Immunomodulatory and antitumor effects of shilajit and glycine: Natural metabolic modulators targeting breast and liver cancer

Abstract

Cancer cells frequently rewire serine/glycine one-carbon metabolism to sustain nucleotide synthesis, redox balance, and epigenetic regulation, pathways that also shape antitumor immunity within the tumor microenvironment. Here, we review two natural agents, shilajit (a fulvic-acid-rich exudate) and glycine, with convergent immunomodulatory and antitumor activities relevant to breast and liver cancers. Shilajit exhibits antioxidant and anti-inflammatory effects and has shown selective cytotoxicity and anti-migratory activity in preclinical cancer models, including when incorporated into nanoformulations. Glycine supports glutathione-dependent redox defense, attenuates inflammatory signaling, and intersects with amino-acid metabolic programs that influence tumor cell fitness and immune cell function. We synthesize mechanistic and preclinical evidence to position shilajit and glycine as complementary, low-cost candidates for modulating tumor immunity and stress responses. Finally, we outline translational priorities, including standardized formulations, pharmacokinetics, safety, biomarker-guided patient stratification, and rational combinations with established systemic therapies.

Key Words: Shilajit; Glycine; Immunometabolism; One-carbon metabolism; Tumor microenvironment; Breast cancer; Hepatocellular carcinoma; Natural products

Core Tip: Shilajit and glycine are low-cost natural agents with convergent immunometabolic activity relevant to breast cancer and hepatocellular carcinoma. This minireview summarizes how shilajit fractions and glycine-dependent redox and one-carbon pathways may modulate oxidative stress, inflammatory signaling, and immune-cell polarization within the tumor microenvironment. We highlight practical translational priorities, including extract standardization, pharmacokinetic constraints, biomarker-guided patient stratification, and rational combinations with established anticancer therapies.

INTRODUCTION

Breast and liver cancers are among the most formidable challenges in oncology, both in incidence and mortality. According to the Global Cancer Observatory (GLOBOCAN), breast cancer is now the most commonly diagnosed malignancy worldwide, with an estimated 2.3 million new cases annually, overtaking lung cancer in incidence but not yet in mortality[1]. Hepatocellular carcinoma (HCC), the dominant form of primary liver cancer, remains the third-leading cause of cancer-related deaths globally, accounting for more than 800000 deaths each year[2]. Together, these cancers reflect the dual burden of rising incidence and poor clinical outcomes, particularly in regions with limited access to early detection and advanced therapies.

Despite remarkable advances in surgery, targeted therapy, and immunotherapy, the prognosis remains unsatisfactory for many patients. In breast cancer, the advent of targeted agents such as trastuzumab for human epidermal growth factor receptor 2-positive disease and cyclin-dependent kinase 4/6 inhibitors for hormone receptor-positive subtypes has significantly improved survival; however, resistance invariably emerges[3]. Similarly, in triple-negative breast cancer, immune checkpoint inhibitors (ICIs) have recently been integrated into clinical practice, yet response rates are modest and often transient[3].

In liver cancer, systemic therapy has evolved from single-agent sorafenib to more effective combinations, such as atezolizumab plus bevacizumab, which significantly improve overall survival[4]. Still, many patients present with advanced disease, cirrhosis, or comorbidities that limit therapeutic options. Moreover, both breast and liver tumors are frequently characterized by primary or acquired resistance to immunotherapy, underscoring the urgent need for complementary strategies that remodel the tumor microenvironment (TME)[5].

A central barrier to effective therapy is the immunosuppressive TME, a complex milieu of stromal cells, immune infiltrates, cytokines, and extracellular matrix that collectively enables tumor immune evasion[6]. Breast tumors often recruit tumor-associated macrophages (TAMs), which secrete vascular endothelial growth factor (VEGF), matrix metalloproteinases, and interleukin-10 (IL-10), thereby fostering angiogenesis and immunosuppression[7]. In HCC, chronic inflammation driven by hepatitis B or C infection, alcohol, or non-alcoholic fatty liver disease leads to the accumulation of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and immunosuppressive cytokines, such as transforming growth factor (TGF)-β. Together, these components limit cytotoxic T lymphocyte (CTL) activity and blunt the efficacy of checkpoint blockade[8].

Beyond cellular composition, the TME is defined by metabolic competition that constrains antitumor immunity. Rapidly proliferating tumor cells consume glucose and amino acids (including serine and glycine) and reshape the local milieu through lactate accumulation, hypoxia, and oxidative stress, thereby impairing T-cell effector function and promoting suppressive phenotypes (e.g., Tregs and MDSCs). These constraints create actionable vulnerabilities: Interventions that rebalance redox pressure, restore immune-cell metabolic fitness, or dampen nuclear factor kappa-light-chain-enhancer of activated B cells/signal transducer and activator of transcription 3 (NF-κB/STAT3)-driven inflammatory circuits may enhance responsiveness to chemotherapy and immunotherapy. This framework is particularly relevant when considering shilajit and glycine as adjuncts that act on redox, inflammatory signaling, and amino-acid-linked immune fitness. Although biologically distinct, breast cancer and HCC share clinically relevant mechanisms that make them ideal paired models for immunometabolic intervention. Both frequently develop an immunosuppressive TME enriched in Tregs, myeloid suppressor cells, and inhibitory cytokines, which limit durable responses to immunotherapy. In parallel, both malignancies adapt to redox stress and rewire the serine/glycine one-carbon pathways to support proliferation and immune evasion. Therefore, evaluating shilajit- and glycine-centered strategies across these two cancers provides a coherent conceptual testbed for modulating inflammation, oxidative stress, and antitumor immunity in high-burden disease settings.

In parallel with pharmaceutical innovations, interest in natural compounds with immunomodulatory and anticancer properties has surged. Natural agents are attractive not only for their multi-target mechanisms but also for their accessibility and relatively favorable safety profiles[9]. Historically, traditional medicine systems have employed natural products as “rasayana”, or rejuvenators, to enhance immunity and resilience. Modern pharmacological studies now validate some of these claims, linking plant- and mineral-derived compounds to modulation of apoptosis, angiogenesis, oxidative stress, and immune checkpoints[10].

