Abstract
Objective
Gastric cancer (GC) is one of the most prevalent malignant tumors of the digestive system. The advanced metastasis of gastric cancer severely limits the conventional approaches for its treatment, while certain traditional Chinese medicinal compounds have been reported to possess promising abilities in inhibiting tumor metastasis. Such as Poria (PA), known as Fu Ling in Chinese, is a commonly used traditional Chinese medicinal herb derived from Poria cocos, a fungus belonging to the polyporaceae family.
Methods
The proliferation capacity of cells was measured using the MTT assay, while the invasion and migration abilities of cells after treatment with different concentrations of PA were evaluated through wound healing assay and Transwell assay. The differential expression of mRNA was analyzed using qPCR. The in vivo growth of tumors was assessed by subcutaneous tumor formation in mice.
Results
Both in vivo and in vitro experiments have demonstrated that PA significantly inhibits the proliferation of GC. Moreover, in vitro experiments have revealed that PA not only suppresses the invasion and migration of GC cells but also reverses TNF-β-induced EMT. Further experiments have revealed that PA inhibits cell invasion, migration and EMT by inducing ferroptosis in GC cells.
Conclusion
In brief, the present study shows that PA inhibits tumor metastasis by inducing ferroptosis in GC cells. Our findings suggest that PA may have therapeutic potential in GC.
Supplementary Information
The online version contains supplementary material available at 10.1186/s40001-024-02110-0.
Keywords: Poria, Gastric cancer, EMT, Ferroptosis, Tumor metastasis
Introduction
Gastric cancer (GC) ranks as the second leading cause of cancer-related mortality globally, with nearly half of the worldwide cases of hereditary cystic diseases occurring in China [1]. Most GC patients receive diagnoses at advanced stages, resulting in elevated mortality rates worldwide. Despite employing combined treatment strategies such as adjuvant chemotherapy or radiotherapy, GC patients diagnosed at advanced stages and undergoing surgical resection still exhibit dismal prognoses and low 5-year survival rates. Hence, the identification of novel chemopreventive and antitumor agents with enhanced efficacy and reduced toxicity is paramount in GC treatment research.
Epithelial–mesenchymal transition (EMT) denotes a biological process wherein epithelial cells transition to mesenchymal cell types [2], losing epithelial traits like polarity and intercellular connections while acquiring mesenchymal characteristics such as increased migratory and invasive abilities [3]. While EMT plays pivotal roles in normal physiological processes like embryonic development, organogenesis, and wound healing, it also contributes to the onset and progression of various diseases, particularly cancer metastasis and drug resistance [4]. Consequently, investigating EMT holds significant implications for comprehending cancer initiation, progression, and treatment. Efforts are underway to explore interventions targeting the EMT process to hinder cancer metastasis and enhance treatment outcomes [5].
Ferroptosis, a newly recognized form of non-apoptotic cell death, is associated with intracellular ferroptosis accumulation and oxidative stress [6]. Initially described in 2012, ferroptosis has gained widespread attention in recent years [7]. Excessive intracellular ferroptosis can induce elevated oxidative stress, resulting in damage to biomolecules such as lipids, proteins, and nucleic acids, ultimately triggering cell death pathways [8]. Although the precise signaling pathways of ferroptosis remain incompletely understood, it is known to differ from apoptosis signaling pathways. Studies indicate that ferroptosis may implicate multiple signaling pathways, including ferroptosis-dependent hydrogen peroxide generation, ROS-mediated mitochondrial damage, and activation of related cell death proteins. Ferroptosis is implicated in the onset and progression of certain diseases such as certain types of cancer, neurodegenerative diseases, and cardiovascular diseases. In cancer, ferroptosis may influence the survival and death decisions of tumor cells, but further research is necessary to elucidate the specific mechanisms.
