MiR-200a-3p/ZEB1/IRF1-mediated PANoptosis prompts Xiangshaliujunzi decoction to overcome 5-fluorouracil resistance in gastric cancer

Abstract

BACKGROUND

One of the main challenges in treating gastric cancer is chemoresistance, particularly when using 5-fluorouracil (5-FU). Traditional Chinese medicine Xiangshaliujunzi decoction (XSLJZD) has been widely used for managing chemotherapy-related side effects; however, its potential antitumor effects remain unexplored. We aimed to study the role of XSLJZD in addressing 5-FU resistance in gastric cancer cells.

AIM

To investigate whether XSLJZD overcomes 5-FU resistance in gastric cancer by regulating miR-200a-3p/ZEB1/IRF1-mediated PANoptosis.

METHODS

BGC-823/5-FU cell line was constructed. ZEB1 and IRF1 knockdown cell lines were constructed by transfecting lentivirus vectors containing shRNAs targeting ZEB1 and IRF1. Cell viability and proliferation were detected by CCK8 and colony formation assays. Cell apoptosis was determined by flow cytometry. Cell necroptosis and pyroptosis were measured using fluorescence staining. The molecular mechanism of XSLJZD was further explored using western blot, RNA immunoprecipitation, co-immunoprecipitation, and dual-luciferase reporter assays. A xenograft tumor nude mouse model was constructed by subcutaneously injecting the gastric cancer cells.

RESULTS

XSLJZD significantly inhibited cell viability and proliferation while promoting PANoptosis in BGC-823/5-FU cells (5-FU-resistant cells). ZEB1 knockdown upregulated pyroptosis-, apoptosis-, and other programmed cell death (PCD)-related proteins. Simultaneous knockdown of ZEB1 and IRF1 suppressed the expression of pyroptosis-, apoptosis- and PCD-related proteins. The combination of XSLJZD and 5-FU promoted miR-200a/ZEB1/IRF1-mediated PANoptosis in transplanted tumor tissues from mice.

CONCLUSION

Our findings suggest that XSLJZD sensitizes gastric cancer cells to 5-FU by modulating the miR-200a-3p/ZEB1/IRF1 pathway, offering a potential therapeutic strategy to overcome chemoresistance.

Key Words: Xiangshaliujunzi decoction; Chemoresistance; Gastric cancer; MiR-200a-3p; ZEB1; miRNA

Core Tip: Xiangshaliujunzi decoction (XSLJZD) overcomes 5-fluorouracil (5-FU) resistance in gastric cancer by inducing PANoptosis via the miR-200a-3p/ZEB1/IRF1 pathway. In vitro, XSLJZD inhibits viability and promotes cell death. In vivo, combination with 5-FU enhances this effect. This provides a novel traditional Chinese medicine strategy for chemoresistance, though limitations include lack of standalone controls and generalizability validation.

INTRODUCTION

Gastric cancer is the fifth most common malignancy worldwide with high mortality rates[1]. It has a poor prognosis, mainly because it is often diagnosed at a late stage. The global 5-year survival rate is approximately 20%, except in Japan and South Korea[2]. For resectable gastric cancer, the standard treatment involves surgical resection in combination with perioperative chemotherapy. For nonresectable or metastatic gastric cancer, systemic chemotherapy is the primary treatment modality[3]. 5-Fluorouracil (5-FU)-based regimen is one of the most commonly used treatment regimens for gastric cancer. However, drug resistance remains a challenge, adversely affecting clinical outcomes[4]. Therefore, innovative therapeutic strategies are needed to address 5-FU resistance and improve the prognosis of gastric cancer.

PANoptosis is a type of regulated cell death characterized by the coordinated activation of apoptosis, pyroptosis, and necroptosis, potentially involving a multiprotein structure termed the PANoptosome[5]. Increasing evidence has revealed the significance of PANoptosis in gastric cancer. A PANscore model has been developed to quantify PANoptosis patterns in patients with gastric cancer, associating low scores with good immunotherapy response and prognosis[6]. Research showed that the ubiquitination-mediated degradation of YBX1 can reduce oxaliplatin resistance by promoting PANoptosis in gastric cancer[7]. Thus, induce PANoptosis in gastric cancer cells might enhance their sensitivity to chemotherapy.

Traditional Chinese medicine (TCM) is widely employed in China in combination with chemotherapy and radiotherapy to decrease the adverse events linked to cancer therapies[8,9]. Integrating TCM with conventional antitumor treatments can improve the overall survival of patients with lung cancer. For instance, TCM users showed a 32% reduction in the risk of death compared with non-TCM users[10]. Xiangshaliujunzi decoction (XSLJZD) is a classic TCM formula composed of eight herbs: Rhizoma Atractylodis macrocephalae, Panax ginseng, Pericarpium Citri Reticulatae, Radix Glycyrrhizae, Pinellia Tuber, Poria, Fructus Amomi, and Radix Aucklandiae[11]. The efficacy of XSLJZD in alleviating chemotherapy-related adverse events has been gradually explored. However, the potential antitumor effects of combining XSLJZD with chemotherapeutic drugs remain unknown. XSLJZD enhances the antitumor immune response by regulating miRNA expression[12]. As one of the miR-200 family members, miR-200a-3p suppresses tumor development and is downregulated in gastric cancer[13]. MicroRNA-200a contributes to the development of chemoresistance in breast[14], ovarian[15], and lung[16] tumors. Zinc finger E-box binding homeobox 1 (ZEB1), a gene expressed in various cancer cells, has been associated with the development of chemotherapy resistance[17]. The miR-200 family regulates ZEB1 expression and contributes to cancer progression[18]. Downregulation of miRNA-200a promotes tumor growth via ZEB1/ZEB2 in gastric cancer[19]. In this study, miR-200a-3p was selected because of its downregulation in gastric cancer and its role in modulating chemoresistance by targeting ZEB1. MiR-200a-3p overexpression promotes NLRP3 expression, which plays a critical role in inducing PANoptosis[20,21]. However, whether XSLJZD regulates miR-200a-3p and ZEB1 expression is still unknown. This study explored how XSLJZD regulates 5-FU resistance in gastric cancer and clarified the associated mechanisms. Our results demonstrated that XSLJZD suppressed cell viability, inhibited cell proliferation, and induced PANoptosis in 5-FU-resistant cells. Furthermore, we showed that XSLJZD induced PANoptosis by upregulating miR-200a-3p expression and inhibiting ZEB1 translation.

