Abstract
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.
Highlights
Hepatocellular carcinoma (HCC) is one of the most common human malignancies worldwide, especially in Asian courtiers
In an effort to identify the synergistic effect of ASH and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HCC cell apoptosis, we examined whether ASH in combination with TRAIL could induce apoptotic cell death in a HepG2 cell
HepG2 cell. (A) The cleavage of caspases and poly (ADP-ribose) polymerase-1 (PARP-1) in HepG2 cells were analyzed via Western cell. (A) The cleavage of caspases and PARP-1 in HepG2 cells were analyzed via Western blotting after blotting after following 4–24 h treatment with 2.5 μM ASH and/or 50 ng/mL TRAIL, and the control following 4–24 h treatment with 2.5 μM ASH and/or 50 ng/mL TRAIL, and the control group received group received an equal amount of vehicle (PBS + 0.01% bovine serum albumin (BSA) + 0.1% dimethyl sulfoxide (DMSO)). (B) Western blotting an equal
Summary
Hepatocellular carcinoma (HCC) is one of the most common human malignancies worldwide, especially in Asian courtiers. A recent study found that shikonin (10 μM) might reduce cells’ survival rate and promote apoptosis in cholangiocarcinoma cells, and the suppressive effects could be enhanced by TRAIL treatment through reactive oxygen species (ROS)-mediated caspases activation [7]. P53/PUMA/Bax activation may be involved in the cell-intrinsic apoptosis pathway by ASH and exhibit synergistic effects with TRAIL-induced cell-extrinsic apoptosis in HCC cells. ROS production, DNA double-strand breaks and p53-induced PUMA and Bax activation may contribute to the sensitization effects of ASH on TRAIL-induced apoptosis. We considered that both cellular intrinsic (p53/PUMA/Bax) and extrinsic (TRAIL) pathways contributed to HepG2 cells’. Apoptosis via the synergistic antitumor effects of ASH and TRAIL
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