Inhibition of TLR4/TRIF/IRF3 Signaling Pathway by Curcumin in Breast Cancer Cells

- Purpose: Toll-like receptor 4 (TLR4) is over-expressed in breast tumors and thus contributing to the tumor progression and metastasis. Natural products have drawn attention in cancer immunotherapy due to their various biological activities. Curcumin is well investigated in different types of cancer. However, the mechanisms underlying its anti-inflammatory actions have not been extensively elucidated. For this purpose, we explored the inhibitory effects of curcumin on lipopolysaccharide (LPS)-induced TLR4 dependent TRIF signaling pathway in two subtypes of breast cancer cell lines (MCF-7 and MDA-MB-231) in this study. Methods: In this context, the cytotoxicity of curcumin and LPS alone and the combination of curcumin with LPS on these cells was evaluated by WST-1 assay. The expression level of TLR4 and the release of type I interferon (IFN) levels were determined after treatment with curcumin and/or LPS by RT-PCR and ELISA analysis, respectively. Furthermore, the subcellular localization of TLR4 and interferon regulatory factor 3 (IRF3) were detected by immunofluorescence analysis. Results : Curcumin treatment suppressed breast cancer cells viabilities and the activation of TLR4-mediated TRIF signaling pathway by the downregulation of TLR4 and IRF3 expression levels and the inhibition of type I IFN (IFN-α/β) levels induced by LPS. However, curcumin was more efficient in MDA-MB-231 cells than MCF-7 cells owing to its greater inhibitory efficacy in the LPS- enhanced TLR4 signaling pathway. Furthermore, IFN-α/β levels induced by TLR4 and IRF3 were decreased in these cells following curcumin treatment. Conclusions: Consequently, these results demonstrated that the activation of LPS stimulated TLR4/TRIF/IRF3 signaling pathway was mediated by curcumin in breast cancer cells, in vitro . However, more studies are necessary to examine the curcumin’s anti-inflammatory activities on TLR4/MyD88/NF-κB as well as other signaling pathways downstream of TLRs in breast cancer.


INTRODUCTION
Toll-like receptors (TLRs) play a crucial role in immune response by recognizing pathogenassociated molecular patterns (PAMPs) associated with cancer and/or other diseases through different intracellular signaling pathway (1)(2)(3). TLRs display different effects in tumor progression and treatment according to the activation of TLRs or TLR associated signaling cascades (4). In tumor progression, TLRs are overexpressed in different types of cancers including lung, prostate and breast and promote survival of cancer cells (5)(6)(7)(8)(9). Toll-like receptors 4 (TLR4) is one of the most important member of the TLR family and is stimulated by lipopolysaccharide (LPS) (10). TLR4, which is highly expressed in breast cancer cells, exhibits a tumor-promoting role, and promotes resistance to apoptosis and metastasis. For example, TLR4 and myeloid differentiation primary response gene 88 (MyD88) expression levels in MCF-7 breast cancer cells are increased by LPS stimulation while the knockdown of TLR4 signaling inhibits the survival of breast cancer cells (11,12).
Curcumin is isolated from Curcuma longa L. rhizomes and many studies have shown that curcumin exerts the anti-inflammatory, anti-viral, anti-carcinogenic, anti-proliferative and anti-oxidant properties. In the literature, curcumin can potentially inhibit the proliferation of cancer cells and enhance chemotherapeutic drugs sensitivity by inducing apoptosis (17)(18)(19)(20). Curcumin can suppress the inflammation via the down-regulation of the extracellular TLRs activation and the inflammatory genes and/or transcription factors (21,22). There has been a number of studies indicating the suppressive effect of different natural products (shinbaro3, curcumin, tanshinones, phloretin, xanthohumol, celastrol, garlic compounds etc.) on the different TLRs signaling (14,(23)(24)(25)(26). Additionally, several promising natural compounds (shinbaro3, curcumin, tanshinones) exert anti-inflammatory activities by suppressing TLR4/MyD88/NF-κB pathway. However, there has been no study that investigated the potential effects of curcumin on the activation of TLR4/TRIF/IRF3 signaling pathway in breast cancer, to our knowledge. For this purpose, we explored the anti-inflammatory effects of curcumin on LPS stimulated TLR4/TRIF/IRF3 signaling pathway in two different types of breast cancer cell lines.

