Anti-carcinogenic effects and mechanisms of actions of Citrus limon fruit peel hydroethanolic extract and limonene in diethylnitrosmine/2-acetylaminofluorene-induced hepatocellular carcinoma in Wistar rats.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death and disability in the world. Citrus species and their constituents have many biological activities including antioxidant, anti-inflammatory and anti-carcinogenic properties. This study aimed to assess the anti-carcinogenic effects and postulate the possible mechanisms of action for Citrus limon fruit peel hydroethanolic extract (CLFPHE) and limonene in diethylnitrosamine (DEN)/2-acetylaminofluorene (2AAF)-induced HCC in male Wistar rats. For analysis and characterization of CLFPHE, gas chromatography-mass spectrum (GC-MS) and high performance liquid chromatography (HPLC) methods were applied. A HCC was elaborated by DEN intraperitoneal injection (150 mg/kg/week) for two weeks followed by oral delivery of 2AAF (20 mg/kg) four times a week for three weeks. The DEN/2AAF-administered rats were treated with CLFPHE (50 mg/kg) and limonene (20 mg/kg) by oral gavage every other day for 24 weeks. CLFPHE and limonene significantly attenuated the harmful effects of DEN on liver function. Histopathological analysis confirmed that both treatments inhibited DEN/2AAF-induced tumorigenesis in association with the suppression of serum tumor markers including AFP, CEA, and CA19.9 and liver proliferator indicator (Ki-67). Moreover, CLFPHE and limonene prevented the oxidative stress and enhanced the antioxidant defenses in DEN/2AAF-administered rats. These ameliorations were manifested by decreases in liver lipid peroxidation, increases in GSH, SOD and GPx levels and upregulation of Nrf2. The treatments also abated inflammation by suppressing TNF-α and IL-1β levels and IL-8 and NF-κB expression. CLFPHE and limonene substantially decreased hepatic BCL-2, IQGAP1, IQGAP3, HRAS, KRAS and Ki-67 while they elevated BAX, P53, PDCD5 and IQGAP2 expressions. Our findings suggest that CLFPHE and limonene may abate HCC development via enhancement of apoptotic, antioxidant, cell anti-proliferatory and anti-inflammatory effects.