Molecular hydrogen in cancer: reprogramming oncogenic signaling pathways and emerging pharmacological potential

1 Introduction

Cancer remains a significant global health burden, accounting for over 10 million deaths annually, with incidence and mortality expected to rise substantially over the coming decades (Bray et al., 2024; Deo et al., 2022). Although numerous organic, inorganic, and naturally occurring compounds have been developed as anticancer agents, the long-term success of conventional chemotherapy and radiotherapy is frequently limited by systemic toxicity, therapeutic resistance, off-target effects and collateral damage to healthy tissues (Zou et al., 2025; Kee et al., 2025; Ribeiro and Correia, 2024; Kozieł et al., 2024; Jiang et al., 2026; Banti et al., 2024). These limitations have intensified the search for mechanistically active, low-toxicity auxiliary interventions capable of improving therapeutic responsiveness while minimizing treatment-associated adverse effects ⁽ Kee et al., 2025; Crans and Miller, 2025; Banti et al., 2024; Bates et al., 2025). Among these emerging approaches, H₂ has gained attention for its ability to achieve broad tissue distribution, modulate redox homeostasis, oncogenic signaling, and tumor-associated immune responses.

H₂ is a small, colorless, non-toxic and odorless gas. Initially investigated for its selective interactions with cytotoxic reactive oxygen species (ROS). H₂ is now increasingly recognized for its ability to selectively neutralize highly cytotoxic oxidants while preserving physiologically relevant redox signaling species. Through this redox-selective behavior, H₂ has been implicated in the oxidative stress responses, inflammatory cascades, epigenetic remodeling, mitochondrial proteostasis and cancer-associated survival pathways. Furthermore, the mild reductive properties of H₂ appear to contribute to broader antioxidant, anti-inflammatory and immunomodulatory actions within the tumor microenvironment. H₂ can therefore mitigate chemotherapy and radiotherapy induced tissue damage while influencing critical molecular mediators linked to tumor proliferation, apoptosis and metastatic progression. In view of these expanding mechanistic insights, this review examines the chemical-biological basis of H₂ mediated anticancer activity, with particular emphasis on epigenetic regulation, mitochondrial stress signaling, tumor suppressive cell death, immunomodulation and its emerging role as a pharmacological adjuvant in cancer therapy.

2 Milestones in the development of cancer therapeutics and the need for low-toxicity auxiliary strategies

Cancer is an ancient disease, evidence of which was found in Egyptian and Peruvian mummies dating to around 1500 BC. Early clinical distinctions between benign and malignant tumor were described by Gabriele Fallopius, based on characteristics such as firmness, irregularity, and tissue adhesion (Faguet, 2015). But later, Louis Bard proposed that cancer arises from defects in cellular differentiation, leading to uncontrolled proliferation. For centuries, surgery was the primary treatment, though its effectiveness was limited by the lack of anesthesia and antisepsis which was modified in the late 19th century enabled more successful interventions, exemplified by William Stewart Halsted who developed radical mastectomy for breast cancer. Around the same time, early immunotherapy approaches were explored by William B. Coley using bacterial toxins, despite initial skepticism. A major breakthrough came with Larry Einhorn whose cisplatin-based combination regimens significantly improved survival in testicular cancer in the 20th century (McAleer, 2022). By the 1990s, multimodal approaches integrating surgery, radiotherapy and chemotherapy had become the standard of care. Now, in this present century the cancer therapeutic approach has become more modern, including T-cell immunotherapy to stem cell therapy with high level of success rate.

3 H₂ as a bioactive gas in cancer pharmacology

H₂ is the smallest, lightest and most abundant molecule available on earth, composed of 2 H atoms linked through a non-polar covalent bond. It is colorless, odorless, non-toxic gas which is highly stable in normal physiological condition.

3.1 Physiochemical and biological properties of H₂

H₂ has certain beneficial properties which makes it a good therapeutic agent. As it is lighter than air so it can easily disperse through blood and can reach tissues. Due to strong covalent bonds, it is highly stable. 1t can easily absorb porous metal nanoparticles. So, this property will be useful for selective delivery of it to the tumor environment keeping the healthy cells untouched. It is a strong reducing agent which can react with oxygen, halogen and other oxidizing elements. Although it is highly inflammable, still the inhalation of certain dose is completely safe for human health (Levikhin and Boryaev, 2024; Leachman et al., 2009).

3.2 Selective redox modulation and cellular penetrability

H₂ has a unique combination of selectivity to highly harmful reactive radicals and permeability which makes it effective for oxidative stress related issues like cancer. H₂ behaves as a strong anti-oxidant by scavenging highly toxic radicals like hydroxyl radical (^**.**OH) and peroxynitrite (ONOO^**−**) without affecting the essential metabolic oxygen species. Whereas, other anti-oxidant doesn’t enter through blood brain barrier (BBB), due to high diffusion coefficient and small nature of it can easily cross BBB (Xie et al., 2016). So, it maintains healthy cellular redox balance by penetrating through cell membranes to nucleus, cytoplasm and mitochondria.

3.3 H₂ delivery modalities in experimental oncology

For delivering H₂, it is mainly focused on its low solubility in water and rapid dissipation characteristics. Primarily H₂ is delivered to the body through inhalation, H₂ rich water and H₂ rich saline. H₂ is taken by inhalation in the form of mixture with oxygen (66.7% H₂ and 33.35 O₂) which is considered safe and it distributes rapidly in our system. Oral consumption of H₂ rich water prepared by dissolving it in water, which is safe and convenient for chronic conditions. For the mitigation of liver injury caused by chemotherapy, H₂ rich water is used as adjuvant treatment. For a more precise dose of H₂, intraperitoneal and intravenous injection of H₂ rich saline is used (Jin et al., 2025).

Nowadays, for targeted delivery of H₂ to the tumor microenvironment (TME) different nanoplatforms are used. Biodegradable magnesium -based material like magnesium wire is used which react with water in tumor micro environment and produce H₂ continuously in situ for more than 24 h that increase oncogenic p53 expression to induce apoptosis (Zan et al., 2022; He et al., 2020). Due to the smallest nature of H₂, it is absorbed by lots of metals. Palladium hydride nanocrystals act as H₂ carriers which release H₂ in NIR (near infra-red) stimulation. So, it is a synergistic approach of H₂ and photo-thermal therapy. Mesoporous silica is loaded with ammonia borane which releases H₂ in the acidic microenvironment of the tumor. Also, synthesized hydrogel is used to carry H₂ precursors like MgH₂ to sustain local release of H₂ (Barzgar et al., 2025).

4 Epigenetic remodeling and non-coding RNA regulation by H₂

Histone methylation was once considered irreversible due to the strong thermodynamic stability of the N−CH₃ bond. Subsequent research, however, revealed that certain genes can dynamically regulate the methylation and demethylation of histone proteins. This reversible modification enables histones to function as either transcriptional activators or repressors, depending on the site of methylation and the number of methyl groups present. In this section, we focus on the methylation status of H3K27 and its implications in cancer development and progression (Table 1) (De Majo and Calore, 2018; Greco and Condorelli, 2015).

TABLE 1. Histone modification and its effect (Zhang et al., 2015).

4.1 JMJD3-H3K27 signaling and chromatin reprogramming

Jumonji C domain-containing protein 3 (JMJD3), a member of the lysine demethylase (KDM) family, displays context-dependent roles, acting as either a tumor suppressor or promoter depending on the cellular environment. Structurally, JMJD3 features a β-barrel domain with a catalytic site that requires Fe²⁺ and α-ketoglutarate as cofactors (Lagunas-Rangel, 2021). Through histone demethylation, JMJD3 regulates gene expression, representing a key epigenetic mechanism in cancer. JMJD3 exerts tumor-suppressive effects in several malignancies. In prostate cancer, it upregulates the miRNA cluster miR-99a/let-7c/125b which represses androgen signaling and modulates the EZH₂/JMJD3 axis, thereby inhibiting cell proliferation. In acute myeloid leukemia (AML), JMJD3 targets C/EBPβ and RIPK3, promoting myeloid differentiation, cell-cycle arrest, and apoptosis, restoring cells to a more differentiated, normal-like state. In colorectal and colon cancers, JMJD3 induces tumor suppressors such as P15INK4b, CST5/Cystatin D, CDH1/E-Cadherin, and CYP24A, represses EMT regulators ZEB1 and SNAI1, suppresses Wnt/β-catenin signaling, and activates chemokines CXCL9 and CXCL10, enhancing antitumor immunity (Sanchez et al., 2021). In other cancers, JMJD3 modulates critical pathways to limit malignancy. In ERα-positive breast cancer, it upregulates the anti-apoptotic gene BCL-2 to maintain cell survival and degrades PHF20 to reduce stemness (Svotelis et al., 2011). In pancreatic adenocarcinoma, JMJD3 mediates KRAS-induced senescence, activates CEBPA, induces HKDC1, disrupts cytoskeletal integrity, and suppresses tumor growth (Yang et al., 2023). In non-small cell lung cancer, JMJD3 inhibits proliferation and migration while activating intrinsic apoptosis via FOXO1 phosphorylation (Zhang et al., 2019). In hypoxic malignant pleural mesothelioma, JMJD3 increases lactate and KDM6B levels, restores ERβ expression, promotes epithelial phenotypes, elevates E-cadherin, and inhibits tumor progression (Farzaneh et al., 2022). Collectively these findings highlight JMJD3 as a versatile epigenetic regulator that modulates differentiation, apoptosis, immune signaling, and metastatic potential across diverse cancer types.

H₂ enhances the expression of the histone demethylase JMJD3 environment (Figure 1), resulting in modifications of the H3K27 methylation landscape. Specifically, H₂ decreases H3K27me2 levels while increasing H3K27me1, which subsequently elevates Kcnc3 mRNA expression, a gene sensitive to H3K27 methylation. Through JMJD3 activation, H₂ also induces mitochondrial heat shock protein 70 (HSP70), a key mediator of the mitochondrial unfolded protein response (mtUPR), linking epigenetic regulation to mitochondrial stress adaptation (Havalová et al., 2021).

Methylation of histone H3 at lysine 27 (H3K27me3) recruits the polycomb repressive complexes (PRC2 and PRC1) to nucleosomes, maintaining gene silencing by restricting nucleosome mobility (Martinez-Garcia and Licht, 2010). This epigenetic modification suppresses transcription of numerous genes, influencing cancer progression. Accumulation of H3K27me3 dampens inflammatory responses, whereas its demethylation enhances the expression of pro-inflammatory cytokines such as IL-6, IL-2, and ICAM. H3K27me3 also inhibits ferroptosis, further contributing to tumor survival.