MATERIALS AND METHODS

Culture of tumor cell and establishment of 5-FU-resistant cell line

Gastric adenocarcinoma cell line BGC-823 was purchased from Shanghai Huiying Biotechnology Co., Ltd. BGC-823, a well-established human gastric adenocarcinoma model with moderate aggressiveness, is commonly used in studies on chemoresistance and exhibits reliable 5-FU resistance. The cells were maintained in RPMI-1640 medium (Gibco, China, 11875093) with 10% fetal bovine serum (FBS) (Excell Bio, China, FSD500) in a humidified incubator with 5% CO₂, at 37 °C. A cell line resistant to 5-FU (BGC-823/5-FU) was developed as follows: Cells were first cultured for 24 hours in RPMI-1640 medium containing 5 mg/L 5-FU, then washed and cultured in a complete medium without the drug for another 24 hours. Once the cells resumed growth, 5-FU was reintroduced at increasing concentrations (incrementing by 5 mg/L each time), and the cells were cultured for another 24 hours. The above operation was repeated eight times until the concentration of 5-FU reached 40 mg/L. Following the above steps, the BGC-823/5-FU cell line was cultured in a medium containing 10 mg/L 5-FU and utilized for subsequent experiments 2 weeks after drug withdrawal (maintained after 10 passages without 5-FU). Resistance was verified by IC₅₀ (IC50 BGC-823/5-FU approximately 40 mg/L vs parental approximately 5 mg/L, approximately 8-fold).

Preparation of drug-containing serum

The herbs were added to distilled water according to their respective dosages and decocted to prepare an herbal decoction. Following concentration, precipitation, filtration, and low-temperature drying, the decoction was processed into a paste, cooled, dried, and ground into a powder. The powder was added to the distilled water to achieve a final concentration of 10 g/L, and the solution was passed through a sterile 0.22 μm filter. Wistar rats aged 8 weeks were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (China). Twenty-four rats were cataloged into four groups (control group and XSLJZD low-dose group, XSLJZD medium-dose group, and XSLJZD high-dose group), with equal numbers of each gender. The rats in the XSLJZD groups were treated with different concentrations of XSLJZD [1.55 g/(kg·day) in the low-dose group, 3.1 g/(kg·day) in the medium-dose group, and 6.2 g/(kg·day) in the high-dose group]. These doses were based on human equivalent from a previous study[22]. The rats in the control group were treated with distilled water at 10 mL/kg. All the rats were treated twice daily for 3 consecutive days. Serum was obtained by collecting and centrifuging blood 1 hour after the final dose was administered. The serum was then heated to 56 °C for 30 minutes, sterilized by microporous membrane filtration, and aliquoted before being stored at -20 °C in a refrigerator for subsequent experiments. For the in vitro experiment, the sera from the low-, medium-, and high-dose groups were added to the cell culture medium (final concentration was 0.25 mL/mL in each group). For the in vitro experiments, the cells in the XSLJZD groups were cultured in serum-containing medium for 24 hours, after which the serum-containing medium was replaced by normal culture medium. The in vitro doses were correlated with in vivo via serum pharmacology, ensuring absorbed component consistency. The Ethics Committee of Liaoning University of Traditional Chinese Medicine approved the animal care and use procedures. The ethical use of animals was conducted in accordance with all applicable institutional and governmental regulations.

Transfection of miR-200a-3p inhibitor, mimic, and the negative control

The miR-200a-3p sequence information was retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/gene). The sequences of the miR-200a-3p inhibitor, mimic, and the negative control (NC) were designed and synthesized by Shanghai GenePharma Co., Ltd. Detailed sequence data are available in Supplementary Table 1. The transfection of miR-200a-3p mimics, inhibitors, and their NC sequences was performed following the manufacturer’s instructions.

Construction of ZEB1 and IRF1 knockdown cell lines

ShRNAs targeting ZEB1 and IRF1 were designed according to their coding sequences (CDS, assessed via the NCBI database). Plasmid pLKO.1, which contains the shRNA construct, was used as the vector. Recombinant plasmids pLKO.1-sh-ZEB1 and pLKO.1-sh-IRF1 were engineered by Shanghai GenePharma Co., Ltd. Detailed information about the vector and shRNA sequences is available in Supplementary Table 2 and Supplementary Figure 1. Lentivirus vectors containing the recombinant plasmids pLKO.1-sh-ZEB1 (Lv-sh-ZEB1) and pLKO.1-sh-IRF1 (Lv-sh-IRF1) were subsequently generated by Sangon Biotech (Shanghai) Co., Ltd. (Supplementary Table 2). The cells were incubated for 12 hours in a six-well plate. Lipofectamine 3000 (Invitrogen, United States) was used to transfect the lentiviruses into the gastric cancer cells.

Cell viability assay

The cells were maintained in a 96-well plate at a concentration of 2000 cells per well at 37 °C with 5% CO₂ for 8 hours. Afterward, the cells were handled according to their specific experimental groups and then cultured for 0, 24, 48, or 72 hours. After each designated culture period, CCK-8 reagent (10 µL/well) (BA00208, Bioss, China) was added and left to incubate for 2 hours, followed by the determination of the optical density at 450 nm.

Cell colony formation assay

After being incubated for 8 hours in a six-well plate, the cells were treated according to their experimental groups. Following the treatment, the cells were cultured for 2 weeks. The medium was then removed, and the cells were fixed with methanol for 20 minutes. The cells were stained using 0.2% crystal violet for 5 minutes. After staining, the cells were rinsed with running water. The colonies were counted. Colony formation rate was computed as follows: Colony formation rate = (number of colonies/number of seeded cells) × 100%.

Apoptosis assay

The cells were incubated in RPMI-1640 medium without FBS for 12 hours at 37 °C with 5% CO₂ and then treated according to the experimental groups. After being washed using phosphate-buffered saline (PBS), the cells were subsequently resuspended in 1 × annexin V binding solution (C1062 L, Beyotime, China) to obtain a cell solution with concentration of 1 × 10⁶ cells/m. The cell suspension (100 μL), annexin V-FITC (5 μL), and PI solution (5 μL) were mixed in a tube and then incubated in the dark for 15 minutes. Afterward, 1 × annexin V binding solution (400 μL) was introduced into the tube, and Attune NxT flow cytometry (Thermo Fisher Scientific, United States) was used for analysis.

