Pyroptosis- and Cuproptosis-Targeting Natural Compounds as a Promising Approach for Hepatocellular Cancer Treatment

Abstract

Background/Objectives: Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide, posing significant health challenges due to its high morbidity and mortality rates. Emerging evidence indicates that non-apoptotic cell death mechanisms, including pyroptosis and cuproptosis, play crucial roles in HCC progression. Natural compounds have been widely investigated as potential primary or adjunct therapies targeting these cell death pathways. This review summarizes recent advances on the application of natural products that influence pyroptosis and cuproptosis in HCC. Methods: A comprehensive critical review of in vitro and in vivo studies examining the effects of natural compounds on HCC was conducted. Results: Several natural molecules demonstrate cytotoxic and cytostatic effects against HCC by modulating pyroptosis and cuproptosis pathways. Conclusions: Natural products exhibit promising potential as adjuvant therapies in hepatocellular carcinoma by selectively inducing or modulating pyroptosis and cuproptosis pathways. These mechanisms contribute to enhanced cancer cell death, underscoring the therapeutic value of targeting non-apoptotic cell death processes in HCC management.

1. Introduction

Hepatocellular carcinoma (HCC) is recognized as the most prevalent form of primary liver cancer, presenting a substantial global health challenge due to its high rates of morbidity and mortality [1,2]. Currently, surgical intervention remains the sole curative option for HCC patients, whereas alternative treatments are constrained by limited response rates and emerging drug resistance [3,4]. Consequently, there is an urgent need to identify novel therapeutic strategies aimed at the effective elimination of liver cancer cells.

From a histological perspective, liver cancer encompasses several distinct entities, including focal nodular hyperplasia, cholangiocellular carcinoma, hepatocellular adenoma, hepatocellular carcinoma (HCC), and combined hepatocellular carcinoma. Among these, HCC stands out as the most prevalent form, representing nearly 85% of all diagnosed liver cancer cases [5]. The pathogenesis of HCC is multifactorial, involving environmental, genetic, and immunological determinants. Various cell death mechanisms, encompassing both programmed and non-programmed pathways, critically regulate HCC development and progression [6]. Over recent decades, multiple programmed non-apoptotic cell death modalities have been characterized and extensively investigated, including necroptosis, ferroptosis, pyroptosis and cuproptosis. In particular, HCC tumor biology involves dysregulated cell death pathways that enable evasion of apoptosis while exploiting inflammatory forms like pyroptosis and emerging copper-dependent cuproptosis, contributing to tumor progression, immune modulation, and therapy resistance. Apoptosis resistance is a hallmark early event in hepatocarcinogenesis, whereas pyroptosis and cuproptosis exhibit dual roles—potentially suppressing tumors through immune activation or promoting progression via chronic inflammation and metabolic rewiring [7]. Current research indicates that these cell death pathways are integral to the pathophysiological landscape of HCC, with complex molecular interactions driving their initiation and propagation, and even though they are initiated and sustained by distinct molecular mechanisms, they exhibit substantial crosstalk and interaction.

Molecules of natural origin have been extensively tested for their possible use as main or adjuvant therapy against HCC, especially targeting the cell death pathways [8]. The ongoing search for novel naturally derived compounds aims to identify new chemotherapy agents that are not only more effective but also exhibit reduced side effects, offering promising advances in cancer treatment. In particular, the use of natural products as anti-cancer agents targeting necroptosis and ferroptosis has been extensively reviewed, [9,10]. Moreover, there are rapidly accumulatinged data about cuproptosis and pyroptosis. In this context, this review summarizes recent knowledge concerning the use of natural products against HCC with direct impact on cuproptosis and pyroptosis cell death mechanisms.

2. Pyroptosis

Pyroptosis is a distinct form of programmed cell death described for its proinflammatory nature and lytic process. Pyroptosis is characterized by activation of inflammasomes and subsequent activation of inflammatory caspases (caspase-1/caspase-11). Inflammasomes which depend on caspase-1 are classified as classical, whereas inflammasomes which depend on caspase-11 are classified as non-classical. Once activated, these inflammasomes cleave gasdermin D (GSDMD) into a 31 kDa N-terminal fragment (GSDMD-N) and a 22 kDa C-terminal fragment (GSDMD-C). The GSDMD-N fragment rapidly translocates to the inner leaflet of the plasma membrane where it binds to phospholipids and forms pores, leading to cell swelling, membrane rupture, and the release of inflammatory cytokines like IL-1β and IL-18 [11]. Conversely, GSDMD-C acts as an inhibitor of GSDMD-N’s pore-forming activity. While caspase-3 is primarily associated with apoptosis, it is also found that upon chemotherapy treatment, caspase-3 induces pyroptosis by cleaving gasdermin E (GSDME), thus promoting pore formation in the cell membrane [12]. This mechanism not only facilitates the rapid clearance of infected or damaged cells but also promotes an immune response by releasing danger signals that recruit and activate other immune cells.

The role of pyroptosis in HCC progression is controversial. Hypoxic HCC cells can induce pyroptosis through the upregulation of proinflammatory factors including IL-1β and IL-18 and the activation of caspase-1, leading to invasion and metastasis [13]. On the other hand, induction of pyroptosis through the caspase-1-mediated pathway can inhibit HCC progression [14]. As pyroptosis and its associated molecules exhibit both pro- and anti-tumorigenic effects, manipulating pyroptosis by using anti-tumor natural products can serve as a promising strategy for HCC treatment.

Natural Products Against HCC Mediating Pyroptosis

Numerous studies have tested the potential cytotoxic and/or cytostatic properties of natural products against HCC targeting the cell death mechanism of pyroptosis. Studies were identified using Boolean strings—(“hepatocellular carcinoma” OR HCC) AND (“pyroptosis” OR “gasdermin” OR GSDMD OR GSDME OR “caspase-1” OR NLRP3 OR “inflammasome”) AND (“natural product*” OR “plant extract*” OR “phytochemical*” OR “herbal”)—while the filters used included English, original research/reviews, and in vitro/in vivo HCC models. The databases searched were PubMed/MEDLINE, Web of Science, Scopus, and Google Scholar with no time frame. Abstracts were screened independently by two reviewers, and studies demonstrating natural products affecting pyroptosis in HCC cells or HCC models were included (

Table 1. In vitro studies of natural products targeting pyroptosis in HCC.