In various cancers, H3K27me3 cooperates with EZH₂ to regulate oncogenic pathways. For example, it modulates TPM1 to promote proliferation and angiogenesis in colorectal cancer and downregulates miR-138-5p in hepatocellular carcinoma (Di and Zhu, 2023), facilitating tumor growth. H3K27 methylation activates Wnt/β-catenin signaling, suppresses negative regulators such as DUSP5, and downregulates tumor suppressors including p53, p15, RUNX3, and E-cadherin, supporting proliferation, EMT and metastasis. In gastric cancer, H3K27me3 represses miR-34a, activating c-Met and Snail1 to promote EMT. It also confers resistance to TRAIL-induced apoptosis by inhibiting DR4 transcription and suppresses miR-143, blocking BAX-dependent caspase-3 signaling. In colon cancer, H3K27me3 reduces TH1-type chemokines CXCL9 and CXCL10, impairing T-cell infiltration and limiting the efficacy of immunotherapy (Chen et al., 2020).

4.2 MALAT1/miR-124-3p/EZH₂ axis in metastatic suppression

Studies on gastric cancer cell lines have demonstrated that H₂ can modulate key epigenetic regulators by downregulating the long non-coding RNA (lncRNA) MALAT1 (Figure 2), upregulating microRNA miR-124-3p, and reducing EZH₂ protein levels (Zhu et al., 2020). These factors interact to influence tumorigenesis. The miR-124-3p generally acts as a tumor suppressor by regulating transcripts involved in cell proliferation, migration, and invasion. For instance, in hepatocellular carcinoma, MALAT1 binds miR-124-3p, promoting the expression of Snail family proteins which suppress the epithelial marker E-cadherin and facilitate cancer cell migration (Fu et al., 2020). In glioma, miR-124-3p inhibits the p62 oncogene and reduces SDCBP expression, limiting tumor progression. In non-small cell lung cancer, it suppresses metastasis via exosomal transport and PI3K/AKT signaling, while in gastric cancer, it downregulates HRCT1, inactivating ERBB2- MAPK signaling. In colorectal cancer, miR-124-3p inhibits proliferation by targeting AKT3 (Gourishetti et al., 2023).

Conversely, MALAT1 can act as a molecular sponge, sequestering miR-124-3p and diminishing its tumor-suppressive activity. MALAT1 also directly interacts with EZH₂, the catalytic subunit of PRC2, enhancing H3K27 trimethylation and silencing tumor suppressor genes such as E-cadherin, DAB2IP and BRACHYURY (Wang et al., 2015). EZH₂ assembles with β-catenin directly to form a complex that controls the expression of FTO genes and increases the expression of c-Myc to promote tumor cell growth (Liu and Yang, 2023). EZH₂ also triggers metastasis through the epigenetic silencing of Ras-GAP gene function, which disables Homolog 2 interacting Protein to activate Ras and the NF-кB pathway, promoting prostate tumor growth and progression (Chang and Hung, 2012). In triple-negative breast cancer, EZH₂ forms a complex with Forkhead box M1 (Fox M1) to induce MMP expression, leading to cancer cell invasion and metastasis. In ovarian cancer, EZH₂ inhibits the expression of TIMP2 via H3K27me3 and DNA methylation, leading to MMP expression and cancer cell migration and invasion. EZH₂ interacts with SNAIL1 and downregulates the expression of E-cadherin to promote EMT, an important factor in cancer cell metastasis (Gan et al., 2018 ). EZH₂ is also a direct target of miR-124-3p. Thus, lncRNA MALAT1 and miR-124-3p antagonize each other and competitively regulate EZH₂ expression to modulate different cancer cell proliferation, invasion, and migration (Figure 2).

4.3 Epigenetic determinants of H₂ responsiveness

H₂ has shown to regulate different gene expression like histone modifications, DNA methylation and non-coding RNA by modulating it. These histone deacetylation and methylation lead to activation of mitochondrial Unfolded Protein responses (mtUPR). It prevents oxidative damage of Histone protein and DNA by which it protects genomic stability and affects Histone code to alter gene accessibility. Also, it affects chromatin modified enzymes that regulate gene responsiveness and control stress response. In this way by changing the methylation of the promoter region, H₂ acts as an epigenetic modulator. The factors such as diet and age-related oxidative stress influence the oxidative landscape that determine the sensitivity of a cell to H₂ (Zhiling et al., 2022).

5 Regulation of proteostasis, mitochondrial stress and tumor suppressive cell death

Aberrant regulation of transcription and translation leads to the production of mutant proteins, which represent a major driving force in cancer initiation and progression. H₂ has been reported to modulate the expression and function of several cancer-associated mutant proteins. The following section will focus on key proteins influenced by H₂ and their relevance to tumorigenesis (Song et al., 2021; Silvera et al., 2010).

5.1 HSP70/UPR and protein homeostasis

H₂ maintains protein homeostasis by regulating ER stress and modulating chaperones like Heat Shock Protein HSP70. HSP70 is a molecular chaperone that maintains quality of proton by folding newly synthesized protein, refolding of misfolded protein and targeting damaged protein to send in Ubiquitin-Proteasome system (UPS) (O’Brien et al., 2014; Larburu et al., 2020; Rosenzweig et al., 2019). When this unfolded protein is stored in endoplasmic reticulum that will create ER stress which is mitigated by H₂, leads to apoptosis and triggered UPR mediated removal of pathogenic cells (Casey and Orth, 2018; Pustovaya et al., 2026; Voos and Röttgers, 2002).

In the “allosteric model”, ER localized binding immunoglobulin protein (BiP) dissociates from the IRE1 receptor when misfolded protein binds to BiP. H₂ stabilizes this system by assisting BiP in managing folding and maintaining UPR at a homeostatic level rather than a pro-apoptotic state (Perera and Ron, 2023).

5.2 CD47/CDC42 signaling in apoptosis resistance and metastasis

Cluster of Differentiation 47 (CD47) is a ∼50 kDa cell-surface glycoprotein, also referred to as integrin-associated protein (IAP). As a member of the immunoglobulin superfamily, CD47 functions as a key antiphagocytic signal by interacting with thrombospondin-1 (TSP-1) and signal regulatory protein-α (SIRPα) expressed on macrophages. This interaction delivers a “don’t eat me” signal that enables cancer cells to evade immune-mediated clearance and contributes to resistance to apoptosis (Huang et al.,2020). Notably, H₂ treatment has been shown to downregulate CD47 expression, along with cell division control protein 42 (CDC42), in lung cancer cells (Figure 3). This reduction is associated with suppressed cellular proliferation, invasion, and migration, accompanied by enhanced apoptotic activity. Collectively, these findings indicate that H₂ directly targets antiphagocytic signaling pathways and exerts a significant antitumor effect in lung cancer cells.

Post-translational modification of CD47, particularly the formation of an N-terminal pyroglutamate residue, is essential for its recognition by the extracellular immunoglobulin-like ligand-binding domain of signal regulatory protein-α (SIRPα) expressed on multiple macrophage populations (Huang et al., 2022). Engagement of CD47 with SIRPα triggers phosphorylation of two tyrosine residues within the immunoreceptor tyrosine-based inhibitory motif (ITIM) located in the cytoplasmic domain of SIRPα. This phosphorylation event recruits and activates the downstream phosphatases SHP-1 and SHP-2, initiating a signaling cascade that results in dephosphorylation of myosin IIA. Consequently, cytoskeletal rearrangement is inhibited, thereby suppressing macrophage phagocytosis of target cells and facilitating immune evasion by cancer cells.

In addition to SIRPα, CD47 interacts with thrombospondin-1 (TSP-1), a ligand secreted primarily by platelets and macrophages. The CD47–TSP-1 interaction stimulates intracellular signaling pathways involving calcium ions (Ca²⁺), cyclic adenosine monophosphate (cAMP), and other secondary messengers that collectively regulate cellular responses to stress, including cell proliferation, migration, survival, and tissue damage repair (Catalán et al., 2020).

Overexpression of CD47 has also been shown to activate Rac and cell division control protein 42 (CDC42), a member of the Rho family of small GTPases. According to the COSMIC database, at least 41 mutations in CDC42 have been identified in human cancers. CDC42 functions as a molecular switch, becoming active upon GTP binding, and subsequently regulates a broad array of downstream effector proteins, including ACK1, p21-activated kinases (PAKs), mixed-lineage kinase 3 (MLK3), IQ motif-containing GTPase-activating proteins (IQGAPs), neural Wiskott–Aldrich syndrome protein (N-WASP), and phosphoinositide 3-kinases (PI3Ks). These signaling pathways collectively promote tumor cell proliferation, invasion, and metastasis (Xiao et al., 2018). Mutant forms of CDC42 further enhance mitochondrial glutamine metabolism, supporting metabolic reprogramming of tumor cells and driving malignant progression. In gastric cancer, CDC42 regulates epidermal growth factor receptor (EGFR) expression, thereby stimulating tumor cell proliferation, while in colorectal cancer, it modulates PAK-dependent tight junctions and adhere junction complexes (AJCs), contributing to uncontrolled cell growth. CDC42 also governs anti-apoptotic signaling by influencing key oncogenic and survival pathways involving p53, anti-apoptotic Bcl-2 family members, c-MYC and fatty acid synthase, collectively supporting cancer cell survival. Moreover, CDC42-mediated activation of PAKs and IQGAP1 promotes epithelial–mesenchymal transition (EMT), a critical driver of angiogenesis and metastatic dissemination. CDC42 further enhances nuclear factor-κB (NF-κB) signaling, leading to increased transcription and accumulation of pro-tumorigenic cytokines such as interleukin-6 (IL-6) and interleukin-8 (IL-8), thereby fostering tumor growth and angiogenesis (Fu et al., 2022).

Importantly, emerging evidence indicates that H₂ directly suppresses CDC42 expression, thereby inhibiting CDC42-driven metastatic signaling and exerting a protective anti-tumor effect (Meng et al., 2020).

5.3 SMC3, GP73, XIAP, BIRC3, and related survival proteins

Structural Maintenance of Chromosomes 3 (SMC3) is a core component of the cohesin multiprotein complex, which is essential for chromosome condensation, sister chromatid cohesion, DNA damage repair, and homologous recombination. Dysregulated expression and pathogenic mutations of SMC3 have been reported across multiple cancer types, implying cohesion dysfunction in tumorigenesis. In a xenograft mouse model using lung cancer cell lines, treatment with H₂ rich water was shown to significantly reduce SMC3 protein levels by enhancing its ubiquitination (Figure 4). In parallel, H₂ also downregulated other chromosomal maintenance proteins, including NIPBL, SMC5, and SMC6, which are involved in chromosome organization and condensation. Notably, these effects were not observed in lung epithelial cells derived from healthy mice, indicating that H₂ selectively targets tumor cells without exerting cytotoxic effects on normal tissues.

