The Antitriple Negative Breast cancer Efficacy of Spatholobus suberectus Dunn on ROS-Induced Noncanonical Inflammasome Pyroptotic Pathway

Breast cancer (BCa) is the leading cause of women's death worldwide; among them, triple-negative breast cancer (TNBC) is one of the most troublesome subtypes with easy recurrence and great aggressive properties. Spatholobus suberectus Dunn has been used in the clinic of Chinese society for hundreds of years. Shreds of evidence showed that Spatholobus suberectus Dunn has a favorable outcome in the management of cancer. However, the anti-TNBC efficacy of Spatholobus suberectus Dunn percolation extract (SSP) and its underlying mechanisms have not been fully elucidated. Hence, the present study is aimed at evaluating the anti-TNBC potential of SSP both in vitro and in vivo, through the cell viability, morphological analysis of MDA-MB-231, LDH release assay, ROS assay, and the tests of GSH aborted pyroptotic noninflammasome signaling pathway. Survival analysis using the KM Plotter and TNM plot database exhibited the inhibition of transcription levels of caspase-4 and 9 related to low relapse-free survival in patients with BCa. Based on the findings, SSP possesses anti-TNBC efficacy that relies on ROS-induced noncanonical inflammasome pyroptosis in cancer cells. In this study, our preclinical evidence is complementary to the preceding clinic of Chinese society; studies on the active principles of SPP remain underway in our laboratory.


Introduction
There are seldom chemotherapeutic medications that can gain moderate success in triple-negative breast cancer (TNBC) management. Breast cancer (BCa) is the leading cause of women's death worldwide. Among them, TNBC accounts for almost 10-15% of all BCs, which refers to the absence of estrogen and progesterone receptors and overexpression of human epidermal growth receptor 2 [1,2]. It is one of the most troublesome subtypes of BCa because of its easy recurrence and highly aggressive properties. Although some basic investigations related to immunotherapy and targeted therapy have shown great potential to inhibit the development of cancer [3,4], they still seldom chemotherapeutic drugs, which can offer positive pathologic complete response for the management of TNBC in the clinic [5].
Earlier researchers paid great attention to investigating programmed cell death: apoptosis, which is believed not to trigger inflammation [22]. It is considered an essential component of several processes such as normal cell turnover, growth, the function of the immune system, embryonic development, and chemical-induced cell death [23]. Recently, pyroptosis is another kind of programmed cell death, which is varied in the mechanisms of apoptosis, and proved to be crucial for clearing dangerous infections [24,25]. Pyroptosis is generally lytic cell demise accompanied by rapid cell membrane rupture [26], in which pores are initially formed in the membrane of the cell, causing water influx and cell swelling, causing cell-membrane damage. Hence, pyroptosis is believed to be more inflammatory and immunogenic than apoptosis [27]. Several pilot studies have also clarified that pyroptosis may trigger inflammation and recruit immune cells to the pyroptotic area [28,29]. Currently, mounting research related to pyroptosis is extensively studied in cancer.
The relationship between ROS and tumor cell pyroptosis has been well established [30]. Low doses of ROS normally stimulate cell proliferation in a wide variety of cancer cell types [31,32]. However, elevated ROS triggers tumor cell pyroptosis-dependent caspases [33]. Some chemotherapeutic drugs are addressed to induce tumor cell pyroptosis dependent on caspase-3 [34,35]. Mechanistically, there are two different kinds of signaling pathways involved in pyroptosis. The first one is the caspase-1-dependent process, named "canonical" inflammasome activation, which is mediated by a dynamic mediator, gasdermin D (GSDMD) [36]. The dynamic caspases-1, -4, -5, and -11 generally cleave GSDMD within a linker between the domains of amino and carboxy-terminal. After the breakdown, the Nterminal generates pores in the cell wall to cause pyroptosis resulting in transmembrane ion flux, cytoplasmic swelling, and osmotic lysis [37]. Secondly, a "noncanonical" inflammasome activation has been termed as a pathway that is ROS/caspases axis-dependent, which is also mediated by GSDMD or/and gasdermin E (GSDME) [38][39][40][41]. Nevertheless, there are seldom reports on TCM that can trigger pyroptosis in cancer management. Therefore, the present study is aimed at evaluating the anti-TNBC efficacy of SSP on TNBC cell lines by analyzing cellular characteristics including cell viability, cell morphology changes, LDH release assay, ROS assay, and glutathione (GSH) aborted pyroptotic noninflammasome signaling pathway.

