
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12634-5
10.1016/j.heliyon.2024.e36603
e36603
Research Article
Miltirone induces GSDME-dependent pyroptosis in colorectal cancer by activating caspase 3
Zheng Guangwei ab
Fang Zhipeng ab
Lin Zhenlv ab
Guan Guoxian fjxhggx@163.com
cd⁎
a Department of Emergency Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, 350005, China
b Department of Emergency Surgery, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, 350212, China
c Department of Colorectal Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, 350005, China
d Department of Colorectal Surgery, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, 350212, China
⁎ Corresponding author. Department of Colorectal Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China. fjxhggx@163.com
20 8 2024
30 8 2024
20 8 2024
10 16 e3660313 5 2024
19 8 2024
19 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Colorectal cancer (CRC) is a common and malignant tumor, ranking as the third most common cancer in men and the second most common cancer in women. Pyroptosis, a recently described programmed cell death mechanism mediated by the GSDM family, has emerged as an immunogenic mechanism for chemotherapy drugs in tumor treatment. In this study, we discovered that Miltirone has the ability to reduce the viability of CRC cells (SW620 and HCT116) and cause the proteolytic cleavage of gasdermin E (GSDME) in CRC cells. It was also observed that inhibiting GSDME prevented pyroptotic cell death induced by Miltirone in SW620 and HCT116 cells. Furthermore, the main active component of Miltirone was found to effectively bind with caspase 3. SiRNA-mediated caspase 3 silencing and specific caspase 3 inhibitor Z-DEVD-FMK were shown to weaken Miltirone-induced GSDME-dependent cell death. The findings of the study suggest that Miltirone has the potential to inhibit the growth of CRC tumors in vivo by inducing pyroptotic cell death. This indicates that Miltirone could be a viable therapeutic agent for the treatment of CRC through GSDME-dependent pyroptosis. These results offer a promising new option for the clinical treatment of CRC.

Keywords

Colorectal cancer
Pyroptosis
Miltirone
GSDME
Caspase 3
==== Body
pmc1 Introduction

Colorectal cancer (CRC) is a prevalent malignant neoplasm, ranking third in incidence among males and second among females [1].According to recent statistics, the year 2020 witnessed an estimated 1.9 million new cases and 900,000 fatalities across the globe, with significant incidence and mortality rates observed on a global scale [2]. In recent years, there has been a rise in the occurrence of colorectal cancer (CRC), which constitutes approximately 10 % of all cancer cases and ranks as the second most common cause of cancer-related mortality. This information is supported by Ref. [3]At present, the primary modalities employed in the management of CRC comprise surgical resection, radiotherapy, and chemotherapy. However, the effective treatment of CRC continues to pose a significant obstacle [4]. Consequently, Colorectal Cancer (CRC) has emerged as a significant and complex worldwide public health concern, and the pursuit of novel treatment modalities has become a focal point in CRC investigation.

Pyroptosis is a form of programmed cell death that is mediated by the GSDM family (GSDMs) and is known to be immunogenic [5,6]. Upon activation by the activated caspase family and granule enzyme B, GSDMs are triggered to release their N-terminal domain, which possesses the ability to form pores in the cell membrane. This process results in intracellular and extracellular osmotic imbalances, ultimately leading to cellular swelling and subsequent demise. This phenomenon has been documented in literature [7]. The GSDME pathway's induction of pyroptosis, resulting in the extracellular release of lactate dehydrogenase (LDH) and HMGB1, has garnered significant attention due to its association with adaptive immunity in anti-tumor therapy [8,9]. Pyroptosis is a kind of cell death process accompanied by inflammatory response, and there is a complex and important connection between it and colorectal cancer.Up to now, several studies have linked pyroptosis with colon cancer [[10], [11], [12], [13]]. In this study, it demonstrated that GSDME mediates the apoptin-induced pyroptosis through the mitochondrial apoptotic pathway in HCT116 cells [11].Most CRC cells exhibit limited gasdermin E (GSDME) expression due to abnormal methylation-mediated silencing. This data showed radiation increased the activation of caspase-3 but did not induce pyroptosis in HT29 cells showed silenced expression of GSDME. However, radiation-induced pyroptosis was obvious in HT29 cells transfected with GSDME-expressing lentivirus, but was inhibited in the presence of Z-DEVD-FMK, a caspase-3 inhibitor that prevented cleavage of GSDME [12].

Moreover, Photodynamic therapy(PDT)induced pyroptosis in CRC via the ROS/p38/CASP3/GSDME axisin vitro and in vivo. and IR700DX-6T-PDT could sensitize MSS-CRC to PD-1 blockade treatment decitabine, a classic methyltransferase inhibitor, increased GSDME expression through demethylation and induced dramatic tumor shrinkage and immune cell infiltration when used in combination with and anti-PD-1 antibody in CRC cells with low endogenous GSDME expression [13].

On the one hand, pyroptosis-related signaling pathways and molecules may play a key role in the pathophysiological process of colorectal cancer. For example, the abnormal expression of some pyroptosis-related genes may affect the proliferation, survival and invasion ability of tumor cells. On the other hand, the inflammatory response triggered by pyroptosis may reshape the tumor microenvironment and have a profound impact on the progression and prognosis of colorectal cancer. A growing number of studies have shown that by regulating pyroptosis-related mechanisms, it is expected to open up new ways for the diagnosis, treatment and prognosis assessment of colorectal cancer. In-depth exploration of the relationship between colorectal cancer and pyroptosis helps us to better understand the nature of colorectal cancer and provides an important theoretical basis for the development of more effective treatment strategies.

Miltirone, derived from Danshen, a traditional Chinese medicine, possesses biological properties including antioxidant, anti-inflammatory, and anti-angiogenic activities, and demonstrates anti-neoplastic effects [14,15]. According to recent research, Miltirone has been discovered to possess anti-tumor properties through various mechanisms. One such mechanism involves the induction of apoptosis in cisplatin-resistant lung cancer cells by augmenting caspase 3/8 activity, increasing the levels of pro-apoptotic proteins such as BAX, apoptosis-inducing factor (AIF), P53, and poly ADP-ribose polymerase (PARP) protein, and inhibiting the expression of matrix metalloproteinase MMP2/9 protein [16]. According to the study, Miltirone induces endoplasmic reticulum stress, aggravates cytoplasmic calcium overload, disrupts the regulation of intracellular calcium homeostasis, and demonstrates significant cytotoxicity against colon cancer cells [17]. According to the study, Miltirone has been found to cause disruption in the mitochondrial membrane potential, stimulate the generation of reactive oxygen species (ROS), induce DNA damage, result in G2/M phase arrest, and exhibit significant cytotoxicity against CCRF-CEM cells [18]. According to research findings, Miltirone has been observed to augment the cytotoxicity of doxorubicin towards liver cancer cells through the inhibition of P-gp-mediated drug efflux. This suggests that Miltirone may have potential as an adjunctive therapeutic agent in liver cancer treatment [19].

