Gastric cancer, also known as stomach cancer, is a leading cause of cancer-related deaths worldwide, particularly in East Asian countries, such as Korea, Japan, and China (Jin et al., 2024; Miliotis et al., 2024). The high incidence and mortality rates associated with gastric cancer can be attributed to several factors, including dietary habits, Helicobacter pylori infection, and genetic predispositions (Linhares et al., 2023; Zhu et al., 2024). Despite advancements in medical treatments, including surgery, chemotherapy, and targeted therapies, the prognosis for patients with advanced gastric cancer remains poor (Fonseca et al., 2022). This is largely due to the aggressive nature of the disease, late-stage diagnosis, and the development of resistance to conventional therapies (Ghazvinian et al., 2023). Consequently, there is a critical need for novel therapeutic agents that are both effective and have minimal side effects. Plant-derived natural compounds have been widely studied for their potential health benefits and therapeutic properties (Kim et al., 2022; Kim et al., 2020). Pandanus tectorius, commonly known as the hala tree or screw pine, is a tropical plant traditionally used in various cultures for its medicinal properties (Wu et al., 2015). The plant has been utilized in folk medicine for its anti-inflammatory, antioxidant, antimicrobial, and wound-healing properties. The leaves, fruits, and roots of P. tectorius contain various bioactive compounds, including flavonoids, tannins, and phenolic acids, that are believed to contribute to its therapeutic effects (Cheng et al., 2022). Although previous studies have indicated the potential anti-cancer properties of P. tectorius extract, its specific effects on gastric cancer cells have not been thoroughly investigated (Del Mundo et al., 2020; Cheng et al., 2022; Anirudhan et al., 2023). The Hippo signaling pathway plays a crucial role in regulating organ size, cell proliferation, apoptosis, and stem cell renewal (Pan et al., 2024). Dysregulation of the Hippo pathway, particularly through over-activation of its downstream effectors, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), has been implicated in the development and progression of various cancers, including gastric cancer. YAP and TAZ function as transcriptional coactivators that promote the expression of genes involved in cell growth and survival (Riffet et al., 2021; Cheng et al., 2024). Elevated levels of YAP and TAZ are often associated with increased tumor aggressiveness and poor prognosis in cancer patients. The Notch signaling pathway is another critical regulator of cell fate determination, differentiation, and proliferation (Kaur et al., 2021; Pan et al., 2024). Aberrant activation of the Notch pathway has been linked to several types of cancer, including gastric cancer (Benjakul et al., 2022). Components of the Notch pathway, such as NOTCH1, HES1, and HES5, are involved in maintaining the balance between cell proliferation and apoptosis (Kim et al., 2022; Xia et al., 2022). Dysregulation of this pathway can lead to uncontrolled cell growth and tumorigenesis (Yousefi et al., 2022).

This study aims to evaluate the anti-cancer effects of P. tectorius extract on AGS gastric cancer cells. Specifically, we investigated the effects of the extract on cell proliferation, migration, and key molecular pathways involved in cancer progression, including the Hippo and Notch signaling pathways. By elucidating the mechanisms underlying the anti-cancer properties of P. tectorius, we hope to identify a potential natural therapeutic agent for gastric cancer.

Cell culture

AGS gastric cancer cells were obtained from the American Type Culture Collection (ATCC) and cultured in the RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ in a humidified atmosphere containing 5% CO₂. The cells were routinely passaged to maintain exponential growth and used for experiments during the logarithmic growth phase.

Preparation of the P. tectorius extract

The powdered P. tectorius was placed in a tube, and methanol was added, maintaining a solid-to-solvent ratio of 1:5 (w/v). The mixture was thoroughly stirred using a magnetic stirrer and then left to stand at room temperature for 48 hours. During this period, the tube was intermittently shaken to facilitate the extraction process. P. tectorius extract was purchased from the Korea Plant Extract Bank (Cheongju, Korea).

