Curcumin Induces MCF-7 Cells Pyroptosis Via Autophagy/CTSB/NLRP3/Caspase-1/GSDMD Signaling Pathway In Vitro And Vivo

Curcumin, as a lipid-lowering drug, has been reported to be effective in the treatment of breast cancer. However, the underlying molecular mechanisms have not been completely investigated. MTT assay was used to determine the effect of curcumin on survival rate of MCF-7 cells. The effects of curcumin on tumor growth were observed in animal models of breast cancer. The positive reactions of Caspase-1, IL-1β and IL-18 were detected by immunohistochemistry. LC3, p62, CTSB, ASC, Pro-Caspase-1, GSDMD, NLRP3, Caspase-1, GSDMD-N, IL-1β and IL-18 were determined by Western blot in vitro and vivo. The release of extracellular IL-1β and IL-18 was determined by ELISA. LDH release was measured. The expression level of CTSB in cytoplasm were determined by immunouorescence assay. Cell proliferation, cell migration and tube formation assays were used to determine the abilities of cells. In this study, NLRP3 inammasome inhibitor MCC950, cathepsin B inhibitor CA-074 ME and autophagy inhibitor 3-MA were used to act on cells to investigate the role of NLRP3 inammasome, cathepsin B and autophagy in curcumin-induced pyroptosis of MCF-7 breast cancer cells. the NLRP3 inammasome by inducing autophagy in MCF-7 breast cancer cells, thus causing pyroptotic cell death via Autophagy/CTSB/NLRP3/Caspase-1/GSDMD signaling pathway. We also demonstrate that the molecular mechanism of NLRP3 inammasome activation underlies the autophagy and the release of CTSB from autolysosome caused by curcumin treatment. In addition, this study provides a new perspective on curcumin in the treatment of breast cancer. This knowledge may complement the treatment strategy for breast cancer and further conrm the bright future of curcumin in cancer treatment.


Introduction
Breast cancer is the most common malignancy in females and ranks second among causes for cancer related death in women [1] . Lifestyle is considered an increasingly signi cant contributing factor to breast cancer etiology. Obesity, overweight, hypercholesterolemia, metabolic syndrome and alcohol represent risk factors for breast cancer [2] . Chemotherapy has been used as a routine treatment for breast cancer and other cancers. However, chemotherapy is prone to drug resistance and has high side effects. For many years clinicians and researchers have been exploring and examining various therapeutic modalities for breast cancer. Finding natural anticancer compounds with low toxicity and high selectivity has always been the mainstream direction of cancer research.
Curcumin, a lipid-lowering drug is the main component of turmeric, derived from the roots of plant Curcuma longa [3] . It has been widely studied for its anti-cancer, antioxidant, anti-in ammatory, antiangiogenic and wound healing effects for its medicinal properties in Chinese and Indian systems of medicine [4] . And the main mechanisms of action by which curcumin exhibits its unique anticancer activity include inducing apoptosis and inhibiting tumor proliferation and invasion by suppressing various cellular signaling pathways [5] . Several studies reported that curcumin may regulate multiple signaling pathways, including PI3K/AKT, nuclear factor (NF)-κB, MAPK and JAK/STAT [6] . Recent studies have shown that curcumin can also inhibit the phosphorylation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR), decreased B-cell lymphoma 2 (BCL2) and promoted BCL-2-associated X protein (BAX) and cleavage of caspase 3, subsequently inducing apoptosis of breast cancer cells [7] . However, the mechanism of curcumin in breast cancer has not been totally investigated and lack of in vivo experiments.
Pyroptosis, a proin ammatory form of regulated cell death that depends on the enzymatic activity of in ammatory proteases belong to the caspase family, especially caspase-1 [8] . Pyroptosis is generally accompanied by plasma membrane rupture, cytoplasmic swelling, DNA cleavage, NLRP3 in ammasome activation and release of proin ammatory cell contents [9] . Additionally, it needs lipopolysaccharide (LPS) to upregulate,NLRP3 and pro-IL-1β are also essential. In ammasomes are multi-protein signaling complexes that trigger the activation of in ammatory caspases and the maturation of IL-1β [10] . Nucleotide oligomerization domain-like receptor proteins (NLRPs), especially NLRP3, interact with ASC and pro-caspase-1 to active caspase-1. Caspase-1 is activated within the in ammasome, and active caspase-1 processes gasdermin D (GSDMD) and cytokines such as pro-IL-1β and pro-IL-18. Upon permeabilization of the plasma membrane by GSDMD pores, cells undergo a lytic, pro-in ammatory cell death (pyroptosis) that promotes the release of mature IL-1β and IL-18 [11] . Currently, it has been reported that the release of lysosomal cathepsin B induces the activation of NLRP3 in ammasome [12] . Cathepsin B (CTSB) is an intracellular cysteine protease, mainly localized in the lysosome [13] . In addition, the level of CTSB in cytoplasm is related to autophagy ux. When autophagy level increases, autophagic lysosomal degradation leads to cytoplasmic release of CTSB, that related to activation of NLRP3 in ammatosomes [14][15][16] .
Autophagy, a lysosomal dependent catabolism process by eukaryotic cells degraded longevity proteins and their organelles, and is involved in the development, differentiation and homeostasis of cells under various physiological and pathological conditions [17] . Autophagy plays a key role in cancer. In recent years, few studies have indicated that curcumin can induces autophagy [18][19][20] .
In our study, we hypothesized that curcumin might induce autophagy, the cytosolic release of lysosomal contents (CTSB), activation of NLRP3 in ammasome and Caspase-1 and GSDMD-dependent pyroptosis.

