The Herbal Formula JI017 Induces ER Stress via Nox4 in Breast Cancer Cells

Chemotherapy is a powerful anti-tumor therapeutic strategy; however, resistance to treatment remains a serious concern. To overcome chemoresistance, combination therapy with anticancer drugs is a potential strategy. We developed a novel herbal extract, JI017, with lower toxicity and lesser side effects. JI017 induced programmed cell death and excessive unfolded protein response through the release of intracellular reactive oxygen species (ROS) and calcium in breast cancer cells. JI017 treatment increased the expression of endoplasmic reticulum (ER) stress markers, including p-PERK, p-eIF2α, ATF4, and CHOP, via the activation of both exosomal GRP78 and cell lysate GRP78. The ROS inhibitors diphenyleneiodonium and N-acetyl cysteine suppressed apoptosis and excessive ER stress by inhibiting Nox4 in JI017-treated breast cancer cells. Furthermore, in paclitaxel-resistant breast cancer cell lines, MCF-7R and MDA-MB-231R, a combination of JI017 and paclitaxel overcame paclitaxel resistance by blocking epithelial-mesenchymal transition (EMT) processes, such as the downregulation of E-cadherin expression and the upregulation of HIF-1α, vimentin, Snail, and Slug expression. These findings suggested that JI017 exerts a powerful anti-cancer effect in breast cancer and a combination therapy of JI017 and paclitaxel may be a potential cancer therapy for paclitaxel resistant breast cancer.


Introduction
Breast cancer is a leading cause of cancer-related deaths in women worldwide [1]. Breast cancer is often divided in non-invasive and invasive breast cancer, and invasive breast cancer has several subtypes [2]. Non-invasive breast cancer (ductal carcinoma), a rare type of breast cancer, is found in the ducts of the breast and cannot spread outside the breast. Conversely, invasive breast cancer is the most common type and can spread outside the breast. Inflammatory breast cancer is a rare type of invasive breast cancer and makes the skin of the breast look red and feel warm [3]. Triple-negative breast cancer is a subtype of breast cancer that lacks estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2) and spread faster than the other types [4]. Breast cancer is treated using a combination of surgery, chemotherapy, hormone treatment, and radiotherapy [5]. Chemo-resistance is a well-known phenomenon that arises due to many factors, such as epigenetic changes, genetic mutations, and molecular mechanisms [6]. Chemotherapy and radiotherapy, which involves the use of X-rays and gamma rays, are potential tumor therapy strategies; however, the problems of resistance are encountered in many patients. Although chemotherapy and radiotherapy can be effective against many cancer types, including non-small-cell lung cancer, prostate cancer, skin cancer, cervical cancer, and neck and head carcinoma, they are not as efficacious against other cancers, such as bladder cancer, glioblastoma, and breast cancer [7]. Radiation and anti-tumor reagents targeted at killing tumor cells can also kill normal cells in the process; therefore, incorporating herbal medicine as a part of a combination therapy may be a potential strategy to reduce the adverse effects [8]. Paclitaxel (brand name: Taxol), which is found in 2.5. LDH Assay MCF-7 and MDA-MB-231 human breast cancer cell lines were seeded into a 96-well plate with growth medium and grown for 24 h, followed by treatment with JI017 for 24 h. LDH cytotoxicity assay (LDH cytotoxicity assay) was performed according to the manufacturer's protocols. The fluorescence was determined by measuring the absorbance of the samples at 490 or 492 nm using the ELISA reader (SpectraMax190, Microplate Reader, Molecular Devices, Sunnyvale, CA, USA).

Caspase-3 Activity Assays
MCF-7 and MDA-MB-231 human breast cancer cell lines were seeded into a 6-well plate with growth medium and grown for 24 h. Caspase-3 activity assay (the Biovision colorimetric caspase-3 assay kit) was performed according to the manufacturer's protocols. The fluorescence was analyzed at 405 nm using a spectrophotometer (Molecular Devices, San Jose, CA, USA).

