Qige San regulates paclitaxel resistance in esophageal cancer by targeting ferroptosis

Abstract

BACKGROUND

Abnormal iron metabolism plays a critical role in paclitaxel (PTX) resistance in esophageal cancer cells. Qige San (QG) is a traditional Chinese herbal formula that is reported to improve short-term therapeutic effects of esophageal cancer.

AIM

To investigate the effects and regulatory mechanisms involved in QG-targeted PTX-resistant esophageal cancer cells.

METHODS

Cell viability was assessed using the Cell Counting Kit-8 assay. Ferroptosis was evaluated by analyzing lipid reactive oxygen species accumulation and the Fe²⁺ concentration in PTX-resistant esophageal cancer cells. Expression of ferroptosis regulators was measured by western blot. Network pharmacology analysis was employed to identify potential targets of QG in PTX-resistant esophageal cancer cells.

RESULTS

Treatment with QG significantly suppressed the viability, proliferation, and migration of PTX-resistant esophageal cancer cells and simultaneously induced ferroptosis. The network pharmacology analysis identified the phosphoinositide 3-kinase (PI3K)/protein kinase B signaling pathway as the potential target of QG in PTX-resistant esophageal cancer cells. Activation of the PI3K pathway notably reversed the ferroptosis of PTX-resistant esophageal cancer cells that was induced by QG.

CONCLUSION

QG could repress the resistance of esophageal cancer cells to PTX via targeting the PI3K signaling pathway.

Key Words: Esophageal cancer; Paclitaxel resistance; Qige San; Network pharmacology; Ferroptosis

Core Tip: Abnormal iron metabolism plays a critical role in paclitaxel-resistance of esophageal cancer cells. Qige San (QG) is a traditional Chinese herbal formula that is reported to improve short-term therapeutic effects of esophageal cancer. We observed that QG could repress the resistance of esophageal cancer cells to paclitaxel via targeting the phosphoinositide 3-kinase/protein kinase B signaling pathway.

INTRODUCTION

Esophageal cancer is a common malignant tumor of the digestive tract that poses a serious threat to human health[1]. According to statistical reports, esophageal cancer currently ranks as the sixth-leading cause of cancer-related deaths worldwide with a 5-year overall survival rate of only 20%-40%[1]. China is a high-incidence area for esophageal cancer, and esophageal squamous cell carcinoma (ESCC) accounts for 95% of all esophageal cancer cases in the country[2,3]. Although advances in early diagnosis, radical surgery, and postoperative adjuvant therapies have improved patient outcomes in recent years, high mortality rates and distant organ metastasis continue to limit the effectiveness of clinical treatments[4-6]. Therefore, esophageal cancer treatment remains a significant challenge.

Chemotherapy can reduce the tumor size to some extent and induce cancer cell death. Unfortunately, long-term chemotherapy use may lead to drug resistance and tumor recurrence. The development of drug resistance not only severely limits the clinical application of chemotherapeutic agents but also enhances tumor metastasis[6]. More importantly, cancer cells can acquire cancer stem cell-like properties during the process of developing drug resistance, which plays a critical role in chemotherapy-resistant relapse and the progression of cancer[6].

Ferroptosis, an iron-dependent programmed cell death driven by lipid peroxidation, has emerged as a key mechanism influencing chemotherapy resistance in ESCC[7-9]. Recent studies revealed that overexpression of glutathione peroxidase 4 (GPX4) and SLC7A11, critical regulators of ferroptosis, correlated with paclitaxel (PTX) resistance in clinical ESCC specimens[10]. Conversely, iron chelation therapy partially restored chemosensitivity[11], suggesting that targeting ferroptosis could overcome drug resistance. Notably, GPX4 is upregulated in 68% of ESCC tumors compared with adjacent normal tissues, and its inhibition synergizes with cisplatin to suppress growth[12]. Similarly, SLC7A11-mediated cystine uptake sustains glutathione synthesis, protecting ESCC cells from ferroptotic death[13]. These findings position ferroptosis modulation as a promising strategy for resistant ESCC.

