6-Shogaol induces apoptosis in human hepatocellular carcinoma cells and exhibits anti-tumor activity in vivo through endoplasmic reticulum stress

Abstract

6-Shogaol is an active compound isolated from Ginger (Zingiber officinale Rosc). In this work, we demonstrated that 6-shogaol induces apoptosis in human hepatocellular carcinoma cells in relation to caspase activation and endoplasmic reticulum (ER) stress signaling. Proteomic analysis revealed that ER stress was accompanied by 6-shogaol-induced apoptosis in hepatocellular carcinoma cells. 6-shogaol affected the ER stress signaling by regulating unfolded protein response (UPR) sensor PERK and its downstream target eIF2α. However, the effect on the other two UPR sensors IRE1 and ATF6 was not obvious. In prolonged ER stress, 6-shogaol inhibited the phosphorylation of eIF2α and triggered apoptosis in SMMC-7721 cells. Salubrinal, an activator of the PERK/eIF2α pathway, strikingly enhanced the phosphorylation of eIF2α in SMMC-7721 cells with no toxicity. However, combined treatment with 6-shogaol and salubrinal resulted in significantly increase of apoptosis and dephosphorylation of eIF2α. Overexpression of eIF2α prevented 6-shogaol-mediated apoptosis in SMMC-7721 cells, whereas inhibition of eIF2α by small interfering RNA markedly enhanced 6-shogaol-mediated cell death. Furthermore, 6-shogaol-mediated inhibition of tumor growth of mouse SMMC-7721 xenograft was associated with induction of apoptosis, activation of caspase-3, and inactivation of eIF2α. Altogether our results indicate that the PERK/eIF2α pathway plays an important role in 6-shogaol-mediated ER stress and apoptosis in SMMC-7721 cells in vitro and in vivo.

Figure 1. Effect of 6-shogaol on viability and apoptosis in HCC cells.

(A) The chemical structure of 6-shogaol. (B) SMMC-7721, BEL-7404 and HepG2 cells were treated with 5, 10, 15, 20, 25, 30, 40 and 50 µM 6-shogaol for 24 h respectively, and the cell viability was determined by MTT assay. (C) SMMC-7721 cells were treated with 5, 10, 15, 20, 25 and 30 µM 6-shogaol for 24, 48, and 72 h respectively, and the cell viability was examined by MTT assay. (D) Cells (1×10⁶) were treated with 20 µM 6-shogaol for 24 h and stained with Hoechst 33258. Original magnification ×400. (E) Cells were treated with 20 µM 6-shogoal for 12 and 24 h. DNA frangemntation was determined. (F and G) SMMC-7721 cells were treated with 20 µM 6-shogaol for 3, 6, 12 and 24 h. The cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. (H) Normal human liver HL-7702 cells were treated with 20, 40, and 80 µM 6-shogaol for 24 h. Apoptosis was determined using flow cytometry.

Figure 2. 2-DE images of total cellular proteins extracted from 6-shogaol-treated SMMC-7721 cells.

(A) SMMC-7721 cells were treated with or without 20 µM 6-shogaol for 24 h. Differentially expressed spots are shown by the arrows. (B) The expanded region of altered protein spots, the proteins with arrow are the differentially expressed proteins identified by MS. (C) The intensity of mitochondria-related and other proteins. (D) The intensity of ER-related proteins. (E) Western bolt imaging of GRP94, GRP78, HSP70 and HSP60. For Western blot analysis, each lane was loaded with 30 µg of protein. Blots were subsequently stripped and reprobed with antibody against β-actin to ensure equivalent loading and transfer. Two additional studies yielded equivalent results.

Figure 3. Effects of 6-shogaol on ER stress signaling proteins.

The cells were treated with or without 20 µM 6-shogaol for 1, 3, 6, 12, and 24 h. Total cellular extracts were prepared and subjected to Western blot analysis using antibodies against PARP, caspase-3 and ER stress-related molecular chaperones (A) and ER stress related proteins (C). CF stands for cleavage fragments of PARP. The caspase-3 activity (B) was assayed according to Caspase-3 Activity Assay Kit. *Values for cells treated with 6-shogaol were significantly greater than the control group by Student’s t-test (P<0.05); **(P<0.01).

Figure 4. Effects of co-administration of salubrinal and 6-shogaol on apoptosis and phosphorylation of eIF2α.

SMMC-7721 cells were pretreated with 5 µM salubrinal for 2 h, followed by treatment with 20 µM 6-shogaol for 3, 6, 12, and 24 h. (A) Apoptosis was determined using flow cytometry. *Values for cells treated with salubrinal and 6-shogaol in combination were significantly greater than those for cells treated with 6-shogaol alone by Student’s t-test; P<0.05. Total protein extracts were prepared and subjected to Western blot assay using antibodies against PARP and caspase-3 (B), and eIF2α pathway related proteins (C). CF stands for cleavage fragments of PARP.

Figure 5. The role of eIF2α in 6-shogaol-induced apoptosis.

SMMC-7721 cells were stably transfected with an empty vector (pWPI) or over-expression form of eIF2α as described in Materials and methods. All cells were then treated with 20 µM 6-shogaol for 24 h. (A) After treatment, apoptosis was determined using flow cytometry. **P<0.01. Total cellular extracts were prepared and subjected to (C) caspase-3 activity assay and Western blot assay using antibodies against PARP, caspase-3 (B) and PERK/eIF2α signaling proteins (D). SMMC-7721 cells were also stably transfected with an empty vector pLKO.1-SH or eIF2α siRNA (two clones siRNA-1 and siRNA-2). Cells were then treated with 20 µM 6-shogaol for 24 h. (E) After treatment, apoptosis was determined using flow cytometry. **P<0.01. Total cellular extracts were prepared and subjected to (G) caspase-3 activity assay and Western blot analysis using antibodies against PARP, caspase-3 (F) and PERK/eIF2α signaling proteins (H). CF stands for cleavage fragments of PARP.

Figure 6. In vivo antitumor effects of 6-shogaol in SMMC-7721 xenografts.

SCID mice were inoculated with SMMC-7721 cells (3×10⁶ cells/mouse, s.c.) and randomly divided into three groups (8/group) treated with 6-shogaol (10 mg/kg and 50 mg/kg, i.p., daily) or vehicle control solvent. (A) Average tumor volume in vehicle control mice and mice treated with 10 mg/kg and 50 mg/kg 6-shogaol. ** P<0.01. (B) Body weight changes of mice during the 28 days of study. (C) Representative photographs of biopsy samples from mice treated with 6-shogaol (50 mg/kg) and vehicle control. Original magnification ×400. (D) Expression of p-PERK, eIF2α, and p-eIF2α in tumor tissues of SMMC-7721 xenograft mouse model treated with 6-shogaol (50 mg/kg) and vehicle control.