Natural Product	Cell Line	Concentration Duration	Mechanism	Result	Organism	Ref.
Euxanthone (flavonoid)	Hep3B, SMMC 7721	5, 10 μM for 24, 48 h	NLRP3 ↑ caspase-1 ↑ IL-1β ↑, IL-18 ↑	↑ cell death ↑ pyroptosis ↓ cell proliferation ↓ invasion ↓ metastasis	Polygala caudata (plant)	[15]
Miltirone (phenanthrene quinone derivative)	HepG2 Hepa1-6	40 μmol/L for 24 h	N-GSDME ↑ GSDME ↑ caspase-3 ↑ PARP ↑ pMEK, ERK 1/2↓ ↑ ROS	↓ cell proliferation ↑ pyroptosis ↑ apoptosis	Salvia miltiorrhiza (plant)	[16]
Alpinumisoflavone (flavonoid)	HepG2,SMMC 7721, Huh7, Bel7402	0–20 μM for 24, 48 h	LDH ↑ NLRP3↑ caspase-1 ↑ IL-1β, IL-18 ↑	↓ cell proliferation ↓ migration ↓ invasion ↑ pyroptosis ↑ autophagy	Derris eriocarpa (plant)	[17]
Curcumin (polyphenol)	HepG2	10, 20, 30 μM for 12 h	ROS ↑, LDH ↑ GSDME-N ↑	↑ apoptosis ↑ pyroptosis	C. longa C. zedoaria (plant)	[18]
Mallotucin D (clerodane diterpenoid)	HepG2	0–20 μM for 24 h	GSDMD, NLRP3, GSDMD-N/GSDMD-F ↑ IL-1β/pro-IL-1β ↑ LDH ↑, ROS ↑	↓ cell proliferation ↓ angiogenesis ↑ apoptosis ↑ pyroptosis ↑ mitophagy	Croton crassifolius (plant)	[19]
Neobavaisoflavone (isoflavone)	HepG2 HCCLM3	50 μM for 24 h	TOM20 ↑ ROS ↑, Bax ↑ caspase-3 ↑, N-GSDME ↑	↑ pyroptosis	Psoralea sp. (plant)	[20]
Psoralidin (coumarin)	HepG2 Hepa1-6, H22	0–160 μM for 24 h	LDH ↑ caspase-3 ↑ ROS ↑	↑ pyroptosis ↑ macrophage pyroptosis ↑ NK cell activation	Psoralea sp. (plant)	[21]
Ajmalicine (alkaloid)	H22	5, 10, 15 μM for 24 h	N-GSDME ↑ IL-1β ↑, IL-6 ↑ TNF-α ↑, LDH ↑ ROS ↑	↑ pyroptosis	Rauvolfia verticillata (plant)	[22]
Ellagic acid (polyphenol)	HEK-293T, HepG2, Huh-7	10, 25, 50, 100, 200 μM for 48 h	NLRP3 ↑ GSDMD ↑ N-GSDMD ↑ ASC ↑, IL-18 ↑ caspase 1-20 ↑ IL-1β ↑, ESR1 ↑	↓ cell proliferation ↓ migration ↑ pyroptosis	Punica granatum, Rubus sp. (plant)	[23]
Glycyrrhetinic acid (triterpenoid)	Hepa1-6	20 μM for 24 h	caspase-3 ↑ GSDME ↑, LDH ↑ PERK ↑	↓ cell proliferation ↑ pyroptosis	Glycyrrhiza glabra (plant)	[24]
Cannabidiol (cannabinoid)	HepG2, Huh7, HCCLM3, MHCC97H	20 and 40 μM for 24 h	P-EIF2 ↑, ATF4 ↑ CHOP ↑ Bax, Bak ↑ caspase-3 ↑ N-GSDME ↑	↓ cell proliferation ↓ cell glycolysis ↑ pyroptosis ↑ mitochondrial stress	Cannabis sativa (plant)	[25]
Jinglinzi powder	MHCC-97L	0.1, 0.2 mg/mL for 12, 24 h.	NLRP3 ↑, GSDMD ↑ IL-1β ↑ IL-18 ↑	↓ cell proliferation ↓ migration ↑ pyroptosis	Alpinia oxyphylla andCorydalis yanhusuo (plant)	[26]
Icaritin (flavonoid)	HepG2, MHCC97H, HCCLM3, Huh7	20, 40 µM for 48 h	caspase-1 ↑, caspase-3 ↑ GSDMD-NT, GSDME ↑ IL-18 ↑, IL-1β ↑	↓ cell proliferation ↑ pyroptosis ↑ proinflammatory macrophage phenotype	Epimedium sp. (plant)	[27]
Schisandrin B (lignan)	HepG2+ NK 92	0–60 μM for 24 h	GSDME ↑ N-GSDME ↑ caspase-3↑, LDH ↑ perforin ↑ granzyme B ↑	↓ cell proliferation ↑ apoptosis ↑ pyroptosis	Schisandra chinensis (plant)	[28]
Polyphyllin II (saponin) + IR780 in PLGA nanoparticles	HepG2	0.75 μg/mL for 12 h	ROS ↑, NLRP3 ↑ caspase-1 ↑ IL-1β/IL-18 ↑	↓ cell proliferation ↓ metastasis ↑ pyroptosis	Rhizoma paridis (plant)	[29]
Puerarin (isoflavone)	Huh7 SMMC-7721 Hep3B	10 μmol for 48 h	NLRP3 ↓ caspase-1 ↓ ASC ↓, IL-1β ↓	↓ cell proliferation ↓ pyroptosis	Pueraria lobata (plant)	[30]
Reuterin (hydroxypropionaldehyde)	HCC-LM9, Huh7, SK-Hep1, HUCC T1, RBE, Hep3B	50, 100μM for 24 h	caspase-1 ↑ GSDMD ↑, IL-1β ↑ caspase-8 ↑	↑ mitophagy ↑ pyroptosis	Lactobacillus reuteri (bacterium)	[31]
Cordycepin (purine nucleoside)	MHCC97H, PLC/PRF/5	350, 500 μM for 4 days	ROS ↑	↓ cell proliferation ↑ pyroptosis	Cordyceps militaris (fungus)	[32]
4,4′-Secalonic acid D (xanthone)	HepG2	12 μM for 48 h	N-GSDME↑, LDH ↑ ROS ↑, PARP1 ↓	↑ pyroptosis ↑ apoptosis	Paraconiothyrium sp. AC31 (fungus)	[33]
Cinobufotalin (bufadienolide)	Hep3B, Huh-7, LM3, HepG2 LO2	0.5 mM for 12, 24, 48, 72 h	NLRP3 ↑, caspase-1 ↑ ASC ↑, IL-1β ↑, GSDMD-N ↑, ROS ↑, H2O2 ↑	↓ cell proliferation ↓ migration ↓ invasiveness ↑ pyroptosis	Bufo sp. (amphibian)	[34]

Abbreviations: NLR Family Pyrin Domain-Containing 3 (NLRP3); Interleukin-1 beta (IL-1β); Interleukin 18 (IL-18); gasdermin D (GSDMD); gasdermin D 31 kDa N-terminal fragment (GSDMD-N); gasdermin E (GSDME); Poly(ADP-ribose) polymerase (PARP); phosphorylated Mitogen-activated protein Kinase (pMEK); Extracellular signal-regulated kinase 1 and 2 (ERK 1/2); Lactate Dehydrogenase (LDH); Translocase of the Outer Mitochondrial membrane 20 (TOM20); Reactive Oxygen Species (ROS); Tumor Necrosis Factor (TNF); Apoptosis-associated Speck-like protein containing a CARD (ASC); Estrogen Receptor 1 (ESR1); Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK); Phosphorylated Eukaryotic Initiation Factor 2 (P-EIF2); Activating Transcription Factor 4 (ATF4); C/EBP Homologous Protein (CHOP); Bcl-2-associated X protein (Bax); Bcl-2 homologous antagonist/killer (Bak); Granulocyte colony-stimulating factor. ↑: up-regulation, ↓: down-regulation.

and

Table 2. In vivo studies of natural products targeting pyroptosis in HCC.

