Embelin downregulated cFLIP in breast cancer cell lines facilitate anti-tumor effect of IL-1β-stimulated human umbilical cord mesenchymal stem cells

Breast cancer is the leading cause of cancer-related death for women. In breast cancer treatment, targeted therapy would be more effective and less harmful than radiotherapy or systemic chemotherapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in cancer cells but not in normal cells. Mesenchymal stem cells have shown great therapeutic potential in cancer therapy owing to their ability of homing to tumor sites and secreting many kinds of anti-tumor proteins including TRAIL. In this study, we found that IL-1β-stimulated human umbilical cord-derived mesenchymal stem cells (hUCMSCs) enhance the expression of membrane-bound and soluble TRAIL. Cellular FADD-like IL-1β-converting enzyme inhibitory protein (cFLIP) is an important regulator in TRAIL-mediated apoptosis and relates to TRAIL resistance in cancer cells. Previous studies have shown that embelin, which is extracted from Embelia ribes, can increase the TRAIL sensitivity of cancer cells by reducing cFLIP expression. Here we have demonstrated that cFLIPL is correlated with TRAIL-resistance and that embelin effectively downregulates cFLIPL in breast cancer cells. Moreover, co-culture of IL-1β-stimulated hUCMSCs with embelin-treated breast cancer cells could effectively induce apoptosis in breast cancer cells. The combined effects of embelin and IL-1β-stimulated hUCMSCs may provide a new therapeutic strategy for breast cancer therapy.

