Atractylenolide-I suppresses tumorigenesis of breast cancer by inhibiting toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway

Background: Toll-like receptor 4 (TLR4) is an essential sensor related to tumorigenesis, and overexpression of TLR4 in human tumors often correlates with poor prognosis. Atractylenolide I (AT-I) is a major bioactive component from Rhizoma Atractylodes Macrocephalae. Emerging evidence suggests that AT-I exerts anti-tumor effects on various cancers such as colorectal cancer, bladder cancer and melanoma. Nevertheless, the effects of AT-I on mammary tumorigenesis remain unclear. Methods: In order to ascertain the correlation of TLR4/NF-κB pathway with breast cancer, the expression of TLR4 and NF-κB in normal breast tissues and cancer tissues with different TNM-stages was detected by human tissue microarray (TMA) and immunohistochemistry technology. The effects of AT-I on tumorigenesis were investigated by cell viability, colony formation, apoptosis, migration and invasion assays in two breast cancer cells (MCF-7 and MDA-MB-231), and N-Nitroso-N-methylurea (NMU) induced rat breast cancer models were developed to evaluate the anti-tumor effects of AT-I in vivo. The possible underlying mechanisms were further explored by western blot and ELISA assays after a series of LPS treatment and TLR4 knockdown experiments. Results (cid:0) We found that TLR4 and NF-κB were signicantly up-regulated in breast cancer tissues, and was correlated with advanced TNM-stages. AT-I could inhibit TLR4 mediated NF-κB signaling pathway and decrease NF-κB-regulated cytokines in breast cancer cells, thus inhibiting cell proliferation, migration and invasion, and inducing apoptosis of breast cancer cells. Furthermore, AT-I could inhibit NMU-induced rat mammary tumor progression through TLR4/NF-κB pathway. Conclusions: Our ndings demonstrated that TLR4 and NF-κB were over expressed in breast cancer, and AT-I could suppress tumorigenesis of breast cancer via inhibiting TLR4-mediated NF-κB signaling pathway in breast cancer, we assessed the expression of TLR4 and NF-κB among normal breast tissues and different TNM-stages breast cancer tissues using tissue microarray and immunohistochemistry technology. The results showed that TLR4 and NF-κB were over expressed in breast cancer, and correlated with the TNM-stages. Furthermore, these differentially expressed levels of TLR4 and NF-κB were also detected in breast cancer cells and mammary epithelial cells. These results indicated that TLR4 and NF-κB were up-regulated in breast cancer, and might function as tumor promoters in breast cancer.


