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<p>Cantharidin (CTD), a traditional Chinese medicinal with a long history of use, exhibits broad-spectrum antitumor properties. However, its clinical application is severely limited by its inherent toxicity. Cytochrome P450 (CYP) enzymes are major phase I drug-metabolizing enzymes in clinical settings. To date, however, no enzymes responsible for the metabolism of CTD have been reported yet. In this study, specific CYP450 isoforms involved in CTD metabolism were identified and validated. We first performed in vitro experiments using Huh-7 cells to screen for enzymes capable of metabolizing CTD. Subsequent in vivo studies were conducted by tail vein injection of adeno-associated virus (AAV8-CYP2C19) to achieve CYP2C19 overexpression in mice. Mice were then administered CTD (1.5&#xa0;mg/kg) by gavage, and survival was monitored. Cardiac injury was evaluated using Hematoxylin–Eosin (HE) staining and Masson staining, while cardiac function was assessed by echocardiography. Myocardial apoptosis was examined by TUNEL staining and Western blotting. The expression levels of genes encoding related enzymes were measured by Real-Time quantitative PCR. GC–MS was employed to quantify CTD concentrations in cell culture medium, mouse blood, and mouse liver tissues. Additionally, single-nucleotide polymorphism (SNP) analysis was performed on human samples. Our in vitro results identified CYP2C19 as a key enzyme involved in CTD metabolism. Consistent with this, AAV8-CYP2C19‑mediated overexpression in mice significantly accelerated CTD metabolism. CTD administration induced cardiac dysfunction, particularly within 6&#xa0;h of gavage, as evidenced by a rapid decline in cardiac output. Histological analysis revealed myocardial damage characterized by inflammatory cell infiltration and collagen fiber deposition. TUNEL staining and Western blotting further confirmed increased cardiomyocyte apoptosis following CTD exposure. GC–MS analysis demonstrated reduced CTD concentrations in the blood and liver of AAV8‑CYP2C19‑treated mice, which corresponded to alleviated myocardial injury in the AAV8-CYP2C19 + CTD group. In human samples, liver samples from patients who died of CTD-induced toxicity were found to carry the CYP2C19*2 SNP, a variant associated with the slow-metabolizer phenotype. Collectively, our findings demonstrate, for the first time, that CTD is metabolized by CYP2C19. These results suggest that genetic testing for CYP2C19 polymorphisms could be performed prior to CTD-based cancer therapy, thereby facilitating precision medicine approaches that minimize toxicity and improve therapeutic efficacy.</p>

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CYP2C19 as a key enzyme in the metabolism of cantharidin in Huh-7 cells and mice

  • 期刊论文

  • 发表日期：

    2026年3月31日

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  • Jie Zhang,
  • Feng Lyu,
  • Tongtong Tian,
  • Jinghong Ma,
  • Yijia Liu,
  • Yifan Wu,
  • Kaiyin Song,
  • Xiaowei Zhou,
  • Qing K. Wang,
  • Yufeng Yao,
  • Liang Liu

摘要

Cantharidin (CTD), a traditional Chinese medicinal with a long history of use, exhibits broad-spectrum antitumor properties. However, its clinical application is severely limited by its inherent toxicity. Cytochrome P450 (CYP) enzymes are major phase I drug-metabolizing enzymes in clinical settings. To date, however, no enzymes responsible for the metabolism of CTD have been reported yet. In this study, specific CYP450 isoforms involved in CTD metabolism were identified and validated. We first performed in vitro experiments using Huh-7 cells to screen for enzymes capable of metabolizing CTD. Subsequent in vivo studies were conducted by tail vein injection of adeno-associated virus (AAV8-CYP2C19) to achieve CYP2C19 overexpression in mice. Mice were then administered CTD (1.5 mg/kg) by gavage, and survival was monitored. Cardiac injury was evaluated using Hematoxylin–Eosin (HE) staining and Masson staining, while cardiac function was assessed by echocardiography. Myocardial apoptosis was examined by TUNEL staining and Western blotting. The expression levels of genes encoding related enzymes were measured by Real-Time quantitative PCR. GC–MS was employed to quantify CTD concentrations in cell culture medium, mouse blood, and mouse liver tissues. Additionally, single-nucleotide polymorphism (SNP) analysis was performed on human samples. Our in vitro results identified CYP2C19 as a key enzyme involved in CTD metabolism. Consistent with this, AAV8-CYP2C19‑mediated overexpression in mice significantly accelerated CTD metabolism. CTD administration induced cardiac dysfunction, particularly within 6 h of gavage, as evidenced by a rapid decline in cardiac output. Histological analysis revealed myocardial damage characterized by inflammatory cell infiltration and collagen fiber deposition. TUNEL staining and Western blotting further confirmed increased cardiomyocyte apoptosis following CTD exposure. GC–MS analysis demonstrated reduced CTD concentrations in the blood and liver of AAV8‑CYP2C19‑treated mice, which corresponded to alleviated myocardial injury in the AAV8-CYP2C19 + CTD group. In human samples, liver samples from patients who died of CTD-induced toxicity were found to carry the CYP2C19*2 SNP, a variant associated with the slow-metabolizer phenotype. Collectively, our findings demonstrate, for the first time, that CTD is metabolized by CYP2C19. These results suggest that genetic testing for CYP2C19 polymorphisms could be performed prior to CTD-based cancer therapy, thereby facilitating precision medicine approaches that minimize toxicity and improve therapeutic efficacy.