Western blot

The cells were subjected to treatments based on the experimental groups. The RIPA buffer containing 0.5 mL PMSF was used to lyse the cells to obtain the lysate. After the lysate was centrifuged for 10 minutes at 4 °C and 12000 × g, the supernatant was collected. Protein concentration was assessed using the BCA protein assay kit. The protein was loaded into the wells of a 12% SDS-PAGE gel (BioFroxx, China) (40 μg/well) and separated at 80 V for 30 minutes, followed by 120 V until the protein was fully resolved. The proteins were transferred onto a PVDF membrane, which was then blocked for 15 minutes using a blocking solution and incubated overnight at 4 °C with the following primary antibodies: Anti-pro-caspase-1 (1:1000, ab179515, Abcam, United Kingdom), anti-GSDMD (1:1000, ab210070, Abcam, United Kingdom), anti-cleaved caspase-3 (1:500, ab32042, Abcam, United Kingdom), anti-cleaved caspase-7 (1:1000, ab256469, Abcam, United Kingdom), anti-cleaved caspase-8 (1:1000, #98134, CST, United States), anti-RIPK1 (1:1000, AB300617, Abcam, United Kingdom), anti-p-RIPK1 (1:1000, ab316923, Abcam, United Kingdom), anti-MLKL (1:2000, ab184718, Abcam, United Kingdom), anti-p-MLKL (1:1000, ab187091, Abcam, United Kingdom), anti-GSDME-N (1:1000, ab215191, Abcam, United Kingdom), anti-IRF1 (1:1000, #8478, CST, United States), anti-ZEB1 (1:1000, ab203829, Abcam, United Kingdom), and anti-β-actin (1:1000, ab8226, Abcam, United Kingdom). The membrane was washed with TBST and incubated with anti-rabbit/mouse IgG secondary antibodies (1:20000, Bioss, China) for 2 hours at room temperature. ECL solution (NCM Biotech, China) was added to the membrane to visualize the proteins using the JP-K6000 system (Shanghai Jiapeng, China). Protein expression levels, including phosphorylated and cleaved forms, were quantified using ImageJ software and normalized to β-actin as the internal reference.

Real-time quantitative PCR

The cells were treated in a six-well plate following the experimental group protocols. The FastPure^® Cell/Tissue Total RNA Isolation Kit V2 (RC112, Vazyme, China) was used to extract the total RNA. The MiPure Cell/Tissue miRNA Kit (RC201, Vazyme, China) was used to extract the miRNA. RNA concentration and purity were assessed using the Nano-600 Ultra Micro Nucleic Acid and Protein Analysis System manufactured by Shanghai Jiapeng, China. cDNA was synthesized following the instructions of the HiScript III 1^st Strand cDNA Synthesis Kit (R312) and the miRNA 1^st Strand cDNA Synthesis Kit (MR101). Quantitative PCR (qPCR) was performed following the guidelines of the Taq Pro Universal SYBR qPCR Master Mix (Q712) and the miRNA Universal SYBR qPCR Master Mix (MQ101). The sequences of the primers are listed in Supplementary Table 3.

Enzyme-linked immunosorbent assay

The cells were treated according to the experimental groups. The culture medium from different groups was collected and centrifuged at 1000 × g for 20 minutes. Thes concentration of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-18 were measured using the Human IFN-γ enzyme-linked immunosorbent assay (ELISA) Kit (D711044-0096, Sangon, China), Human TNF-α ELISA Kit (PT518, Beyotime, China), Human IL-1β ELISA Kit (PI305, Beyotime, China), and Human IL-18 ELISA Kit (PI558, Beyotime, China) following the manufacturer’s instructions. Tumor tissue from the mice was treated with liquid nitrogen and thawed at 2-8 °C. After homogenization with a homogenizer, the tissue was centrifuged at 3000 rpm for 20 minutes. The concentrations of IFN-γ, TNF-α, IL-1β, and IL-18 in the supernatant were determined using the Mouse IFN-γ ELISA Kit (D721025-0096, Sangon, China), Mouse TNF-α ELISA Kit (PT512, Beyotime, China), Mouse IL-1β ELISA Kit (PI301, Beyotime, China), and Mouse IL-18 ELISA Kit (PI553, Beyotime, China).

Fluorescence staining for apoptosis, necroptosis, and pyroptosis detection

Apoptosis, necroptosis, and pyroptosis were detected using the YO-PRO-1/PI Apoptosis and Necrosis Detection Kit (C1075S, Beyotime, China). YO-PRO-1/PI working solution was prepared by adding YO-PRO-1 (1000 ×, 1 µL) and PI (1000 ×, 1 µL) to 998 µL of buffer. Following treatment in a six-well plate, the cells were rinsed with PBS buffer. The working solution (1 mL/well) was added to the wells, and the plate was incubated at 37 °C for 15 minutes. A fluorescence microscope was used to observe fluorescence. YO-PRO-1-positive cells (green fluorescence) were indicative of early apoptosis or necroptosis, PI-positive cells (red fluorescence) were indicative of necroptosis or pyroptosis, and double-positive cells were indicative of late apoptosis or necrosis. Although YO-PRO-1/PI provides general indicators, specific validation was performed using western blot (WB) for markers such as GSDMD cleavage (pyroptosis) and p-MLKL (necroptosis).

Dual-luciferase reporter assay

PmirGLO-ZEB1 3'UTR-WT and PmirGLO-ZEB1 3'UTR-mut reporter plasmids were constructed on the basis of the predictive binding sites of miR-200a-3p and ZEB1 (Sangon Co., Ltd., Shanghai, China). The plasmid structure information is provided in Supplementary Figure 2. The cells were cotransfected with either miR-200a-3p mimics or NC, along with PmirGLO-ZEB1 3’UTR-WT and PmirGLO-ZEB1 3’UTR-mut reporter plasmids, following the manufacturer’s instructions. A dual-luciferase reporter assay system (RG027, Beyotime, China) was applied to evaluate the Firefly and Renilla luciferase activities 48 hours after transfection.