Background
Breast cancer, a malignancy stemming from mammary epithelial tissues, stood as the most common cancer in women, and the second contributory factor of cancer related women death all over the world [1].
Despite recent improvements in surgical excision, chemotherapy and radiotherapy of breast cancer, patients are often a icted by various complications such as axillary vein thrombosis, neuropathy and cardiovascular diseases [2]. Furthermore, the current available treatments with two selective ER modulators (SERMs), tamoxifen and raloxifene, approved by the FDA for breast cancer chemoprevention, remain unsatisfactory due to the drug resistance and side-effects such as hepatic injury [3]. Therefore, it is crucial to identify the underlying mechanisms involved in tumorigenesis of breast cancer, and facilitate the nding of more effective treatment strategies against breast cancer.
Toll-liked receptors (TLRs), mainly expressing in human immune related cells, plays a crucial role in the rst line of host defense by recognizing pathogen-associated molecular patterns (PAMPs) [4,5]. TLRs could also adjust in ammatory microenvironment, which is vital to tumorigenesis, and multiple effects had been identi ed by activating TLRs. Various TLRs agonists are currently under investigation for their ability in anticancer immunotherapy [6][7][8]. While several other studies provided evidence that TLRs ligands such as lipopolysaccharide (LPS) was associated with epithelial-to-mesenchymal transition (EMT) and accelerated metastatic tumor growth through TLR4 [9,10]. In addition, the expression of TLR4 was the highest among other TLRs in human breast cancer, and TLR4 activation could subsequently activate nuclear factor-κB (NF-κB) and produce pro-in ammatory cytokines, ultimately stimulating in ammation [11,12].
Last few years, naturally existed chemicals have attracted widespread attentions for being identi ed as new preventive agents against cancer, with high anti-in ammatory e cacy and low toxicity. Rhizoma Atractylodis Macrocephalae, one of the traditional Chinese crude materials, had signi cant gastrointestinal tract protective, neuroprotective and immunomodulatory activities [13], and numerous lines of evidence showed that it also exerted anti-tumor, anti-in ammatory and antioxidant effects [13,14]. Atractylenolide I (AT-I), the major bioactive component from Rhizoma Atractylodes Macrocephalae, also had multiple therapeutic activities including anti-in ammatory [15,16] and anti-tumor effects [17][18][19]. Speci cally, AT-I could ameliorate LPS-induced lung damages in mouse model [15], and suppressed LPS-induced NO release and diminished pro-in ammatory cytokines levels in BV-2 cells [16]. AT-I had the anti-tumor effects against many cancers, such as colorectal cancer [17], non-small cell lung cancer [18] and melanoma [19], and it also had a binding site similar to LPS and served as a novel TLR4antagonizing agent [20]. Moreover, a randomized pilot study of AT-I on gastric cancer cachexia patients showed that AT-I could inhibit pro-in ammatory cytokines and proteolysis-inducing factor (PIF) proteolysis, and then alleviating symptoms of gastric cancer cachexia patients [21]. However, the effects of AT-I on mammary tumorigenesis remain largely unknown.
In this study, we investigated the effects of AT-I on mammary tumorigenesis, and explored the possible mechanisms. We found that TLR4 and NF-κB were over expressed in human breast cancer tissues, and activation of TLR4/NF-κB pathway was correlated with advanced TNM-stages. We also found that AT-I could inhibit cell proliferation, migration, invasion and induce apoptosis. Further investigation revealed that AT-I could inhibit TLR4/NF-κB pathway, resulting in decreased proin ammatory factors expression, and these chemopreventive effects of AT-I were TLR4-dependent. Furthermore, in vivo studying demonstrated that AT-I suppressed tumorigenesis of breast cancer via inhibiting TLR4/NF-κB pathway.
All these results suggested that AT-I could be a potential agent in suppressing tumorigenesis of breast cancer.
To con rm that TLR4 could mediate the tumorigenesis inhibitory effects of AT-I, the shTLR4 plasmid and lipofectamine ® 3000 (Invitrogen, Carsbad, CA, USA) were used to conduct the transfection. The knockdown studies were performed at 48 h after transfection.

Cell migration assay
Wound healing assay was adopted to detect the migration abilities of MCF-7 and MDA-MB-231 cells. Brie y, the cells were seeded and grew to 80% con uence in 24-well plates, and we creat a wound by scratching cells with a sterile 200 µL pipette tip. Then, cells were continued to incubate in the medium in the absence or presence of 25, 50 and 100 µM AT-I for 48 h. The cells grew into wound surface were considered as migrated cells, and photographed by microscope (Nikon, Japan). The wound width difference of 0 h and 48 h was used to calculated the rate of wound healing.

Cell invasion assay
Transwell chambers (Millipore, Billerica, MA, USA) with 8.0 μm pore membranes were coated with matrigel (BD, Franklin Lakes, NJ, USA) and used for invasion analysis. In brief, cells were treated with 0, 25, 50 and 100 µM AT-I. After for 48 h, cells were collected by trypsin and resuspended in 200 μL serumfree medium, and then seeded on the upper chamber (5×10 4 cells/well). 600 μL complete medium was added to the lower chamber as a chemoattractant. After 24 h incubation, the cells remaining at the upper surface of the membrane were removed, and the cells on the lower surface, regarded as invasive cells.
After xing with 4% paraformaldehyde, the cells were stained with 0.5% crystal violet solution. The invasive cells were photographed and counted under microscope.
Colony formation assay MCF-7 and MDA-MB-231 cells were seeded in 6-well plates (500 cells/well), and cultivated in medium containing AT-I (0, 25, 50, and 100 µM) for 48 h. Cells were maintained in the well for 14 days to form colony, and the colonies were xed and stained with 0.5% crystal violet solution (30% methanol) for 30 minutes at room temperature. The numbers of colonies with ≥ 50 cells were counted under microscope.

