Alantolactone promotes ER stress‐mediated apoptosis by inhibition of TrxR1 in triple‐negative breast cancer cell lines and in a mouse model

Abstract Triple‐negative breast cancer (TNBC) is a subtype of breast cancer with poor clinical outcome and currently no effective targeted therapies are available. Alantolactone (ATL), a sesquiterpene lactone, has been shown to have potential anti‐tumour activity against various cancer cells. However, the underlying mechanism and therapeutic effect of ATL in the TNBC are largely unknown. In the present study, we found that ATL suppresses TNBC cell viability by reactive oxygen species (ROS) accumulation and subsequent ROS‐dependent endoplasmic reticulum (ER) stress both in vitro and in vivo. Thioredoxin reductase 1 (TrxR1) expression and activity of were significantly up‐regulated in the TNBC tissue specimens compare to the normal adjacent tissues. Further analyses showed that ATL inhibits the activity of TrxR1 both in vitro and in vivo in TNBC and knockdown of TrxR1 in TNBC cells sensitized ATL‐induced cell apoptosis and ROS increase. These results will provide pre‐clinical evidences that ATL could be a potential therapeutic agent against TNBC by promoting ROS‐ER stress‐mediated apoptosis through partly targeting TrxR1.

metastases, recurrence and worse mortality rates despite systemic therapy. 2,3 So far, due to molecular characteristics of TNBC, no efficient targeted therapy is available and surgery, radiotherapy, chemotherapy are major choices of the systemic treatment. 4,5 However, these common systemic therapies accompany with high relapse rate and significantly affect patient's quality of life. 6 For example, generally used chemotherapeutic drugs, such as taxol and doxorubicin, exhibit high dose-limiting toxicity to tumour cells as well as normal cells, which limit their clinical usage. 7,8 Although several clinical trials targeting TNBC specific molecules have been conducted, including poly ADP-ribose polymerase (PARP) and epidermal growth factor receptor (EGFR), but no significant improvements were observed in patients with TNBC. 9 Therefore, there is an urgent need to discover more favourable and effective anti-cancer drugs that improve therapeutic effects and prognosis of patients with TNBC.
Using plants treatment of cancers has a long history and there are more than 3000 plant species have been reported. 10 Alantolactone (ATL), main bioactive compounds that are presented in many medicinal plants such as Inula helenium, Inula racemosa, L. Inula japonica, Aucklandia lappa and Radix inulae. Various pharmacological actions of ATL have been found, including anti-inflammatory, antimicrobial and anti-cancer activities with no significant toxicity. 11 Over the past decade, ATL has been reported to inhibit cancer cell proliferation in liver cancer, 12 lung squamous cell carcinoma (SCC), 13 breast cancer, 14 cervical cancer 15 and colorectal cancer. 16 One recent study has shown that ATL inhibits MDA-MB-231 cell growth by induction of reactive oxygen species (ROS) and ROS-mediated apoptosis. 17 However, detailed anti-cancer mechanism of ATL against TNBC is unclear, especially its direct target and effect in vivo are unknown.
Thioredoxin (Trx)/thioredoxin reductase (TrxR) system has been reported to involve in oxidative stress-induced apoptosis in cancer cells. 18,19 Increased thioredoxin levels contribute to the cancer cell growth and resistance to chemotherapy. 19 Various anti-cancer drugs have been reported to directly or indirectly inhibit TrxR to reverse malignant characteristics. 20 Thioredoxin reductase 1 (TrxR1) is a major redox regulator in mammalian cells and has been reported to be overexpressed in many cancer cells, such as cervical, hepatoma, pancreatic, gastric, lung and breast cancers. [21][22][23][24][25][26] Therefore, TrxR1 has emerged as a promising biomarker and drugable target for cancer therapy. This raises the questions whether TrxR1 is also overexpressed in TNBC, and whether ATL targets TrxR system to generate ROS.
In this study, we have examined the therapeutic effect and the anti-cancer mechanism of ATL on TNBC cells both in culture and in xenografts. Our study showed that ATL suppresses TNBC cell viability through inducing apoptosis and causing cell cycle arrest by ROSdependent endoplasmic reticulum (ER) stress pathway both in vitro and in vivo. In addition, TrxR1 expression and activity were significantly increased in TNBC specimens compared to the normal breast specimens. We also found that ATL can effectively inhibit TrxR1 in vitro and in vivo. The present study provides strong evidences that ATL can be a clinical candidate drug for the treatment of TNBC. Treatment of TNBC cells with ATL decreased Bcl-2 ( Figure 1E) while increased the levels of Bax. As caspase-3 is a crucial component of the apoptotic machinery, and the cleavage of caspase-3 is a central event in the process of apoptosis, we further examined the expression of cleaved caspase-3. As shown in Figure 1E, the cleaved forms of caspase-3 were increased in TNBC cells exposed to ATL. These data suggest that ATL could induce apoptosis of TNBC cells.   Figure 2C). Furthermore, colony formation assay also showed that ATL significantly prevented colony formation of TNBC cells at 5 and 10 μmol/L levels ( Figure 2D).