The present investigation employed Miltirone as a potential therapeutic agent to elucidate its pyroptosis-triggering impact on colorectal cancer cells (SW620 and HCT116). Under normal conditions, caspase 3 is present in an inactive proenzyme form in the cytoplasm and is cleaved by aspartic acid, functioning as a cysteine protease. This information has been documented in literature [20]. Prior research has posited that caspase 3 is a pivotal protein associated with the process of programmed cell death, also known as apoptosis [21]. Recent research has demonstrated that the cleavage and activation of caspase 3 can lead to the subsequent cleavage and activation of the pyroptosis key protein GSDME. This process results in the release of the GSDME-N terminal fragment, which possesses cell membrane and mitochondrial pore-forming capabilities. Consequently, the release of cell membrane contents, such as LDH and HMGB1, occurs, leading to osmotic imbalance within and outside the cell and ultimately inducing cell pyroptosis [8,22]. It has been established that Miltirone exhibits anti-cancer effects on colorectal cancer (CRC) by promoting pyroptosis through the targeting of caspase 3 via GSDME. The findings of this study could potentially offer a novel avenue for the clinical management of colorectal cancer (CRC).

2 Materials and methods

2.1 Cell lines and cell culture

Two frequently utilized colorectal cancer cell lines are SW620 and HCT116. Their cell culture conditions and procedures are as follows: In DMEM (Dulbecco's Modified Eagle Medium) with 10 % fetal bovine serum (FBS; Thermo Fisher Science, Waltham, MA, USA), penicillin (100 U/mL), and streptomycin (100 mg/mL; HyClone, USA), SW620 and HCT116 cells are both grown. At 37 °C and 5 % CO2, SW620 and HCT116 cells are grown. In order to prevent uncontrolled development and apoptosis, cell density should be kept below 80 %. Every two to three days, the medium should be replaced, and cell shape and growth status should be monitored frequently. Cell passage should be carried out when SW620 and HCT116 cells are 80–90 % dense.

2.2 Lentivirus infection and siRNA transfection

To initiate the experiment, obtain 2nd generation SW620 and HCT116 cells that are in optimal condition and in the logarithmic growth phase. Inoculate these cells in a 6-well plate at a density of 5 × 104 cells/mL. Allow the cells to adhere to the wall completely for a duration of 24 h. Each group is assigned two identical wells. Discard the previous substrate from the Petri dish. It is imperative to adhere to the guidelines for lentivirus transfection reagents. The quantity of lentivirus infection reagent necessary for each well should be determined based on the MOI value of SW620 and HCT116 cells. Subsequently, the corresponding lentivirus infection reagent and infection enhancement reagent should be added in accordance with the grouping. Following a 12-h transfection period, it is recommended to replace the complete medium and proceed with cell culture. Following a 72-h period of transfection, it is recommended to observe the transfection efficiency of each cell group utilizing a fluorescence microscope. Upon achieving a transfection efficiency of over 90 %, it is recommended to culture the cells in a complete medium supplemented with 2 μg/mL puromycin for a duration of 2 days. Following this, it is advised to maintain the culture in a complete medium containing 1 μg/mL puromycin for any subsequent experiments. In this study, all lentiviruses were procured from GeneChem, a company based in Shanghai, China. To perform siRNA knockdown, 96-well or 6-well plates were utilized to seed SW620 and HCT116 cells. Following a 24-h incubation period, the cells were subjected to transfection with caspase3-specific siRNAs, Gsdmd-specific siRNAs, and control siRNAs using identical conditions. The siRNAs utilized in the study were procured from GenePharma. The process of transfection was executed utilizing the Lipofetamine2000 transfection reagent in accordance with the guidelines provided by the manufacturer. Following a 24-h period, SW620 and HCT116 cells that had undergone transfection were subjected to treatment with 40 mM Miltirone (HY–N1951, MemChemExpress, USA) in preparation for subsequent analysis. The powder Z-DEVD-FMK (HY-12466, MemChemExpress, USA) was solubilized in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to generate stock solutions of 1 mM and 10 mM, correspondingly. The ultimate concentration of dimethyl sulfoxide (DMSO) within the solution did not surpass 0.1 % (v/v).

2.3 MTT assay

The logarithmic growth phase of SW620 and HCT116 cells were subjected to trypsin digestion and subsequently inoculated into a 96-well plate at a density of 1 × 103 cells per well. Following a culture period of 24, 48, and 72 h, 20 μl of MTT solution should be introduced to each well, and the samples should be maintained in the incubator for an additional 4 h. The supernatant should be removed and 150 μl of DMSO should be added to each well. The mixture should then be incubated at room temperature for a duration of 10 min. The absorbance value (OD) at 490 nm should be measured subsequently.

2.4 ELISA and LDH activity assays

Administer Miltirone (HY–N1951,MedChemExpress, United States) to the cells for a predetermined duration. Retrieve the culture medium and subject it to centrifugation in order to eliminate any cellular debris or suspended cells. Quantify the amount of IL-1β that has been discharged into the culture medium by employing the Human IL-1β ELISA Set II (RUO-557953, BD Biosciences, United States). Quantify the LDH activity present in the culture medium by employing the LDH activity assay kit (88954, Thermo Scientific, Rockford, IL, USA). Utilize Western blotting to ascertain the protein concentration of LDHA that has been discharged into the culture medium.

2.5 COIP assay

The total protein samples that were extracted underwent purification in accordance with the operational guidelines of the Protein A/G immunomagnetic beads provided by MedChemExpress. The magnetic beads underwent pretreatment for antibody binding as per the provided instructions. Subsequently, 30 μl of beads were supplemented with a single antibody (IgG, GSDME, or caspase 3 antibody) in accordance with the grouping, with a final concentration of 10 μg/ml. Following thorough mixing of the antibody and beads, the mixture was subjected to incubation at ambient temperature for a duration of 1 h. Subsequently, the beads underwent a washing process and were subjected to enrichment. Following this, they were meticulously blended with the total cell protein sample that had been extracted, and left to incubate at a temperature of 4 °C for the duration of the night. The beads underwent four rounds of washing with binding/washing buffer, followed by centrifugation at a speed of 8000 revolutions per minute for a duration of 30 s. The beads were then allowed to settle for approximately 2 min, after which the supernatant was discarded. The sediment underwent two rounds of washing with a TBS solution that had a volume 20 times greater than that of the beads, and subsequently, the supernatant was eliminated. Subsequently, the beads underwent a single wash with a solution of 0.1 mol/L glycine HCL (pH 3.5), with the two components being mixed for a duration not exceeding 20 min. The resulting supernatant was discarded, and 30 μl of 5 × loading buffer was introduced to the sample. Following this, the sample was subjected to denaturation in a metal bath for a period of 10 min, after which it was subjected to Western blotting detection.