Animal and experimental procedures
Twenty-four female Sprague-Dawley (SD) rats (Dashuo, Chengdu, China) were randomly distributed into four groups. After pretreatment with vehicle or AT-I (100 mg/kg and 200 mg/kg) for 3 days, a single dose of NMU (75 mg/kg) was intraperitoneal injected to the rats at 21 days of age, and continued to treat with vehicle or AT-I daily for 9 weeks. Then they were sacri ced, and cancer samples were collected and kept at -80 °C for western blot and ELISA analysis.
Protein lysate preparation and western blot assay The total protein was isolated from cell lysates using RIPA buffer (Beyotime, Shanghai, China) according to the manufacturer's instructions. Equal amounts of protein were separated by gel electrophoresis, and then transferred to a polyvinylidene uoride membrane (Bio-Rad, Hercules, CA, USA). After blocked with 5% BSA, the membrane was incubated with primary antibody (Table 1) overnight at 4 °C, washed three times, and subsequently incubated with horseradish peroxidase-conjugated secondary antibody (ZSGB, Beijing, China) for 2 h at 25 °C. The density analysis of each band was conducted using Image Lab 5.0 software (Bio-Rad).

ELISA assay
For measurements of TNF-α, IL-6 and IL-1β, the supernatants of cell culture medium and rat tissue lysis were collected to performe ELISA analysis according to the manufacturer's protocol.

Statistical analysis
The data was expressed as the mean ± SEM and analyzed using one-way analysis of variance (ANOVA) followed by the Tukey test. The statistical analysis was performed using SPSS 16.0 software. Values of p < 0.05 were considered statistically signi cant.

Results
TLR4 and NF-κB were up-regulated in human breast cancer tissues and correlated with TNM-stages To study the role of TLR4/NF-κB pathway in breast cancer, we assessed the expression of TLR4 and NF-κB in normal breast tissues and different TNM-stages breast cancer tissues using tissue microarray and immunohistochemistry technology. The results showed that TLR4 and NF-κB levels in breast cancer tissues were signi cantly higher than that in normal breast tissues ( Fig. 1a-b). Furthermore, we found that the expression of TLR4 and NF-κB in high TNM stages was signi cantly higher than that in low TNMstages of breast cancer ( Fig. 1a-b). In addition, we detected the expression of TLR4 and NF-κB in MCF-7, MDA-MB-231 and MCF 10A cells by western blot assay. As shown in Fig. 1c-d, TLR4 and NF-κB levels in breast cancer cells were signi cantly higher than that in mammary epithelial cells. These results indicated that the expression of TLR4 and NF-κB were up-regulated in human breast cancer tissues and cells, and their high expression was correlated with advanced TNM-stages in breast cancer patients.

AT-I inhibited cell growth and induced apoptosis
Cell viability was determined by MTT assay after AT-I treatment, and the results (Fig. 2a) (Fig. 2c-d).

