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Introduction

Breast cancer (BC) is the most prevalent malignant tumor in women worldwide. According to the World Health Organization, BC accounts for the majority of cancer related mortalities among women globally, which seriously impacts the physical and psychological health of patients (1–3). The 5-year survival rate of BC has reached 90% because of advances in drug therapy and the establishment of a precise diagnosis, with the treatment level of BC improving annually (4,5). Chemotherapy remains an essential component of the comprehensive treatment of BC because it can substantially increase the 5-year survival rate and improve prognosis (6). However, issues with treatment such as drug resistance, drug-related toxicity and poor tolerance to traditional chemotherapeutic drugs must be resolved. Therefore, the development of new drugs has become a priority.

Pyroptosis is a novel form of programmed cell death (PCD) mediated by members of the gasdermin (GSDM) family that occurs after autophagy and apoptosis (7). It is morphologically characterized by cell swelling, dissolution and vesicular protrusions, followed by cell membrane rupture, which leads to the release of proinflammatory cytokines and cell contents into the extracellular space. These include high mobility group B protein (HMGB)14, IL-18, IL-1, adenosine 5′-triphosphate (ATP) and HMGB1, all of which trigger an inflammatory response (7–9). This is closely associated with immune system activation (10). The GSDM family of proteins, which are effectors of pyroptosis, can be cleaved by the caspase family and granzyme (GZM) to release the active, membrane pore-forming N-terminal domain and split the junction domain between the N- and C-terminus (9). The N-terminus of GSDM family proteins forms large pores in the cell membrane, breaking osmotic pressure causing cell expansion and producing large vacuoles (11–13).

GSDME, a protein belonging to the GSDM family, is distinct from GSDMD and specifically cleaved by caspase-3 (14,15). Previously, GSDME has been found to serve a role in the occurrence and development of malignant tumors, including lung cancer, colorectal cancer and BC (16–18). In addition, paclitaxel accelerates pyroptosis in small cell lung cancer cells, and increasing the expression of GSDME can effectively enhance their sensitivity to paclitaxel (19). There appears to be an association between low survival rate and low expression of GSDME in patients with BC, indicating that GSDME might serve as a tumor suppressor in BC (20).

GSDME is highly expressed in normal cell lines; however, most tumor cell lines have methylated promoters that lower GSDME expression compared with that in normal cells, making it more difficult to activate pyroptosis in tumor cells (14,15). As a result, when treating malignant tumors, appropriate chemotherapy drugs could be chosen based on the expression level of GSDME. Drugs which can increase GSDME expression may increase the sensitivity of cells to chemotherapy drugs and achieve a more potent anticancer effect (21). The DNA methyltransferase inhibitor, decitabine (DAC; Fig. 1A), can induce functional re-expression of the abnormally silenced GSDME gene in cancer (22,23); therefore, DAC can be used to increase the expression of GSDME in BC cells with low GSDME expression, thereby enhancing the killing of tumor cells through pyroptosis. Therefore, the combination of DAC and GSDME-inducing pyroptosis agents for cancer treatment may be a promising strategy for BC treatment.

Figure 1. SNS-032 inhibits cell growth in breast cancer cells. (A) Chemical structures of SNS-032 and decitabine. (B) MCF-7 and 4T1 cells were treated with SNS-032 (0–32 µmol/l) for 24 h, and cell viability was analyzed by the CCK-8 assay. (C) MCF-7 and 4T1 cells were treated with SNS-032 (10 and 15 µmol/l, respectively) for different lengths of time, and cell viability was analyzed using the CCK-8 assay. The cells were exposed to 0.1% dimethyl sulfoxide as a control. Data are presented as mean ± standard deviation (n=3). ***P<0.001 and ****P<0.0001 vs. control group. ns, not significant; CCK-8, Cell Counting Kit-8.

SNS-032 (formerly BMS-387032; Fig. 1A) was originally designed to obtain a selective cyclin-dependent kinase 2 (CDK2) inhibitor by the Bristol-Myers Squibb Pharmaceutical Research Institute (Stamford, USA) (24). Subsequent studies have found that the anticancer activity of this compound is mainly dependent on the inhibition of CDK7 and CDK9 (24–26), which effectively kill chronic lymphocytic leukemia (CLL) cells in vitro by blocking RNA polymerase II phosphorylation and inhibiting RNA synthesis (24). Several studies have demonstrated that SNS-032 has a strong anticancer effect on hematological malignancies (24–27) and solid tumors, such as uveal melanoma (25), cervical cancer (28) and non-small cell lung cancer (29). In addition, SNS-032 has been reported in phase I clinical studies in patients with metastatic refractory solid tumors (30), advanced CLL and multiple myeloma (31). However, to date, there have been no further reports of SNS-032 in clinical trials. SNS-032 not only inhibits tumor proliferation through the cell cycle, but also induces apoptosis, leading to tumor cell death in melanoma (25), BC (32) and esophageal squamous cell carcinoma (33). However, no studies have reported the activation of the pyroptosis pathway to inhibit tumors.

SNS-032 combined with the demethylating drug, DAC, is a promising treatment strategy for patients with BC. The present study aimed to establish the mechanism of SNS-032 to inhibit tumors and to determine whether the combination with DAC can improve inhibition of tumors, providing a new approach for BC treatment.