In addition to cohesin disruption, H₂ treatment suppresses the expression of key cell-cycle regulators such as cyclin D1, CDK4, and CDK6, resulting in reduced tumor cell proliferation and enhanced apoptotic activity through G2/M phase cell-cycle arrest. Under physiological conditions, SMC3 undergoes acetylation at lysine residues K105 and K106 during the S phase to stabilize cohesion between replicated sister chromatids. This modification is subsequently reversed during telophase by histone deacetylase 8 (HDAC8), allowing recycling of the cohesin complex for subsequent cell-cycle progression(Wilson et al.,2010). It has been proposed that H₂ may interfere with HDAC8 expression or activity, leading to the accumulation of acetylated SMC3, prolonged G2/M checkpoint arrest, and consequent inhibition of tumor cell proliferation (Sang et al., 2024).

Also, H₂ may influence SMC3 function indirectly by reducing Reactive oxygen species, thereby limiting oxidative DNA damage and preserving chromosomal stability. Additionally, H₂ mediated modulation of cellular redox balance and epigenetic states could affect cohesin dynamics and SMC3 activity, particularly in the context of cancer and genome maintenance (Wang and Yi, 2008).

In studies using highly invasive liver cancer cell lines, H₂ rich water has been found to slow cancer cell growth by altering key molecular signals (Figure 5). Specifically, treatment with H₂ rich water lowered both GP73 mRNA and protein levels, factors linked to cancer metastasis. While, elevating the mRNA and protein expression of TGF-β and TNF-α, which are known to function as tumor suppressors. These molecular changes suggest that H₂ rich water can exert inhibitory effects on hepatocellular carcinoma cell proliferation.

Golgi protein 73 (GP73) is predominantly expressed in biliary epithelial cells and is markedly upregulated in hepatocellular carcinoma (HCC) as well as other pathological liver conditions. GP73 functions as an anti-apoptotic factor and facilitates tumor cell proliferation, migration, invasion, and metastasis through multiple signaling mechanisms that vary with cellular context. For instance, in adenocarcinoma and breast cancer cells, GP73 enhances the activation of matrix metallopeptidase-13 (MMP-13) by increasing the expression of cAMP response element-binding protein (CREB). CREB transcriptionally stimulates MMP-13, resulting in extracellular matrix (ECM) remodeling and the release of ECM-associated growth and angiogenic factors, thereby promoting tumor cell invasion and proliferation (Jin et al., 2015).

In melanoma, pancreatic and prostate cancer cells, GP73 drives cell proliferation by modulating the phosphoinositide 3-kinase (PI3K/AKT) signaling pathway (Wang and Wan, 2020). GP73 also regulates the secretion of alpha-fetoprotein (AFP), an oncoprotein that suppresses the tumor suppressor PTEN and interacts with caspase-3 to inhibit apoptosis in HCC. Furthermore, GP73 induces the expression of metastasis-related genes, including K19, EPCAM, MMP2, MMP9, and CXCR4, thereby enhancing HCC cell migration. By binding to its receptor, AFPR, GP73 activates intracellular Ca²⁺ and cAMP signaling cascades that further stimulate HCC cell proliferation (Liu et al., 2021).

Downregulation of GP73 using H₂ decreases the Bcl-2/Bax ratio, elevates cytochrome c and caspase-3 levels and promotes apoptotic cell death. GP73 silencing also reduces the expression of N-cadherin and E-cadherin while increasing vimentin levels, leading to inhibition of epithelial–mesenchymal transition (EMT) and suppression of tumor cell proliferation. Additionally, GP73 overexpression induces caveolin-1 (Cav-1), a key structural protein of lipid rafts, which sequesters transforming growth factor-β receptors (TβRs) within lipid rafts and promotes their degradation. This process results in reduced TGF-β expression, a known tumor suppressor (Yang et al., 2019). TGF-β acts as a potent anti-mitogenic factor by inducing cyclin-dependent kinase (Cdk) inhibitors that block late G1-phase Cdk activation, thereby preventing retinoblastoma protein (pRb) phosphorylation and subsequent entry of tumor cells into the S phase of the cell cycle (Zhang et al., 2014). The transforming growth factor-β (TGF-β) signaling pathway plays a critical role in regulating cellular differentiation, migration, invasion and apoptosis through the SMAD family of transcriptional mediators. Upon ligand binding, TGF-β engages type II receptors, which subsequently recruit and phosphorylate type I receptors. Activated type I receptors then phosphorylate receptor-regulated SMADs (R-SMADs) enabling them to associate with the common SMAD. Through these mechanisms, H₂ may help suppress abnormal SMAD activation associated with inflammation, fibrosis and cancer progression, highlighting its potential as a modulatory therapeutic agent. We are investigating the mechanistic pathway how the H₂ molecule regulates GP73 mRNA and protein expression. This R-SMAD/co-SMAD complex translocate into the nucleus, where it regulates the transcription of specific target genes by interacting with promoter regions, thereby mediating tumor suppressive effects (Wang et al., 2023).

In the context of Immunotherapy which is an emerging therapeutic strategy for cancer treatment: T cells, which are white blood cells known as lymphocytes, play a crucial role in killing cancer cells through their receptors (TCR) binding with specific antigens. Cluster of differentiation 8 (CD8) is a transmembrane glycoprotein that functions as a co-receptor for the TCR, and together they mediate tumor cell killing through multiple intracellular signaling pathways. Once activated, CD8⁺ T cells induce direct cytotoxicity by releasing perforin and granzymes and trigger apoptosis through the surface expression of death ligands, primarily Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL). In addition, these cells secrete cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-α(TNF-α), and TNF-β, which coordinate antitumor immune responses involving both innate and adaptive immunity (Farhood et al., 2019; Raskov et al., 2021).

Despite their potent effector functions, prolonged TCR stimulation can drive CD8⁺ T cells into a state of functional exhaustion. Tumors exhibiting systemic immunosuppressive properties (SIP) release atypical, non-proteinaceous factors that impair mitochondrial energy metabolism in CD8⁺ T cells, thereby promoting exhaustion (Figure 6) (Zhang et al., 2022a). Sustained antigen stimulation leads to the accumulation of depolarized mitochondria, further exacerbating this exhausted phenotype (Yu et al., 2020). Mitochondrial dysfunction disrupts cellular metabolism and alters chromatin accessibility, resulting in aberrant gene expression patterns characteristic of exhausted T cells (Lisci and Griffiths, 2022; Vardhana and Hwee, 2020).

Moreover, impaired mitochondria generate excessive mitochondrial reactive oxygen species (mtROS), which damage DNA, proteins, and lipids, further compromising T-cell function. Elevated ROS levels sustain chronic NFAT signaling, inducing the transcription factors TOX and NR4A and promoting the expression of exhaustion-associated genes. Excess ROS also suppresses NF-κB phosphorylation, reduces mTORC1 signaling, and diminishes T-cell activation. Additionally, ROS activates the caspase cascade, ultimately increasing T-cell apoptosis (Yang et al., 2024).

Programmed cell death protein-1 (PD-1) is an inhibitory immune checkpoint receptor expressed on lymphocytes, including T and B cells, where it suppresses T-cell inflammatory responses and maintains immune self-tolerance. Although PD-1 signaling is essential for preventing autoimmunity, it simultaneously diminishes immune cytotoxicity and is widely recognized as a hallmark of exhausted T cells. Mechanistically, PD-1 associates with the SH₂-domain containing tyrosine phosphatase SHP-2, which dephosphorylates proximal activating signaling molecules and suppresses T-cell receptor (TCR) signaling (Ahn et al., 2018).

Effector CD8⁺ T cells have high energetic demands that are largely met through glycolysis, a metabolic feature shared with rapidly proliferating tumor cells. Immune checkpoint molecules such as PD-1 and cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) suppress glycolytic flux by inducing glucose deprivation, thereby contributing to T-cell exhaustion. PD-1 signaling further impairs mitochondrial oxidative phosphorylation (OXPHOS) by downregulating metabolic gene expression, exacerbating the exhausted phenotype. H₂ has been shown to reduce the proportion of exhausted PD-1⁺ CD8⁺ T cells while enhancing the population of functional PD-1^- CD8⁺ T cells through activation of peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), a key regulator of mitochondrial biogenesis and mitochondrial repair (Akagi and Baba, 2019). So, through this mechanism, H₂ promotes mitochondrial function, improves oxidative metabolism, and may protect against metabolic disorders and cancer-related mitochondrial dysfunction.

PGC-1α, encoded by the PPARGC1α gene, serves as a master transcriptional regulator of mitochondrial biogenesis. Intracellular hydroxyl radicals initiate lipid peroxidation chain reactions that generate reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE). Elevated 4- HNE restores Akt phosphorylation, which in turn (Enoch et al., 1976) phosphorylates forkhead box protein O1 (FoxO1) and cAMP response element-binding protein (CREB), leading to repression of PGC-1α transcription. H₂ selectively scavenges hydroxyl radicals, interrupting lipid peroxidation cascades, reducing 4-HNE levels, and thereby restoring PGC-1α expression (Figure 6) (Kamimura et al., 2016).

Activated PGC-1α induces a network of nuclear transcription factors, including nuclear respiratory factors-1 and 2 (NRF-1, NRF- 2) and estrogen-related receptor-α (ERR-α), which collectively enhance the expression of mitochondrial transcription factor A (TFAM), a protein essential for mitochondrial DNA (mtDNA) replication and transcription. MtDNA-encoded genes are translated into proteins with the help of specific translation factors like mitochondrial translation initiation factors 2 and 3 (mtIF2 and mtIF3), elongation factors Tu, Ts, and G1 (mtEFTu, mtEFTs, and mtEFG1), translation releasing factor-1-like (mtRF1L), and recycling factors (mtRRF1 and mtRRF2). Mitochondrial protein synthesis is further regulated by translational activator of cytochrome c oxidase 1 (TACO1), which binds mitochondrial RNA and facilitates efficient translation of mitochondrial proteins (Popov, 2020).

In addition to promoting mitochondrial biogenesis, PGC-1α enhances the clearance of damaged mitochondria through transcriptional activation of PTEN-induced kinase 1 (PINK1). Upon mitochondrial membrane depolarization, PINK1 accumulates on the outer mitochondrial membrane, recruits and activates the E3 ubiquitin ligase PARKIN, and initiates polyubiquitination of mitochondrial proteins. These ubiquitinated mitochondria are recognized by the autophagy receptor optineurin (OPTN), which interacts with microtubule-associated protein light chain 3 (LC3) to form autophagosomes that subsequently fuse with lysosomes for mitochondrial degradation (Chen et al., 2022). PGC-1α also limits excessive mitochondrial fission by modulating dynamin-related protein-1 (Drp1) expression, thereby preserving mitochondrial integrity.

Collectively, H₂ mediated upregulation of PGC-1α supports mitochondrial biogenesis promotes mitophagy of dysfunctional mitochondria, and restores metabolic fitness in exhausted CD8⁺ T cells (Abu Shelbayeh et al., 2023). The functional reactivation of these T cells enhances their cytotoxic capacity, ultimately facilitating tumor cell apoptosis.

