Synergistic antitumor effects of quercetin and triptolide in hepatocellular carcinoma via modulation of PI3K/AKT/mTOR and Nrf2 signaling pathways

Abstract

Phytochemicals derived from traditional Chinese medicine have demonstrated therapeutic potential in liver cancer. Among these, quercetin has been reported to modulate immunosuppressive mechanisms and attenuate liver injury and carcinogenesis. This review focuses on the synergistic effects of triptolide (TP) and quercetin, particularly their co-inhibitory actions on cell proliferation and the mammalian target of rapamycin signaling pathway in preclinical studies. Quercetin administration (80 mg/kg) reduced serum alanine aminotransferase and aspartate aminotransferase levels, as well as biomarkers for oxidative stress. It also mitigated TP-induced inhibition of the protein kinase B/mammalian target of rapamycin pathway and reversed excessive ferroptosis, as evidenced by decreased Fe²⁺ accumulation and lipid peroxidation (malondialdehyde). In contrast, hepatic glutathione and superoxide dismutase levels were elevated, along with enhanced nuclear expression of nuclear factor erythroid 2-related factor 2. Moreover, mRNA expression of heme oxygenase-1, nicotinamide adenine dinucleotide (phosphate) hydrogen dehydrogenase (quinone) 1, and glutamate-cysteine ligase catalytic subunit was upregulated. Collectively, these findings suggest that quercetin attenuates TP-induced immunological liver injury via activation of the nuclear factor erythroid 2-related factor 2/antioxidant response element signaling pathway. Compared with monotherapy, the combination of TP and quercetin shows enhanced antitumor effects by inhibiting cell proliferation, inducing apoptosis through transcriptional regulation, and suppressing migration and invasion.

Key Words: Phytochemical; Polyphenol; Liver; Proliferation; Carcinoma

Core Tip: The combination of quercetin and triptolide demonstrates enhanced antitumor effects by reducing tumor growth and oxidative stress while modulating the protein kinase B/mammalian target of rapamycin signaling pathway. Plant-derived bioactive compounds and herbal formulations have shown promise in hepatocellular carcinoma therapy, particularly in combination regimens that exert anticancer and anti-inflammatory effects through modulation of the tumor microenvironment. Quercetin may also inhibit triptolide-induced liver injury and carcinogenesis by regulating the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathway, which is associated with cell proliferation.

INTRODUCTION

Phytochemicals derived from traditional Chinese medicine (TCM) and nutritional supplements have shown therapeutic potential in liver cancer[1,2]. Particular attention has been given to compounds that reduce liver injury and are used in combination therapies for cancer treatment[2]. Overall survival is influenced by systemic health parameters, including tumor volume, body weight, nutritional status, and detoxification processes related to oxidative stress[1,3]. Recent studies highlight quercetin as a multifunctional flavonoid with immunomodulatory, hepatoprotective, and antioxidant properties, particularly in combination with triptolide (TP), a diterpenoid epoxide derived from the Thunder God Vine or Tripterygium wilfordii Hook F[1,2]. Quercetin has demonstrated both in vitro and in vivo efficacy in mouse models of polycystic kidney disease and pancreatic cancer, as well as protective effects against TP-induced injury[2]. It also exhibits antimicrobial activity against Helicobacter pylori[4]. Furthermore, quercetin and its metal complexes (e.g., zinc and iron) attenuate hepatocellular and renal cell damage induced by arsenic exposure[5]. Evidence from multiple animal models, including nude mice[1] and rats, indicates that quercetin modulates immunosuppressive pathways, thereby reducing liver injury and carcinogenesis[5]. In addition, quercetin enhances the efficacy of chemotherapeutic agents such as 5-fluorouracil, cisplatin, doxorubicin, and paclitaxel, while mitigating the adverse effects of chemotherapy, partly through modulation of immune checkpoint pathways[6] in cancer cells that are clinically related to colitis/gastritis[4].