Materials and methods

Cell culture

MCF-7 cells, a human BC cell line from Procell Life Science & Technology Co., Inc., were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.). The mouse BC cell line 4T1, derived from BALB/c mice, were from cell bank of the Typical Culture Preservation Committee of the Chinese Academy of Sciences (Beijing, China) and cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.). All media were supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (cat. no G4003; Wuhan Servicebio Technology Co., Ltd.). All cell lines were cultured in a humidified incubator at 37°C and 5% CO2. The cells were seeded 24 h later. Trypsin/EDTA at 0.25% (w/v) and phosphate-buffered saline (PBS) were purchased from Thermo Fisher Scientific, Inc.

Materials

SNS-032 (cat. no S1145) and DAC (cat. no S1200) were purchased from Selleck Chemicals and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mM. The pan-caspase-specific inhibitor Z-VAD-FMK (cat. no HY-16658B), necroptosis inhibitor Necrostatin-1 (cat. no HY-15760) and specific cell caspase-3 inhibitor Z-DEVD-FMK (cat. no HY-12466) were purchased from MedChemExpress and dissolved in DMSO at concentrations of 10, 5 and 10 mM, respectively. The lactate dehydrogenase (LDH) Release Assay kit (cat. no C0017), bicinchoninic acid assay (cat. no P0009) and ATP Assay kit (cat. no S0027) were purchased from Beyotime Institute of Biotechnology. The Annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Test kit (cat no. 556547) was purchased from BD Biosciences.

Cell proliferation assay

The Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Inc.) was used to determine the viability of the BC cell lines. MCF-7 and 4T1 cells were pretreated with or without DAC (5 µmol/l) for 24 h at 37°C seeded in 96-well plates at 3,000 cells/well and treated with different doses (0, 1, 2, 4, 8, 16 and 32 µmol/l) of SNS-032. According to the manufacturer's instructions, 100 µl working solution was added to each well after 24 h SNS-032 treatment and incubated for 2 h. The absorbance at 450 nm was measured using a Synergy 2 microplate reader (BioTek; Agilent Technologies, Inc.) and cell viability was determined using the following formula: Cell viability (%)=(experimental group OD450-blank control OD450)/(control group OD450-blank control OD450) ×100.

Colony formation assay

MCF-7 and 4T1 cells were seeded in six-well plates (1,000 cells/well), incubated overnight and treated with DAC (5 µmol/l) and SNS-032 (10 µmol/l in MCF-7 and 15 µmol/l in 4T1 cells) at 37°C. The culture was allowed to continue for 7 days, with medium changes every 3 days. The cells were fixed with 4% paraformaldehyde for 10 min (cat. no S1101; Wuhan Servicebio Technology Co., Ltd.), stained with 0.1% crystal violet for 15 min and thoroughly washed at room temperature. Finally, the number of colonies (>50 cells/colony) in each well was counted using ImageJ (V1.8.0.112; National Institutes of Health) and the colony formation efficiency was calculated as a percentage relative to the control (100%).

LDH assay

LDH release was detected using an LDH release Assay Kit. The supernatants were collected by centrifugation of cells at 400 × g for 5 min at 4°C after treatment with SNS-032 (10 µmol/l in MCF-7 and 15 µmol/l in 4T1 cells) for 24 h with or without DAC (5 µmol/l) pretreatment for 24 h at 37°C. According to the manufacturer's protocol, after the working detection reagent was prepared, 60 µl LDH detection reagent and 100 µl sample supernatant were added into 96 wells quickly. After 30 min incubation, a Synergy 2 microplate reader was used to measure the absorbance at 490 nm in the dark and the data were processed according to the following formula: LDH release (%)=(experimental group OD490-blank control OD490)/(maximum release group OD490-blank control OD490) ×100.

ATP assay

An ATP Assay Kit was used for quantitative determination of ATP. The medium was discarded and the lysis buffer was added to the BC cells after treatment with SNS-032 (10 µmol/l in MCF-7 and 15 µmol/l in 4T1 cells) for 24 h with or without DAC (5 µmol/l) pretreatment for 24 h at 37°C. The cell lysate was collected and centrifuged at 12,000 × g for 5 min at 4°C to obtain the supernatants. According to the manufacturer's protocol, 100 µl ATP detection reagent was added to a black 96-well plate and incubated at room temperature for 5 min. Subsequently, 20 µl each sample supernatant was added immediately into the black 96-well plate at room temperature. Finally, a Synergy 2 microplate reader was used to detect the luminescence of samples immediately and ATP levels were calculated as follows: ATP cell viability (%)=(experimental group luminescence/control group luminescence) ×100.

Annexin V/PI double staining

BC cell lines, MCF-7 and 4T1, were seeded into 12-well plates overnight at a density of 1×105 cells/well and treated with different concentrations of SNS-032 for 24 h in the presence or absence of DAC, Z-VAD-FMK (20 µmol/l), Necrostatin-1 (10 µmol/l) or Z-DEVD-FMK (20 µmol/l) at 37°C. Cells were collected, centrifuged at 300 × g for 5 min at 4°C and washed twice with cold PBS. The following staining procedure was performed according to the manufacturer's protocol. Resuspended cells were mixed with 200 µl 1× Binding Buffer and incubated with 5 µl Annexin V-FITC and 5 µl PI at room temperature for 10 min. SNS-032-induced cell death was monitored with Annexin V-FITC/PI and detected with CytoFLEX flow cytometer (Beckman Coulter, Inc.). All data were analyzed using FlowJo V10.8.1 (BD Biosciences).