RNA immunoprecipitation assay

After being treated according to the experimental protocol, 1 × 10⁷ cells were collected and spun for 5 minutes at a speed of 1000 × g. The polysome lysis buffer (900 μL) was mixed with the protease inhibitor (9 μL) and the RNase inhibitor (4.5 μL). DNA was removed from the lysate. The mixture was used to resuspend the cell lysate. Anti-Ago2 antibodies or control IgG was immobilized on magnetic beads. Immunoprecipitation was performed at 4 °C. The recovered RNA was subsequently amplified. The HiScript III 1^st Strand cDNA Synthesis Kit (R312, Vazyme, China) was used to synthesize cDNA. qPCR was performed using the Taq Pro Universal SYBR qPCR Master Mix (Q712, Vazyme, China). The primer information is shown in Supplementary Table 3.

Co-immunoprecipitation

Protein was extracted using the RIPA buffer. Co-immunoprecipitation (CO-IP) assays were carried out using the Magnetic IP/Co-IP Kit (P2179M, Beyotime, China) following the manufacturer's guidelines. Protein A agarose (5 μL) and Protein G agarose (5 μL) were mixed with 500 μL of cell lysate and left to incubate for 1 hour. The supernatant was collected. An additional 500 μL of lysate was incubated with anti-ZEB1 or anti-IRF1 antibodies for 12 hours at 4 °C. Protein A/G magnetic beads were then added and incubated overnight. After centrifugation at 12000 × g and 4 °C for 1 minute, the antigen-antibody complexes were recovered. Following washing, the antigen-antibody complexes were eluted using an elution buffer. WB was employed to detect the expression levels of ZEB1 and IRF1.

In vivo xenograft tumor model

Twenty-four male BALB/c nude mice (approximately 4 weeks old, Vital River Laboratory Animal Technology Co., Ltd., China) were used. Approximately 5 × 10⁷ BGC-823 cells were subcutaneously administered into the right flank of each mouse. When the tumor grew to 100-150 mm³, the mice received treatment based on the following groupings (six mice in each group): Control group, receiving saline gavage (once daily for 4 weeks) and intraperitoneal injection of saline (every 3 days for 4 weeks); 5-FU group, receiving saline gavage (once daily for 4 weeks) and intraperitoneal injection of a 5-FU solution (50 mg/kg, every 3 days for 3 weeks); XSLJZD group, receiving XSLJZD [6.2 g/(kg·day), once daily for 3 weeks][22] and intraperitoneal injection of saline (every 3 days for 4 weeks); and 5-FU + XSLJZD group, receiving XSLJZD [6.2 g/(kg·day), once daily for 3 weeks] and intraperitoneal injection of a 5-FU solution (20 mg/kg, every 3 days for 3 weeks). After treatment, the mice were euthanized, and tumors were excised. Tumor samples were either snap-frozen in liquid nitrogen for WB analysis or preserved in 4% formaldehyde and embedded in paraffin as a preparatory step.

Statistical analysis

ImageJ software was applied to analyze the images. GraphPad Prism 9 (Version 9.5.1) was used for data analysis. Data were presented as mean ± SD from three independent replicates (n = 3). Homogeneity of variance was confirmed using Levene’s test (P > 0.05). Differences were assessed by one-way ANOVA followed by Tukey’s post-hoc test, with significance at P < 0.05. Difference was considered statistically significant if P < 0.05.

RESULTS

XSLJZD suppressed cell viability and proliferation and promoted apoptosis

We treated BGC-823/5-FU cells with XSLJZD at low, medium, and high doses to evaluate its effects on 5-FU-resistant cells. XSLJZD significantly reduced cell viability in a dose-dependent manner, with the high-dose group exhibiting lower viability compared with the control group at 24, 48, and 72 hours (Figure 1A). Colony formation assays indicated that XSLJZD treatment dose-dependently decreased colony formation rates, with the high-dose group showing fewer colonies compared with the control (Figure 1B). Flow cytometry analysis of apoptosis revealed that XSLJZD dose-dependently increased the percentage of apoptotic cells (annexin V-FITC/PI positive), with the high-dose group showing the highest apoptosis rate (Figure 1C). These findings collectively confirm that XSLJZD inhibits cell viability and proliferation while promoting apoptosis in BGC-823/5-FU cells.

Figure 1 Xiangshaliujunzi decoction suppressed cell viability, cell proliferation and promoted apoptosis in a dose-dependent manner. A: Cell viability detected by CCK8 assays; B: Cell proliferation detected by colony formation assays; C: Cell apoptosis detected by flow cytometry. L-Xiangshaliujunzi decoction (XSLJZD): 1.55 g/(kg·day); M-XSLJZD: 3.1 g/(kg·day); H-XSLJZD: 6.2 g/(kg·day). The vertical axis represents optical density at 450 nm from CCK-8 assays (A), normalized to the control at 0 hour (set as 1). ^aP < 0.05, ^bP < 0.01 vs control. One-way ANOVA with Tukey's post-hoc test was used. XSLJZD: Xiangshaliujunzi decoction.

XSLJZD induced apoptosis, necroptosis, and pyroptosis

We used YO-PRO-1/PI staining to further assess XSLJZD’s impact on various forms of programmed cell death (PCD) in BGC-823/5-FU cells. Figure 2A shows representative fluorescence microscopy images, where YO-PRO-1-positive cells (green fluorescence) indicate early apoptosis or necroptosis due to initial membrane permeability, YO-PRO-1/PI double-positive cells (green/red fluorescence) suggest late apoptosis or necrosis, and PI-positive cells (red fluorescence) indicate necroptosis or pyroptosis due to rapid membrane rupture. XSLJZD treatment dose-dependently increased YO-PRO-1-positive and PI-positive cell populations. Quantification of relative fluorescence intensity revealed that the high-dose XSLJZD group showed significantly higher fluorescence for YO-PRO-1 and PI compared with the control group, confirming the enhanced induction of apoptosis, necroptosis, and pyroptosis.