Cell viability assay

Cell viability was assessed via water-soluble tetrazolium salt (WST)-1 assay. AGS cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and allowed to adhere overnight. The cells were treated with various concentrations of P. tectorius extract (200, 400, 600, 800, and 1,000 μg/mL) for 24 h. After treatment, the WST-1 reagent was added to each well, and the plates were incubated for an additional 2 h. Absorbance was measured at 450 nm using a microplate reader to determine cell viability. The results are expressed as a percentage of the control values, and the data were analyzed to assess the dose-dependent effects of the extract on cell proliferation.

Wound healing assay

AGS cells were seeded in 6-well plates and cultured until they reached confluence. A linear scratch was created in the cell monolayer using a sterile 200-μL pipette tip. Cells were washed with PBS to remove the detached cells and debris and treated with P. tectorius extract at a concentration of 600 μg/mL in a serum-free medium. Wound images were captured at 0, 6, and 12 h using an inverted microscope. The wound area was measured using the ImageJ software to assess cell migration. The rate of wound closure was calculated as a percentage of the initial wound area to determine the inhibitory effect of the extract on cell migration.

Transwell migration assay

Cell migration was evaluated using transwell chambers with an 8-μm pore size membrane. AGS cells were seeded in the upper chambers at a density of 1 × 10⁵ cells/well in serum-free medium, and the lower chambers were filled with medium containing 10% FBS as a chemoattractant. After 24 h of incubation with P. tectorius extract at a concentration of 600 μg/mL, the cells on the upper surface of the membrane were removed using a cotton swab. Cells that migrated to the lower surface of the membrane were fixed with methanol, stained with crystal violet, and counted under a microscope. The number of migrating cells is expressed as a percentage of the control value to assess the effect of the extract on cell migration.

Western blotting analysis

Protein expression levels of YAP, TAZ, C-Myc, TEA domain transcription factor 4 (TEAD4), HES1, HES5, NOTCH1, and β-actin were analyzed by western blotting. AGS cells were treated with P. tectorius extract at concentrations of 0, 300, and 600 μg/mL for 24 h. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors, and protein concentrations were determined using a BCA protein assay kit. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in PBS-T (PBS with 0.1% Tween 20) for 1 h at room temperature and incubated overnight at 4℃ with primary antibodies against YAP, TAZ, C-Myc, TEAD4, HES1, HES5, NOTCH1, and β-actin. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system and quantified using the ImageJ software.

Quantitative polymerase chain reaction (PCR)

Total RNA was extracted from treated and control cells using TRIzol reagent (Invitrogen), according to the manufacturer's instructions. Reverse transcription was performed using a cDNA synthesis kit to synthesize first-strand cDNA from 1 μg of total RNA. qPCR was performed using the SYBR Premix Ex Taq (TAKARA) on an ABI Prism 7500 Real-Time PCR System (TAKARA). Specific primers for YAP, TAZ, TEAD4, c-Myc, HES1, HES5, NOTCH1, and β-actin were designed using PrimerBLAST (NCBI). The relative expression levels of the target genes were normalized to those of β-actin and calculated using the 2^-ΔΔCt method. Each experiment was performed in triplicate.

Inhibition of cell proliferation by P. tectorius extract

The effects of the P. tectorius extract on AGS gastric cancer cell proliferation were evaluated using the WST-1 assay. The cells were treated with various concentrations of the extract (0, 200, 400, 600, 800, and 1,000 μg/mL) and cell viability was measured at different time points. The results demonstrated significant dose-dependent inhibition of cell proliferation by the extract. AGS cells treated with the P. tectorius extract exhibited a marked reduction in cell proliferation compared to the control group. At lower concentrations (200 and 400 μg/mL), the extract did not significantly affect cell viability. Significantly at concentrations of 600 μg/mL and above, there was a notable decrease in cell proliferation (Fig. 1A). We also investigated the time-dependent effects of P. tectorius extract on cell proliferation at two different time points. The results indicated a clear time-dependent reduction in cell proliferation with increasing extract concentrations (Fig. 1B). These findings suggest that the P. tectorius extract effectively inhibits the proliferation of AGS cells in a dose- and time-dependent manner. The ability of the extract to significantly reduce cell viability at higher concentrations and for longer exposure times indicated its potential as a potent anti-cancer agent for gastric cancer treatment.