| ATL increases ROS levels in human TNBC cells
Reactive oxygen species accumulation has been reported to induce apoptosis in several cancer cells. 27,28 To examine whether apoptosis and reduced viability in TNBC cells following ATL treatment are involved in increased ROS levels, we further treated cells using 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA), which is rapidly oxidized by ROS to produce fluorescence 2',7'-dichlorofluorescein We next wanted to check whether the negative growth signals from ATL can be attenuated by reduction of ROS levels. Pre-treatment of cells with NAC (1 mmol/L) was able to attenuate ATL-induced apoptosis ( Figure 3F; Figure S1A,B) and cell cycle arrest

| ROS-dependent ER stress pathway contributes to ATL lethality in TNBC cells
Increased ROS levels and imbalance of the intracellular redox status increase unfolded proteins in the ER and induce ER stress response. 29   in MDA-MB-231 cells. And the expression levels of these ER stress-related proteins reached the peak about 9 h following treatment ( Figure 4A). We further noted that dose-dependent activation of ER stress by ATL treatment in TNBC cells ( Figure 4B; Figure S2A). Moreover, pre-treatment of cells with NAC (1 mmol/ L) markedly attenuated the induction of these ER stress related proteins ( Figure 4C; Figure S2B). We next investigated the effect of ATL on the morphology of ER in MDA-MB-231 cells. Electron microscopy showed that a 9 h ATL challenge is conspicuous to cause ER swelling, while pre-treatment with NAC totally reversed this morphological alteration ( Figure 4D). Electron microscopy indeed revealed swollen mitochondria with disrupted cristae in cells exposed to ATL ( Figure S3A). To determine ATL treatment indeed causes ER stress and UPR response in TNBC cells, we evaluated XBP1S and ATF6 expression in TNBC cells after treatment with ATL. The IRE1α-XBP1S pathway has been reported to enhance or suppress cancer progression in different contexts. 30,31 On the other hand, ATF6α-dependent UPR has shown cytoprotective functions leading to oncogenic roles in tumourigenesis. 32,33 We found that ATL treatment markedly increased XBP1S expression ( Figure S2C), but decreased ATF6 expression in MDA-MB-231 cells ( Figure S2D) indicating that ER stress and a UPR response are indeed activated after treatment with ATL. Next, knockdown analysis was performed to further investigate that sustained ER stress indeed contributes to the ALT-induced cell death.
Knockdown of ATF4 by siRNA markedly reduced ATF4 expression in MDA-MB-231 cells ( Figure 4E), accordingly we observed that reduced apoptotic cells in ATF4 knockdown cells treated with ALT ( Figure 4F,G). These findings indicate that ALT-induced cell death is at least in part a result of sustained ER stress.