2.6 Transwell assay

The cells from each group were collected and subsequently resuspended in serum-free medium. The cell count was determined, and 5 × 104 cells/well were seeded in the upper chamber. Dispense 700 μL of complete medium into the lower chamber and subject to incubation within a cell culture incubator for a duration of 24 h. The methodology involves utilizing a cotton swab to eliminate non-permeating cells from the small chamber, subsequently stabilizing the small chamber with 4 % paraformaldehyde, applying 0.1 % crystal violet stain, capturing images under a microscope, and quantifying cells with the aid of ImageJ software. In the context of the invasion experiment, it is recommended to pre-add Matrigel that has been diluted with serum-free medium to the upper chamber and incubate it for a duration of 3 h. The subsequent steps involved in this experiment are identical to those of the migration experiment.

2.7 Colony formation assay

Retrieve cellular samples from each respective group and proceed to re-suspend them. Culture 1000 cells per well in a 6-well plate and maintain the culture for a period of 14–21 days. Following the completion of the culture, it is recommended to rinse it with phosphate-buffered saline (PBS) and subsequently fix it with methanol for a duration of 15 min. The fixative should then be discarded and a crystal violet staining solution added, allowing it to stand for a period of 10 min. Finally, the culture should be rinsed with distilled water. Utilize an inverted microscope to visually examine a colony consisting of over 50 cells, and proceed to quantify the quantity of cell colonies that have been produced.

2.8 Wound healing assay

The cellular clusters were re-suspended and subsequently placed in a 6-well plate, with each well containing 5 × 105 cells per unit volume. Upon reaching confluence, a scratch was generated via a 100 μl pipette tip, followed by a PBS wash of the remaining cells, and subsequent culturing in fresh medium. The width of the scratch was quantified and captured through photographic means at two time points, namely 0 h and 24 h post-scratch. Subsequently, the rate of cellular migration was determined.

2.9 Microscopy imaging

In order to examine the morphology of pyroptotic cells, SW620 and HCT116 cells were cultured in a 6-well plate at a confluence of approximately 60 %. Following the administration of Miltirone, the cells were subjected to imaging using an Olympus IX53 microscope (Olympus Corporation, Tokyo, Japan) under bright-field conditions. A control solution of DMSO at a concentration of 0.1 % was employed.

2.10 Apoptosis

Cells of SW620 and HCT116 varieties were subjected to treatment with Miltirone, followed by PBS washing and subsequent collection through centrifugation. Subsequently, the cells were subjected to staining with Annexin V and PI as per the guidelines provided by the staining kit (Yisheng, Shanghai, China). The detection of stained cells was performed utilizing a flow cytometer manufactured by Miltenyi Biotec. The study established the excitation and detection wavelengths for Annexin V and PI, and subsequently documented the apoptosis status of individual cells. The rate of apoptosis in the cells was determined by analyzing the data obtained from flow cytometry.

2.11 Western blotting

The cellular specimens were gathered and subjected to lysis through the utilization of RIPA buffer. The micro-BCA protein assay (Rockford, Pierce, USA) was utilized to determine the protein concentration. A quantity of 40 μg of protein per lane was subjected to separation using a 12 % SDS-PAGE technique, and subsequently transferred onto nitrocellulose membranes obtained from Amer sham Pharmacia in Germany. The membranes were blocked using 5 % skim milk and subsequently incubated with primary antibodies against GSDMD (1:1000, 20770-1-AP, Proteintech, USA), N-GSDMD (1:1000, ab215203, abcam, USA), GSDME (1:1000, 13075-1-AP, Proteintech, USA), N-GSDME (1:1000, ab215191, abcam, USA), caspase 3 (1:1000, 66470-2-Ig, Proteintech, USA), cleaved-caspase 3, (1:1000, E83-77, abcam, USA), cleaved-PARP (1:1000, ab32064, abcam, USA), PARP (1:1000, 13371-1-AP, Proteintech, USA), LDHA (1:1000, 21799-1-AP, Proteintech, USA), and β-actin (1:1000, 81115-1-RR, Proteintech, USA) at 4 °C overnight. Following incubation with the primary antibody, the membranes underwent incubation with a secondary antibody (SA00001-2, Proteintech, USA) at a dilution of 1:1000, and were subsequently visualized using an enhanced chemiluminescence (ECL) kit sourced from Amersham Pharmacia, UK.

2.12 Animal study

A cohort of female nude mice (BALB/c, 4 weeks old) were subcutaneously injected with 1 × 106 SW620 cells in the right flank. The rodents were maintained in a controlled environment free from specific pathogens. Upon the tumor volume reaching approximately 100 mm3, the mice were subjected to random allocation into three treatment cohorts (n = 4) and administered intraperitoneal injections twice a week with either Miltirone solution (20 or 100 mg/kg body weight) or vehicle (5 % ethanol in PBS). The dimensions of the tumors were assessed biweekly utilizing calipers. The formula utilized to determine the volume (V) of the tumor involved the calculation of 1/a multiplied by b2, where a represents the long axis and b represents the short axis. Upon completion of the experiment, the mice were subjected to euthanasia, and the xenograft tumors were quantified. Pyroptosis markers in the xenograft tumors were identified through the utilization of Western blotting.The animal experimental procedures were approved by the Animal Ethics Committee of Fujian Medical University (No.IACUCFJMU2023-0308).

2.13 Collection of chemical components and targets

Using the PubChem database (https://pubchem.ncbi.nlm.nih.gov), the SMILES (Simplified Molecular Input Line Entry System) of the compounds were obtained and imported into the SwissTargetPrediction database (http://www.swisstargetprediction.ch) for predicting their effective targets.

2.14 Prediction of disease targets

By entering the keywords “Colorectal cancer, pyroptosis” into the GeneCards (https://www.genecards.org/) and OMIM (https://www.omim.org/) databases for search, the genes related to the disease were obtained. The “Export” button was clicked to download all relevant information in excel format, and the Gene Symbol information corresponding to the disease was obtained. The “Gene Symbol, Description, Category” columns were selected for preliminary screening. OMIM is a comprehensive and authoritative summary of human genes and genetic phenotypes, and the full-text reference summaries in OMIM contain information on all known Mendelian diseases and over 16,000 genes. OMIM focuses on the relationship between phenotype and genotype. The disease-related targets were obtained by searching for keywords in the OMIM database, entering the disease name in the search box on the “Gene Map” page, and downloading the corresponding excel file. The disease-related targets were integrated into excel, and duplicate genes were removed to obtain the disease targets for this study.

