The Emerging Role of Herbal Medicines in Cancer by Interfering with Posttranslational Modifications

Roles and anticancer effects of herbal therapy

Traditional herbal medicines are reported to successfully play prominent roles in the strategy to contain and treat various human diseases, such as mental disorder, cardiovascular diseases, skin diseases, diabetes, jaundice, and hypertension. Despite the accumulated collected information into pharmacological properties and written evidence dating back over 5,000 years, the major use of herbal medicines and/or their formulae is for human health promotion and therapy for chronic conditions. During the past two decades, the use of herbal medicine has also greatly increased public interests in developed countries with their own manifestation in different cultures. Around 80% of population were utilized agents from natural plants or nutrients concurrently with traditional chemotherapy and/or radiation therapy (Amrati et al., 2021; Torre et al., 2015; Ullah et al., 2022), and about 50% of approved antitumor drugs that exhibit significant anticancer efficacy were obtained from natural products (Talib et al., 2022).

Herbal therapy has been globally practiced for many years. According to origin, evolution, and forms of usage, herbal medicines (also called herbalism, phytomedicine, or phytotherapy) are classified into four categories by the World Health Organization, containing (1) indigenous herbal medicines, (2) herbal medicines in systems, (3) modified herbal medicines, and (4) imported products with a herbal medicine base (Sammons et al., 2016). So far, a series of their bioactivations have been demonstrated, such as antiviral, antioxidant, hypoglycemic, anti-inflammatory, and immunostimulatory response, cardioprotective, and nephroprotective (Balsano and Alisi, 2009; Guo et al., 2022; Koehler et al., 2020; Zhang et al., 2021a). Their importance and popularity right now are recognized as important complementary and alternative remedies in modern clinical practice. Astragalus membranaceus (traditionally known as Huang qi) is commonly used for preventing acute respiratory tract infections in children, diabetes, cardiovascular disease, and renal disease (Ahmed et al., 2007; Gong et al., 2018; Huang et al., 2019; Su et al., 2016). The Nobel Prize in physiology and medicine was awarded to Youyou Tu in 2015 for the antimalarial drug artemisinin (qinghaosu) as a treatment for fever and malaria (Tu, 2011). Radix Astragali, Ginkgo biloba, Ginseng, Gynostemma pentaphyllum, and Ganoderma lucidum are recognized as antiaging phytotherapeutics (Phu et al., 2020). Quercetin, wogonin and rutaecarpine are the main bioactive components of Er Miao San in the treatments of human rheumatoid arthritis (RA), which plays an essential role in anti-inflammation via suppressing inflammatory cytokines (Guo et al., 2022).

Although clinically determined and improved safety, efficacy, and quality, there are also some concerns about the underlying mechanism of herbal medicines in cancer treatment. Considering that the majority of herbal medicines were prepared from decoction, some researchers also reported that herbal medicines can initiate toxic effects (Chaachouay et al., 2021), and most studied plants comprise toxic substances (Ishii et al., 1984). In addition, the general anticancer treatment should possess substantial capabilities of altering proliferation, migration, and apoptosis involved in a multitude of biological mechanisms. Therefore, in-depth and systematic studies of herbal medicines should be elusive to explain their molecular mechanism underlying herbal medicine recognized therapeutic benefits against cancers (Fig. 1).

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FIG. 1.

Systematic study of herbal treatment cancers in posttranslational modifications (PTMs). Active ingredients extracted from herbal medicines (such as baicalein/wogonoside) with mild and broad-spectrum efficacy present a potential treasure trove of cancer treatment strategy through interacting and modulating PTMs in cellular and physiological processes. They have central roles in the development of various diseases through defending against tumor progression, contributing to an improved quality of life.