Poria (PA) [9], commonly known as Fu Ling in Chinese, is a widely used traditional Chinese medicinal herb derived from Poria cocos, a fungus belonging to the Polyporaceae family [10]. PA primarily grows in China, especially in regions like Hunan, Hubei, Anhui, and Jiangxi. Traditional Chinese medicine has demonstrated that PA can regulate spleen and stomach functions, enhance digestive absorption capacities, and improve digestive system issues such as poor appetite and abdominal distension [11]. PA can also modulate immune system function, boost organismal resistance, and enhance overall immunity [12]. In this study, we employed the GC cell line SGC-7901 and nude mice to assess the efficacy of PA in GC treatment and explore its underlying mechanisms. This endeavor may provide novel insights into the potential therapeutic effects of PA in GC.
Materials and methods
Materials
PA (purity 97% by HPLC) was obtained from Shanghai Yuanye Biotechnology Co., Ltd., China. PA was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored at − 20 °C. SGC-7901 cell-specific culture medium; phosphate-buffered saline (PBS); trypsin; Fetal Bovine Serum (FBS); Transwell permeable supports, 8.0 μm polycarbonate membrane; MTT; E-cadherin, N-cadherin, vimentin, and β-actin antibodies; TIMP-1; anti-rabbit IgG, HRP-conjugated antibody were purchased from Thermo.
Cell culture
The human gastric cancer cell line SGC-7901 and Human Gastric mucosal Epithelial Cells GES1 was obtained from China Procell Life Science & Technology Co., Ltd. (Catalog number: CL-0021). The cells were cultured in SGC-7901 cell-specific culture medium at 37 °C and monitored for growth status over time in an incubator with 5% CO2. When the cell confluence exceeded 80%, the cells were dissociated using trypsin, and passaged every 2 to 3 days.
Cell viability assay
SGC-7901 and GES1 cells in the logarithmic growth phase were suspended as single cells after trypsin digestion and seeded at a density of 3 × 103 cells per well in a 96-well plate. Subsequently, the cells were treated with PA at final concentrations of 0, 25, 50, 100 and 500 μM. Following a 24-h incubation period in a CO2 incubator, 10 μL of 5% MTT solution was added to each well, and the plate was further incubated for 4 h. After discarding the supernatant, 100 μL of DMSO was added to each well. Absorbance values at 490 nm were measured for each well using an automatic microplate reader.
Wound healing assay
SGC-7901 cells in the logarithmic growth phase were digested with trypsin and seeded at a density of 3 × 106 cells per well in 6-well plates. Cultured at 37 °C in a CO2 incubator, when cellular confluence exceeded 80%, a scratch was made on the monolayer using the tip of a 200 μL pipette, followed by PBS washing to remove debris. Subsequently, cells were treated with PA at concentrations of 0, 50, and 100 μM, and incubated further. Images of the scratch area were captured at 0 and 24 h using an inverted microscope, and scratch width was quantified using ImageJ software.
Transwell chamber assay
Matrigel was diluted eightfold in serum-free culture medium and added to Transwell chambers, which were then incubated at 37 °C for 4 h for gel solidification. SGC-7901 cells in the logarithmic growth phase, suspended after trypsin digestion, were seeded at a density of 2 × 104 cells per well in the upper chamber and treated with PA at concentrations of 0, 50, and 100 μM in the lower chamber. Following 24 h of culture, cells were washed with PBS, fixed in methanol, and stained with crystal violet. The number of migrated cells was counted in 5 randomly selected areas per well using an inverted microscope. The remaining steps were consistent with the Transwell invasion assay.
Immunostaining
After SGC-7901 cells treated with specified concentrations of PA for 48 h, cells were fixed with 4% paraformaldehyde. After incubation with 0.1% Triton X-100 for 30 min, cells were blocked with 1% FBS and then incubated with primary antibodies at 4 °C for 12 h. Then, cells were incubated with secondary antibodies for 2 h. Cell nuclei were stained with DAPI for 10 min. Mitochondria and cell nuclei were visualized under a fluorescence microscope, and signal intensity was quantified using ImageJ software.