Western blotting analysis

Total cell protein was extracted from BC cells after treatment with SNS-032 (10 µmol/l in MCF-7 and 15 µmol/l in 4T1 cells) for 24 h with or without DAC (5 µmol/l) pretreatment for 24 h or Z-DEVE-FMK (20 µmol/l) pretreatment for 3 h at 37°C using radioimmunoprecipitation lysis buffer (cat. no G2002; Wuhan Servicebio Technology Co., Ltd.) for western blotting. The buffer contained a 1% protease inhibitor cocktail (cat. no G2006; Wuhan Servicebio Technology Co., Ltd.). The cell samples were incubated in cold lysis buffer for 20 min, centrifuged at 13,600 × g for 20 min at 4°C. Protein concentration was measured using the bicinchoninic acid assay. Equal amounts of protein sample (30 µg) were loaded and separated by SDS-polyacrylamide gel electrophoresis (Dakewe Biotech Co., Ltd.) and transferred to polyvinylidene difluoride (PVDF) membranes (MilliporeSigma). The PVDF membranes were blocked with 5% bovine serum albumin (cat. no GC305006; Wuhan Servicebio Technology Co., Ltd.) for 1 h at room temperature and incubated with the primary antibody at 4°C overnight. The following antibodies were used: Anti-GSDME (1:1,000; cat. no ab215191; Abcam), anti-caspase-3 (1:1,000; cat. no #9662; Cell Signaling Technology, Inc.), anti-Bcl-2-associated X protein (BAX; 1:2,000; cat. no ab32503; Abcam), anti-B-cell lymphoma-2 (BCL-2; 1:2,000; cat. no ab182858; Abcam), anti-mouse cleaved N-terminal GSDMD (Asp275; 1:1,000; cat. no #36425; Cell Signaling Technology, Inc.), anti-cleaved N-terminal GSDMD (1:1,000; cat. no ab215203; Abcam) and anti-GAPDH (1:5,000; cat. no A19056; ABclonal Biotech Co., Ltd.). The PVDF membrane was exposed to anti-rabbit secondary antibody (1:20,000; cat. no ANT020; Wuhan Antejie Biotechnology Co., Ltd.) at room temperature for 1 h after washing three times with TBST (0.1% Tween-20 in Tris-HCl buffer). Protein bands were visualized using a multimode chemiluminescence system (Bio-Rad Laboratories, Inc.) and ImageJ software (version 1.5) for densitometry analysis.

Statistical analysis

All data presented were obtained from ≥3 independent experiments and are expressed as mean ± standard deviation. Data were analyzed using GraphPad Prism 8 (Dotmatics). The significance of the differences was assessed using one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference in all comparisons.

Results

SNS-032 can affect the cell viability of BC cells

To evaluate the anticancer effect of SNS-032 on BC cells, a CCK-8 assay was used to assess the effects of SNS-032 on the activity of human and mouse BC cell lines, MCF-7 and 4T1. Results from CCK-8 experiments (Fig. 1B) demonstrated that SNS-032 caused significant decreases in cell viability in a dose-dependent manner in BC cells compared with the control. The half-maximal inhibitory concentrations of the two BC cell lines were calculated to be 9 µmol/l (MCF-7) and 13.5 µmol/l (4T1). Based on these results, the following concentrations were used in the subsequent time-course investigations: 10 µmol/l for MCF-7 and 15 µmol/l for 4T1. SNS-032 significantly decreased the viability of BC cells in a time-dependent manner as shown by CCK-8 assays (Fig. 1C). These results indicate that the inhibitory effect of SNS-032 on BC cells was dose- and time-dependent, and the results were significantly different compared with the untreated control or 0 h timepoint (P<0.001), with the exceptions being the 0.5 µmol/l concentration in 4T1 cells, and the 6 and 12 h timepoints in both MCF-7 and 4T1 cells. To further support these results, colony formation experiments were performed on MCF-7 and 4T1 cells, as shown in Fig. 2B and C, which demonstrated that SNS-032 significantly inhibited the cloning of BC cells (P<0.001). In summary, these data show that SNS-032 treatment reduces the viability of BC cells.

Figure 2. DAC can promote the anticancer effect of SNS-032. (A) After 24 h treatment with or without DAC (5 µmol/l), MCF-7 and 4T1 cells were treated with SNS-032 (10 and 15 µmol/l, respectively) for 24 h and cell viability was analyzed using a CCK-8 assay. (B) After 24 h treatment with or without DAC (5 µmol/l), the results of a colony formation experiment were obtained after adding SNS-032 (10 and 15 µmol/l, respectively) to MCF-7 and 4T1 cells for 24 h. Representative images are shown. (C) Quantification of colony formation in MCF-7 and 4T1 cells treated with SNS-032. The cells were exposed to 0.1% dimethyl sulfoxide as a control. Data are presented as mean ± standard deviation (n=3). ***P<0.001 and ****P<0.0001 vs. control. ns: not significant; DAC, decitabine; SNS, SNS-032; Con, control; D + S, decitabine + SNS-032; CCK-8, Cell Counting Kit-8.

