Anticancer activities of the bioactive compounds from Citrus unshiu leaf: molecular targets and mechanisms

Abstract

ABSTRACT Citrus leaves are reputedly general tonics for gastrointestinal disorders and fevers. The leaf extract is consumed as an herbal tea and the leaf hydrosol from numerous Citrus species is used for some traditional dishes in Tunisian cuisine. The appropriate tree canopy for Citrus unshiu should be maintained by skirting and pruning. Therefore, Citrus unshiu leaves are readily available agricultural byproducts. Although there are reports on the biological effects and chemical compositions of certain species of citrus leaves, very limited information about the anticancer properties of Citrus unshiu leaves has been reported. Therefore, Citrus unshiu leaves usages as anticancer reagents in food and pharmaceutical industries has been neglected. In CHAPTER II, we assessed antiproliferative activities of the methanolic extract of Citrus unshiu leaves (MECL) using several cancer cell lines and marked cytotoxicity was observed in AGS human gastric cancer cells. Treatment with MECL induced non-apoptotic cell death and increased formation of the acidic vesicular organelles and LC3 puncta. MECL-induced cell death was significantly reduced by pretreatment with an autophagy inhibitor, 3-methyladeinine. Gas chromatographymass spectrometry identified the major compound of MECL to be phytol (47.35%). In CHAPTER III, we investigated cytotoxic effects of phytol, the major component of MECL, on cancer cells. Phytol induces apoptosis in AGS cells, as evidenced by an increase of the sub-G1 population, activation of caspases, and depolarization of the mitochondrial membrane. Co-treatment with chloroquine (CQ), a lysosomal inhibitor, strongly enhanced phytol-induced apoptosis in AGS cells, suggesting that phytol could induce protective autophagy. Furthermore, N-acetyl-Lcysteine, a reactive oxygen species (ROS) scavenger, increased the phytol-induced cytotoxicity, but decreased the levels of p62 and the formation of the acidic vesicular organelles, suggesting that the Nrf2 pathway was induced by phytol. In CHAPTER IV, we fractionated methanolic extract of Citrus unshiu leaves by using n-hexane, chloroform, ethyl acetate, n-butanol and water; we found 2,4-ditert-butylphenol (DTBP) as the most potent candidate for anticancer agent in Citrus unshiu leaves. DTBP increased acetylation of α-tubulin, p21 and Rb expression, but reduced β-catenin expression and cell proliferation. Increase of α-tubulin acetylation by DTBP resulted in tubulin polymerization, consequently, inducing aberrant mitosis.