Natural Product	Animal Model	Concentration Duration	Mechanism	Result	Organism	Ref.
Euxanthone (flavonoid)	BALB/c nude mice	20 mg/kg 40 mg/kg for 24 days	NLRP3 ↑, caspase-1 ↑ IL-1β ↑, IL-18 ↑	↓ tumor growth ↑ pyroptosis	Polygala caudata (plant)	[15]
Miltirone (phenanthrene quinone derivative)	C57BL/6 male mice	1 mg/kg, 3 mg/kg, 6 mg/kg for 27 days	LDH ↑ P-N-GSDME ↑ caspase-3 ↑	↓ tumor growth	Salvia miltiorrhiza (plant)	[16]
Alpinumisoflavone (flavonoid)	Nude mice	20 mg/kg 40 mg/kg for 30 days	NLRP3 ↑ caspase-1 ↑ IL-1β, IL-18 ↑ GSDMD-N ↑	↓ tumor growth ↑ pyroptosis	Derris eriocarpa (plant)	[17]
Mallotucin D (clerodane diterpenoid)	BALB/c nude mice	5 mg/kg 15 mg/kg for 15 days	TOM20 ↑ ROS ↑ caspase-3 ↑ GSDMD ↑	↓ tumor growth ↑ apoptosis	Croton crassifolius (plant)	[19]
Neobavaisoflavone (isoflavone)	BALB/c mice	15 mg/kg and 30 mg/kg for 25 days	TOM20 ↑, ROS ↑ Bax ↑ caspase-3 ↑ N-GSDME ↑	↓ tumor growth ↑ pyroptosis	Psoralea sp. (plant)	[20]
Psoralidin (coumarin)	BALB/c mice	20 mg/kg for 14 days	caspase-3 ↑ GSDME ↑	↓ tumor growth ↑ pyroptosis ↑ immune cell infiltration	Psoralea sp. (plant)	[21]
Ajmalicine (alkaloid)	nude mice	5, 10, 15 mg/kg for 15 days	GSDME ↑ caspase-3 ↑	↓ tumor growth	Rauvolfia verticillata (plant)	[22]
Ellagic acid (polyphenol)	BALB/c nude mice	200 mg/kg for 3 weeks	ESR1	↓ tumor growth	Punica granatum, Rubus sp. (plant)	[23]
Glycyrrhetinic acid (triterpenoid)	C57BL/6J mice	20, 40, 60 mg/kg for 15 days	GSDME ↑ PERK ↑ cytoplasmic Ca2+ ↑	↑ pyroptosis ↑ immune cell infiltration	Glycyrrhiza glabra (plant)	[24]
Cannabidiol (cannabinoid)	Nude mice	40 mg/kg for 14 days	caspase-3 ↑, N-GSDME ↑ ATF4 ↑, CHOP ↑	↓ tumor growth ↑ pyroptosis	Cannabis sativa (plant)	[25]
Icaritin (flavonoid)	WT C57BL/6N mice	70 mg/kg twice daily for 17 days	caspase-1 caspase-3 ↑ GSDMD ↑ GSDME ↑ granzyme B ↑ IL-6 ↓, G-CSF↓ KC ↓ PD-L1 ↓, CD8+ ↑	↓ tumor growth ↑ pyroptosis ↑ immune cell infiltration	Epimedium sp. (plant)	[27]
Polyphyllin II (saponin) + IR780 in PLGA nanoparticles	C57BL/6 mice	5 mg/kg for 14 days	caspase-1 ↑ GSDMD-N ↑ IL-1β ↑	↓ tumor growth ↑ apoptosis ↑ pyroptosis ↑ immune cell infiltration	Rhizoma paridis (plant)	[29]
Naringenin (flavonoid)	Fischer rats	100 mg/kg for 17 days	caspase-1 ↓ GSDMD-N ↓ NLRP3 ↓	↓ pyroptosis	Citrus sp. (plant)	[35]
Cordycepin (purine nucleoside) Cordycepin + atezolizumab	BALB/c-NU/NU C57BL/6 mice	150 mg/kg once every 3 days for 15 days	TXNIP ↑ NLRP3 ↑ GSDMD ↑ LDH ↑	↓ tumor growth ↑ pyroptosis ↑ CD8+ T-cellinfiltration	Cordyceps militaris (fungus)	[32]
Cinobufotalin (bufadienolide)	BALB/c-NU/NU mice	8 mg/kg for 14 days	NOX4 ↑ NLRP3 ↑ caspase-1 ↑ IL-1β ↑ CB proteins ↑	↓ tumor growth ↓ metastasis ↑ pyroptosis	Bufo sp. (amphibian)	[34]

Abbreviations: NLR Family Pyrin Domain-Containing 3 (NLRP3); Interleukin-1 beta (IL-1β); Interleukin 18 (IL-18); gasdermin D (GSDMD); gasdermin D 31 kDa N-terminal fragment (GSDMD-N); Lactate Dehydrogenase (LDH); Translocase of the Outer Mitochondrial membrane 20 (TOM20); Reactive Oxygen Species (ROS);; Estrogen Receptor 1 (ESR1); Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK);; Activating Transcription Factor 4 (ATF4); C/EBP Homologous Protein (CHOP); Bcl-2-associated X protein (Bax);; Granulocyte colony-stimulating factor (G-CSF); Keratinocyte-Derived Chemokine (KC); Programmed cell death 1 ligand 1 (PD-L1); Thioredoxin-Interacting Protein (TXNIP); NADPH oxidase 4 (NOX4). ↑: up-regulation, ↓: down-regulation.

).

Chen et al. were the first to investigate the capacity of euxanthone (Eu), a flavonoid extracted from Polygala caudata, to elicit anti-cancer effects via the induction of pyroptosis. Human hepatocellular carcinoma (HCC) cell lines Hep3B and SMMC 7721 were exposed to euxanthone concentrations of 0, 5, and 10 μM for 24 and 48 h, while BALB/c nude mice in the in vivo arm of the study received daily administrations of 20 mg/kg and 40 mg/kg for 24 days. Εuxanthone robustly activated the canonical inflammasome pathway in HCC cells, with upregulation of NLRP3, cleaved caspase-1, and mature IL-1β/IL-18 observed both in vitro and in tumor-bearing mice. This finding established that euxanthone triggers caspase-1-dependent pyroptotic cell death in HCC [15].

In contrast, miltirone, a quinone derivative from Salvia miltiorrhiza, was found to provoke pyroptosis via the apoptotic caspase-3/GSDME pathway. Treatment of HepG2 human and Hepa1-6 murine HCC cells with 40 μmol/L miltirone for 24 h led to an upsurge in N-GSDME, a pivotal marker for pyroptosis, alongside augmentation of caspase-3, PARP, and GSDME expression and a concomitant decrease in MEK and ERK1/2. The findings implicate the MEK/ERK1/2 axis in miltirone-induced pyroptosis alongside enhanced ROS generation observed post treatment. These effects were corroborated in vivo by the accumulation of GSDME-N in treated mouse tumors, further supporting the role of inflammasome-mediated pyroptosis [16].

Treatment with alpinumisoflavone (AIF), the principal bioactive molecule sourced from Derris eriocarpa, further supports the role of inflammasome-mediated pyroptosis. Multiple HCC cell lines were treated with 0–20 μM AIF for intervals of 24 and 48 h, which resulted in increased LDH release and elevated mRNA and protein levels of NLRP3, cleaved caspase-1, cleaved IL-1β, and IL-18. Consistent pyroptotic marker induction was also observed in xenograft models, with enhanced expression of NLRP3, cleaved caspase-1, mature IL-1β, IL-18, and GSDMD-N, indicating pyroptosis induction predominantly through the NLRP3-caspase-1-IL-1β and IL-18 pathways in HCC cells [17].

Likewise, exposure of HepG2 cells to curcumin (a polyphenol from Curcuma longa and Curcuma zedoaria) led to characteristic pyroptotic changes including cell swelling, membrane lysis, and abundant formation of “pyroptotic bodies,” accompanied by heightened LDH release and the appearance of cleaved GSDME fragments, confirming that curcumin augments pyroptosis in HCC cells [18].