2.15 Obtaining intersection targets of active compounds and diseases

Using the Venny software (https://bioinfogp.cnb.csic.es/tools/venny/), the intersection targets of the active compounds of traditional Chinese medicine and the disease were obtained as potential key targets for treating the disease by regulating the cell necrosis pathway.

2.16 “Component-Target-Disease” network analysis

The compound gene “network” file and Type file were prepared, and the Cytoscape3.9.0 software was used to import the relevant files for network topology analysis. The target graph shape, color, transparency, and size were adjusted according to the Degree value (the number of gene connections), and the “Traditional Chinese Medicine Component-Target-Disease” network diagram was constructed. This study used the Cytoscape software (version 3.9.0) to construct the “Component-Target-Disease” network.

2.17 PPI network construction and network topology analysis

The intersection genes were imported into the String platform (https://string-db.org/) to obtain protein interaction relationships, with the object set as “homo sapiens” and the highest confidence level set to 0.900, and the free gene nodes were hidden. The results were imported into the Cytoscape3.9.0 software, and the “network Analyzer” was selected to obtain the network topology parameters. The downloaded TSV file was imported into the Cytoscape software to create a PPI graph, and the top 10 core targets were selected based on the Degree value.

2.18 Molecular docking

The large and small molecules with topological parameters were subjected to molecular docking. The protein crystal structure was obtained from the RCSB PDB database (https://www.rcsb.org/) or the alphafold database (https://alphafold.ebi.ac.uk/). The small molecule library for docking was obtained from the TCMSP database (old.tcmsp-e.com/tcmsp.php) by searching for traditional Chinese medicine and establishing it. AutodockTools1,2 was used to remove water and add hydrogen to the protein crystal structure and prepare the receptor structure. Open Babel and Autodock programs were used to prepare the small molecule library for docking. Autodock program was used for docking, and the results were imported into pymol for visualization of the docking results.

2.19 Statistical analysis

The statistical analysis was performed using the SPSS 21.0 software. The data is displayed in the format of mean value along with its corresponding standard deviation. The study employed t-tests to compare differences between two groups, and analysis of variance was utilized to analyze differences among multiple groups. A statistically significant threshold was set at a P value of less than 0.05.

3 Results

3.1 The effect of miltirone on proliferation, invasion, and migration of colorectal cancer cells

In order to examine the potential anti-cancer properties of Miltirone on colorectal cancer, the authors utilized SW620 and HCT116 cell lines for their analysis. These cell lines were treated with varying doses of Miltirone (10, 20, and 40 μM) for a range of time periods (0–72 h). The results of the study indicated that as the treatment time and dosage of Miltirone increased, there was a corresponding decrease in the viability of colorectal cancer cells (Fig. 1A). Additionally, the number of colony-forming units of colorectal cancer cells treated with Miltirone decreased in a dose-dependent manner (Fig. 1B). To investigate the impact of Miltirone on colorectal cancer cell migration and invasion, the authors employed the transwell assay. The data gathered from the experiment displayed that Miltirone could inhibit the migration and invasion of SW620 and HCT116 cells in a dose-dependent manner (Fig. 1C). In addition, the migration rate of colorectal cancer cells decreased as the dose of Miltirone increased (Fig. 1D). These findings suggest that Miltirone effectively restrains the proliferation, invasion, and migration of colorectal cancer cells.Fig. 1 The effect of Miltirone on proliferation, invasion, and migration of colorectal cancer cells. (A) SW620 and HCT116 cells were administrated with 0∼40 μM Miltirone for 0–72 h. MTT assays were performed for cell viability assessment. (B) SW620 and HCT116 cells were treated with 0∼40 μM Miltirone for 72 h. Following a two-week incubation at 37 °C, colony formation assay was conducted and colony numbers were calculated accordingly. (C) SW620 and HCT116 cells were treated with 0∼40 μM Miltirone for 72 h. Transwell assays were conducted, migrating and invasive cell number was calculated. (D) In SW620 and HCT116 cells, scratches were made in culture plates and wound healing was calculated. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 1

3.2 Miltirone induced pyroptotic cell death in colorectal cancer cells

The authors first investigated the impact of Miltirone on apoptosis levels in SW620 and HCT116 cells. The results of Annexin V/PI staining showed that Miltirone did not induce apoptosis in these cells (Fig. 2A). Nevertheless, the researchers observed cell death in SW620 and HCT116 cells under a microscope. Additionally, the cells treated with Miltirone displayed cytoplasmic swelling and membrane rupture (Fig. 2B).The morphological changes in SW620 and HCT116 cells suggested that cell pyroptosis occurred after Miltirone treatment. ELISA analysis showed that Miltirone-treated SW620 and HCT116 cells could significantly release the IL-1β cytokine (Fig. 2C). LDH activity assay showed that the LDH activity in the culture supernatant of Miltirone-treated SW620 and HCT116 cells increased (Fig. 2D). Western blot analysis also confirmed the abundant expression of LDHA protein in the culture medium of SW620 and HCT116 cells (Fig. 2E). Overall, these results indicate that Miltirone induces pyroptotic cell death in colorectal cancer cells.Fig. 2 Miltirone induced pyroptotic cell death in colorectal cancer cells. (A) Apoptotic cell frequencies treated without or with Miltirone (40 μM) for 24 h were determined by annexin V/PI assays in SW620 and HCT116 cells. (B) Morphological alterations induced by Miltirone (40 μM) treatment. Arrow showed cell swelling and rupture. (C) IL-1β released into culture medium was detected by ELISA assays. (D) LDH activity assays. LDH activity in culture mediums of cells treated without or with Miltirone (40 μM) for 24 h were measured by LDH kit. (E) Release of LDHA proteins to culture mediums were detected by Western blot assays. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2

3.3 GSDME mediates the effect of miltirone on pyroptosis, proliferation, invasion, and migration of colorectal cancer cells