Within this framework, shilajit and glycine stand out as particularly intriguing. Shilajit, a tar-like organic exudate found in high-altitude rocks, has been used for centuries in Ayurvedic and Central Asian medicine to promote vitality, aid bone healing, and combat cognitive decline[11,12]. Its primary bioactive constituents, fulvic acids, humic substances, and dibenzo-α-pyrones, have shown antioxidant, anti-inflammatory, and cytoprotective properties in recent studies[13]. Importantly, in vitro studies suggest that shilajit selectively induces apoptosis in breast and liver cancer cells while sparing normal tissues[14,15].

Glycine, in contrast, is a simple, nonessential amino acid with a disproportionately broad biological influence. It is a critical precursor to glutathione, the master cellular antioxidant, and plays essential roles in nucleotide synthesis, collagen formation, and redox balance[16]. In immune regulation, glycine suppresses NF-κB activation and reduces the secretion of pro-inflammatory cytokines[17,18]. At the same time, cancer cells frequently hijack serine/glycine metabolism to support proliferation, implicating glycine as both a nutritional immunomodulator and a metabolic vulnerability[19,20].

Emerging insights into cancer immunometabolism underscore the therapeutic potential of targeting amino acid pathways. Tumor cells depend on serine/glycine one-carbon metabolism for nucleotide synthesis, methylation, and redox control[20]. Lv et al[19] demonstrated that glycine consumption rates correlate with proliferation across cancer cell lines, while Heylen et al[21] identified NKX2-1 as a transcriptional driver of serine/glycine addiction, creating vulnerabilities to serine hydroxymethyltransferase (SHMT) inhibitors. Radiotherapy further increases dependence on this pathway, and combining SHMT inhibitors such as sertraline with irradiation produces synergistic tumor control[22].

Shilajit’s redox-modulating properties may intersect with this metabolic framework. By stabilizing mitochondrial function in healthy cells while increasing oxidative stress in malignant cells, shilajit could act as a metabolic adjuvant, thereby improving the therapeutic index of glycine-targeted interventions. Together, these agents align with a vision of integrative oncology that combines traditional compounds with modern metabolic and immunological strategies[15]. Both shilajit and glycine exhibit antitumor activity by inducing apoptosis and enhancing tumor cell death in breast and liver cancer models. This apoptotic mechanism is illustrated in Figure 1, which shows how these compounds target tumor cells and promote programmed cell death.

Figure 1 Shilajit and glycine-induced apoptosis in breast and liver cancer cells.

In this review, we synthesize current evidence on the immunomodulatory and antitumor activities of shilajit and glycine, with a focus on breast and liver cancers. We highlight their mechanisms, spanning apoptosis and angiogenesis to NF-κB suppression and metabolic reprogramming, and explore their translational potential as adjuncts to modern therapies. By bridging traditional knowledge with precision oncology, this work aims to provide a roadmap for future research and clinical integration.

IMMUNOLOGICAL LANDSCAPE OF BREAST AND LIVER CANCER

TME: A dynamic ecosystem

The TME is not merely a passive background but an active participant in cancer development, progression, and therapeutic resistance. It comprises tumor cells, stromal fibroblasts, endothelial cells, infiltrating immune cells, extracellular matrix components, and soluble mediators, including cytokines and chemokines. In both breast and liver cancers, the TME is profoundly immunosuppressive, creating conditions that shield malignant cells from immune surveillance[6].

In breast cancer, particularly triple-negative subtypes, the TME is characterized by abundant TAMs, which may constitute up to 50% of the tumor mass[23]. These TAMs exhibit an M2-like phenotype, producing IL-10, TGF-β, and VEGF, thereby suppressing CTLs and enhancing angiogenesis. Similarly, in HCC, chronic inflammation driven by hepatitis B or C infection, alcoholic liver disease, or non-alcoholic steatohepatitis results in persistent recruitment of immunosuppressive cell types, including Tregs and MDSCs[2]. This immune milieu not only promotes tumor progression but also dampens responses to ICIs.

Tregs and immune evasion

Tregs are critical for maintaining self-tolerance under physiological conditions, but in cancer, they are co-opted to suppress effective antitumor immunity. Elevated Treg infiltration has been associated with poor prognosis in breast cancer, particularly in human epidermal growth factor receptor 2-positive and triple-negative subtypes[24]. In HCC, Tregs inhibit natural killer (NK) cell activity and dampen interferon-γ production, thereby further weakening innate immune surveillance[25]. The immunosuppressive effects of Tregs are mediated not only by the secretion of IL-10 and TGF-β but also by direct cell-cell contact, which inhibits effector T cell proliferation.

MDSCs

MDSCs are another hallmark component of the suppressive microenvironment. These immature myeloid cells expand in response to chronic inflammation and accumulate in both breast and liver cancers[26]. They promote tumor growth by suppressing T-cell activation through the induction of arginase-1, inducible nitric oxide synthase, and reactive oxygen species (ROS). In HCC, MDSCs are recruited by tumor-secreted chemokines, such as C-C motif chemokine ligand 2 and C-X-C motif chemokine ligand 5, which blunt responses to ICIs. Targeting MDSCs is an emerging strategy to restore antitumor immunity.

NK cells and exhaustion

NK cells provide frontline defense against tumors by recognizing stress ligands on malignant cells. However, in both breast cancer and HCC, NK cells often exhibit reduced cytotoxicity and an “exhausted” phenotype, characterized by diminished secretion of granzyme B and perforin[27]. Factors such as hypoxia, lactic acidosis, and TGF-β in the TME further impair NK cell activity. Restoring NK function through metabolic support or checkpoint blockade is a promising avenue of research.