RT-qPCR
Total RNA was extracted using Trizol (Invitrogen, USA) according to the manufacturer’s instructions. cDNA was synthesized using the Prime-Script RT reagent kit from Thermo Fisher (USA). Quantitative polymerase chain reaction (qPCR) was performed using the SYBR Green PCR kit (QIAGEN, Dusseldorf, Germany) to determine gene expression levels. β-Actin was used as an internal control. The relative mRNA levels of target genes were normalized to β-actin using the 2−ΔΔCT method.
Western blot
Proteins in equal volumes were electrophoresed on SDS-PAGE gels before being transferred to NC membranes. After blocking with 5% skim milk in Tris-buffered saline, the membranes were incubated with the corresponding primary antibodies overnight at 4 °C, followed by an additional 2-h incubation at room temperature on a rotating shaker. Signals were developed using ECL (GE, USA). The grayscale values of the bands were analyzed using image analysis software (Image J), and protein bands were imaged using a gel analysis system.
Animal model
All animal experiments were approved by the Animal Ethics Committee and conducted following the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. Five-week-old female BALB/C nude mice (18–20 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd., China. Prior to tumor establishment, SGC-7901 cells were treated with specified concentrations of PA for 48 h. Euthanasia was performed on animals, and tumors were excised and weighed on day 90 post-implantation for tumor size analysis.
Statistical analysis
All data were obtained from at least three independent experiments. Graph Prism 8.0 software was used for one-way analysis of variance (ANOVA) to determine statistical significance. *P < 0.001 was considered statistically significant.
Results
Inhibition of GC cell growth by PA
The chemical structure of PA is shown in Fig. 1A. Under an inverted microscope, SGC-7901 cells treated with PA exhibited morphological changes, as shown in Fig. 1B. Results indicated that cells in the control group maintained integrity and good adhesion, whereas those in the PA-treated group started to detach, decreased in volume, and some cells collapsed and floated. To assess the in vivo effects of PA, GC cells pre-treated with specified concentrations of PA for 48 h were used to construct a xenograft animal model. SGC-7901 cells were subcutaneously injected into the right flank of nude mice, and after 3 months, secondary tumors from all injected mice were observed and analyzed. As shown in Fig. 1C, D, significant reductions in tumor volume and weight were observed in the PA pre-treated group before the construction of the animal xenograft model, which decreased with increasing PA concentration. This indicates that GC cells pre-treated with PA exhibited weaker tumorigenic and lethal properties, and PA treatment effectively inhibited in vivo GC growth.
Fig. 1.
Inhibition of gastric cancer proliferation by PA in vitro and in vivo. A Molecular structure of PA. B Effects of PA on the morphology of GC cells SGC-7901. C Volume of tumor xenograft model in C57BL/6 mice. D Weight of tumor xenograft model in C57BL/6 mice. All experiments were conducted independently three or more times, and all results are presented as mean ± SD of the control group. Scale bar, 100 μm. *P < 0.001
Inhibition of GC cell proliferation, invasion, and migration by PA
To further elucidate the effect of PA on GC cells, we co-cultured PA with SGC-7901 cells at three concentration gradients of 0, 50, and 100 μM. As shown in Fig. 2A, cell viability gradually decreased with increasing concentrations of PA. After 24 h of culture with 500 µM PA, GES-1 cells were completely dead. This indicates that high concentrations of PA have excessive cytotoxicity. Therefore, we chose 50 and 100 µM for subsequent studies. Meanwhile, we co-cultured 50 µM PA with SGC-7901 and GES-1 cells for 3 days, and cell viability gradually decreased over time. Further analysis of the effect of PA on SGC-7901 cell invasion, as shown in Fig. 2B, C, revealed significant inhibition of cell invasion with increasing PA concentration. Finally, the effect of PA on SGC-7901 cell migration was analyzed. Experimental results, as shown in Fig. 2D, E, demonstrated significant inhibition of cell migration with increasing PA concentration. Thus, PA can inhibit the proliferation, invasion, and migration of SGC-7901 and GES-1 cell cells in a concentration-dependent manner.
Fig. 2.
