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Piperlongumine, a Novel TrxR1 Inhibitor, Induces Apoptosis in Hepatocellular Carcinoma Cells by ROS-Mediated ER Stress

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Piperlongumine, a Novel TrxR1 Inhibitor, Induces Apoptosis in Hepatocellular Carcinoma Cells by ROS-Mediated ER Stress

Qianqian Zhang et al. Front Pharmacol.

Abstract

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related deaths globally. Despite advances in diagnosis and treatment, the incidence and mortality of HCC continue to rise. Piperlongumine (PL), an alkaloid isolated from the fruit of the long pepper, is known to selectively kill tumor tissues while sparing their normal counterparts. However, the killing effects of PL on HCC and the underlying mechanism of PL are not clear. We report that PL may interact with thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, and induce reactive oxygen species (ROS)-mediated apoptosis in HCC cells. Our results suggest that PL induces a lethal endoplasmic reticulum (ER) stress response in HCC cells by targeting TrxR1 and increasing intracellular ROS levels. Notably, PL treatment reduces TrxR1 activity and tumor cell burden in vivo. Additionally, TrxR1 is significantly upregulated in existing HCC databases and available HCC clinical specimens. Taken together, these results suggest PL as a novel anticancer candidate for the treatment of HCC. More importantly, this study reveals that TrxR1 might be an effective target in treating HCC.

Keywords: endoplasmic reticulum stress; hepatocellular carcinoma; piperlongumine; reactive oxygen species; thioredoxin reductase 1.

Figures

Figure 1
Figure 1
PL inhibits cell growth and induces ROS accumulation in HCC cells. (A) Chemical structure of PL. (B) The effect of PL on the proliferation of HCC cells. Cells were incubated with increasing doses of PL for 24 h, respectively. Cell viability was determined by MTT assay. (C) Intracellular ROS generation in HUH-7 cells was determined in a time- and dose-dependent manner using the redox-sensitive dye DCFH-DA (10 μM). HUH-7 cells were treated with PL (15 μM) for the indicated times. HUH-7 cells were preincubated with or without 5 mM NAC for 2 h before exposure to PL at the indicated concentrations for 30 min. Intracellular ROS generation was measured by flow cytometry. (D) Quantification of 2’-,7’dichlorofluorescein (DCF) fluorescence data from (C). (E) Intracellular ROS generation induced by PL was measured by fluorescence microscopy. Magnification, 200×. Bar, 100 µm. HUH-7 cells were preincubated with or without 5 mM NAC for 2 h before exposure to PL (15 μM) for 30 min. Then, intracellular ROS generation was measured by fluorescence microscopy. (F) Effect of PL treatment on colony formation. Cells were preincubated with or without 5 μM NAC for 1 h before exposure to PL at the indicated concentration for 5 h and then stained with crystal violet on day 8. Data represent similar results from three independent experiments. Error bars represent the S.E.M. of triplicate experiments (*p < 0.05, **p < 0.01).
Figure 2
Figure 2
PL-induced apoptosis is dependent on intracellular ROS generation in HCC cells. (A–C) PL treatment induces apoptotic characteristics in HCC cells. Magnification, 200×. Bar, 100 µm. HCC cells were preincubated with or without 5 mM NAC for 2 h before exposure to PL (15 μM) for 24 h. Cell morphology was observed using an inverted microscope after Hoechst and PI staining. (D) Two HCC cell lines were preincubated with or without 5 mM NAC for 2 h before exposure to PL at the indicated concentration for 24 h, and apoptosis-related protein expression was determined by western blotting. Data represent similar results from three independent experiments. (E) Western blot results from (D) were calculated and compared with the BAX or caspase3. Western blot results were calculated and represent the percentage of the control (*p < 0.05, **p < 0.01). All images shown here are representative of three independent experiments with similar results.
Figure 3
Figure 3
PL induces -induced cell cycle arrest is dependent on intracellular ROS generation in HCC cells. (A) HUH-7 and HepG2 cells were preincubated with or without 5 mM NAC for 2 h before exposure to PL at the indicated concentrations for 16 h. The cell cycle distribution was analyzed by flow cytometry. (B and C) Representative histogram from the cell cycle analysis shown in panel (A). (D) Expression of G2/M phase-related proteins CyclinB1 and CDC2 in HCC cells exposed to the indicated concentration of PL with or without NAC (5 mM) for 20 h. GAPDH was used as an internal control. Data represent similar results from three independent experiments. Error bars represent the S.E.M. of triplicate experiments (*p < 0.05, **p < 0.01).
Figure 4
Figure 4
The ER stress pathway is involved in PL-induced apoptosis by promoting the accumulation of ROS. (A) HUH-7 and HepG2 cells were treated with PL (15 μM) for the indicated times, and the protein levels of p-eIF2α and ATF4 were determined by western blotting. GAPDH and eIF2α were used as internal controls. (B) Western blot results from (A) were calculated and compared with the control. (C) HUH-7 and HepG2 cells were pretreated with or without 5 mM NAC for 2 h before exposure to PL at the indicated concentrations. Six hours later, ATF4 and p-EIF2α expression was detected by western blot. GAPDH and eIF2α were used as internal controls. (D) Western blot results from (C) were calculated and compared with the control. (E) HUH-7 cells were transfected with siRNA against ATF4. Cells were then exposed to 15 μM PL and apoptotic cells were determined by acridine orange and ethidium bromide dual staining. Data represent similar results from three independent experiments. Western blot results were calculated and represent the percentage of the control (*p < 0.05, **p < 0.01).
Figure 5
Figure 5
Upregulation of TrxR1 expression in LIHC. (A) TrxR1 levels in LIHC liver hepatocellular carcinoma and NATs normal adjacent tissues. (B) Higher increased TrxR1 protein expression predicts decreased survival. (C) Representative immunohistochemical staining for TrxR1 in LIHC and NATs. Bar, 100 µm. (D) Summary of immunohistochemical staining results. (E) HUH-7 cells transfected with TrxR1 siRNA and treated with 15 µM PL. Apoptotic cells were determined by acridine orange and ethidium bromide dual staining. Three independent experiments were performed.
Figure 6
Figure 6
PL inhibits HUH-7 xenograft tumor growth accompanied by increasing ROS levels and decreasing TrxR1 activity. PL treatment inhibited tumor volume (A–B) and tumor weight. (C) of HUH-7 HCC xenografts in nude mice, but did not affect the body weight. (D) of the mice. (E) H&E staining images of kidney, liver, and heart tissues from the two groups showing no significant alterations. Bar, 100 µm. (F) Western blot analysis of ATF4, CHOP, and cleaved caspase-3 levels in resected tumor specimens. Bar, 100 µm. GAPDH and caspase-3 were used as loading control. (G) Immunohistochemical staining of tumor specimens for the cell proliferation marker Ki-67, the apoptosis marker cleaved caspase-3 and Bcl-2. (H) Levels of the oxidative stress marker MDA in the tumor tissues. (I) TrxR1 enzyme activity was measured with/without PL treatment in vitro. (J) TrxR1 activity of TrxR1 in tumor tissue lysates as determined by an endpoint insulin reduction assay. Data represent similar results from three independent experiments. Error bars represent the S.E.M. of triplicate experiments (*p < 0.05, **p < 0.01).
Figure 7
Figure 7
Schematic illustration of the underlying mechanism of the anticancer activity of PL.

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