Figure 2 Xiangshaliujunzi decoction induced apoptosis, necroptosis and pyroptosis in a dose-dependent manner. A: Apoptotic and necrotic cells were identified by green fluorescence (YO-PRO-1 positive), while necrotic and pyroptotic cells were identified by red fluorescence (PI positive); B: Bar chart of relative fluorescence intensity. The fluorescence intensity of the control group in the first replicate was set as the reference (value = 1), and intensities of the second and third replicates, as well as all Xiangshaliujunzi decoction (XSLJZD)-treated groups, were normalized to this reference. L-XSLJZD: 1.55 g/(kg·day); M-XSLJZD: 3.1 g/(kg·day); H-XSLJZD: 6.2 g/(kg·day). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control, determined by one-way ANOVA with Tukey's post-hoc test. XSLJZD: Xiangshaliujunzi decoction.

XSLJZD promoted PANoptosis-related protein expression

WB analysis was performed to investigate the effect of XSLJZD on PANoptosis-related protein expression in BGC-823/5-FU cells. Figure 3A shows representative WB bands for pyroptosis-related proteins (cleaved caspase-1, GSDMD-FL, GSDMD-N, GSDME-FL, and GSDME-N), apoptosis-related proteins (cleaved caspase-3, cleaved caspase-7, and cleaved caspase-8), and other PCD-related proteins (p-RIPK1 and p-MLKL). Figure 3B demonstrates the relative protein expression levels. All these proteins were up-regulated in the cells exposed to XSLJZD. Their levels increased with the dose of XSLJZD. By contrast, the levels of pro-caspase-1, pro-caspase-3, pro-caspase-7, pro-caspase-8, RIPK1, and MLKL were unaffected by XSLJZD treatment.

Figure 3 Xiangshaliujunzi decoction promoted the expression of PANoptosis-related proteins. In BGC-823/5-fluorouracil cells treated with Xiangshaliujunzi decoction (XSLJZD), the expression levels of pyroptosis-related proteins (cleaved caspase-1, GSDMD-FL, GSDMD-N, GSDME-FL, GSDME-N), apoptosis-related proteins (cleaved caspase-3, cleaved caspase-7, and cleaved caspase-8), and programmed cell death-related proteins (p-RIPK1, p-MLKL) increased in a dose-dependent manner. A: Western blot bands of PANoptosis proteins; B: Bar chart of relative protein expression levels. L-XSLJZD: 1.55 g/(kg·day); M-XSLJZD: 3.1 g/(kg·day); H-XSLJZD: 6.2 g/(kg·day). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control. XSLJZD: Xiangshaliujunzi decoction.

Secretion of inflammatory factors was increased in cells treated with XSLJZD

We measured the concentration of IFN-γ, TNF-α, IL-1β, and IL-18 in the cell culture medium of different XSLJZD treatment groups and control groups using ELISA. The IFN-γ levels in the culture medium dose-dependently increased with XSLJZD treatment, with the high-dose group exhibiting the highest concentration (Figure 4A). A similar dose-dependent increase was observed for TNF-α secretion (Figure 4B). Figure 4C illustrates elevated IL-1β levels, with significant increases in the medium- and high-dose groups. Figure 4D shows that IL-18 secretion was significantly enhanced in all XSLJZD-treated groups, with the high-dose group showing the greatest increase. These increases in PANoptosis-associated cytokines support the induction of apoptosis, pyroptosis, and necroptosis, as corroborated by YO-PRO-1/PI staining (Figure 2) and WB analysis of specific PCD markers (Figure 3). However, direct evidence of PANoptosome formation was not assessed in this study.

Figure 4 Interferon-γ, tumor necrosis factor-α, interleukin-1β, and interleukin-18 secreted were increased in correlation with the concentration of Xiangshaliujunzi decoction in BGC-823/5-fluorouracil cells treated with Xiangshaliujunzi decoction. A: Interferon-γ; B: Tumor necrosis factor-α; C: Interleukin (IL)-1β; D: IL-18. L-Xiangshaliujunzi decoction (XSLJZD): 1.55 g/(kg·day); M-XSLJZD: 3.1 g/(kg·day); H- XSLJZD: 6.2 g/(kg·day). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control. XSLJZD: Xiangshaliujunzi decoction; IFN: Interferon; TNF: Tumor necrosis factor; IL: Interleukin.

XSLJZD inhibited ZEB1 expression through the up-regulation of miR-200a-3p

On the basis of prior reports of miR-200a-3p downregulation in gastric cancer and its role in chemoresistance via ZEB1, we investigated its regulation by XSLJZD in BGC-823/5-FU cells. We examined the expression of miR-200a-3p and ZEB1 in BGC-823/5-FU cells exposed to XSLJZD. MiR-200a-3p expression was significantly increased after treatment with XSLJZD, whereas ZEB1 expression was decreased. The influence of XSLJZD on miR-200a-3p and ZEB1 expression was dose dependent (Figure 5A and B). Inhibiting miR-200a-3p in the XSLJZD group partially restored ZEB1 expression (Figure 5C). These data imply that ZEB1 expression is influenced by miR-200a-3p.

Figure 5 Xiangshaliujunzi decoction inhibited ZEB1 expression by up-regulation of miR-200a-3p. MiR-200a-3p expression was increased in the Xiangshaliujunzi decoction (XSLJZD) group, while ZEB1 expression was significantly decreased. The effect of XSLJZD on miR-200a-3p and ZEB1 expression was dose-dependent. Inhibiting miR-200a-3p expression in the XSLJZD group partially restored ZEB1 expression. A: MiR-200a-3p and ZEB1 mRNA expression; B: ZEB1 protein expression in different treatment groups; C: MiR-200a-3p and ZEB1 mRNA expression in XSLJZD-treated BGC-823/5-fluorouracil cells transfected with miR-200a-3p or ZEB1 knock down. L-XSLJZD: 1.55 g/(kg·day); M-XSLJZD: 3.1 g/(kg·day); H- XSLJZD: 6.2 g/(kg·day). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control. ^dP < 0.05, ^eP < 0.01 vs XSLJZD + in-negative control (NC). ^fP < 0.01 vs XSLJZD + sh-NC. XSLJZD: Xiangshaliujunzi decoction; NC: Negative control.

