Hexokinase (HK), the first rate-limiting enzyme in glycolysis, catalyzes the conversion of glucose to glucose-6-phosphate [G-6-P (23, 74)], a pivotal intermediate in glycolysis, the pentose phosphate pathway (PPP), and glycogen synthesis. Among glycolytic enzymes, HK is considered the most critical regulatory node in glucose metabolism. Mammalian cells express four HK isoforms: HK1, HK2, HK3, and HK4 (24).

Several HK inhibitors have demonstrated anticancer activity, with the most extensively studied being 3-bromopyruvate (3-BrPA), lonidamine (LN), 2-deoxy-D-glucose (2-DG), and metformin. 3-BrPA directly inhibits HK2, thereby suppressing glycolytic flux in cancer cells (75). In addition to its glycolytic inhibition, 3-BrPA enhances the cytotoxicity of chemotherapeutic agents and mitigates multidrug resistance (MDR), a major mechanism of therapeutic failure in cancer by promoting drug efflux (25, 76). Lonidamine (LN), an adenine nucleotide translocator (ANT) ligand, inhibits mitochondrial complexes I and II and promotes the formation of mitochondrial permeability transition pores (77, 78). It is a novel glycolysis-targeting agent currently undergoing clinical evaluation for the treatment of various cancers, including ovarian, breast, and lung malignancies (26). 2-Deoxy-D-glucose (2-DG), a glucose analog, interferes with glycolysis and ATP synthesis, leading to energy depletion and cancer cell death. In CRC, 2-DG has shown antitumor efficacy both as monotherapy and in combination with chemotherapy and radiotherapy (26, 27). Metformin, another HK2 inhibitor, exerts its effects primarily by activating AMP-activated protein kinase (AMPK), which subsequently suppresses the mTOR pathway, thereby reducing glycolysis and cell proliferation (79). In CRC models, metformin decreases glucose uptake and lactate production, ultimately inhibiting tumor growth and enhancing chemosensitivity (28).

Several natural compounds also inhibit HK2 and exert pro-apoptotic effects in cancer cells. Curcumin, a polyphenolic compound derived from Curcuma longa, has been widely investigated for its anticancer potential (29, 30). By downregulating HK2, curcumin induces mitochondrial dysfunction and the release of cytochrome c, activating caspases and promoting apoptosis in CRC cells (31). Epigallocatechin gallate (EGCG), a major polyphenol in green tea, inhibits the anchorage-independent growth of CRC cells by disrupting the interaction between HK2 and mitochondria. This mitochondrial disruption impairs energy metabolism and induces apoptosis (32). Arsenic trioxide (As₂O₃), an established therapeutic agent for acute promyelocytic leukemia (APL) (33), has also been shown to inhibit HK2 expression and glycolysis in cancer cells. By downregulating glucose metabolism and inducing apoptosis, As₂O₃ offers a unique mechanism for suppressing tumor growth (80).

PFK, the second major rate-limiting enzyme in glycolysis, catalyzes the conversion of fructose-6-phosphate (F-6-P) to fructose-1,6-bisphosphate (F-1,6-BP) (81). PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3) is a critical glycolytic regulator that promotes the synthesis of fructose-2,6-bisphosphate (F-2,6-BP), a potent allosteric activator of PFK1. PFKFB3 is frequently overexpressed in various cancers and is associated with lymph node metastasis and poor survival outcomes (34).

Several PFKFB3 inhibitors—including 3PO, PFK15, and PFK158—have been identified as potential therapeutic agents. Administration of 3PO leads to a rapid reduction in glucose uptake, lactate production, and ATP synthesis. Additionally, 3PO can reprogram the metabolic profile of patient-derived tumor organoids to favor oxidative phosphorylation. In vivo, neoadjuvant treatment with 3PO promotes vascular normalization, alleviates hypoxia, and enhances tumor necrosis (35). PFK15 and PFK158, derivatives of 3PO, have also demonstrated efficacy in attenuating glycolytic activity and suppressing tumor growth in colorectal cancer (CRC) models. Notably, PFK15 exhibits approximately 100-fold greater inhibitory potency against PFKFB3 compared to 3PO (82). PFK15 significantly reduces F-2,6-bisphosphate (F-2,6-BP) levels in xenograft tumors and induces apoptosis in transformed cancer cells in both in vivo and in vitro settings (82). PFK158 is currently being evaluated in a Phase I, dose-escalation, multicenter clinical trial (NCT02044861) aimed at assessing its safety, tolerability, and pharmacokinetics in patients with advanced solid tumors. Preliminary findings revealed antitumor activity in 6 of 19 evaluable patients. Although the trial did not meet the desired efficacy endpoints, it underscored the therapeutic potential of targeting PFKFB3 in cancer treatment (36, 83).

