GC is a major global health challenge, frequently diagnosed at a late stage, leading to limited choices for therapy and poor prognosis(27). Although endoscopy and biopsy are widely used clinically, their use in population screening is limited. Targeted therapy for GC shows potential for specific subtypes but is constrained by the variation in molecular characteristics and the evolution of drug resistance, necessitating improvement in universal therapeutic efficacy(28). The rate of survival after five years for GC is lower than that of most other cancers, highlighting the need for more effective diagnostic and treatment methods(29). Biomarkers offer potential solutions by providing non-invasive, cost-effective tools for early detection, prognosis, and monitoring of treatment response. For instance, artesunate, originally developed to treat malaria, inhibits GC cell growth in vitro and in vivo. Studies by Zhou X et al. showed that artesunate inhibits tumor growth by inducing calcium overload and regulating vascular endothelial growth factor and calpain-2 expression(30). Similarly, Yan X et al. found that baicalein, a key flavonoid in skullcap, inhibits GC cell migration and invasion by suppressing the p38 signaling pathway(31). Zhao C et al. also demonstrated that ginsenosides, particularly 25-OH-PPD and 25-OCH3-PPD, exhibit significant antitumor effects on GC cells by inducing apoptosis(32). These studies on traditional Chinese medicine ingredients have deepened our understanding of GC pathogenesis and revealed their potential as therapeutic agents for GC.
Catechins, as important polyphenolic compounds in tea, are widely considered to have health benefits, particularly in GC research. Existing studies have highlighted the role of EGCG in inhibiting various biological processes in GC cells. For example, Zhu BH et al. demonstrated that EGCG can downregulate vascular endothelial growth factor (VEGF) expression in GC cells via preventing the signal transducer and activator of STAT3 from functioning, which then inhibits tumor angiogenesis(33). Furthermore, Zhou M et al. found that using chitosan gelatin nanoparticles as a carrier to deliver EGCG can silence the long non-coding RNA TMEM44-AS1, activate the P53 signaling pathway, and significantly enhance the sensitivity of GC cells to the chemotherapy drug 5-fluorouracil (5-FU)(34). In our study, we observed that catechins attenuated the viability of GC cells, increased LDH release, and inhibited cell migration and invasion. These results are in line with the findings of Zhao Y et al., who stated that EGCG inhibits GC cell growth and promotes apoptosis in a dose-dependent manner by downregulating long non-coding RNA LINC00511, upregulating miR-29b, and downregulating histone demethylase KDM2A(35). These studies underscore the potential of catechins in GC treatment and management, particularly as an adjunct to conventional therapies. The ability of catechins to modulate multiple molecular pathways highlights their promise in enhancing the efficacy of existing treatments and improving patient outcomes.
In multicellular organisms, apoptosis, commonly referred to as programmed cell death, is a tightly controlled process that is essential for preserving tissue homeostasis and removing unhealthy or undesired cells(36). Cells go through several morphological changes during apoptosis, such as membrane blebbing, nuclear disintegration, chromatin condensation, and shrinking of the cell(37). This process is orchestrated by molecular events involving various signaling pathways and regulatory proteins, such as death receptors, Bcl-2 family proteins, and caspases. According to Tao K et al.'s research, miR-147b targets CPEB2 to control GC cell proliferation and apoptosis via the PTEN pathway, emphasizing its association with the development and progression of GC(38). Similarly, Zu X et al. found that 2,6-DMBQ, an mTOR inhibitor from fermented wheat germ extract, targets the mTOR pathway in vitro and in vivo, induces apoptosis, and inhibits GC cell growth, demonstrating its potential application in GC treatment(39). Additionally, Zulueta A et al. showed that resveratrol promotes apoptosis and inhibits tumor progression in GC, underscoring its therapeutic potential(40). Consistent with these studies, our findings indicated that Catechins regulate the expression of apoptotic proteins via inducing dose-dependent apoptosis in GC cells. This was evidenced by increased levels of pro-apoptotic proteins Bax, caspase-9, and caspase-3, and decreased levels of the anti-apoptotic protein Bcl-2. These results emphasize the medicinal possibilities of catechins in inducing programmed cell death in GC cells and provide a molecular basis for novel therapeutic strategies for GC.