Intracellular Ca 2+ Assays
MCF-7 and MDA-MB-231 human breast cancer cell lines were seeded into a 96-well plate with growth medium and grown for 24 h, Then, the cells were treated with JI017. Intracellular calcium assays were performed using a Ca 2+ Assay Kit (Colorimetric) (Abcam; ab102505), as described in the supplier's manual. The fluorescence was measured and analyzed at 575 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).

Intracellular ROS Assays
MCF-7 and MDA-MB-231 human breast cancer cell lines were seeded and incubated into a 96-well plate with growth medium and grown for 24 h, followed by treatment with JI017. After the treatment, cells were incubated with the cell permeant 2 7 -dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA, Invitrogen, Waltham, MA, USA) for 30 min at 37 • C, as described in the supplier's protocol. The fluorescence was measured and analyzed at 495 nm (Ex)/525 nm (Em) using a microplate reader (Molecular Devices, San Jose, CA, USA).

Establishment of Paclitaxel-Resistant MCF-7 and MDA-MB-231 Cell Lines
MCF-7 and MDA-MB-231 human breast cancer cell lines were seeded into 60 mm dishes and exposed to increasing doses of paclitaxel for 6 months. To establish paclitaxelresistant cell lines, MCF-7R and MDA-MB-231R, paclitaxel (2 nM, half of the IC 50 ) treatment was initiated and gradually increased to 100 nM over a period of 6 months.

Colony Formation Assay
Human breast cancer cells and paclitaxel resistant human breast cancer cells (MCF-7, MCF-7R, MDA-MB-231, and MDA-MB-231R) were seeded in 60 mm dishes with growth medium and grown for 24 h at 37 • C in a CO 2 incubator. Cells were incubated for 10 days for colony formation, and then the colonies were stained with 0.5% crystal violet (Amresco, Solon, OH, USA). To calculate the survival fraction, the number of colonies formed was divided by the number of seeded colonies formed on the control plate.

Isolation of Total RNA and Protein
Total RNA (10 µg) from human breast cancer cell lines MCF-7 and MDA-MB-231 in a 100 mm cell culture dish was prepared using TRIzol reagent according to the manufacturer's instructions (Invitrogen). Protein (15 µg) from the cell lysates was collected using the radioimmunoprecipitation assay lysis buffer (Bio-Rad, Hercules, CA, USA). The supernatant was analyzed to quantify the collected protein using the bicinchoninic acid (BCA) method (Thermo Scientific, Rockford, Waltham, MA, USA).

Exosome Isolation
MCF-7 and MDA-MB-231 human breast cancer cell lines were treated with JI017 at the doses shown, and exosomes were obtained from the supernatant of JI017-treated MCF-7 and MDA-MB-231 cells according to the manufacturer's protocol (Total Exosome Isolation Reagent (for cell culture media), Thermo Fisher Scientific, Rockford). Protein concentration was measured using the BCA method (Thermo Scientific). The protein loading samples (10 µg) were also quantified by Ponceau S staining and subjected to Western blot analysis. Positive exosomes were identified using the exosome marker CD63.

Animals
For animal study, 5-week-old, female, athymic BALB/c nude mice (nu/nu) were purchased from OrientBio, Inc. (Daejeon, Korea), and maintained for 1 week with ad libitum access to sterile standard mouse chow (NIH-7 open formula) and water before use. Mice were housed randomly at 50 ± 20% humidity and approximately 21 ± 2 • C on a 12 h light-dark cycle (n = 10 mice/group). All animal experimental procedures were performed according to the National Institutes of Health guidelines and a protocol approved by the Institutional Animal Care and Use Committee of Kyung Hee University.

