Dual regulating of mitochondrial fusion and Timp-3 by leflunomide and diallyl disulfide combination suppresses diethylnitrosamine-induced hepatocellular tumorigenesis in rats

Life Sci. 2022 Apr 1:294:120369. doi: 10.1016/j.lfs.2022.120369. Epub 2022 Feb 1.


Aims: Hepatocellular carcinoma (HCC) is considered one of the main causes of cancer-related death globally. Combination therapy targeting different pathways can improve the efficacy of HCC management. Mitofusin 2 (Mfn2), a mitochondrial fusion protein, and a tissue inhibitor of matrix metalloproteinase 3 (Timp-3) were found to be downregulated in various cancers, including HCC. Our study aimed to evaluate the possible antineoplastic effect of a novel combination in the treatment of HCC through targeting mitochondrial fusion and metastatic proteins.

Main methods: HCC induction was performed using a single intraperitoneal dose of diethylnitrosamine (200 mg/kg), followed by adding phenobarbital sodium (0.05%) to the drinking water for successive 18 weeks. Then, leflunomide (LF, 10 mg/kg) was administered orally for 28 days. Diallyl disulfide (DADS, 50 mg/kg) was also given orally for 28 days, either alone or in combination with LF.

Key findings: Treatment with LF or DADS could alleviate the HCC- induced histological and biochemical variations, including liver enzyme activities (ALT, AST), alpha-fetoprotein, Bax, cyclin D1, Ki67, malondialdehyde, and reduced glutathione. They could shift the mitochondrial dynamics toward mitochondrial fusion through upregulating the expression of Mfn2 and also exhibited antimetastatic activity through upregulating the expression of Timp-3 and decreasing hepatic MMP9 content.

Significance: the treatment with a combination of LF and DADS displayed a more potent effect than the treatment with each drug alone. Our results suggest that the combined use of LF and a naturally occurring DADS can be used as a promising novel combination in managing HCC.

Keywords: Diallyl disulfide; HCC; Leflunomide; Mitochondrial fusion; Mitofusin 2; Timp-3.

MeSH terms

  • Alkylating Agents / toxicity
  • Allyl Compounds / pharmacology*
  • Animals
  • Antineoplastic Agents / pharmacology
  • Carcinoma, Hepatocellular / chemically induced
  • Carcinoma, Hepatocellular / metabolism
  • Carcinoma, Hepatocellular / pathology
  • Carcinoma, Hepatocellular / prevention & control*
  • Diethylnitrosamine / toxicity
  • Disulfides / pharmacology*
  • Drug Therapy, Combination
  • Gene Expression Regulation, Neoplastic / drug effects*
  • Immunosuppressive Agents / pharmacology
  • Leflunomide / pharmacology*
  • Liver Neoplasms, Experimental / chemically induced
  • Liver Neoplasms, Experimental / metabolism
  • Liver Neoplasms, Experimental / pathology
  • Liver Neoplasms, Experimental / prevention & control*
  • Male
  • Mitochondrial Dynamics / drug effects*
  • Rats
  • Rats, Wistar
  • Tissue Inhibitor of Metalloproteinase-3 / genetics
  • Tissue Inhibitor of Metalloproteinase-3 / metabolism*


  • Alkylating Agents
  • Allyl Compounds
  • Antineoplastic Agents
  • Disulfides
  • Immunosuppressive Agents
  • Tissue Inhibitor of Metalloproteinase-3
  • Diethylnitrosamine
  • diallyl disulfide
  • Leflunomide