| TRXR1 is up-regulated in clinical TNBC cancer tissues and inactivated by ATL
Thioredoxin reductase 1 overexpression has been reported in several malignancies. Overexpression of TrxR1 was associated with aggressive tumour growth and poor survival. In addition, it has been reported that TrxR system contributes to tumour cell resistance to oxidative stress. 18 We firstly examined TrxR1 mRNA expression in TNBC. GSE59590 dataset showed that  sues. Further, we tested the TrxR1 enzyme activity by using the 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) assay. As shown in Figure 5E and F, the activities of TrxR1 in tumours were significantly up-regulated compared to the corresponding normal breast specimens. All together, these findings indicate that TrxR1 might play pivotal functions in TNBC carcinogenesis.
We next want to know whether TrxR1 is a target of ATL in TNBC cells. A recent study showed that ATL inhibits the recombinant TrxR1 in HeLa cells. 34 To investigate the structural mechanism of ATL binding to the TrxR1 protein, we performed a molecular simulation of ATL-TrxR1 complex using AutoDock. Our result showed that ATL not only can insert into the C-terminal active site of TrxR1 but also form a strong covalent bond (Figure 5G). It has been reported that the redox motif containing Cys-497, Sec-498 plays a vital role in enzyme inactivation, thus competitive inhibition to these residues could significantly desensitize the enzyme. 35,36 During the docking process, the alkenyl in ATL was detected as Michael acceptor to form a hard covalent bond with Cys-497 while the cyclohexane part inserted into the hydrophobic pocket. Thus occupying the redox active centre may block the nature enzymatic recognition. This docking study suggests that TrxR1 is the potential target of ATL and blocking the critical residues in redox centre could inhibit its enzyme activity.
We further tested the direct inhibitory effects of ATL on TrxR1 enzyme activity by using DTNB assay. When lysates prepared from MDA-MB-231 cells were incubated with various concentrations of ATL for 2 h, the DTNB reducing activity of TrxR1 decreased in a dose-dependent manner ( Figure 5H). We confirmed these results by measuring TrxR1 enzyme activity in pre-   Figure 6G). We next assessed ATL-induced apoptosis by cleaved caspase-3 levels through both Western blot analyses and immunohistochemistry. We found that cleaved caspase-3 levels were increased in tumour tissues following treatment with ATL ( Figure 6G,H), while cell proliferation (ki-67 levels) was decreased ( Figure 6H). Importantly, NAC treatment reversed the ALT-induced alterations of cleaved caspase 3 and ki-67 levels ( Figure 6G-H We have found that ATL promotes ER stress by ROS induction resulting in apoptotic cell death. To better understand how ATL F I G U R E 3 Alantolactone (ATL) increases ROS accumulation in human triple-negative breast cancer (TNBC) cells. (A,B) Intracellular ROS generation was assessed by DCF fluorescence in MDA-MB-231 cells following exposure to ATL for different time periods (A, 30 μmol/L) and different concentrations (B). Quantification of data in (B) is shown in panel (C) [*P < 0.05 compared to DMSO control] (n = 3). D, MDA-MB-231 cells with or without 1 mmol/L NAC were exposed to ATL (30 μmol/L) for 1 h and intracellular ROS levels were measured by DCF fluorescence. Quantification of data in (D) is shown in panel (E) (*P < 0.05 compared to DMSO control) (n = 3). F, Apoptosis induction in cells exposed to ATL (24 h), with or without pre-treatment with 1 mmol/L NAC for 1 h. Figure showing flow cytometry histograms. (***P < 0.001, ****P < 0.0001 compared to DMSO). Representative figures are shown in Figure S3B. G, G2/M phase accumulation in cells exposed to ATL (12 h), with or without pre-treatment with 1 mmol/L NAC for 1 h. Figure showing flow cytometry histograms. (*P < 0.05, **P < 0.01 compared to DMSO) (n = 3). Representative figures are shown in Figure S3C and D. H, Western blot analysis of proteins in cells pre-treated with 1 mmol/L NAC prior to 30 μmol/L ATL exposure. For cell cycle phase proteins, ATL exposure was carried out for 16 h and for apoptosisrelated protein, exposure was for 18 h. I, Colony formation in cells exposed to 30 mμmol/L ATL (24 h), with or without pre-treatment with 1 mmol/L NAC for 1 h influences this signalling pathway, we intend to find the target gene of ATL which involved in ROS-ER stress-mediated cell death.