Scientific Reports | (2021) 11:14720 | https://doi.org/10.1038/s41598-021-94006-w www.nature.com/scientificreports/ studies show that hBMMSCs could also be recruited to tumor site where they transform into tumor-associated fibroblasts (TAFs) which then enhance tumor growth 9 . Unlike hBMMSCs, human umbilical cord-derived mesenchymal stem cells (hUCMSCs) did not transform to TAFs in the presence of breast and ovarian cancer 9 . In addition to the ability to be easily isolated, hUCMSCs also hold an advantage over hBMMSCs in tumor suppression. It has been found that the growth of breast cancer cells can be attenuated by naïve hUCMSCs 10 . According to these findings, hUCMSCs are more suitable for cell therapy 11 . Many reports demonstrated that microvesicles derived from MSCs induced cell apoptosis and cell cycle arrest 12,13 . MSCs have been engineered as a transmitter to deliver anti-tumor proteins like interferons, interleukins and soluble tumor necrosis factor-related apoptosis-inducing ligands (soluble TRAIL) [14][15][16] . TNF-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand, is a protein belonging to the tumor necrosis factor (TNF) ligand family. Most of the normal cells express membrane form TRAIL and also secrete soluble form TRAIL. Previous studies have shown that TRAIL could induce cell apoptosis in many kinds of cancer cells 17,18 .
Apoptosis has two main pathways: the intrinsic and extrinsic pathways. The intrinsic pathway is initiated through cell stress or damage and is controlled by the Bcl 2 family. Regulation of apoptosis occurs through perforation of the mitochondrial membrane to release cytochrome c into the cytoplasm and combine with pro-caspase-9 to form the apoptosome. Then the apoptosome activated caspase-3 leads to cell destruction 19 . The extrinsic way is triggered by ligands like Fas and TRAIL binding to cell surface death receptors. The binding of ligand and receptor changes the Fas-associated death domain protein (FADD) to activate caspase-8/10 and caspase-3 20 . Both extrinsic and intrinsic pathways are involved in TRAIL induced apoptosis. First, TRAIL binds to the death receptors TRAILR1 (DR4) and TRAILR2 (DR5), initiating the extrinsic pathway to activate caspase-8 and caspase-3, leading to cell death. Subsequently, the activated caspase-8 initiates the apoptotic intrinsic pathway.
IL-1β is involved in many kinds of cellular activities including migration, proliferation, and apoptosis 21,22 . Studies have shown that IL-1β could enhance the immunosuppressive properties of hBMMSCs in cell therapy 23 . In our previous research, we found that pre-activating hUCMSCs with IL-1β could induce membrane-bound TRAIL expression and enhance apoptosis of MDA-MB-231, MCF-7 and MDA-MB-453 breast cancer cell lines (unpublished data). In this study, we further investigated whether treatment of IL-1β on hUCMSCs could increase the expression of soluble TRAIL.
Cellular FADD-like IL-1β-converting enzyme inhibitory protein (cFLIP), a homolog of caspase-8, forms heterodimers with caspase-8 and blocks the activation of caspase-8 to regulate the death receptor-related extrinsic apoptosis pathway 24 . In humans, there are three isoforms of cFLIP: cFLIP L , cFLIP S and cFLIP R . cFLIP L is similar to pro-caspase-8 but lacks a functional caspase domain. cFLIP S and cFLIP R have two death-effector domains. Many studies had demonstrated that cFLIP is highly expressed in various human cancers, such as colorectal cancer, bladder urothelial cancer and breast cancer [25][26][27] , and the level of cFLIP expression was correlated with poor clinical outcomes. High expression of cFLIP L has been found in most of the malignant cancer cells, and cFLIP s was found in glioblastoma cells 28 . cFLIPR is specifically expressed in a number of cell lines and primary human T cells 29 . In addition, downregulation of FLIP enhances the FAS-or TRAIL-mediated cell apoptosis and the TRAIL sensitivity of cancers 27,30 . Given these findings, cFLIP is considered to be the master of the antiapoptotic regulator of the FAS-or TRAIL-mediated apoptosis pathways and the resistant factor of cancers. Here we wanted to know the relationship between cFLIP and TRAIL resistance in breast cancer cell lines.
Embelin (2, 5-dihydroxy-3-undecyl-1, 4-benzoquinone) is a natural benzoquinone which is isolated from the Embelia ribes. Embelin has exhibited anti-inflammatory and anti-tumor effects in various cancer cells 31 . Embelin has also been found to be a cell-permeable, small-molecular weight inhibitor of X-linked inhibitor of apoptosis (XIAP), a target of apoptosis-resistance of cancer cells, and could hold great potential in cancer therapy 32,33 . The antitumor effect of embelin might manifest via inhibiting tumor growth, activating tumor suppressor gene p53 and inducing apoptosis [34][35][36] . Some studies have shown that embelin inhibited XIAP enhances TRAIL sensitivity in breast cancer cells and downregulates cFLIP S to induce TRAIL-mediated cell apoptosis in malignant glioma cells 37,38 . We wondered if embelin could downregulate cFLIP to enhance the TRAIL sensitivity of breast cancer cells.
In this study, we investigated the relationship between the expression of cFLIP L and TRAIL resistance in three different breast cancer cell lines. We further examined whether embelin could effectively downregulate the expression of cFLIP L . After co-culture with IL-1β-stimulated hUCMSCs, the effects of the secreted soluble TRAIL-mediated apoptosis in breast cancer cells was investigated. This study may provide a new strategy in breast cancer therapy.

Results
The impact of embelin on breast cancer cells. We used an MTT assay to examine the cytotoxic impact  39 . We wanted to know if embelin could downregulate cFLIP in breast cancer cells. Here we used two normal human mammary epithelial breast cell lines (H184B5F5/M10 and MCF10A) and three breast cancer cell lines to investigate the relationship between cFLIP L , cFLIP S , and TRAIL resistance in these cell lines. The previous study already found different TRAIL resistance in three breast cancer cell lines we used including TRAIL-sensitive MDA-MB-231, TRAIL-low resistant MCF-7, and TRAIL-high resistant MDA-MB-453 40 . The western blot data demonstrated that normal human breast epithelial cells expressed cFLIP L and cFLIP S. cFLIP L expression was increased as TRAIL-resistance increased and cFLIP S was nonsignificant in three breast cancer cell lines (Fig. 2a,b). It also showed that cFLIP L was correlated with the TRAIL resistance of three breast cancer cell lines. Then we treated breast cancer cells with 25 and 50 μM embelin for 24 h to investigate the effect of embelin on cFLIP L expression. As shown in the western blot and immunofluorescence data, the expression of cFLIP L exhibited a significant decrease in 50 μM embelin treatment of three breast cancer cell lines ( Fig. 2c-i). Moreover, we detected the effect of embelin on two normal breast epithelial cell lines. The data showed that embelin did not downregulate the expression of cFLIP L in H184B5F5/M10 and MCF10A cell lines (see Supplementary Fig. S1).  www.nature.com/scientificreports/ cleaved caspase-9, the activated form of caspase-9. Cleaved caspase-9 significantly increased in 50 μM embelin treatment of three breast cancer cell lines. Therefore, we conclude thar embelin could induce the apoptosis of breast cancer cells through the intrinsic and extrinsic apoptotic pathways ( Fig. 3m-r).