PA inhibits proliferation, invasion, and migration of GC cells. A Viability analysis of SGC-7901 and GES-1 cells under different concentrations of PA treatment. B, C Representative images and invasive cell counts of SGC-7901 and GES-1 cells under treatment with different concentrations of PA. D, E Representative images and migration rates of SGC-7901 and GES-1 cells under treatment with different concentrations of PA. All experiments were conducted independently three or more times, and all results are presented as mean ± SD of the control group. Scale bar, 100 μm. *P < 0.001
Inhibition of GC cell EMT by PA
The metastasis of tumor cells is associated with EMT. Previous experiments demonstrated that PA can inhibit the migration of GC cells; therefore, we sought to analyze the relationship between PA and EMT in tumor cells. TNF-β is a tumor necrosis factor that can induce EMT in tumor cells by inducing AKT phosphorylation [13]. After co-culturing SGC-7901 cells with TNF-β for 24 h, a decrease in E-cadherin protein expression and an increase in Vimentin protein expression were observed (Fig. 3A). This indicates that the cells were undergoing EMT transformation, and the addition of PA reversed this behavior (Fig. 3B, C).
Fig. 3.
PA reverses TNF-β-induced EMT. A Immunofluorescence staining of E-cadherin and Vimentin in SGC-7901 cells. B Statistical analysis of fluorescence intensity of E-cadherin and Vimentin proteins. C Expression analysis of E-cadherin and Vimentin by PCR. All experiments were conducted independently three or more times, and all results are presented as mean ± SD of the control group. Scale bar, 25 μm. *P < 0.001
Induction of GC cell ferroptosis by PA
Previous experiments demonstrated that when PA was co-cultured with GC cells, changes in cell morphology were observed under bright field microscopy, including decreased mitochondrial size, increased membrane density, and decreased cristae. These morphological changes are highly consistent with the phenomenon of ferroptosis. Therefore, we further analyzed the relationship between PA and ferroptosis. As shown in Fig. 4A–C, with increasing PA concentration, cell GSH and p53 expression increased, while the expression of SLC7A11 and GPX4 decreased, indicating the accumulation of ferroptosis in cells. Additionally, ROS staining also supported this conclusion. After adding PA, cellular ROS increased, while the addition of the ferroptosis inhibitor Fer-1 significantly reduced cellular ROS (Fig. 4D, E). Additionally, we performed WB analysis to detect the expression of ferroptosis-related proteins GPX4 and SLC7A11, as shown in Fig. 4F, G. The results demonstrate that the expression of GPX4 and SLC7A11 significantly decreased after PA treatment, and this effect could be reversed by the addition of a ferroptosis inhibitor. This indicates that PA can induce cell ferroptosis, and Fer-1 can inhibit this phenomenon.
Fig. 4.
PA induces ferroptosis in gastric cancer cells. Expression analysis by qPCR of key factors in ferroptosis: A GSH, B SLC7A11, C GPX4. D Representative images of ROS staining and E statistical analysis of fluorescence intensity in GC cells treated with different concentrations of PA and Fer-1. F WB and G statistical analysis of fluorescence intensity in GC cells treated with different concentrations of PA and Fer-1. All experiments were conducted independently three or more times, and all results are presented as mean ± SD of the control group. Scale bar, 100 μm. *P < 0.001
Inhibition of proliferation, invasion, and migration of GC cells by PA through induction of ferroptosis
To confirm that PA inhibits invasion and migration by inducing ferroptosis, we co-cultured GC cells with the ferroptosis inhibitor Fer-1 and PA. According to MTT data, cells co-cultured with Fer-1 and PA exhibited a much higher survival rate compared to the control group (Fig. 5A). Cell scratch assays and Transwell migration assays showed that the invasion and migration abilities of PA-treated SGC-7901 cells were significantly reduced. Particularly, addition of Fer-1 promoted cell invasion (Fig. 5B, C) and migration (Fig. 5D, E). To further understand how PA regulates cell invasion and migration through ferroptosis, we performed E-cadherin and Vimentin protein staining. As shown in Fig. 5F, G, addition of Fer-1 reversed EMT in cells. These findings suggest that PA reverses EMT and inhibits invasion and migration by inducing ferroptosis.
Fig. 5.