Qige San (QG) is a traditional Chinese herbal formula first recorded in the book “Yixue Xinwu.” Its primary functions are moistening dryness, relieving stagnation, resolving phlegm, and counteracting upward rebellion[14]. It is traditionally used to treat dysphagia and obstruction symptoms, such as difficulty swallowing, vomiting after eating, morning food intake followed by evening vomiting, gastric distension and pain, red tongue with little moisture, and dry stools. The main ingredients of QG include Radix Glehniae (Sha Shen, rich in tanshinones with demonstrated proferroptotic activity in lung and liver cancers), Salvia miltiorrhiza (Dan Shen), Poria (Fu Ling), Bulbus Fritillariae Cirrhosae (Chuan Bei Mu, core removed), Curcuma aromatica (Yu Jin), Amomum Shell (Sha Ren Ke), Lotus Pedicel (He Ye Di), and Bran (Chu Tou Kang)[15]. This formula was prioritized based on both clinical tradition and modern compound screening: Tanshinone IIA from Salvia miltiorrhiza directly binds to GPX4 to inhibit its activity; and emodin (present in Rheum officinale, a common QG additive) enhances iron accumulation via nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy[16].

In cancer treatment QG has shown potential clinical applications. When used in combination with chemotherapy for esophageal cancer, it has been found to improve short-term therapeutic effects and enhance patient quality of life[16]. Compared with chemotherapy alone, the combination of QG and chemotherapy demonstrated better efficacy[17]. Additionally, QG and its modified formula have shown promise in preventing and managing radiation esophagitis as they can reduce the severity of esophagitis, delay its onset, and shorten symptom duration, thereby ensuring the continuity of treatment[17]. The antitumor effects of QG are believed to be mediated through immune regulation. In vitro studies have explored its immunomodulatory effects, providing objective evidence for its use in cancer treatment. Research indicates that QG and its components can inhibit tumor angiogenesis by suppressing growth factor-PLCγ1 signaling, thereby reducing tumor cell proliferation and tumor growth[17]. However, whether QG can reverse PTX resistance in ESCC by inducing ferroptosis, and whether this process involves key signaling pathways such as phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), remains unclear.

This study aimed to investigate the effects of QG on the induction of ferroptosis in PTX-resistant ESCC cells, validate the involvement of the PI3K/Akt pathway in QG-mediated chemosensitization, and identify the bioactive components of QG using network pharmacology.

MATERIALS AND METHODS

Cell culture

The esophageal cancer cells TE-1 and EC109 were incubated in DMEM medium supplemented with 10% fetal bovine serum. Cells were maintained in a humidified incubator at 37 °C with 95% air and 5% CO₂. PTX-resistant cell lines, TE-1/PTX and EC109/PTX, were established by our laboratory. Briefly, TE-1 and EC109 cells were initially exposed to 1 nM of PTX for 1 week. When cells returned to a normal growth rate after the period of recovery, the concentrations of PTX were gradually increased (5 nM, 10 nM, 20 nM, and 40 nM) until cells became resistant to 40 nM of PTX after approximately 10 months. The obtained PTX-resistant esophageal cancer cells were treated with QG (50 μmol/L), PI3K activator (740 Y-P, 20 μmol/L), and Ferrostatin-1 (Fer-1) (5 μmol/L) for 24 h.

Cell viability

The proliferation of PTX-resistant esophageal cancer cells was evaluated using the Cell Counting Kit-8 (CCK-8; Beyotime, China) according to the manufacturer’s instructions. Cells were seeded into 96-well plates at a density of 5000 cells/well and incubated overnight. After the specified treatments 10 μL of CCK-8 reagent was added to each well. The absorbance at 450 nm (OD450) was measured 24 h post-treatment using a microplate reader (Thermo, United States).

