Pekinenin E Inhibits the Growth of Hepatocellular Carcinoma by Promoting Endoplasmic Reticulum Stress Mediated Cell Death

Abstract

Hepatocellular carcinoma (HCC) is a malignant primary liver cancer with poor prognosis. In the present study, we report that pekinenin E (PE), a casbane diterpenoid derived from the roots of Euphorbia pekinensis, has a strong antitumor activity against human HCC cells both in vitro and in vivo. PE suppressed the growth of human HCC cells Hep G2 and SMMC-7721. In addition, PE-mediated endoplasmic reticulum (ER) stress caused increasing expressions of C/EBP homologous protein (CHOP), leading to apoptosis in HCC cells both in vitro and in vivo. Inhibition of ER stress with CHOP small interfering RNA or 4-phenyl-butyric acid partially reversed PE-induced cell death. Furthermore, PE induced S cell cycle arrest, which could also be partially reversed by CHOP knockdown. In all, these findings suggest that PE causes ER stress-associated cell death and cell cycle arrest, and it may serve as a potent agent for curing human HCC.

Keywords: ER stress; apoptosis; cell cycle arrest; hepatocarcinoma; pekinenin E.

FIGURE 1

PE inhibits human hepatocellular carcinoma cell growth without affecting normal control cells. (A) Chemical structure of PE. (B–D) Hep G2 and SMMC-7721 cells were incubated with PE at various concentrations for 24, 48, and 72 h, respectively. The cell viability was determined by MTT assay. Values were expressed as mean ± SD of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01 versus control cells cultured with 0.1% DMSO by one-way ANOVA and post hoc tests. (E,F) The effect of PE on cell viability in non-malignant cells was determined by MTT assay. H9c2 and BEAS-2B cells were incubated with PE at various concentrations for 48 h. Values were expressed as mean ± SD of three independent experiments.

FIGURE 2

Effects of PE on cell apoptosis in hepatocellular carcinoma cell lines. (A) SMMC-7721 cells were seeded in six-well plate and incubated with 0, 3, 10, and 30 μM PE for 24 h. The apoptosis of SMMC-7721 cells was determined by Annexin V/PI staining. Values were expressed as mean ± SD of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01 versus control cells cultured with 0.1% DMSO by one-way ANOVA and post hoc tests. (B) Representative transmission electron micrographs of SMMC-7721 cells treated with or without PE. After treated with 30 μM PE for 24 h, SMMC-7721 cells exhibited the characteristic ultrastructural morphology of apoptosis, such as vacuolation in cytoplasm, chromatin condensation, and apoptotic body. (C) SMMC-7721 cells were incubated with various concentrations of PE for 24 h, immunoblots against Cleaved Caspase-9, Caspase-9, Cleaved PARP, PARP, and β-actin were detected. β-Actin was taken as control. Blots are representative of three independent experiments. (D) SMMC-7721 cells were incubated with 30 μM PE for 0, 6, 12, and 24 h, Cleaved Caspase-9, Caspase-9, Cleaved PARP, PARP, and β-actin were determined by western blotting. β-Actin was taken as control. Blots are representative of three independent experiments. (E) Hep G2 cells were incubated with various concentrations of PE for 24 h; immunoblots against Caspase-9, Cleaved PARP, PARP, and β-actin were detected. β-Actin was taken as control. Blots are representative of three independent experiments. (F) Hep G2 cells were incubated with 30 μM PE for 0, 6, 12, and 24 h, Caspase-9, Cleaved PARP, PARP, and β-actin were determined by western blotting. β-Actin was taken as control. Blots are representative of three independent experiments.