XSLJZD affected cell functions via the miR-200a-3p/ZEB1 axis

Cell viability and proliferation were significantly suppressed after treatment with XSLJZD (Figure 6A and B). By contrast, cell apoptosis was significantly increased in the XSLJZD-treated group (Figure 6C). In addition, cell apoptosis/necroptosis (YP1 positive) and necroptosis/pyroptosis (PI positive) were elevated in the XSLJZD-treated group (Figure 6D). The XSLJZD-induced effects were partially abolished after miR-200a-3p inhibitors were transfected into the 5-FU-resistant cells. Moreover, knocking down ZEB1 expression in 5-FU-resistant cells with miR-200a-3p inhibition restored the effects of XSLJZD on cell viability, proliferation, and PANoptosis (Figure 6) to some extent. These results demonstrate that the impacts of XSLJZD on cell viability, proliferation, and apoptosis are mediated partially by the miR-200a-3p/ZEB1 axis.

Figure 6 The effect of Xiangshaliujunzi decoction on cell functions was mediated by the miR-200a-3p/ZEB1 axis. Cell viability and proliferation were significantly decreased, while apoptosis was significantly increased in the Xiangshaliujunzi decoction (XSLJZD)-treated group. Cell apoptosis/necroptosis (YP1 positive) and necroptosis/pyroptosis (PI positive) were also elevated in the XSLJZD-treated group. Transfection of miR-200a-3p inhibitors into BGC-823/5-fluorouracil (5-FU) cells partially reversed the effects of XSLJZD. Knocking down ZEB1 expression in BGC-823/5-FU cells transfected with miR-200a-3p inhibitors partially restored the effects of XSLJZD. A: Cell viability detected by CCK8 assays; B: Cell proliferation detected by colony formation assays; C: Cell apoptosis detected by flow cytometry; D: Cell PANoptosis detected by YO-PRO-1/PI staining. ^bP < 0.01, ^cP < 0.001 vs control. ^dP < 0.05, ^eP < 0.01 vs XSLJZD + in-negative control (NC). ^fP < 0.05 vs XSLJZD + sh-NC. XSLJZD: Xiangshaliujunzi decoction; NC: Negative control.

XSLJZD affected PANoptosis-related proteins and inflammatory factors level via the miR-200a-3p/ZEB1 axis

XSLJZD promoted the expression of pyroptosis-, apoptosis-, and PCD-related proteins without affecting the expression of pro-caspase-1, pro-caspase-3, pro-caspase-7, pro-caspase-8, RIPK1, and MLKL. In addition, XSLJZD upregulated the secretion of IFN-γ, TNF-α, IL-1β, and IL-18 in BGC-823/5-FU cells. The transfection of miR-200a-3p inhibitors into BGC-823/5-FU cells partially abolished the effects of XSLJZD on the expression of PANoptosis-related proteins and inflammatory factors. Furthermore, knocking down ZEB1 n in BGC-823/5-FU cells with miR-200a-3p inhibition partially restored the effects of XSLJZD on PANoptosis-related proteins and inflammatory factor expression (Figure 7).

Figure 7 The effect of Xiangshaliujunzi decoction on the expression of PANoptosis-related proteins and inflammatory factors was mediated by the miR-200a-3p/ZEB1 axis. Xiangshaliujunzi decoction (XSLJZD) enhanced the expression levels of pyroptosis-related proteins, apoptosis-related proteins, and programmed cell death (PCD)-related proteins. XSLJZD upregulated the secretion of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-18 in BGC-823/5-fluorouracil (5-FU) cells. Transfection of miR-200a-3p inhibitors into BGC-823/5-FU cells partially abolished the effects of XSLJZD on the expression of PANoptosis-related proteins and inflammatory factors. Knocking down ZEB1 expression in BGC-823/5-FU cells transfected with miR-200a-3p inhibitors partially restored the effects of XSLJZD on PANoptosis-related proteins and inflammatory factor expression. A: Expression levels of pyroptosis-related proteins, apoptosis-related proteins, and PCD-related proteins; B: IFN-γ, TNF-α, IL-1β, and IL-18 in cell culture medium detected by ELISA. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs Control. ^dP < 0.05, ^eP < 0.01 vs XSLJZD + in-negative control (NC). ^fP < 0.05 vs XSLJZD + sh-NC. XSLJZD: Xiangshaliujunzi decoction; IFN: Interferon; TNF: Tumor necrosis factor; IL: Interleukin.

MiR-200a-3p inhibited ZEB1 expression by binding to ZEB1 3’UTR

According to the dual-luciferase reporter assay results, WT ZEB1 3’UTR reduced luciferase activity, whereas MUT ZEB1 3’UTR had no impact (Figure 8A). RNA immunoprecipitation (RIP) assay was conducted to explore how miR-200a-3p interacts with ZEB1. The enrichment of miR-200a-3p and ZEB1 was observed in the RNA-induced silencing complex after immunoprecipitation using the Ago2 antibody (Figure 8B). Knocking down miR-200a-3p led to an elevation of ZEB1 expression, whereas overexpressing miR-200a-3p decreased ZEB1 expression (Figure 8).

Figure 8 MiR-200a-3p inhibited ZEB1 expression by binding to the ZEB1 3’UTR. A: Interaction between ZEB1 3’UTR and ZEB1 detected by dual-luciferase reporter assays; B: The interaction between miR-200a-3p and ZEB1 detected by RIP assays; C: ZEB1 mRNA expression; D: ZEB1 protein expression. ^aP < 0.01 vs negative control (NC) mimics. ^bP < 0.05 vs anti-IgG. ^cP < 0.05, ^dP < 0.01 vs in-NC. XSLJZD: Xiangshaliujunzi decoction; NC: Negative control.

PANoptosis regulation of ZEB1 was mediated by IRF1

Co-IP revealed that IRF1 was present in the ZEB1 immunoprecipitation complex, and ZEB1 was conversely detected in the IRF1 immunoprecipitation complex, indicating a direct interaction between these two proteins (Figure 9A). Conversely, knocking down ZEB1 expression increased the expression of IRF1 (Figure 9B). Knocking down IRF1 expression in ZEB1 knockdown BGC-823/5-FU cells led to the partial recovery of ZEB1 expression compared with sh-ZEB1 alone, suggesting a negative feedback loop (Figure 9C). Knocking down ZEB1 upregulated the pyroptosis-related proteins (cleaved caspase-1, GSDMD-FL, GSDMD-N, GSDME-FL, and GSDME-N), apoptosis-related proteins (cleaved caspase-3, cleaved caspase-7, and cleaved caspase-8), and other PCD-related proteins (p-RIPK1 and p-MLKL). However, simultaneous knockdown of ZEB1 and IRF1 suppressed the expression of these proteins (Figure 9D). Moreover, the levels of IFN-γ, TNF-α, IL-1β, and IL-18 were increased in the culture medium of ZEB1 knockdown cells but decreased when ZEB1 and IRF1 were knocked down (Figure 9E).