Cell proliferation

For the colony formation assay, cells (1 × 10³) were first mixed with culture medium (1.5 mL) and placed onto a 6-well plate. After 3 weeks of incubation, colonies were stained with crystal violet (SolarBio, China). A microscope (Nikon, Japan) was used to visualize and count the colonies.

Cell migration

For the cell invasion assay 5 × 10⁴ cells in serum-free media were placed in the top chamber (354480, BD Biosciences, San Jose, CA, United States), while complete media was added to the bottom chamber. After incubation for 24 h at 37 °C, the medium was removed, and the chambers were fixed with methanol for 30 min. Subsequently, the chambers were stained with crystal violet for another 30 min.

Detection of ferroptosis

The intracellular iron and Fe²⁺ levels were quantified using an Iron Assay Kit (ab83366) following the manufacturer’s instructions. Malondialdehyde (MDA) is the end product of lipid peroxidation and was assessed using a lipid peroxidation (MDA) assay kit (#MAK085, Sigma). Cells were homogenized on ice in MDA lysis buffer, followed by centrifugation at 13000 × g for 3 min. The MDA in the cell lysate reacted with thiobarbituric acid solution to form an MDA-thiobarbituric acid adduct, which exhibited colorimetric absorbance at 532 nm. The intensity of the color was proportional to the MDA concentration present in the sample.

Western blot assay

Total protein was extracted from cells using RIPA lysis buffer containing protease and phosphatase inhibitors (Roche), and protein concentrations were determined using a bicinchoninic acid assay kit (Pierce). Equal amounts of protein (30 μg per lane) were resolved by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore). Membranes were blocked with 5% skim milk in TBST for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: Anti-GPX4 (Abcam, ab125066, 1:2000); anti-SLC7A11 (Abcam, ab175186, 1:2000); anti-acyl-CoA synthetase long chain family member 4 (ACSL4) (Proteintech, 22401-1-AP; 1:1000); anti-transferrin receptor 1 (TFR1) (Abcam, ab84036, 1:2000); anti-NCOA4 (Santa Cruz, sc-373739, 1:500); anti-phospho-Akt (Ser473) (Cell Signaling Technology, #4060, 1:1000); and anti-total Akt (Cell Signaling Technology, #4691, 1:1000). After washing three times with TBST, membranes were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit, Sigma, A0545, 1:20000; goat anti-mouse, Sigma, A6154, 1:2000) for 1 h at room temperature. Protein bands were visualized using ECL detection reagent (Bio-Rad) on a ChemiDoc Imaging System (Bio-Rad), and band intensities were quantified using ImageJ software (National Institutes of Health, United States). Phospho-Akt levels were normalized to total Akt.

Network pharmacology analysis

To identify the active ingredients of the drug, we used the Traditional Chinese Medicine Systems Pharmacology database, applying the filtering criteria of oral bioavailability ≥ 30% and drug-likeness ≥ 0.18. The UniProt database was then utilized to remove duplicates, standardize target information, and obtain gene abbreviations. Disease-related targets were retrieved from the GeneCards, Therapeutic Target Database, and Online Mendelian Inheritance in Man databases. The common targets shared between disease-related targets and traditional medicine-related targets were visualized using a Venn diagram. These common targets were imported into the STRING database with “Homo sapiens” as the protein species, while the other parameters remained default. A protein-protein interaction network for potential drug targets was constructed. This network was further imported into Cytoscape, where the degree value was used to filter key targets. Gene Ontology (biological process, cellular component, and molecular function) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed using the R package clusterProfiler. A P value < 0.05 was considered statistically significant. The top enriched pathways were visualized in graphical form.