Preparation of Spatholobus suberectus Percolation (SSP)
Extract. SSP extract was prepared in accordance with EMA guidelines as described previously with some modifications [42]. Briefly, dried SSD stems, which were provided by Guangdong Kangmei Pharmaceutical Co., Ltd. (Guangdong Province, China), were ground into coarse powder, and it was extracted using a percolating device with 10 times volume (v/w) of 60% ethanol. The filtrate was then concentrated under reduced pressure by a rotary evaporator (IKA RV 10, IKA-Werke GmbH & Co. KG, Darmstadt, Germany). The obtained percolation powder was then freezedried (percent yield 20%) and stored at 4°C for further use.

Cell
2.6. Morphological Analysis by Scanning Electron Microscopy. Morphological analysis was performed as described earlier [30]. Cells were treated with SSP (100 μg/ml) for 24 h and were fixed with 2.5% glutaralde-hyde (Sigma-Aldrich, St. Louis, MO, USA) overnight. The cells were rinsed thrice using phosphate buffer saline, and the critical point of the drying procedure was carried out. Samples were dehydrated through a graded series of ethanol (30,50,70,95, and 100%) and dried in a Critical Point Dryer using liquid carbon dioxide. The dried specimens were mounted on specimen holders (aluminium stubs) for scanning electron microscopy (SEM), using double-sided adhesive tape, glue, colloidal silver, or colloidal carbon. Then, a thin layer (100-200 Å) of the metallic film was coated on the specimen surface for electrical conduction using either a sputter coater or a vacuum evaporator. Gold, gold-palladium, platinum, aluminium, or carbon was commonly used for the preparation of the thin conducting film. Image with a Hitachi S-3400 N scanning electron microscope was operated for the present study at 20 kV.

Animals.
Female (BALB/c) nude mice (6-7 weeks old) were purchased from Harlan Laboratories, Indianapolis, IN, USA, that were housed and maintained in Laboratory Animal Unit, the University of Hong Kong, a specific pathogen-free and climate-controlled room (22 ± 2°C, 50 ± 10% relative humidity) with a 12 h light/dark cycle and provided with diet and water ad libitum. The xenograft assay was performed as described before with some modifications [43,44]. MDA-MB-231 cells (2 × 10 6 /site) were implanted subcutaneously into the bilateral flank of each mouse. Palpable and measurable tumors were initially found 10 days after cell injection. Then, the animals were randomly assigned into four groups that were receiving the following treatments: the vehicle control group (n = 5) received Milli-Q water; the SSP-L group (n = 5) received SSP (0.4 g/kg/p.o, daily); the SSP-H (n = 5) group received SSP (0.8 g/kg/p.o, daily); the DTX group (n = 5) received docetaxel (5 mg/kg/i.p. week). The tumor size was calculated using the formula: 0:5 × length × width2. All experiments were approved by the Institutional guidelines of Laboratory Animal Care and Committee on the Use of Live Animals in Teaching and Research (CULATR No.: 4484-17).

Acute Toxicity Study.
Acute toxicity studies were performed to determine the short-term adverse effects of a drug when administered in a single dose or multiple doses during 24 hours in two rodent species. The acute oral toxicity study was evaluated as per OECD guidelines. The studies were carried out in BALB/c mice (20-30 g) and Sprague-Dawley rats (150-180 g), respectively, using a single dose or multiple doses, which were treated orally. Thirty animals, divided into respective 5 groups, were designed for the study of acute toxicity via the oral route. Each group contains 6 animals (3 males and 3 females) receiving a single oral dose of 2, 4, 8, and 10 g/kg body weight of SSP extract, while the control group was administrated with distilled water. The general behavior of the animal and signs of toxicity were observed continuously for 1 h after the oral administration and then intermittently for 4 h and thereafter for 24 h. The animals were further observed once a day up to 14 days following treatment for behavioral changes and signs of toxicity and/or death and the latency of death. The LD 50 value was 3 Oxidative Medicine and Cellular Longevity determined according to the method described by Kharchoufa et al. [45].