Immune checkpoints: Beyond programmed cell death protein 1 and cytotoxic T-lymphocyte-associated protein 4

The clinical success of checkpoint inhibitors underscores the pivotal role of immune checkpoints in tumor evasion. Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are the most extensively studied, but emerging checkpoints such as lymphocyte activation gene-3, T-cell immunoglobulin and mucin domain-containing protein 3, and T-cell immunoreceptor with Ig and ITIM domains are also implicated in breast and liver cancer[28]. These inhibitory receptors contribute to T cell exhaustion and limit the durability of antitumor responses[29]. Combination therapies targeting multiple checkpoints are under clinical investigation, though balancing efficacy with autoimmune toxicity remains challenging.

Inflammation as a driver of tumorigenesis

Chronic inflammation is a unifying feature of both breast and liver carcinogenesis. In the liver, persistent hepatitis infection or metabolic injury induces cycles of hepatocyte death and regeneration, promoting fibrogenesis and mutagenesis[2]. In breast cancer, obesity and metabolic syndrome contribute to chronic low-grade inflammation, with adipocyte-derived cytokines such as IL-6 and tumor necrosis factor-α (TNF-α) creating a permissive environment for tumor initiation and progression[30]. These insights underscore the importance of targeting inflammatory pathways in comprehensive cancer therapy. The TME comprises effector and suppressor immune subsets that either contribute to tumor killing or to immune evasion. As illustrated in Figure 2, cytotoxic subsets such as Th1 cells, NK cells, and M1 macrophages mediate granzyme/perforin-dependent tumor cell killing, whereas Tregs, M2 macrophages, and MDSCs promote immunosuppression by secreting IL-6, IL-10, and TGF-β.

Figure 2 Immune cells in the tumor microenvironment. NKM1: Natural killer cell, type 1; Th1: T helper cell, type 1; ILC1: Group 1 innate lymphoid cell; IFN: Interferon; TNF: Tumor necrosis factor; DC: Dendritic cell; N1: Neutrophil, type 1; M1: Macrophage, type 1; MDSC: Myeloid-derived suppressor cell; IL: Interleukin; CCL: CC chemokine ligand; TGF: Transforming growth factor.

Current status of immunotherapy

Immunotherapy has reshaped oncology, but its impact on breast and liver cancer has been mixed. In triple-negative breast cancer, atezolizumab combined with nab-paclitaxel showed significant benefit in the IMpassion130 trial, leading to regulatory approval; however, subsequent trials reported variable outcomes based on PD-L1 expression[3,31]. In HCC, the IMbrave150 trial demonstrated that atezolizumab plus bevacizumab improved survival compared with sorafenib, establishing a new standard of care[4]. Nonetheless, only a fraction of patients derive durable benefit, highlighting the need for adjunctive approaches to overcome resistance.

Rationale for integrating natural compounds

Given these challenges, natural agents that modulate oxidative stress, inflammation, and immune cell function offer intriguing potential. Shilajit, with its ability to suppress NF-κB signaling, inhibit angiogenesis, and induce apoptosis, may reshape the TME to enhance immune activation[14,32]. Glycine, by dampening pro-inflammatory cytokine production and supporting glutathione synthesis, may counteract TME-driven immune suppression[17,18]. By acting at multiple levels, metabolic, immunological, and redox, these agents could serve as cost-effective adjuncts to checkpoint blockade or standard chemotherapy[33].

SHILAJIT: ORIGIN, COMPOSITION, AND MECHANISMS OF ACTION

Origin and traditional use

Shilajit, also known as Mumio or mineral pitch, is a tar-like exudate that forms in rocks in high-altitude mountain ranges such as the Himalayas, the Altai, and the Caucasus. Formed over centuries through microbial decomposition and humification of organic matter, it has long been revered in Ayurvedic and Central Asian medicine as a rejuvenating tonic. Traditionally, Shilajit was prescribed for ailments ranging from digestive disorders to memory decline, reflecting its recognition as a “rasayana”, or restorative compound[11,12].

Chemical composition

Modern analyses reveal that shilajit is a complex matrix of organic and inorganic constituents. Fulvic and humic acids are its dominant organic fractions, conferring antioxidant and redox-modulating activity[11]. Dibenzo-α-pyrones and their conjugates protect mitochondrial function by acting as electron shuttles. Shilajit also contains a rich array of minerals, including selenium, zinc, iron, magnesium, and copper, which contribute to its immunomodulatory and cytoprotective properties[13]. This chemical heterogeneity underlies its wide-ranging biological effects but also complicates reproducibility and standardization.

ANTITUMOR MECHANISMS

Apoptosis and cell cycle arrest

Preclinical studies show that shilajit selectively induces apoptosis in cancer cells while sparing normal tissues. In breast cancer cell lines, shilajit activates caspases 3 and 9, triggers DNA fragmentation, and reduces mitochondrial membrane potential[14,34]. In HCC models, it suppresses proliferation by modulating pro-apoptotic and anti-apoptotic signaling, including upregulation of B-cell lymphoma 2 (Bcl-2)-associated X protein and downregulation of Bcl-2[15].

Oxidative stress and redox modulation

Oxidative stress is a double-edged sword in oncology: Moderate ROS promotes tumor survival, whereas excessive ROS induces apoptosis. Shilajit’s fulvic acid fraction reduces oxidative stress in normal tissues but augments ROS-mediated apoptosis in cancer cells. Konnova et al[35] demonstrated that shilajit increases intracellular ROS in HCC cells, thereby tipping the balance toward apoptosis. Its ability to modulate glutathione pools is particularly relevant to glycine metabolism and immunometabolism.