AT-I inhibited breast cancer cell migration and invasion
We explored the effects of AT-I on cell migration and invasion, and the wound healing assay suggested that the migration of MCF-7 and MDA-MB-231 cells was signi cantly inhibited by 50 μM or 100 μM AT-I treatment for 48 h (Fig. 3a). The transwell invasion assay showed that the number of cells invading the lower chamber was markedly decreased after 50 μM or 100 μM AT-I treatment, compared with the control cells (Fig. 3b). In addition, as dose increased, AT-I has stronger inhibitory effects on the migration and invasion of MCF-7 and MDA-MB-231 cells. Taken together, these results strongly demonstrated that AT-I treatment resulted in effective inhibition of migration and invasion in breast cancer cells.
AT-I inhibited TLR4/NF-κB pathway in breast cancer cells AT-I was reported to be a novel TLR4-antagonizing agent [20], and we used western blot to detect the effects of AT-I on TLR4/NF-κB pathway in MCF-7 and MDA-MB-231 cells. After AT-I treatment, the expression of TLR4, MyD88, p-NF-κB p65, p-IκBα and p-IKKα/β was signi cantly down-regulated in a dose-dependent manner in both cells (Fig. 4a-b). Furthermore, we used LPS (TLR4 agonist) induced cell in ammation to measure the levels of NF-κB related pro-in ammatory cytokines by ELISA assays. The results showed that the levels of the NF-κB-regulated cytokines (i.e., TNF-α, IL-6 and IL-1β) were all decreased after AT-I treatment. These data suggested that AT-I could inhibit TLR4/NF-κB pathway, and down-regulate the downstream pro-in ammatory cytokines in breast cancer cells.
The tumorigenesis inhibitory effects of AT-I is mediated by TLR4 To con rm that TLR4 was essential in the tumorigenesis inhibitory effects of AT-I, the shTLR4 plasmid was adopted to transfect MCF-7 and MDA-MB-231 cells, and then western blot and ELISA assays were used to evaluate the changes of related protein levels after AT-I treatment. We found that the expression of TLR4, MyD88, p-NF-κB p65, p-IκBα and p-IKKα/β were signi cantly down-regulated after AT-I or shTLR4 treatment in LPS-induced MCF-7 and MDA-MB-231 cells. However, no inhibitory effects of AT-I on these protein levels were observed after shTLR4 transfection in LPS induced cells (Fig. 5a-b). To clearly demonstrate the mechanisms, the levels of secreted pro-in ammatory cytokines TNF-α, IL-6 and IL-1β were determined by ELISA, and the results were consistent with western blot assays (Fig. 5c-d).
To further examine the role of TLR4 in the migration and invasion of breast cancer cells after AT-I treatment, the LPS induced MCF-7 and MDA-MB-231 cells were used for wound healing and transwell invasion assays. The results indicated that LPS could induce the migration and invasion activities of both MCF-7 and MDA-MB-231 cells, and treatment with AT-I or shTLR4 could decrease cell migration and invasion. However, in TLR4 knockdown cells, no inhibitory effects of AT-I on cell migration and invasion were observed, which indicated that TLR4 is essential for AT-I's inhibitory effects on cell migration and invasion ( Fig. 6a-d).

AT-I inhibited NMU-induced mammary tumor progression in rats
The previous studies indicated that AT-I exerted tumorigenesis inhibition in vitro, and we next investigated these effects in vivo. We employed the NMU-induced rat breast cancer model, which is commonly accepted for candidate chemopreventive agents evaluation [23,24]. We rstly found that NMU treatment could signi cantly decrease the body weights of rats, while AT-I could revert these effects (Fig. 7a). In addition, the rst palpable mammary tumors in the NMU-treated group appeared after 5 weeks of NMU treatment, but it did not appear until 6 and 7 weeks in 100 mg/kg and 200 mg/kg AT-I group, and all rats had tumors at 9 weeks (Fig. 7b). Averagely there were 3.67, 1.83 and 1.33 tumors monitored in the NMU, 100 mg/kg and 200 mg/kg AT-I treatment groups, respectively (Fig. 7c). Moreover, the mean tumor volume was signi cantly decreased in the 100 mg/kg and 200 mg/kg AT-I treatment group (Fig. 7d). These results indicated that AT-I treatment could inhibit the mammary tumorigenesis in rats.
To further elucidate the underlying mechanisms of these effects, we detected the in uence of AT-I on the TLR4/NF-κB pathway and its downstream proin ammatory factors in the NMU-induced mammary carcinogenesis. As shown in Fig. 6e-i, NMU treatment alone could induce TLR4, MyD88 and p-NF-κB p65 expression, and then increase its downstream proin ammatory factors TNF-α, IL-6 and IL-1β levels. However, AT-I treatment could reduce the activation of TLR4/NF-κB pathway induced by NMU, and then decrease the TNF-α, IL-6 and IL-1β level. Our ndings suggested that AT-I could inhibit NMU-induced mammary tumor progression in rats through inhibiting TLR4/NF-κB pathway.