Collection and Analysis of Biological Information.
The association between caspase-4, caspase-9, and overall survival was performed by the online tool KM plot (http:// kmplot.com/) [46] with the Affymetrix ID: 213596_at and 237451_x_at, respectively. Differential gene expression analyses of the tumor, normal, and metastatic tissues were conducted by the online tool TNMplot (https://www.tnmplot .com/) with the genes' symbols based on RNA-Seq data offered by the database [47].
2.11. Statistical Analysis. Nonlinear regression was operated with GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) choosing log(inhibitor) vs. response-variable slope (four parameters) as the equation. All data were expressed as mean ± standard deviation. Tukey's multiple comparison test was carried out on data from at least three independent experiments. The differences between the two groups were performed using two-tailed Student's t-test, and significance was established at p ≤ 0:05.   (Figure 4(e)). There were significant body weight changes in the treated groups during the study period (Figure 4(b)). And treatment of SSP-L, SSP-H (0.4 and 0.8 g/kg/p.o, daily), and DTX (5 mg/kg/i.p. week) significantly inhibited the growth of tumors in the animals when compared to the vehicle group (Figures 4(c) and 4(e)). At the endpoint, tumor volume was significantly different ( * p = 0:0151) between the SSP-H-treated group and the vehicle group. In addition, DTXtreated animals were also significantly ( * p = 0:0435) reducing the growth of tumors (Figure 4(c)).  Oxidative Medicine and Cellular Longevity 3.4. Evaluation of Acute Toxicity. The acute toxicity study was conducted to determine the harmful effects of SSP to the animals administered as a single or short-term exposure. This investigation assessed the changes in the behavior, sign, body weight, mortality, and other changes in the overall well-being of the animals. In the present study, the acute toxicity evaluation showed that the oral LD 50 value of SSP was 10 g/kg b.w. (Table 1).

Generation of Intracellular ROS by Treatment with SSP
in MDA-MB-231 Cell Lines. To determine the effect of SSP on ROS generation, ROS was detected using CM-H2DCFDA for general oxygen species and dihydroethidium staining, which facilitated to show the expression levels of superoxide and hydrogen peroxide. As shown in Figure 5, the Generation of ROS, specifically superoxide and hydrogen peroxide, were measured in a 24 h cultured plate containing MDA-MB-231 cells, which was shown in a dosedependent manner of SSP (25, 50, and 100 μg/ml). The outcomes showed that SSP treatment upregulated ROS generation in which the number of cells was stained using CM-H2DCFDA and dihydroethidium in MDA-MB-231 cells.

MB-231 cells after 24 h treatment with SSP (100 μg/ml).
The western blot analysis of inflammasome protein showed caspase-4 cleaved GSDME that permeabilized into the cell membrane and might trigger pyroptosis, a form of inflammatory programmed cell death (Figure 7). The full-length GSDME (F-GSDME) degraded into an N-terminal fragment of GSDME (N-GSDME) by caspase-4 that transported into the cell membrane and lysed the cells (Noncanonical pathway). Moreover, caspase-1 did not involve in the cleaving of GSDMD in which there were no products of GSDMD-N and therefore, this mechanism of the inflammasome was a ROS-dependent noncanonical pathway (Figure 7).  (Figures 8(a) and 9(a)). Similarly, phase-contrast microscopic observation demonstrated that MDA-MB-231 and 4T1 cells treated with GSH followed by the administration of SSP showed less pyroptotic features, whereas SSP treatment showed flattened cells with fried egg-like morphology (Figures 8(b) and 9(b)). Western blot assay revealed GSH aborted pyroptotic signaling upon SSP treatment. Cleaved caspase-3, caspase-4, and GSDME were involved in the inflammasome signaling pathway (Figures 8(c) and 9(c)).

The Relationship between Caspase-4/9 and Overall
Survival of the Patients. Confirming prognostic or projecting candidate genes in suitably powered BCa cohorts is of greatest interest nowadays. Based on the online Kaplan-Meier plotter tool, we drew survival plots, which were used to assess the relevant expression levels of caspase-4 and caspase-9 genes on the clinical outcome of BCs individuals. Using the selected parameters, the analysis was operated on caspase-4 (Affy ID: 233596, 3951 patients) and caspase-9 (Affy ID: 237451_x, 1751 patients). Based on the median of participants, the relevant expression levels were demonstrated at the lower or higher risks of 1978  (25,50, 100 μg/ml). The expression of caspase-4 cleaved GSDME that triggers pyroptosis. The full-length GSDME (GSDME-F) degraded into N-terminal fragment of GSDME (N-GSDME) that directly transferred to the plasma membrane and lysed the cells (noncanonical pathway). However, caspase-1 did not involve in the cleaving of GSDMD-F (canonical pathway) in which there were no products of GSDMD-N, and hence this inflammasome mechanism was a ROS-dependent noncanonical pathway. Data were shown as mean ± SD (n = 3). * * p = 0:0031, * * * p < 0:001.   (Figures 10(a) and 10(b)).