In traditional Chinese medicine (TCM), oleanolic acid (OA) has emerged as a potential therapeutic candidate for gastric cancer (37). OA, a triterpenoid compound abundant in plants of the Oleaceae family, modulates aerobic glycolysis and tumor cell proliferation. Specifically, OA inhibits gastric cancer cell growth and reduces intracellular lactate levels by suppressing glucose uptake and utilization through downregulation of HIF-1α, HK2, and PFK1 expression (84).

Glucose transporters (GLUTs) are integral membrane proteins responsible for facilitating glucose entry into cells. Among the best-characterized subtypes are GLUT1, GLUT2 (SLC2A2), GLUT3 (SLC2A3), and GLUT4 (SLC2A4), each exhibiting distinct regulatory mechanisms and kinetic properties, thereby playing specialized roles in maintaining cellular and systemic glucose homeostasis (85, 86). GLUT1 is implicated in chemoresistance via its regulation of glycolysis (87), while elevated expression of GLUT2 and GLUT3 correlates with poor prognosis in CRC (38). Consequently, GLUTs represent attractive therapeutic targets for disrupting glucose metabolism in CRC.

Several small-molecule inhibitors targeting GLUTs have shown preclinical promise, including STF-31, WZB117, BAY-876, and CG-5. STF-31 selectively induces apoptosis in cancer cells without affecting normal tissues, thereby reducing CRC cell viability and proliferation (39). WZB117 triggers CRC cell death, particularly when delivered via hypoxia-responsive nanoparticles (88), and has also been shown to resensitize 5-fluorouracil (5-FU)-resistant colon cancer cells to chemotherapeutic agents, supporting its potential as an adjuvant for overcoming drug resistance (40). BAY-876 is a potent and selective GLUT1 inhibitor with high metabolic stability in vitro and favorable oral bioavailability in vivo; it has demonstrated antitumor efficacy in several cancers, including ovarian and triple-negative breast cancer (41, 42). CG-5, a thiazolidinedione derivative, inhibits GLUT-mediated glucose transport in T cells, disrupts glycolysis, and impairs Th1 and Th17 cell differentiation while promoting Treg cell development and reducing CD4+ T cell proliferation (43).

Natural compounds have emerged as promising GLUT inhibitors with potential anticancer effects in CRC. Key examples include cytochalasin B, phloretin, apigenin, trehalose, silibinin, and genistein. Cytochalasin B, the first identified GLUT1 inhibitor, has provided critical insights into CRC metabolism (44). By inhibiting GLUT1, it reduces intracellular glucose availability, thereby limiting substrates for glycolysis and oxidative phosphorylation (45). Phloretin, a natural GLUT2 inhibitor, suppresses GLUT2 expression by blocking the transcription factor HNF6. It also activates the p53 pathway, promoting cell cycle arrest and apoptosis—mechanisms that collectively inhibit tumor progression and enhance CRC cell sensitivity to other therapies (46). Apigenin, a flavonoid found in parsley, celery, onions, and chamomile (47), inhibits CRC cell proliferation and induces apoptosis through downregulation of GLUT1, partially via HIF-1α inhibition (48). Furthermore, Apigenin reduces VEGF secretion under both normoxic and hypoxic conditions, highlighting its anti-metastatic potential (49). Trehalose, a non-reducing disaccharide composed of two glucose units linked via an α,α-1,1-glycosidic bond (50), impedes glucose transporter activity and induces a starvation-like state characterized by ATP depletion. This metabolic stress activates autophagy through AMPK-dependent signaling, contributing to its anticancer effects (51). Silibinin, a flavonoid derived from Silybum marianum (52) (milk thistle), rapidly induces oxidative stress in CRC cells. It disrupts energy homeostasis by inhibiting the PI3K-Akt-mTOR pathway and activating ERK1/2, leading to metabolic reprogramming (53). Genistein, an isoflavone abundant in soybeans and legumes (54), suppresses GLUT1 and HK2 by downregulating HIF-1α (55). It also induces cell cycle arrest and reduces invasion capacity in CRC cells (54).