Statistical analysis

The data in this study are presented as mean ± standard error of the mean and analyzed using GraphPad Prism version 7.0 software. The differences between groups were assessed using the Student’s t-test for two groups and one-way analysis of variance for comparisons among multiple groups. A P value of < 0.05 was considered statistically significant. For western blot quantification normalized band intensities (phospho-Akt/total Akt ratio) were analyzed as continuous variables.

RESULTS

QG regulated PTX resistance in esophageal cancer

To investigate the effects of QG on PTX resistance of esophageal cancer, we established drug-resistant esophageal cancer cell lines (TE-1/PTX and EC109/PTX). CCK-8 results verified that TE-1/PTX and EC109/PTX showed notable resistance to PTX treatment compared with the parental TE-1 and EC109 cell lines (Figure 1A). Next, results from CCK-8 and colony formation demonstrated that QG treatment significantly enhanced the in vitro anti-growth effects of PTX against the PTX-resistant cells (Figure 1B and C). Moreover, QG treatment also suppressed the migration of esophageal cancer cells treated with PTX (Figure 1D).

Figure 1 Qige San regulated paclitaxel resistance in esophageal cancer. A: The viability of TE-1/paclitaxel (PTX) and EC109/PTX and parental cancer cells treated with PTX; B: Viability of TE-1/PTX and EC109/PTX cells that treated with PTX and Qige San (QG); C: Proliferation of TE-1/PTX and EC109/PTX cells that treated with PTX and QG; D: Migration of TE-1/PTX and EC109/PTX cells that treated with PTX and QG. Data are mean ± standard error of the mean (n = 3). ^aP < 0.05; ^cP < 0.001 vs control; ^fP < 0.001 vs QG (one-way analysis of variance with Tukey’s test); QG: Qige San; PTX: Paclitaxel; Cont: Control.

QG regulated ferroptosis in esophageal cancer

Next, we analyzed the effects of QG on ferroptosis of drug-resistant esophageal cancer cells. As shown in Figure 2A, treatment with PTX alone did not significantly alter the accumulation of total iron and Fe²⁺ (Figure 2A and B), production of MDA (Figure 2C), and the expression of ferroptosis regulators GPX4 and SLC7A11 (Figure 2D). QG alone enhanced the levels of total iron, Fe²⁺, and MDA and reduced the expression of GPX4 and SLC7A11, suggesting the induction of ferroptosis. The combination of QG and PTX elevated ferroptosis induced by QG. Western blot analysis further confirmed that QG treatment upregulated key ferroptosis drivers (ACSL4, TFR1, NCOA4) while downregulating the p-Akt/t-Akt ratio in both TE-1/PTX and EC109/PTX cells (Supplementary Figure 1), indicating simultaneous activation of ferroptosis and inhibition of PI3K/Akt signaling.

Figure 2 Qige San regulates ferroptosis in esophageal cancer. A: Level of total iron; B: Level of Fe²⁺; C: Level of malondialdehyde; D: Protein expression of glutathione peroxidase 4 and SLC7A11. Data are mean ± standard error of the mean (n = 3). ^cP < 0.001 vs control; ^fP < 0.001 vs Qige San (one-way analysis of variance with Tukey’s test); QG: Qige San; PTX: Paclitaxel; Cont: Control; MDA: Malondialdehyde; GPX4: Glutathione peroxidase 4.

Network pharmacologic analysis of potential targeted signaling pathways of QG in esophageal cancer

Subsequently, we conducted a network pharmacology analysis to explore the potential regulatory mechanisms influenced by QG. Initially, we utilized the Traditional Chinese Medicine Systems Pharmacology database to identify the active compounds and corresponding target genes (Figure 3A). The top five active compounds, selected based on oral bioavailability ≥ 30% and drug-likeness ≥ 0.18, included tanshinone IIA, emodin, curcumin, baicalin, and ginsenoside Rg3, along with their respective targets (Supplementary Table 1). We then leveraged databases such as GeneCards, Therapeutic Target Database, and Online Mendelian Inheritance in Man to identify disease-related targets (Figure 3B). By combining the disease targets from these databases with the drug targets, we identified 40 common genes (Figure 3C).

