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Chalcone-Induced Apoptosis through Caspase-Dependent Intrinsic Pathways in Human Hepatocellular Carcinoma Cells.

ABSTRACT: Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers worldwide. Chemoprevention of HCC can be achieved through the use of natural or synthetic compounds that reverse, suppress or prevent the development of cancer progression. In this study, we investigated the antiproliferative effects and the mechanism of action of two compounds, 2,3,4'-trimethoxy-2'-hydroxy-chalcone (CH1) and 3'-bromo-3,4-dimethoxy-chalcone (CH2), over human hepatoma cells (HepG2 and Huh-7) and cultured mouse hepatocytes (HepM). Cytotoxic effects were observed over the HepG2 and Huh-7, and no effects were observed over the HepM. For HepG2 cells, treated separately with each chalcone, typical apoptotic laddering and nuclear condensation were observed. Additionally, the caspases and Bcl-2 family proteins activation by using Western blotting and immunocytochemistry were studied. Caspase-8 was not activated, but caspase-3 and -9 were both activated by chalcones in HepG2 cells. Chalcones also induced reactive oxygen species (ROS) accumulation after 4, 8 and 24 h of treatment in HepG2 cells. These results suggest that apoptosis in HepG2 was induced through: (i) a caspase-dependent intrinsic pathway; and (ii) by alterations in the cellular levels of Bcl-2 family proteins, and also, that the chalcone moiety could be a potent candidate as novel anticancer agents acting on human hepatomas.

SUBMITTER: Ramirez-Tagle R

PROVIDER: S-EPMC4783989 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Project description:The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown strong and explicit cancer cell-selectivity, which results in little toxicity toward normal tissues, and has been recognized as a potential, relatively safe anticancer agent. However, several cancers are resistant to the apoptosis induced by TRAIL. A recent study found that shikonin b (alkannin, 5,8-dihydroxy-2-[(1S)-1-hydroxy-4-methylpent-3-en-1-yl]naphthalene-1,4-dione) might induce apoptosis in TRAIL-resistant cholangiocarcinoma cells through reactive oxygen species (ROS)-mediated caspases activation. However, the strong cytotoxic activity has limited its potential as an anticancer drug. Thus, the current study intends to discover novel shikonin derivatives which can sensitize the liver cancer cell to TRAIL-induced apoptosis while exhibiting little toxicity toward the normal hepatic cell. The trypan blue exclusion assay, western blot assay, 4',6-diamidino-2-phenylindole (DAPI) staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as well as the 'comet' assay, were used to study the underlying mechanisms of cell death and to search for any mechanisms of an enhancement of TRAIL-mediated apoptosis in the presence of ASH. Herein, we demonstrated that non-cytotoxic doses of acetylshikonin (ASH), one of the shikonin derivatives, in combination with TRAIL, could promote apoptosis in HepG2 cells. Further studies showed that application of ASH in a non-cytotoxic dose (2.5 μM) could increase intracellular ROS production and induce DNA damage, which might trigger a cell intrinsic apoptosis pathway in the TRAIL-resistant HepG2 cell. Combination treatment with a non-cytotoxic dose of ASH and TRAIL activated caspase and increased the cleavage of PARP-1 in the HepG2 cell. However, when intracellular ROS production was suppressed by N-acetyl-l-cysteine (NAC), the synergistic effects of ASH and TRAIL on hepatocellular carcinoma (HCC) cell apoptosis was abolished. Furthermore, NAC could alleviate p53 and the p53 upregulated modulator of apoptosis (PUMA) expression induced by TRAIL and ASH. Small (or short) interfering RNA (siRNA) targeting PUMA or p53 significantly reversed ASH-mediated sensitization to TRAIL-induced apoptosis. In addition, Bax gene deficiency also abolished ASH-induced TRAIL sensitization. An orthotopical HCC implantation mice model further confirmed that co-treated ASH overcomes TRAIL resistance in HCC cells without exhibiting potent toxicity in vivo. In conclusion, the above data suggested that ROS could induce DNA damage and activating p53/PUMA/Bax signaling, and thus, this resulted in the permeabilization of mitochondrial outer membrane and activating caspases as well as sensitizing the HCC cell to apoptosis induced by TRAIL and ASH treatment.

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Chalcone-Induced Apoptosis through Caspase-Dependent Intrinsic Pathways in Human Hepatocellular Carcinoma Cells.

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