Anticancer effects of herbal medicines by interfering with posttranslational modifications

Proteins are elaborately modified by the covalent addition of functional moieties to the amino side chain, which facilitates their activity, stability, function, subcellular localization, and interacting network (Jensen, 2004) in the crowded cell microenvironment. Numerous cases have proved that these modifications play an important role in the dynamic regulation of protein–protein interactions (PPIs) to spatially organize diverse complexes with other cellular molecules, such as proteins, lipids, metabolites, and nucleic acids (Fig. 2). As a result, over 1 million human proteins have been identified within the last few decades, outnumbering the 20,000–25,000 human genes, in which one single gene encodes multiple proteins to expand the coding capacity. It is no surprise that a wide range of posttranslationally modified proteins and their interactions are implicated in various human diseases with herbal remedies. Collectively, scientists have discovered over 500 types of posttranslational modifications (PTMs) with the development of high-sensitivity mass spectrometry (MS), gradually deepening the complete and quantitative proteomes as well as their interactome (Aebersold and Mann, 2016). These PTMs have profound yet poorly understood effects on their physiological functions in human. In this section, we thus focus on the understanding of how herbal medicines regulate the cancer cellular pathways and disease processes via several popularly representative PTMs (ubiquitination, SUMOylation, phosphorylation, and acylation).

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FIG. 2.

Proteins can be edited by a wide variety of posttranslational modification (PTM) mechanisms and their functional significance in the cellular microenvironment. Cellular PTMs provide a dynamic mechanism for the metabolic regulation, chromatin regulation, cancer signaling, immunomodulation, protein conformation, and interactions.

Ubiquitination

Ubiquitination refers to an important type of stepwise PTM that the direct modification of ubiquitin (Ub) is covalently installed on the target protein, tightly catalyzed at different levels by a range of indicated enzymes (including Ub-activating enzyme E1, Ub-conjugating enzyme E2, and Ub ligase E3, and an array of Ub-degrading enzymes DUBs) (Fig. 3A). Strikingly, the indirect regulation of ubiquitination is crucial for ensuring the stability of endogenous proteins, the activity of enzymes, and the facilitation of PPIs across a variety of cellular functions (Cai et al., 2018). Thereby notably functional herbal medicines targeting the Ub enzymes of the ubiquitin–proteasome system (UPS) are considered to be important for cancer treatment and prevention. In this part, we discuss the ubiquitination enzymes and targets in the herbal-treatment cancers and highlight several major clinical herbal medicines that regulate the UPS.

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FIG. 3.

The signaling mechanisms and cellular functions of ubiquitination/SUMOylation pathways. (A) Sumoylation is a cascade of enzymatic steps that involve an E1 activating enzyme, an E2 conjugating enzyme, and an E3 SUMO ligase. Mature SUMO is immature and cleaved to expose the C-terminal Gly–Gly motif; then, mature SUMO requires ATP to conjugate E1 at its cysteine residue, transfer to the cysteine residue of E2 that recognizes the lysine residue of the substrate protein under E3 SUMO ligases. Dynamic SUMOylation affects the protein functions in various manners, including protein interaction, oncogenic activity, and protein stability. (B) Shikonin promotes ubiquitination and degradation of cIAP1/2-mediated apoptosis and necrosis. After treatment with Shikonin, cIPAs, such as E3-like ubiquitination enzyme, enter into autoubiquitination process and are degraded by proteasome, which triggers the activation of the noncanonical pathway, NIK (NF-κB-inducing kinase)-Tumor necrosis factor (TNF) signaling.

Glioblastoma (GBM) is a type of fast-growing, low-survival, and aggressive brain tumor in children and adults, but there is a lack of significantly effective therapies. In a study by Sun et al., acevaltrate, an active component from the plant Valeriana jatamansi Jones, suppressed USP10-mediated deubiquitylation on cyclin D1 (CCND1) for CCND1 degradation, caused GBM cell cycle arrest at G1 phase and induced GBM cell apoptosis (Sun et al., 2021), because USP10 stabilizes CCND1 by preventing K48-linked polyubiquitination in GBM. Polyphyllin I (PPI) from Polyphylla rhizomes displays anticancer activity in many kinds of cancers. In a study by Liu et al., Polyphyllin I (PPI) selectively inhibited the unfolded protein response (UPR)-induced glucose-regulated protein 78 (GRP78) levels to prevent C/EBP homologous protein from the GRP78-mediated ubiquitination and degradation in the non-small-cell lung cancer (NSCLC) cell (Liu et al., 2021). Shikonin, a naphthoquinone from Arnebiae Radix, has triggered both necrosis and apoptosis in a triple-negative breast cancer (TNBC) cell, in which shikonin promoted the autoubiquitination and degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) and cIAP2 but induced the ubiquitination and inactivation of the receptor-interacting protein 1, one substrate of cIAP1 and cIAP2 (Wang et al., 2021) (Fig. 3B). In addition, herbal-mediated ubiquitination has also been used to treat and prevent atherosclerotic cardiovascular and cerebrovascular diseases. Choi et al. found the first molecular evidence for the antihypercholesterol activity of platycodin D, a triterpenoid saponin from Platycodon grandiflorum (Choi et al., 2020). Platycodin D increased the half-life of cell surface low-density lipoprotein receptor (LDLR) protein by reducing the E3 Ub ligase named inducible degrader of the LDLR (IDOL) ubiquitination in HepG2 cell. Zhang et al. discovered one natural small-molecule eupalinolide B to allosterically inhibit Ub-specific protease (USP7), blocking microglial activation and the resultant neuron injury (Zhang et al., 2022b). USP7 inhibition caused a Keap1 degradation to activate the antineuroinflammation genes in microglia of dementia and Parkinson’s disease mouse models. Another example is ginsenosides from the plant Panax with low toxicity, popularly used in food and traditional herbal medicine for more than 1000 years. Previous studies have demonstrated their inhibitory effects on the 26S proteasome or Ub enzyme in UPS (Chang et al., 2014).