Evaluation of the cell death pathways of SNS-032 in BC cells

To explore the cell death pathway of SNS-032 in BC cells, cells were pretreated for 3 h with pan-caspase inhibitor, Z-VAD-FMK (20 µmol/l), necroptosis inhibitor Necrostatin-1 (10 µmol/l) or specific cell caspase-3 inhibitor Z-DEVD-FMK (20 µmol/l). MCF-7 and 4T1 cells were treated with SNS-032 for 24 h to observe cellular changes. Annexin V-FITC and PI were used to stain the cells for apoptosis studies, and the efficacy of the three inhibitors in reducing cell death caused by SNS-032 was assessed, as shown in Figs. 3 and 4B. Based on the results of flow cytometry, Necrostatin-1 had little effect on inhibiting SNS-032 induced-BC cell death, Z-VAD-FMK could partially inhibit the cell death of BC cells induced by SNS-032, while Z-DEVD-FMK was able to more markedly inhibit the SNS-032-induced cell death of BC cells, indicating the effect of the caspase-3 specific inhibitor was superior. Therefore, the possibility that cell death induced by SNS-032 is due to necroptosis can be excluded. Additionally, the results suggest that the cell death triggers may be related to the caspase family and caspase-3.

Figure 3. Different cell death inhibitors dissimilarly inhibit SNS-032-induced BC cell death. Annexin V-FITC/PI assay was performed to analyze pyroptosis and apoptosis of cells after pretreatment with SNS-032 for 24 h with or without Z-VAD-FMK (20 µmol/l) and Nec-1 (10 µmol/l) for 3 h in (A) MCF-7 and (B) 4T1 cells. Representative images are shown. The percentage of late-stage cell apoptosis in (C) MCF-7 and (D) 4T1 cells was quantified with SNS-032 treatment with or without Z-VAD-FMK and Nec-1. The cells were exposed to 0.1% dimethyl sulfoxide as a control. Data are presented as mean ± standard deviation (n=3). ****P<0.0001 vs. control. ns, not significant; SNS, SNS-032; Con, control; Nec-1, Necrostatin-1; FITC, fluorescein isothiocyanate.

Figure 4. Caspase-3 is required for SNS-032-dependent pyroptosis. (A) Representative microscopic images of MCF-7 and 4T1 cells pretreated with SNS-032 (10 and 15 µmol/l, respectively) with or without Z-DEVD-FMK (20 µmol/l) for 3 h. The white arrows indicate pyroptotic cells. Scale bar, 20 µm. (B) Annexin V-FITC/PI assay was performed to analyze pyroptosis and apoptosis of cells after pretreatment with SNS-032 for 24 h with or without Z-DEVD-FMK (20 µmol/l) for 3 h. The percentage of late-stage cell apoptosis in cells was quantified. Representative images are shown. (C) Cleaved-caspase-3 and GSDME proteins were detected after SNS-032 pretreatment with or without Z-DEVD-FMK (20 µmol/l) for 3 h by western blotting in MCF-7 and 4T1 cells. Semi-quantitative western blot analysis of cleaved caspase-3 in (D) MCF-7 and (E) 4T1 cells and GSDME-N in (F) MCF-7 and (G) 4T1 cells. The cells were exposed to 0.1% dimethyl sulfoxide as a control. Data are presented as mean ± standard deviation (n=3). *P<0.05, **P<0.01 and ****P<0.0001 vs. control. ns, not significant; FITC, fluorescein isothiocyanate; Con, control; ZDF, Z-DEVD-FMK; SNS, SNS-032; Z + S, Z-DEVD-FMK + SNS-032; GSDME, gasdermin E; CASP-3, caspase-3; FL, full-length; N, N-terminus.

SNS-032-induced pyroptosis is dependent on activation of caspase-3

MCF-7 and 4T1 cells were treated with four different treatments. During 24 h SNS-032 treatment, swelling, plasma membrane fragmentation and morphological alterations were observed (Fig. 4A). There were no discernible morphological changes in the control group, indicating that SNS-032 acts as an anticancer agent by inducing pyroptosis in BC cells. These morphological changes were consistent with the typical morphological changes of pyroptosis rather than with the classical changes observed during apoptosis, including shrinkage, rounding, volume reduction, deformation and maintenance of membrane integrity. As pyroptosis results in the destruction of the cell membrane, which is comparable to that of late-stage apoptotic cells, the potential of SNS-032 treatment to promote pyroptosis was evaluated by detecting the percentage of Annexin V-FITC+/PI+ cells using flow cytometry in BC cells. The results demonstrated that following exposure to SNS-032, the populations of Annexin V+/PI+ cells in MCF-7 and 4T1 cells significantly increased, resulting in a notably higher rate of late-stage apoptosis (Figs. 3A, C, 4B). These results demonstrate that SNS-032 is associated with BC cell death.