Animal experiment
Ten six-year-old female BALB/c-Nude mice (weight:20g) were purchased from Beijing Vital River, raised in the Institute of Genome Engineered Animal Models for Human Disease of Dalian Medical University (SPF level). The mice were kept under sterile conditions and fed a sterilized mouse diet and water. All mice were anaesthetized via inhalation of iso urance and a tumor cell suspension of 10 7 MCF-7 cells in 0.2 ml DMEM was injected subcutaneously into the inguinal of each mouse. When tumors reached about 30-60mm 3 at 1 week, the mice were randomly seperated into two groups (n = 5/group). The mice were treated with curcumin 200 ug/kg or saline (control) by intraperitoneal injections every day for 4 weeks. Tumor size was measured every week in two perpendicular dimensions with vernier calipers and converted to tumor volume using the formula: a*b*b (a:longer, b:shorter). At the end of the experimental period, all mice were euthanized and tumors were segregated and weighed.

Immunohistochemical assays
The tissue samples of the tumors, preserved in 2.5% glutaraldehyde-polyoxymethylene solution, were dehydrated and embedded in para n following routine methods. The para n sections were dewaxed and hydrated following routine methods. Rinsed the para n sections in PBS-T (3×5 min before each following steps), and then blocked with 3% peroxide-methanol at room temperature for endogenous peroxidase ablation. Afterwards, the sections were immersed in a boiling sodium citrate buffer for 15min, cooled down to room temperature. After that, incubated with blocking buffer (normal goat serum at room temperature for 20 min. Then incubated with primary antibody Caspase1(A nity, AF4005, 1:100), IL-1β (Wanleibio, WL00891, 1:100), IL-18 (Wanleibio, WL01127, 1:100) at 4℃ overnight. After that, the sections were incubated with the Secondary Goat anti-rabbit-IgG at 37℃ for 30min.Then incubated with the S-A/ HRP at 37°C for 30 min. Colored with 3,3-diaminobenzidin (DAB), and kept at room temperature without light for 10 min. After rinsing adequately with water, the sections were stained with hematoxylin for 5s, then dehydrated and sealed with neutral resins. We observed the sections under an upright microscope.

Lactate dehydrogenase (LDH) release assays
To access the toxic effect of curcumin, the LDH release of MCF-7 cells were measured using an LDH Cytotoxicity Assay Kit (Beyotime, C0016). MCF-7 cells were seeded in 96 well plates to desired con uence and treated with inhibitor for 4h then treated with curcumin for 48h. One hour before the end of the treatment, we added 10% of the LDH release reagent and the original culture volume when the sample showed maximum enzyme activity. The culture supernatants were collected, and the absorbance was read at 490 nm with a microplate reader (Thermo Fisher Scienti c). The percentage of LDH released was calculated as the percentage of the total release amount and considered to be the sum of the enzyme activity in the cell lysate and the enzyme activity in the medium.