Differential Gene Expression Analysis of GSDME and
Caspase-4 in Tumor, Normal, and Metastatic Tissues. Genes generally show differential expression in either tumor or metastatic tissues, which can be beneficial to envisage tumor formation and to facilitate cancer management as a biomarker. Using the TNM plot tool, based on an integrated dataset that was documented in the RNA sequencing data of normal (n = 113), tumor (n = 1097), and metastatic (n = 07) tissues, we compared the differential expression levels of selected genes in normal, tumor, and metastatic tissues. The expression of caspase-4 and GSDME was significantly inhibited in the tumor tissues. The fold changes of caspase-4 from tumor to normal and from metastasis to the tumor were about 0.76 and 1.41, respectively (Figure 10(c)). The analysis of GSDME exhibited fold changes from tumor to normal (0.61) and from metastasis to tumor (1.14) (Figure 10(d)).

Discussion
SSD is a traditional medicinal plant normally used in China for its hematopoietic and antiviral properties [48,49]. Mounting research has been conducted in vitro and in vivo of SSD showing as a promising traditional medicinal drug in the management of various cancers [15,17,20,21]. Presently, physicians from TCM have utilized SSD as a potential therapy for BCa patients and accomplished greater positive outcomes [19]. Studies have further suggested that SSD directly suppresses various molecular signaling pathways, upregulates apoptotic signaling, inhibiting LDH and arresting the cell cycle, and is thereby proved as a potential anticancer compound [19,21,50]. In addition, SSD protects against various effects of oxidative stress, cerebral ischemia, radiation, and diabetic complications [51][52][53]. However, the anticancer efficacy of SSP and its protective mechanism against the most fetal and invasive subtype of BCa, TNBC have not been completely revealed.

