SH003 Causes ER Stress-mediated Apoptosis of Breast Cancer Cells via Intracellular ROS Production

Abstract

Background/aim: Breast cancer is one of the most common cancers in women all over the world and new treatment options are urgent. ER stress in cancer cells results in apoptotic cell death, and it is being proposed as a new therapeutic target. SH003, a newly developed herbal medicine, has been reported to have anti-cancer effects. However, its molecular mechanism is not yet clearly defined.

Materials and methods: Microarray was performed to check the differential gene expression patterns in various breast cancer cell lines. Cell viability was measured by MTT assays to detect cytotoxic effects. Annexin V-FITC and 7AAD staining, TUNEL assay and DCF-DA staining were analyzed by flow cytometry to evaluate apoptosis and ROS levels, respectively. Protein expression was examined in SH003-breast cancer cells using immunoblotting assays. The expression of C/EBP Homologous Protein (CHOP) mRNA was measured by real-time PCR. The effects of CHOP by SH003 treatment were investigated using transfection method.

Results: Herein, we investigated the molecular mechanisms through which SH003 causes apoptosis of human breast cancer cells. Both cell viability and apoptosis assays confirmed the SH003-induced apoptosis of breast cancer cells. Meanwhile, SH003 altered the expression patterns of several genes in a variety of breast cancer cell lines. More specifically, it upregulated gene sets including the response to unfolded proteins, independently of the breast cancer cell subtype. In addition, SH003-induced apoptosis was due to an increase in ROS production and an activation of the ER stress-signaling pathway. Moreover, CHOP gene silencing blocked SH003-induced apoptosis.

Conclusion: SH003 causes apoptosis of breast cancer cells by upregulating ROS production and activating the ER stress-mediated pathway. Thus, our findings suggest that SH003 can be a potential therapeutic agent for breast cancer.

Keywords: Breast cancer; CHOP; ER stress; ROS; SH003.

Figure 1. SH003 induces apoptosis in breast cancer cells. (A) SH003 effect on breast cancer cell viability. HCC-1419, MCF-7 and MBA-MD-231 cells were treated with 0, 50, 100, 250 and 500 μg/ml of SH003 for 48 h. (B) Annexin V-FITC and 7-AAD double-staining assays. Breast cancer cells were treated with SH003 at 500 μg/ml for 48 h. Representative Annexin V-FITC and 7-AAD double-staining data show SH003-induced apoptotic cell death. Bar graphs show apoptotic cell numbers from those analyses. (C) Western blots for cleaved forms of Caspase-9, Caspase-8, Caspase-7, and poly (ADP-ribose) polymerase (PARP). Actin was blotted as the internal loading control. (D) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Representative histograms show SH003-induced apoptotic cell death. Bar graphs show apoptotic cell numbers. All bar graphs are shown as mean and standard deviation of three independent experiments. *p<0.05, Student’s t-test.

Figure 2. SH003 alters global gene expression patterns in breast cancer cells. (A) Heatmap produced by hierarchical clustering analysis shows SH003-altered gene expression profiles in various breast cancer cell lines. (B) Genes altered by SH003 in all breast cancer cell lines (All), triple negative breast cancer (TNBC) cell lines, human epidermal growth factor receptor 2 (HER2)+ cell lines, and estrogen receptor (ER)+ cell lines. (C) Venn diagrams representing the number of differentially expressed genes.

Figure 3. SH003 causes apoptosis of breast cancer cells through ER stress. (A) SH003 induces ER stress in breast cancer cells. The cells were treated with SH003 at 500 μg/ml for 24 h. Protein levels of binding immunoglobulin protein (BIP), p-PKR-like endoplasmic reticulum kinase (PERK), PKR-like endoplasmic reticulum kinase (PERK), p-eukaryotic translation initiation factor 2A (eIF2α), eukaryotic translation initiation factor 2A (eIF2α), p-inositol-requiring enzyme 1α (IRE1α), inositol-requiring enzyme 1α (IRE1α), p-c-Jun n-terminal kinase (JNK), c-Jun n-terminal kinase (JNK) and C/EBP homologous protein (CHOP) were examined by western blot. Actin was detected as the internal loading control. (B) SH003-induced endoplasmic reticulum (ER) stress results in apoptotic cell death. The cells were pretreated with 4-PBA at 1 mM for 2 h and then treated with SH003 at 500 μg/ml for another 24 h. Protein levels of BIP, CHOP, cleaved Caspase-7 and cleaved PARP were examined by western blot. (C) Apoptotic cell death was confirmed by Annexin V-FITC and 7-AAD double-staining assay. The cells were pretreated with 4-Phenylbutyric acid (4-PBA) at 1 mM for 2 h and then treated with SH003 at 500 μg/ml for another 24 h. Bar graphs presented as mean and standard deviation indicate apoptotic cell numbers. *p<0.05. One-way ANOVA followed by the Bonferroni post hoc test.

Figure 4. SH003-induced ROS production results in ER stress. (A) SH003 increases intracellular reactive oxygen species (ROS) production. Breast cancer cells were pretreated with N-acetyl cysteine (NAC) at 5 mM for 30 min and then treated with SH003 at 500 μg/ml for 4 h. Intracellular ROS levels were measured by DCF-DA staining assay. Bar graphs indicating mean and standard deviation from three independent experiments show intracellular ROS levels. (B) SH003-increased ROS levels are accumulated by blocking ER stress. Breast cancer cells were pretreated with 4-PBA at 1 mM for 2 h and then treated with SH003 at 500 μg/ml for 4 h. Intracellular ROS levels were measured by DCF-DA staining assay. Bar graphs indicating mean and standard deviation from three independent experiments show intracellular ROS levels. (C) Inhibition of intracellular ROS production ameliorates SH003-induced ER stress. Protein levels of p-eIF2α, eIF2α and CHOP were examined by western blot. (D) Relative levels of p-eIF2α, eIF2α and CHOP. Bar graphs indicate mean and standard deviation. *p<0.05. One-way ANOVA followed by the Bonferroni post hoc test.

Figure 5. SH003 requires CHOP for apoptosis of breast cancer cells. (A) SH003 induces CHOP mRNA expression. The cells were treated with SH003 at 500 μg/ml for 48 h and then CHOP mRNA levels were measured by quantitative real-time PCR. (B) CHOP gene silencing inhibits SH003-induced apoptosis. The cells transfected with either control siRNAs (siControl) or CHOP siRNAs (siCHOP) were treated with SH003 for 48 h, and then protein levels of CHOP, cleaved Caspase-7 and cleaved PARP were detected by western blot. (C) Relative protein levels of CHOP, cleaved Caspase-7 and cleaved PARP were measured. Bars indicate mean and standard deviation. (D) SH003-induced apoptosis requires CHOP. The cells transfected with either control siRNAs or CHOP siRNAs were treated with SH003 for 48 h, and then subjected to Annexin V-FITC and 7-AAD staining assays. Bar graphs shown as mean and standard deviation indicate apoptotic cell numbers. *p<0.05, one-way ANOVA followed by the Bonferroni post hoc test.

Figure 6. Schematic image representing a mechanism of SH003-induced apoptosis of breast cancer cells. SH003 induces intracellular ROS production followed by ER stress, which results in apoptotic cell death independently of breast cancer subtype.