Other phytochemicals also provoke pyroptotic cell death in HCC. For instance, mallotucin D (MLD), a clerodane diterpenoid isolated from Croton crassifolius, induces pyroptosis in HepG2 cells treated with 0–20 μM for 24 h by activating the GSDMD pathway. MLD significantly increased inflammasome-associated markers (NLRP3, cleaved IL-1β, etc.) and GSDMD cleavage in cells, ultimately causing pyroptosis and suppressing cell proliferation. In MLD-treated tumor-bearing mice, these effects translated into marked tumor regression, underlining the potent anti-HCC impact of GSDMD-driven pyroptosis [19].

Similarly, two compounds from Psoralea species—neobavaisoflavone (NBIF) and psoralidin—were shown to engage the caspase-3/GSDME axis to induce pyroptotic death in HCC. Li et al. explored neobavaisoflavone (NBIF) in HepG2 and HCCLM3 cells treated with 50 μM NBIF for 24 h in vitro and BALB/c mice administered 15 or 30 mg/kg NBIF for 25 days in vivo. NBIF consistently enhanced TOM20 via ROS, fostered Bax translocation to mitochondria, and activated the caspase-3-GSDME pathway, thereby inducing pyroptosis in hepatoma cells [20]. Wang et al. conducted an investigation into the anti-cancer mechanisms of psoralidin, a coumarin, against HepG2 cells and mouse hepatoma cell lines Hepa1–6 and H22. The experimental results demonstrated an elevation in lactate dehydrogenase (LDH) release, proteolytic cleavage of gasdermin E (GSDME), and activation of caspase-3, collectively indicating the induction of pyroptosis in HCC via the caspase-3/GSDME signaling axis. BALB/c mice also received psoralidin treatment at a dosage of 20 mg/kg for 14 consecutive days, resulting in a significant reduction in tumor growth, attesting to pyroptosis-mediated anti-tumor efficacy in vivo [21].

In the same context, Sun et al. tested ajmalicine (AJM), an alkaloid from Rauvolfia verticillata roots. Mouse H22 cells were exposed to 5, 10, or 15 μM AJM for 24 h, and mice received 5, 10, or 15 mg/kg for 15 days. AJM has been shown to induce pyroptosis through the ROS-mediated caspase-3/GSDME cascade. Treatment with ajmalicine caused high LDH release and increased TNF-α, IL-1β, and IL-6 in HCC cell cultures, alongside accumulation of GSDME-N fragments, indicating GSDME-driven pyroptosis. Correspondingly, ajmalicine-treated tumor-bearing mice exhibited elevated cleaved caspase-3 and GSDME-N in tumor tissues, correlating with suppressed tumor growth via pyroptotic cell death [22].

Wang et al. assessed ellagic acid (EA), a polyphenol with multifaceted bioactivities, in vitro using HEK-293T, HepG2, and Huh-7 cells treated with 10–200 μM EA for 48 h and in vivo with BALB/c nude mice given 200 mg/kg EA for three weeks. In both cell-based and mouse xenograft experiments, ellagic acid upregulated NLRP3, ASC, cleaved caspase-1 (p20 subunit), IL-18, IL-1β, and GSDMD and its N-terminal fragment, indicating full assembly of the pyroptotic machinery. Notably, the tumor-suppressive impact of EA-induced pyroptosis was shown to depend on estrogen receptor-1 (ESR1), suggesting a link between hormonal signaling and inflammasome activity in HCC [23].

Bian et al. explored the efficacy of glycyrrhetinic acid (GA), a triterpenoid from Glycyrrhiza glabra, in vitro at 20 μM for 24 h in Hepa1-6 cells and in C57BL/6J mice at 20, 40, or 60 mg/kg intraperitoneally for 15 days. GA induced pyroptosis via the endoplasmic reticulum (ER) stress pathway. GA treatment activated the PERK/eIF2α unfolded protein response in HCC cells, which led to mitochondrial apoptotic events (upregulating Bax/Bak) and culminated in caspase-3 activation and GSDME-mediated pyroptosis. Consistently, GA administration in vivo elevated cleaved caspase-3 and GSDME levels and produced typical pyroptotic morphology in tumors, reinforcing that ER stress-induced apoptosis can lead to pyroptosis in HCC [24].

Cannabidiol (CBD), a phytochemical constituent of Cannabis sativa, has also been investigated for its anti-tumor properties in hepatocellular carcinoma. Multiple HCC cell lines were exposed to CBD at concentrations of 20 and 40 μM for 24 h and nude mice were treated with 40 mg/kg CBD for 14 days. CBD exposure in HCC cells led to phosphorylation of eIF2α and upregulation of the downstream transcription factors ATF4 and CHOP. This triggered the mitochondrial apoptotic pathway (increasing Bax/Bak) and subsequent caspase-3 activation, resulting in the cleavage of GSDME and the formation of membrane pores characteristic of pyroptosis. Consistently, in vivo treatment with CBD caused significant tumor growth suppression accompanied by GSDME fragment accumulation in tumor tissues [25].

Furthermore, traditional herbal formulations have demonstrated pyroptotic effects: Jinglinzi Powder (JLZP), composed of Alpinia oxyphylla and Corydalis yanhusuo, induced marked pyroptosis in HCC cells. JLZP treatment upregulated NLRP3 and promoted the maturation and release of IL-1β and IL-18, along with cleavage of GSDMD, thereby activating the canonical pyroptotic cell death pathway in hepatoma cells. This suggests that multi-component herbal remedies can target inflammasome signaling to exert anti-cancer effects [26].

In addition to directly killing tumor cells, some natural compounds modulate the immune microenvironment while inducing pyroptosis. Jiao et al. explored the mechanism by which icaritin (ICT), a bioactive compound derived from the dried leaves of the genus Epimedium, induces pyroptosis in HCC using multiple cell lines including HepG2, MHCC97H, HCCLM3, and Huh7. The cells were exposed to 20 and 40 µM concentrations of ICT for 48 h. Icaritin simultaneously activated the canonical caspase-1/GSDMD pathway and the apoptotic caspase-3/GSDME pathway in HCC cells, evidenced by increased cleavage of caspase-1 and -3 and the formation of both GSDMD-N and GSDME-N fragments. Notably, in co-culture systems combining HCC cells with immune cells, icaritin prompted the release of IL-1β and IL-18 and drove macrophages toward a proinflammatory phenotype, indicating that pyroptosis induction by icaritin can stimulate an immune response. In their in vivo experiments, wild-type C57BL/6N mice were treated with ICT at 70 mg/kg twice daily over 17 days. The study revealed increased levels of cleaved caspase-1, cleaved caspase-3, and the N-terminal fragments of GSDMD and GSDME, confirming the activation of pyroptosis pathways. Furthermore, ICT treatment resulted in decreased proinflammatory cytokines IL-6, G-CSF, and KC, alongside elevated infiltration of CD8+ T cells and granzyme B expression, further supporting an immune-potentiating effect alongside tumor pyroptosis [27]. Meanwhile, schisandrin B (Sch B), a lignan from Schisandra chinensis, demonstrated a unique immune context for pyroptosis. HepG2 cells were cultured with and without NK-92 cells and treated with 0–60 μM Sch B for 24 h. Sch B alone could decrease cell viability and induce apoptosis in HepG2 cells. However, in HCC cells co-cultured with NK immune cells, Sch B converted what would be apoptosis into pyroptosis via a perforin/granzyme B mechanism that activated caspase-3 and led to GSDME-mediated cell lysis. This immunogenic activation of pyroptosis by Sch B highlights how immune cell interactions can influence cell death mode [28].