Angiogenesis and inflammatory pathways

Shilajit suppresses angiogenesis by downregulating VEGF and inhibiting endothelial cell proliferation[11]. In parallel, it attenuates NF-κB signaling, thereby reducing transcription of inflammatory cytokines and survival genes[36]. These dual actions impair both tumor growth and the recruitment of immunosuppressive immune subsets within the TME. Shilajit exerts anticancer effects by modulating multiple molecular pathways. Direct signaling interfaces (NF-κB/STAT3): Mechanistically, shilajit’s fulvic-acid-rich and dibenzo-α-pyrone fractions can be framed as upstream redox modulators that shift the activation threshold of inflammatory transcriptional programs. By buffering or amplifying intracellular ROS in a context-dependent manner, these constituents may modulate inhibitor of NF-κB kinase/inhibitor of NF-κB dynamics, thereby reducing NF-κB nuclear translocation and downstream expression of survival and cytokine genes. In parallel, suppression of inflammatory cytokine loops (e.g., IL-6 family signaling) provides a plausible route to dampened Janus kinase/STAT3 activation, a central node linking tumor proliferation, angiogenesis, and immune suppression. Explicitly positioning shilajit as a redox-inflammation regulator clarifies how it can affect both tumor-intrinsic fitness and immune-cell polarization. As shown in Figure 3, it induces apoptosis via Bcl-2-associated X protein/Bcl-2 regulation, inhibits angiogenesis by downregulating VEGF, and suppresses inflammation by inhibiting the NF-κB pathway.

Figure 3 Molecular mechanisms of shilajit in cancer modulation. Bax: B-cell lymphoma/Leukemia-2 family-associated X protein; Bcl-2: B-cell lymphoma/Leukemia-2 family; NF-κB: Nuclear factor-kappa B; VEGF: Vascular endothelial growth factor.

MOLECULAR MECHANISMS OF SHILAJIT IN CANCER MODULATION

Nanomedicine applications

Recent research has explored the use of shilajit in nanotechnology. Perumal et al[37] reported the green synthesis of zinc oxide nanoparticles using an aqueous shilajit extract. These nanoparticles were spherical and well characterized by scanning electron microscopy and X-ray diffraction, exhibiting potent cytotoxicity against HeLa cervical cancer cells (IC₅₀: Approximately 38.6 μg/mL). These findings highlight the potential of shilajit not only as a therapeutic agent but also as a reducing and capping agent in nanoparticle fabrication. Nanocarriers incorporating shilajit fractions may improve delivery, enhance selectivity, and minimize toxicity.

Synergy with chemotherapy and radiotherapy

Preclinical studies suggest that shilajit may act synergistically with conventional therapies. Wilson et al[38] observed that Shilajit enhances the efficacy of chemotherapeutic drugs, possibly by sensitizing cancer cells to apoptosis. Its antioxidant properties may also protect non-malignant tissues from chemotherapy- or radiotherapy-induced oxidative injury while amplifying cytotoxic stress within tumors. This duality, protecting normal tissues and sensitizing malignant cells, makes Shilajit particularly attractive as an adjunctive agent[39].

Immunomodulatory effects

Shilajit influences both innate and adaptive immunity. Dang et al.[40] reported immunosuppressive effects in overactive immune states, whereas later studies highlighted its capacity to stimulate macrophage activity and enhance antibody production in immunodeficient conditions. This bidirectional immunomodulation may be particularly useful in cancer, where immune suppression dominates the TME, yet therapy-induced inflammation may need to be dampened to avoid systemic toxicity. Shilajit exerts its anticancer effects through multiple mechanisms, including induction of apoptosis, redox modulation, anti-angiogenesis, and NF-κB suppression. These pathways and their potential clinical implications are summarized in Table 1.

Table 1 Mechanistic overview of shilajit in cancer.

Mechanism	Molecular target/pathway	Preclinical evidence	Potential clinical relevance
Apoptosis induction	Caspase-3, Bax/Bcl-2	Shilajit extracts trigger apoptosis in breast and liver cancer cells	Enhances cytotoxicity, tumor selectivity
ROS modulation	Mitochondria, glutathione	Increased ROS in malignant cells, reduced oxidative stress in normal cells	Tumor-specific redox modulation
Anti-angiogenesis	VEGF, endothelial proliferation	Inhibition of angiogenesis in vitro	Prevents tumor vascularization
NF-κB suppression	NF-κB p65 subunit	Reduced transcription of IL-6, TNF-α	Immunomodulation, sensitization to ICIs
Immunomodulation	Macrophages, antibodies	Bidirectional immune regulation	May reduce therapy toxicity, enhance immunity

Bax: B-cell lymphoma/Leukemia-2 family-associated X protein; Bcl-2: B-cell lymphoma/Leukemia-2 family; NF-κB: Nuclear factor-kappa B; VEGF: Vascular endothelial growth factor; ROS: Reactive oxygen species; TNF: Tumor necrosis factor; IL: Interleukin; ICI: Immune checkpoint inhibitor.

Toxicology and safety considerations

Despite centuries of use, the clinical translation of shilajit is hampered by safety concerns. Raw shilajit can be contaminated with heavy metals, free radicals, or fungal toxins unless properly purified[12]. Controlled animal studies suggest it is well-tolerated at therapeutic doses; however, standardized toxicological evaluations are lacking. Regulatory frameworks must establish clear quality-control markers; fulvic acid and dibenzo-α-pyrones could serve as benchmarks to ensure consistency across preparations[11,13].

Preclinical evidence in breast and liver cancer

Cumulative evidence underscores the promise of shilajit in breast and liver oncology. In MCF-7 breast cancer cells, shilajit extracts reduce proliferation, induce apoptosis, and impair mitochondrial function[14]. In HepG2 and Huh-7 liver cancer cell lines, shilajit triggers cell cycle arrest, ROS accumulation, and inhibition of NF-κB[15,41]. Kloskowski et al[32] demonstrated selective cytotoxicity in bladder cancer, reinforcing the tumor-specific nature of its effects. Together, these findings provide a robust preclinical foundation for advancing Shilajit into translational cancer research.

GLYCINE: ROLE IN CELLULAR METABOLISM AND IMMUNE MODULATION

Biological importance of glycine

Glycine, the simplest amino acid, is traditionally classified as non-essential because it can be synthesized endogenously from serine, threonine, and choline. However, its demand often exceeds its endogenous capacity, making it conditionally essential under stress, injury, or rapid proliferation[16,42]. Glycine is a critical component of structural proteins, such as collagen, accounting for one-third of collagen residues and thereby contributing to tissue integrity, wound repair, and fibrosis regulation[40]. In cellular metabolism, glycine is indispensable for nucleotide biosynthesis, heme formation, and particularly for glutathione synthesis, the tripeptide antioxidant composed of glutamate, cysteine, and glycine. Through glutathione, glycine protects cells from oxidative stress and supports the detoxification of xenobiotics. Its versatility as both a metabolic building block and a signaling molecule underlies its broad physiological significance[43,44].