Tumor Xenograft Mouse Models
For mice xenograft experiment, mice, aged 6 weeks, were inoculated with MDA-MB-231 human breast cancer cell line by subcutaneously (sc) implanting 1 × 10 7 cultured cells into the right thigh. Mice were grouped randomly (n = 10 per group) 6 day later, and JI017 (400 or 600 mg/kg) was administered intraperitoneally (ip) once a day for 2 days. Tumor sizes on two axes (L, longest axis; W, shortest axis) were measured three times per week using Vernier calipers. Tumor volume was calculated as (L × W 2 )/2 (mm 3 ).

Statistical Analysis
All results were confirmed using at least three independent experiments. Student's t-test was used to compare the means of quantitative data between groups, and a p value < 0.05 was considered statistically significant.

Anticancer Effects of JI017 In Vitro and In Vivo in Breast Cancer Cells
To study the anti-cancer effect of JI017 in breast cancer cell lines, including MCF-7, T-47D, SK-BR-3, MDAM-MB-231, HCC-1419, and HT-20, we tested the cell viability and LDH cytotoxicity using WST-1 and LDH assays, respectively, in a dose-dependent manner ( Figure 1A,B). Compared with the control treatment, which involved DMSO, JI017 treatment resulted in reduced cell viability and increased LDH cytotoxicity in a dose-dependent manner in breast cancer cell lines ( Figure 1A,B). To identify the effects of JI017 in vivo, a breast cancer xenograft mice model was constructed with MDA-MB231 cells. Mice in the 400 mg/kg and 600 mg/kg JI017 groups showed lower tumor volumes than the control group and exhibited dose-dependent efficacy ( Figure 1C). The body weight did not significantly differ among the groups ( Figure 1D). To confirm the anticancer effect of JI017, we tested the cell viability, cell cytotoxicity, and caspase-3 activity using WST-1, LDH cytotoxicity, and caspase-3 activity assays, respectively, in a time-dependent manner ( Figure 1E-G). JI017 treatment induces the reduction in cell viability and increase in LDH production, and caspase-3 activity in a time-dependent manner in the breast cancer cell lines MCF-7 and MDA-MB-231 ( Figure 1E-G). On performing Western blot, we found that JI017 treatment mediates caspase-3 and -9 cleavage in a time-dependent manner ( Figure 1H). To study whether JI017 regulates caspase-dependent apoptosis in breast cancer cells, we performed a pharmacological inhibitor experiment using caspase inhibitor Z-VAD-FMK. Z-VAD-FMK alone did not change cell viability, LDH cytotoxicity, and caspase-3 activity; however, JI017 alone reduced cell viability and increased LDH production, and caspase-3 activity. JI017 in combination with Z-VAD-FMK markedly inhibited the decrease in cell viability and increase in LDH cytotoxicity and caspase-3 activity ( Figure 1I-K). Furthermore, Western blot analyses indicated that the combination of JI017 and Z-VAD-FMK blocked caspase-3 cleavage to a greater extent than JI017 alone ( Figure 1L). These results suggested that JI017 treatment induces apoptosis in breast cancer cells. Cell viability was determined using the WST-1 assay; cell cytotoxicity was monitored using the LDH assay, and caspase-3 activity was assessed using the caspase-3 activity assay; *, p < 0.05, #, p < 0.01. To identify caspase-3 cleavage, Western blot assay performed. β-actin was used as a protein loading control.

JI017 Treatment Mediates ER Stress Response in Breast Cancer Cells
Ca 2+ is a potential regulator of cell death and cell survival, and it induces apoptosis by regulating the early and late stages of apoptosis, UPR, and protein folding in various WST-1 assay; cell cytotoxicity was monitored using the LDH assay, and caspase-3 activity was assessed using the caspase-3 activity assay; *, p < 0.05, #, p < 0.01. To identify caspase-3 cleavage, Western blot assay performed. β-actin was used as a protein loading control.