Previous studies have reported that pyroptosis is a caspase-dependent PCD pathway in which GSDME is cleaved by active caspase-3, releasing its N-terminal domain to penetrate the cell membrane, leading to cell swelling, rupture and ultimately cell death (11,12). To assess this, BC cells were treated with the caspase-3 inhibitor Z-DEVD-FMK. Subsequently, western blotting and semi-quantitative analysis were performed to evaluate the changes of GSDME full-length (GSDME-FL) and N-terminus domain (GSDME-N) expression levels in BC cells treated using different approaches (Fig. 4C). Western blotting revealed that the N-terminus of GSDME could barely be detected after Z-DEVD-FMK inhibited caspase-3 activation (Fig. 4C-E). SNS-032 treatment alone significantly increased cleavage and activation of caspase-3, with a corresponding significant increase in GSDME-N compared with those in the control and Z-DEVD-FMK treatment groups. However, after SNS-032 treatment of BC cells pretreated with Z-DEVD-FMK, activated caspase-3-induced GSDME-N production was significantly reduced compared with SNS-032 treatment alone, providing further evidence that activated caspase-3 is required for GSDME cleavage (Fig. 4C-G). BC cells pretreated with Z-DEVD-FMK also showed significantly fewer FITC/PI double-positive cells compared with SNS-032 treatment alone (Fig. 4B). In addition to pyroptosis, the caspase family serves an important role in cell apoptosis (34–36). Z-VAD-FMK alone had no effect on cell death compared with the control in either of the cells and could inhibit BC cell death induced by SNS-032 in the present study (Fig. 3A and C). These results suggest that SNS-032 induces pyroptosis by activating caspase-3 cleavage and GSDME in BC cells. This indicates that caspase-3 serves a crucial role in the process of BC cell death induced by SNS-032 and provides an important basis for further exploration of the mechanism of cell death and the development of associated therapeutic targets.

DAC can enhance the pyroptosis induced by SNS-032 in BC cells

DAC, a DNA methyltransferase inhibitor that exhibits anticancer activity in various types of cancer (37), can demethylate BC cells and increase GSDME expression (38,39). In the present study, in combination with the CDK inhibitor SNS-032, DAC was used as a pretreatment agent in BC cells. According to the results shown in Fig. S1A, the expression of GSDME-FL was assessed in two BC cell lines following a 48 h treatment with DAC at concentrations of 2.5, 5 and 7.5 µmol/l. GSDME-FL expression was significantly increased when the cells were treated with 5 µmol/l DAC. Therefore, pretreatment with DAC at 5 µmol/l in the dark for 24 h was chosen before adding SNS-032. To evaluate the effects of selected concentrations of DAC on BC cells, cell viability was assessed using CCK-8 and colony formation assays. The cell viability of BC cells was not significantly affected with DAC treatment compared with the control group, as shown in Fig. 2A-C. Compared with SNS-032 monotherapy, the combination of DAC and SNS-032 demonstrated significantly reduced viability of BC cells (P<0.001). Marked pyroptotic morphological changes, such as large vacuoles and membrane balloon-like changes, occurred more frequently in BC cells following combination treatment with DAC and SNS-032 (Fig. 5A) compared with morphological changes in cells treated with SNS-032. Compared with the control group and single-drug treatment groups, BC cells treated with DAC and SNS-032 exhibited a significantly greater proportion of late-stage apoptotic cells (>60%), as illustrated in Fig. 5C.

Figure 5. SNS-032 can cause the release of breast cancer cell contents and DAC can enhance the effect. (A) Representative microscopic images of MCF-7 (10 µmol/l) and 4T1 (15 µmol/l) cells treated with SNS-032 for 24 h with or without pretreatment with DAC (5 µmol/l). White arrowheads indicate the large vacuoles, cell membrane ruptures and organelle edema. Scale bar, 20 µm. (B) Quantification of the release of LDH after treatment with SNS-032 for 24 h with or without DAC (5 µmol/l) pretreatment for 24 h. (C) An Annexin V-FITC/PI assay was performed to analyze pyroptosis and apoptosis of cells after treatment with SNS-032 for 24 h with or without pretreatment with DAC (5 µmol/l) for 24 h in MCF-7 (10 µmol/l) and 4T1 (15 µmol/l) cells. Representative images are shown. (D) Quantification of ATP levels to assess cell viability after treatment with SNS-032 with or without DAC (5 µmol/l) pretreatment for 24 h. Data are presented as mean ± standard deviation (n=3). The cells were exposed to 0.1% dimethyl sulfoxide as a control. *P<0.05 and **P<0.01 vs. control. DAC, decitabine; SNS, SNS-032, D + S, decitabine + SNS-032; Con, control; ATP, adenosine 5’-triphosphate; LDH, lactate dehydrogenase; FITC, fluorescein isothiocyanate.

During pyroptosis, cells disintegrate and lose membrane integrity, releasing large quantities of ATP and LDH. As illustrated in Fig. 5B and D, upon exposure to SNS-032, intracellular ATP levels were significantly decreased and LDH release levels were significantly increased (P<0.01) in BC cells compared with those in the control group. Furthermore, Fig. 5B and D demonstrate that there was a significant decrease in intracellular ATP levels and a significant increase in LDH release levels in MCF-7 and 4T1 cells with DAC and SNS-032 treatment compared with the control and SNS-032 treatment alone. By contrast, the DAC group demonstrated little effect compared with that of the control group, corroborating the suggestion that combined DAC treatment could accelerate SNS-032-triggered cell pyroptosis. A similar pattern was observed in subsequent experiments.

