ABT-737 efficiently inhibits hepatocellular carcinoma cell activity via regulating PANoptosis.

Introduction
Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumors worldwide. The majority of patients present with advanced-stage disease at diagnosis, resulting in limited therapeutic options and poor clinical outcomes [1]. The prognosis for HCC patients remains dismal [2], and the development of effective therapeutic strategies represents an urgent clinical need. A hallmark characteristic of HCC and many other cancers is the overexpression of anti-apoptotic members of the Bcl-2 family, which enables cancer cells to evade programmed cell death and maintain sustained proliferation [3, 4].
ABT-737 is a rationally designed small-molecule BH3 (Bcl-2 homology domain 3) mimetic that specifically targets and inhibits the anti-apoptotic Bcl-2 family proteins, including Bcl-2, Bcl-xL, and Bcl-w [5, 6]. Previous studies have demonstrated that ABT-737 exerts its anticancer activity primarily through antagonizing these pro-survival mediators, leading to the induction of apoptosis in various cancer types [7, 8].
PANoptosis represents a recently identified form of inflammatory programmed cell death that uniquely integrates three distinct cell death pathways: pyroptosis, apoptosis, and necroptosis [9, 10]. This complex cell death modality is characterized by extensive molecular crosstalk between traditionally separate death pathways. Emerging evidence indicates that PANoptosis plays a critical role in various pathological conditions, including microbial infections, inflammatory diseases, and malignant tumors [11, 12].
Recent studies have demonstrated a strong association between PANoptosis-related gene signatures and both the progression and prognosis of hepatocellular carcinoma patients [13]. Furthermore, activation of the PANoptosis pathway has shown potential for enhancing the antitumor effects of oncogenic kinase inhibitors and may contribute to sustained clinical responses in HCC treatment [14]. Despite these promising observations, the ability of ABT-737 to induce PANoptosis in HCC cells has not been investigated to date.
Therefore, the present study was designed to investigate whether ABT-737 could induce PANoptosis in hepatocellular carcinoma cells, evaluate its effects on cellular proliferation, migration, and invasion, and elucidate the underlying molecular mechanisms responsible for these antitumor effects.

Materials and methods

Cell lines and culture conditions
Two human hepatocellular carcinoma cell lines were employed in this study. SK-HEP-1 cells were obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). BEL-7402 cells were purchased from Nanjing Kaiji Biotechnology Development Co., Ltd. (Nanjing, China).
Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells at passages 3–8 were used for all experiments to ensure consistency and reproducibility.

Reagents and antibodies
ABT-737 was purchased from Selleck Chemicals (Houston, TX, USA) and dissolved in DMSO to prepare a 10 mM stock solution, which was stored at −20 °C. The pan-caspase inhibitor Z-VAD-FMK was obtained from MedChemExpress (Monmouth Junction, NJ, USA). The necroptosis inhibitor Necrostatin-1 (Nec-1) and the caspase-1 inhibitor VX-765 were purchased from MedChemExpress (USA).
Primary antibodies against RIPK1, RIPK3, MLKL, phospho-MLKL (Ser358), caspase-3, cleaved caspase-3, Bax, Bcl-2, NLRP3, ASC, caspase-1, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). HRP-conjugated secondary antibodies were obtained from Proteintech (Wuhan, China).

Cell viability assay
Cells were seeded in 96-well plates at a density of 5,000 cells per well and allowed to adhere overnight. The following day, cells were treated with ABT-737 at concentrations of 0, 5, 10, and 20 µM for 24 and 48 h. Prior to each MTS assay, cell morphology was examined and photographed under an inverted phase-contrast microscope (Olympus, Japan) at 100× magnification to document changes in cell morphology, including cell shrinkage, membrane blebbing, and cell detachment (Supplementary Figure S1). After the designated treatment periods, 20 µL of MTS reagent (Promega, Madison, WI, USA) was added to each well. Plates were incubated at 37 °C for 2 h, and absorbance was measured at 490 nm using a microplate reader (Thermo Fisher Scientific, USA). Cell viability was calculated as a percentage of the control group. Each experiment was performed in triplicate and repeated three times independently.

BrdU incorporation assay
To specifically assess cell proliferation, BrdU (5-bromo-2’-deoxyuridine) incorporation assays were performed using the BrdU Cell Proliferation Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Cells were seeded in 96-well plates at a density of 5,000 cells per well and treated with ABT-737 at concentrations of 0, 5, 10, and 20 µM for 24 h. BrdU labeling solution (10 µM) was added to the culture medium 4 h before the end of the treatment period. Cells were then fixed and DNA was denatured. After incubation with anti-BrdU antibody and HRP-conjugated secondary antibody, the colorimetric reaction was developed using TMB substrate, and absorbance was measured at 450 nm. The percentage of BrdU-positive cells was calculated relative to the control group.