METHODS
Culture conditions for cells proliferation MCF-7 and MDA-MB-231 cells were used in this study. These cells were incubated in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific, USA) with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, USA) and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific, USA). All cell lines were cultured in a humidified 37 °C incubator (Thermo Fisher Scientific, USA) with 5% CO2. All cells were counted using Countes II Cell Counter (Thermo Fisher Scientific, USA) before further analysis.
WST-1 analysis Equal number (2x10 4 cells/well) of these cells were cultured in 96-well culture plates, then treated alone with different curcumin concentrations (5-100 µM) and LPS (2 µM) and the combination of curcumin (5, 10 and 25 µM) plus LPS (2 µM) for 24 and 48 h. LPS was used to stimulate TLR4 signaling pathway. Following incubation, the cells were then incubated by 10 µL of WST-1 reagent (Biovision, San Francisco, CA, USA) at 37°C for 30 minutes in the dark conditions. After incubation, the cells were examined at 450 nm with 96-well plate reader (Allsheng, China). Each experiment was performed three replicates. Real-Time PCR (Applied Biosystems, Foster City, CA). β-actin was utilized as the endogenous reference gene. Each experiment was performed in three replicates.

Immunofluorescence analysis
Equal number (5x10 5 cells/well) of breast cancer cells were cultured on slides and after 24 hours, the cells incubated with LPS (2 µM), curcumin (5, 10 and 25 µM) and the combination of curcumin plus LPS for 48 hours. Then, the cancer cells were fixed with 4% cold-paraformaldehyde for 15 minute and were incubated in 5% goat serum and 1% Triton X-100 in 1× PBS for 1 h at room temperature. Then, the cells were treated with primary antibody of TLR4 (1:2000, Santa Cruz Biotechnology, CA, USA) and IFN regulatory factor 3 (IRF3) (1:2000, Santa Cruz Biotechnology, CA, USA) for 4 hours and incubated with secondary antibody (1:1000, Bio-Rad, Hercules, California, USA) for 1 h at room temperature. Afterwards, the nucleus of cells was stained with DAPI. Fluorescent images were photographed and examined with an EVOS FL Cell Imaging System (Thermo Fisher Scientific, USA).

ELISA analysis
Conditioned medium was collected from breast cancer cells incubated alone with different concentrations of curcumin (5, 10 and 25 µM) and LPS (2 µM) and the combination of curcumin plus LPS for 48 hours. Concentrations of interferon-α (IFN-α) and interferon-β (IFN-β) were analyzed by ELISA kits (Elabscience, USA). Each experiment was conducted in three replicates. Obtained data were shown as mean pg of cytokine ±S.D. normalized per 100 cells.
Acridine orange (AO) and ethidium bromide (EtBr) staining Equal number (5x10 5 cells/well) of cells were cultured in 6-well culture plates and treated alone with LPS (2 µM), curcumin (5, 10 and 25 µM) and the combination of curcumin plus LPS. Then, all cells were fixed with 4% cold-paraformaldehyde for 15 minute and the cells were stained by adding 500 µL of AO/EtBr for 30 minute in the dark. The cells were examined with EVOS FL Cell Imaging System (Thermo Fisher Scientific, USA).

STATISTICAL ANALYSIS
All data was statistically evaluated by SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Data were expressed as a mean ± SD of the three experiments. The Analysis of Variance (ANOVA) test by Post-Tukey test was conducted for multiple comparisons. p<0.05 was considered to be statistically significant. Furthermore,https://dataanalysis.qiagen.com/pcr/arr ayanalysis.php software was used for gene expression analysis.