Glycine in one-carbon metabolism and cancer

Cancer cells use glycine for one-carbon metabolism, a central hub linking amino acid catabolism to nucleotide synthesis and methylation. Jain et al[45] showed that glycine uptake correlates strongly with cancer cell proliferation, with rapidly dividing cells exhibiting heightened glycine consumption. In HCC, glycine decarboxylase (GLDC) is frequently upregulated, channeling glycine into the folate cycle to support nucleotide biosynthesis and epigenetic reprogramming[20]. Recent mechanistic studies have highlighted the role of transcription factors, such as NKX2-1, in driving serine/glycine synthesis pathways, thereby creating a state of metabolic “addiction”[21]. This dependency renders tumors sensitive to inhibition of SHMT, an enzyme central to the interconversion of glycine and serine. Notably, Sánchez-Castillo et al[22] demonstrated that radiotherapy exacerbates this metabolic demand and that inhibiting SHMT with repurposed sertraline enhances radiosensitivity in non-small cell lung cancer. These findings position glycine metabolism as a targetable vulnerability in multiple malignancies, including breast and liver cancers.

Anti-inflammatory and immunomodulatory effects

Beyond its metabolic role, glycine serves as an anti-inflammatory mediator. It activates glycine-gated chloride channels on immune and epithelial cells, resulting in membrane hyperpolarization and reduced calcium influx. This mechanism dampens NF-κB activation and cytokine release[18]. Schaumann et al[17] reported that glycine reduces IL-6 and IL-8 secretion in gingival epithelial cells, while Iimuro et al[46] demonstrated that it protects hepatocytes from endotoxin-induced injury by inhibiting Kupffer cell activation. These findings highlight glycine’s unique ability to modulate innate immunity while preserving tissue integrity. In cancer contexts, glycine’s immunomodulatory properties may mitigate therapy-induced inflammation. For instance, chemotherapy and radiotherapy generate oxidative and inflammatory stress in normal tissues. Supplementation with glycine could protect against collateral tissue injury while maintaining antitumor activity, a hypothesis that warrants further exploration[47].

Protective role in organ injury and fibrosis

Glycine supplementation has been extensively studied in models of ischemia-reperfusion injury, hepatic fibrosis, and toxin-induced organ damage. In experimental liver injury, dietary glycine has been shown to attenuate fibrosis and reduce hepatocarcinogenesis[47]. Zhuang et al[48] demonstrated that glycine suppresses the growth of HCC cells by inducing mitochondrial apoptosis. In cardiovascular research, glycine has been shown to enhance endothelial function and mitigate vascular inflammation, suggesting systemic protective roles that may indirectly affect cancer progression[49]. Glycine exerts pleiotropic biological effects, including redox regulation, cytoprotection, and immune modulation. As depicted in Figure 4, glycine contributes to glutathione synthesis and ROS detoxification, protects hepatocytes from fibrosis and therapy-induced damage, suppresses NF-κB-driven inflammation, and modulates macrophage and T-cell responses.

Figure 4 Glycine’s roles in redox balance, cytoprotection, and immune regulation. NF-κB: Nuclear factor-kappa B; Th1/2: T helper cell, type 1/2; IL: Interleukin.

Glycine in breast and liver cancer

Evidence directly implicating glycine in breast and liver cancer is growing. In murine breast cancer models, dietary glycine has been shown to reduce tumor growth and metastasis, in part by decreasing angiogenesis and enhancing immune function[50]. In HCC, glycine supplementation has been shown to reduce inflammatory cytokine production, inhibit fibrosis, and delay carcinogenesis[51]. Importantly, these effects were achieved without significant toxicity, reinforcing glycine’s potential as a safe adjunct in oncology. Glycine plays a dual role in cancer, serving as both a metabolic substrate that supports tumor proliferation and an immunomodulatory agent that protects host tissues. The significant roles of glycine in cancer biology are outlined in Table 2.

Table 2 Roles of glycine in cancer biology.

Role	Pathway/target	Evidence	Implication
Metabolic substrate	One-carbon metabolism (SHMT, GLDC)	Supports nucleotide synthesis and methylation	Tumor proliferation, metabolic vulnerability
Redox defense	Glutathione synthesis	Maintains antioxidant pools	Protects normal cells; tumor paradox
Immunomodulation	Glycine receptor, NF-κB	Suppresses IL-6/IL-8, reduces inflammation	Mitigates therapy-induced toxicity
Tissue protection	Anti-fibrosis, ischemia-reperfusion	Prevents fibrosis, protects the liver/kidney	Beneficial in HCC and comorbidities
Tumor suppression	Anti-angiogenesis, apoptosis	Reduced metastasis in breast cancer models	Adjuvant potential

SHMT: Serine hydroxymethyltransferase; GLDC: Glycine decarboxylase; NF-κB: Nuclear factor-kappa B; HCC: Hepatocellular carcinoma; IL: Interleukin.

Clinical and translational considerations

Although preclinical data are compelling, translation into clinical oncology remains in its infancy. Glycine is generally regarded as safe, widely available as a dietary supplement, and inexpensive. However, its dual role complicates therapeutic application: In some contexts, glycine supplementation may protect normal tissues and enhance immunity, but in tumors with high GLDC expression, it could theoretically fuel proliferation. This paradox underscores the importance of biomarker-driven approaches, such as stratifying patients by tumor GLDC, SHMT2, or NKX2-1 status[52]. A key translational issue for glycine is its context-dependent “glycine paradox”: Glycine may support host protection by buffering inflammation and oxidative stress, yet in specific tumors with high serine-glycine one-carbon flux, it could, in theory, sustain anabolic growth programs. Therefore, throughout this review, we emphasize biomarker-guided application, highlighting one-carbon pathway markers (e.g., GLDC/SHMT2-associated activity) as potential boundaries for distinguishing settings in which glycine is more likely to be host-beneficial, and for when caution and monitoring are warranted. Future clinical trials should investigate the use of glycine both as a preventive intervention (e.g., to reduce fibrosis-driven HCC) and as an adjunctive therapy (e.g., to protect normal tissues during chemotherapy or radiotherapy). Combining glycine modulation with metabolic inhibitors (such as SHMT or GLDC inhibitors) may further exploit tumor vulnerabilities while maintaining systemic protection.