Combining a natural product with a photothermal agent can amplify pyroptosis. Polyphyllin II (PPII), a steroidal saponin from Rhizoma paridis, notably induced pyroptosis in HepG2 cells treated with 0.75 μg/mL PPII combined with 0.3 μg/mL IR780 in PLGA nanoparticles for 12 h. This regimen promoted ROS generation, activation of the NLRP3 inflammasome, caspase-1 action, GSDMD cleavage, and IL-1β/IL-18 release. In vivo, C57BL/6 mice received treatment with PPII (5 mg/kg) and IR780 (2.5 mg/kg) co-loaded in PLGA nanoparticles every 2 days, resulting in significant tumor suppression through pyroptosis induction, as observed by increased levels of caspase-1, GSDMD-N, and IL-1β, as well as immune cell infiltration [29].

Interestingly, not all natural compounds promote pyroptosis; some can mitigate this pathway. For example, puerarin (PR), an isoflavone from Pueraria lobata, was found to inhibit the pyroptotic cascade in HCC cells. Puerarin treatment downregulated key inflammasome components (NLRP3, ASC, and caspase-1) and IL-1β expression, correlating with reduced pyroptosis and the suppression of proinflammatory signaling in HCC cultures. This suppression of the NLRP3/caspase-1/IL-1β axis by puerarin suggests that dampening pyroptosis-associated inflammation can also exert anti-tumor effects [30]. In an inflammation-driven liver cancer model, certain natural products have shown protective effects by suppressing pyroptosis. Naringenin (NAR), a citrus flavonoid, was tested in a diet-plus-carcinogen rat model of steatohepatitis-induced early HCC. Rats were subjected to a hepatopathogenic diet and hepatotoxins (carbon tetrachloride (400 mg/kg, i.p. and diethylnitrosamine 40 mg/kg, i.p.) for 16 weeks, followed by daily administration of 100 mg/kg NAR for 17 days. NAR treatment markedly inhibited NLRP3 inflammasome activation and pyroptosis in the liver, as evidenced by lower levels of cleaved caspase-1 and GSDMD-N in treated rats. This reduction in pyroptotic activity is associated with naringenin’s attenuation of inflammatory damage, thereby potentially impeding HCC development in that context [35].

Microbial-derived natural products have also been shown to induce pyroptosis in HCC. Reuterin (RT), a metabolite produced by Lactobacillus reuteri, was investigated by Cui et al. for its effect on pyroptosis in HCC models. HCC-LM9, HuH7, SK-Hep1, HuCCT1, RBE, and Hep3B cells treated with 50 or 100 μM for 24 h displayed increased activation of caspase-1 and GSDMD, elevated IL-1β, and upregulated caspase-8, which antagonized necroptosis, overall implicating the STING pathway in reuterin-induced pyroptosis [31]. Cordycepin (COR), an active compound isolated from the fungus Cordyceps militaris, was tested in MHCC97H and PLC/PRF/5 cells at concentrations of 350 and 500 μM for 4 days. BALB/c-nu/nu and C57BL/6 mice were also orally administered 150 mg/kg COR every 3 days for 15 days. Cordycepin caused mitochondrial membrane disruption and ROS accumulation in HCC cell lines, precipitating GSDMD-mediated pyroptosis and subsequent inhibition of cell proliferation. In mice, cordycepin treatment led to upregulation of TXNIP (an NLRP3 inflammasome activator) along with robust cleavage of GSDMD and the release of LDH, confirming pyroptosis induction in vivo. Notably, combining cordycepin with the immune checkpoint inhibitor atezolizumab (anti-PD-L1) further enhanced anti-tumor efficacy, significantly reducing tumor burden compared to monotherapy—an effect attributed to increased CD8⁺ T-cell infiltration in the tumor microenvironment [32].

Finally, even certain toxic metabolites and animal venoms have been repurposed to trigger pyroptosis in HCC. Zhang et al. investigated the anti-proliferative effects of 4,4′-secalonic acid D, isolated from the endophytic fungus Paraconiothyrium sp. AC31, on HepG2 and Huh7 cells treated with 12 μM for 48 h. Treated cells exhibited high caspase-3 activity and abundant GSDME-N release, along with elevated LDH and ROS levels, collectively indicating membrane rupture and lytic cell death; concurrently, PARP1 activity was inhibited, reinforcing the link to pyroptotic cell demise [33]. Meanwhile, cinobufotalin (CB), a bufadienolide from toad (Bufo sp.) venom, was shown to inhibit HCC progression by inducing pyroptosis. In vitro, HCC cell lines Hep3B, Huh-7, LM3, HepG2, and LO2 were treated with 0.5 μM CB for up to 72 h, while BALB/c-nu/nu mice received 8 mg/kg CB for 14 days in vivo. CB robustly activated the canonical NLRP3 inflammasome pathway as HCC cells and xenograft models showed a dose-dependent increase in NLRP3, ASC, cleaved caspase-1, IL-1β, and GSDMD-N, conclusively demonstrating pyroptosis initiation via caspase-1. The treatment also triggered a surge in oxidative stress markers (NOX4, H₂O₂), suggesting that ROS generation contributes to CB’s pyroptotic and anti-tumor effects [34].

Taken together, these studies illustrate that a wide array of natural compounds—ranging from plant polyphenols and alkaloids to microbial metabolites and animal toxins—can selectively induce pyroptosis in HCC cells. By activating either the inflammasome (caspase-1/GSDMD) or the apoptotic (caspase-3/GSDME) pyroptotic pathways (Figure 1), these agents effectively cause inflammatory cell death and often curb tumor growth in vivo, highlighting pyroptosis as a promising therapeutic target in hepatocellular carcinoma.

3. Cuproptosis

Copper is an indispensable metal nutrient required by all living organisms and involved in numerous biological activities. It serves as an essential cofactor for maintaining cellular processes and has been linked to cancer progression [36]. Recently, a novel form of cell death termed cuproptosis was discovered, which differs mechanistically from apoptosis, necroptosis, and ferroptosis. Cuproptosis is characterized by the direct binding of accumulated copper ions to lipoylated proteins within the mitochondrial tricarboxylic acid (TCA) cycle, leading to toxic protein aggregation and cell death. This process is specifically triggered by oxidative stress and mitochondrial dysfunction, representing a promising target for anti-cancer therapy by modulating copper metabolism [37]. The liver’s role is pivotal to copper metabolism—being responsible for uptake, storage, and elimination—and is thus vulnerable to copper imbalances that may contribute to the onset and progression of hepatic diseases [38].

The molecular mechanisms underlying cuproptosis have been extensively characterized in preclinical research, and the potential for translating these findings into therapeutic strategies for human diseases is substantial. The primary morphological characteristics of cuproptosis include plasma membrane rupture, mitochondrial contraction, and damage to the endoplasmic reticulum [39]. In affected cells, the abnormally accumulated copper in the mitochondria binds to lipoylated proteins, inducing their aggregation, which generates proteotoxic stress and leads to cell death. Ferredoxin 1 (FDX1) supplies electrons to lipoic acid synthase (LIAS) to initiate a free radical chain reaction essential for the synthesis of lipoyl cofactors, acting as a crucial mediator of cuproptosis. FDX1 works alongside LIAS to catalyze the formation of a disulfide bond in dihydrolipoamide S-acetyltransferase (DLAT), a critical step triggering its abnormal oligomerization. This oligomerization disrupts the pyruvate dehydrogenase complex (PDC), impairing the tricarboxylic acid (TCA) cycle and promoting ROS generation. Additionally, copper interferes significantly with the mitochondrial [4Fe-4S] cluster assembly, decreasing Fe-S cluster protein production through competition for metal-binding sites. During mitochondrial respiration, Cu+ directly binds to lipoylated TCA cycle components, causing the accumulation of lipid-acylated proteins and reduced synthesis of iron–sulfur cluster proteins, ultimately resulting in proteotoxic stress and cell death. These findings strongly highlight the direct link between copper-induced toxicity and mitochondrial dysfunction in cuproptosis [40].