Another protein Stearoyl-CoA desaturase-1 (SCD1) is an endoplasmic reticulum localized transmembrane enzyme that catalyzes the desaturation of saturated fatty acids (SFAs) into Δ-9 monounsaturated fatty acids (MUFAs). Specifically, SCD1 converts palmitic acid (C16:0) to palmitoleic acid (C16:1) and stearic acid (C18:0) to oleic acid (C18:1). By generating MUFAs, SCD1 plays a pivotal role in lipid biosynthesis, providing phospholipid components required for cell membrane assembly during mitotic progression (Hess et al., 2010).

Over the past decade, SCD1 has attracted considerable interest in oncology and has been identified as a potential therapeutic target across multiple tumor types (Hao et al., 2004; Von Roemeling et al., 2014). Suppression of SCD1 disrupts membrane lipid synthesis, leading to impaired cancer cell proliferation and the induction of apoptotic cell death (Figure 7) (Scaglia and Igal, 2005).

Dysregulated activation of the PI3K/AKT/mTOR signaling cascade is closely associated with tumorigenesis. A major mechanism by which AKT/mTOR drives cancer progression is through the metabolic reprogramming of tumor cells, particularly by enhancing lipogenic pathways (Bakan and Laplante, 2012). Upon activation, mTOR stimulates the transcription and maturation of sterol regulatory element-binding protein-1 (SREBP-1), which in turn upregulates the expression of key lipogenic enzymes, including ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), and stearoyl-CoA desaturase-1 (SCD1). Multiple studies have demonstrated that hyperactivation of the AKT/mTOR axis leads to elevated SCD1 expression in various cancers (Calvisi et al., 2011; Luyimbazi et al., 2010).

Conversely, SCD1 is a critical downstream effector required for AKT/mTOR-mediated tumor cell proliferation (Figure 7). Both genetic knockdown and pharmacological inhibition of SCD1 have been shown to suppress AKT phosphorylation and reduce cell growth in prostate cancer models (Fritz et al., 2010). Furthermore, silencing SCD1 impairs oncogenic signaling by attenuating Akt activation and altering the activity of glycogen synthase kinase-3β (GSK3β), a downstream substrate of Akt, ultimately leading to reduced cancer cell proliferation. Emerging evidence indicates that H₂ suppresses colorectal cancer (CRC) cell proliferation by inhibiting the AKT/SCD1 signaling axis. Clinical observations have shown that SCD1 is markedly overexpressed in CRC patients from eastern China, suggesting that H₂ may represent a potential therapeutic option for CRC cases characterized by elevated SCD1 expression. Several studies has consistently demonstrated that H₂ treatment significantly reduces phosphorylated AKT (pAKT) levels across the three distinct CRC cell lines (Figure 7).

Notably, dose-dependent effects of H₂ have been reported. A recent study revealed that low H₂ concentrations (30%) were sufficient to induce cell death in SW480 cells, whereas higher concentrations (>50%) were required to inhibit proliferation in RKO and HCT116 cells. These differences may be attributed to tumor heterogeneity, as SW480 cells are derived from rectal carcinoma, while RKO cells originate from colon adenocarcinoma, indicating that distinct CRC subtypes exhibit variable sensitivity to H₂ exposure. Furthermore, Holder et al. demonstrated that PI3K/AKT signaling enhances SCD1 expression in breast cancer cells (Luyimbazi et al., 2010). Based on these findings, it has been proposed that H₂ exerts its antitumor effects in colorectal cancer by suppressing SCD1 expression through the downregulation of AKT phosphorylation.

AKT, also referred to as protein kinase B (PKB), is a 57 kDa serine/threonine kinase that serves as a central regulator of growth factor–mediated cell survival (Hu et al., 2019). Elevated levels of phosphorylated AKT (pAKT) are observed in the majority of malignancies, reflecting its pivotal role in oncogenic signaling. AKT activation contributes to tumor progression by promoting de novo lipid synthesis, fatty acid oxidation, and very-low-density lipoprotein (VLDL) assembly and secretion in rapidly proliferating cells. In addition, phosphorylation-dependent activation of AKT suppresses apoptotic pathways and enhances tumor cell proliferation, making pAKT an attractive therapeutic target in cancer (Song et al., 2019).

Notably, H₂ has been shown to exert context-dependent effects on AKT signaling. While a previous study demonstrated that high concentrations of H₂ ameliorated myocardial ischemia–reperfusion injury in mice by increasing pAKT1 expression (Chen et al., 2017), other investigations have reported that H₂ does not alter total AKT levels but significantly reduces pAKT in cancer cells. These findings suggest that H₂ may inhibit colorectal cancer (CRC) cell proliferation by modulating AKT phosphorylation at Ser473. AKT levels but significantly reduces pAKT in cancer cells. These findings suggest that H₂ may inhibit colorectal cancer (CRC) cell proliferation by modulating AKT phosphorylation at Ser473 (Figure 7).

To validate this hypothesis, CRC cells were treated with the AKT activator SC79 in the presence of H₂. Activation of AKT by SC79 effectively reversed the H₂ induced suppression of cell proliferation across all three CRC cell lines examined, confirming that H₂ mediated inhibition of tumor cell survival occurs through downregulation of pAKT. Based on these observations, it has been proposed that H₂ interfere with AKT phosphorylation and may also reduce specific lipid species involved in lipid metabolism, thereby attenuating AKT activity (Siess and Leonard, 2019). Furthermore, accumulating evidence indicates that H₂ suppresses the expression of stearoyl-CoA desaturase-1 (SCD1) by reducing pAKT levels. Consistent with this mechanism, treatment of CRC cells with SC79 significantly increased SCD1 expression, counteracting the effects of H₂. Collectively, these findings support the conclusion that H₂ reduces CRC cell survival by targeting the pAKT/SCD1 signaling axis and identify SCD1 as a potential molecular mediator of H₂ based therapeutic strategies.

The effects of H₂ on colorectal cancer (CRC) cells appear to be cell type specific, reflecting differential modulation of the PI3K/AKT signaling pathway. H₂ treatment has been shown to increase PI3K expression in RKO cells, while reducing PI3K levels in SW480 and HCT116 cells indicating heterogeneous cellular responses. In SW480 and HCT116 cells, H₂ suppresses both PI3K and phosphorylated AKT (p-AKT), thereby inhibiting PI3K/AKT signaling and reducing tumor cell proliferation. In contrast, the elevated PI3K expression observed in RKO cells may represent a compensatory or resistance mechanism to counteract H₂ induced inhibition of AKT phosphorylation. Overall, decreased AKT phosphorylation is likely to impair downstream oncogenic signaling cascades in CRC cells.

Stearoyl-CoA desaturase-1 (SCD1) is frequently overexpressed in colorectal cancer tissues and contributes to cancer stem cell maintenance and tumor progression. Given its central role in CRC pathogenesis, SCD1 has emerged as a promising molecular target for anticancer therapy. H₂ mediated suppression of the PI3K/AKT/SCD1 axis may therefore reduce tumor growth, recurrence and metastasis, particularly in CRC patients exhibiting high SCD1 expression (Figure 7) (Zhang et al., 2022b).

Inhibitor of apoptosis proteins (IAPs) are key modulators of programmed cell death and play a central role in suppressing apoptotic signaling through both intrinsic and extrinsic pathways. A defining feature of all IAP family members is the presence of one to three baculovirus IAP repeat (BIR) domains, which mediate interactions with caspases and other regulatory proteins. Among these, the X-linked inhibitor of apoptosis protein (XIAP) is the most potent and extensively studied anti-apoptotic IAP. XIAP inhibits apoptosis by directly binding and suppressing caspase-9 through its BIR3 domain, as well as caspases-3 and -7 via its BIR2 domain. Second mitochondria-derived activator of caspases (SMAC/Diablo) serves as a natural antagonist of XIAP and is released from mitochondria during apoptotic signaling. SMAC/Diablo disrupts the interaction between XIAP and caspases by competing for binding to the BIR2 and BIR3 domains, thereby restoring caspase activity and promoting apoptosis. Based on this mechanism, SMAC mimetics have been developed to selectively trigger apoptosis in cancer cells or to enhance the efficacy of conventional anticancer therapies (Obexer and Ausserlechner, 2014).

Apoptosis is essential for maintaining cellular homeostasis and regulating cell survival (Fulda et al., 2010). However, this process is frequently dysregulated in cancer, particularly during adaptation to therapeutic stress. Such dysregulation facilitates the clonal expansion of aggressive tumor populations that exhibit resistance to hypoxia, radiation, chemotherapy, and other adverse conditions (Reed, 1999; Pazarentzos and Bivona, 2015). Accumulating evidence indicates that elevated XIAP expression contributes to resistance against chemotherapy and radiotherapy while also modulating anti-tumor immune responses. As a result, substantial efforts over the past decade have focused on developing XIAP- targeted therapeutic strategies, including RNA-based approaches, pan-IAP peptidomimetics (Amantana et al., 2004; LaCasse, 2013; Cao et al., 2004; Devi et al., 2005; Arora and Devi, 2004), and small-molecule inhibitors (Abbas and Larisch, 2020; Shahar and Larisch, 2020; Panayotopoulou et al., 2017). Notably, XIAP overexpression has been associated with poor prognosis and reduced overall survival across a wide range of malignancies, including hepatocellular carcinoma, gastric adenocarcinoma, head and neck squamous cell carcinoma, esophageal cancer, and hematological malignancies such as leukemia (Tamm et al., 2000; Shi et al., 2008; Dizdar et al., 2018; Gao et al., 2019).

In addition to their direct inhibitory interactions with caspases, IAP family members facilitate caspase ubiquitination and subsequent proteasomal degradation through their RING-finger domains, which confer E3 ubiquitin ligase activity (Yang et al., 2000). In multiple cancer types, XIAP has been strongly associated with the development of resistance to both chemotherapy and radiotherapy (Sapi et al., 2004; Muris et al., 2005; Alvero et al., 2006). Consequently, XIAP has emerged as an attractive therapeutic target for circumventing treatment resistance. Supporting this concept, numerous preclinical studies have demonstrated that pharmacological inhibition of XIAP effectively enhances tumor cell sensitivity across a broad range of malignancies (Holcik et al., 2000).

In this context, H₂ has emerged as a functional “small-molecule inhibitor” capable of inducing apoptosis in the lung adenocarcinoma cell line A549. H₂ treatment has been shown to markedly suppress the expression of X-linked inhibitor of apoptosis protein (XIAP) and baculoviral IAP repeat-containing protein 3 (BIRC3) in A549 cells. In a study conducted by Zhang et al., A549 cell suspensions were subcutaneously implanted into nude mice, followed by exposure to 60% H₂ inhalation. The H₂ treated group exhibited significantly reduced levels of XIAP and BIRC3 compared with control animals. These findings indicate that XIAP and BIRC3, as key members of the IAP family, may play an important role in H₂ mediated apoptotic signaling in A549 cells. Notably, IAP family proteins are frequently overexpressed in a wide range of human cancers and possess strong regulatory control over cell death and survival pathways, thereby contributing to chemoresistance, tumor progression, and unfavorable clinical outcomes (Mizutani et al., 2007; Yang et al., 2012).