Fig. 1. Effect of the Pandanus tectorius extract on AGS cell proliferation. (A) AGS cells were treated with various concentrations of P. tectorius extract (200, 400, 600, 800, and 1,000 μg/mL) for 24 h. Cell viability was assessed using the water-soluble tetrazolium salt (WST)-1 assay, and the results are expressed as the cell proliferation ratio relative to the control. (B) Time-dependent effects of the P. tectorius extract on AGS cell proliferation at 1 h (blue line) and 2 h (black line). Cell viability was measured using the WST-1 assay, and the results are expressed as the cell proliferation ratio relative to the control. Data are represented as the mean ± standard error of the mean (SEM) of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control group.

Inhibition of AGS cell migration by P. tectorius extract

To investigate the effect of P. tectorius extract on the migratory ability of AGS gastric cancer cells, a wound-healing assay was performed. The assay involved creating a uniform scratch in a confluent monolayer of AGS cells and then treating the cells with 600 μg/mL of P. tectorius extract. Wound closure was monitored and imaged at 0, 6, and 12 h to assess the cell migration rate. The data show wound healing progression at different time points. In the control group, cells rapidly migrated to the wound area, leading to significant wound closure. At 0 h, the initial wound areas were similar between the control and treated groups. After 6 h, control cells demonstrated substantial migration into the wound area, significantly reducing the wound gap. After 12 h, the wound area in the control group was nearly closed, indicating the high migratory capacity of AGS cells (Fig. 2A). In contrast, cells treated with 600 μg/mL of P. tectorius extract exhibited a markedly slower wound closure rate. At 6 h, the wound area was significantly larger than that of the control, indicating reduced cell migration. After 12 h, the treated group showed a considerable wound area, demonstrating that the extract effectively inhibited cell migration over time (Fig. 2A). We examined the wound-healing areas at 0, 6, and 12 h. At 0 h, the percentage of wound area was similar between the control and treated groups, confirming the uniformity of the initial scratch. At 6 h, the wound healing area in the control group was significantly reduced to approximately 15%, whereas the treated group retained approximately 25% of the wound area (**P < 0.01). After 12 h, the control group exhibited a further reduction in the wound area to below 10%, whereas the wound area of the treated group was approximately 20% (**P < 0.01), indicating sustained inhibition of cell migration (Fig. 2B). These results suggest that P. tectorius extract significantly impaired the migratory ability of AGS cells. The inhibitory effect of the extract on cell migration was evident both qualitatively, through the reduced wound closure observed in the images, and quantitatively, through the significant differences in the wound healing area at 6 and 12 h compared to the control. This indicated that the extract has potent anti-migratory properties, making it a potential therapeutic agent for inhibiting cancer cell metastasis.

Fig. 2. Effect of the P. tectorius extract on AGS cell migration in a wound healing assay. (A) Representative images of wound healing in AGS cells treated with 600 μg/mL of the P. tectorius extract compared to that in the control group at 0, 6, and 12 h. Images show the initial wound area and progression of wound closure over time. (B) Quantitative analysis of wound healing area expressed as a percentage at 0, 6, and 12 h. Data are represented as the mean ± SEM of three independent experiments. **P < 0.01 compared to the control group.

Effect of P. tectorius extract on cell migration

To further evaluate the effect of P. tectorius extract on the migratory capacity of AGS cells, a transwell migration assay was performed. This assay provides a quantitative measure of the ability of cells to migrate through the cell membrane towards a chemoattractant. We demonstrated that AGS cells migrated through the transwell membrane in both the control and treatment groups. The control group, which was not exposed to the extract, contained a moderate number of cells that had successfully migrated to the lower membrane. In contrast, the group treated with 600 μg/mL of P. tectorius extract showed a lower number of migrated cells, indicating an enhancement in cell migration inhibited by the extract. We illustrated the number of migrated cells per field in both the control and treatment groups. In the control group, the average number of migrating cells per field was approximately 1200. Treatment with 600 μg/mL of P. tectorius extract resulted in a significant decrease in cell migration, with an average of approximately 600 cells per field (*P < 0.05). This significant decrease suggests that the extract has anti-migratory effect on AGS cells, the typical expectation of anti-cancer agents that usually inhibit migration (Fig. 3B). These results indicate that, while P. tectorius extract exhibits antiproliferative properties, it diminishes the migratory capacity of AGS cells. The anti-migratory effect observed in the transwell migration assay is similar to the inhibitory effect on migration observed in the wound healing assay, suggesting that the extract is involved in the migration of cancer cells.