Cuproptosis profoundly influences HCC tumor biology by disrupting copper homeostasis, which drives both tumor-promoting “cuproplasia” (copper addiction for growth/metastasis) and anti-tumor cell death through mitochondrial proteotoxic stress. Dysregulated copper transporters like SLC31A1 (↑) and ATP7B (↓) elevate intra-tumoral copper, fueling glycolysis, lipid metabolism, angiogenesis, and immune evasion, while excessive copper triggers FDX1-mediated reduction of Cu²⁺ to toxic Cu⁺, aggregating lipoylated TCA enzymes (DLAT/PDHA1/DLD/LIAS); low FDX1/LIAS expression in HCC subtypes correlates with resistance and poor survival. Thus, targeting cuproptosis exploits HCC’s copper dependency and can induce tumor cell death [41].

Natural Products Against HCC Mediating Cuproptosis

A number of studies have tested the potential cytotoxic and/or cytostatic properties of natural products against HCC targeting the cell death mechanism of cuproptosis. Studies were identified using Boolean strings—(“hepatocellular carcinoma” OR HCC) AND (“cuproptosis” OR “copper-dependent cell death” OR “Cu-induced cell death”) AND (“natural product*” OR “plant extract*” OR “phytochemical*” OR “herbal”)—while the filters used included English, original research/reviews, and in vitro/in vivo HCC models. The databases searched were PubMed/MEDLINE, Web of Science, Scopus, and Google Scholar with no time frame. Abstracts were screened independently by two reviewers and studies demonstrating natural products affecting cuproptosis in HCC cells or HCC models were included (

Table 3. In vitro studies of natural products targeting cuproptosis in HCC.

Natural Product	Cell Line	Concentration Duration	Mechanism	Result	Organism	Ref.
Curcumin (polyphenol)	PLC, KMCH Huh7	25 μM for 72 h	LIAS ↓ DLAT ↓	CPI ↓ in KMCH	Curcuma longa (plant)	[42]
(1E, 6E)-1,7-Bis(3,4-dihydroxyphenyl)-1,6-heptadiene-3,5-dione) (curcumin derivative)	HepG2	1–30 μM for 48 h	GSH ↓ DLAT, FDX1 ↓	↓ cuproptosis	Curcuma longa (plant)	[43]
Taxifolin (flavonoid)	Huh7, HepG2	0–1000 μM for 48 h	Cu2+ ↑ ROS levels ↑ SLC31A1, FDX1 ↑ LIAS, DLAT ↑	↓ cell proliferation ↑ cuproptosis	Larix gmelinii (plant)	[44]
Quercetin (flavonoid)	HCC-LM3 Huh7 HCC-LM3-LR Huh7-LR	2–40 μM for 24–48 h	Cu2+ ↑ FDX1 ↑ lipoylation of DLAT and LIAS ROS levels ↑	↑ cuproptosis	Malus sp. Vaccinium sp. Lactuca sativa (plant)	[45]
Epigallocatechin gallate (polyphenol)	HepG2, SMMC-7721	100 μM for 24 h	Cu2+ ↑ HSP70 ↑ aggregation of DLAT MTF1 ↓ ATP7B ↓	↓ cell proliferation ↑ cuproptosis	Camellia sinensis (plant)	[46]
Plumbagin (naphthoquinone)	Huh7, PLC	0–64 μM for 24 h	DNMT1 ↓ miR-302a-3p ↑ ATP7B ↓ Cu2+ ↑ DLAT oligomerization LIAS ↓	↓ cell proliferation ↑ cuproptosis ↑ oxidative stress	Plumbago zeylanica (plant)	[47]
Tian Yang Wan (combined plant extract)	HepG2	0–5 μM for 24–72 h	CCDC43 ↓	↓ cell proliferation ↓ migration ↓invasion ↑ apoptosis cuproptosis immune cell infiltration	Rehmannia, Morindaofficinalis, Cistanche, Cynomorium songaricum, Cornus, Yam (plants)	[48]

Abbreviations: cuproptosis potential index (CPI); glutathione (GSH); dihydrolipoamide S-acetyltransferase (DLAT); ferredoxin 1 (FDX1); lipoic acid synthase (LIAS), Solute Carrier Family 31 Member 1 (SLC31A1); Heat Shock Protein 70 (HSP70); Metal Regulatory Transcription Factor 1 (MTF1); ATPase copper transporting beta (ATP7B); DNA methyltransferase 1 (DNMT1); Coiled-coil domain-containing protein 43 (CCDC43). ↑: up-regulation, ↓: down-regulation.

and

Table 4. In vivo studies of natural products targeting cuproptosis in HCC.

Natural Product	Animal Model	Concentration Duration	Mechanism	Result	Organism	Ref.
(1E, 6E)-1,7-Bis(3,4-dihydroxyphenyl)-1,6-heptadiene-3,5-dione) curcumin derivative	BALB/c-nude mice	5 mg/kg for 18 days	DLAT ↓ FDX1 ↓	↓ tumor growth	Curcuma longa (plant)	[43]
Taxifolin (flavonoid)	BALB/c nude mice	200 mg/kg for 7 days	Ki67 ↓	↓ tumor volume	Larix gmelinii (plant)	[44]
Quercetin (flavonoid)	BALB/c nude mice	20 mg/kg for 3 weeks	Cu2+ ↑ FDX 1 lipoylation of DLAT and LIAS ROS levels ↑	↓ tumor growth ↑ cuproptosis	Malus sp. Vaccinium sp. Lactuca sativa (plant)	[45]
Epigallocatechin gallate (polyphenol)	BALB/c-nude mice	10 mg/kg for 3 weeks	Cu2+ ↑ HSP70 ↑ aggregation of DLAT MTF1 ↓ ATP7B ↓	↑ cuproptosis	Camellia sinensis (plant)	[46]
Plumbagin (naphthoquinone)	BALB/c nude mice	2 mg/kg for 2 weeks	DNMT1 ↓ miR-302a-3p ↑ ATP7B ↓ Cu2+ ↑ DLAT oligomerization LIAS ↓	↓ tumor growth ↑ cuproptosis ↑ oxidative stress	Plumbago zeylanica (plant)	[47]

Abbreviations: dihydrolipoamide S-acetyltransferase (DLAT); ferredoxin 1 (FDX1); lipoic acid synthase (LIAS), Heat Shock Protein 70 (HSP70); Metal Regulatory Transcription Factor 1 (MTF1); ATPase copper transporting beta (ATP7B); DNA methyltransferase 1 (DNMT1);. ↑: up-regulation, ↓: down-regulation.

, Figure 1).