Figure 9 Regulation of ZEB1 to PANoptosis was mediated by IRF1 was present in the ZEB1 immunoprecipitation complex, and conversely, ZEB1 was detected in the IRF1 immunoprecipitation complex. Knocking down IRF1 expression in ZEB1 knockdown BGC-823/5-fluorouracil (5-FU) cells resulted in an increased expression of ZEB1. Knocking down ZEB1 expression also led to an increased expression of IRF1 ZEB1 knockdown in BGC-823/5-FU cells upregulated the expression of PANoptosis-related proteins. Simultaneous knockdown of both ZEB1 and IRF1 suppressed the expression of PANoptosis-related proteins. Levels of interferon (IFN)-γ, tumor necrosis factor (TNF-α), interleukin (IL)-1β, and IL-18 were increased in the culture medium of ZEB1 knockdown cells but decreased when both ZEB1 and IRF1 were knocked down. A: Immunoprecipitation complex of ZEB1 and IRF detected by co-immunoprecipitation; B: IRF1 expression in ZEB1 knock down and ZEB1/IRF1 knock down cells; C: ZEB1 expression in ZEB1 knock down and ZEB1/IRF1 knock down cells; D: PANoptosis-related proteins expression in ZEB1 knock down and ZEB1/IRF1 knock down cells; E: IFN-γ, TNF-α, IL-1β, and IL-18 secretory in culture medium of ZEB1 knock down and ZEB1/IRF1 knock down cells. ^aP < 0.05, ^bP < 0.01 vs sh-negative control (NC). ^dP < 0.05, ^eP < 0.01 vs sh-ZEB1 + sh-NC. XSLJZD: Xiangshaliujunzi decoction; NC: Negative control.

Combination of XSLJZD and 5-FU promoted miR-200a/ZEB1/IRF1-mediated PANoptosis in transplanted tumor tissues from mice

Tumor tissues from BALB/c nude mice (n = 6 per group) were excised and snap-frozen in liquid nitrogen for protein extraction. The levels of miR-200a-3p, ZEB1, and IRF1 mRNA in tumor samples were analyzed by real-time quantitative PCR. In contrast to the control group, the 5-FU and XSLJZD treatment groups exhibited higher miR-200a-3p and IRF1 mRNA expression and lower ZEB1 mRNA expression. In addition, the combination treatment group (5-FU + XSLJZD) showed even higher levels of miR-200a-3p and IRF1 mRNA and lower ZEB1 mRNA compared with the individual 5-FU and XSLJZD groups (Figure 10A). Further analysis of PANoptosis-related proteins and inflammatory cytokines in tumor tissues revealed that the 5-FU and XSLJZD groups had significantly increased expression of cleaved caspase-1, GSDMD-FL, GSDMD-N, GSDME-FL, GSDME-N, cleaved caspase-3, cleaved caspase-7, cleaved caspase-8, p-RIPK1, and p-MLKL. The combination treatment group (5-FU + XSLJZD) showed even further increased levels of these proteins compared with the 5-FU and XSLJZD groups. However, the expression of the precursor forms of these proteins, including pro-caspase-1, pro-caspase-3, pro-caspase-7, pro-caspase-8, RIPK1, and MLKL, did not show significant changes across the groups (Figure 10B). Moreover, the concentrations of IFN-γ, TNF-α, IL-1β, and IL-18 were significantly increased in the 5-FU and XSLJZD groups. The combination treatment group (5-FU + XSLJZD) further increased the levels of these inflammatory cytokines compared with the 5-FU and XSLJZD groups (Figure 10C).

Figure 10  The combination of Xiangshaliujunzi decoction and 5-fluorouracil promoted miR-200a/ZEB1/IRF1-mediated PANoptosis in transplanted tumor tissues in mice. The combination treatment group [5-fluorouracil (5-FU) + Xiangshaliujunzi decoction (XSLJZD)] showed higher levels of miR-200a-3p and IRF1 mRNA and lower ZEB1 mRNA compared to the individual 5-FU and XSLJZD groups. The combination treatment group (5-FU + XSLJZD) showed further increased levels of PANoptosis-related proteins and inflammatory cytokines [interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-18] compared to the 5-FU and XSLJZD groups individually. A: MiR-200a-3p, IRF1 mRNA and lower ZEB1 mRNA expression; B: PANoptosis-related proteins expression; C: IFN-γ, TNF-α, IL-1β, and IL-18 expression. ^aP < 0.05, ^bP < 0.01 vs control/sh-negative control. ^dP < 0.05, ^fP < 0.001 vs 5-FU. ^gP < 0.05 vs XSLJZD. XSLJZD: Xiangshaliujunzi decoction; NC: Negative control; 5-FU: 5-fluorouracil.

DISCUSSION

In China, the traditional Chinese decoction XSLJZD has been widely used to alleviate treatment-related adverse events in patients with cancer, particularly chemotherapy-induced nausea and vomiting[23]. However, its antitumor effects are rarely reported. In this study, we found that XSLJZD exerts an antitumor effect on 5-FU-resistant gastric cells by suppressing cell viability and proliferation and promoting PANoptosis in a dose-dependent manner. Our findings suggest that XSLJZD may help increase sensitivity to 5-FU in gastric cancer. In vitro assays demonstrated that XSLJZD dose-dependently inhibited cell viability and induced PANoptosis in BGC-823/5-FU cells, primarily through miR-200a-3p upregulation and ZEB1 downregulation. By contrast, in vivo experiments revealed pronounced miR-200a-3p upregulation in the combination group, likely attributable to the tumor microenvironment’s influence, including the immune modulation and systemic factors absent in the cell culture. This discrepancy highlights the complementary nature of these approaches: In vitro models isolate molecular pathways, whereas in vivo settings capture holistic responses. Our findings introduce a novel IRF1-mediated mechanism, emphasizing XSLJZD’s unique role in TCM-integrated therapy. Clinical evidence supports XSLJZD’s benefits, including improved quality of life, reduced nausea, and prolonged progression-free survival in advanced gastric cancer when combined with chemotherapy. An ongoing clinical study is exploring the effects of XSLJZD combined with S-1 in gastric and colorectal cancer and will further validate the antitumor effects of XSLJZD in a clinical setting[24]. Our study also demonstrates that XSLJZD may overcome 5-FU-resistance by promoting PANoptosis. The involvement of PANoptosis in chemotherapy resistance has been previously reported. A recent work indicated that lncRNA FLJ20021 enhances the transcriptional stability of CDK1 mRNA in the nucleus, which subsequently promotes ZBP-1-mediated PANoptosis, helping overcome resistance to cisplatin in liver cancer cells[24]. In colorectal cancer, the PANoptosis-related lncRNA SNHG7 contributes to the development of irinotecan resistance[25]. These previous studies on the relationship between PANoptosis and chemotherapy resistance are consistent with our findings.