Experimental evidence obtained from immunohistochemistry and flow cytometry indicates that exposure to high concentrations of H₂ (40–60%) significantly suppresses XIAP expression in lung cancer A549 cells. Multiple studies have demonstrated that XIAP plays a critical role in regulating mitochondrial membrane permeabilization, a process whose disruption facilitates cytochrome c release into the cytosol and subsequent activation of caspase-dependent apoptotic pathways (Gogada et al., 2011; Hamacher-Brady et al., 2014; Hamacher-Brady and Brady, 2015). These findings suggest that H₂ may interfere with mitochondrial and caspase-associated signaling cascades, thereby promoting apoptosis in tumor cells.

Recent investigations have highlighted two emerging hallmarks of cancer: metabolic reprogramming and immune evasion (Hanahan and Weinberg, 2011). Rapidly proliferating tumor cells require a highly adaptive energy metabolism, with mitochondria serving as central regulators of bioenergetic supply. Consistent with this, elevated hydrogen exposure has been associated with reduced expression of BIRC3 in A549 lung cancer cells. Supporting the oncogenic role of BIRC3, Xiaojie Jiang et al. reported that cIAP2/BIRC3 is highlyexpressed in gallbladder carcinoma and correlates with poor patient prognosis (Jiang et al., 2017). Similarly, Nagata et al. demonstrated that BIRC3 is overexpressed in oral squamous cell carcinoma and is closely linked to patient survival, identifying it as a potential therapeutic target (Nagata et al., 2011). Notably, suppression of BIRC3 expression markedly enhances apoptosis in cancer cells.

Inflammatory signaling mediated by XIAP is primarily regulated through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) isoforms, which interact directly with the XIAP promoter and control its transcription. Co-immunoprecipitation studies have confirmed binding of NF-κB p65 to the XIAP promoter in BRCA1-mutated cells, suggesting that dysregulated NF-κB signaling contributes to elevated XIAP expression. Further analyses revealed significant upregulation of activated NF-κB subunits, including p65 phosphorylated at Ser536 and p50, as well as its precursor p105, in BRCA1-mutated UWB1289 cells. These observations indicate that NF-κB activation, rather than increased protein abundance, is responsible for enhanced XIAP expression. Accordingly, pharmacological inhibition of NF-κB leads to reduced XIAP mRNA and protein levels through both transcriptional and post-transcriptional mechanisms, underscoring the central role of NF-κB in XIAP regulation.

5.4 NLRP3-mediated pyroptosisand apoptotic reprogramming

Certain pathological subtypes of endometrial cancer characterized by deep myometrial invasion and a high risk of distant metastasis frequently exhibit resistance to radiotherapy and chemotherapy, largely due to enhanced tolerance to apoptosis and necrosis. In this context, pyroptosis, a pro-inflammatory and lytic form of programmed cell death, has emerged as a promising alternative therapeutic strategy. Experimental evidence indicates that H₂ promotes pyroptotic cell death in endometrial cancer cells by activating tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) signaling pathways, alongside inducing both cellular and mitochondrial reactive oxygen species (ROS) production. Elevated ROS levels subsequently trigger activation of the NLRP3 inflammasome and caspase-1, leading to cleavage and activation of the pyroptosis executor protein Gasdermin D (GSDMD). Activated caspase-1 cleaves GSDMD into a C-terminal p20 fragment and an N-terminal p30 domain. The N-terminal fragment translocate to the plasma membrane, where it oligomerizes into ring-shaped pore structures, as visualized by negative-stain electron microscopy. These oligomers form membrane pores approximately 10–20 nm in diameter, a defining event in pyroptosis. Pore formation results in calcium release from preloaded liposomes, potassium efflux, osmotic cell swelling, membrane rupture, and release of intracellular contents. These events are accompanied by nuclear chromatin condensation, DNA cleavage and fragmentation, and the extracellular release of pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18), collectively culminating in robust pyroptotic death of cancer cells. These findings highlight H₂ induced pyroptosis as a potential therapeutic mechanism for overcoming apoptosis-resistant endometrial cancer (Yang et al., 2020). Reactive oxygen species (ROS) activate the NLRP3 inflammasome through multiple mechanisms that are highly dependent on cellular context. Among these, mitochondrial-derived ROS are widely recognized as a principal trigger of NLRP3 inflammasome activation (Dominic et al., 2021). A key regulatory pathway involves thioredoxin-interacting protein (TXNIP) and its redox partner thioredoxin-1 (Trx1). Under basal conditions, TXNIP associates with Trx1, limiting its antioxidant capacity and permitting intracellular ROS accumulation. Upon oxidative stress, TXNIP dissociates from oxidized Trx1 and directly interacts with the leucine-rich repeat (LRR) and NACHT domains of NLRP3, thereby promoting inflammasome assemblyand activation (Tschopp and Schroder, 2010). Beyond its inflammatory role, NLRP3 inflammasome activation exhibits context-dependent anti-tumorigenic functions across distinct cancer phenotypes. Activation of NLRP3 leads to the production of interleukin-18 (IL-18), which enhances Fas ligand (FasL) expression on natural killer (NK) cells, thereby augmenting their cytotoxic activity and suppressing colorectal cancer liver metastasis. IL-18 further exerts tumor-suppressive effects by inducing interferon-γ (IFN-γ) production and STAT1 signaling, while concurrently downregulating IL-22 binding protein (IL-22BP), a modulator of colorectal carcinoma progression. In contrast, in melanoma, NLRP3 inflammasome activation has been shown to impair anti-tumor immunity by promoting the recruitment and migration of myeloid-derived suppressor cells (MDSCs), underscoring the dual and tumor-type–specific roles of NLRP3 signaling in cancer biology (Hamarsheh and Zeiser, 2020).

6 Redox dependant signaling, inflammatory cascades and metabolic reprogramming

H₂ acts as a signaling modulator and balances the redox state of cells. It attenuates inflammation by modulating various transcription factors and immune cells. Also, it has been shown to reprogram metabolic pathways, particularly in the liver and during conditions of high metabolic demand (Zhang et al., 2020; Wang and Zhao, 2022).

6.1 NF-кβ and cytokine driven oncogenic inflammation

NF-κB activation is well known to induce anti-apoptotic genes such as XIAP, enabling cells to survive inflammatory and physiological stress. In parallel, NF-κB regulates immune responses by inducing cytokines including TNF-α, IL-1, IL-6, and IL-8, as well as adhesion molecules that facilitate leukocyte recruitment. Beyond innate immunity, NF-κB signaling plays key roles in cell proliferation (Guttridge et al., 1999; La Rosa et al., 1994) and apoptosis (Perkins, 1997), highlighting its broad regulatory functions in cellular homeostasis. Chronic inflammation and sustained NF-κB activation contribute to tumor initiation and progression through mechanisms involving ROS mediated DNA damage and mutagenesis (Liou and Storz, 2010). Moreover, NF-κB signaling promotes cancer progression by regulating epithelial–mesenchymal transition and metastasis, processes often accompanied by increased matrix metalloproteinase expression that facilitates extracellular matrix degradation and tumor cell invasion (Figure 8) (Huber et al., 2004).

Aberrant NF-κB activation may arise from direct mutations within NF-κB pathway components or oncogenes that constitutively activate this signaling axis. Alternatively, tumor cells may acquire elevated NF-κB activity through cytokine-rich tumor microenvironments (Ben-Neriah and Karin, 2011). NF-κB activation can be triggered by factors such as TNF-α stimulation, AKT pathway activation, or excessive accumulation of ROS. Notably, ROS and NF-κB engage in reciprocal regulation where ROS can activate NF-κB, while NF-κB controls the expression of genes involved in ROS detoxification, alongside pro-oxidant enzymes such as nitric oxide synthases. This intricate feedback loop underscores the complex interplay between oxidative stress and NF- κB signaling in cancer biology (Morgan and Liu, 2011).

ROS mediated oxidative stress represents a key pathogenic factor in acute pancreatitis. Ren et al. demonstrated that excessive ROS generation during acute pancreatitis enhances NF-κB activation and increases the production of pro-inflammatory cytokines, including TNF-α and IL-1β. Importantly, these pathological changes were significantly attenuated following treatment with H₂ rich saline.

Based on these findings, it can be hypothesized that H₂ may similarly regulate aberrant NF-κB activation in cancer by scavenging ROS and downregulating XIAP expression, thereby exerting potential anti-tumor effects (Figure 8). (Tao et al., 2019) Elevated intracellular ROS levels also function as signaling molecules that activate redox-sensitive transcription factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which in turn induce the expression of genes that drive cancer cell proliferation. Hydrogen peroxide (H₂O₂), in particular, enhances vascular endothelial growth factor (VEGF) expression and matrix metalloproteinase (MMP) activity, facilitating angiogenesis and rapid tumor expansion (Wang and Yi, 2008). Furthermore, ROS activate all three major mitogen-activated protein kinase (MAPK) pathways like extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAPK which are critically involved in tumor cell proliferation, migration, and invasion. ROS also stimulate the phosphoinositide 3-kinase (PI3K)/AKT signaling cascade, leading to the upregulation of cell cycle–associated genes such as cdc2, PRC1, and PCNA, thereby further promoting cancer cell proliferation (Moldogazieva et al., 2018).

Collectively, H₂ confers cellular protection by attenuating the accumulation of cytotoxic reactive oxygen species and preserving intracellular redox balance. Through its antioxidant activity, H₂ limits oxidative damage to biomolecules, thereby reducing the risk of malignant transformation and contributing to the maintenance of cellular homeostasis.

6.2 Hydroxyl radical, peroxynitrite and ROS mediated cellular injury

Reactive oxygen species (ROS) are highly reactive oxidative molecules generated within living cells, and their tight regulation is essential for maintaining normal cellular function. Under physiological conditions, ROS participate in redox signaling and metabolic processes. During mitochondrial electron transport and other metabolic reactions, various flavoproteins and heme-containing proteins can leak electrons due to inefficiencies in electron transfer (Klaunig et al., 1998). These leaked electrons interact with molecular oxygen or nitric oxide to generate reactive oxygen species (ROS) and reactive nitrogen species (RNS) through multiple pathways and intermediates. Representative ROS and RNS include superoxide anion (O₂•^-), hydroxyl radical (•OH), peroxyl (ROO•), alkoxyl (RO•), hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl), ozone (O₃), singlet oxygen (¹O₂), and peroxynitrite (ONOO⁻). Excessive accumulation of these reactive species leads to oxidative stress, a pathological condition that contributes to the initiation and progression of numerous diseases (Yang et al., 2018; Halliwell, 1994; Zhang et al., 2023).