| ATL inhibits the growth of MDA-MB-231 cell xenograft in vivo, accompanied with decreased TRXR1 activity and increased ROS level
Recently, one research article has shown that ATL is an inhibitor of TrxR induces generation of ROS in HeLa cells. 34  F I G U R E 4 ROS-dependent endoplasmic reticulum (ER) stress pathway is involved in alantolactone (ATL)-induced triple-negative breast cancer (TNBC) cell's growth inhibition. A, MDA-MB-231 cells were exposed to 30 μmol/L ATL for the indicated times. Protein levels of ATF4, phosphorylated eIF2α and CHOP were determined by western blot. GAPDH served controls. B, Western blot analysis of ER-stress pathway associated proteins in MDA-MB-231 cells exposed to various doses of ATL for 9 h. C, MDA-MB-231 cells were pre-treated with 1 mmol/L NAC prior to 30 μmol/L ATL exposure. Endoplasmic reticulum-stress pathway associated proteins levels were detected by western blot. D, Electron microscopy images of MDA-MB-231 cells exposed to ATL (10 000× and 20 000× shown]. Cells were exposed to 30 μmol/L ATL for 9 h, with or without pre-treatment with 1 mmol/L NAC for 1 h. E, MDA-MB-231 cells were transfected with ATF4 siRNA or control siRNA, ATF4 expression in MDA-MB-231 cells was determined by Western blotting after treating with ATL (30 μmol/L) for 9 h. F, Effect of ATF4 knockdown on ATL-induced apoptosis was determined by annexin V/propidium iodide (PI) staining and flow cytometry. MDA-MB-231 cells were transfected with ATF4 siRNA or control siRNA for 24 h and then exposed to ATL (30 μmol/L) for 24 h. G, Quantification of annexin V/PI staining presented as the percentage of apoptotic cells following treatment (*P < 0.05, **P < 0.01 and***P < 0.001 compared to DMSO control) (n = 2) observed in various malignancies including breast cancer. [21][22][23][24][25][26]38 In addition, it has been demonstrated that TrxR1 plays an important function in tumour growth, progression, metastasis and chemotherapy resistance. [39][40][41] Our patient sample analyses, in agreement with publicly available GSE59590 dataset validated that TrxR1 expression and its activity were significantly increased in TNBC tissue samples compared to the NAT and other types of breast cancer samples. In addition, our study first time showed that ATL can suppress TrxR1 activity in the TNBC cell line MDA-MB-231 cells and its xenograft models. ATL inhibits TrxR1 activity, suppresses TNBC cell growth in xenografts by increasing accumulation of ROS in tissues, and leading to ROS-dependent ER stress. Taken together, our study indicates that TrxR1 may serve as a promising biomarker and target molecule for TNBC therapy.
In conclusion, our study showed the potential usefulness and novel molecular mechanism of ATL in the treatment of TNBC. We found that ATL treatment resulted in significant ROS accumulation by inhibition of TrxR1 activity. ROS accumulation caused the activation of the ER stress-mediated apoptotic pathway both in vitro and in vivo. Considering no efficient targeted therapy in TNBC and ATL effectively inhibits tumour growth of TNBC in vivo without obvious side effects, ATL might be a potential anti-tumor drug in TNBC.

ACKNOWLEDG EMENTS
This work was supported by National Natural Science Foundation of China (81672305 to Ri Cui, 81622043 to Guang Liang, 81372380 to Ouchen Wang).

CONFLI CTS OF INTEREST
The authors declare no potential conflicts of interest.

AUTHORS' CONTRIBUTI ONS
Ri Cui and Guang Liang conceived the idea and designed the research. Changtian Yin, Xuanxuan Dai, Xiangjie Huang, Qiulin Zhou, F I G U R E 7 Schematic illustration of the proposed anti-cancer mechanism of alantolactone (ATL) in triple-negative breast cancer (TNBC). We show first time that ATL significantly inhibits thioredoxin reductase 1 (TrxR1) activity leading to increased ROS levels in the TNBC cells and its xenograft models. ROS accumulation causes the activation of the endoplasmic reticulum (ER) stress pathway/unfolded protein response and contributes the ROS-induced cell death by activating caspases in TNBC cell lines and in a mouse pre-clinical model F I G U R E 6 Alantolactone (ATL) inhibits MDA-MB-231 cell xenograft growth in vivo, accompanied with decreased Thioredoxin reductase 1 (TrxR1) activity and increased ROS level. (A,B) Tumour volume in vehicle and ATL with or without NAC treated mice. Injected MDA-MB-231 cells in the flanks of nude mice and tumours were allowed to develop for 12 d. Mice were then treated with ATL at 15 or 30 mg/kg interperitoneally for 20 d. NAC (0.5 g/L) was administered in the drinking water for one group mice with 30 mg/kg ATL for the same days. P < 0.01]. Images showing tumour tissues at day 32 (A) and the final tumour volume (B). Tumour volumes were calculated as described in the methods section. (*P < 0.05). C, Body weight of the mice in four groups (n = 6). D, Hematoxylin and eosin staining of heart, liver and kidney specimens (magnification 200×). E, Levels of malondialdehyde (MDA) in tumour tissue lysates (*P < 0.05, **P < 0.01 compared to vehicle treated group) (n = 3). F, Fluorescence images of tumour specimens stained with DHE (red) and DCFH-DA (green) and tissue sections were counterstained with 4′,6-diamidino-2-phenylindole (blue). Increased fluorescence intensity is indicative of increased ROS levels (magnification 200×). G, Western blot analysis of ATF4, CHOP and Cleaved-caspase3 levels in tumour tissues. GAPDH was used as loading control. H, Representative immunohistochemical staining images of cell proliferation marker (Ki-67) and apoptosis marker (Cleaved caspase-3) in tumour tissues. I, Activity of TrxR1 in tumour tissue lysates as determined by end-point insulin reduction assay. (*P < 0.05, **P < 0.01, compared to vehicle treated mice) (n = 3) Chengguang Zhao and Peng Zou performed in vitro experiments.