Phosphorylation

Phosphorylation direct modification is enacted by kinases that target specific amino acids (commonly serine, threonine, or tyrosine) in proteins for phosphate group transfer, and phosphatases that remove these groups. On the contrary, indirect regulation of phosphorylation does not directly engage in the addition or removal of phosphate groups but influences the phosphorylation process through a variety of mechanisms. Reversible protein phosphorylation is one of the most prevalent and well-studied PTMs tightly associated with many protein activities and most signaling pathways (mainly including Wnt/β-catenin, Notch, nuclear factor kappa-B (NF-κB), phosphoinositide 3-kinase (PI3K)/AKT (PI3K/Akt), mitogen-activated protein kinase (MAPK), The Janus kinase/signal transducer and activator of transcription (JAK/STAT), Transforming growth factor beta (TGF-β)/Smad (TGFβ/SMAD), and Hedgehog signaling pathways). It is estimated that more than two-thirds of the 21,000 proteins encoded by the human genome, also one-third of human proteome, are the phosphorylation substrates (Li et al., 2013). Phosphorylation principally occurs at the side chains of serine, threonine, or tyrosine residues with the nucleophilic (-OH) group that attacks the terminal phosphate group on the universal phosphoryl donor adenosine triphosphate (ATP), which is reversibly and specifically mediated by kinases and phosphatase, respectively. Therefore, multiple proteins and their modulations have been strongly linked to phosphorylation signaling pathways in herbal-treatment cancers (Fig. 4).

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FIG. 4.

Herbal-modulated phosphorylation. Herbal medicines are able to affect the phosphorylation of key signaling proteins involved in cellular proliferation and survival pathways. Phosphorylation of tyrosine (Tyr), serine (Ser), and threonine (Thr) residues in proteins, mainly catalyzed by kinases, can be modulated by herbal compounds, resulting in critical signaling cascades, including cell proliferation and survival. For example, apigenin modulates the Wnt/β-catenin pathway, apigenin and ginkgolic acids modulate NF-κB, melanin, berberine, apigenin, curcumin, and wogonin modulate the MAPK/ERK pathway, Qingre Jiedu and apigenin modulate the JAK/STAT pathway, and apigenin, rapamycin, and OP16 modulate the PI3K/Akt pathway.

Apigenin (APG) is not only a common dietary flavonoid but also used in alternative medicine for asthma, Parkinson’s, neuralgia, and intransigent insomnia. In the last few decades, APG has been proven as an adjuvant chemotherapeutic agent for various cancer therapies by multiple signaling pathways, including PI3K/AKT, MAPK/ERK, JAK/STAT, NF-κB, and Wnt/β-catenin pathways (Yan et al., 2017). Birt et al. first reported APG’s antitumor efficacy in 1986 (Birt et al., 1986). In the A549 human lung cancer cell, APG suppressed the migration and invasion by inhibiting the phosphorylation of AKT and modulating the PI3K/Akt signaling pathway (Zhou et al., 2017). In three kinds of colorectal adenocarcinoma cell lines (SW480, DLD-1 and LS174T), APG decreased the phosphorylation of AKT Ser473 and ATK Thr308 to upregulate transgelin and downregulate matrix metallopeptidase-9 (MMP-9) expression for the inhibition of tumor growth and metastasis to the liver and lung (Chunhua et al., 2013). High deposition of visceral adipose tissue (VAT) known as visceral obesity is directly associated with metabolic syndrome, breast cancer, and endometrial cancer. Su et al. investigated that APG bound to nonphosphorylated STAT3 but reduced the phosphorylation of STAT3 and transcriptional activity in VAT, in contrast to high-fat diet-increased STAT3 phosphorylation in VAT (Su et al., 2020). Consequently, APG reduced the expression of STAT3 target gene cluster of differentiation 36 (CD36) and peroxisome proliferator-activated receptor gamma, leading to reduced body weight and VAT. Chen et al. have reported that bear powder significantly inhibited STAT3 phosphorylation and regulated Bcl-2. Cyclin D1, VEGF-4, and CDK4 resulted in cell apoptosis, cell proliferation, and vessel density in hepatocellular carcinoma tumor tissues (Chen et al., 2020).