GSDMD, a member of the gasdermin protein family, has been recognized as a caspase 1/4/5/11 substrate. The cleavage of GSDMD can result in the release of its N-terminal domain onto the cell membrane, thereby triggering cell pyroptosis. This has been reported in literature [23]. Despite the expression of GSDMD in SW620 and HCT116 cells, the administration of Miltirone did not result in the liberation of N-terminal GSDMD (GSDMD-NT), as depicted in Fig. 3A. Furthermore, the siRNA-mediated knockdown of GSDMD in SW620 and HCT116 cells (Fig. 3B) did not exhibit any impact on the cell death and LDH release induced by Miltirone (Fig. 3C and D). Thus, it can be inferred that GSDMD does not play a role in the process of cell death induced by Miltirone in SW620 and HCT116 cells. The effector GSDME, which induces cell pyroptosis, has been identified in recent research and its function has been investigated in multiple studies [24]. The results of the Western blot analysis indicate that the expression of cleaved-caspase 3 and cleaved-PARP(poly ADP-ribose polymerase, PARP) in colorectal cancer SW620 and HCT116 cells was induced in a dose-dependent manner by Miltirone (Fig. 3E). The administration of Miltirone exhibited a dose-dependent effect on the discharge of N-GSDME in SW620 and HCT116 cells, thereby signifying the occurrence of pyroptosis. The findings were in line with the cleaved-caspase 3 pattern, as depicted in Fig. 3E.Fig. 3 GSDMD is not involved in Miltirone-induced cell death in SW620 and HCT116 cells. (A) SW620 and HCT116 cells were treated with Miltirone (40 μM) for 24 h, total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against GSDMD and β-actin. (B–D) SW620 and HCT116 cells were transfected with siRNA targeting GSDMD or control siRNA for 24 h, total cellular extracts were prepared and subjected to (B) Western blotting analyses using antibodies against GSDMD and β-actin, (C) cell viability was analyzed by MTT assay, and (D) LDH-release was analyzed using LDH assay kit. (E) SW620 and HCT116 cells were treated with Miltirone (40 μM) for 24 h, total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against caspase 3, PARP, GSDME, and β-actin. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

The researchers employed lentiviral short hairpin RNAs (shRNAs) to induce the suppression of gasdermin E (GSDME) expression. The results of the Western blot analysis indicate that the GSDME protein levels were significantly reduced in SW620 and HCT116 cells upon treatment with two shRNAs targeting GSDME (Fig. 4A). The results of colony formation analysis indicate that the absence of GSDME facilitated the viability of cancer cells that were subjected to Miltirone treatment (Fig. 4B). The results obtained from Transwell assays indicate that the absence of GSDME facilitated the invasion of cancer cells that were subjected to treatment with Miltirone (Fig. 4C). The deficiency of GSDME was observed to enhance the migration rate of cancer cells when subjected to treatment with Miltirone (Fig. 4D). The prevention of pyroptotic cell death induced by Miltirone in SW620 and HCT116 cells was observed upon inhibition of GSDME. The results indicated that GSDME-silenced cells exhibited reduced frequency of cytoplasmic swelling and membrane rupture upon treatment with Miltirone (Fig. 4E). It was observed that the inhibition of GSDME in SW620 and HCT116 cells resulted in a decrease in the release of IL-1β and LDH into the culture medium upon treatment with Miltirone (Fig. 4F–G). The findings suggest that GSDME plays a crucial role in the induction of pyroptosis in colorectal cancer cells triggered by Miltirone.Fig. 4 GSDME mediates the effect of Miltirone on pyroptosis, proliferation, invasion, and migration of colorectal cancer cells. (A) SW620 and HCT116 cells were treated with Miltirone (40 μM) for 24 h, total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against GSDME and β-actin. (B) It was administrated with Miltirone (40 μM) for 24 h in GSDME-silenced or negtive cells. Cell survival was measured by colony formation assay. (C) Transwell assays were conducted, migrating and invasive cell number was calculated. (D) Scratches were made in culture plates and wound healing was calculated. (E) Morphological features of cells treated with Miltirone (40 μM) showed reduced pyroptotic cell death in GSDME-silenced cells. (F) LDH activity assays. (G) IL-1β released into culture medium was detected by ELISA assays.

Fig. 4

3.4 Caspase 3 mediates the effect of miltirone on pyroptosis, proliferation, invasion, and migration of colorectal cancer cells

The study employed network pharmacology to identify the principal constituents linked to cell pyroptosis (Fig. 5A). Additionally, molecular docking was conducted to validate the binding efficacy of caspase 3 to the primary active constituent of Miltirone (Fig. 5B). Studies conducted recently have demonstrated that GSDME has the ability to generate pores in the plasma membrane subsequent to its cleavage by caspase 3 [25,26]. Thus, an investigation was conducted to analyze the correlation existing between caspase 3 and GSDME. The results depicted in Fig. 5C demonstrate the presence of GSDME/caspase 3 binding in cell lysates that were subjected to caspase 3 or GSDME immunoprecipitation. Given that cleaved caspase-3 is the primary enzyme responsible for processing GSDME into the N-GSDME fragment [27], our study aimed to explore the potential of inhibiting caspase 3 activation as a means of mitigating Miltirone-induced cell pyroptosis. Following treatment with the caspase 3 specific inhibitor Z-DEVD-FMK [28] or lentiviral shRNAs, the expression levels of cleaved-caspase 3, cleaved-GSDME, and cleaved-PARP, which were induced by Miltirone, were observed to be downregulated (Fig. 5D–E).Fig. 5 GSDME/caspase 3 binds in colorectal cancer cells. (A) Network pharmacology approach to detect genes related to pyroptosis from colorectal cancer and Miltirone. (B) Molecular docking of caspase 3 with major components of Miltirone. (C) Co-IP assays were performed with caspase 3 or GSDME antibody in SW620 cells following caspase 3 or GSDME detection by WB. (D) SW620 and HCT116 cells were treated with Miltirone (40 μM) in the absence or presence of Z-DEVD-FMK (20 mmol/L) for 24 h, total cellular extracts were prepared and subjected to Western blotting analyses using antibodies against caspase 3, PARP, GSDME, and β-actin. (E) SW620 and HCT116 cells were treatedwith Miltirone (40 mmol/L) in the absence or presence of caspase 3-shRNA for 24 h, total cellular extracts were prepared and subjected toWestern blotting analyses using antibodies against caspase 3, PARP, GSDME, and β-actin.

