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.
Breast cancer is the second most frequent cancer in women (1). Breast cancer subtypes are classified by expression patterns of three receptor proteins: estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) (2, 3). Meanwhile, chemotherapeutic agents for breast cancer show side effects and are very expensive (4). Moreover, drug resistance is a hurdle in treating this type of cancer. Herbal medicine is one of the options to solve those problems (5, 6).
SH003 consists of 1:1:1 ratio of Trichosanthes kirilowii Maxim., Astragalus membranaceu, and Angelica gigas (7). Using network pharmacological approaches, active compounds of SH003 were found to be functionally involved in various pathways that are associated with tumorigenesis and development of cancer. SH003 has been investigated in various cancer cell types to test its anti-cancer effects. In brief, SH003 suppressed tumor growth and metastasis both in cell line experiments in vitro and in mouse xenograft studies (7-14). SH003 and classical anti-cancer drugs such as doxorubicin, docetaxel and paclitaxel synergistically inhibited cancer cell growth (9, 14-16). SH003 also repressed tumor angiogenesis by blocking VEGF binding to VEGR2 (17). Thus, SH003 is likely to target various cell types in the tumor microenvironment. Accordingly, we now run clinical studies for SH003 (18-20). More recently, we revealed that SH003 blocked docetaxel-induced neuropathic pain and ameliorated cyclophosphamide-induced immunosuppression in mouse models (21, 22). Therefore, it is plausible that SH003 plays multifaceted roles in cancer.
Alterations of endoplasmic reticulum (ER) function lead to the accumulation of unfolded or incorrectly folded proteins, a specific condition called ER stress (23). ER stress is involved in the unfolded protein response, an adaptive reaction that maintains the function by reducing the unfolded or misfolded protein load (24). Unfolded protein response (UPR) signaling pathway is mediated by three ER-transmembrane stress sensors: Protein kinase RNA-like ER kinase (PERK), inositol requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6) (25). Especially, the PERK-eIF2α-CHOP pathway results in apoptosis (26, 27). Cancer cells undergo prolonged ER stress, which activates the UPR signaling to restore ER homeostasis (28). Thus, UPR signaling is either cytotoxic or cytoprotective in a cell state-dependent manner (29, 30). However, in cancer cells with excessive ER stress, UPR signaling pathway leads to apoptosis (31). In the process for apoptotic cell death, CHOP is crucial for ER stress-induced apoptosis (26, 32).
We herein investigated the mechanism though which SH003 causes apoptosis of breast cancer cells, independently of the human breast cancer subtype. Our present work shows that SH003 induces apoptotic cell death through intracellular reactive oxygen species (ROS) production and ER stress. Supportively, we revealed that CHOP gene silencing rescued SH003-induced apoptosis.
Materials and Methods
Cell culture and transfection. MCF-7, T47D, ZR-75-1, SKBR-3, HCC-1419, MDA-MB-453, HCC-1569, BT-474 and MDA-MB-231 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle’s Medium (Welgene, Seoul, Republic of Korea) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Welgene). HCC-38 and HCC-70 cells were obtained from the Seoul National University Cell Bank (Seoul, Republic of Korea) and cultured in Roswell Park Memorial Institute Medium-1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Breast cancer cell lines were seeded at a density of 2×105 cells with antibiotics-free media in 6-well plates. The next day, cells were transfected with 10nmol/mL of either CHOP siRNAs or control siRNAs (Bioneer, Daejeon, Republic of Korea) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Medium was replaced with fresh medium including antibiotics at 16 h after transfection, and the cells were treated with SH003 for another 48 h.