IL-1β induces soluble TRAIL expression in hUCMSCs.
In our previous studies, the TRAIL expression of hUCMSCs can be highly induced by treatment with IL-1β (100 ng/ml) (unpublished data). First, we investigated the effect of IL-1β on hUCMSCs cytotoxicity and apoptosis by using MTT assay and flow cytometry. hUCMSCs were treated with 100 ng/ml IL-1β for 24 h. The result showed that IL-1β treatment had no significant effect on cell viability and apoptosis of hUCMSCs (see Supplementary Fig. S2). Furthermore, there was a finding that soluble TRAIL had higher apoptosis-inducing activity than membrane-bound TRAIL in cancer therapy 43 .
Here we examined whether hUCMSCs could secrete soluble TRAIL and also be enhanced by treatment with 100 ng/ml IL-1β. hUCMSCs were treated with 100 ng/ml IL-1β for different durations (0, 3, 6, 12, 24, 36, 48, 60 h) and then the expression of soluble and membrane-bound TRAIL was detected by western blot. As the data show, soluble TRAIL emerged at 6 h and was significantly increased at 24 h (Fig. 4a,c). The membrane-bound TRAIL was continuously expressed and significantly increased at 12 h ( Fig. 4a,b).

Embelin-treated breast cancer cells and IL-1β-stimulated hUCMSCs enhance apoptosis.
According to our results, three breast cancer cell lines treated with 50 μM embelin for 24 h downregulated cFLIP L expression ( Fig. 2c-i) and increased caspase proteins activation (Fig. 3). hUCMSCs with 100 ng/ml IL-1β treatment for 24 h highly expressed soluble TRAIL (Fig. 4a). The transwell co-culture system was used to confirm the apoptosis rate of embelin-treated breast cancer cells and IL-1β-stimulated hUCMSCs. The apoptosis of breast cancer cells were detected using Annexin V-FITC and PI staining. Positive results for Annexin V-   (Fig. 5b,d,f). Co-culturing embelin-treated breast cancer cells with hUCMSCs or with IL-1βstimulated hUCMSCs led to more apoptotic cells than co-culturing untreated breast cancer cells with hUCMSCs or with IL-1β-stimulated hUCMSCs in all three cell lines (Fig. 5b,d,f). Elevated apoptotic cell population was found in embelin-treated breast cancer cells co-cultured with IL-1β-stimulated hUCMSCs and the apoptotic rate was nearly 30% in MDA-MB-231 cell line, and 40% in MCF-7 and MDA-MB-453 cell lines (Fig. 5b,d,f). Furthermore, we calculated the combination index (CI) 44 to determine the extent to which embelin with IL-1β-