Annexin V/PI flow cytometry analysis
Cell death was directly assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, USA). Cells were treated with ABT-737 (0, 5, 10, and 20 µM) for 24 h, then harvested by trypsinization and washed twice with cold PBS. Cells were resuspended in 100 µL of binding buffer and incubated with 5 µL of Annexin V-FITC and 5 µL of PI for 15 min at room temperature in the dark. An additional 400 µL of binding buffer was added, and samples were analyzed immediately using a flow cytometer (BD FACSCanto II). Data were analyzed using FlowJo software to determine the percentages of viable cells (Annexin V-/PI-) and total apoptotic cells (Annexin V+/PI- plus Annexin V+/PI+).

Wound healing assay
Cells were seeded in 6-well plates and cultured until they reached approximately 90% confluency. A sterile 200-µL pipette tip was used to create a straight scratch wound through the cell monolayer. Cells were washed twice with PBS to remove detached cells and debris, then cultured in serum-free medium supplemented with ABT-737 (10 µM) or vehicle control (DMSO). The concentration of 10 µM was selected for migration assays as it represents a sub-lethal dose that minimizes excessive cell death while still exerting biological effects on cell motility, as confirmed by preliminary dose-response studies. Images of the wound area were captured at 0 and 24 h using an inverted microscope. Wound closure was quantified using ImageJ software, and the migration rate was calculated as follows: Migration rate (%) = [(Initial wound width - Final wound width)/Initial wound width] × 100.

Transwell invasion assay
Cell invasion capability was assessed using Transwell chambers (8-µm pore size, Corning, USA) coated with Matrigel. Cells (5 × 10⁴) suspended in serum-free medium containing either ABT-737 (10 µM) or DMSO vehicle control were seeded in the upper chamber. The lower chamber was filled with complete medium containing 10% FBS as a chemoattractant. Similar to the migration assay, 10 µM ABT-737 was used to minimize proliferation-related confounding effects while maintaining sufficient biological activity to assess invasion capacity. After 24 h of incubation at 37 °C, non-invasive cells on the upper surface of the membrane were gently removed with a cotton swab. Cells that had invaded through the Matrigel and membrane to the lower surface were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 15 min. Invading cells were counted in five random fields per membrane under a light microscope at 200× magnification. Each experiment was performed in triplicate.

Combination treatment experiments
To investigate the involvement of specific cell death pathways in ABT-737-mediated cytotoxicity, combination treatment experiments were performed. Cells were pre-treated with pathway-specific inhibitors for 1 h: Z-VAD-FMK (pan-caspase inhibitor, 10 µM) for apoptosis, Necrostatin-1 (necroptosis inhibitor, 30 µM) for necroptosis, or VX-765 (caspase-1 inhibitor, 10 µM) for pyroptosis. Subsequently, cells were treated with 20 µM ABT-737 for 24 h. The higher concentration of 20 µM was specifically selected for combination treatment experiments to induce robust activation of multiple cell death pathways, thereby enabling clear detection of pathway-specific inhibitor effects. Cell viability was then assessed using the MTS assay as described above.