ER stress occurs when the ER organelle responsible for protein folding becomes overwhelmed with unfolded or misfolded proteins. GRP78 serves as a master regulator, controlling the activation of ER stress pathways(41). One key pathway involves PERK, which phosphorylates eIF2α, thereby reducing protein synthesis to alleviate ER stress(42). Another pathway leads to the upregulation of CHOP, a transcription factor that promotes apoptosis under prolonged ER stress(43). TG, a SERCA inhibitor, induces ER stress by depleting ER calcium stores and triggering the unfolded protein response(44). Additionally, disturbances in calcium homeostasis increase cytosolic calcium levels, further exacerbating ER stress(45). Together, GRP78, PERK, CHOP, TG, and calcium dysregulation represent key elements of the ER stress response, highlighting the intricate network governing cellular proteostasis and apoptosis. Research by Kuang Y et al. revealed that HAND1, a methylated tumor suppressor gene in GC, triggers ER stress and apoptosis via CHOP and BAK, particularly potentiated by cisplatin, suggesting a promising therapeutic target for GC(46). Additionally, Liu L et al. found that evodiamine induces cytotoxicity in human GC cells through the TRPV1/Ca²⁺ pathway, involving ROS generation, mitochondrial dysfunction, and ER stress activation(47). Similarly, Kim TW et al. discovered that cinnamaldehyde causes GC cells to experience ER stress-mediated cell death through the PERK-CHOP axis and Ca2+ release, while also promoting autophagy-mediated cell death through G9a inhibition(48). In our study, catechin-induced ER stress in GC cells led to increased intracellular Ca²⁺ levels. This stress-activated ER stress-related proteins, indicating its cytotoxic effects. Additionally, catechin-mediated apoptosis involved GRP78-containing exosomes, which increased GRP78 expression. Co-treatment with TG enhanced apoptosis and upregulated CHOP, p-PERK, GRP78, and cleaved caspase-3 levels. Focusing on ER stress proteins like GRP78, PERK, and CHOP inhibited catechin-induced apoptosis, indicating their involvement in regulating ER stress responses and cell survival in GC cells.
ROS are highly reactive molecules that play crucial roles in cell signaling and homeostasis(49). However, excessive ROS production leads to oxidative stress and damage to cellular components, contributing to various diseases(50). Cells employ antioxidant defense mechanisms to regulate ROS levels. NAC and DPI have commonly used antioxidants that scavenge ROS(51). NOX4 is an enzyme that generates ROS as a byproduct of its catalytic activity(52). DPI inhibits NOX4 activity, thereby reducing ROS levels, while NAC acts as a ROS scavenger(53). Studies by Wang L et al. reveal that NOX4 drives GC progression by increasing ROS levels, promoting tumor invasiveness and proliferation(54). Manipulating NOX4 alters ROS production, influencing cancer cell behavior. Crucially, NOX4-overexpressing cells exhibit heightened responsiveness to inducers of ferroptosis, suggesting a potential therapeutic strategy for GC. Tang CT et al. indicate that NOX4-generated ROS regulates GC cell proliferation and apoptosis via the GLI1 pathway, highlighting NOX4 as a potential therapeutic target(55). Additionally, Lu Y et al. demonstrate that myricetin induces ferroptosis in GC by targeting NOX4, suggesting its potential as a therapeutic agent(56). Our study found that catechins induced GC cell apoptosis by increasing ROS levels and inducing ER stress. NAC and DPI inhibited catechin-induced ROS elevation and ER stress, thereby maintaining cell viability. Additionally, catechins promoted ER stress-mediated apoptosis through NOX4-induced ROS in GC cells. This suggests that understanding the interactions between ROS, DPI, NAC, and NOX4 is crucial for elucidating their roles in GC and developing therapeutic interventions.