Previously, the process of pyroptosis has been divided into GSDMD-mediated classical and non-classical pathways, and other GSDME-mediated pathways (7). As shown in Fig. S1C, the expression of the N-terminus domain of GSDMD was evaluated in BC cells after exposure to SNS-032 treatment to determine whether SNS-032-triggered pyroptosis. The results demonstrated that no GSDMD N-terminus domain expression was observed in SNS-032-treated BC cells. Western blotting was performed on MCF-7 and 4T1 BC cells to assess the expression of specific proteins triggered by pyroptosis. As shown in Fig. 6A, GSDME-FL was visibly increased in the DAC treatment group compared with the control group. The levels of cleaved GSDME-N were significantly increased in the drug combination group compared with SNS-032 alone (Fig. 6E). Consequently, the expression of apoptosis-related proteins in MCF-7 and 4T1 cells was assessed (Fig. 6B). As shown in Fig. 6D and F, following SNS-032 treatment, the expression of pro-apoptotic protein, BAX, was significantly increased whereas the expression of the anti-apoptotic protein, BCL-2, was significantly decreased. This suggested that SNS-032 also induces apoptosis in MCF-7 and 4T1 BC cells. There were no significant changes in the expression levels of cleaved caspase-3, BAX or BCL-2 between the DAC and control groups. The pro-apoptotic proteins, BAX and cleaved caspase-3, were significantly increased in SNS-032 treated groups and the anti-apoptotic protein BCL-2 was significantly decreased. Treatment with DAC and SNS-032 increased the expression of cleaved caspase-3 further and significantly decreased the expression of BCL-2 compared with SNS-032 alone (Fig. 6C and F). Based on the aforementioned results, it can be concluded that BC cells exposed to DAC demonstrate increased susceptibility to pyroptosis induced by SNS-032, due to the upregulated expression of GSDME. These results indicated that SNS-032 has anticancer effects on BC cells by inducing pyroptosis and that GSDME, but not GSDMD, is involved in pyroptosis induced by SNS-032 through caspase-3 activation.

Figure 6. DAC enhances the protein expression changes of SNS-032-treated breast cancer cells. (A) Western blotting of cleaved-caspase-3 and GSDME proteins after SNS-032 treatment in MCF-7 (10 µmol/l) and 4T1 (15 µmol/l) cells with or without DAC (5 µmol/l) pretreatment for 24 h. (B) BAX and BCL-2 proteins were detected after SNS-032 treatment with or without DAC (5 µmol/l) pretreatment for 24 h by western blotting in MCF-7 (10 µmol/l) and 4T1 (15 µmol/l) cells. (C) Semi-quantitative western blot analysis of cleaved caspase-3 in MCF-7 and 4T1 cells. (D) Semi-quantitative western blot analysis of BAX in MCF-7 and 4T1 cells. (E) Semi-quantitative western blot analysis of GSDME-N in MCF-7 and 4T1 cells. (F) Semi-quantitative western blot analysis of BCL-2 in MCF-7 and 4T1 cells. The cells were exposed to 0.1% dimethyl sulfoxide as a control. Representative images are shown. Data are presented as mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. ns, not significant; DAC, decitabine; SNS, SNS-032; D + S, decitabine + SNS-032; BCL-2, B-cell lymphoma-2; BAX, Bcl-2-associated X protein; CASP-3, caspase-3; GSDME, gasdermin E; FL, full-length; N, N-terminus; Con, control.

Discussion

Over the past several decades, progress has been achieved in the prevention, detection and treatment of cancer. In women, BC is the most commonly diagnosed cancer and the leading cause of cancer-associated mortalities (1,3). Despite the development of several targeted drugs, effective BC therapeutic drugs are still limited (2,4). As reported in previous studies, more CDK-specific inhibitors are being developed with encouraging results and potential to become available therapeutic targets for malignant tumors (40,41).

The present study reported the effects of the CDK inhibitor, SNS-032, on BC cells. SNS-032 is a specific and potent anticancer drug that inhibits CDKs 2, 7 and 9 (24,25), blocks RNA polymerase II phosphorylation and inhibits RNA synthesis (26,27). SNS-032 is a promising anticancer drug for a number of cancers (25–29), nevertheless its anticancer mechanisms remain unclear. In the present study, it was experimentally validated that SNS-032 may induce GSDME-mediated pyroptosis, which is activated via caspase-3 pathway, in BC cell lines.