Xenograft mouse model
Six-week-old male BALB/c nude mice (weight 18–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were housed under specific pathogen-free (SPF) conditions with free access to food and water, and maintained on a 12-hour light/dark cycle. All animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Ethics Committee of Guangxi University (No. 202502AL) and conducted according to Guide for Care and Use of Laboratory Animals (8th edition, National Research Council). In accordance with the ethical approval granted by our Institutional Review Board (IRB), which stipulates a maximum permitted tumor burden of 2000 cubic millimeters, we confirm that all tumors included in this study were within this limit.
For tumor cell inoculation, mice were anesthetized with 3% isoflurane for induction and maintained under anesthesia with 1.5–2% isoflurane delivered via nose cone throughout the inoculation procedure. SK-HEP-1 or BEL-7402 cells (5 × 10⁶ cells in 100 µL PBS) were subcutaneously injected into the right flank of each mouse. Mice were randomly divided into treatment groups (n = 5 per group) after tumors became palpable (approximately 7 days post-inoculation). Treatment groups included: [1] vehicle control (PBS, i.v.) [2], ABT-737 alone (6 mg/kg, i.v., twice weekly) [3], ABT-737 + Z-VAD-FMK (6 mg/kg ABT-737 i.v. + 3 mg/kg Z-VAD-FMK i.v., twice weekly) [4], ABT-737 + Necrostatin-1 (6 mg/kg ABT-737 i.v. + 6 mg/kg Necrostatin-1 i.p., twice weekly), and [5] ABT-737 + VX-765 (6 mg/kg ABT-737 i.v. + 4 mg/kg VX-765 i.p., twice weekly).
Tumor volumes were measured twice weekly using digital calipers and calculated using the formula: Volume (mm³) = (Length × Width²)/2. Tumor growth curves were plotted to monitor treatment efficacy over time. At the end of the experiment (day 21), mice were euthanized by deep anesthesia with 5% isoflurane for 2–3 min followed by cervical dislocation. Tumors were excised, photographed, and weighed immediately using a precision balance. Tumor weights were recorded for each group to provide quantitative assessment of treatment effects. A portion of each tumor was fixed in 4% paraformaldehyde for histological analysis, while the remaining tissue was snap-frozen in liquid nitrogen and stored at −80 °C for Western blot analysis.

Immunohistochemistry and histological analysis
Tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm thickness. For Ki-67 immunohistochemistry, sections were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 3% H₂O₂, and sections were incubated with anti-Ki-67 antibody (1:200, Cell Signaling Technology) overnight at 4 °C. After washing, sections were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. DAB substrate was used for visualization, and sections were counterstained with hematoxylin. Ki-67-positive cells were quantified by counting stained nuclei in five random high-power fields (200× magnification) per section, and the percentage of positive cells was calculated.
For necrosis assessment, adjacent sections were stained with hematoxylin and eosin (H&E) following standard protocols. Whole slide images were captured using a digital slide scanner. Necrotic areas, characterized by nuclear pyknosis, karyolysis, loss of cellular detail, and eosinophilic cytoplasm, were identified and quantified using ImageJ software. The percentage of necrotic area relative to total tumor area was calculated for each section.

Western blot analysis
Cultured cells or tumor tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies against RIPK1, RIPK3, MLKL, phospho-MLKL (Ser358), caspase-3, cleaved caspase-3, Bax, Bcl-2, NLRP3, ASC, caspase-1, and β-actin (all diluted 1:1000). After washing, membranes were incubated with HRP-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) reagent and imaged using a ChemiDoc imaging system (Bio-Rad). Band intensities were quantified using ImageJ software and normalized to β-actin. For Western blot analysis in cultured cells, a concentration of 10 µM ABT-737 was used for 24 h to evaluate protein expression changes under conditions that induce moderate cell death while maintaining sufficient viable cells for protein extraction and analysis.

Statistical analysis
All data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical comparisons between groups were performed using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. A P-value < 0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism 8.0 software.

Results

ABT-737 inhibits HCC cell proliferation, migration, and invasion
Phase-contrast microscopy revealed dose-dependent morphological changes characteristic of cell death, including cell shrinkage, rounding, and detachment from the culture surface (Supplementary Figure S1). MTS assays demonstrated that compared with the control group, ABT-737 treatment groups showed significantly decreased cell viability in a concentration- and time-dependent manner, with more pronounced effects at 48 h than at 24 h (Fig. 1A).
BrdU incorporation assays were performed to distinguish between reduced proliferation and increased cell death. Compared with the control group, ABT-737 5 µM treatment group showed significantly reduced BrdU incorporation rate (P < 0.01). Compared with the control group, ABT-737 10 µM and 20 µM treatment groups showed further reduced BrdU incorporation rate (P < 0.001), demonstrating dose-dependent inhibition (Fig. 1B).
Wound healing assays showed that compared with the control group, ABT-737 (10 µM) treatment group exhibited significantly reduced migration rate in both SK-HEP-1 and BEL-7402 cells (P < 0.001) (Figs. 1C–D). Transwell invasion assays revealed that compared with the control group, ABT-737 treatment group showed significantly reduced number of invaded cells (P < 0.001) (Fig. 1E).

ABT-737 induces cell death in HCC cells
Annexin V/PI flow cytometry was performed to directly assess cell death. Compared with the control group, ABT-737 5 µM treatment group showed significantly increased total apoptosis rate (P < 0.01). Compared with the control group, ABT-737 10 µM and 20 µM treatment groups showed further increased total apoptosis rate (P < 0.001), demonstrating dose-dependent induction of cell death in both cell lines (Fig. 2A–B).