Fig. 5

The results of the colony formation analysis indicate that the absence of caspase 3 facilitated the viability of cancer cells that were subjected to Miltirone treatment (Fig. 6A). The results obtained from Transwell assays indicated that the absence of caspase 3 facilitated the invasion of cancer cells that were subjected to treatment with Miltirone (Fig. 6B). The results indicate that the migration rate of cancer cells treated with Miltirone was enhanced in the absence of caspase 3 (Fig. 6C). The prevention of pyroptotic cell death induced by Miltirone in SW620 and HCT116 cells was observed upon inhibition of caspase 3. The findings indicate that caspase 3 silenced cells exhibited reduced instances of cytoplasmic swelling and membrane rupture when subjected to Miltirone treatment (Fig. 6D). It was observed that the inhibition of caspase 3 in SW620 and HCT116 cells resulted in a decrease in the release of IL-1β and LDH into the culture medium upon treatment with Miltirone (Fig. 6E–F). In general, the impact of Miltirone on pyroptosis, proliferation, invasion, and migration of colorectal cancer cells is facilitated by caspase 3.Fig. 6 Caspase 3 mediates the effect of Miltirone on pyroptosis, proliferation, invasion, and migration of colorectal cancer cells. (A–F) SW620 and HCT116 cells were treated with Miltirone (40 μM) in the absence or presence of caspase 3-shRNA for 24 h. (A) Cell survival was measured by colony formation assay. (B) Transwell assays were conducted, migrating and invasive cell number was calculated. Scratches were made in culture plates and wound healing (C) was calculated. (D) Morphological features of cells treated with Miltirone (40 μM) showed reduced pyroptotic cell death in GSDME-silenced cells. (E) LDH activity assays. (F) IL-1β released into culture medium was detected by ELISA assays. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6

3.5 Miltirone inhibits tumor growth and induces pyroptotic cell death in vivo

Since the above results showed that Miltirone induces pyroptosis in vitro, we further investigated whether Miltirone reduces tumor volume and weight in vivo. SW620 colorectal cancer cells were implanted subcutaneously into the right flank of BALB/C nude mice. When the xenograft tumors grew to approximately 100 mm3, mice were treated with Miltirone solution (20 or 100 mg/kg body weight) twice a week via intraperitoneal injection. The results showed that the xenograft tumors treated with Miltirone had significantly smaller tumor volume and weight than the control group (Fig. 7A–C). In addition, Miltirone treatment significantly induced the formation of cleaved-caspase 3, cleaved-PARP, and GSDME-NT (Fig. 7D). Therefore, these results suggest that Miltirone may inhibit the growth of CRC tumors in vivo by inducing pyroptotic cell death.Fig. 7 Miltirone inhibits tumor growth and induces pyroptotic cell death in vivo. (A) Tumor images formed in nude mice after different treatments. (B) Tumor weight measured in xenograft mouse models in the groups shown. (C) Tumor volume measured in xenograft mouse models in the groups shown. (D) Western blot analyzing protein expression of cleaved Caspase-3, cleaved PARP, and GSDME in tumors derived from treatment with Miltirone or vehicle. n = 4. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7

4 Discussion

Colorectal cancer is a prevalent malignancy that affects the digestive system [29]. The malignancy's elevated frequency of occurrence and propensity for metastasis render it a formidable adversary that poses a significant threat to human well-being [30]. Thus, it is imperative to discover pharmacological agents that are more efficient and less harmful. Several clinical studies have demonstrated the efficacy of traditional Chinese medicine and its active constituents in the prevention and treatment of tumors. These studies have reported favorable outcomes in ameliorating bone marrow suppression, reversing drug resistance, mitigating chemotherapy-related adverse effects, augmenting immune function, and prolonging patient survival [31,32]. The maturation of biotechnology has led to the advancement of isolating and extracting essential elements from traditional Chinese medicine. Consequently, the modernization of traditional Chinese medicine has become an inevitable trend [33]. Danshen, a traditional Chinese medicine, has been the subject of extensive research. Numerous chemical components, primarily lipophilic diterpenoid compounds, have been identified and found to possess various pharmacological activities, including anti-inflammatory, anti-thrombotic, antioxidant, and anti-tumor effects. These effects are attributed to intricate mechanisms of action [34]. Although Miltirone has been found to exhibit anti-tumor effects through various pathways, its usage in the management of colorectal cancer is not widely practiced [16,19]. Hence, it is of significant importance to actively investigate the pathogenesis of Miltirone in the management of colorectal cancer and provide appropriate intervention therapy.

Pyroptosis is a distinct form of cellular demise that is characterized by unique mechanisms of formation and cellular morphology, setting it apart from both apoptosis and necrosis [35]. Prior research has indicated a strong correlation between the Gasdermin protein family and pyroptosis. Among the various pyroptosis mechanisms studied in the realm of tumors, GSDME-dependent cell pyroptosis has received the most attention [5]. The gene GSDME, also referred to as DFNA5, is associated with deafness. It consists of two domains, namely GSDME-C and GSDME-N, which have the ability to inhibit each other upon binding. This information has been documented in literature [9]. Research findings indicate that the expression of GSDME in Bel-7402 liver cancer cells is notably low. However, administering Triptolide can stimulate the activation of GSDME, leading to the formation of GSDME-N, which triggers pyroptosis in liver cancer cells [36]. Furthermore, it has been observed that GSDME exhibits low expression levels in Lovo cells affected by colorectal cancer. However, administration of Oxaliplatin has been found to enhance the expression of GSDME, thereby triggering pyroptosis in colorectal cancer cells [37]. The aforementioned research suggests that the occurrence of GSDME-mediated cellular pyroptosis is a significant factor in the progression of tumors.

This study has revealed that GSDME plays a mediating role in the impact of Miltirone on various cellular processes such as pyroptosis, proliferation, invasion, and migration in colorectal cancer cells. By conducting a comprehensive analysis of potential targets associated with cell pyroptosis and colorectal cancer, utilizing GeneCards, OMIM database, and Cytoscape software, we established a protein-protein interaction network (PPI) database in the String database. Our findings indicate a significant association between Miltirone and caspase 3. Caspase 3 is a type of cysteine protease which undergoes cleavage through aspartic acid specificity. Typically, it is present as an inert zymogen within the cellular cytoplasm [20]. Prior research has indicated that caspase 3 is a pivotal protein associated with the process of apoptosis. Upon activation of caspase 3, cells initiate the apoptosis pathway [20]. Recent research has indicated that the process of caspase 3 cleavage and activation can lead to the subsequent cleavage and activation of the crucial pyroptosis protein GSDME. The resulting GSDME-N terminal fragment is capable of creating pores in both the cell membrane and mitochondria. This finding has been documented in various studies [24]. The formation of pores in the cellular membrane has the potential to result in the discharge of cellular components, including LDH and HMGB1. Furthermore, the disruption of the balance between intracellular and extracellular osmotic pressure can trigger cell pyroptosis [8]. This has been documented in literature. In conjunction with the antecedent experimental results expounded in this article, the induction of cleaved-caspase 3 expression in a dose-dependent manner by Miltirone has led to the identification of caspase 3 as the pivotal node for subsequent experiments. Simultaneously, the experiment validated that the impact of Miltirone on pyroptosis, proliferation, invasion, and migration of colorectal cancer cells is mediated by caspase 3.