Fig. 3. Effect of the P. tectorius extract on AGS cell migration in a transwell migration assay. (A) Representative images of AGS cells migrating through the transwell membrane in the control group and the group treated with 600 μg/mL of the P. tectorius extract. Images were taken at 20× magnification. (B) Quantitative analysis of cell migration, expressed as the number of migrated cells per field. Data are represented as the mean ± SEM of three independent experiments. *P < 0.05 compared to the control group.

Modulation of oncogenic protein and gene expression levels by P. tectorius extract

To investigate the molecular mechanisms underlying the anti-cancer effects of P. tectorius extract, we examined its effect on the expression of key oncogenic proteins and genes in AGS gastric cancer cells. This study focused on com-ponents of the Hippo signaling pathway, such as YAP, TAZ, and TEAD4, as well as the oncogene c-Myc, which are crucial for regulating cell proliferation, survival, and tumorigenesis (Yuan et al., 2020; Jin et al., 2022). The Hippo signaling pathway controls organ size and suppresses tumor formation by regulating cell proliferation and apoptosis (Heng et al., 2020). YAP and TAZ are downstream effectors of this pathway that, when activated, translocate to the nucleus to promote the expression of growth-related genes in cooperation with transcription factors, such as TEAD4 (Yuan et al., 2020; Lo Sardo et al., 2021). The oncogene C-Myc is integral to cell cycle regulation and metabolism, frequently overexpressed in cancers, driving increased cell proliferation and tumor growth (Giacomini et al., 2020). Quantitative PCR analysis demonstrated that treatment with P. tectorius extract resulted in a significant downregulation of YAP, TAZ, TEAD4, and C-Myc gene expression in AGS cells. Specifically, the extract markedly reduced YAP expression, indicating suppression of this key regulator of the Hippo pathway. Similarly, TAZ expression was significantly decreased, suggesting the inhibition of this critical coactivator in cancer cell proliferation and survival. The expression of TEAD4, which interacts with YAP and TAZ, was also reduced, further supporting the inhibitory effect of the extract on the Hippo signaling pathway. Additionally, the oncogene c-Myc was significantly downregulated, highlighting the potential of the extract to disrupt crucial oncogenic signaling pathways (Fig. 4A). Western blotting corroborated these findings at the protein level. The protein levels of YAP and TAZ were notably lower in the extract-treated group than in the control group, indicating the effective inhibition of these critical components of the Hippo pathway. TEAD4 protein levels were also reduced, further confirming the downregulation of the Hippo pathway activity. The expression of c-Myc protein significantly decreased, demonstrating that the extract effectively targeted and inhibited this potent oncogene (Fig. 4B). Overall, these results show that P. tectorius extract exerts its anti-cancer effects by modulating key oncogenic signaling pathways in AGS cells. Downregulation of YAP, TAZ, TEAD4, and c-Myc at both the mRNA and protein levels suggests that the extract interferes with the Hippo pathway and other oncogenic mechanisms, thereby inhibiting cell proliferation and promoting apoptosis.