Enzyme-linked immunosorbent assays (ELISA)
IL-1β and IL-18 levels were measured by using ELISA kits (Lengton, BPE10083, BPE10092) according to the manufacturer's instructions. MCF-7 cells were seeded in 96-well plates to desired con uence and treated with inhibitor for 4h then treated with curcumin for 48h. The 96-well plates were centrifuged at 3000rpm for 20 min at 4 °C. Finally, the supernatants were collected. A total of 50μl of serially diluted samples and standard were added to the ELISA plate wells and incubated with horseradish peroxidaseconjugated speci c antibodies for IL-1β and IL-18 for 60 min at 37 °C. The OD values were detected at 450nm by using microplate reader. The linear regression equation of the standard curve was calculated according to the concentration of the standard corresponding OD value. Finally, the sample concentration was calculated on the linear regression equation according to its OD value.

Cell migration assays
For the transwell migration assays, MCF-7 cells were treated with various treatments. After indicated treatment, cells were resuspended at serum-free medium then seeded onto the upper chambers with noncoated membrane (24-well insert; 8-mm pore size). After that, we lled the lower chambers with 600ml DMEM containing 10% FBS. After 24 hours of incubation, non-invading cells on the upper surface of the upper chamber were utterly removed by using cotton swabs. Other cells on the lower surface of lters were xed with methanol for 30 min then stained with 0.1% crystal violet for 30 min. The number of invaded cells was counted under an upright microscope.

Matrigel tube formation assay
Via a precooled pipette, 50 µL of freeze thawing liquid Matrigel (Corning, 354248, USA) was embedded into a 96-well plate at 4 °C. HUVECs (2 × 10 4 cells per well) were seeded onto the solidi ed matrigel 96well plate and cultured for 12 h at 37°C in 5% CO 2 . After the HUVECs were overlaid, we replaced the culture medium of HUVECs with MCF-7 cell culture medium treated by curcumin and inhibitor. Capillarylike structures were evident and counted using a phase-contrast microscope and the networks formed by HUVECs were quanti ed with ImageJ.
2.11 Plate clone formation assay MCF-7 cells were cultured and seeded onto 6-well plates (1000 cells per well) to prepare the cells for the plate clone formation assay. Different wells were treated variously. When the cells in 6-well plates growth to a density of 50% per cluster, which were washed with the phosphate-buffered saline(PBS) and xed by 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Giemsa stain (Sigma-Aldrich) was used to stain the cells. Eventually, cell clones were counted and analyzed.

Statistical analysis
All Data were expressed as means ± standard deviation (SD) from at least three independent experiments performed in triplicates and analyzed by using the SPSS 20.0. Signi cance was determined using one-way analysis of variance (ANOVA) or t-test, and P value < 0.05 was considered statistically signi cant.

Curcumin inhibited proliferation and induced cell death in MCF-7 breast cancer cells
In order to assess the effects of curcumin on proliferation and cell death of MCF-7 cells, we performed in vitro assays. As shown in Fig. 1

Curcumin inhibited the tumour growth in mouse model of breast cancer
In vivo assays, we established a mouse model of breast cancer, to comprehend the role of curcumin on tumors' growth. Results obtained by detection of mice body indicated that curcumin has no toxicity effect on mice. As shown in Fig. 2, tumors' volumes decreased signi cantly (P<0.05) after 4 weeks of curcumin treatment as compared with control.

Curcumin induced pyroptosis in vitro and vivo
To con rm the effect of curcumin in tumor, protein expression levels of LC3, p62, CTSB, ASC, pro-Caspase-1, GSDMD, NLRP3, Caspase-1, GSDMD-N, mature IL-1β and IL-18 were measured. As shown in  (Fig. 3CD). In cell experiments (Fig. 4) we found that curcumin treatment(0~128μM) similarly decreased the expression of p62 and increased the expression of LC3, CTSB, ASC, pro-Caspase-1, GSDMD, NLRP3, Caspase-1, GSDMD-N, mature IL-1β and IL-18 compared with control in LPS-primed cells, with a dose-dependent manner. Notably, there was no signi cant difference between the LPS group and the control group. Together, these results suggest that curcumin is able to induce pyroptosis in MCF-7 cells and tumors.