Liu et al. investigated the role of curcumin in modulating cuproptosis within HCC cells, leveraging its well-documented pharmacological profile. Treatment of PLC, KMCH, and Huh7 HCC cell lines with 25 μM curcumin for 72 h revealed a cell type-specific response, as evidenced by a significant reduction in the cuproptosis potential index (CPI) exclusively in KMCH cells, whereas PLC and Huh7 cells showed no notable CPI changes [42].

In a related study, Yang et al. explored the effects of a novel curcumin polyphenol derivative, ((1E, 6E)-1,7-Bis(3,4-dihydroxyphenyl)-1,6-heptadiene-3,5-dione), designated bm-Cur, on HepG2 cells. Cells were treated with bm-Cur at concentrations ranging from 1 to 30 μM for 48 h. The results indicated concentration-dependent depletion of intracellular glutathione (GSH) and downregulation of key cuproptosis-related proteins DLAT and FDX1, culminating in cuproptosis induction. In vivo, BALB/c nude mice treated with bm-Cur (5 mg/kg) for 18 days demonstrated significant tumor growth inhibition, concomitant with decreased DLAT and FDX1 expression within tumor tissues, reinforcing bm-Cur’s potential as an anti-HCC therapeutic agent [43].

Taxifolin, a flavonoid extracted from the stem bark of Larix gmelinii (Dahurian larch), was evaluated in Huh7 and HepG2 cells treated with concentrations of 0–1000 μM for 48 h. Taxifolin treatment inhibited cell proliferation, concomitantly elevating intracellular Cu²⁺ levels, ROS, and the expression of cuproptosis markers SLC31A1, FDX1, LIAS, and DLAT. Correspondingly, oral administration of 200 mg/kg taxifolin to BALB/c nude mice for 7 days resulted in reduced tumor volume and Ki67 expression, suggesting that taxifolin promotes SLC31A1-mediated cuproptosis via intracellular copper accumulation [44].

Quercetin, a ubiquitous flavanol found in fruits and vegetables known for its antioxidant, anti-inflammatory, and anti-cancer attributes, was examined by Yang et al. for its capacity to sensitize lenvatinib-resistant HCC cells to copper-induced cytotoxicity. Parental and lenvatinib-resistant HCC-LM3 and Huh7 cells were treated with 2–40 μM quercetin, while BALB/c nude mice received 20 mg/kg quercetin combined with 5 mg/kg ES-Cu complex. Quercetin enhanced intracellular Cu²⁺ accumulation and activated cuproptosis through upregulation of FDX1 and increased lipoylation of DLAT and LIAS, thereby restoring the sensitivity of resistant HCC cells to ES-Cu-induced cell death [45].

Fu et al. studied the effect of Epigallocatechin gallate (EGCG), a major green tea polyphenol, on cuproptosis induction in HepG2 and SMMC-7721 cell lines treated with 100 μM for 24 h and BALB/c nude mice administered 10 mg/kg EGCG for 3 weeks. The results indicated elevated intracellular copper, upregulation of HSP70, DLAT aggregation, and reduced levels of MTF1 and ATP7B, implicating EGCG in facilitating cuproptosis via regulation of the MTF1/ATP7B axis [46].

Plumbagin (PLB), a quinoid compound from Plumbago zeylanica roots, was assessed in Huh7 and PLC cells with 0–64 μM PLB treatment for 24 h and BALB/c nude mice treated with 2 mg/kg PLB for 2 weeks. Both in vitro and in vivo experiments showed that PLB reduced DNMT1 levels, leading to increased miR-302a-3p expression, downregulation of ATP7B, and subsequent intracellular copper accumulation, triggering cuproptosis. The cuproptotic response was further confirmed by DLAT oligomerization, LIAS depletion, and copper accumulation [47].

Tian Yang Wan (TYW), a traditional Chinese medicinal formulation comprising Rehmannia sp., Morinda officinalis, Cistanche sp., Cynomorium songaricum, Cornus sp., and Dioscorea sp. (Yam), was tested on HepG2 cells with 0–5 μM TYW treatment over 24–72 h, resulting in significant downregulation of CCDC43. Bioinformatic analyses correlated CCDC43 expression strongly with multiple cuproptosis-related genes, including ABCC1, ATG5, TP53, and PDHA1, suggesting CCDC43 as a candidate therapeutic target mediating cuproptosis in HCC [48].

4. Discussion

Natural compounds that target critical carcinogenic pathways while exhibiting minimal adverse effects may serve as valuable chemotherapeutic or chemopreventive agents, providing an alternative or complementary strategy for cancer treatment. In this article, recent data concerning the use of natural products against HCC were summarized focusing on the relatively newly discovered non-apoptotic mechanisms of pyroptosis and cuproptosis.

Many natural products enhance HCC cell death through pyroptosis or cuproptosis induction. Previous studies in cancer models have also highlighted that the activation of pyroptosis or cuproptosis may serve as a potent target for cancer therapy. In particular, miltirone has been reported to reduce the viability of colorectal cancer cells (CRCs), causing the proteolytic cleavage of gasdermin E (GSDME) and effectively binding with caspase-3. These findings suggest that miltirone has the potential to inhibit the growth of CRC tumors in vivo by inducing pyroptotic cell death [49]. Furthermore, in certain cancer contexts, curcumin promotes pyroptosis by stabilizing NLRP3 through the inhibition of Smurf2-mediated ubiquitination. Molecular docking studies support curcumin’s direct binding to several pyroptosis-associated proteins, including NLRP3, AMPK, caspase-1, and Smurf2. These context-dependent regulatory effects underscore the therapeutic potential of curcumin as a pyroptosis inducer in cancer [50].

On the other hand, puerarin and naringenin are proposed to enhance HCC cell death through pyroptosis inhibition. Even though there are not much data concerning the effects of puerarin on cancer cells’ pyroptosis mechanisms, numerous studies have demonstrated that puerarin inhibits pyroptosis in various types of diseases. In particular, puerarin effectively inhibits the pyroptosis signaling pathway during diabetic cardiomyopathy as it is shown to block NLRP3-caspase-1-GSDMD-mediated pyroptosis in H9C2 cells and RAW264.7 cells, alleviating cellular inflammation. The protective effects of puerarin are related to the P2X7 receptor as the molecular docking results indicate key binding activity between the P2X7 receptor and puerarin [51]. Puerarin also alleviates cerebral ischemia–reperfusion by inhibiting pyroptosis through the caspase-1/GSDMD axis [52] and restrains pyroptosis of cells induced by NLRP3 inflammasome to abate acute lung injury [53] as it mitigates renal inflammatory damage by modulating the SIRT1/NLRP3/caspase-1 pathway [54]. Accordingly, naringenin alleviates renal ischemia–reperfusion injury by suppressing ER stress-induced pyroptosis and apoptosis through activiation of the Nrf2/HO-1 signaling pathway [55].