To counterbalance oxidative stress, cells possess endogenous antioxidant defense systems, including enzymes such as superoxide dismutase (SOD) and catalase, which neutralize reactive species. However, mutations or functional impairments in these antioxidant enzymes reduce their protective capacity, resulting in elevated intracellular ROS levels and increased cellular damage (Sies, 1997).

H₂ functions as a potent reducing agent capable of alleviating cellular oxidative stress. Owing to its small molecular size and nonpolar nature, H₂ readily diffuses across biological membranes, enabling efficientintracellular distribution, a property that distinguishes it from many conventional antioxidants. Notably, H₂ selectively reacts with the most highly cytotoxic reactive species, particularly the hydroxyl radical (•OH) and peroxynitrite (ONOO⁻), which are strongly associated with DNA damage, including single-and double-strand breaks, base modifications, and chromosomal rearrangements. Such DNA damage can subsequently induce epigenetic alterations and contribute to carcinogenesis. Importantly, no endogenous enzymatic systems exist to specifically neutralize these highly reactive radicals. (Franco et al., 2008).

A key advantage of H₂ as an antioxidant is its selectivity; it does not interact with essential redox couples or metabolic intermediates such as NAD⁺, FAD, or cytochrome c, thereby preserving normal cellular metabolic processes (Ohsawa et al., 2007). In addition to its direct radical scavenging activity, H₂ enhances endogenous antioxidant defense by activating nuclear factor erythroid 2–related factor 2 (Nrf2)–dependent transcription, leading to increased expression of antioxidant enzymes such as superoxide dismutase (SOD) and elevated glutathione levels. This dual mechanism contributes to the reinforcement of the cellular antioxidant system (Tian et al., 2021).

The hydroxyl radical is an extremely reactive species that interacts with nearly all classes of organic biomolecules at rates approaching diffusion control. It has been shown to activate oncogenes such as C-Raf-1 and K-Ras through N-terminal deletions and to induce point mutations within GC base pair of DNA. Cells harboring mutant p53 or lacking functional p53 are particularly susceptible to hydroxyl radical–mediated damage, as the absence of effective G1-phase cell cycle arrest compromises DNA repair capacity. This results in replication errors, inactivation of tumor suppressor genes, further oncogene activation, and ultimately malignant transformation.

In addition to its effects on DNA, the hydroxyl radical readily modifies cellular proteins and nucleic acids. It preferentially attacks pyrimidine bases at the C5 and C6 positions of cytosine and thymine, generating C5-OH and C6-OH adduct radicals (Dreher and Junod, 1996). The hydroxyl radical also selectively targets the 2′-deoxyribose moiety of DNA, producing C-5′ radicals that undergo cyclization with the C-8 position of purine bases, leading to the formation of 8,5′-cyclopurine-2′-deoxynucleosides. Through these diverse reactions, hydroxyl radicals induce single- and double-strand DNA breaks as well as inter- and intra-strand crosslinks, thereby promoting mutagenesis and carcinogenesis (Halliwell et al., 2021; Zahra et al., 2021).

Peroxynitrite (ONOO⁻) is generated through the rapid, near equimolar reaction between nitric oxide (NO) and superoxide anion. As a potent nitrating and oxidizing species, peroxynitrite readily permeates cellular membranes and gains access to the nucleus, where it interacts with genomic DNA. Among DNA bases, guanine is particularly vulnerable to oxidative and nitrative damage due to its relatively low reduction potential (E₀ ≈ 1.29 V). Reaction of guanine with peroxynitrite preferentially yields 8-nitroguanine at mildly alkaline pH (∼8), along with additional oxidation products such as 2,5-diamino-4H-imidazol-4-one (Iz) and 8-oxoguanine. It undergoes heterolytic cleavage to generate nitrosonium ion (NO⁺) and a highly reactive intermediate, –NOOH, which drives the formation of 8-nitroguanine. This DNA lesion is strongly mutagenic, as it rapidly depurinates to create apurinic sites that promote guanine-to-thymine (G→T) transversion mutations. In addition, peroxynitrite oxidizes 2′-deoxyguanosine to produce 4,5-dihydro-5-hydroxy-4-(nitrosooxy)- 2′-deoxyguanosine (nox-dG). It can also react with multiple deoxyribonucleosides to generate 2-thiobarbituric acid (TBA)–reactive substances, further contributing to DNA damage and genomic instability. It is a potent activator of poly (ADP-ribose) polymerase-1 (PARP-1), a key member of the PARP enzyme family involved in the cellular DNA damage response. Upon sensing DNA strand breaks, PARP-1 binds to damaged DNA and forms homodimers, initiating the catalytic cleavage of nicotinamide adenine dinucleotide (NAD⁺) into nicotinamide and ADP-ribose. This reaction results in the synthesis of poly (ADP-ribose) chains on acceptor proteins, including histones. Excessive PARP-1 activation leads to rapid depletion of intracellular NAD⁺ and ATP pools, culminating in cellular energy failure, cell death, and tissue dysfunction (Beckman et al., 1990; Yermilov et al., 1995; Misonou et al., 2006; Douki and Cadet, 1996; Tsai et al., 2000).

In addition, PARP-1 plays a critical role in cell death signaling by regulating the mitochondrial release and nuclear translocation of apoptosis-inducing factor (AIF), which triggers chromatin condensation and extensive DNA fragmentation. Beyond its role in cell death, PARP-1 also promotes inflammatory responses by activating transcription factors that drive the expression of pro-inflammatory genes. Peroxynitrite induces DNA strand breaks through an initial H₂ abstraction step, followed by oxygen attack at the C4′ or C5′ positions of the deoxyribose moiety. Oxidative modification at the C4′ site promotes strand scission via cleavage of the C3′–phosphate–oxygen bond or through simultaneous disruption of the C3′–C4′ bond and the C1′–ring oxygen linkage. Alternatively, strand breakage may occur through cleavage of the C4′–C5′ bond, accompanied by oxidative damage at the C5′ position. These peroxynitrite-mediated alterations compromise DNA replication fidelity and contribute to mutagenesis and carcinogenesis (Islam et al., 2015).

6.3 Oxidative protein and lipid damage in tumor progression

ROS preferentially attack the lipid components of cellular membranes, which are rich in polyunsaturated fatty acids. The initial products of ROS induced lipid peroxidation are lipid hydroperoxides (LOOH), which are normally detoxified and removed through glutathione S-transferase dependent pathways. Disruption of this regulatory balance results in the accumulation of secondary lipid peroxidation products, particularly malondialdehyde (MDA), a highly reactive aldehyde with strong mutagenic potential. Another major byproduct of lipid peroxidation is 4-hydroxy-2-nonenal (HNE), a highly reactive α,β-unsaturated aldehyde. Malondialdehyde (MDA) readily reacts with amino (–NH₂) groups of biomolecules, including proteins, phospholipids, and nucleic acids, forming inter- and intramolecular amino-3-iminopropene crosslinks. These covalent modifications can induce DNA strand breaks and compromise genomic integrity. Due to its high diffusibility, MDA can traverse cellular membranes and accumulate in the nucleus, where it forms adducts with nucleotide bases such as guanine and cytosine, generating DNA crosslinks that interfere with replication and promote mutagenesis. (Esterbauer et al., 1991).

Similarly, HNE rapidly modifies proteins by reacting with thiol (-SH) and amino (-NH₂) groups of amino acid residues, particularly lysine and histidine, through Michael addition reactions. These modifications alter protein structure and function, contributing to cellular dysfunction and disease progression (Ayala et al., 2014).

ROS induce a wide range of oxidative modifications in cellular proteins, including the oxidation of methionine residues to methionine sulfoxide and methionine sulfone, as well as the conversion of cysteine thiol (–SH) groups into sulfenic acid (–SOH), disulfide bonds (–S–S–), sulfinic acid (-SO₂H) or sulfonic acid (–SO₃H). In nucleic acids, ROS preferentially oxidize guanine to form 8-oxo-7,8-dihydroguanine (8-oxodG), a highly mutagenic lesion that promotes guanine-to-thymine (G→T) transversion mutations, thereby contributing to oncogene activation and tumor initiation. (Marnett, 2002).

6.4 HIF-associated glycolytic modulation and cancer metabolism

Cancer cells exhibit elevated rates of glycolysis to meet their increased energy requirements. A defining metabolic feature of malignant cells is their preference for converting glucose to lactate regardless of oxygen availability, a process that occurs under both aerobic and anaerobic conditions. In contrast, normal cells predominantly rely on lactate production only when oxygen levels are limited, such as during hypoxia. This distinct metabolic reprogramming in cancer cells is known as the Warburg effect (Figure 9).

This metabolic pathway generates lactate and protons that readily diffuse across the plasma membrane, leading to acidification of both the intracellular and extracellular environments. Consequently, cancer cells sustain continuous energy production through enhanced glycolysis, while limiting the entry of pyruvate into the mitochondrial tricarboxylic acid (TCA) cycle (Harris and Johnson, 2014).

Hypoxia-inducible factor (HIF) is a key transcriptional regulator that is markedly upregulated in tumor cells, where it controls the expression of genes involved in glycolytic metabolism. HIF is a heterodimeric complex composed of an oxygen-sensitive α subunit (HIF- 1α, HIF-2α, or HIF-3α) and a constitutively expressed β subunit. Among these isoforms, HIF-1α plays a dominant role in regulating glycolysis under hypoxic conditions. Under normoxia, HIF- 1α undergoes rapid degradation through oxygen-dependent proline hydroxylation at Pro402 and Pro564. In contrast, hypoxic conditions stabilize HIF-1α, allowing it to accumulate in the cytoplasm and translocate into the nucleus, where it dimerizes with HIF-1β/aryl hydrocarbon receptor nuclear translocator (ARNT). This complex binds to hypoxia-responsive elements (HREs) within target gene promoters, thereby initiating transcription (Kierans and Taylor, 2021). HIF-1–dependent transcription induces the expression of glucose transporters such as GLUT1 and GLUT3, as well as multiple glycolytic enzymes, including phosphofructokinase-1 (PFK-1), aldolase (ALDA), phosphoglycerate kinase-1 (PGK-1), enolase (ENOL), lactate dehydrogenase-A (LDHA), and pyruvate dehydrogenase kinase-1 (PDK-1) (Semenza et al., 1994). LDHA facilitates the conversion of pyruvate to lactate while regenerating NAD⁺, thereby sustaining glycolytic flux. Additionally, HIF-1α regulates cytochrome c oxidase subunit composition, which limits mitochondrial ROS production and protects tumor cells from oxidative stress–induced apoptosis.