Curcumin-induced MCF-7 pyroptosis depends on activation of the NLRP3 in ammasome
To determine the role of NLRP3 in ammasome components in activation of caspase-1 under curcumin stress, we inhibited the expression of NLRP3 using NLRP3-speci c inhibitor MCC950. The MCF-7 cells were pretreated with MCC950 (5 μM) and LPS (1 μg/ml) prior to treatment with 8 μM curcumin for 48 h.
Before that, we detected the effect of curcumin on the viability of MCF-7 cells. As shown in Fig. 5AB, NLRP3, Caspase-1, GSDMD-N, mature IL-1β and IL-18 expression were increased by curcumin treatment. However, MCC950 restrained Caspase-1, GSDMD-N expression and IL-1β, IL-18 production. Moreover, MCC950 inhibited the release of mature IL-1β, IL-18 and LDH caused by curcumin (Fig. 5CD). Besides, in functional experiments, MCC950 reversed the amount of MCF-7 cell migration decreased by curcumin treatment (Fig. 5E), as well as the matrigel tube formation (Fig. 5F) and plate clone formation (Fig. 5G). In all, these ndings indicated that curcumin-induced pyroptosis is dependent on NLRP3 in ammasome activation.

Curcumin-induced NLRP3 in ammasome activation and pyroptosis are mediated by cytoplasmic CTSB
It has been indicated that NLRP3 in ammasome activation was associated with a variety of upstream factors, including the release of lysosomal CTSB. As shown, CTSB expression was up-regulated in cells and tumors after curcumin treated. Therefore, we pretreated cells with CA-074 Me, an inhibitor of CTSB, to measure the role of CTSB in curcumin-induced activation of the NLRP3 in ammasome. As shown in Fig.  6AB, the increased expression level of CTSB, NLRP3, Caspase-1, GSDMD-N, mature IL-β, IL-18 proteins caused by curcumin were inhibited by CA-074 Me compared with control. Besides, CA-074 Me inhibited the release of mature IL-1β, IL-18 and LDH caused by curcumin (Fig. 6CD). Moreover, in functional experiments, CA-074 Me reversed the amount of MCF-7 cell migration decreased by curcumin treatment (Fig. 6E), as well as the matrigel tube formation (Fig. 6F) and plate clone formation (Fig. 6G). As observed by microscopy, immuno uorescence results showed that curcumin treatment could cause MCF-7 intracellular lysosomes to rupture and release CTSB into the cytoplasm, and CA074 Me treatment could signi cantly reverse this situation (Fig. 6H). Together, the results indicated that curcumin-induced NLRP3 in ammasome activation and cell pyroptosis was CTSB-dependent. The increase in cytosolic CTSB levels is associated with lysosomal degradation. The results show an increase in autophagic ux in MCF-7 cells. To con rm the change of autophagic ux, we used the autophagy inhibitor 3-MA in MCF-7 cells. As shown in Fig. 7AB, the up-regulation of LC3 and the downregulation of p62 by curcumin were reversed by 3-MA since 3-MA inhibited the fusion of autophagosome and autolysosome. The protein LC3 could be transformed from a soluble form (LC3-I) to a lipidized form (LC3-II) when autophagy was activated. And p62 is a multifunctional protein regulated by the balance between its transcriptional regulation and post-translational autophagic degradation [21] . The LC3 conversion and p62 protein are regard as the most credible biochemical markers of autophagy [22] . These results suggested that curcumin induced up-regulation of MCF-7 autophagy. In addition, the expression of cytosolic CTSB, NLRP3, Caspase-1, GSDMD-N, mature IL-1β, IL-18 were up-regulated by curcumin and down-regulated by 3-MA (Fig. 7AB). Moreover, 3-MA inhibited the release of mature IL-1β, IL-18 and LDH caused by curcumin (Fig. 7CD). Furthermore, in functional experiments, 3-MA reversed the amount of MCF-7 cell migration decreased by curcumin treatment (Fig. 7E), as well as the matrigel tube formation (Fig. 7F) and plate clone formation (Fig. 7G). These results demonstrated that curcumin induced up-regulation of MCF-7 autophagy which was associated with the increase in cytosolic CTSB, activation of NLRP3 in ammasome and pyroptotic cell death.

