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Retrochalcone Echinatin Triggers Apoptosis of Esophageal Squamous Cell Carcinoma via ROS- And ER Stress-Mediated Signaling Pathways

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Retrochalcone Echinatin Triggers Apoptosis of Esophageal Squamous Cell Carcinoma via ROS- And ER Stress-Mediated Signaling Pathways

Ah-Won Kwak et al. Molecules.

Abstract

Esophageal squamous cell carcinoma (ESCC) is a poor prognostic cancer with a low five-year survival rate. Echinatin (Ech) is a retrochalone from licorice. It has been used as a herbal medicine due to its anti-inflammatory and anti-oxidative effects. However, its anticancer activity or underlying mechanism has not been elucidated yet. Thus, the objective of this study was to investigate the anti-tumor activity of Ech on ESCC by inducing ROS and ER stress dependent apoptosis. Ech inhibited ESCC cell growth in anchorage-dependent and independent analysis. Treatment with Ech induced G2/M phase of cell cycle and apoptosis of ESCC cells. It also regulated their related protein markers including p21, p27, cyclin B1, and cdc2. Ech also led to phosphorylation of JNK and p38. Regarding ROS and ER stress formation associated with apoptosis, we found that Ech increased ROS production, whereas its increase was diminished by NAC treatment. In addition, ER stress proteins were induced by treatment with Ech. Moreover, Ech enhanced MMP dysfunction and caspases activity. Furthermore, it regulated related biomarkers. Taken together, our results suggest that Ech can induce apoptosis in human ESCC cells via ROS/ER stress generation and p38 MAPK/JNK activation.

Keywords: Echinatin; Esophageal squamous cell carcinoma; Reactive oxygen species; c-Jun N-terminal kinase; p38.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effects of echinatin (Ech) on cell growth. (a) Structure of Ech. (b–f) Ech inhibited KYSE 30, KYSE 70, KYSE 410, KYSE 450, and KYSE 510 ESCC cells growth at 5, 10, or 15 µM for 24 h or 48 h. Cell viability was examined using MTT assay. (g) Representative pictures (left panel) and graph (right panel) of KYSE 30 and KYSE 450 cells colonies treated with Ech (0, 5, 10, 15 µM) for 14 days. Colonies were counted using a microscope. The graph shows the percentage compared to the control group. Data represent mean ± SD. Asterisk (*) denotes p < 0.05 compared to the control.
Figure 2
Figure 2
Effects of Ech on cell cycles and apoptosis. (a) Ech arrested G2/M phase of cell cycle and (b) induced sub-G1 population in KYSE 30 and KYSE 450 cells. (c) Ech increased apoptotic population of KYSE 30 and KYSE 450 cells. Viable cells (Annexin V negative/7-AAD negative) are shown in the lower left; Early apoptotic cells (Annexin V positive/7-AAD negative) are shown in the lower right; Late apoptotic cells (Annexin V positive/7-AAD positive) are shown in the upper right; Necrotic cells (Annexin V negative/7-AAD positive) are shown in the upper left. Cells were treated with Ech at 0, 5, 10, or 15 µM for 48 h, stained with 7-AAD for the cell cycle or Annexin V/7-AAD for apoptosis, and analyzed with Muse™ Cell Analyzer. Asterisk (*) denotes p < 0.05 compared to the control.
Figure 3
Figure 3
Effects of Ech on cell cycle and cell death related signals. (a) Ech induced p21 and p27 expression but decreased cyclin B1 and cdc2 expression. (b) Ech induced p-JNK and p-p38 expression, although total proteins levels of JNK or p38 were not changed. KYSE 30 and KYSE 450 cells were treated with Ech (0, 5, 10, 15 µM) for 48 h. The expression was examined with Western blot. β-actin was used as a loading control.
Figure 4
Figure 4
Effects of Ech on intracellular ROS induction. (a) Ech induced ROS formation in ESCC cells. ROS formation was measured with a Muse™ Cell Analyzer (materials and method). M1 refers to the fraction of ROS negative population while M2 refers to the region of ROS positive population. Average percentages of ROS positive cells are shown in the M2 region. (b) NAC rescued Ech induced cell death through ROS scavenging. Cells were pretreated with 6 mM NAC for 3 h and then exposed to 15 μM Ech for 48 h. Data are presented as mean ± SD of three independent experiments (* p < 0.05 vs. untreated control; # p < 0.05 vs. Ech-treated cells). (c) Ech increased ROS-mediated signal proteins. Expression levels of GRP78, CHOP, DR4, and DR5 were determined with Western blot. β-actin was used as a loading control.
Figure 5
Figure 5
Effect of Ech on MMP activities and apoptosis-related proteins. (a) Ech induced MMP dysfunction. Cells were treated with Ech (0, 5, 10, or 15 µM) for 48 h, stained with MitoPotential Dye and 7-AAD, and analyzed with a Muse™ Cell Analyzer. Percentages in the left top show depolarized/dead cells. Percentages in the left bottom show depolarized/live cells. Experiments were done at least three times in triplicate. Data are presented as means ± SD. (b) Ech decreased Bid, Bcl-2, and cyto C (mitochondria) expression but increased Bax, cyto C (cytosol), Apaf-1, and c-PARP expression. Western blotting was performed to determine expression levels. β-actin, α-tubulin (cytosol), and COX4 (mitochondria) were used as loading controls.
Figure 6
Figure 6
Effects of Ech on multi-caspases activity in KYSE 30 and KYSE 450 cells. After cells were treated with Ech at various concentrations for 48 h, percentages of KYSE 30 and KYSE 450 cells with caspases activities were assessed. X-axis means the degree of caspase activity and y-axis shows the fluorescence of 7-AAD. Each proportion in the figure represents multi-caspases positive/live cells (lower right) or multi-caspases positive/dead cells (upper right). All tests were performed in triplicate. Values are expressed as mean ± SD.
Figure 7
Figure 7
Schematic showing Ech-induced cell death. Ech induces ROS production and activates p38 and JNK MAPK kinases, or stimulates ER stress. p38 and JNK MAPK kinases signaling cascades increase death receptor expression and caspase-8/Bid activation, BAX expression, Bcl2 reduction, and cytochrome c release (cytosol), which then induces Apaf-1/caspase9 or caspase-3 activation, thus resulting in cell apoptosis. On the other hand, ER stress with increasing GRP78 and CHOP expression will induce cancer cell apoptosis.

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