Gene expression profiling. RNAs from twelve breast cancer cells (MCF-7, T47D, ZR-75-1, SKBR-3, HCC-1419, MDA-MB-453, HCC-1569, BT-474, MDA-MB-231, HCC-38, HCC70) were isolated using the easy-BLUE RNA Extraction kit (iNtRON Biotech, Sungnam, Republic of Korea), and cDNAs were synthesized and hybridized onto a Human HT12 genome-wide expression profiling chip (Illumina, San Diego, CA, USA). Detection of p-Values greater than 0.01 was considered meaningful for analyses, and expression levels greater than 1 were adjusted to 1; both fold changes over 5 and delta difference over 50 with a t-test p-Value lower than 0.05 were selected. A heat-map was generated for the expression patterns of significant genes. Hierarchical trees were produced by Pearson correlation. Analyses were conducted in Genome Studio software (Illumina), and images were produced in Multiexperimental Viewer (Boston, MA, USA).
Western blot. Cells were lysed in 2× Laemmli sample buffer and boiled at 100°C for 10 min. Equal quantities of samples were loaded on SDS-PAGE gels (10-15%) and transferred onto nitrocellulose membranes. The membranes were then incubated with primary antibodies. The used primary antibodies were as follows: cleaved Caspase-7 (#9491), cleaved Caspase-8 (#9496), cleaved Caspase-9 (#7237), cleaved PARP (#9541), BIP (ab21685), PERK (#5683), p-PERK (#3197), eIF2α (#9722), p-eIF2α (#3398), IRE1α (#3294), p-IRE1α (PA1-16927), JNK (#3708), p-JNK (#4668), CHOP (#2895) and β-actin (#3700). BIP was obtained from Abcam (Cambridge, UK) and p-IRE1α was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
MTT assay. Cells cultured in 96 well plates were treated with different concentrations of SH003 (0, 50, 100, 250 and 500 μg/ml) for 48 h and then incubated with 5 mg/ml of thiazolyl blue tetrazolium bromide solution for 2 h at 37°C in the dark. After removing the medium, formazan crystals were solubilized in dimethyl sulfoxide and an absorbance was measured at 575 nm in an ELISA microplate reader.
Annexin V and 7-AAD double staining assay. The rate of apoptotic cells was measured using staining with Annexin V-fluorescein isothiocyanate (FITC) and 7-Aminoactinomycin D (7-AAD). Cells were washed with PBS, trypsinized, re-washed twice with PBS and incubated with FITC-conjugated Annexin-V for 15 min in the dark and 7-AAD for 15 min at room temperature. Next, the samples were measured using BD FACSCalibur flow cytometry. Data were analyzed using Cell Quest software (BD Biosciences, San Jose, CA, USA).
DCF-DA staining assay. Levels of intracellular ROS production were quantified using 2′,7′-dichlorofluorescein diacetate (DCF-DA). Cells were plated at 70~80% confluency in 60mm culture dishes. Cells were treated by SH003 at 500 ug/ml for 4 h after N-acetyl-L-cysteine (NAC) treatment for 2 h. The cells were incubated with 10 μM DCF-DA in media for 30 min at 37°C. Then, the cells were washed with phosphate buffered saline (PBS), trypsinized, resuspended in 0.5 ml of PBS and analyzed by flow cytometry.
TUNEL assay. Apoptotic cells were detected by transferase-mediated deoxyuridine triphosphate (dUTP)-fluorescein nick end-labeling (TUNEL) assay kit (ab66110, Abcam). Forty-eight hours after SH003 treatment, cells were washed with Dulbecco’s phosphate buffered saline (DPBS), fixed with 4% paraformaldehyde for 15 min, washed twice with DPBS and incubated with 70% ethanol. TUNEL reaction was then performed according to the manufacturer’s protocol.
RNA isolation and real-time PCR. Cells were seeded in 60mm culture dishes. After SH003 treatment for 48 h, total RNA was isolated using the RNA Easy-blue kit (Qiagen, Germantown, MD, USA) and cDNA was synthesized by reverse transcriptase PCR using the cDNA synthesis kit (Takara, Kusatsu, Shiga, Japan). The SYBR Green PCR Master Mix was used to perform quantitative real-time PCRs. Primers were as follows: CHOP-forward: 5-TGG AAA GCA GCG CAT GAA-3, CHOP-reverse: 5-AAA GGT GGG TAG TGT GGC-3. GAPDH-forward: 5′-TGG ACT CCA CGA CGT ACT CA-3′, GAPDH-reverse: 5′-AAT CCC ATC ACC ATC TTC CA-3′.