Beyond protein-coding genes, HIF also modulates the expression of several long non-coding RNAs (lncRNAs) implicated in glycolytic regulation, including lincRNA-p21, lncHIFCAR, and lncRNA-UCA1. LincRNA-p21 enhances LDHA and GLUT1 expression, thereby increasing glucose uptake and lactate generation. LncHIFCAR is essential for the transcriptional activation of LDHA and PDK-1, while lncRNA-UCA1 promotes glycolysis by upregulating hexokinase-2 (HK-2), particularly in bladder cancer cells. Elevated lactate accumulation within the tumor microenvironment disrupts immune homeostasis. Increased lactate levels promote histone lactylation, which enhances gene expression associated with immunosuppressive macrophage phenotypes and alters macrophage polarization, ultimately impairing natural killer (NK) cell activation and survival. Furthermore, the high glycolytic demand of tumor cells restricts glucose availability for immune cells, leading to suppressed T-cell activity and weakened antitumor immune responses (Brand et al., 2016).

Recent studies have demonstrated that H₂ effectively lowers HIF−1α mRNA expression in cellular models, animal studies, and clinical settings (Peng et al.,2023). Additional research indicates that H₂ can reverse the metabolic reprogramming of tumor cells by inhibiting glycolytic enzyme activity while enhancing mitochondrial oxidative phosphorylation (OXPHOS) (Niu et al.,2020). Consequently, H₂ suppresses the elevated glycolytic flux characteristic of cancer cells by downregulating HIF-1α and modulating key enzymes involved in metabolic pathways.

7 Crosstalk between warburg effect, PGC-1α and chromatin modifications

Cancer cells commonly suppress PGC-1α expression to maintain the Warburg phenotype, thereby favoring aerobic glycolysis over mitochondrial oxidative phosphorylation (OXPHOS). This metabolic reprogramming supports rapid glucose utilization for the generation of biosynthetic precursors required for sustained proliferation rather than efficient ATP production. Consequently, glycolysis-derived metabolites, including lactate and acetyl-CoA, accumulate and serve as substrates or cofactors for chromatin-modifying enzymes. These metabolic alterations promote aberrant epigenetic remodeling, characterized by increased histone acetylation and altered DNA and histone methylation patterns, ultimately repressing tumor suppressor genes and sustaining a proliferative phenotype (Tzeng et al., 2026).

H₂ has been proposed to function as a mild, selective redox modulator capable of reshaping the metabolic epigenetic landscape of cancer cells. By attenuating excessive oxidative stress, H₂ indirectly promotes PGC-1α activation, thereby stimulating mitochondrial biogenesis and restoring mitochondrial oxidative metabolism. This metabolic shift suppresses aerobic glycolysis, enhances OXPHOS and reduces pathological lactate accumulation. Re-establishment of mitochondrial respiration also alters the availability of key metabolic intermediates, such as acetyl-CoA and α-ketoglutarate, that regulate the activity of epigenetic enzymes. Consequently, H₂ may normalize aberrant histone acetylation and methylation patterns, leading to reprogramming of gene expression from a tumor-promoting state toward a more differentiated and less proliferative phenotype (Figure 10) (Xing et al., 2024; Renaud et al., 2021).

8 H₂ and disulfidptosis cell death pathway

The dual role of SLC7A11 in regulating two distinct forms of programmed cell death, ferroptosis and disulfidptosis, highlighting its paradoxical function as both a protector against oxidative stress and a source of metabolic vulnerability in cancer cells. It discusses the underlying molecular mechanisms, the contribution of metal dyshomeostasis, and the clinical significance of multi-omic prognostic signatures in predicting patient outcomes and immunotherapy responses. The review also examines how disulfidptosis may enhance antitumor immunity and overcome therapy resistance, emphasizing the SLC7A11 axis as a promising therapeutic target for precision oncology (Tolbatov and Marrone, 2026).

H₂ has been proposed to function as a protective redox modulator in this context. Owing to its rapid diffusion across cellular membranes, H₂ can attenuate excessive oxidative stress by reducing ROS, thereby limiting oxidative damage to intracellular thiol (-SH) groups and preventing the formation of abnormal disulfide bonds. By alleviating oxidative stress, H₂ may help preserve intracellular NADPH availability and glutathione (GSH) levels, supporting endogenous disulfide-reducing systems responsible for converting cystine back to cysteine. Maintenance of intracellular thiol redox balance may prevent excessive intermolecular cross-linking of actin proteins, thereby preserving cytoskeletal integrity and potentially suppressing the progression of disulfidptosis (Xiao et al., 2024; Liu et al., 2025).

9 Selectivity of H₂ towards cancer cell

As an inert, non-polar, and non-toxic molecule, H₂ preferentially scavenges highly cytotoxic radicals such as ^**.**OH and ONOO^**−**while largely sparing H₂O₂. Although H₂ is a reducing agent and H₂O₂ is an oxidizing agent, spontaneous reaction between them is thermodynamically and kinetically unfavorable because of the high activation energy required to cleave their respective chemical bonds. Consequently, H₂ does not directly interfere with H₂O₂ mediated physiological signaling in healthy cells (Yang et al., 2024; Di Marzo et al., 2018; Forman et al., 2010).

Therefore, H₂ does not directly regulate signaling pathways in the manner of gaseous signaling molecules such as nitric oxide (NO) or hydrogen sulfide (H₂S). Instead, it indirectly modulates cellular signaling by reducing excessive oxidative stress within the tumor microenvironment (TME). The tumor microenvironment is a highly dynamic and dysregulated ecosystem consisting of cancer cells, cancer-associated fibroblasts (CAFs), immune cells, endothelial cells, abnormal ECM and soluble signaling molecules. The TME is typically characterized by hypoxia, acidic pH, chronic inflammation, elevated reactive oxygen species (ROS), abnormal angiogenesis and immune suppression. These features promote tumor growth, metabolic reprogramming, invasion, metastasis and resistance to chemotherapy and immunotherapy, distinguishing the TME from the tightly regulated environment of normal tissues.By attenuating oxidative stress-induced activation of inflammatory and mitochondrial stress pathways, H₂ subsequently influences downstream signaling networks, including NF-κB, AMPK, NRF2, HIF-1α, FOXO, and AKT. These indirect, context-dependent effects provide a possible explanation for the preferential actions of H₂ in cancer cells while largely preserving normal cellular physiology. (Oshawa et al., 2007; Sies and Jones, 2020; Sies, 2021).

10 Interaction of H₂ with transition metal center and molecular mechanism

Several hypotheses have been proposed to explain how the small, non-polar diatomic molecule H₂ initiates cellular signaling despite its chemical inertness. Owing to its small size and non-polar nature, H₂ rapidly diffuses across plasma and mitochondrial membranes by passive diffusion. One proposed mechanism suggests that H₂ interacts with metalloproteins containing specialized catalytic centers, such as [Ni-Fe] and [Fe-Fe] hydrogenases, where H₂ undergoes heterolytic cleavage into protons and electrons. In these enzymes, surrounding iron sulfur (Fe-S) clusters function as molecular wires that facilitate rapid electron transfer. The catalytic center consists of Ni and Fe atoms bridged by cysteine sulfur atoms. Ni is considered the primary H₂ binding site, whereas the Fe atom is coordinated by strong field ligands, including carbon monoxide (CO) and cyanide (CN^**−**), which is maintained it in a low-spin, catalytically restricted state. Although mammalian cells do not possess classical hydrogenases, these observations have inspired the hypothesis that H₂ may interact with Fe–S containing metalloproteins or transition metal centers to influence cellular redox regulation. Fe–S cluster containing proteins are highly sensitive to oxidative damage, and disruption of these clusters compromises their enzymatic activity and DNA binding functions. By limiting oxidative injury, H₂ may preserve the structuralintegrity and regulatory activity of Fe–S proteins, thereby maintaining redox signaling, cellular adaptation to oxidative stress, and mitochondrial homeostasis. Furthermore, modulation of electron flow through Fe–S centers may reduce electron leakage, thereby attenuating ROS generation while preserving mitochondrial bioenergetics (Ohta, 2021).

Another proposed mechanism involves the Rieske iron–sulfur protein (RISP), a redox-sensitive subunit of mitochondrial Complex III. H₂ has been hypothesized to promote activation of the mitochondrial protease LONP1, resulting in transient degradation of RISP and temporary suppression of Complex III activity. This mild mitochondrial stress is proposed to activate the mitochondrial unfolded protein response (mtUPR), a hormetic quality control pathway that stimulates mitochondrial repair and biogenesis. Subsequent restoration and upregulation of RISP improve mitochondrial function and enhance resistance to oxidative stress (Negishi et al., 2025).

A complementary hypothesis proposes that H₂ is activated within the ubiquinone (Q) chamber of mitochondrial Complex I, where it donates electrons to the terminal N2 Fe–S cluster. This process may facilitate forward electron transport while suppressing reverse electron transport (RET), thereby decreasing electron leakage and reducing mitochondrial ROS production. H₂ has also been proposed to reduce semiquinone intermediates to fully reduced quinol, improving electron transfer to Complex III. In parallel, a modest reduction in mitochondrial membrane potential (ΔΨm), resulting from altered quinone redox cycling, may further limit excessive ROS generation without significantly compromising ATP synthesis. Collectively, these effects could stabilize mitochondrial bioenergetics, reduce oxidative DNA damage, and attenuate oncogenic signaling, thereby contributing to the selective anticancer effects of H₂ while preserving normal cellular function (Ishihara et al., 2020).

It should be emphasized that these mechanisms remain hypotheses based on emerging experimental evidence. Although they provide plausible explanations for the upstream molecular actions of H₂, definitive molecular sensors and direct binding targets in mammalian cells have yet to be conclusively identified.

11 H₂ as an adjuvant to conventional cancer therapy

Conventional chemotherapy and radiotherapy are frequently associated with significant adverse effects that negatively impact patients’ quality of life. Common complications of chemotherapeutic agents such as cisplatin, 5-fluorouracil (5-FU), and doxorubicin include nephrotoxicity, ototoxicity, hepatotoxicity, and cardiotoxicity (Elmorsy et al., 2024; Bygrave et al., 1998; Rawat et al., 2021). For instance, cisplatin is well known for inducing renal injury, while doxorubicin is associated with cardiac dysfunction, cardiomyopathy, and liver damage. Similarly, radiotherapy can cause radiation dermatitis, oral mucositis, and persistent fatigue as a result of unintended exposure of surrounding healthy tissues to high-energy radiation. These toxicities are largely mediated by excessive oxidative stress and overproduction of reactive oxygen species (ROS), which damage normal cells and tissues.