JI017 Treatment Mediates ER Stress Response in Breast Cancer Cells
Ca 2+ is a potential regulator of cell death and cell survival, and it induces apoptosis by regulating the early and late stages of apoptosis, UPR, and protein folding in various conditions [34]. To identify if JI017 regulates Ca 2+ production, intracellular Ca 2+ assay was performed. When MCF-7 and MDA-MB-231 cells were treated with JI017, they showed approximately 6-8 fold higher intracellular Ca 2+ release than the controls in a time-dependent manner ( Figure 2A). We performed RT-PCR to study the mRNA expression of ER stressrelated genes, such as ATF4 and CHOP, in JI017-treated MCF-7 and MDA-MB-231 cells in a time-dependent manner. JI017-treated cells showed upregulated ATF4 and CHOP when comparison compared with control cells ( Figure 2B). We performed Western blot analyses to confirm the protein expression of ER stress markers, including GRP78, p-PERK, PERK, p-eIF2α, eIF2α, ATF4, and CHOP, in JI017-treated MCF-7 and MDA-MB-231 cells in a time-dependent manner. JI017-treated cells showed higher expression levels of GRP78, p-PERK, p-eIF2α, ATF4, and CHOP than control cells ( Figure 2C). Many reports suggest that GRP78 regulates cell survival and cell death via cell-cell communication in exosome isolates. To study if JI017 treatment mediates GRP78 in MCF-7 and MDA-MB-231 exosome fractions, we isolated the exosome from the culture medium of JI017-treated breast cancer cells and performed Western blot analyses for JI017-treated exosomal proteins. JI017 treatment upregulated exosome GRP78 and exosome marker CD63 in a time-dependent manner ( Figure 2D). To determine if JI017 treatment induces apoptosis via ER stress response, we co-treated the cells with JI017 and the ER stress inducer TG. TG in combination with JI017 reduced cell viability and enhanced Ca 2+ production to a greater extent than JI017 or TG alone ( Figure 2E,F). Combination treatment with TG and JI017 mediated the increase in ATF4 and CHOP mRNA levels and GRP78, p-PERK, ATF4, CHOP, and caspase-3 cleavage protein levels in MCF-7 and MDA-MB-231 cells to a greater extent than TG or JI017 alone ( Figure 2G,H). Our findings indicated that JI017 treatment mediates apoptosis through the PERK-ATF4-CHOP signaling pathway in breast cancer cells. breast cancer cells and performed Western blot analyses for JI017-treated exosomal proteins. JI017 treatment upregulated exosome GRP78 and exosome marker CD63 in a timedependent manner ( Figure 2D). To determine if JI017 treatment induces apoptosis via ER stress response, we co-treated the cells with JI017 and the ER stress inducer TG. TG in combination with JI017 reduced cell viability and enhanced Ca 2+ production to a greater extent than JI017 or TG alone ( Figure 2E,F). Combination treatment with TG and JI017 mediated the increase in ATF4 and CHOP mRNA levels and GRP78, p-PERK, ATF4, CHOP, and caspase-3 cleavage protein levels in MCF-7 and MDA-MB-231 cells to a greater extent than TG or JI017 alone ( Figure 2G,H). Our findings indicated that JI017 treatment mediates apoptosis through the PERK-ATF4-CHOP signaling pathway in breast cancer cells.