Discussion
TRAIL is a potent inducer of apoptosis in various cancer cells but not in normal cells. Due to its ability to selectively induce cancer cell death, there is growing interest in TRAIL for use in cancer therapy 43,45 . TRAILmediated apoptosis initiates at the extrinsic apoptosis pathway by binding to death receptors DR4 and DR5 46 . It has been found that embelin, the XIAP inhibitor, induces TRAIL-mediated apoptosis by upregulating DR4   47 . In our previous studies, we found that embelin could upregulate DR4 and DR5 expression and enhance TRAIL-mediated apoptosis in breast cancer cell lines (unpublished data). The role of cFLIP L in apoptosis is connected with the death receptor-related extrinsic apoptosis pathway. Accumulating reports showed that cFLIP is correlated with TRAIL-resistance and a regulator of TRAIL-mediated apoptosis. We show that the expression level of cFLIP L is related to TRAIL-resistance in three different TRAILresistant breast cancer cell lines (Fig. 2a,b). The expression of cFLIP S is unrelated to the TRAIL resistance in these cell lines. Embelin has been reported to enhance TRAIL-mediated apoptosis not only by upregulating the death receptors DR4 and DR5 in breast cancer cells, but also by downregulating cFLIP S in malignant glioma cells. In this study, we found that embelin significantly downregulated cFLIP L in breast cancer cell lines (Fig. 2c-i). In addition, embelin caused dose-dependent death of breast cancer cells and upregulated cleaved caspase-8, cleaved caspase-9 and cleaved caspase-3 expression (Figs. 1, 3).
Because of the antitumor effect and the homing ability of MSCs, stem cell therapy is considered to have great potential for cancer therapy. Various antitumor effects of hUCMSCs have been observed such as inhibition of proliferation, modulation of the cell cycle, and inducement of apoptosis through the secretion of related proteins. Research has found that the conditioned medium of hUCMSCs could induce cell apoptosis in human lung cancer cell lines, human hepatocellular cancer cell lines 12 , and human glioma cell lines 48 . In our previous studies, we had found that naïve hUCMSCs expressed membrane-bound TRAIL; the expression of membrane-bound TRAIL was upregulated after being treated with IL-1β (unpublished data). Here, we have also provided evidence that hUCMSCs expressed membrane-bound TRAIL and soluble TRAIL, and that both can be enhanced by IL-1β treatment (Fig. 4). The function of soluble TRAIL secreted from hUCMSCs in embelin treated breast cancer cells was further investigated by co-culturing these cells. In three breast cancer cell lines, the group of co-cultured embelin-treated breast cancer cells and IL-1β induced hUCMSCs, enhanced nearly 40% of apoptotic cells including early apoptosis and late apoptosis. These data suggested that embelin downregulating cFLIP L could enhance In summary, our data demonstrated that the cFLIP L is correlated with TRAIL resistance in breast cancer cells. Embelin downregulated cFLIP L to enhance the TRAIL sensitivity of breast cancer cells and induced the caspase proteins activation in the extrinsic and intrinsic apoptotic pathways. In addition, hUCMSCs expressed the membrane form and soluble form of TRAIL protein induced by IL-1β treatment. Co-cultured embelin-treated breast cancer cells and IL-1β-stimulated hUCMSCs exert a synergistic effect in increasing TRAIL-mediated apoptosis. Above all, this research presents an effective way of utilizing embelin and IL-1β-stimulated hUCMSCs in cancer therapy of different TRAIL-resistant breast cancer cells.  www.nature.com/scientificreports/ Transwell co-culture system and apoptosis detection. Human breast cancer cells were seeded 2 × 10 5 cells on 16 mm glass coverslips in 6 well plates with the culture medium for 24 h and then starved in serumfree DMEM-F12 for 16 h. hUCMSCs were seeded 1 × 10 5 cells in 0.4 μm pore, 24 mm transwell inserts with the culture medium for 24 h and starved for 16 h in DMEM-LG medium with 1% FBS. Then breast cancer cells were pretreated with 50 μM embelin and hUCMSCs were pretreated with100 ng/ml IL-1β for 24 h. After pretreatment, the transwell inserts were moved into 6 well plates to co-culture breast cancer cells and hUCMSCs for 24 h in DMEM-LG with 1% FBS. The coverslips were removed and Annexin V-FITC Apoptosis Detection Kit (Strong Biotech Corporation, TPE, ROC) were used to detect apoptosis. Breast cancer cells were stained with 2% Annexin V-FITC (AV) and 2% Propidium Iodide (PI) in a binding buffer for 10 min in the dark. After staining, the images were immediately captured by using a Fluorescent Microscope (Leica DM6000B, Wentzler, Germany). The apoptotic rate in Fig. 5 was determined by calculating the proportion of cells that have undergone apoptosis (cell numbers of Annexin V+/PI−, Annexin V+/PI+ expressing cells) in relation to the total number of cells in five randomly selected fields of three independent experiments.

Methods
Statistical analysis. All data were represented as mean ± SEM from three independent experiments. Statistical analysis was analyzed by one-way ANOVA using Prism 5 software. P values < 0.05 were considered statistically significant by Dunnett's test. The combination index (CI) was calculated with the apoptotic rate using following equation:

Data availability
The data used to support the findings of this study are included within the article.