Previously, pyroptosis has been implicated in inflammatory PCD (8,42). Pyroptosis has emerged as a promising research topic in the context of traditional chemotherapy resistance. In cancers that develop resistance to conventional chemotherapy, pyroptosis represents a novel cell death pathway. Conventional chemotherapy relies predominantly on inducing apoptosis; however, when tumor cells acquire resistance to apoptosis, the pyroptotic pathway may still be effective. For instance, multiple drug-resistant tumor cells are sensitive to drugs that induce pyroptosis (43), thereby offering new insights for overcoming resistance. Moreover, studying different drug-induced pyroptosis mechanisms can further optimize the design of drugs and enhance their specificity and efficacy. Pyroptosis-inducing drugs can be used in combination with existing therapies such as chemotherapy drugs, to overcome tumor cell resistance (44), and immunotherapy drugs, to strengthen the immune response (45). In addition, the tumor cells of different patients vary in their gene expression and signaling pathways, leading to different sensitivities to pyroptosis-inducing drugs (46). Analyzing patient tumor samples to determine the expression of pyroptosis-related proteins and potential targets is beneficial for improving the accuracy of treatments and reducing drug side effects.

In eukaryotic cells, pyroptosis is defined as the creation of 10–20 nm pores in the cell membrane, followed by nuclear concentration, cell enlargement and the release of cytoplasmic contents into the extracellular environment. The formation of pores in the cell membrane (47,48) promotes the release of several components, including the inflammatory cytokine IL-18 (49). Pyroptosis enhances the immune response against cancer cells. When cancer cells undergo pyroptosis, they release certain damage-associated molecular patterns (DAMPs) (10). These DAMPs can activate antigen-presenting cells in the immune system, such as dendritic cells, which subsequently trigger the activation and recruitment of immune cells, such as T cells. This process strengthens the immune surveillance and killing ability of the body towards cancer cells. This property is crucial in the current context of the rapid development of immunotherapy, particularly in cancer types such as BC, and in the application of stereotactic body radiotherapy in metastatic disease. Inducing pyroptosis in combination with immunotherapy could improve the therapeutic effect in clinical trials.

Pyroptosis occurs through four signaling pathways: Typical and atypical inflammasome pathways, GZM-based systems and caspase-mediated pathways of apoptosis (13). The GSDM family is the final enforcer of these signaling cascades, which require upstream caspases or GZMs to cleave them (9). Most studies to date have focused on the roles of GSDMD and GSDME in pyroptosis. A previous study demonstrated caspase-3 can cleave GSDME during chemotherapy to generate GSDME-N, which then penetrates the plasma membrane and leads to pyroptosis (14). Pyroptosis stimulation is challenging in BC cells because of the markedly decreased expression of GSDME compared with expression in normal cells (14,15,22). Endogenous GSDME expression was profiled in 57 cancer cells from the National Cancer Institute-60 cell lines screen, which includes numerous BC cell lines. And MCF-7 cells exhibited the highest level of endogenous GSDME expression, indicating a higher probability of inducing GSDME-mediated pyroptosis (14). Therefore, this cell line was chosen as the human BC cell line for the present study.

It has been reported that DAC is capable of augmenting the expression of GSDME protein in 4T1 cells (50). Pyroptosis has the capacity to activate the body's immune system, which potentially responds to the tumor-killing effect of the drugs. To optimize the anticancer effect of SNS-032-induced pyroptosis in BC, the BC cell line 4T1 was chosen, which is derived from BALB/c mice. The 4T1 cells can be utilized to construct immunocompetent BC mouse models in BALB/c mice, thereby paving the way for further investigations into the anticancer efficacy of SNS-032 in vivo. In addition, the DNA methyltransferase inhibitor, DAC, was utilized to induce functional re-expression of the abnormally silenced GSDME gene in cancer (22,23), and elevate the expression of GSDME in BC cells with low GSDME expression, thus promoting the anticancer ability through cell pyroptosis. Based on previous research (39,51) and the results of the present study, the optimal DAC concentration was determined to be 5 µmol/l. The optimal concentration of SNS-032 was determined to be 10 µmol/l in MCF-7 cells and 15 µmol/l in 4T1 cells for the subsequent experiments in the present study.

Initially, SNS-032 was shown to inhibit in the viability of BC cells using CCK-8 and colony formation assays, and the combination of DAC and SNS-032 further enhanced this effect. Furthermore, the morphology of MCF-7 and 4T1 cells following SNS-032 treatment revealed swelling and generation of large vacuoles on the cell membrane, which is a characteristic morphological manifestation of pyroptosis.

Previous studies have shown that pyroptosis and apoptosis can be differentiated by the positivity of Annexin V/PI staining. Generally, pyroptosis occurs more rapidly than apoptosis. For double-staining with Annexin V and PI, in the early stage of pyroptosis, the integrity of the cell membrane is compromised, leading to the externalization of phosphatidylserine, to which Annexin V can specifically bind. Concurrently, due to the increased membrane permeability, PI can enter the cell through pores formed on the cell membrane and stain the nucleus (34). In flow cytometric analysis, a cell population that is positive for both Annexin V and PI will be detected. In the early stage of apoptosis, although phosphatidylserine also externalizes on the cell membrane, the integrity of the cell membrane remains intact initially. At this point, cells exhibit Annexin V-positive and PI-negative staining. In the present study, BC cells were pretreated with Z-VAD-FMK, Necrostatin-1 or Z-DEVD-FMK, and the proportion of cell death was detected using Annexin V/PI staining. The findings of present study suggested that cell death induced by SNS-032 is not due to necroptosis. However, the pan-caspase inhibitor, Z-VAD-FMK, significantly inhibited late-stage apoptosis induced by SNS-032, while Z-DEVD-FMK almost completely inhibited its anticancer effect. Meanwhile, SNS-032 combined with DAC could further increase the proportion of late-stage apoptotic cells. In addition, in BC cells, SNS-032 significantly increased LDH release and decreased intracellular ATP, and these effects were markedly enhanced in combination with DAC. Z-DEVD-FMK was used to inhibit the activation of caspase-3 in BC cells treated with SNS-032. Consequently, the characteristic morphological features of pyroptosis in MCF-7 and 4T1 cells were notably alleviated and the protein expression levels of cleaved caspase-3 and GSDME-N were markedly decreased compared with SNS-032 treatment alone. Therefore, pretreatment with Z-DEVD-FMK effectively reduced pyroptosis in BC cells treated with SNS-032.