Targeting ER Stress Suppresses JI017-Mediated Cell Death in Breast Cancer Cells
To confirm whether JI017 treatment regulates the ER stress master regulator GRP78, GRP78-specific siRNAs were transfected into MCF-7 and MDA-MB-231 cells, which were then treated with JI017. In a knockdown experiment, GRP78 siRNAs blocked the decrease in cell viability and increase in LDH production, Ca 2+ production, and caspase-3 activity to a greater extent in JI017-treated MCF-7 and MDA-MB-231 cells ( Figure 3A,B). Western blot analyses revealed that GRP78 inhibition downregulated GRP78, p-PERK, PERK, p-eIF2α, eIF2α, CHOP, and caspase-3 cleavage levels in JI017-treated MCF-7 and MDA-MB-231 cells to a greater extent ( Figure 3C). Furthermore, in exosomes isolated from JI017-treated MCF-7 and MDA-MB-231 cells, GRP78 knockdown downregulated the expression levels of exosome GRP78 in JI017-treated MCF-7 and MDA-MB-231 cells ( Figure 3D). Our findings suggest that the inhibition of GRP78 suppresses ER stress and apoptosis in JI017-treated breast cancer cells. To check whether JI017 treatment regulates ER stress and cell death, PERK-and CHOP-specific siRNAs were transfected into MCF-7 and MDA-MB-231 cells, and these cells were then treated with JI017. Unlike transfection of control siRNA, transfection of PERK and CHOP siRNAs blocked the decrease in cell viability and increase inCa 2+ production in JI017-treated MCF-7 and MDA-MB-231 cells ( Figure 3E,G). Western blot analyses revealed that PERK and CHOP knockdown reduced p-PERK, PERK, ATF4, CHOP, and caspase-3 cleavage levels in JI017-treated MCF-7 and MDA-MB-231 cells to a greater extent ( Figure 3F,H). In real-time RT-PCR, CHOP knockdown decreased CHOP levels in JI017-treated MCF-7 and MDA-MB-231 cells to a greater ( Figure 3H). Our findings suggest that targeting PERK and CHOP blocks ER stress and apoptosis by JI017 treatment in breast cancer cells.

JI017 Treatment Mediates ER Stress and Cell Death by Releasing ROS in Breast Cancer Cells
To confirm if JI017 treatment regulates ROS release in breast cancer cells, intracellular ROS assay was performed. JI017 induces intracellular ROS release in a time-dependent manner ( Figure 4A). To study if ROS inhibitors DPI and NAC suppress JI017-treated ROS release and apoptosis in MCF-7 and MDA-MB-231 cells, we performed the WST-1 assay, LDH assay, intracellular ROS assay, and intracellular Ca 2+ assay. The combination of JI017 with DPI and with NAC inhibited the decrease in cell viability and increase in LDH release, ROS production, and Ca 2+ release to a greater extent than JI017 treatment alone ( Figure 4B-E). Western blot analyses revealed that co-treatment of JI017 with DPI or NAC blocked p-PERK, ATF4, CHOP, and caspase-3 cleavage expression to a greater extent than JI017 treatment alone ( Figure 4F). Our findings indicate that J017 treatment mediates ER stress and apoptosis by producing ROS in breast cancer cells.
inCa production in JI017-treated MCF-7 and MDA-MB-231 cells ( Figure 3E,G). Western blot analyses revealed that PERK and CHOP knockdown reduced p-PERK, PERK, ATF4, CHOP, and caspase-3 cleavage levels in JI017-treated MCF-7 and MDA-MB-231 cells to a greater extent ( Figure 3F,H). In real-time RT-PCR, CHOP knockdown decreased CHOP levels in JI017-treated MCF-7 and MDA-MB-231 cells to a greater ( Figure 3H). Our findings suggest that targeting PERK and CHOP blocks ER stress and apoptosis by JI017 treatment in breast cancer cells.

