Fig. 4: BCAT2 participates in the mechanisms for sulfasalazine synergizing with sorafenib to induce ferroptosis in vivo.

A Schematic representation of the in vivo sorafenib and sulfasalazine combination anticancer effect in Panc02 subcutaneous tumor model. C57BL/6 mice were injected subcutaneously with 1 × 10⁶ Panc02 cancer cells and treated with DMSO (control group), sorafenib (10 mg/kg/i.p., every 2 days), sulfasalazine (100 mg/kg/i.p., every 2 days), or sorafenib (10 mg/kg/i.p., every 2 days) + sulfasalazine (100 mg/kg/i.p., every 2 days) for 2 weeks (n = 5 mice/group). B Representative photographs of isolated tumor tissues at day 14 after treatment. C Tumor volume was calculated every 2 days. D Schematic representation of the in vivo sorafenib and sulfasalazine combination anticancer effect in H22 orthotopic xenograft tumor model. 1 × 10⁶ H22 cells were injected into left lobe of C57BL/6 mice livers and following treated with DMSO (control group), sorafenib (30 mg/kg/i.g., every 2 days), sulfasalazine (300 mg/kg/i.g., every 2 days), or sorafenib (10 mg/kg/i.g., every 2 days) + sulfasalazine (300 mg/kg/i.g., every 2 days) for 2 weeks (n = 10 mice/group). E Representative photographs of isolated tumor tissues following various treatments. F Animal survival was calculated every day for 2 months (Kaplan–Meier survival analysis). G Schematic representation of established PDX hepatocellular carcinoma models was treated with DMSO (control group), sorafenib (10 mg/kg/i.p., every 2 days), sulfasalazine (100 mg/kg/i.p., every 2 days), or sorafenib (10 mg/kg/i.p., every 2 days) + sulfasalazine (100 mg/kg/i.p., every 2 days) for 2 weeks. H Representative photographs of isolated tumor tissues at day 40 after treatment. I Tumor weight of isolated tumor tissues at day 40 after treatment. J Tumor volume was calculated every 3 days. K MDA levels in isolated tumors at day 40 after treatment were assayed. K MDA levels in isolated tumors at day 40 after treatment were assayed. L Western blot analysis of the protein expression level of BCAT2 in isolated tumor tissues at day 40 after treatment. M qRT-PCR analysis of mRNA-expression level of BCAT2 in isolated tumor tissues at day 40 after treatment. N Immunohistochemistry analysis of the expression of BCAT2 in isolated tumor tissues at day 40 after treatment. MDA represents malondialdehyde; i.p. represents intraperitoneal injection; i.g. represents intragastrical administration; SOR represents sorafenib; SAS represents sulfasalazine. Experiments were repeated three times, and the data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group. Statistical analysis was performed using Student’s t test.