Discussion
It is well established that tumorigenesis is a multi-step process caused by various factors, such as environmental carcinogens, in ammatory mediators, and tumor promoters. Multiple effects had been identi ed of TLR4 in tumor progression [11,[25][26][27][28]. In breast cancer cells and primary breast cancer tissues from patients, TLR4 expression was found to be up-regulated both at mRNA and protein levels, and signi cantly correlated with the high incidence of lymph node metastasis [11,26]. Moreover, as TLRs ligands, LPS was reported to increase breast cancer metastasis in vitro and in vivo [27], and acquiring of high metastatic potential upon the TLR4-elicited activation of NF-κB in breast cancer cells was associated with integrin αvβ3, TPM1 and maspin [28]. However, Connelly et al. suggested that inhibition of NF-κB might lead to the increased tumor latency and decreased tumor burden and numbers of lung metastases during breast cancer development in mice [29]. All these studies suggested that TLR4/NF-κB pathway played a critical role in the mammary tumorigenesis, but the underlying mechanisms need to be further elucidated.
To study the important role of TLR4/NF-κB pathway in breast cancer, we assessed the expression of TLR4 and NF-κB among normal breast tissues and different TNM-stages breast cancer tissues using tissue microarray and immunohistochemistry technology. The results showed that TLR4 and NF-κB were over expressed in breast cancer, and correlated with the TNM-stages. Furthermore, these differentially expressed levels of TLR4 and NF-κB were also detected in breast cancer cells and mammary epithelial cells. These results indicated that TLR4 and NF-κB were up-regulated in breast cancer, and might function as tumor promoters in breast cancer.
AT-I is a novel TLR4-antagonizing agent [20], and it exerted anti-tumor effects on colorectal cancer, bladder cancer and melanoma [17][18][19], then we investigated whether it could suppress tumorigenesis in breast cancer via inhibiting TLR4/NF-κB pathway. Firstly, we found that AT-I could inhibit cell growth, proliferation, migration and invasion, and induce apoptosis in breast cancer cells. Then, we detected the effects of AT-I on the TLR4/NF-κB pathway in breast cancer cells. Previous studies have demonstrated that activation of TLR4 could lead to its dimerization, activation of the MyD88-dependent or -independent NF-κB signaling pathway, thus promoting tumor growth and invasion by regulating tumor immune and in ammatory response [30,31]. Furthermore, TLR4 could activate the downstream IκB kinase (IKK) complex, and then phosphorylate IκB, an NF-κB inhibitor that could prevent nuclear translocation of NF-κB. Upon phosphorylation, IκB was degraded and released NF-κB, which then entered nucleus and mediated the expression of in ammatory cytokines [32,33]. In the present study, we found that the expression of TLR4, MyD88, p-NF-κB p65, p-IκBα and p-IKKα/β in breast cancer cells was signi cantly down-regulated after AT-I treatment. Furthermore, we used the LPS (TLR4 agonist) treatment as cell in ammation model, and measured the levels of secreted pro-in ammatory cytokines regulated by NF-κB.
The results showed that the secretion of TNF-α, IL-6 and IL-1β were all decreased after AT-I treatment, and we concluded that AT-I could suppress tumorigenesis in breast cancer via inhibiting TLR4/NF-κB pathway, and down-regulate downstream pro-in ammatory cytokines.
TLR4, in the frame of our investigation, played a pivotal function role in the tumorigenesis inhibition of AT-I by regulating NF-κB signaling pathway. We found that LPS could induce cell migration and invasion, and co-treatment with AT-I or shTLR4 could decrease the migration and invasion. However, in TLR4 knockdown cells, no inhibitory effects of AT-I on cell migration and invasion were observed, which indicated that TLR4 was essential in AT-I's effects. To further elucidate the underlying mechanisms, we examined the effects of TLR4 on the NF-κB signaling pathway and downstream pro-in ammatory cytokines, and we found that the expression of TLR4, MyD88, p-NF-κB p65, p-IκBα and p-IKKα/β and downstream pro-in ammatory cytokines TNF-α, IL-6 and IL-1β were signi cantly down-regulated after AT-I or shTLR4 treatment in LPS induced breast cancer cells. Similarly, in TLR4 knockdown cells, no inhibitory effects of AT-I on protein expression were observed, which indicated that AT-I's effects on these protein levels were TLR4-dependent. The results above demonstrated that AT-I suppressed tumorigenesis in breast cancer cells via inhibiting TLR4-mediated NF-κB signaling pathway.
We further con rmed these effects and mechanisms in vivo using NMU-induced rat breast cancer model [23], which shared a lot similarities with human mammary carcinomas including histopathology [34, 35]. Previously, we had used this model to explore the inhibitory effects of AT-II on breast cancer through regulating Nrf2/ARE pathway [24]. In the present study, we found that AT-I could inhibit the mammary tumorigenesis in rats, and the underlying mechanisms were further elucidated by detecting the effects of AT-I on TLR4/NF-κB pathway, and its downstream proin ammatory factors in breast cancer rats. The results indicated that TLR4, MyD88 and p-NF-κB p65 were up-regulated in NMU-induced breast cancer, along with its downstream pro-in ammatory factors. While AT-I treatment could down-regulate TLR4/NF-κB pathway, and then decrease the TNF-α, IL-6 and IL-1β levels in NMU-induce rats. Our ndings suggested that AT-I could inhibit NMU-induced mammary tumor progression in rats by inhibiting of TLR4/NF-κB pathway.