ROS in oxidative stress and redox signaling

Aerobic respiration is a highly efficient ways of energy production to develop all eukaryotes, in which the generation of reactive oxygen species (ROS) is an inevitable by-product that confers reactivity to different targets. ROS contain the superoxide anion (O₂ ^–), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH⋅) produced as natural by-products of cellular metabolism (Finkel, 2003). If the imbalance between oxidative and antioxidative systems of cells and tissues is disrupted, overproduction of oxidative-free radicals and the associated ROS leads to oxidative stress, which is also a major apoptotic stimulus in cancer cells. Therefore, ROS are widely considered the second messenger in the processes of cell proliferation, differentiation, and apoptosis (Freinbichler et al., 2011).

Notably, many herbal medicines such as flavonoids, polyphenols, and alkaloids, have been found to possess antioxidant or anti-inflammatory properties to reduce the ROS levels and alleviate oxidative stress. For example, curcumin, the active compound in turmeric, has been used to scavenge ROS and activate antioxidant enzymes, to attenuate oxidative stress-induced carbonylation, nitrosylation, and phosphorylation. Rutin, a flavonoid, exhibits antioxidant effects and can scavenge ROS to attenuate oxidative stress-induced glycation and lipid peroxidation for anticancer. In this part, we focus on the research of herbal potential to modulate ROS and ROS-associated PTMs to open new avenues for the understanding and treatment of human cancer pathologies (Fig. 6).

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FIG. 6.

Herbal-regulated oxidative stress and protein modification in cells. Various factors such as mitochondria, UV/radiation, inflammation, metabolism, air pollution, smoking, and NADPH oxidase can promote the generation of ROS, thereby exacerbating oxidative stress. However, antioxidant regulation can counteract oxidative stress and modulate critical cellular processes such as proliferation, cell death, cell differentiation, immune evasion, genetic alterations, and cell survival. The ROS-related posttranslational modifications depicted include lipid-associated prenylation and palmitoylation, electrophile/aldehyde-related Michael addition and alkylation, irreversible modifications such as sulfenylation and sulfonylation, as well as reversible modifications, including S-cysteinylation, S-glutathionylation, disulfide formation, sulfenylation, nitrosylation, and sulfhydration. ROS, reactive oxygen species.

ROS-associated PTMs

During oxidative stress and redox signaling processes, ROS could modify redox-sensitive residues in proteins. For instance, H₂O₂ mediated oxidation of cysteine residues on proteins (Rhee, 2006), of which thiolate anions (Cys-S^-) are more susceptible to oxidation at physiological pH. ROS can also be detoxified by several kinds of antioxidants, based on both enzymatic and nonenzymatic mechanisms, for example, superoxide dismutase (SOD), catalase, peroxiredoxin, glutathione peroxidase, and sulfiredoxin. These common ROS-induced PTMs (oxidation, phosphorylation, carbonylation, nitrosylation, and glycation) have significant effects on cellular processes, PPIs, and disease processes, while excessive ROS levels with its induced PTMs can contribute to some pathological conditions in the cancer cellular context.

