Eunji Im1, Changhwan Yeo1, Eun-Ok Lee2. 1. Department of Cancer Preventive Material Development, Graduate School, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea. 2. Department of Cancer Preventive Material Development, Graduate School, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea; College of Korean Medicine, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea. Electronic address: leook@khu.ac.kr.
Abstract
AIMS: Luteolin, a naturally occurring flavonoid, possesses anti-cancer effects including induction of apoptosis. This study investigated the involvement of osteopontin (OPN) in luteolin-induced apoptosis in human hepatocellular carcinoma (HCC) SK-Hep-1 cells with high OPN expression. MAIN METHODS: MTT assay was used to determine the cell viability. Cell cycle analysis was performed to identify apoptosis. Apoptosis was confirmed by detecting cytoplasmic histone-associated-DNA-fragments using a cell death detection ELISAPLUS kit. The expression of proteins was evaluated by Western blot. Reverse transcriptase-polymerase chain reaction was employed to detect the expression of mRNA. KEY FINDINGS: Cytotoxic effect of luteolin was higher in cancer cell line SK-Hep-1 cells than in normal cell line AML12 cells. Luteolin led a significantly increase in apoptosis accompanied by activation of caspase 8, -9 and -3 and cleavage of poly (ADP-ribose) polymerase (PARP), which was completely blocked by Z-VAD-FMK, a pan caspase inhibitor. Luteolin significantly downregulated the expression of X-linked inhibitor of apoptosis (XIAP), Mcl-1 and Bid. Furthermore, luteolin effectively decreased OPN expression at both mRNA and protein level. Exogenous OPN markedly blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of XIAP and Mcl-1 in luteolin-treated cells. Luteolin impaired the AKT pathway by inhibiting the phosphorylation of AKT. SC79, an AKT activator, blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of OPN, XIAP, Mcl-1 and Bid in luteolin-treated cells. SIGNIFICANCE: These results demonstrated that luteolin inhibits the AKT/OPN pathway, thereby inducing caspase-dependent apoptosis in human HCC SK-Hep-1 cells with little toxicity.
AIMS: Luteolin, a naturally occurring flavonoid, possesses anti-cancer effects including induction of apoptosis. This study investigated the involvement of osteopontin (OPN) in luteolin-induced apoptosis in humanhepatocellular carcinoma (HCC) SK-Hep-1 cells with high OPN expression. MAIN METHODS:MTT assay was used to determine the cell viability. Cell cycle analysis was performed to identify apoptosis. Apoptosis was confirmed by detecting cytoplasmic histone-associated-DNA-fragments using a cell death detection ELISAPLUS kit. The expression of proteins was evaluated by Western blot. Reverse transcriptase-polymerase chain reaction was employed to detect the expression of mRNA. KEY FINDINGS:Cytotoxic effect of luteolin was higher in cancer cell line SK-Hep-1 cells than in normal cell line AML12 cells. Luteolin led a significantly increase in apoptosis accompanied by activation of caspase 8, -9 and -3 and cleavage of poly (ADP-ribose) polymerase (PARP), which was completely blocked by Z-VAD-FMK, a pan caspase inhibitor. Luteolin significantly downregulated the expression of X-linked inhibitor of apoptosis (XIAP), Mcl-1 and Bid. Furthermore, luteolin effectively decreased OPN expression at both mRNA and protein level. Exogenous OPN markedly blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of XIAP and Mcl-1 in luteolin-treated cells. Luteolin impaired the AKT pathway by inhibiting the phosphorylation of AKT. SC79, an AKT activator, blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of OPN, XIAP, Mcl-1 and Bid in luteolin-treated cells. SIGNIFICANCE: These results demonstrated that luteolin inhibits the AKT/OPN pathway, thereby inducing caspase-dependent apoptosis in human HCC SK-Hep-1 cells with little toxicity.
Authors: Heba R Al Refaey; Al-Sayeda A Newairy; Mayssaa M Wahby; Chris Albanese; Mohamed Elkewedi; Muhammad Umer Choudhry; Ahmed S Sultan Journal: Biol Res Date: 2021-05-28 Impact factor: 5.612