TRANSLATIONAL AND INTEGRATIVE POTENTIAL

Complementary mechanisms of action

Shilajit and glycine exert distinct yet complementary biological effects. Shilajit, rich in fulvic acids and dibenzoyl-α-pyrones, exhibits tumor-selective cytotoxicity by inducing apoptosis, inhibiting angiogenesis, and suppressing NF-κB-driven inflammatory signaling[14]. Glycine, on the other hand, regulates glutathione synthesis, stabilizes redox balance, and reduces pro-inflammatory cytokine release via activation of the glycine receptor[17]. Together, these compounds provide a dual mechanism: Direct antitumor activity (shilajit) and systemic immunoprotective effects (glycine). This complementarity suggests their potential as adjuvants to existing therapies, improving the therapeutic index by simultaneously weakening tumor defenses and protecting host tissues. Shilajit and glycine exhibit distinct yet complementary biological activities: Shilajit primarily acts through antioxidant and pro-apoptotic pathways, while glycine acts through glutathione synthesis and the control of inflammation. Their overlapping mechanisms, including regulation of Bcl-2 and caspases, are illustrated in Figure 5, highlighting their shared and unique roles in immune modulation and tumor suppression.

Figure 5 Comparative and overlapping mechanisms of shilajit and glycine in cancer modulation. MDSC: Myeloid-derived suppressor cell; Bcl-2: B-cell lymphoma/Leukemia-2 family.

Intersection with cancer immunometabolism

Cancer immunometabolism is increasingly recognized as a determinant of treatment success. Tumors with high proliferation rely heavily on the serine/glycine one-carbon pathway for nucleotide biosynthesis and redox control[20]. NKX2-1 has been identified as a transcriptional driver of this addiction, rendering cancers sensitive to SHMT inhibition[21]. Moreover, radiotherapy induces dependence on glycine metabolism, and SHMT inhibition synergizes with radiotherapy to improve tumor control[22]. Shilajit’s ability to modulate mitochondrial oxidative stress complements this vulnerability. By sensitizing cancer cells to ROS-mediated death while protecting normal cells, shilajit could enhance the efficacy of metabolic inhibitors or radiotherapy. In this sense, shilajit and glycine interventions are not traditional “alternative therapies” but rather agents that integrate seamlessly with the metabolic vulnerabilities targeted in modern oncology.

Opportunities for combination therapy

Both compounds could be incorporated into rational combination strategies: With ICIs, Shilajit may reduce immunosuppressive cytokines (IL-6, TNF-α, IL-10), thereby lowering barriers to T cell infiltration. Glycine may enhance systemic tolerance by mitigating therapy-induced inflammation, reducing fatigue, and supporting antioxidant defense mechanisms[53]. In chemotherapy, shilajit’s fulvic acid fraction protects normal tissues from oxidative injury while increasing cytotoxic stress in malignant cells[54]. Glycine supplementation could reduce mucositis and hepatotoxicity, which are common side effects of cytotoxic regimens. Radiotherapy-induced metabolic stress increases reliance on SHMT2 and glycine metabolism. Modulating this pathway with glycine supplementation or inhibition, while incorporating shilajit’s redox effects, could optimize radiosensitization[22]. Using nanomedicine platforms, zinc oxide nanoparticles synthesized from shilajit extracts have demonstrated enhanced anticancer effects[37]. Similar strategies could be used to deliver glycine in a controlled-release format, enabling site-specific modulation.

Biomarker-guided precision oncology

The paradoxical role of glycine in cancer underscores the need for biomarker-guided stratification. Patients with tumors showing high GLDC or SHMT2 expression may benefit from inhibiting the glycine pathway rather than supplementing it[55,56]. Conversely, patients with chronic inflammation, fibrosis, or therapy-induced tissue injury may benefit from glycine supplementation. Similarly, biomarkers of oxidative stress and NF-κB activation could guide the development of shilajit-based interventions.

The integration of omics technologies, including metabolomics, transcriptomics, and single-cell immune profiling, will be essential for identifying predictive signatures. Patient stratification based on metabolic and immunological phenotypes will prevent indiscriminate use and maximize therapeutic benefit. Shilajit and glycine converge on shared molecular and metabolic pathways to modulate immune subsets and enhance therapeutic efficacy. As depicted in Figure 6, Shilajit primarily targets NF-κB, STAT3, and VEGF signaling, whereas glycine regulates ROS and glutathione metabolism. Together, they influence Tregs, CD8+ T cells, and MDSCs, providing opportunities to integrate with chemotherapy, immunotherapy, and biomarker-driven omics stratification.

Figure 6 Integrative mechanisms of shilajit and glycine in cancer therapy. NF-κB: Nuclear factor-kappa B; VEGF: Vascular endothelial growth factor; STAT3: Signal transducer and activator of transcription 3; MDSC: Myeloid-derived suppressor cell; ROS: Reactive oxygen species.

Safety and standardization challenges

Clinical translation requires rigorous safety data. Shilajit preparations are highly variable, and raw samples may be contaminated with heavy metals or fungal toxins[57]. Quality control is essential: Fulvic acid and dibenzo-α-pyrones should be established as standardization markers[58]. Animal toxicological studies suggest good tolerability; however, phase I human trials are still needed. Glycine, in contrast, has an excellent safety record and is widely available as a nutritional supplement. However, long-term supplementation in cancer patients has not been systematically studied. Careful monitoring will be required in patients with advanced tumors in which glycine metabolism is hyperactivated.

