The C57BL/6J mice were received 100 mg/kg vandetanib and/or 30 mg/kg tannic acid for 4 weeks. (F) Representative M-mode echocardiogram images. (G) Quantifications of Ejection fraction and Fractional shortening ratios. (H) (Upper photos) Representative photographs of heart tissues. Scale bar, 500 μm. (Lower photos) H&E staining of heart tissues. Scale bar, 50 μm. (I) Representative images of immunohistochemistry for cleaved Caspase 3 staining in heart tissues. Scale bar: 50 μm. (J) Total RNA was extracted from mice hearts and the expression levels of Myh6, Myh7, Nppa and Nppb were measured by qRT-PCR. Data are represented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. One way ANOVA followed by Tukey post hoc test for (B), (E), (G) and (J).