PA suppresses proliferation, invasion, and migration of GC cells by inducing ferroptosis. A Viability analysis of SGC-7901 cells treated with 50 μM PA and Fer-1. B, C Representative images and quantification of cell invasion assays in SGC-7901 cells treated with 50 μM PA and Fer-1. D, E Representative images and quantification of cell migration assays in SGC-7901 cells treated with 50 μM PA and Fer-1. F Immunofluorescence staining of E-cadherin and Vimentin in SGC-7901 cells treated with 50 μM PA and Fer-1. G Statistical analysis of ROS intensity in GC cells treated with different concentrations of PA and Fer-1. All experiments were conducted independently three or more times, and all results are presented as mean ± SD of the control group. Scale bar, 100 μm. *P < 0.001
Discussion
It is generally believed that traditional Chinese medicine (TCM) possesses the characteristics of “multi-component” and “multi-target” [14]. However, TCM comprises numerous ineffective and unidentified components, posing significant challenges in elucidating its mechanism and active ingredients, as well as ensuring its quality and stability. Hence, there is a pressing need to develop TCM formulations composed of identified components [15].
In recent years, various studies have shown that the main bioactive component extracted from PA [16]. PA, has effective anti-tumor effects by inducing cell death and inhibiting cell proliferation in various cancers [17]. However, little is known about PA in GC. In this study, we investigated the anti-tumor effects of PA in the SGC-7901 cell line. PA significantly inhibited the proliferation, invasion, and migration of SGC-7901 cells in a concentration-dependent manner. Furthermore, our in vivo experimental data demonstrated that PA could inhibit tumor growth, indicating its strong anti-tumor effect.
EMT is a phenotype transformation that promotes embryonic development, initially described by developmental biologists [18]. EMT is the process by which epithelial cells lose their connections and polarity during embryogenesis, resulting in the generation of migrating mesenchymal cell types (such as the mesoderm and neural crest) [19, 20]. Next, we analyzed the effects of PA on the migration and invasion of GC cells and conducted related verification through the EMT pathway. Our study showed that after TNF-β treatment, the expression of E-cadherin in the cells decreased, while the expression of Vimentin increased. This behavior could be reversed by adding PA [21]. Meanwhile, an increasing number of studies indicate that EMT is closely related to tumor metastasis.
Increasing scientific evidence suggests that the development of cancer is closely related to uncontrolled cell proliferation and insufficient cell death. Ferroptosis, a recently emerging form of programmed cell death, is also considered a common method of programmed removal of damaged and unnecessary cells in the body [22]. Inducing cell ferroptosis is generally considered an ideal approach for treating various cancer diseases. Our study shows that PA promotes the expression of ferroptosis-related genes GPX4 and SLC7A11 and increases cellular ROS levels, while co-culturing with a ferroptosis inhibitor and PA can reverse this effect. On one hand, PA regulates the growth, migration, and EMT of GC cells by activating ferroptosis in GC cells. On the other hand, the reversal of PA’s regulatory effect by ferroptosis inhibitors indicates a targeted regulatory relationship between PA and ferroptosis. In conclusion, our data suggest that PA inhibits the proliferation, migration, invasion, and EMT of SGC-7901 and GES-1 cells by activating ferroptosis, providing new ideas and directions for the treatment of gastric cancer.
Supplementary Information
Author contributions
G.Z. and X.L. wrote the main manuscript text and were co-first authors and A.A. prepared Figs. 1, 2, 3, 4 and 5 and H.Y. and S.H prepared the experiments and W.J revised the manuscript.
Funding
This study was supported by supported by Xinjiang Uygur Autonomous Region “Tianshan Talent” High-level Medical and Health Talent Training Plan (No. TSYC202301B105).
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
All animal studies have been approved by the Ethics Committee of the Fifth Affiliated Hospital of Xinjiang Medical University (ID: XYDWFYLSk-2023-11) and performed in accordance with the ethical standards.
Competing interests
The authors declare no competing interests.
Footnotes
Guangtao Zheng and Xiaoyan Liu are co-first authors.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
No datasets were generated or analysed during the current study.