Studies in pancreatic cancer cell lines (BxPC-3, PANC-1) and hepatocellular carcinoma (HCC) cell lines (HepG2, Huh-7) further support its anticancer potential. Moreover, numerous phytochemicals, such as kaempferol, curcumin, resveratrol, apigenin, luteolin, and quercetin, have demonstrated multifaceted antitumor effects in HCC through the regulation of proliferation, apoptosis, invasion, migration, angiogenesis, and tumor microenvironment (TME) dynamics in both in vitro and animal studies[3,4,7-9].

SYNERGISM

A recent study by Tong et al[1] highlighted the synergistic effects of TP and quercetin via co-inhibition of cell proliferation and modulation of the mammalian target of rapamycin (mTOR) signaling pathway in preclinical models, with potential relevance to metastatic cancer. Quercetin administration (80 mg/kg) significantly reduced serum alanine aminotransferase and aspartate aminotransferase levels, as well as biomarkers associated with TP-inhibited protein of the protein kinase B (AKT)/mTOR pathway[1,2,10]. In addition, quercetin attenuated TP-induced excessive ferroptosis, as evidenced by decreased Fe²⁺ accumulation and reduced lipid peroxidation (malondialdehyde). Antioxidant defenses were enhanced, with increased levels of glutathione and superoxide dismutase in liver tissues[11,12], along with upregulated nuclear expression of nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore, quercetin stimulated mRNA expression of heme oxygenase-1[12,13], nicotinamide adenine dinucleotide (phosphate) hydrogen dehydrogenase (quinone) 1, and glutamate-cysteine ligase catalytic subunit[12]. Despite these promising findings, the clinical application of quercetin remains challenging owing to its poor water solubility and low oral bioavailability.

Within the framework of TCM, therapeutic strategies such as strengthening the spleen, replenishing qi, fortifying the body, alleviating depression, detoxifying, and resolving symptoms are believed to exert clinical benefits through targeting and modulating various pathophysiological processes[13]. Numerous natural products and bioactive compounds with anticancer potential, such as curcumin, gallic acid, and quercetin, have been identified from TCM and investigated in cancers such as prostate cancer[14]. In HCC, plant-derived bioactive compounds, single herbs, and composite formulations (e.g., capsules, Shenqi injection, and Compound Kushen injection) have been widely studied for their ability to inhibit cell proliferation and induce apoptosis. These natural compounds and secondary metabolites can enhance the efficacy of conventional chemotherapy and are often applied in combination regimens (Table 1)[1,2,4,7-9,15-37]. Examples include monotherapies[3], autophagy-targeting Chinese medicine monomers[38], curcumin-mediated inhibition of invasion[9], and other combinations of natural products that modulate the TME[9,13]. Additionally, plant-derived compounds such as Ashwagandha[15], curcumin, and thymoquinone have demonstrated synergistic effects with chemotherapeutic agents, including cisplatin, against HCC[18]. Lignans are also known for their broad anticancer activity and effects against skeletal diseases[26]. Many flavonoids and chemomodulatory agents have been shown to suppress carcinogenesis or improve anticancer efficacy in combination with phenethyl isothiocyanate (Table 1; Xanthohumol[37]). Furthermore, antimicrobial activity has been reported for compounds such as d-glucaro-1,4-lactone, resveratrol, and the widely studied quercetin[3,7].

Table 1 Phytochemicals and traditional Chinese medicine-derived compounds used in the treatment of hepatocellular carcinoma.