Fig. 4. Effects of the P. tectorius extract on oncogenic protein and gene expression levels in AGS cells. (A-D) Quantitative polymerase chain reaction (qPCR) analysis showing the relative gene expression levels of Yes-associated protein (YAP) (A), transcriptional co-activator with PDZ-binding motif (TAZ) (B), TEA domain transcription factor 4 (TEAD4) (C), and C-Myc (D) in AGS cells treated with 600 μg/mL of the P. tectorius extract compared to those in the control. Data are represented as the mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 compared to the control group. (E) Western blotting analysis showing the protein levels of YAP, TAZ, TEAD4, and C-Myc in AGS cells treated with the P. tectorius extract. β-actin was used as a loading control. The results indicate the significant reduction in the protein levels of these oncogenic markers in the treated group.

Modulation of Notch signaling pathway by P. tectorius extract

To further elucidate the molecular mechanisms by which P. tectorius extract exerts its anti-cancer effects, we investigated its impact on the Notch signaling pathway in AGS gastric cancer cells. The Notch signaling pathway is pivotal in the regulation of cell fate determination, proliferation, and apoptosis (BeLow and Osipo, 2020). Dysregulation of this pathway is frequently associated with various cancers, including gastric cancer (Tyagi et al., 2020; Zakiryanova et al., 2021). Key components of this pathway include NOTCH1, HES1, and HES5, which play significant roles in maintaining the balance between cell proliferation and apoptosis (Kim et al., 2020). Quantitative PCR analysis revealed that treatment with P. tectorius extract significantly modulated the expression of genes associated with the Notch signaling pathway. Specifically, the extract downregulated the expression levels of HES1 and HES5 in AGS cells compared to those in the control group. HES1 is a transcriptional regulator that controls cell differentiation and proliferation, and HES5 functions similarly in these pathways. Although NOTCH1 expression was reduced, the change was not significant, indicating a more nuanced regulatory effect of the extract (Fig. 5A). We found that the protein levels of HES1 and HES5 were significantly lower in AGS cells treated with P. tectorius extract than in the control group, confirming the inhibitory effect of the extract on these transcription factors. The protein levels of NOTCH1 were also reduced, but this change was less pronounced, suggesting that the primary targets of the extract within the Notch pathway were the downstream effectors HES1 and HES5 (Fig. 5B). Overall, these results demonstrate that P. tectorius extract modulates the Notch signaling pathway in AGS gastric cancer cells by downregulating key components, such as HES1 and HES5. This modulation likely contributes to the anti-cancer effects of the extract, including inhibition of cell proliferation and induction of apoptosis.

Fig. 5. Effect of the P. tectorius extract on the Notch signaling pathway in AGS cells. (A-C) qPCR analysis showing the relative gene expression levels of HES1 (A), HES5 (B), and NOTCH1 (C) in AGS cells treated with 600 μg/mL of the P. tectorius extract compared to those in the control. Data are represented as the mean ± SEM of three independent experiments. *P < 0.05 compared to the control group. (D) Western blotting analysis showing the protein levels of HES1, HES5, and NOTCH1 in AGS cells treated with the P. tectorius extract. β-actin was used as a loading control. The results indicate the significant reduction in the protein levels of these markers in the treated group.