5 Conclusion

This article utilized colorectal cancer cells as the subject of investigation. Through both in vitro and in vivo experimentation, it was determined that Miltirone exhibits concentration-dependent inhibition of colorectal cancer cell activity. Additionally, Miltirone induces the manifestation of pyroptosis-specific morphology and LDH release, while also targeting the cleavage and activation of GSDME protein levels mediated by caspase 3. The present study has confirmed that the activation of the caspase 3/GSDME pathway is a mechanism underlying the anti-colorectal cancer effects of Miltirone (Fig. 8). This finding suggests that Miltirone may represent a novel therapeutic option for the treatment of colorectal cancer.Fig. 8 A schematic summary of this study, showing that the activation of the caspase 3/GSDME pathway is a mechanism underlying the anti-colorectal cancer effects of miltirone. CRC, Colorectal cancer; GSDME, Gasdermin E; GSDMD, Gasdermin D; PARP, Poly ADP-ribose polymerase; LDH, Lactate dehydrogenase.

Fig. 8

Funding

The work was supported by Grants from Fujian Provincial Health Technology Project, China [No.2020GGB030 ].

Ethics statement

This project was approved by the Animal Ethics Committee of Fujian Medical University (No.IACUCFJMU2023-0308).

Data availability statement

Data will be made available on request.

CRediT authorship contribution statement

Guangwei Zheng: Writing – original draft, Supervision, Software, Project administration, Formal analysis, Data curation, Conceptualization. Zhipeng Fang: Visualization, Software, Resources, Methodology, Data curation. Zhenlv Lin: Supervision, Project administration, Conceptualization. Guoxian Guan: Writing – review & editing, Supervision, Project administration.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Guoxian Guan reports financial support was provided by Grants from 10.13039/501100017686 Fujian Provincial Health Technology Project . Guoxian Guan has patent issued to no. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
==== Refs
References