Global health and accessibility

One of the most compelling arguments for advancing the use of shilajit and glycine is their low cost and accessibility. Many cancer patients worldwide lack access to expensive biologics or targeted therapies. If validated, natural compounds could serve as adjuncts that improve quality of life and enhance responses to standard therapies. Integrating them into global cancer care could help address equity gaps in oncology, aligning with the World Health Organization’s calls for affordable cancer care[59].

FUTURE PROSPECTS

Standardization and quality control of shilajit

One of the primary challenges in developing shilajit as a therapeutic agent is the heterogeneity of its natural sources. Variability in geographic origin, collection methods, and processing can result in significant differences in chemical composition[11,12]. This lack of standardization complicates reproducibility and hampers regulatory approval. Establishing international quality-control markers, such as quantification of fulvic acid and dibenzo-α-pyrones, will be essential to ensure consistent pharmacological activity. Modern analytical techniques, including high-resolution mass spectrometry and nuclear magnetic resonance, could be used to develop a chemical fingerprint of Shilajit preparations[40]. Moreover, collaborations among pharmacognosy experts, oncologists, and regulatory agencies may accelerate the development of standardized formulations suitable for clinical trials.

Optimizing delivery systems

The bioavailability of shilajit’s active components and the systemic distribution of glycine remain underexplored. Advances in drug delivery could enhance their clinical potential. For instance, nanoparticle-based systems, such as zinc oxide nanoparticles synthesized with shilajit extract, have already demonstrated improved cytotoxicity against cervical cancer cells[37]. Liposomal encapsulation or polymeric carriers could protect fulvic acids from degradation, while targeted delivery systems might increase accumulation in tumor tissues. Similarly, glycine could be incorporated into controlled-release dietary supplements or intravenous formulations tailored for oncology settings, particularly for patients with metabolic or hepatic dysfunction[60,61].

Precision oncology and biomarker-guided use

Both shilajit and glycine intersect with highly context-dependent pathways. Glycine supplementation may benefit patients with inflammation-driven cancers, but it could theoretically support tumor growth in cancers with upregulated GLDC or in those with altered serine-glycine one-carbon metabolism[20]. Precision oncology frameworks should therefore integrate biomarkers such as GLDC expression, NKX2-1 status, and systemic glycine flux to stratify patients most likely to benefit from targeted therapies. Similarly, biomarkers of oxidative stress and NF-κB activation may guide the rational use of shilajit in patient subgroups with inflammatory TMEs[62]. Conceptual decision framework for glycine use (precision boundary): (1) More likely beneficial when systemic inflammation/oxidative stress is prominent and there is no evidence of strongly upregulated tumor one-carbon glycine flux; in such settings, glycine may support glutathione buffering and reduce pro-inflammatory cytokine signaling; (2) Use with caution/monitoring when tumors demonstrate signatures consistent with elevated serine-glycine one-carbon activity (e.g., high GLDC/SHMT2-associated programs) or rapid anabolic growth, where exogenous glycine could theoretically support biomass and redox adaptation; and (3) Prefer biomarker-enriched trials where glycine exposure is paired with metabolomic/transcriptomic monitoring (glycine flux surrogates, redox markers) to define safe, effective contexts rather than indiscriminate supplementation. Prospective studies using metabolomics, transcriptomics, and immune profiling are urgently needed to establish predictive signatures of treatment response[63].

Combination strategies with existing therapies

The pleiotropic effects of shilajit and glycine make them attractive candidates for combination therapies. Preclinical evidence suggests that glycine supplementation reduces angiogenesis and fibrosis, processes that often limit the efficacy of chemotherapy and immunotherapy[64,65]. Shilajit’s antioxidant properties could protect normal tissues from therapy-induced toxicity while enhancing apoptosis in malignant cells[66]. The potential to combine these compounds with ICIs, such as PD-1/PD-L1 or CTLA-4 antibodies, is particularly promising. Additionally, radiotherapy-induced dependence on serine/glycine metabolism[22] raises the prospect of combining glycine modulators with radiotherapy and SHMT inhibitors to achieve synergistic tumor control. Such rational combinations should be systematically evaluated in preclinical models before progressing to early-phase clinical trials.

Addressing safety and toxicology

Despite centuries of traditional use, rigorous toxicological data on shilajit remain scarce. Heavy metal contamination, adulteration, and inconsistent dosing are legitimate safety concerns[12]. Controlled animal toxicology studies and phase I clinical trials in humans will be required to establish safe dosage ranges. Glycine, in contrast, is generally regarded as safe and well-tolerated; however, long-term supplementation in cancer patients should be carefully monitored, given its dual roles in metabolism and immunity. Special attention should be paid to vulnerable populations, such as patients with compromised liver or kidney function, where altered amino acid handling may influence therapeutic outcomes[67].

Integrating natural compounds into cancer care

Finally, the broader challenge is to bridge the gap between traditional remedies and modern oncology[68]. Patients frequently use complementary and alternative medicine during cancer treatment, often without clinical guidance[9]. Developing shilajit and glycine into evidence-based adjuncts could meet patient demand while ensuring safety and efficacy. Educational initiatives for healthcare providers, coupled with integrative oncology clinics, may help translate emerging scientific data into real-world practice.

Actionable research roadmap

Preclinical study design: In an orthotopic or chemically induced murine HCC model, evaluate anti-PD-1 (or anti-PD-L1) ± a standardized shilajit fraction, with or without controlled glycine supplementation. Primary endpoints should include tumor growth control and survival; mechanistic endpoints should include CD8+ T-cell infiltration, Treg/MDSC frequencies, macrophage polarization markers, and cytokine profiling (e.g., IL-6, TNF-α, IL-10, TGF-β).

Pharmacokinetic and quality-control priorities: Shilajit requires batch-to-batch standardization using quantifiable markers (e.g., fulvic acid and dibenzo-α-pyrones), contaminant screening (heavy metals and mycotoxins), and basic pharmacokinetic characterization of key fractions to inform dosing and formulation.

Biomarker-guided stratification concept: For glycine-centered interventions, stratify by tumor expression/activity signatures of glycine/one-carbon metabolism (e.g., GLDC, SHMT2, and related folate-cycle genes) and by host inflammatory/tissue-injury context. This approach enables rational selection between glycine supplementation (host-protective setting) and metabolic inhibition strategies (tumor-addicted setting).