We further explored the regulatory mechanism of XSLJZD on PANoptosis. Previous studies showed that miRNAs contribute to the formation of 5-FU resistance in gastric cancer. Some miRNAs play an oncogenic role, promoting 5-FU resistance. For instance, miR-BART20-5p promotes 5-FU resistance by downregulating BAD in gastric cancer cells[26]. Similarly, miR-193-3p enhances 5-FU resistance by inhibiting PTEN expression[27], and miR-17 induces resistance by suppressing DEDD expression[28]. Some miRNAs function as tumor suppressors and help overcome 5-FU resistance. For example, miR-429 decreases resistance to 5-FU by silencing Bcl-2 expression[29], and miR-BART15-3p sensitizes cancer cells to 5-FU by suppressing Tax1-binding protein 1 expression[30]. We found that XSLJZD upregulated miR-200a-3p in gastric cancer cells. Inhibiting miR-200a-3p abolished the therapeutic effect of XSLJZD in XSLJZD-treated gastric cancer cells. These results suggest that XSLJZD overcomes, at least partially, 5-FU resistance in gastric cancer cells in a miR-200a-3p-dependent manner, indicating that miR-200a-3p acts as a tumor suppressor in the context of 5-FU resistance in gastric cancer. By binding to the 3’UTR of the mRNA, miRNA regulates gene expression and thereby influences the biological processes of cancer cells. Through online predictive software, ZEB1 was identified as one of the potential targets of miR-200a-3p. We further confirmed the interaction between ZEB1 and miR-200a-3p using dual luciferase and RIP assays. Previous studies pointed out that ZEB1 induces chemoresistance by promoting the clearance of chemotherapy-induced DNA damage[31]. The high ZEB1 expression in patients with breast cancer has been associated with poor response to chemotherapy[32]. Similarly, ZEB1 expression is elevated in cisplatin-resistant ovarian cancer cells, and its knockdown increases sensitivity to cisplatin[33].

We demonstrated that XSLJZD overcomes 5-FU resistance by upregulating miR-200a-3p. MiR-200a-3p inhibition or ZEB1 knockdown diminished the effect of XSLJZD, suggesting that XSLJZD reverses 5-FU resistance via the miR-200a-3p/ZEB1 axis. These results align with previous reports on the role of the miR-200a-3p/ZEB1 axis in promoting chemoresistance. How miR-200a-3p/ZEB1 regulates PANoptosis warrants further investigation. IRF1 plays a critical role in PANoptosis by regulating the formation of the NLRP12-PANoptosome, which mediates PANoptosis development in response to TNF and IFNγ[34,35]. In respiratory virus infection, ZEB1 silences IRF1 expression[36]. However, our study revealed that a ZEB1/IRF1 complex is present in 5-FU-resistant cells. Knocking down ZEB1 led to an increase in IRF1 Levels. Interestingly, silencing IRF1 also elevated ZEB1 expression, suggesting the presence of a regulatory loop between ZEB1 and IRF1. The precise mechanisms underlying this mutual regulation remain unknown and need further exploration.

In summary, our study demonstrated that XSLJZD helps reduce 5-FU resistance in gastric cancer. The underlying mechanism involves XSLJZD promoting miR-200a-3p expression, which in turn induces PANoptosis through the miR-200a-3p/ZEB1/IRF1 axis. However, several limitations must be acknowledged. First, the standalone effects of miR-200a-3p inhibition or ZEB1 knockdown without XSLJZD were not evaluated, potentially overlooking their independent contributions to resistance. Second, the unequal 5-FU doses in vivo (50 mg/kg monotherapy vs 20 mg/kg combination) may confound the interpretations of synergy vs additivity. Third, pharmacodynamic data, including tumor growth curves, weights, and survival rates, were not comprehensively collected because of resource constraints. Finally, PANoptosome assembly was not directly confirmed, and no specific inhibitors were used to dissect individual cell death pathways. Future studies should address these gaps by incorporating equivalent doses, additional models, pathway-specific inhibitors, and histological analyses to strengthen mechanistic insights and clinical translation. The efficacy of XSLJZD in overcoming 5-FU resistance must also be validated through prospective, randomized controlled clinical trials.

CONCLUSION

Our study demonstrates that XSLJZD overcomes 5-FU resistance in gastric cancer by upregulating miR-200a-3p, which in turn suppresses ZEB1 expression, thereby relieving the ZEB1-mediated inhibition of IRF1 and inducing PANoptosis. In vitro experiments showed that XSLJZD inhibits cell viability and proliferation while promoting apoptosis, necroptosis, and pyroptosis in 5-FU-resistant BGC-823/5-FU cells. This effect is mediated through the miR-200a-3p/ZEB1/IRF1 axis. In vivo xenograft results supported these findings with molecular evidence of increased levels of PANoptosis-related proteins and cytokines. However, limitations include the lack of in vivo pharmacodynamic data, standalone effects of miR-200a-3p/ZEB1 manipulations, and direct PANoptosome confirmation due to resource constraints. Future clinical trials are essential to validate XSLJZD as a therapeutic strategy for overcoming chemoresistance in gastric cancer.

ACKNOWLEDGEMENTS

The authors express their appreciation to staff in the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, for their technical assistance.