Statistical analysis. All experiments were conducted at least three times. Data were presented as mean and standard deviation, and the analyses were done using Student’s t-test and one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. All analyses were done in GraphPad Prism 7 software (San Diego, CA, USA).
Results
SH003 induces apoptosis in breast cancer cells. To evaluate the cytotoxic effect of SH003 on each subtype of breast cancer cells, we performed MTT assays. The breast cancer cell lines HCC-1419 (HER2-positive), MCF-7 (ER-positive) and MDA-MB-231 (triple-negative) were treated with SH003 (0, 50, 100, 250, 500μg/ml) for 48 h. SH003 decreased the viability of HCC-1419, MCF-7 and MDA-MB-231 cells in a dose-dependent manner (Figure 1A). Accordingly, Annexin V-FITC and 7-AAD double staining assays showed that SH003 causes apoptosis (Figure 1B). SH003 also increased the levels of cleaved Caspase-9, cleaved Caspase-8, cleaved Caspase-7, and cleaved PARP (Figure 1C). Consistently, SH003 increased apoptotic cell number in TUNEL assays (Figure 1D). Thus, our data indicate that SH003 induces apoptosis independently of the breast cancer cells line.
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.
SH003 alters expression patterns of gene in breast cancer cells. To investigate the genes affected by SH003 in breast cancer cells, we analyzed differential gene expression patterns between non-treated and SH003-treated breast cancer cells. Various breast cancer cell lines representing different breast cancer subtypes were used in the experiment as follows: ER-positive (MCF-7, T47D and ZR-75-1), HER2-positive (SKBR-3, HCC-1419, MDA-MB-453, HCC-1569 and BT-474) and triple-negative (MDA-MB-231, HCC-38 and HCC70). A heatmap for the expression levels of 31,823 genes is shown in Figure 2A. SH003 upregulated 111 genes and downregulated 27 genes independently of subtypes (cut-off over 2-fold change, p-Value <0.5) (Figure 2B, C, and Table I). SH003 subtype-specifically affected 84 genes in triple-negative breast cancer cells, 25 genes in HER2-positive breast cancer cells and 174 genes in ER-positive breast cancer cells (Figure 2B and C, Table II, Table III, and Table IV). Next, we conducted gene ontology analysis for gene set enrichment. SH003 upregulated gene sets including ‘response to unfolded protein’ in biological process, ‘transcription corepressor activity’ in molecular function and ‘nucleus’ in cellular component, and downregulated ‘receptor tyrosine kinase binding’ in molecular function (Table V). While SH003 altered gene sets in ER-positive breast cancer cells, it did not alter gene sets in triple-negative and HER2-positive breast cancer cells. KEGG analysis for biological pathway showed that SH003 altered 20 pathways including the phosphatidylinositol signaling system, MAPK signaling pathway and apoptosis (Table VI).
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.
List of differentially expressed genes in all breast cancer cells.
List of differentially expressed genes in triple-negative breast cancer cells.
List of differentially expressed genes in HER2+ cells.
List of differentially expressed genes in ER+ cells.
Candidate enriched gene sets for the differentially expressed genes in breast cancer cells.
Biological pathway analysis of differentially expressed genes.