ABT-737-induced cell death involves multiple pathways (PANoptosis)
MTS assays showed that compared with the control group, ABT-737 (20 µM) treatment group exhibited significantly decreased cell viability (P < 0.001). Compared with the ABT-737 alone group, Z-VAD-FMK, Necrostatin-1, and VX-765 co-treatment groups all showed partially restored cell viability (P < 0.05), but none completely rescued cell viability (Fig. 3A and C). The partial rescue by each individual pathway inhibitor, rather than complete rescue, indicates that ABT-737-induced cell death involves simultaneous activation of multiple cell death pathways characteristic of PANoptosis.
In xenograft models, compared with the control group, ABT-737 treatment group showed significantly reduced tumor volume throughout the experimental period (P < 0.001). Compared with the ABT-737 alone group, each pathway inhibitor co-treatment group showed partially increased tumor volume (P < 0.05) (Fig. 3E). At endpoint, compared with the control group, ABT-737 treatment group showed significantly reduced tumor weight (P < 0.001), which was partially reversed by pathway inhibitors (P < 0.05) (Fig. 3B, D and F).

ABT-737 reduces proliferation and increases necrosis in xenograft tumors
Ki-67 immunohistochemistry showed that compared with the control group, ABT-737 treatment group exhibited significantly reduced Ki-67 positive rate (P < 0.001). Compared with the ABT-737 alone group, pathway inhibitor co-treatment groups showed partially increased Ki-67 positive rates (P < 0.05 or P < 0.01), but still significantly lower than the control group (Figs. 4A–B).
H&E staining revealed that the control group showed minimal necrosis. Compared with the control group, ABT-737 treatment group showed significantly increased necrotic area (P < 0.001). Compared with the ABT-737 alone group, pathway inhibitor co-treatment groups showed significantly reduced necrotic areas (P < 0.05 or P < 0.01) (Figs. 4C–D).

ABT-737 modulates expression of PANoptosis-related proteins in cultured cells
Western blot analysis was performed to examine PANoptosis-related protein expression. For necroptosis markers, compared with the control group, ABT-737 treatment group showed significantly increased expression of RIPK1, RIPK3, and MLKL (P < 0.01). Importantly, compared with the control group, ABT-737 treatment group showed significantly increased pMLKL (Ser358) expression and pMLKL/MLKL ratio (P < 0.001), confirming necroptosis activation (Figs. 5A–B).
For apoptosis markers, compared with the control group, ABT-737 treatment group showed significantly increased cleaved caspase-3 (P < 0.001) and Bax expression (P < 0.01), while Bcl-2 expression was significantly decreased (P < 0.001) (Figs. 5C–D).
For pyroptosis markers, compared with the control group, ABT-737 treatment group showed significantly increased expression of NLRP3 (P < 0.01), ASC (P < 0.01), and caspase-1 (P < 0.001) (Figs. 5E–F).

ABT-737 activates PANoptosis pathways in xenograft tumor tissues
Western blot analysis of xenograft tumor tissues confirmed the in vitro findings. Compared with the control group, ABT-737 treatment group showed significantly increased expression of RIPK1, RIPK3, MLKL, and pMLKL (P < 0.001). The pMLKL/MLKL ratio was also significantly elevated (P < 0.001). Compared with the ABT-737 alone group, Necrostatin-1 co-treatment group showed partially reduced pMLKL level (P < 0.05) (Figs. 6A–B).
Compared with the control group, ABT-737 treatment group showed significantly increased cleaved caspase-3 and Bax expression (P < 0.001), while Bcl-2 was significantly decreased (P < 0.001) (Figs. 6C–D).
Compared with the control group, ABT-737 treatment group showed significantly increased NLRP3, ASC, and caspase-1 expression (P < 0.001) (Figs. 6E–F).