11.1 H₂ treatment in combination with chemotherapy

In preclinical and clinical observations, H₂ administration has been shown to mitigate cisplatin induced nephrotoxicity, as reflected by reductions in serum creatinine and blood urea nitrogen (BUN) levels (Qu et al., 2012). Likewise, H₂ rich saline has demonstrated protective effects against doxorubicin-induced cardiotoxicity and hepatotoxicity (Gao et al., 2016). Beyond its cytoprotective role in normal tissues, H₂ therapy may also enhance the therapeutic efficacy of certain chemotherapeutic agents. For example, in colorectal cancer models, the combination of H₂ rich saline with 5-fluorouracil has been reported to produce superior antitumor outcomes compared to 5-fluorouracil alone. These findings suggest a potential synergistic interaction, whereby H₂ not only reduces treatment-related toxicity but also supports anticancer activity (Tigu et al., 2020). H₂ combined with XELOX chemotherapeutic agent in colorectal cancer, shows improvement of overall survival and progression free survival. The common side effects like nausea, vomiting, dizziness, and low platelet counts are mitigated by H₂ treatment. As H₂ selectively deceases harmful toxic radicals without compromising the therapeutic toxicity of chemotherapeutic drugs, it serves as a promising agent for adjuvant treatment.

11.2 H₂ treatment in combination with radiotherapy

During radiotherapy excessive reactive oxygen is produced in normal cells that damage DNA and cellular structures. Here H₂ plays its positive role by scavenging those toxic radicals, increasing the patient’s quality of life. Studies shows that patient with advanced stage receiving intensity modulated radiation therapy (IMRT), reduce bone marrow damage by inhalation H₂ gas, limits the reduction of white blood cells and platelets (Hirano et al., 2021). Some clinical and experimental studies suggest that H₂ patients receiving radiotherapy for liver tumors, consumption of H₂ rich water during treatment significantly reduced oxidative stress markers and improved patients’ quality of life compared with placebo-treated patients, while the antitumor efficacy of radiotherapy remained unchanged. H₂ commonly administered as H₂ gas at 1.35 atm pressure or as H₂ rich saline before and after the radio sessions. A widely cited study by Shigeo Ohta and colleagues demonstrated that patients who drank H₂ rich water during radiotherapy experienced reduced biological oxidative reactions and maintained better appetite and overall wellbeing during treatment (Ohta, 2014; Kang et al., 2011). This, combined treatment is considered as promising, low-cost approach with minimal adverse reactions for increasing the therapeutic approach of conventional radiotherapy (Hua et al., 2020; Qian and Shen, 2022).

11.3 Protection against treatment associated toxicity

Radio and chemo therapy is efficient treatment procedure for removing malignant cells. However, these treatments also generate excessive ROS, inflammatory mediators, and oxidative stress which can damage healthy tissues and organs. Such adverse effects often include hepatotoxicity, nephrotoxicity, cardiotoxicity, neurotoxicity, fatigue, mucositis, and gastrointestinal complications. The protective mechanism of H₂ is mainly associated with its antioxidant, anti-inflammatory and anti-apoptotic properties. Patients receiving H₂ rich water experienced less fatigue, appetite loss, and taste disorders during treatment. This study highlighted the clinical potential of H₂ as a supportive therapeutic agent for minimizing radiation-associated adverse effects. Patients receiving H₂ rich water experienced less fatigue, appetite loss, and taste disorders during treatment. This study highlighted the clinical potential of H₂ as a supportive therapeutic agent for minimizing radiation-associated adverse effects (Spitz et al., 2004).

From a clinical perspective, H₂ therapy possesses several advantages. It is generally considered safe, inexpensive, easy to administer, and capable of being delivered through multiple routes, including H₂ rich water, inhalation gas, and H₂ infused saline. Despite encouraging findings, large-scale randomized clinical trials are still necessary to establish standardized dosing protocols, treatment duration, and long-term safety.

12 Pharmacokinetics and pharmacodyna-mics of H₂

H₂ is a highly bioavailable therapeutic gas that rapidly diffuses across biological barriers owing to its extremely small molecular size and low molecular weight. Depending on the route of administration, H₂ reaches peak blood concentrations within approximately 10–20 min and is subsequently eliminated predominantly through pulmonary exhalation. Several delivery modalities have been explored, including H₂ gas inhalation, H₂ rich water (HRW), H₂ rich saline (HRS), H₂ generating tablets, and direct intratumoral administration to achieve higher local concentrations.

Inhalation of 2–4% H₂ gas results in rapid systemic absorption, with blood concentrations rising quickly but declining within minutes after cessation of inhalation because of continuous expiration through the lungs. Oral administration of HRW (0.5–1.6 ppm) or sustained-release H₂ generating tablets enables H₂ absorption through the stomach and proximal small intestine, followed by transport into the portal circulation and systemic bloodstream, although a substantial proportion is exhaled before reaching the systemic arterial circulation. Intravenous administration of H₂ saturated saline (0.6–0.8 mM) also delivers dissolved hydrogen into the circulation. However, pharmacokinetic studies indicate that most of the dissolved H₂ is rapidly eliminated during passage through the pulmonary circulation, thereby limiting its bioavailability in peripheral tissues (Ge et al., 2017; Shibuya et al., 2025; Bo-Yan et al., 2019).

The pharmacodynamic actions of H₂ are attributed to its rapid diffusion into cells, including mitochondria and nuclei, where it selectively attenuates excessive oxidative stress and helps restore cellular redox homeostasis. Rather than acting as a direct signaling molecule, H₂ indirectly modulates intracellular signaling by preserving mitochondrial function, protecting redox-sensitive metalloproteins, and suppressing oxidative stress-induced activation of multiple signaling cascades. As discussed previously, H₂ scavenges highly reactive hydroxyl radicals (^**.**OH), thereby interrupting lipid peroxidation chain reactions and reducing the formation of reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE). The resulting alterations in the intracellular redox environment subsequently influence signaling pathways regulating PGC-1α expression, ultimately promoting mitochondrial biogenesis and epigenetic remodeling in cancer cells while largely preserving normal cellular physiology (Slezák et al., 2024; Ge et al., 2024).

Although H₂ exhibits a short biological half-life and transient tissue retention, accumulating experimental evidence suggests that even brief exposure may initiate sustained adaptive cellular responses, including activation of antioxidant defense systems, mitochondrial quality-control pathways, and redox-sensitive signaling networks. Nevertheless, the minimum effective intratumoral concentration and exposure duration required to induce durable antitumor and epigenetic effects remain to be established through quantitative pharmacokinetic and pharmacodynamic studies (Li et al., 2024).

13 Clinical evidence and emerging human studies of H₂ based interventions

There are so many in vivo and clinical studies of application of H₂ as cancer therapy has been done all over the world with good successful results in different stages of cancer. Somes are presented here.

13.1 In vivo studies of hyperbaric H₂ treatment on mice

Here in this study hairless albino mice with squamous cell carcinoma were exposed to a mixture of 2.5 percent O₂ and 97.5 percent H₂ at a total pressure of 8 atm for periods up to 2 weeks in a gas chamber (Figure 11). Here O₂ percentage is maintained at that level where it is free of risk of explosion and also the partial pressure of O₂ is same as that for atmosphere so that mice can live in this environment. Inside the chamber, there was a CO₂ scrubber containing soda lime, circulation through the scrubber was created by a small induction motor fan and the temperature was maintained at 32 °C. So, here is the delivery of the hyperbaric hydrogen treatment on mice by inhalation and the hydrogen concentration kept in large so that it penetrates to all parts of the body (Table 2) (Dole et al., 1975).

TABLE 2. Change in volume of tumor in mice after treating with Hyperbaric Hydrogen (2.5% oxygen and 97.5% hydrogen at a pressure of 8 atm for 2 weeks period).

13.2 Clinical study on advanced stage cancer patients treatment with H₂ gas inhalation

In this study which was carried out mainly in the Guangdong Xukecheng Health Care Studio in China, total 82 advanced stage cancer patients taken for treatment (Figure 12). These patients fall in some of these criteria like they failed to respond in conventional treatment, had other symptoms such as poor appetite, fatigue, insomnia and pain, existing tumors or abnormal tumor markers, aged 20-80, without obvious cardiac, cerebral, pulmonary, renal failure and mental abnormalities who can able to describe symptoms. They treated with H₂ using Hydrogen-Oxygen nebulizer (license No: AMS-H-01, Shanghai Asclepius Meditec Co., Ltd., Shanghai, China). They remained seated or recumbent, inhaling a mixture of H₂ (66.7%) and O₂ (33.3%) with spontaneous breathing in a nasal tube or mask and a gas flow rate of 3000 mL/min. The hydrogen inhalation should be continued for more than 3 h per day for at least 3 consecutive months. The response of the treatment was evaluated by measuring the size of tumor detected by computed tomography or magnetic resonance imaging. Also, the patient’s quality of life was evaluated by 5 functional subscales (physical function, role function, cognitive function, emotional function and social function) and 3 symptom subscales (fatigue, pain, nausea/vomiting). This Evaluation was conducted every other week before and after hydrogen inhalation. The final result of these 3 months treatment of H₂ gas inhalation is given here as graphical form (Table 3) (Chen et al., 2019).

TABLE 3. Summarization of in vitro and in vivo studies of molecular hydrogen on different types of cancer and its effect.

14 Conclusion and future perspective

H₂ has emerged as a potentially valuable adjunct in cancer therapy owing to its broad antioxidant, anti-inflammatory, cytoprotective, and tumor-modulatory properties. Current preclinical and limited clinical evidence suggests that H₂ may reduce the adverse effects associated with chemotherapy and radiotherapy while preserving, and in some contexts enhancing, antitumor efficacy through the regulation of oxidative stress, inflammatory signaling, and cellular survival pathways. Despite these encouraging findings, the precise molecular basis of H₂ mediated interactions with cancer-associated proteins, non-coding RNAs, and intracellular signaling networks remains to be fully elucidated. Further mechanistic dissection, dose-standardization studies, and well-designed clinical trials are therefore essential to establish its long-term safety, pharmacodynamic profile, and therapeutic applicability in modern oncologic practice. Taken together, these observations support the continued investigation of H₂ based interventions as a mechanistically distinct and pharmacologically adaptable complementary platform for improving therapeutic efficacy and reducing treatment-associated toxicity in cancer management.

The growing body of evidence on the small molecule H₂ suggest that future investigations should focus on defining the molecular determinants that govern H₂ responsiveness across distinct tumor types, particularly its influence on protein homeostasis, RNA regulatory circuits, mitochondrial stress adaptation, and tumor-immune communication. Clarification of optimal delivery modalities, dosing kinetics, and combinatorial interactions with existing chemotherapeutic and radiotherapeutic regimens will be critical for translational advancement. In addition, identification of H₂ sensitive biomarkers may help stratify patients most likely to benefit from H₂ based interventions. Ongoing investigations, including those in our laboratory, are aimed at elucidating these fundamental principles by which H₂ regulates cancer cell proliferation, survival, and metastatic progression. With continued mechanistic refinement and clinical validation, H₂ based therapeutics may evolve into a scientifically grounded complementary platform capable of improving the efficacy of existing anticancer regimens while reducing treatment-associated toxicity in integrative cancer management.

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Footnotes

Edited by: Tiziano Marzo, University of Pisa, Italy

Reviewed by: Iogann Tolbatov, University of Sassari, Italy

Anastasios Tasiopoulos, University of Cyprus, Cyprus