SH003 causes apoptosis by activating ER stress. Among 138 genes, SH003 strongly altered the expression levels of SNORD3C, ATF3, SNORD3D, SNORD3A, FOS, DDIT3 (also called GADD153 or CHOP), NR1H4 (FXR or RIP14), GADD45B (MyD118), ARC (ARG3.1) and SESN2 in terms of fold-change >5, p-Value <0.05 by paired t-test and Total Number of Misclassifications (TNoM) (Table I). Considering the top 10 genes upregulated by SH003 (Table I) and data from gene ontology and KEGG (Table V and Table VI), we hypothesized that SH003 might cause apoptotic cell death by activating ER stress. Therefore, we further examined this possibility. We found that SH003 increased the protein expression levels of BIP and CHOP, and phosphorylation levels of PERK, eIF2α, IRE1α and JNK, independently of the subtype of breast cancer cells (Figure 3A). To confirm that SH003 causes ER stress-induced apoptosis, HCC-1419 (HER2-positive), MCF-7 (ER-positive) and MDA-MB-231 (triple-negative) breast cancer cells were pretreated with an inhibitor of ER stress pathway (4-PBA) for 2 h, and then treated with SH003 for another 24 h. 4-PBA reduced levels of BIP, CHOP, cleaved Caspase-7 and cleaved PARP increased by SH003 (Figure 3B), suggesting that SH003 could cause apoptosis of breast cancer cells via ER stress. Consistently, 4-PBA reduced SH003-increased apoptotic cell number (Figure 3C). Therefore, SH003 causes apoptosis via ER stress, independently of the subtype of breast cancer cells.
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.
SH003 causes ER stress via the increase of intracellular ROS level in breast cancer cell lines. Increase of intracellular reactive oxygen species (ROS) level results in ER stress followed by apoptosis and vice versa (33, 34). Moreover, we recently found that SH003 induces intracellular ROS production in MDA-MB-231 cells (11). Therefore, we examined whether SH003 commonly causes an increase of intracellular ROS level in breast cancer cell lines. HCC-1419, MCF-7 and MDA-MB-231 breast cancer cells were pretreated with NAC, an inhibitor of ROS production, for 30 min followed by SH003 treatment for another 4 h. SH003 significantly increased intracellular ROS level independently of breast cancer subtype, and NAC failed to reduce SH003-induced intracellular ROS production (Figure 4A), suggesting that SH003 might overcome NAC inhibition of ROS production. However, 4-PBA rather slightly increased intracellular ROS production even under SH003 treatment (Figure 4B). Those data suggested that the intracellular ROS production by SH003 might induce ER stress. Thus, we next investigated whether SH003-induced ROS production causes ER stress. When we examined ER stress markers, NAC reduced SH003-induced eIF2α phosphorylation and CHOP expression (Figure 4C and D). Therefore, it is plausible that SH003 causes ER stress-mediated apoptosis via intracellular ROS production in breast cancer cells, subtype-independently.
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.
CHOP is required for SH003-induced apoptosis. CHOP is a key player for ER stress-induced apoptosis (35, 36). SH003 increased CHOP protein expression levels (Figure 3A), which was blocked by 4-PBA (Figure 3B). As SH003 induces ER stress (Figure 3), we assumed that SH003 might increase CHOP gene expression. As expected, SH003 increased CHOP mRNA levels (Figure 5A), which is consistent with the SH003 increase of CHOP protein levels (Figure 3A). Therefore, we further examined whether SH003-induced apoptosis requires CHOP. When CHOP gene expression was silenced with CHOP siRNAs, CHOP gene silencing inhibited SH003-induced apoptosis (Figure 5B-D). CHOP knockdown also repressed SH003-mediated increase of cleaved PARP and cleaved Caspase-7 (Figure 5B and C). Accordingly, CHOP knockdown rescued SH003-induced apoptosis (Figure 5D). Therefore, SH003 requires CHOP to cause apoptosis of breast cancer cells.
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.
Discussion
Herbal medicine named SH003 was originally developed for targeting highly metastatic triple-negative breast cancer cells based on the theory of the traditional Korean medicine (7). Our previous works revealed that SH003 causes apoptosis of triple-negative breast cancer cells (7, 8, 11). However, our first report showed that SH003 subtype-independently reduces a viability of breast cancer cells, although it mainly focused on triple-negative breast cancer cells (7). Moreover, our serial works revealed that SH003 causes apoptosis in various cancer cell types including breast, lung, gastric, prostate, and cervical cancer cells (7-15). However, we still need to clearly draw its mode of action in biochemical and cell biological views, although clinical studies for SH003 is ongoing and preclinical good laboratory practice toxicity tests have shown its safety (11, 18-20). Herein, we revealed that SH003 causes apoptosis of breast cancer cells through intracellular ROS production and ER stress in a breast cancer subtype-independent manner.