JI017 Induces ER Stress and Apoptosis via Nox4 and ROS Release in Breast Cancer Cells
To identify whether JI017 treatment regulates Nox4 expression and ROS production, Nox4-specific siRNAs were transfected into MCF-7 and MDA-MB-231 cells, and these cells were then treated with JI017. Knockdown experiments of Nox4 indicated higher cell viability and lower LDH release, caspase-3 activity, intracellular ROS release, and intracellular Ca 2+ release in JI017-treated MCF-7 and MDA-MB-231 cells in comparison to transfection of control siRNA with JI107 treatment (Figure 5A-E). Western blot analyses indicated that Nox4 knockdown decreased Nox4 and CHOP levels to a greater extent in JI017-treated MCF-7 and MDA-MB-231 cells than in control cells ( Figure 5F). Our findings suggest that Nox4 is involved in ER stress and apoptotic cell death by ROS production in JI017-treated breast cancer cells. release and apoptosis in MCF-7 and MDA-MB-231 cells, we performed the WST-1 assay, LDH assay, intracellular ROS assay, and intracellular Ca 2+ assay. The combination of JI017 with DPI and with NAC inhibited the decrease in cell viability and increase in LDH release, ROS production, and Ca 2+ release to a greater extent than JI017 treatment alone (Figure 4B-E). Western blot analyses revealed that co-treatment of JI017 with DPI or NAC blocked p-PERK, ATF4, CHOP, and caspase-3 cleavage expression to a greater extent than JI017 treatment alone ( Figure 4F). Our findings indicate that J017 treatment mediates ER stress and apoptosis by producing ROS in breast cancer cells.

JI017 Induces ER Stress and Apoptosis via Nox4 and ROS Release in Breast Cancer Cells
To identify whether JI017 treatment regulates Nox4 expression and ROS production, Nox4-specific siRNAs were transfected into MCF-7 and MDA-MB-231 cells, and these cells were then treated with JI017. Knockdown experiments of Nox4 indicated higher cell viability and lower LDH release, caspase-3 activity, intracellular ROS release, and intracellular Ca 2+ release in JI017-treated MCF-7 and MDA-MB-231 cells in comparison to  Figure 5F). Our findings suggest that Nox4 is involved in ER stress and apoptotic cell death by ROS production in JI017-treated breast cancer cells.  with Nox4 siRNAs and treated with JI017 (300 µg/mL, 24 h). Next, WST-1 assay, LDH cytotoxicity assay, caspase-3 activity assay, intracellular ROS assay, and intracellular Ca 2+ assay were performed along with Western blot analysis for Nox4 and CHOP; *, p < 0.05, #, p < 0.01. β-actin was used as the protein loading control.

Paclitaxel in Combination with JI017 Overcome Paclitaxel Resistance by Blocking EMT in Paclitaxel-Resistant Breast Cancer Cells
To study whether the combination of paclitaxel and JI017 overcomes paclitaxel resistance in breast cancer cells, paclitaxel-resistant MCF-7R and MDA-MB-231R cells were established by exposing MCF-7 and MDA-MB-231 cells to paclitaxel and then performing the colony formation assay, WST-1 assay, LDH assay, Western blot analysis, and RT-PCR. The results indicated that JI017 reduces surviving fraction levels depending on the paclitaxel dose (2, 10, Figure 6D,E). Specifically, the combination of paclitaxel and JI017 showed more potent inhibitory effects on EMT than JI017 alone in MCF-7R and MDA-MB-231R cells ( Figure 6D,E). Therefore, these results suggest that the combination of paclitaxel and JI017 overcomes paclitaxel resistance via the suppression of EMT in paclitaxel-resistant breast cancer cells.  (Figure 6D,E). Specifically, the combination of paclitaxel and JI017 showed more potent inhibitory effects on EMT than JI017 alone in MCF-7R and MDA-MB-231R cells ( Figure 6D,E). Therefore, these results suggest that the combination of paclitaxel and JI017 overcomes paclitaxel resistance via the suppression of EMT in paclitaxel-resistant breast cancer cells.