Here are three examples of ROS-associated PTMs not mentioned above: Oxidation, ROS can directly oxidize residues in proteins, such as cysteine oxidation (disulfide bond formation, sulfenic acid [SOH], sulfinic acid [SO2H], or sulfonic acid [SO3H]), methionine oxidation, and tyrosine nitration. Nitrosylation, nitric oxide (NO) and peroxynitrite (ONOO−), can react with reactive nitrogen species to conjugate a nitric oxide group (−NO) to the cysteine thiol groups in proteins (Nathan, 2023). Glycation, is associated with some destructive chemical reactions that generate intermediates, including Schiff base (aldimine) and Amadori species to produce ROS, leading to the advanced glycation end-products (AGEs) (Alaei et al., 2024; Cepas et al., 2020). ROS-associated PTMs can either promote or modulate cellular responses to oxidative stress, depending on the context and extent of ROS production, so studying these PTMs and their functional consequences provides insights into the complex interplay between ROS and cellular processes under herbal medicines.

Antioxidants in herbal medicines

Herbal medicines often contain various natural compounds that exhibit antioxidant properties to help neutralize ROS and reduce oxidative stress in the body.

Sideritis scardica, an endangered plant species endemic to the central part of the Balkan Peninsula, is known for its antioxidant, neuroprotective, and anti-inflammatory properties (Tasheva et al., 2023). The higher content of polyphenols and flavonoids underlies the higher antioxidant activity with the antitumor effects.

Salidroside, a phenolic glycoside from Rhodiola rosea, has been shown to have some potent pharmacological effects on antioxidative stress, anti-inflammation, anticancer, antifatigue, and myocardial injury (Zhang et al., 2021b). As oxidative stress plays a crucial role in carcinogenesis and metastasis, some studies found that salidroside displayed anticancer effect in A549 lung cancer cell through downregulating the ROS-phospho-p38 signaling pathway and inhibiting oxidative stress in a dose-dependent manner (Wang et al., 2014a), in which salidroside inhibited tumor invasion and suppressed the Snail protein expression to induce apoptosis. Moreover, salidroside could block the formation and growth of U251 glioma cell in vitro and in vivo, as well as improve the tumor microenvironment by inhibiting oxidative stress and astrocyte overgrowth.

Tanshinone IIA (TIIA), extracted from the traditional Chinese herbal medicine Danshen, has potential activity against a variety of cancers, such as liver cancer, leukemia, lung cancer, cervical cancer, bladder cancer, colorectal cancer, and stomach cancer. Yin et al. found that the heat shock protein 27 (HSP27) Ser82 was phosphorylated after TIIA reaction, resulting in ROS production and UPR. TIIA-treated cells induced an overproduction of ROS, which is essential to prevent genomic instability and DNA damage in various cancer types. Elevated ROS production induces antitumor signaling, leading to a variety of biological effects, including cell cycle arrest, apoptosis, and UPR. TIIA can promote cancer cell death by producing ROS and inducing UPR (Yin et al., 2020).

The tumor suppressor protein p53 is a class of transcription factors that are sensitive to redox. Oxidative stress, especially when cellular DNA is damaged, causes upregulation of p53 gene expression, manifested by accelerated translation of mRNA and PTMs. It affects the regulation of many posttranslational levels such as phosphorylation, acetylation, ubiquitination, and SUMOylation to change the conformation, localization, and interaction of p53 proteins, thereby improving its stability and activity (Lavin and Gueven, 2006). In this study, ursolic acid (UA) is an extract of a variety of traditional Chinese medicinal plants such as summer hay, hawthorn, and azalea, and studies have shown that UA has inhibitory effects on a variety of tumor cells. Hemozosis is a new way of death caused by iron-dependent oxidative damage in recent years; UA induces hemozois in ovarian cancer cells by upregulating p53 expression level, downregulating SLC7A11, reducing GSH level, and downregulating GPX4 expression. At the same time, UA can directly stimulate the increase of COX-2, promote the expression of ferritin heavy chain, significantly induce the production of oxidative stress, induce the occurrence of iron osis in cancer cells, and inhibit tumor growth (Ruan et al., 2021).