DISCUSSION

Breast and liver cancers remain formidable clinical challenges worldwide. Breast cancer remains the most commonly diagnosed malignancy, with heterogeneous subtypes and evolving therapeutic targets. At the same time, HCC remains one of the deadliest cancers because of its association with chronic liver disease and late diagnosis. Despite advances in systemic therapy, including targeted agents and ICIs, resistance and limited response rates underscore the need for complementary strategies.

The immunological and metabolic landscapes of these tumors are highly dynamic and immunosuppressive. TAMs, Tregs, and MDSCs converge to inhibit effective cytotoxic T-cell responses, while chronic liver inflammation or adipose-driven cytokine secretion in breast tissue further fosters tumorigenesis. Upregulation of immune checkpoints such as PD-1, PD-L1, and CTLA-4 creates additional barriers to durable immunotherapy responses. At the same time, tumors reprogram serine-glycine one-carbon metabolism to support proliferation, nucleotide biosynthesis, and epigenetic adaptation. These hallmarks of cancer point to vulnerabilities that can be modulated not only by synthetic agents but also by natural compounds with multi-targeted activities.

In this context, shilajit and glycine are particularly intriguing candidates. Shilajit, a mineral-rich organic exudate long used in traditional medicine, contains fulvic acid, humic substances, and dibenzo-α-pyrones that collectively act as antioxidants, mitochondrial stabilizers, and immunomodulators. Modern studies show that shilajit selectively induces apoptosis in breast and liver cancer cells, modulates ROS, suppresses angiogenesis, and inhibits NF-κB signaling. These effects align with tumor-specific vulnerabilities while also providing cytoprotective benefits for non-malignant tissues. Importantly, nanomedicine applications, such as Shilajit-mediated green synthesis of zinc oxide nanoparticles, highlight its potential for integration into advanced delivery systems.

Glycine, though structurally simple, exerts broad and paradoxical effects in cancer biology. As a metabolic substrate, it supports one-carbon metabolism, purine biosynthesis, and redox homeostasis, processes that tumors hijack to sustain growth. However, as an immunomodulator, glycine activates glycine-gated chloride channels, suppresses NF-κB signaling, reduces secretion of IL-6 and IL-8, and stimulates glutathione synthesis to support antioxidant defense. Preclinical studies show that glycine supplementation reduces breast tumor growth, inhibits HCC progression, and protects against fibrosis and tissue injury. These dual roles highlight both the promise and complexity: Glycine can act as a fuel for cancer proliferation under certain oncogenic conditions, but also as a protector of host tissues and immune balance. This duality underscores the importance of biomarker-driven application. Glycine’s role depends on tumor metabolic demand and the host inflammatory state. In tumors with high glycine flux (e.g., elevated GLDC/SHMT2 activity), glycine can support nucleotide synthesis and methylation capacity, potentially favoring proliferation. In contrast, in inflammation- or therapy-injury-dominant settings, glycine’s glycine receptor signaling and glutathione replenishment can reduce NF-κB-driven cytokine production, oxidative damage, and immune exhaustion, thereby supporting host tolerance and potentially improving antitumor immune function. This framework motivates biomarker-guided use - supplementation where tissue injury and inflammation dominate, vs metabolic pathway inhibition where glycine dependence is a tumor liability.

Together, shilajit and glycine are complementary agents at the intersection of immunology and metabolism. Shilajit exerts direct tumoricidal effects by inhibiting angiogenesis, inflammation, and mitochondrial stability, while glycine serves as a systemic modulator of redox and immune responses. Their integration could enhance the therapeutic index of conventional therapies by weakening tumor resilience while protecting normal tissues. Potential applications include: (1) In immunotherapy, shilajit may remodel the TME by reducing suppressive cytokines, while glycine enhances tolerance and reduces systemic inflammation; (2) In chemotherapy, shilajit may protect normal tissues from oxidative damage while sensitizing malignant cells, and glycine may mitigate hepatotoxicity and mucositis; (3) In radiotherapy, shilajit’s redox modulation and glycine’s role in serine-glycine metabolism offer opportunities for radiosensitization; and (4) In nanomedicine, both compounds lend themselves to advanced delivery formats that maximize tumor selectivity and efficacy.

The prospects of these compounds hinge on several key priorities. First, rigorous standardization of shilajit is required to address sourcing variability and ensure reproducible bioactivity. Establishing fulvic acid and dibenzo-α-pyrones as quality control markers will be crucial for regulatory approval. Second, biomarker-guided stratification is essential for glycine use: Patients with tumors showing high GLDC or SHMT2 expression may benefit from inhibition rather than supplementation, while those with inflammation-driven carcinogenesis may benefit from dietary glycine. Third, nanotechnology platforms should be harnessed to deliver shilajit and glycine with controlled release and tumor targeting. Fourth, clinical trials, both preventive (e.g., HCC prevention in patients with cirrhosis) and therapeutic (e.g., adjunct to immunotherapy in breast cancer), are urgently needed to translate preclinical promise into patient benefits. Finally, the global health implications of shilajit and glycine warrant attention. Both agents are low-cost and widely accessible, making them attractive adjuncts in regions where access to biologics or advanced therapeutics is limited. By incorporating these compounds into precision oncology frameworks, it may be possible to bridge the gap between traditional remedies and modern cancer care, aligning with the global mandate for affordable, accessible therapies.

CONCLUSION

In conclusion, shilajit and glycine demonstrate the potential of natural compounds to enhance cancer therapeutics. They are not alternatives to modern treatments but integrative agents that can be woven into the fabric of immunotherapy, chemotherapy, radiotherapy, and nanomedicine. With robust research on safety, biomarker stratification, and clinical efficacy, these agents have the potential to usher in a new era of cancer care that is both scientifically rigorous and globally inclusive.

ACKNOWLEDGEMENTS

The authors are grateful to all personnel associated with the references who participated in this review.