Discussion
This study demonstrates that ABT-737 induces PANoptosis in hepatocellular carcinoma cells through simultaneous activation of apoptosis, necroptosis, and pyroptosis pathways. These findings reveal a mechanism of action for ABT-737 that extends beyond its established function as a Bcl-2 family antagonist.
PANoptosis represents a recently characterized form of inflammatory programmed cell death that integrates apoptosis, necroptosis, and pyroptosis into a coordinated cellular response [9]. This interconnected cell death network has been implicated in various pathological conditions, including cancer progression and therapeutic resistance [15]. Previous studies have established that PANoptosis-related gene signatures correlate with HCC progression and patient prognosis [16, 17], supporting the clinical relevance of this pathway in hepatocellular carcinoma.
The apoptotic effects of ABT-737 observed in our study are consistent with its established mechanism as a BH3 mimetic that selectively inhibits anti-apoptotic Bcl-2 family proteins [18]. The molecular changes observed, including increased cleaved caspase-3 and Bax with decreased Bcl-2, indicate activation of the intrinsic apoptotic pathway, consistent with previous reports in various cancer types [19, 20]. Our study reveals that ABT-737 also activates necroptosis, representing a novel finding. The upregulation of RIPK1, RIPK3, MLKL, and particularly the robust phosphorylation of MLKL at Ser358 provides unequivocal evidence of necroptosis activation [21]. The partial reduction of pMLKL by Necrostatin-1 confirmed the specificity of this activation. While previous studies have primarily focused on ABT-737’s apoptotic effects, emerging evidence suggests that Bcl-2 family proteins may regulate necroptosis through direct interactions with MLKL. Specifically, Bcl-2 has been shown to target BH3-like domains in MLKL to suppress necroptosis [22]. By antagonizing Bcl-2 family proteins, ABT-737 may release this suppression, facilitating necrosome formation. This caspase-independent pathway may provide therapeutic advantages when apoptosis is compromised, as reported in melanoma and small cell lung cancer models [23, 24].
ABT-737 treatment also activated the pyroptosis pathway through upregulation of NLRP3, ASC, and caspase-1, which constitute the canonical NLRP3 inflammasome complex [25]. The mechanism may involve mitochondrial dysfunction following Bcl-2 inhibition, leading to the release of oxidized mitochondrial DNA that binds to and activates the NLRP3 inflammasome [26]. Since Bcl-2 inversely regulates mitochondrial dysfunction and NLRP3 inflammasome activation, ABT-737-mediated Bcl-2 inhibition may promote this pyroptotic cascade.
In addition to inducing cell death, ABT-737 significantly impaired HCC cell migration and invasion at sub-lethal concentrations. Previous studies have reported that ABT-737 can affect migration and invasion through mechanisms independent of apoptosis, including modulation of EMT markers and cytoskeletal remodeling in breast cancer and glioblastoma [27, 28], suggesting Bcl-2 family proteins may influence cell adhesion and motility pathways.

The partial rescue effects observed with pathway-specific inhibitors demonstrate molecular crosstalk between the three cell death pathways. The inability of any single inhibitor to completely block ABT-737’s cytotoxic effects indicates cooperative contribution of multiple pathways (Fig. 7). To our knowledge, this is the first comprehensive demonstration of PANoptosis induction by ABT-737 in hepatocellular carcinoma. Figure 7 Hypothesized schematic of ABT-737’s regulatory mechanism in hepatocellular carcinoma. ABT-737 inhibits anti-apoptotic Bcl-2 family proteins, leading to coordinated activation of apoptosis, necroptosis, and pyroptosis (PANoptosis), resulting in comprehensive suppression of HCC cell survival, proliferation, migration, and invasion.
The in vivo relevance was validated through xenograft studies showing consistent molecular changes, reduced tumor growth, decreased Ki-67 expression, and increased necrosis. The partial reversal by pathway-specific inhibitors corroborated the therapeutic relevance of PANoptosis induction.
These findings have significant therapeutic implications. The simultaneous activation of multiple cell death pathways may reduce therapeutic resistance development, as cancer cells would need to acquire resistance against all three pathways simultaneously.
Several limitations should be acknowledged. First, the complete mechanistic details of how ABT-737 upregulates necroptosis and pyroptosis markers require further investigation through co-immunoprecipitation, ROS measurement, and genetic manipulation studies. Second, xenograft studies in immunodeficient mice limit evaluation of the immunological aspects of PANoptosis. Studies in immunocompetent models would provide valuable insights into immune responses in ABT-737’s antitumor effects.

Conclusion
This study establishes that ABT-737 exerts antitumor effects in hepatocellular carcinoma through PANoptosis induction, involving coordinated activation of apoptosis, necroptosis, and pyroptosis pathways. The decreased cell viability reflected both reduced proliferation (demonstrated by BrdU assays) and increased cell death (demonstrated by flow cytometry), with necroptosis activation definitively confirmed by pMLKL phosphorylation. The simultaneous engagement of multiple cell death mechanisms provides a molecular basis for ABT-737’s therapeutic efficacy and suggests potential strategies for overcoming treatment resistance in HCC. Further investigation of PANoptosis-targeting approaches may yield improved therapeutic options for patients with hepatocellular carcinoma.

Supplementary Information