Discussion
The breast cancer has still been the most common cancer among women worldwide [23] . Recently study indicated that it has a younger trend [24] . Curcumin, a polyphenol extracted from Curcuma longa in 1815, has gained attention from scientists worldwide for its anticancer potential [25] . The phosphoinositide 3kinase (PI3K)/Akt signaling pathway has always been a focus of interest in breast cancer. However, deregulation of the PI3K/Akt signaling pathway including PIK3CA activating mutation is frequently present in breast cancer [26] . Besides, the mechanism of curcumin in the treatment of breast cancer has not been completely investigated. In the present study, we investigated the effect on pyroptosis in curcumin-treated cells. It is worth mentioning that our study provide the rst evidence for curcumin induces cell pyroptosis by activating autophagy/NLRP3/Caspase-1/GSDMD pathway in MCF-7 breast cancer cells. Our study demonstrated that curcumin upregulated the level of autophagy and triggered NLRP3 in ammasome activation, leading to pyroptotic cell death. In addition, curcumin-induced pyroptotic cell death dependent upon the CTSB-mediated activation of NLRP3 in ammasome and curcumin-induced autophagy was implicated in the curcumin-induced release of CTSB.
To date, pyroptosis is a type of programmed cell death mediated by the formation of plasma membrane pores by members of the gasdermin protein family, characterized by the swelling and lysis of cells, and release of many proin ammatory factors [27] . Caspase-1, as a statically determinate proenzyme, is an important component of the in ammasome and is formed by different PRRs through (or without) ASC and Caspase-1 under the stimulation of speci c PAMPs and DAMPs [28] . The in ammasome contains an NLR and adaptor protein ASC, which interacts with procaspase-1 through caspase recruitment domain, and then self-cleavage forms active caspase-1. Caspase-1 mediated the maturation and secretion of proin ammatory (IL-1β and IL-18) and lead to pyroptosis [29] . Gasdermin D (GSDMD) is a direct substrate of caspase-1 which can be specially cleaved by in ammatory caspases and plays a key role in the downstream of in ammatory caspases in pyroptosis [30] . In ammatory caspases cleaved GSDMD to form GSDMD-N, which is capable of forming porelike structures in lipid membranes and thus constitutes the direct and unique effector of pyroptosis [31] . GSDMD-N forms pores in the plasma membrane leading to membrane defects and cytoplasmic protein release [32] . In our study, treatment with curcumin elevated the levels of Caspase-1, GSDMD-N, IL-1β, IL-18 both in vitro and in vivo. Furthermore, the immunohistochemical results of Caspase-1, IL-1β and IL-18 in the tumor tissues of mice were signi cantly positive compared with control. In addition, the release of IL-1β, IL-18 and LDH in the cytoplasm were also increased. These results demonstrated that curcumin induced Caspase-1 and GSDMD dependent pyroptosis in MCF-7 breast cancer cells.
In ammation is involved in the development and progression of tumors, as well as in the antitumor response to treatment. Among the in ammasomes family, NLRP3 in ammasome is the most characterized. As we known that NLRP3 is deubiquitinated and associates to ASC, which then associates to procaspase-1 to form a large multimeric polyprotein complex. Pro-caspase-1 undergoes autoproteolytic cleavage, possibly due to proximal-induced multimerization, resulting in the active form of caspase-1 [33] . The activation of NLRP3 in ammasome induces two primary effects, programmed cell death known as pyroptosis, and/or proin ammatory responses caused by the release of in ammatory cytokines IL-1βand IL-18 [34] . Previous studies have found that curcumin induced NLRP3 in ammasome priming and caspase-1 activation [35] . Interestingly, however, few studies have shown that curcumin suppresses IL-1β secretion through Inhibition of the NLRP3 In ammasome [36] . In our study, we reported that treatment with curcumin elevated the levels of NLRP3, Caspase-1, IL-1β, IL-18, GSDMD and GSDMD-N which was reversed using the NLRP3 inhibitor MCC950 both in vitro and in vivo. These results indicated that curcumin caused activation of the NLRP3 in ammasome and lead to pyroptotic cell death nally.