FIGURE 3

PE induces ER stress in hepatocellular carcinoma cell lines. (A,B) Representative immunoblots against GRP78, ATF6α, ATF4, p-eIF2α, eIF2α, CHOP, and β-actin from cell lysates of SMMC-7721 and Hep G2 treated with PE (0, 3, 10, and 30 μM) for 24 h. β-Actin was taken as control. Blots are representative of three independent experiments. (C,D) SMMC-7721 and Hep G2 cells were treated by 30 μM PE for 0, 6, 12, and 24 h. Immunoblots against GRP78, ATF6α, ATF4, p-eIF2α, eIF2α, CHOP, and β-actin from cell lysates of SMMC-7721 and Hep G2 were detected. β-Actin was taken as control. Blots are representative of three independent experiments. (E) Representative mRNA levels of GRP94, GRP78, ATF4, and CHOP from SMMC-7721 cell treated with PE (0, 3, 10, and 30 μM) for 24 h. GAPDH was used as control. Values were expressed as mean ± SD of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01 versus control cells cultured with 0.1% DMSO by one-way ANOVA and post hoc tests. (F) Representative mRNA levels of GRP94, GRP78, ATF4, and CHOP from cell lysates of SMMC-7721 treated with 30 μM PE for 0, 6, 12, and 24 h. GAPDH was taken as control. Values were expressed as mean ± SD of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01 versus control cells cultured with 0 h by one-way ANOVA and post hoc tests. (G) Fluorescence microscopic images of the SMMC-7721 cells stained with CHOP in both control and PE treated cells. Scale bars, 100 μm.

FIGURE 4

PE induces ER stress mediated apoptosis in hepatocellular carcinoma cell lines. (A) SMMC-7721 cells were seeded in six-well plates for 12 h, transfected with Luc siRNA or CHOP siRNA for 24 h followed by incubation with 0, 3, 10, and 30 μM PE for another 24 h. Cell growth of three independent experiments was shown. Values were expressed as mean ± SD of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01 versus PE group by two-way ANOVA and post hoc tests. (B) SMMC-7721 cells were incubated with PE for 24 h in the absence or presence of TG (1 μM). The protein levels of Cleaved PARP and CHOP were determined by western blotting. β-Actin was taken as control. Blots are representative of three independent experiments. (C–E) SMMC-7721 or Hep G2 cells were transfected with Luc siRNA or CHOP siRNA for 24 h followed by incubation with 30 μM PE for another 24 h. (C,D) The protein levels of Cleaved PARP and CHOP were determined by western blotting. β-Actin was taken as control. Blots are representative of three independent experiments. (E) SMMC-7721 cell apoptosis was detected using TUNEL staining.

FIGURE 5

PE induces S cell cycle arrest via ER stress in SMMC-7721 cells. (A) S cell cycle arrest in PE-treated SMMC-7721 cells. Cells were incubated with indicated concentrations of PE for 24 h. Propidium iodide staining and flow cytometry were used to determine the proportion of cells in various phases of the cell cycle. (B) Representative immunoblots against Cyclin A2, CDK2, and β-actin from SMMC-7721 cell lysates after treated with PE (0, 3, 10, and 30 μM) for 24 h. β-Actin was taken as control. Blots are representative of three independent experiments. Values were expressed as mean ± SD of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01 versus control cells cultured with 0.1% DMSO by one-way ANOVA and post hoc tests. (C) The effects of CHOP knockdown on cell cycle arrest. SMMC-7721 cells were transfected with Luc siRNA or CHOP siRNA for 24 h, followed by incubation with 30 μM of PE for another 24 h. (D) Representative immunoblots against Cyclin A2, CDK2, and β-actin from cell lysates of Hep G2 treated with PE (0, 3, 10, and 30 μM) for 24 h. β-Actin was taken as control. Blots are representative of three independent experiments. Values were expressed as mean ± SD of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01 versus control cells cultured with 0.1% DMSO by one-way ANOVA and post hoc tests.

FIGURE 6

PE inhibits the growth of hepatocellular carcinoma in BALB/c athymic nude mice. 5 × 10⁶ cells were injected subcutaneously into right flank regions of each mouse. PBS (vehicle control) or various concentrations of PE were administrated every day. (A) Body weight during administration of PE. (B) Representative pictures of tumor from mice per group were shown. (C) Tumor weight after 7 days administration of PE. Values were expressed as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01 versus vehicle by one-way ANOVA and post hoc tests. (D,E) Immunoblots against Cleaved PARP and CHOP in tumor samples were determined. β-actin was performed as control. Blots are representative of the 10 mice per group. Values were expressed as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01 versus vehicle by one-way ANOVA and post hoc tests.

FIGURE 7

Mechanism for PE’s effects on hepatocellular carcinoma cells. PE induces ER stress through PERK-p-eIF2α-ATF4 pathway and ATF6 pathway, leading to the overexpression of CHOP, which triggers apoptosis and cell cycle arrest.