This study explored the anti-cancer effects of P. tectorius extract on AGS cells, focusing on its impact on cell proliferation, migration, and key molecular pathways involved in oncogenesis, such as the Hippo and Notch signaling pathways. These results provide compelling evidence that P. tectorius extract possesses significant anti-cancer properties, which may be attributed to its ability to modulate critical oncogenic and tumor-suppressive pathways. The WST-1 assay demonstrated dose- and time-dependent inhibition of AGS cell proliferation upon treatment with P. tectorius extract. Higher concentrations of the extract significantly reduced the cell viability, highlighting its potent cytotoxic effects. This finding is consistent with previous studies that have reported the anti-cancer properties of various plant extracts. The ability of the extract to significantly decrease cell proliferation suggests its potential as a therapeutic agent for the management of gastric cancer, in which uncontrolled cell growth is a primary concern. Wound healing and transwell migration assays provided further insights into the effects of the extract on cancer cell behavior. In the wound healing assay, P. tectorius extract significantly inhibited cell migration, as evidenced by slower wound closure in the treated cells. This was further supported by the transwell migration assay, in which the extract significantly reduced the number of migrated cells. These results indicate that the extract not only inhibits cell proliferation, but also impairs the migratory capacity of cancer cells, which is crucial for metastasis. The inhibition of cell migration is particularly important in cancer therapy because metastasis is a leading cause of cancer-related mortality. At the molecular level, we focused on the Hippo and Notch signaling pathways, which play crucial roles in regulating cell growth, apoptosis, and differentiation. The Hippo pathway controls cell proliferation and apoptosis through its downstream effectors YAP and TAZ. Dysregulation of this pathway, leading to overactivation of YAP and TAZ, is commonly associated with cancer. The study found that P. tectorius extract significantly downregulated the expression of YAP, TAZ, TEAD4, and c-Myc at both the mRNA and protein levels. This study revealed that P. tectorius extract significantly reduced the expression of HES1 and HES5, which are key transcriptional regulators of the Notch pathway. Although NOTCH1 expression was reduced, the change was less pronounced, indicating that the extract might exert its effects more strongly on downstream targets, such as HES1 and HES5. Downregulation of these components likely contributes to the ability of the extract to inhibit cell proliferation and induce apoptosis. The exact mechanisms by which P. tectorius extract exerts its anti-cancer effects remain unclear. However, these findings suggest that the extract may interfere with the transcriptional activity of YAP and TAZ, thereby inhibiting the expression of genes that promote cell growth and survival. Additionally, suppression of the Notch signaling pathway can lead to reduced cell proliferation and increased apoptosis. The bioactive compounds present in P. tectorius extracts, such as flavonoids, tannins, and phenolic acids, are possibly responsible for these effects. These compounds exert antioxidant and anti-inflammatory effects, which may contribute to the overall anti-cancer activities of the extract. Given these promising results, P. tectorius extract has potential as a complementary therapeutic agent for the treatment of gastric cancer. Its ability to inhibit key oncogenic pathways and reduce cell proliferation and migration makes it a potential candidate for further investigation. Future studies should aim to isolate and characterize the specific bioactive compounds responsible for these effects and explore their mechanisms of action in more detail. In addition, in vivo studies and clinical trials are necessary to evaluate the safety and efficacy of P. tectorius extracts in clinical settings. Despite these promising findings, this study had several limitations that should be addressed in future research. The experiments were conducted in vitro using the AGS gastric cancer cell line. Although in vitro studies are essential for understanding the molecular mechanisms of action, they do not fully replicate the complexity of in vivo tumor environments. Therefore, it is crucial to validate these findings in animal models and clinical trials to determine the efficacy and safety of this extract in living organisms. Additionally, we did not identify the specific bioactive compounds responsible for the observed anti-cancer effects. P. tectorius contains various phytochemicals and isolating and characterizing these compounds would provide a clearer understanding of the mechanisms of action of the extract. This could also facilitate the development of targeted therapeutic agents derived from the extracts. This study primarily focused on the Hippo and Notch signaling pathways. While these pathways are critical in cancer biology, other signaling pathways may also play significant roles in mediating the effects of P. tectorius extract. Future studies should explore the impact of the extract on a broader range of molecular pathways to gain a comprehensive understanding of its anti-cancer properties. Furthermore, the potential side effects and toxicity of P. tectorius extract were not evaluated. Assessing the cytotoxicity of the extract in normal and noncancerous cells is essential to ensure its safety for therapeutic use. Long-term toxicity studies in animal models are also necessary to evaluate the adverse effects associated with the prolonged use of the extract.

In conclusion, this study provides strong evidence that P. tectorius extract possesses significant anti-cancer properties against AGS cells. The extract effectively inhibited cell proliferation and migration, and modulated key oncogenic pathways, including the Hippo and Notch signaling pathways. These findings highlight the potential of P. tectorius extract as a novel therapeutic agent for gastric cancer treatment. However, further research is necessary to address the limitations of this study, including in vivo validation, identification of other active compounds, elucidation of underlying molecular pathways, and toxicity assessments, and facilitate the therapeutic application of the P. tectorius extract in clinical settings.

None.

No potential conflict of interest relevant to this article was reported.