Assessment of cell viability
In order to evaluate curcumin's anti-proliferative effects on breast cancer cells, WST-1 assay was performed (Figure 1). Both breast cancer cells proliferation was significantly inhibited by curcumin (p<0.01).
However, the inhibitory effects of curcumin on MDA-MB-231 cells were greater than MCF-7 cells. The IC50 concentration of curcumin 25.60 ± 2.83 µM and 8.05 ± 0.70 µM in MCF-7 and MDA-MB-231 cells, respectively for 48 h ( Figure   1A). The maximum inhibitions were identified at doses ≥25 µmol/l for 48 h with similar viability percentages. Thus, 5, 10 and 25 µM curcumin were selected as suitable doses for further experiments to compare curcumin efficacy in MCF-7 and MDA-MB-231 cells. Then, we investigated the antiinflammatory effects of curcumin on LPS-stimulated TLR4 activation in these cells ( Figure 1B). We determined that curcumin remarkably inhibited the viability of breast cancer cells by blocking LPS activity (p<0.01). Thus, curcumin exhibited potential anti-inflammatory activity in breast cancer cell lines. The subcellular localization of TLR4 and IRF3 in breast cancer cells To explore whether curcumin suppresses the activation of IRF3, the subcellular localizations of TLR4 and IRF3 were observed by immunofluorescence staining (Figure 3). We observed the cytoplasmic TLR4 and the nuclear IRF3 proteins following exposure to LPS in breast cancer cells. Furthermore, curcumin decreased the activation of TLR4 and IRF3 because we observed inactive IRF3, which localized in the cytoplasm. However, the cytoplasmic localization of TLR4 and the nuclear localization of IRF3 were detected after treatment with the combination of curcumin with LPS. These results were consistent with gene expression analysis. Our findings suggested that curcumin reduced the endosomal localization of TLR4 and inhibited the nuclear translocation of IRF3 and thus, curcumin could suppress TLR4/TRIF/IRF3 signaling pathway.

The type I IFN level in breast cancer cells
To further confirm the suppression of IRF3 activity by curcumin, the changes in the level of type I IFN were detected in both breast cancer cells (Figure 4). Treatment of breast cancer cells with LPS alone significantly stimulated the release of IFN-α and IFN-β levels (p<0.01). However, the levels of IFNα and IFN-β considerably decreased following treatment with curcumin or the combination of curcumin with LPS (p<0.01). Furthermore, curcumin reduced IFN-α and IFN-β levels to a greater extent than curcumin plus LPS combination (p<0.01). As a result, curcumin and curcumin plus LPS stimulation resulted in a decrease in the level of IFN-α and IFNβ, and thus curcumin suppressed TLR4/TRIF/IRF3 signaling pathway.

Observation of morphological changes in breast cancer cells
In order to observe morphological changes in breast cancer cell, we used AO/EtBr staining ( Figure 5). The more rounded cells, plasma membrane blebbing, cellular shrinkage, nuclear condensation and fragmentation were observed in both breast cancer cells following incubation with curcumin alone and the combination of curcumin plus LPS. At a concentration of 25 µM curcumin, multiple segregated apoptotic bodies were especially noticed in MDA-MB-231 cells. However, LPS stimulation did not alter overall morphology of MCF-7 and MDA-MB-231 cells. Thus, our findings indicated that curcumin suppressed LPS activity in breast cancer cells by inducing apoptotic cell death.

DISCUSSION
Curcumin interacts with multiple molecular targets through a wide range of biological properties (27)(28)(29). In the current study, the anti-inflammatory effects of curcumin on MyD88 independent-TRIF signaling pathway were investigated in breast cancer cell lines, for the first time. Our findings demonstrated that curcumin could potentially suppress LPS-stimulated TLR4/TRIF/IRF3 signaling pathway through the downregulation of TLR4 and IRF3 activity and the inhibition of type I IFN (IFN-α/β) levels in breast cancer cells.
Curcumin treatment significantly inhibits the proliferation of different subtypes (MCF7, MDA-MB-231 and BT-474 etc.) of breast cancer cells by inducing apoptosis through multiple signaling pathways (30)(31)(32)(33)(34). Additionally, the molecular mechanisms underlying curcumin-mediated apoptotic cell death have been identified in the literature (35)(36)(37)(38). In the current study, curcumin showed higher toxicity in MDA-MB-231 cells than MCF-7 cells. Moreover, we observed typical apoptotic morphological in these cells treating with curcumin. However, only a limited number of studies have been demonstrated for the antiinflammatory effects of curcumin on cancer cells. For this purpose, we evaluated the potential effect of curcumin on TLR4 signaling and especially TLR4/TRIF/IRF3 signaling pathway.
In conclusion, we evaluated that curcumin exerted TLR4 antagonist activity in two different subtypes of breast cancer cells through its antiinflammatory effects and could mediate TLR4/TRIF/IRF3 signaling pathway and IFN responses by suppressing of LPS stimulated inflammation. However, further molecular studies are needed to explore the relationship between curcumin anti-inflammatory action and LPS induced TLR4/TRIF/IRF3 and TLR4/MyD88/NF-κB signaling pathways in breast cancer, in detail. Additionally, the link between curcumin and other signaling pathways via TLRs activation requires further investigation, in vitro.