Natural compound	Classification	Anticancer efficacy in liver treatment	Ref.
Ashwagandha	Plant-derived product	In combination with cisplatin or intermittent fasting; protective against breast cancer and liver damage	[15]
Becium grandiflorum	Diterpene isolates	In combination with doxorubicin	[16]
Berberine	Plant-derived product	In combination with resveratrol	[17,18]
Naringenin (Ceiba pentandra)	Flavonoid	In combination with doxorubicin	[19,20]
Corydalis Saxicola	Alkaloids	ND	[21]
Curcumin	Diarylheptanoid	Modulates non-coding RNA; in combination with cisplatin	[7,9,18,20,22]
d-Glucaro-1,4-lactone	Secondary metabolite	Antibacterial activity; in combination with lenvatinib	[23]
Lime peel extract	NA	Limonin, hesperidin	[24]
Extracts of Viola canescens	NA	Hepatoprotective effect	[25]
Genistein (Soy)	Phytoestrogen	Chemomodulatory effect	[18,22]
Honokiol (Magnolia)	Lignan	Anticancer activity	[26]
Licochalcone A	Chalcone (secondary metabolite)	In combination with magnesium isoglycyrrhizinate	[27,28]
Luteolin	Flavonoid	Anticancer activity	[4,29]
Moringin	Stable isothiocyanate	Avenanthramide 2f	[30]
Polyphillin I	Secondary metabolite	In combination with chloroquine	[31]
Pomgranate root extract	NA	Antimetastatic	[32]
Quercetin	Flavonoid	Alleviates TP-induced toxicity; enhances doxorubicin and gemcitabine efficacy	[1,2,4,8,18,20,33]
Resveratrol	Polyphenol; phytoestrogen	Antimetastatic effects	[7,17,18,22]
Silymarin	Flavanonol	In combination with doxorubicin	[34]
L-THP	Alkaloid	In combination with DPI	[35]
Thymoquinone	Secondary metabolite	In combination with cisplatin	[18,36]
Xanthohumol	Flavonoid	In combination with resveratrol or phenethyl isothiocyanate	[37]

L-THP: Levo-tetrahydropalmatine; NA: Not applicable; ND: Not done; TP: Triptolide; DPI: Diphenyleneiodonium.

MECHANISMS OF TP-INDUCED INJURY AND LIVER DAMAGE

In vivo studies have demonstrated that combination therapy with quercetin and TP exerts beneficial effects on tumorigenesis. Quercetin was administered intraperitoneally twice daily for 10 days[11] or via daily gavage for 7 consecutive days[12], either before or after tumor establishment, while TP was administered at doses of up to 25 nmol/L per mouse[1]. The combined regimen exhibited synergistic antitumor effects, potentially offering therapeutic benefits for patients who are unresponsive or resistant to conventional treatments, particularly through coordinated inhibition of Janus kinase and phosphatidylinositol 3-kinase (PI3K)/mTOR signaling pathways. During a 21-day intervention period after tumor formation, tumor growth (length and width), body weight, food intake, and overall health status of nude mice were monitored at 7-day intervals, followed by histological and pathological evaluation of liver tissues[1]. Quercetin is suspected to inhibit TP-induced immunological liver injury[10,39], potentially through activation of the Nrf2/antioxidant response element signaling pathway[12,13,39].

The antitumor effects of quercetin on TP-induced liver carcinogenesis have been investigated through assays of cell migration, invasion, and pathway-specific functional analyses. In HepG2 cell-based assays on cell viability, apoptosis, and protein expression (via western blotting), it was observed that multiple signaling pathways, including JAK/signal transducer and activator of transcription, mTOR, and glycosphingolipid biosynthesis, were modulated following treatment. Additional pathway alterations were identified in processes related to maturity-onset diabetes of the young, viral life cycle (human immunodeficiency virus type 1), type II diabetes mellitus, and legionellosis.

At the molecular level, the combination of quercetin and TP significantly increased the expression of pro-apoptotic markers, including Bax, p53, and cleaved caspase-3, as well as the Bax/Bcl-2 ratio, while reducing the expression of proliferative-associated proteins compared with control groups. No statistically significant differences were observed in PI3K and AKT expressions. Functional assays, including colony formation, wound healing, and transwell invasion assays, further confirmed enhanced apoptosis and reduced migratory and invasive capacities of HCC cells following combination treatment[1]. Transcriptome analysis highlighted the JAK/signal transducer and activator of transcription and mTOR signaling pathways as key targets uniquely regulated by the combination therapy, supporting its role in suppressing tumor progression through coordinated inhibition of proliferation, migration, and invasion.

