Table 1.
Target sequences of shRNAs used in this study.
Table 2.
List of primers used for quantitative RT-PCR analysis.
Fig 1.
Tunicamycin(TM) induces ER stress, which in turn results in apoptosis of HCC cells.
(A) Hepa 1–6 cells were treated with 0.8 μg/mL of TM for 0, 8, 12, or 24 h. Cells were collected and subjected to total RNA extraction. The mRNA levels of ER stressrelated genes Grp94 and Grp78 were measured by quantitative RT-PCR. All results were normalized with the internal control β-actin. Bars, SD. Data represent3 independent experiments.(B) Hepa 1–6 cells underwent aforementioned treatment were collected and subjected to western blot analyses with specific antibodies directed against Grp78, Grp94, p-eIF2α, eIF2α, c-PARP, or β-actin. The density of the corresponding bands was measured quantitatively using image analysis software and corrected by reference to the value of β-actin. Each data point is the mean ± SD of 3 independent experiments, *P<0.05 or **P<0.01 denotes significant difference from normal control Hepa1-6 cells.
Fig 2.
Autophagy is induced in response to ER stress in HCC cells.
(A) Heap 1–6 cells were treated with TM (0.8 μg/mL) for 0, 8, 12, or 24 h. Cells were collected and subjected to western blot analyses with specific antibodies directed against p-ULK1, LC3B, Beclin1, Atg5, or β-actin. The density of the corresponding bands was measured quantitatively with an aforementioned method. Each data point is the mean ± SD of 3 independent experiments, *P<0.05 or **P<0.01 denotes a significant difference compared with normal control Hepa 1–6 cells. (B) Hepa1-6 cells were treated with TM (0.8 μg/mL) for 24 h and stained with acridine orange (1 mg/mL) for 15 min, followed immediately by detection using flow cytometry. The values were expressed as mean ± SD of 3 independent experiments, *P<0.05 denotes significant difference from normal control Hepa 1–6 cells. (C) Hepa 1–6 cells were treated with TM (0.8 μg/mL) for 12 h or 24 h and harvested for immunofluorescence assay and representative results are shown. Hepa 1–6 cells without or with strvation for 24 h were served as control or positive control, respectively.
Fig 3.
Inhibition of ER stress-induced autophagy aggravates, while activation of ER stress-induced autophagy decreases apoptosis in HCC cells.
(A) The Hepa 1–6 cells were incubated with rapamycin (100 nM) or 3-MA (5 mM) for 2 h, followed by treatment with TM (0.8 μg/mL) for an additional 24 h. Cells were then subjected to WST-1 assay. Data represent the mean ± SD of 3 separate experiments. *P<0.05 or **P<0.01 denotes a significant difference from the indicated control. (B) Hepa 1–6 cells that underwent the same treatment as in Figure 3A were subjected to flow cytometry assay and representative results are shown. The rate of apoptosis in each group was calculated based on PI staining assays. (C) Hepa 1–6 cells that underwent the same treatment as in Figure 3A were collected and subjected to western blot analyses with specific antibodies directed against c-PARP, LC3B, or β-actin. The density of the corresponding bands was measured quantitatively with an aforementioned method. Each data point is the mean ± SD of 3 independent experiments, *P<0.05 or **P<0.01 denotes a significant difference from the indicated control. (D) Hepa 1–6 cells with or without specific knockdown of LC3B were treated with TM (0.8 μg/mL) for 24 h and then harvested for western blot analyses with specific antibodies directed against c-PARP, LC3B II, or β-actin.The density of the corresponding bands was measured quantitatively with an aforementioned method. *P<0.05 or **P<0.01 denotes a significant difference from the indicated control.
Fig 4.
Knockdown of CHOP reduces TM-induced apoptosis in HCC cells.
(A) Hepa 1–6 cells were treated with 0.8 μg/mL of TM for 0, 8, 12, or 24 h. Cells were collected and subjected to total RNA extraction. The mRNA levels of CHOP were measured by quantitative RT-PCR. All results were normalized with the internal control β-actin. Bars, SD. Data show the representative of 3 independent experiments. Meanwhile Hepa 1–6 cells given the aforementioned treatment were also collected and subjected to western blot analyses with specific antibodies directed against CHOP or β-actin. (B) Hepa 1–6 cells with or without knockdown of CHOP were treated with different doses of TM as indicated for 24 h. Cells were collected and subjected to total RNA extraction. The mRNA expression levels of CHOP were measured by RT-qPCR assays. All results were normalized with the internal control, β-actin. Bars, SD. The data are representative of 3 independent experiments. (C) Hepa 1–6 cells with or without knockdown of CHOP were treated with TM (0.8 μg/mL) for 24 h. Cells were collected and subjected to western blot analyses with specific antibodies directed against Grp94, Grp78, p-eIF2α, c-PARP, Bax, CHOP, or β-actin. The density of the corresponding bands was measured quantitatively with an aforementioned method. Each data point is the mean ± SD of 3 independent experiments, *P<0.05 or **P<0.01 denotes significant difference from TM-treated Hepa 1–6 cells with normal level of CHOP. (D) Hepa 1–6 cells underwent the same treatment as in Figure 4C were subjected to western blot analysis with specific antibodies directed against c-Caspase9, Caspase3, or β-actin. (E) Hepa 1–6 cells underwent the same treatment as in Figure 4C were subjected to flow cytometry assay and representative results are shown. The rate of apoptosis in each group was calculated based on Annexin V and PI staining assays. Representative experiments were carried out at least three times, **P<0.01 denotes significant difference from shCtrl group.
Fig 5.
Downregulation of CHOP decreases apoptosis by activation of ER stress-induced autophagy.
(A) Hepa 1–6 cells with or without knockdown of CHOP were treated with TM (0.8 μg/mL) for 24 h. Cells were collected and subjected to western blot analyses with specific antibodies directed against LC3B, Atg5, Beclin1, or β-actin. The density of the corresponding bands was measured quantitatively with an aforementioned method. Each datapoint is the mean ± SD of 3 independent experiments, *P<0.05 denotes significant difference from TM-treated Hepa 1–6 cells with normal levels of CHOP. (B) Hepa 1–6 cells underwent the same treatment as in Figure 5A were subjected to flow cytometry assay to detect the expression of LC3B and representative results are shown. Representative experiments were carried out at least three times, **P<0.01 denotes significant difference from shCtrl group. (C) Hepa 1–6 cells without or with knockdown of CHOP alone, or combined knockdown of CHOP and LC3B, were treated with TM (0.8 μg/mL) for 24 h. Cells were collected and subjected to western blot analyses with specific antibodies directed against c-PARP or β-actin. The density of the corresponding bands was measured quantitatively with an aforementioned method. Each data point is the mean ± SD of 3 independent experiments, *P<0.05 denotes significant difference from normal control Hepa 1–6 cells. (D) Hepa 1–6 cells with or without knockdown of CHOP were treatedt with TM (0.8 μg/mL) for 24 h, or 3-MA for 2 h, and then with TM (0.8 μg/mL) for an additional 24 h and then harvested for western blot analyses with specific antibodies directed against c-PARP or β-actin. *P<0.05 or **P<0.01 denotes significant difference compared to the TM-treated cells.
Fig 6.
Model for CHOP regulation of autophagy and ER stress-induced apoptosis in HCC cells.
TM-induced ER stress resulted in apoptosis as well as autophagy simultaneously in HCC cells (black line).CHOP may promote ER stress-induced apoptosis in HCC cells via inhibition of autophagy (red line).