Conclusions
This study demonstrated that TLR4 and NF-κB were over expressed in breast cancer, and AT-I could suppress tumorigenesis of breast cancer via inhibiting TLR4-mediated NF-κB signaling pathway. These ndings also provided proof that inhibiting TLR4/NF-κB pathway by natural compounds was an effective chemopreventive strategy for breast cancer, and AT-I appeared to have potential value as a novel candidate for breast cancer treatment.          The effects of AT-I on suppressing tumorigenesis was mediated by TLR4. The shTLR4 plasmid was used to transfect breast cancer cells, and then western blot and ELISA assays were used to compare related proteins expression with untransfected cells after cells had been pre-treated in presence or absence AT-I for 48 h during LPS stimulation. (a, b) The expression of TLR4/NF-κB signaling pathway was detected by western blot assay (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, compared with LPS treated cells. (c, d) The levels of TNF-α, IL-6 and IL-1β in the cell supernatants were measured by ELISA assay (n = 3). *P < 0.05, **P < 0.01, compared with LPS treated cells.

Figure 5
The effects of AT-I on suppressing tumorigenesis was mediated by TLR4. The shTLR4 plasmid was used to transfect breast cancer cells, and then western blot and ELISA assays were used to compare related proteins expression with untransfected cells after cells had been pre-treated in presence or absence AT-I for 48 h during LPS stimulation. (a, b) The expression of TLR4/NF-κB signaling pathway was detected by western blot assay (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, compared with LPS treated cells. (c, d) The levels of TNF-α, IL-6 and IL-1β in the cell supernatants were measured by ELISA assay (n = 3). *P < 0.05, **P < 0.01, compared with LPS treated cells.   Rat breast tissues were kept to detect TLR4/NF-κB pathway by western blot assay at the end of experiment. (g-i) TNF-α, IL-6 and IL-1β levels in rat breast tissues were analyzed by ELISA at the end of experiment. *P < 0.05, **P < 0.01, ***P < 0.001, compared with NMU group.