Discussion
In this study, we showed that JI017 or paclitaxel with JI017 potently induces ER stress and apoptotic cell death in paclitaxel resistant breast cancer cells and breast cancer cells. In addition, we suggested evidence that sensitivity of paclitaxel resistant breast cancer cells to this combination treatment correlates with EMT process by blocking HIF-1α, vimentin, Snali, and Slug and by activating E-cadherin in paclitaxel resistant breast cancer cells.
EMT phenomenon processed by the loss of epithelial phenotypes and the acquisition

Discussion
In this study, we showed that JI017 or paclitaxel with JI017 potently induces ER stress and apoptotic cell death in paclitaxel resistant breast cancer cells and breast cancer cells. In addition, we suggested evidence that sensitivity of paclitaxel resistant breast cancer cells to this combination treatment correlates with EMT process by blocking HIF-1α, vimentin, Snali, and Slug and by activating E-cadherin in paclitaxel resistant breast cancer cells.
EMT phenomenon processed by the loss of epithelial phenotypes and the acquisition of mesenchymal characteristics has been associated with chemoresistance on breast cancer therapy, indicating that inhibition of EMT process represents a rational strategy to overcome chemoresistance [35]. Our finding identified this hypothesis by showing that treatment of paclitaxel resistant MCF-7R and MDA-MB-231R cells with JI017 in combination with paclitaxel decreased cell survival and increased apoptotic cell death in association with the reduction in cell viability and the increase in LDH release. Furthermore, reduced expression of EMT-related genes such as HIF-1α, vimentin, Snail, and Slug and enhanced expression of E-cadherin increased these inhibitory effects of cell survival.
The researchers have reported antitumor effects of herbal medicines such as Paris polyhylla, Saussurea lappa and Aucklandia lappa, Oryza officinalis, and Curcuma longa via ER stress response [36]. ER stress pathway response regulates ER homeostasis, and it is initiated by three UPR sensors such as PERK, IRE1α, and ATF6 [37]. The PERK/eIF2α/ATF4/CHOP signaling pathway is a potential therapeutic strategy for sensitizing cancer cells [38]. Here, we showed that JI017 induces ER stress in MCF-7 and MDA-MB-231 cells as indicated by the upregulation of exosome GRP78 and cell lysate ER stress markers including GRP78, p-PERK, p-eIF2α, ATF4, and CHOP. These are first report showing the regulation of JI017-induced ER stress response via exosome release. In MCF-7 and MDA-MB-231 cells treated with JI017, the upregulation of exosome marker CD63 and ER stress marker GRP78 mean that JI017 induces ER stress via exosome and cell lysate. More importantly, our study identified that thapsigargin (TG), in combination with JI017, has synergistic anticancer effects in MCF-7 and MDA-MB-231 cells to a greater extent than JI017 or TG alone. TG blocked Ca 2+ homeostasis via the inhibition of sarco/endoplasmic reticulum (ER) Ca 2+ -ATPase (SERCA) and then induced cell death [39]. The synergistic effect of TG and JI017 co-treatment mean that JI017 also induces ER stress and cell death by inhibiting calcium pump SERCA in breast cancer cells. Moreover, GRP78, PERK, or CHOP knockdown suppressed ER stress and cell death in JI017-treated breast cancer cells, MCF-7 and MDA-MB-231.
ROS exert a potential function to determine cancer cell survival and death and is an important target for anticancer drugs [40,41]. The accumulation of ROS induces by increasing protein-folding in the ER and Nicotinamide adenine dinucleotide phosphate (NADPH) and then it mediates apoptosis via the inhibition of cancer cell growth and thioredoxin reductase, and activation of redox signaling [42,43]. With our results, JI017 induced ER stress and cell death by increasing intracellular ROS production and NADPH oxidase NOX4 activity in MCF-7 and MDA-MB-231 cells. Furthermore, NOX inhibitor DPI and ROS inhibitor NAC blocked ER stress and cell death in JI017-treated MCF-7 and MDA-MB-231 cells. Nox4 binds to p22phox and contributes to cancer development and progression via ROS release and HIF-1α in cancer cells [44].

Conclusions
In conclusion, we suggest that novel herbal formula JI017 inhibits breast tumor growth via ER stress and cell death and blocks EMT process in paclitaxel resistant breast cancer cells and breast cancer cells. Our results indicate important insights into the molecular mechanism of paclitaxel/JI017 in breast cancer combination therapy.