Previous study con rmed that three signaling pathways of the NLRP3 in ammasome activation involving potassium e ux, generation of reactive oxygen species, and cathepsin B release [37] . Cathepsin B is a cysteine protease involved in the regulation of metalloproteinases, intracellular communication, autophagy induction and immune resistance, and plays an important role in cancer progression and anticancer therapy [38] . Recently study indicated that cathepsin B was required for caspase-1 activation induced by many different NLRP3 in ammasome activators [39] . In our study, the similar results were observed. Treatment with curcumin elevated the levels of CTSB, NLRP3, Caspase-1, GSDMD-N, IL-1β and IL-18 both in vitro and in vivo which could reverse by the cathepsin B inhibitor CA074-Me. Furthermore, the result also showed that CA074 Me reduced cytoplasmic cathepsin B in immuno uorescence assay.
Consistent with others [40] , these results demonstrated that the activation of the NLRP3 in ammasome was mediated by cathepsin B.
Autophagy is a process by which proteins and organelles are phagocytosed in autophagosomal vesicles and delivered to the lysosome/vacuole for degradation [41] . Autophagy is a tightly coordinated process that isolates misfolded proteins, damaged or aged organelles, and mutated proteins in double membrane vesicles called autophagosomes that eventually fuse into lysosomes, leading to the degradation of the isolated components [42] . During autophagy, cytoplasmic LC3 protein is processed and recruited to the autophagosomal membranes; the autophagosome then fuses with the lysosome to form the autolysosome. Cytoplasmic LC3-I binds to phosphatidylethanolamine to form LC3-II. LC3-II was then incorporated into the autophagosome membrane [43] . Ubiquitin-associated protein p62 is a classic autophagy receptor that binds to LC3 and recruits the selected cargo to mature autophagosomes. A lipidated form of LC3, LC3-II, p62 has been shown to be an autophagosomal marker in mammals [44] . In our study, treatment with curcumin elevated the levels of LC3, CTSB, NLRP3, Caspase-1, GSDMD-N, IL-1β, IL-18 and decreased the level of p62 both in vitro and in vivo, indicating that the degradation of autophagosomes leads to the release of CTSB into the cytoplasm to activate the NLRP3 in ammasome. Besides, the increased level of CTSB, subsequent activation of NLRP3 in ammasome and Caspase-1 and GSDMD-dependent pyroptotic cell death induced by curcumin were reversed by the autophagy inhibitor 3-MA obviously. These results are consistent with the recent studies [45] [46] . Notably, curcumin did not increase NLRP3 protein expression after treatment with the autophagy inhibitor 3-MA, suggested that curcumin could not directly induce the activation of NLRP3 in ammasome, which was consistent with the anti-in ammatory properties of curcumin. Taken together, these results demonstrate that curcumin induces autophagy in MCF-7 breast cancer cells and then activates the NLRP3 in ammasome.
In conclusion, our study indicates that curcumin activates the NLRP3 in ammasome by inducing autophagy in MCF-7 breast cancer cells, thus causing pyroptotic cell death via Autophagy/CTSB/NLRP3/Caspase-1/GSDMD signaling pathway. We also demonstrate that the molecular mechanism of NLRP3 in ammasome activation underlies the autophagy and the release of CTSB from autolysosome caused by curcumin treatment. In addition, this study provides a new perspective on curcumin in the treatment of breast cancer. This knowledge may complement the treatment strategy for breast cancer and further con rm the bright future of curcumin in cancer treatment.

Conclusion
Curcumin induces MCF-7 breast cancer cells pyroptosis dependent on caspase-1 and GSDMD. Curcumininduced pyroptotic cell death in MCF-7 breast cancer cells depends on the activation of NLRP3 in ammasome. The activation of NLRP3 in ammasome induces by curcumin is mediated by cytoplasmic CTSB. Curcumin induces autophagy in MCF-7 breast cancer cells, which then induces in ammation and eventually leads to pyroptosis.

Declarations
Ethics approval and consent to participate: The animal studies covered in this manuscript have been approved by the Ethics Committee of Dalian medical university. Consent for publication: Not applicable