1 Siegel R.L. Miller K.D. Fuchs H.E. Jemal A. Cancer statistics CA A Cancer J. Clin. 72 2022 7 33 2022 https://10.3322/caac.21708
2 Siegel R.L. Miller K.D. Goding Sauer A. Fedewa S.A. Butterly L.F. Anderson J.C. Cercek A. Smith R.A. Jemal A. Colorectal cancer statistics CA A Cancer J. Clin. 70 2020 145 164 2020 https://10.3322/caac.21601
3 Strickler J.H. Yoshino T. Graham R.P. Siena S. Bekaii-Saab T. Diagnosis and treatment of ERBB2-positive metastatic colorectal cancer: a review JAMA Oncol. 8 2022 760 769 https://10.1001/jamaoncol.2021.8196 35238866
4 Biller L.H. Schrag D. Diagnosis and treatment of metastatic colorectal cancer: a review JAMA 325 2021 669 685 https://10.1001/jama.2021.0106 33591350
5 Wei X. Xie F. Zhou X. Wu Y. Yan H. Liu T. Huang J. Wang F. Zhou F. Zhang L. Role of pyroptosis in inflammation and cancer Cell. Mol. Immunol. 19 2022 971 992 https://10.1038/s41423-022-00905-x 35970871
6 Tsuchiya K. Switching from apoptosis to pyroptosis: gasdermin-elicited inflammation and antitumor immunity Int. J. Mol. Sci. 22 2021 https://10.3390/ijms22010426
7 LaRock D.L. Johnson A.F. Wilde S. Sands J.S. Monteiro M.P. LaRock C.N. Group A. Streptococcus induces GSDMA-dependent pyroptosis in keratinocytes Nature 605 2022 527 531 https://10.1038/s41586-022-04717-x 35545676
8 Fan C.Y. Ye F.H. Peng M. Dong J.J. Chai W.W. Deng W.J. Zhang H. Yang L.C. Endogenous HMGB1 regulates GSDME-mediated pyroptosis via ROS/ERK1/2/caspase-3/GSDME signaling in neuroblastoma Am. J. Cancer Res. 13 2023 436 451 PMID:36895972 36895972
9 Xia Y. Jin Y. Cui D. Wu X. Song C. Jin W. Huang H. Antitumor effect of simvastatin in combination with DNA methyltransferase inhibitor on gastric cancer via GSDME-mediated pyroptosis Front. Pharmacol. 13 2022 860546 https://10.3389/fphar.2022.860546
10 Yu J. Li S. Qi J. Chen Z. Wu Y. Guo J. Wang K. Sun X. Zheng J. Cleavage of GSDME by caspase-3 determines lobaplatin-induced pyroptosis in colon cancer cells Cell Death Dis. 10 2019 193 https://10.1038/s41419-019-1441-4 30804337
11 Liu Z. Li Y. Zhu Y. Li N. Li W. Shang C. Song G. Li S. Cong J. Li T. Xiu Z. Lu J. Ge C. Yang X. Li Y. Sun L. Li X. Jin N. Apoptin induces pyroptosis of colorectal cancer cells via the GSDME-dependent pathway Int. J. Biol. Sci. 18 2022 717 730 https://10.7150/ijbs.64350 35002520
12 Tan G. Lin C. Huang C. Chen B. Chen J. Shi Y. Zhi F. Radiosensitivity of colorectal cancer and radiation-induced gut damages are regulated by gasdermin E Cancer Lett. 529 2022 https://10.1016/j.canlet.2021.12.034
13 Zhou Y. Zhang W. Wang B. Wang P. Li D. Cao T. Zhang D. Han H. Bai M. Wang X. Zhao X. Lu Y. Mitochondria-targeted photodynamic therapy triggers GSDME-mediated pyroptosis and sensitizes anti-PD-1 therapy in colorectal cancer J Immunother Cancer 12 2024 https://10.1136/jitc-2023-008054
14 Feng H. Xi F. Miltirone attenuates reactive oxygen species-dependent neuronal apoptosis in MPP(+)-Induced cell model of Parkinson's disease through regulating the PI3K/Akt pathway Neurochem. Res. 47 2022 3137 3149 https://10.1007/s11064-022-03669-y 35810264
15 Shakeri A. Hafezian T. Kusz N. Hohmann J. Boozari M. Mottaghipisheh J. Emami S.A. Tayarani-Najaran Z. Asili J. Cytotoxicity, apoptosis inducing activity and Western blot analysis of tanshinone derivatives from Stachys parviflora on prostate and breast cancer cells Mol. Biol. Rep. 49 2022 8251 8258 https://10.1007/s11033-022-07541-8 36002657
16 Zhu Z. Miltirone-induced apoptosis in cisplatin-resistant lung cancer cells through upregulation of p53 signaling pathways Oncol. Lett. 15 2018 8841 8846 https://10.3892/ol.2018.8440 29928326
17 Salati S. Genovese E. Carretta C. Zini R. Bartalucci N. Prudente Z. Pennucci V. Ruberti S. Rossi C. Rontauroli S. Enzo E. Calabresi L. Balliu M. Mannarelli C. Bianchi E. Guglielmelli P. Tagliafico E. Vannucchi A.M. Manfredini R. Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in K562 cells expressing CALR mutants Sci. Rep. 9 2019 10558 https://10.1038/s41598-019-46843-z
18 Wu C.F. Efferth T. Miltirone induces G2/M cell cycle arrest and apoptosis in CCRF-CEM acute lymphoblastic leukemia cells J. Nat. Prod. 78 2015 1339 1347 https://10.1021/acs.jnatprod.5b00158 26035463
19 Zhou X. Wang Y. Lee W.Y. Or P.M. Wan D.C. Kwan Y.W. Yeung J.H. Miltirone is a dual inhibitor of P-glycoprotein and cell growth in doxorubicin-resistant HepG2 cells J. Nat. Prod. 78 2015 2266 2275 https://10.1021/acs.jnatprod.5b00516 26339922
20 Asadi M. Taghizadeh S. Kaviani E. Vakili O. Taheri-Anganeh M. Tahamtan M. Savardashtaki A. Caspase-3: structure, function, and biotechnological aspects Biotechnol. Appl. Biochem. 69 2022 1633 1645 https://10.1002/bab.2233 34342377
21 Wang J. Huangfu M. Li X. Han M. Liu G. Yu D. Zhou L. Dou T. Liu Y. Guan X. Wei R. Chen X. Osthole induces apoptosis and caspase-3/GSDME-dependent pyroptosis via NQO1-mediated ROS generation in HeLa cells Oxid. Med. Cell. Longev. 2022 2022 8585598 https://10.1155/2022/8585598
22 Shen X. Wang H. Weng C. Jiang H. Chen J. Caspase 3/GSDME-dependent pyroptosis contributes to chemotherapy drug-induced nephrotoxicity Cell Death Dis. 12 2021 186 https://10.1038/s41419-021-03458-5 33589596
23 Li S. Sun Y. Song M. Song Y. Fang Y. Zhang Q. Li X. Song N. Ding J. Lu M. Hu G. NLRP3/caspase-1/GSDMD-mediated pyroptosis exerts a crucial role in astrocyte pathological injury in mouse model of depression JCI Insight 6 2021 https://10.1172/jci.insight.146852
24 Jiang M. Qi L. Li L. Li Y. The caspase-3/GSDME signal pathway as a switch between apoptosis and pyroptosis in cancer Cell Death Dis. 6 2020 112 https://10.1038/s41420-020-00349-0
25 Yao F. Jin Z. Zheng Z. Lv X. Ren L. Yang J. Chen D. Wang B. Yang W. Chen L. Wang W. Gu J. Lin R. HDAC11 promotes both NLRP3/caspase-1/GSDMD and caspase-3/GSDME pathways causing pyroptosis via ERG in vascular endothelial cells Cell Death Dis. 8 2022 112 https://10.1038/s41420-022-00906-9
26 Zheng X. Zhong T. Ma Y. Wan X. Qin A. Yao B. Zou H. Song Y. Yin D. Bnip3 mediates doxorubicin-induced cardiomyocyte pyroptosis via caspase-3/GSDME Life Sci. 242 2020 117186 https://10.1016/j.lfs.2019.117186
27 Zhang Z. Zhang Y. Xia S. Kong Q. Li S. Liu X. Junqueira C. Meza-Sosa K.F. Mok T.M.Y. Ansara J. Sengupta S. Yao Y. Wu H. Lieberman J. Gasdermin E. Suppresses tumour growth by activating anti-tumour immunity Nature 579 2020 415 420 https://10.1038/s41586-020-2071-9 32188940
28 Xu W.F. Zhang Q. Ding C.J. Sun H.Y. Che Y. Huang H. Wang Y. Wu J.W. Hao H.P. Cao L.J. Gasdermin E-derived caspase-3 inhibitors effectively protect mice from acute hepatic failure Acta Pharmacol. Sin. 42 2021 68 76 https://10.1038/s41401-020-0434-2 32457417
29 Ciardiello F. Ciardiello D. Martini G. Napolitano S. Tabernero J. Cervantes A. Clinical management of metastatic colorectal cancer in the era of precision medicine CA A Cancer J. Clin. 72 2022 372 401 https://10.3322/caac.21728
30 Sinicrope F.A. Increasing incidence of early-onset colorectal cancer N. Engl. J. Med. 386 2022 1547 1558 https://10.1056/NEJMra2200869 35443109
31 Wu K.C. Chu P.C. Cheng Y.J. Li C.I. Tian J. Wu H.Y. Wu S.H. Lai Y.C. Kao H.H. Hsu A.L. Lin H.W. Lin C.H. Development of a traditional Chinese medicine-based agent for the treatment of cancer cachexia J Cachexia Sarcopenia Muscle 13 2022 2073 2087 https://10.1002/jcsm.13028 35718751
32 Hu L. Ma L. Xia X. Ying T. Zhou M. Zou S. Yu H. Yin J. Efficacy of bariatric surgery in the treatment of women with obesity and polycystic ovary syndrome J. Clin. Endocrinol. Metab. 107 2022 e3217 e3229 https://10.1210/clinem/dgac294 35554540
33 Zeng M. Guo D. Fernandez-Varo G. Zhang X. Fu S. Ju S. Yang H. Liu X. Wang Y.C. Zeng Y. Casals G. Casals E. The integration of nanomedicine with traditional Chinese medicine: drug delivery of natural products and other opportunities Mol. Pharm. 20 2023 886 904 https://10.1021/acs.molpharmaceut.2c00882 36563052
34 Li Y. Yao Y. Cao X. Yi N. Chen A. Li J. Wu M. Clinical efficacy of Danshen preparation in the treatment of vascular cognitive impairment: a systematic review and meta-analysis Front. Aging Neurosci. 14 2022 1090665 https://10.3389/fnagi.2022.1090665
35 Zeng X. Liu D. Huo X. Wu Y. Liu C. Sun Q. Pyroptosis in NLRP3 inflammasome-related atherosclerosis Cell Stress 6 2022 79 88 https://10.15698/cst2022.10.272 36304814
36 Zhang H. Liao X. Wu X. Shi C. Zhang Y. Yuan Y. Li W. Wang J. Liu Y. Iridium(III) complexes entrapped in liposomes trigger mitochondria-mediated apoptosis and GSDME-mediated pyroptosis J. Inorg. Biochem. 228 2022 111706 https://10.1016/j.jinorgbio.2021.111706
37 Guo J. Zheng J. Mu M. Chen Z. Xu Z. Zhao C. Yang K. Qin X. Sun X. Yu J. GW4064 enhances the chemosensitivity of colorectal cancer to oxaliplatin by inducing pyroptosis Biochem. Biophys. Res. Commun. 548 2021 60 66 https://10.1016/j.bbrc.2021.02.043 33631675