The anticancer agent adriamycin (ADR) is a commonly used chemotherapy drug to treat various cancers. However, the production of ROS caused by ADR can disturb the normal redox balance and produce huge oxidative stress, thereby stimulating carbonylation of specific protein groups involved in physiological dysfunction and inducing apoptosis of cardiomyocytes, and thus its severe cardiotoxicity greatly limits its clinical application. In addition, Salvia miltiorrhiza (SM) has long been used to treat cardiovascular disease. The water-soluble components extracted from SM, including salvianolic acids A, B and C, have significant antioxidant effects against free radicals. Salvia miltiorrhiza aqueous extract (SMAE) has been widely used in the treatment of coronary heart disease, atherosclerosis, and ischemic cardiovascular disease. Studies have shown that SMAE could prevent oxidative damage by increasing the levels of SOD and catalase in H9C2 cells exposed to ADR. SMAE attenuates ROS-mediated apoptosis by regulating protein carbonylation and antioxidant systems, and in combination with ADR can partially alleviate ADR-induced cardiomyopathy (Hung et al., 2020) without affecting the efficacy of ADR. This finding also proved the advantage of the combination of Chinese herbal medicine with drugs.

Influence of herbal medicines on PTMs as antioxidant and anticancer

The exploration of herbal medicine in cancer therapy has increasingly focused on understanding how natural compounds influence cellular mechanisms to combat cancer. A critical area of interest is how these compounds impact antioxidant defense systems and redox signaling pathways, particularly through PTMs of proteins.

Quercetin, a naturally flavonoid, has been shown to attenuate inflammation-related signaling pathways, including ROS, nitric oxide, and proinflammatory cytokines, in prostate cancer (Pellegrino et al., 2023). In addition, it inhibits the production of AGEs and protein glycation in MCF-7 (Khan et al., 2021) and HCT116 cells (Hafsa et al., 2016). Quercetin directly interacts with SIRT5, leading to the inhibition of PI3K/AKT phosphorylation through the SIRT5-PI3K interaction in NSCLC (Zhou et al., 2023). Furthermore, it enhances the binding activity of the serum response factor transcription factor, thereby increasing the expression of DUSP5 at the transcriptional level (Boonruang et al., 2023). This, in turn, augments ERK phosphorylation in colorectal cancer. Moreover, quercetin has the potential to reverse the hypermethylation of P16INK4a and modulate the activity of DNA methylation enzymes, including DNMTs and histone methyltransferases, in bladder cancer cells and hepatocellular carcinoma (Qadir Nanakali et al., 2023).

Acteoside, a glycosylated phenylpropanoid derived from Syringa vulgaris (Oleaceae), has been reported to modulate DNA methylation levels, specifically increasing the methylation of SEC24D reduces its expression levels, thereby decelerating tumor progression (Du et al., 2023). It also attenuates the phosphorylation of p38 MAPK (Wu et al., 2021a) and reduces ROS production, lipid peroxidation, and protects mitochondrial functions (Sciandra et al., 2023; Zhang et al., 2022a). Furthermore, acteoside enhances the expression of nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase (NQO-1), and the glutamate cysteine ligase catalytic subunit. It concurrently decreases the activity of the NF-κB signaling pathway in A549 cells (Xiao et al., 2022; Zheng et al., 2021). This dual action of inhibiting oxidative stress and the NF-κB pathway suggests a protective role against oxidative damage by mitigating the ROS response.

Silybin, a flavonolignan derived from Silybum marianum, exhibits chemopreventive effects against various cancers through multiple mechanisms. It induces NO generation, apoptosis, and autophagy (Yu et al., 2012), and reduces the elevated levels of AGEs, their receptors, and ROS. Silybin also attenuates the phosphorylation of p38 MAP kinase (Karimi et al., 2022) and inhibits TLR4/NFκB-mediated signaling pathways, leading to the downregulation of proinflammatory mediators (Surai et al., 2024). In addition, it increases the phosphorylation of Jun N terminal kinase/c-Jun N-terminal protein kinases (JNK) and p-p38, significantly reducing the viability of human gastric cancer cells (Kim et al., 2019).

Daphnetin (DAP), a phenolic coumarin derivative sourced from the Daphne species, has been shown to augment the levels of GSH, glutathione S-transferase, SOD, and catalase, while reducing malondialdehyde levels. These actions contribute to the suppression of hepatic cancer by decreasing inflammatory mediators (Li et al., 2022). Furthermore, DAP activates the translocation of Nrf2 and upregulates antioxidant enzymes, including HO-1 and NQO1. It also promotes the phosphorylation of JNK, ERK, and P38, thereby alleviating oxidative stress and mitochondrial dysfunction (Fan et al., 2020; Kumar et al., 2016). This multifaceted mechanism of action aids in inhibiting nephrotoxicity in the kidneys and mammary carcinogenesis.