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Oxidative Medicine and Cellular Longevity genotoxic effects [31,32,[54][55][56][57][58][59]. Some of these major compound(s) is/are responsible for the anti-TNBC efficacy of SSP, which is being studied in our laboratory. The outcome of acute toxicity indicated that the oral LD 50 value of SSP was about 10 g/kg, and this extract is considered as low toxic to the animals. These findings offer preliminary data on the toxic profile of SSP. Hence, further studies (genotoxicity, subchronic toxicity, reproductive toxicity, etc.) are needed to validate the clinical studies of the plant.
In this study, SSP was investigated on three different TNBC cells: MDA-MB-231, 4T1, and BT 549 cell lines. SSP had significant cytotoxic and growth inhibitory effects on all cell lines in a dose-dependent manner. These effects can be mediated by the generation of ROS [60]. Previously, SSD treatment significantly increased cytotoxic effects through the generation of ROS in U266 and U937 cells [15]. ROS plays a critical role in multiple tumor chemother-apy and involves cytotoxicity, autophagy, and apoptosis [61]. There were significant differences in the SSP-treated groups (bodyweight-15.35 g and tumor volume-415.4 mm 3 ) and vehicle group (bodyweight-18.04 g and tumor volume-937.4 mm 3 ), which showed about 55.69% tumor growth inhibition at the endpoint. Thus, SSP inhibits the growth of MDA-MB-231 human TNBC in a xenograft-bearing mouse model. This study was consistent with the earlier study upon the treatment of resveratrol inhibited the gaining of body weight and tumor growth in the animal models [62,63].
In our study, we detected ROS generation upon SSP treatment, which was consistent with earlier investigations [15,17]. The detection of intracellular ROS was based on the presence of CM-H2DCFDA and dihydroethidium staining of MDA-MB-231 cells upon the treatment of SSP (25,50, and 100 μg/ml), and this ROS generation was significantly greater in MDA-MB-231 cells when treated with the higher concentrations of SSP (100 μg/ml). For TNBC management, to date, there is no promising medication. Hence, there is urgent to find novel anti-TNBC strategies or molecular targets. Natural compounds like SSP, in this setting, have many advantages, especially in the clinical practices of TCM or in preclinical research. In our study, SSP could inhibit the growth of TNBC both in vitro and in vivo, and this mechanism has been elucidated through noncanonical pyroptotic pathways. The expression of caspase-4, cleaved caspase-9, GSDME, and the N-fragment of GSDME was upregulated upon SSP administration in TNBC cells.
Earlier, researchers believed only SSD treatment could induce apoptosis [15,17]. However, in the present study, SSP promotes pyroptotic cell death in TNBC cells. Pyroptosis is a process of programmed cell death, mediated by the key factors, GSDMD or GSDME, which can be activated by caspase-4 and/or caspase-3 [64][65][66]. Several caspases can cleave GSDMD or GSDME into the N and C-terminal domain of GSDMD or GSDME, in which the N-terminal fragments have the ability of pore-forming activity in the plasma membrane [37,67]. The significant difference between apoptosis and pyroptosis is the microscopy and cellular osmotic features. The morphological analysis of SSPtreated TNBC cells is of pyroptotic features. The cells are exhibited flattened cells with the "cabbage" or "fried egg"like, and the cell nucleus located in the center. The activation of GSDME causes a loss of membrane integrity and release/discharge of cytosolic LDH, resulting in inflammatory cell death. LDH with other cellular contents is also discharged during the pyroptotic blebs of cellular demise [68]. Interestingly, SSP-treated TNBC cells have neither altered the expression of cleaved GSDMD nor cleaved caspase-1. This activation is performed through the activation of caspase-4 and caspase-3. The complex of N-GSDMEs inserts into the plasma membrane as pores resulting in cell lysis. In this process, canonical inflammasomes are not involved. Thus, the process was regarded as a noncanonical inflammasome pyroptotic signaling pathway.
The mechanisms underlying the events of the noncanonical inflammasome are still being described. Caspase-3, -8, -9, -7, -4, -5, and -11 trigger its activation, as they are recognized as molecular switches or effectors for pyroptotic cells [ 34,65,[69][70][71][72]. These activated caspases then generate GSDME and biologically active executioners, impacting pyroptotic cell death [64]. GSH is an inhibitor of ROS that attenuates SSP-induced ROS generation in the cell and hence rescues pyroptotic cell death. SSP treatment alone could cause pyroptosis through the activation of caspase-4 and caspase-9. These findings indicated that SSP-induced pyroptotic death is ROS-dependent. The present investigation validated that SSP promotes ROS generation in TNBC cells which triggers noncanonical pyroptosis and involves a novel anti-TNBC-based intervention strategy for the treatment of BCa. Noncanonical inflammasome-associated pyroptosis has been reported to play in both pro-and anti-tumor development. The tumor microenvironment is shaped by a chronic inflammation in which polarized macrophages and stromal components promote tumor development [73][74][75]. Thus, non-canonical inflammasome activation and regulation have been vital especially in cancer and other disease management. Non-canonical pro-pyroptotic agents like SSP induce an acute inflammatory immune response that warrants further investigation in a clinical setting. Furthermore, our bioinformatic analysis of caspase -4, -9, and GSDME are well-established as cancer markers that are also involving in non-canonical pyroptotic mechanisms.
Bioinformatic tools are extensively used to evaluate gene expression levels and to explore their possible implications in the development of various cancers [76][77][78]. In the present study, survival analysis using the KM Plotter revealed that the low transcription levels of caspase-4 and 9 are related to low relapse-free survival in BCa. This study was consistent with earlier investigations in which researchers concluded that the caspase family is operated as new prognostic indicators in various cancers, including breast [79], gastric [80], ovary [81], and renal [82]. TNM plot analysis showed that the expression of caspase-4 and GSDME was significantly inhibited in clinical tissues in normal (113), tumor (1097), and metastatic (07) states, which was consistent with the earlier investigation [47]. Based on the study, the ROS-induced pyroptotic pathway which is associated with caspase-4/9 and GSDME that are potential targets of precision therapy for patients with TNBC.

Conclusions
TNBC is one of the most problematic classes of BCa with easy recurrence and considerably assertive type. SSD has been used in the clinic of TCM as a potential therapeutic agent to heal BCa individuals, which accounts for relatively positive responses. However, the anti-TNBC potential of SSP and its evidence-based in vitro and preclinical studies is still deficient. Hence, the present study was evaluated the anti-TNBC potential of SSP through various in vitro and in vivo studies. SSP showed significant growth inhibitory efficacy in both TNBC cell lines and xenograft animal models. Western blot analysis was also encouraged that SSP elevated inflammasome proteins such as caspase-4 and 9, which cleaved GSDME triggering pyroptosis and permeabilizing the cell membrane. Furthermore, cotreatment of GSH and SSP markedly attenuates the SSP-induced ROS generation in the cell and validated the rescuing pyroptotic cell death. Survival analysis using the KM Plotter and TNM plot database exhibited the curved transcription levels of caspase-4 and 9 related to low relapse-free survival in patients with BCa. SSP is comprised of catechin, procyanidin B2, epicatechin, genistein, and formononetin that are recognized as anticancer agents. All findings strongly suggest that SSP possesses anti-TNBC efficacy and continues to be an inspiring and dynamic research niche in the upcoming days with evident antitumorigenesis effects and targets of eradicating BCa cells. However, well-controlled future clinical studies are quite required to advance an understanding of the pharmacological functions of SSP. Such information could be used to categorize effective preventive strategies targeting specific components of TNBC.