Figure 3 Network pharmacological analysis of potential targeted signaling pathways of Qige San in esophageal cancer. A: Active compounds and target genes identified by Traditional Chinese Medicine Systems Pharmacology database; B: Overlapped genes of disease targets from GeneCards, Therapeutic Target Database, and Online Mendelian Inheritance in Man database; C: Overlapped genes of disease targets and drug targets; D: Gene Ontology analysis of the overlapped genes; E: Kyoto Encyclopedia of Genes and Genomes analysis of the overlapped genes; F: HALLMARK analysis of the overlapped genes; G: REACTOME analysis of the overlapped genes; H: Protein-protein interaction analysis of the overlapped genes; I: Gene Ontology analysis of the connected genes; J: Kyoto Encyclopedia of Genes and Genomes analysis of the connected genes; K: HALLMARK analysis of the connected genes; L: REACTOME analysis of the connected genes.

Functional analysis of these common genes revealed that they were predominantly involved in critical cellular processes and pathways, including cell death, apoptosis, the p53 pathway, and the PI3K/Akt pathway (Figure 3D-G). Moreover, we performed a protein-protein interaction network analysis of the overlapping genes (Figure 3H) and observed that these genes were primarily enriched in cellular processes such as response to stimuli, intracellular signal transduction, and stress response (Figure 3I). In addition, these genes were associated with several key signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway, NOD-like receptor signaling, proteasome pathway, and PI3K/Akt pathway (Figure 3I-L). These findings suggest that the PI3K/Akt pathway is a potential regulatory signaling pathway involved in the treatment of esophageal cancer by QG.

QG regulated PTX resistance in esophageal cancer cells by targeting ferroptosis through the Akt signaling pathway

To verify that the PI3K/Akt signaling is involved in PTX-resistant cancer cell ferroptosis induced by QG treatment, we treated PTX-resistant esophageal cancer cells with QG alone or in combination with a PI3K activator or ferroptosis inhibitor (Fer-1). As shown in CCK-8 and colony formation results (Figure 4A and B), activation of PI3K and treatment with Fer-1 significantly recovered the viability and proliferation of PTX-resistant esophageal cancer cells. The PI3K activator and Fer-1 treatment also repressed the total iron and Fe²⁺ accumulation (Figure 4C and D) as well as the MDA level (Figure 4E) induced by QG treatment. Moreover, the reduced expression of GPX4 and SLC7A11 under QG treatment was recovered by PI3K activation and Fer-1 treatment (Figure 4F). Importantly, the PI3K activator abolished QG-induced upregulation of ACSL4, TFR1, and NCOA4 (Supplementary Figure 2A) and restored Akt phosphorylation (Supplementary Figure 2B), whereas Fer-1 only partially reversed these effects. This demonstrates that PI3K/Akt signaling is the dominant pathway mediating the ferroptosis-sensitizing effects of QG in PTX-resistant cells.

Figure 4 Qige San regulated paclitaxel resistance in esophageal cancer cells by targeting ferroptosis through the Akt signaling pathway. A: Cell viability was detected by the Cell Counting Kit-8; B: Cell proliferation measured by colony formation assay; C and D: Total iron and Fe²⁺ level in paclitaxel-resistant esophageal cancer cells; E: Malondialdehyde production in paclitaxel-resistant esophageal cancer cells; F: Protein expression of glutathione peroxidase 4 and SLC7A11. Data are mean ± standard error of the mean (n = 3). ^cP < 0.001 vs control; ^fP < 0.001 vs Qige San (one-way analysis of variance with Tukey’s test); QG: Qige San; PTX: Paclitaxel; Cont: Control; MDA: Malondialdehyde; Fer-1: Ferroptosis inhibitor; act: PI3K activator; GPX4: Glutathione peroxidase 4.