In our gene expression analysis, SH003 altered the expression pattern of 138 genes independently of the subtype of breast cancer cell lines. Interestingly, SH003 differentially altered the expression patterns of genes in different subtypes of breast cancer cells, although we statistically found common genes altered by SH003. This indicates that SH003 as a bundle of natural products targets multiple genes in breast cancer subtype-specific manner. Nevertheless, our previous and present data indicate that SH003 can be used to treat breast cancer independently of breast cancer subtypes and applied together with classical anti-cancer agents (7-9, 11, 15, 16). Therefore, understanding general modules for SH003 effect on cancer will improve our knowledge to convince SH003 effect in clinics.
Our gene expression data further showed that SH003 might alter genes involved in ER stress in a breast subtype-independent manner. ER stress is triggered by oxidative stress and vice versa (37). Our data showed that SH003 increases the level of ROS production independently of breast cancer subtype. Moreover, SH003 overcomes NAC inhibition of ROS generation, but 4-PBA rather increased SH003-triggered ROS production. In addition, NAC inhibited SH003-induced CHOP gene expression and eIF2α phosphorylation. Those data indicate that SH003-induced ROS production is located upstream of ER stress. Moreover, we found that CHOP gene silencing rescued SH003-induced apoptotic cell death. Thus, our data show that SH003 causes apoptosis through the increased ROS production and ER stress in breast cancer cells (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.
Meanwhile, our previous studies show that SH003 inhibits EGFR phosphorylation in breast cancer and lung cancer cells and VEGFR phosphorylation in endothelial cells (7, 11, 14). Thus, SH003 is likely to inhibit the activation of receptor tyrosine kinases (RTKs). It has been known that RTKs activate ROS production and vice versa (38-40). Thus, we could image that SH003 may target RTKs, which is followed by the increase of intracellular ROS production and ER stress. Meanwhile, although SH003 is known to target STAT3 (7, 11, 16), it is unknown whether STAT3 is directly linked to ER stress in SH003-stimulated cancer cells. However, ER stress-related pathway inhibits STAT3-involved pathway and vice versa (41-44). ROS is also known to block STAT3 activation (45, 46). As the present study revealed that SH003 induces ROS production, it is plausible that SH003-induced ROS inhibits STAT3 activation and activates ER stress. Our ongoing study will reveal relations between ROS, STAT3 and ER stress in SH003-treated cancer cells.
We also need to consider multiple components in SH003. It is very plausible that each compound in SH003 may target various molecules within the cells. Although we still need more data to clearly understand the molecular functions of SH003, this study first answers how SH003 causes apoptosis of breast cancer cells independently of their subtype. This is important for understanding the SH003 function in the clinic, as breast cancer is highly heterogeneous (47-50). Our ongoing works will gather actions of all components in SH003 in the cells to draw a mode of action of SH003 holistically.
Conclusion
SH003 as herbal medicine was developed to treat cancer. Although its anti-cancer effects have been confirmed in various cancer cells, the underlying mechanisms are not yet clearly defined. This study shows that SH003 causes apoptotic cell death in various types of breast cancer cell lines by promoting intracellular ROS production and ER stress. Therefore, we suggest that SH003 may be a promising agent against breast cancer.
Acknowledgements
This work was supported by Korea National University of Transportation 2021, the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2017R1D1A1B03034996, 2020R1A5A2019413 and 2021R1F1A1057282) and the Ministry of Education (2021R1A6A1A03046418).
Footnotes
Authors’ Contributions
Lee SY designed the study, performed experiments and wrote the manuscript. Kim TH performed experiments. Ko SG, Choi WG and Chung YH worked on the design of the study together. Cheon C designed the study and managed the experiments. Cho SG designed the study, supervised the experiments, and wrote the manuscript. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors declare no competing interests.
- Received July 4, 2022.
- Revision received October 17, 2022.
- Accepted October 24, 2022.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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