KEY IMPLICATIONS AND FUTURE RESEARCH

Current research efforts on HCC, a deadly disease with nearly 900000 new cases reported in 2022, are primarily directed toward developing nano-delivery systems to overcome the poor water solubility and pharmacological limitations of therapeutic compounds[3]. Among phytochemicals, oroxin B, a flavonoid glucoside, was found to induce apoptosis by downregulating microRNA (miRNA)-221, resulting in inactivation of the phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase/PI3K/AKT pathway in the liver (Figure 1)[40].

Figure 1 Schematic illustration of the interplay between the phosphatidylinositol 3-kinase/protein kinase B/ mammalian target of rapamycin and nuclear factor erythroid 2-related factor 2 signaling pathways. Phosphatidylinositol 3-kinase activation induces protein kinase B phosphorylation, leading to mammalian target of rapamycin signaling and regulation of cell proliferation, survival, and cell cycle progression, while phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase acts as a negative regulator. Mammalian target of rapamycin also influences hypoxia-inducible factor 1-alpha activity. Oxidative stress impairs nuclear factor erythroid 2-related factor 2 nuclear translocation, reducing antioxidant responses. Interactions involving glycogen synthase kinase 3 and nuclear factor kappa B further link these pathways to inflammation and cell fate regulation. PI3K: Phosphatidylinositol 3-kinase; AKT: Protein kinase B; PTEN: Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase; HIF-1α: Hypoxia-inducible factor 1-alpha; GSK3: Glycogen synthase kinase 3; mTOR: Mammalian target of rapamycin; Nrf2: Nuclear factor erythroid 2-related factor 2; NF-κB: Nuclear factor kappa B; NOS: Nitric oxide synthase.

Glycyrrhizic acid has been reported to reduce stem cell pluripotency, whereas blockage of c-Jun N-terminal kinase 1 mitigates the degree of malignancy of HCC[41]. Notably, glycyrrhizic acid effectively enhances the antitumor effects of sorafenib, a multikinase inhibitor, particularly through the induction of cell cycle arrest[13,42]. Moreover, catalpol and panax notoginseng saponins - bioactive compounds containing various sugar moieties - were shown to alleviate TP-induced hepatotoxicity besides regulating cancer stem cells and drug resistance via upregulation of the Nrf2 pathway[43].

Despite these advances, comprehensive safety evaluations remain essential before the clinical implementation of combination therapies. Taken together, combination therapies involving purified phytochemicals show promising chemopreventive and potential senolytic effects, with the capacity to improve patient prognosis. TP has been further developed in the form of its water-soluble prodrug, minnelide, a heat shock protein 70 inhibitor, which has demonstrated clinically relevant activity against pancreatic cancer[44].

The technological advancement of sequencing tools and metabolomics has facilitated the identification of alterations in miRNA deletion. Both TP and quercetin have been shown to regulate miR-142-3p, which targets the 3′ untranslated region of heat shock protein 70, thereby influencing tumor cell proliferation. Notably, overexpression of heat shock protein 70 can rescue miR-142-3p-induced cell death in pancreatic ductal adenocarcinoma[45]. Although miRNA-based therapeutic strategies represent a promising avenue, their application in liver cancer requires further investigation and careful therapeutic regulation.

CONCLUSION

Compared with monotherapy using a single phytochemical, the combined administration of TP and quercetin exhibits enhanced antitumor efficacy in HCC. This combination inhibits cell proliferation by inducing apoptosis, reduces cellular toxicity, and inhibits migration and invasion, highlighting its potential for the development of effective anti-HCC regimens. Beyond its established roles in the treatment of osteoporosis and antimicrobial effects, quercetin has also been implicated in immunomodulatory processes relevant to cancer therapy. Notably, it may enhance immune checkpoint inhibitor-mediated responses by promoting immune cell infiltration within the TME. This effect is associated with increased proliferation and activation of CD8⁺ T cells, thereby contributing to improved antitumor immunity in liver cancer.