In the cases of ellagic acid, schisandrin B, CBD, and cordycepin, data seem controversial. The aforementioned natural products are suggested to serve as potent anti-HCC agents through pyroptosis induction, yet studies in other model diseases suggest their pyroptosis-inhibiting potential. In particular, ellagic acid suppresses ovarian cancer cell viability and proliferation by arresting cells at the G1 phase of the cell cycle through modification of cell death mediated by inflammatory-caused pyroptosis. Ellagic acid downregulates GSDMD and GSDME and suppress the levels of inflammatory markers, including IL-1β and IL-6 [56]. Schisandrin B suppresses NLRP3 inflammasome activation-mediated IL-1β levels and pyroptosis in intestinal epithelial cells of a colitis model through the activation of AMPK/Nrf2-dependent signaling ROS-induced mitochondrial damage, which may be a significant therapeutic approach in the treatment of acute colitis [57]. CBD prevents liver steatosis and oxidative stress in a mice liver injury model induced by ethanol plus a high-fat high-cholesterol diet, as it inhibits macrophage recruitment and suppresses activation of the NFκB-NLRP3–pyroptosis pathway in mice livers. The hepatoprotective property of CBD in the current model is proposed to be a result of inflammation inhibition by alleviating activation of the hepatic NFκB-NLRP3 inflammasome–pyroptosis pathway [58]. Cordycepin also inhibits macrophage pyroptosis by reducing XO activity, suppressing ROS production, and regulating the expression of key molecules in the NLRP3/caspase-1/GSDMD pathway [59]. Nevertheless, most disease models demonstrating the therapeutic potential of pyroptosis inhibition are related to inflammation. Given that the pyroptosis phenotype is closely associated with inflammatory processes, the suppression of pyroptotic cell death and the subsequent reduction in inflammation likely accounts for the observed therapeutic effects in these studies.

In the context of cuproptosis, data from other studies also demonstrate the therapeutic potential of cuproptosis mediation. A study regarding quercetin aimed to elucidate the mechanism by which lenvatinib-resistant HCC cells evade copper-induced cell death and to evaluate whether quercetin enhances ES-Cu-induced cuproptosis by targeting FDX1 and reprogramming mitochondrial metabolism. Lenvatinib-resistant HCC cells display downregulation of cuproptosis-related genes (FDX1, DLAT) and impaired copper accumulation. Quercetin binds with FDX1, enhances mitochondrial OCR, and synergistically increases intracellular copper accumulation with ES-Cu, leading to lipoylated protein aggregation, mitochondrial dysfunction, and copper-induced cell death. In vivo, quercetin plus ES-Cu significantly suppresses tumor growth without evident toxicity, highlighting the therapeutic potential of quercetin and ES-Cu combination treatment as a novel strategy to overcome lenvatinib resistance in HCC [45]. In addition, quercetin is found to suppress cuproptosis, guarding against acute kidney injury [60].

On the other hand, curcumin promotes programmed cell death in colorectal cancer through metabolism control of lipids, RNA, NADH, and NADPH as well as up-regulation of positive cuproptosis mediators [61] and oxidative stress-induced cuproptosis [62]. In addition, nano-curcumin liposomes inhibit the growth and survival of gastric adenocarcinoma cells by interfering with the expression of FDX1, GPX4, SERPINE1, and SLC27A5, which are closely linked to copper-induced oxidative stress, thereby promoting tumor-associated programmed cell death linked to cuproptosis [63].

Pyroptosis, as an inflammatory form of programmed cell death, plays a significant role in the progression and treatment of hepatocellular carcinoma (HCC). Unlike apoptosis, pyroptosis is characterized by cell swelling, lysis, and the release of proinflammatory cytokines such as IL-1β and IL-18, thereby modulating the immune landscape within the tumor microenvironment. Evidence indicates that pyroptosis can both inhibit HCC development by promoting tumor cell death and activating anti-tumor immunity, and, in certain contexts, potentially alter the tumor microenvironment to facilitate tumor progression. In HCC tissues, pyroptosis is often found to be suppressed, and reactivation of the pathway has been shown to reduce cancer cell viability and invasion through the restoration of caspase-1 activity [64]. Thus, understanding and modulating pyroptosis has emerging therapeutic and prognostic significance in HCC, offering novel approaches to enhance anti-cancer immune responses and to overcome resistance to existing treatments.

In addition, recent studies reveal that key regulators such as SEC14L3 promote cuproptosis in HCC cells by enhancing copper toxicity through pathways involving ERK/YY1/FDX1, thereby suppressing tumor cell viability and growth. The dysregulation of cuproptosis-related genes correlates with immune microenvironment alterations and patient prognosis, highlighting the dual role of cuproptosis in modulating tumor progression and immune response [65]. Therapeutically, targeting cuproptosis offers a promising strategy in HCC treatment, as manipulating copper homeostasis or cuproptosis signaling pathways could overcome drug resistance and improve patient outcomes. This growing understanding positions cuproptosis as a critical process influencing HCC pathogenesis and as a novel target for innovative cancer therapies.

Emerging evidence highlights mechanistic crosstalk between pyroptosis and cuproptosis in HCC pathogenesis. Central to this crosstalk is copper-induced ROS overproduction. Excess Cu²⁺, facilitated by transporters like CTR1 and ATP7A/B (often upregulated in HCC), is reduced to cytotoxic Cu⁺ by FDX1. This triggers dual downstream effects: (i) mitochondrial ROS activates the NLRP3 inflammasome, priming pro-caspase-1 cleavage and GSDMD-N pore assembly for pyroptosis; and (ii) direct Cu⁺ ligation to the dihydrolipoamide arms of DLAT/PDHA1 disrupts TCA flux (e.g., α-ketoglutarate accumulation), inducing proteotoxic aggregates and CHOP-mediated ER stress that amplifies both pathways. In HCC cells, this hub is dysregulated: FDX1 downregulation (observed in ~70% of The Cancer Genome Atlas project-HCC samples) promotes copper tolerance and tumor evasion while sustaining low-level ROS that fuels NLRP3 without full pyroptotic commitment, fostering an immunosuppressive tumor microenvironment [41,66,67]. In the HCC context, this integration has prognostic and therapeutic implications. Highly cuproptosis-related gene signatures (e.g., FDX1, LIAS, LIPT1) correlate with “cold” tumors exhibiting PD-L1 overexpression and poor CD8⁺ T-cell infiltration, yet pyroptotic damage-associated molecules (like IL-1β, ATP) from adjacent cells can sensitize to immune checkpoint inhibitors. Preclinical models demonstrate synergy: copper ionophores like elesclomol or disulfiram/Cu²⁺ combinations induce bimodal regulated cell death, enhancing calreticulin exposure and STING activation for superior anti-tumor immunity compared to single modalities [41,68]. Natural compounds further exploit this axis, warranting clinical translation.

Nevertheless, pyroptosis induction in cancer therapy risks excessive inflammation from cytokine storms (IL-1β/IL-18), chronic tissue damage, and off-target pyroptosis in healthy cells, exacerbating chemotherapy toxicity (e.g., doxorubicin-induced cardiotoxicity via caspase-3/GSDME). Cuproptosis, triggered by copper overload, can cause copper toxicity (hepatotoxicity, nephrotoxicity, neurotoxicity), ROS-mediated damage, and paradoxical tumor promotion via cuproplasia at low doses. Thus, therapeutic strategies targeting tumor selectivity (nanoparticles) are necessary to mitigate these side effects [41,69].

As HCC represents a significant global health burden and is the predominant form of primary liver cancer, the use of natural products targeting pyroptosis or cuproptosis is increasingly being recognized as a novel strategy in HCC treatment. Natural products modulating pyroptosis and cuproptosis in hepatocellular carcinoma (HCC), such as curcumin, psoralidin, icaritin, and euxanthone, exhibit promising preclinical potential by targeting the NLRP3/GSDMD or FDX1/LDLRAP1 pathways to induce tumor cell death and enhance immune responses in HepG2/Huh7 and murine models. However, some of the products are relatively newly discovered; so, further studies are needed to elucidate their bioavailability, pharmacokinetics and possible toxicity. Furthermore, additional in vivo investigations are required to determine whether these compounds are effective at physiological concentrations or exhibit synergistic effects when combined with conventional chemotherapeutic drugs, thereby establishing their potential as candidates for clinical trials.