DISCUSSION

We demonstrated that QG treatment could induce ferroptosis and relieve drug resistance of esophageal cancer cells. The network pharmacology analysis on the potential targets of QG in esophageal cancer revealed PI3K/Akt signaling as the potential regulatory mechanism. Subsequent in vitro cell experiments confirmed that activation of the PI3K pathway partially impeded the anti-cancer effects of QG on drug resistant esophageal cancer. Notably, QG uniquely upregulated key ferroptosis drivers (ACSL4, TFR1, NCOA4) while suppressing Akt phosphorylation (Supplementary Figure 1), a dual effect not reported for classical ferroptosis inducers like erastin (system Xc^- inhibitor) or RSL3 (GPX4 inhibitor). This multitarget action may explain the superior efficacy of QG in reversing PTX resistance compared with single-agent inducers.

Chemotherapy resistance is a significant challenge in cancer treatment and severely affects the efficacy of chemotherapy drugs, leading to poor prognosis for cancer patients[18]. Various cell process and signaling pathways are involved in the drug resistance[19]. Ferroptosis is an iron-dependent form of cell death characterized by the accumulation of lipid peroxides and rupture of the cell membrane. Recent research has revealed that ferroptosis plays a significant role in cancer drug resistance[20,21]. The concomitant upregulation of ACSL4 and NCOA4 (Supplementary Figure 1A) suggests that QG simultaneously enhances lipid peroxidation and ferritinophagy, two critical ferroptosis subroutines. This contrasts with erastin, which primarily disrupts glutathione synthesis[22].

The multi-component nature of QG raises questions about potential off-target effects. Our network pharmacology identified tanshinone IIA (from Salvia miltiorrhiza) and emodin (common in traditional Chinese medicine formulas) as top candidates responsible for the observed effects: Tanshinone IIA directly binds GPX4 to inhibit its activity[23], consistent with our findings of GPX4 downregulation. Emodin promotes NCOA4-mediated ferritinophagy[24], aligning with increased iron accumulation. While these components likely drive the primary anti-resistance effects, future studies should isolate individual compounds to delineate their specific contributions and minimize off-target interactions.

Cancer cells activate multiple signaling pathways to resist chemotherapy-induced damage, among which PI3K/Akt, MAPK, Wnt/β-catenin, and nuclear factor kappa B are particularly critical[25,26]. While network pharmacology predicted both PI3K/Akt and MAPK as potential targets of QG, our functional validation identified PI3K/Akt as the dominant mediator. This was supported by the near-complete reversal of the effects of QG upon PI3K activation, including restored cell viability, suppressed ferroptosis markers (ACSL4, TFR1, NCOA4), and recovery of Akt phosphorylation. In contrast the MAPK pathway appeared to play a limited, auxiliary role as suggested by only partial rescue with ferroptosis inhibition. Unlike synthetic ferroptosis inducers that do not directly modulate PI3K/Akt, the ability of QG to suppress this pathway may disrupt survival signaling and metabolic reprogramming essential for PTX resistance[27-31].

CONCLUSION

Our study demonstrated that QG overcame PTX resistance in esophageal cancer through a dual mechanism: First, by inducing ferroptosis as evidenced by the upregulation of ACSL4, TFR1, and NCOA4 alongside suppression of GPX4 and SLC7A11; and second, by inhibiting PI3K/Akt survival signaling, indicated by decreased p-Akt levels and the complete reversal of the effects of QG following PI3K pathway activation. The multi-component nature of QG, particularly the presence of compounds such as tanshinone IIA and emodin, enabled coordinated disruption of iron metabolism and lipid peroxidation, setting it apart from single-target ferroptosis inducers like erastin and RSL3. These findings suggest that QG is a promising multitarget therapeutic strategy for overcoming drug resistance in esophageal cancer although further research is needed to isolate its active constituents and optimize its clinical application.