Western blotting was used to evaluate caspase-3 and GSDME protein expression in BC cells during treatment with SNS-032. The results demonstrated that caspase-3 was activated, GSDME was cleaved and GSDME N-terminus protein was significantly increased compared with the negative control group. Furthermore, DAC treatment enhanced GSDME-FL expression and when combined with SNS-032, the expression of GSDME-N was higher compared with the SNS-032 group. These findings suggest that SNS-032 triggers pyroptosis in MCF-7 and 4T1 cells via the caspase-3/GSDME signaling pathway.

The study indicated that SNS-032 decreased the expression of BCL-2, and increased the expression of BAX, and those changes became more apparent when combined with DAC. These data demonstrate that SNS-032 can trigger the pyroptosis in MCF-7 and 4T1 cells through the caspase-3/GSDME pathway, and DAC can boost the expression of GSDME protein by methylation, thus enhancing pyroptosis induced by SNS-032 in BC cells. In conclusion, the present article demonstrates that the CDK2/7/9 inhibitor SNS-032 can induce pyroptosis and apoptosis of human BC MCF-7 cells and mouse BC 4T1 cells through the caspase-3/GSDME pathway in vitro, thereby reducing the viability of cancer cells, providing a potential new strategy for the treatment of BC.

SNS-032 as a CDK inhibitor, may have potential off-target effects such as the inhibition of other kinases with similar structures or functions. This might lead to unintended consequences in cellular processes which are not associated with the expected target. Another effect of SNS-032 could be its impact on transcriptional regulation which might affect the expression of genes not directly associated with its primary mechanism of action. In addition, it might potentially alter the overall cellular gene expression profile and lead to unforeseen physiological changes or cytotoxicity in cells that are not the target of treatment. Furthermore, there are considerable clinical limitations regarding CDK inhibitors. As low-dose CDK inhibitors are ineffective, highly selective CDK inhibitors require higher doses, making the side effects of CDK inhibitors in clinical trials a limiting factor (52).

CDK inhibitors are tyrosine kinase inhibitors, with a main concern being potential acquired resistance during treatment (53). Future research should utilize a number of strategies to address these potential off-target impacts. Firstly, high-throughput screening techniques, such as proteomics and genomics assays, can be utilized to comprehensively map the interactome of SNS-032. By identifying proteins and genes with which it interacts, on-target and off-target effects can be differentiated more precisely. In order to improve clinical treatment efficacy and prevent needless drug exposure that leads to drug resistance, biomarkers associated with CDK inhibitor resistance should be assessed in patient blood or tumor tissues. These could then be used to screen patient groups that are less likely to develop resistance and are most likely to benefit from SNS-032 treatment. Secondly, structure-activity relationship studies should be conducted. By implementing chemical modifications to SNS-032, analogs with improved selectivity for the intended target may be developed, thereby minimizing interactions with off-target molecules and delaying the emergence of resistance. In addition, SNS-032 can be combined with drugs that have different mechanisms of action, such as targeting tumor angiogenesis, to treat the tumor from multiple directions to diminish the possibility of developing drug resistance (54). Immunomodulatory drugs, for instance immune checkpoint inhibitors, can be used to stimulate the body's immune system, synergizing with the inflammatory response induced by SNS-032-induced pyroptosis, to attack tumor cells that are resistant to treatment. Thirdly, it is imperative to utilize more physiologically relevant in vitro and in vivo models. For instance, patient-derived organoids or genetically engineered mouse models can offer a more precise evaluation of off-target effects, particularly those that are tissue-specific or disease-specific. Finally, long-term follow-up studies in both preclinical and clinical settings are crucial. Monitoring treated subjects over an extended duration can facilitate the detection of any delayed or cumulative off-target effects that may not be immediately evident. This approach allows for the optimization of dosage and treatment regimens aimed at reducing off-target toxicity while maximizing therapeutic efficacy.

The present study established that SNS-032 can cause apoptosis and pyroptosis of BC cells through caspase-3. Further research is required to establish the detailed mechanism underlying SNS-032-induced pyroptosis. Additionally, whether the antitumor activity of DAC in combination with SNS-032 could be utilized in in vivo experiments should be studied in the future.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present work was supported by the National Natural Science Foundation of China (grant no 82172717).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

The present study was proposed and designed by YC and LX. YC and DZ performed the experiments. Data collection and analysis were performed by JL, JW and YS. The manuscript was drafted by YC and checked by LX. YC and DZ confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References