The third rate-limiting step in glycolysis is catalyzed by pyruvate kinase (PK), which converts phosphoenolpyruvate to (89)pyruvate. In mammals, PK exists in four isoforms: PKM1, PKM2, PKR, and PKL (56), among which PKM2 is predominant in cancer cells. Inhibition of PKM2 disrupts glycolysis and promotes apoptosis.

Three principal classes of PKM2 inhibitors have been identified: metformin, vitamin K, and shikonin. Metformin suppresses PKM2 expression and inhibits tumor growth (90) by modulating AMPK and mTOR signaling. In CRC xenograft models, metformin significantly reduces tumor volume through these pathways (57). Vitamin K (VK), a lipophilic naphthoquinone, exhibits isoform-specific inhibition, with VK3 and VK5 more potently targeting PKM2 over PKM1 (91). The combination of VK3 and vitamin C has shown enhanced anticancer efficacy, and clinical studies suggest VK3 can overcome resistance to chemotherapeutics such as doxorubicin and adriamycin (92). VK2 has also been reported to inhibit CRC cell proliferation by suppressing NF-κB signaling and inducing pro-apoptotic proteins (58).

Shikonin, a naturally occurring compound, selectively inhibits PKM2 without affecting PKM1. It reduces glucose uptake and lactate production, underscoring its therapeutic promise in cancer metabolism (59). Similarly, lapachol, a naphthoquinone derived from the bark of Tabebuia avellanedae, inhibits PKM2 activity and reduces ATP levels (60). An active fraction from clove (Eugenia caryophyllata or Syzygium aromaticum), referred to as AFC, has been shown to decrease glucose uptake, lactate production, PK activity, and pyruvate synthesis in CRC cells via PKM2 downregulation, ultimately attenuating aerobic glycolysis (93).

Lactate dehydrogenase (LDH) catalyzes the final step of glycolysis by reversibly converting pyruvate to lactate. The human genome encodes four LDH isoforms—LDHA, LDHB, LDHC, and LDHD (61). Among these, LDHA and LDHB are highly expressed in malignancies, with LDHA predominantly converting pyruvate to lactate and LDHB catalyzing the reverse reaction. Elevated LDHA expression is associated with poor prognosis across multiple tumor types.

FX11, a small-molecule LDHA inhibitor, binds directly to its active site, blocking pyruvate-to-lactate conversion and thereby reducing cancer cell invasiveness and metastatic potential (94). In preclinical xenograft models of human lymphoma and pancreatic cancer, FX11 exhibited significant antitumor activity (95).

The natural compound gossypol, a nonselective LDHA inhibitor, has demonstrated potent anticancer effects in vitro and in animal models (62). In CRC cells, gossypol suppresses LDHA activity, lowering lactate production and glycolytic flux. This metabolic disruption induces bioenergetic and oxidative stress, resulting in cell cycle arrest and apoptosis (96). In CRC xenograft models, gossypol markedly inhibited tumor growth and progression (63). Galloflavin, another LDHA inhibitor, binds to the enzyme’s NADH-binding site, blocking its activity and impeding CRC proliferation (64). Additionally, Galloflavin modulates the tumor inflammatory microenvironment by targeting NLRP3 and downregulating oncogenic c-Myc and P21, further enhancing its antitumor efficacy (65). Oxamate, an LDHA inhibitor, induces mitochondrial apoptosis in CRC cells by suppressing lactate synthesis. In combination with metformin or mTOR inhibitors, it shows synergistic antitumor effects in preclinical models (97).

To meet increased energy and biosynthetic demands, cancer cells often augment oxidative phosphorylation (OXPHOS) (98).Inhibiting OXPHOS suppresses proliferation and tumorigenicity even in glycolysis-competent CRC cells, both in vitro and in patient-derived xenografts (66). Proper electron transport chain (ETC) function is essential for OXPHOS and ATP production, both critical for carcinogenesis. ETC inhibitors—such as metformin, tamoxifen, α-tocopheryl succinate (α-TOS), 3-bromopyruvate (3-BrPA), and ME-series inhibitors—disrupt respiratory complex activity, increase reactive oxygen species (ROS) levels, and trigger apoptosis (67). Tamoxifen, traditionally used as a selective estrogen receptor modulator in breast cancer, also targets mitochondria (68). Its derivative, MitoTam, localizes to mitochondria, inhibits complex I, elevates ROS, and induces cell death in breast cancer cells (69). The small-molecule inhibitor ME-344 effectively inhibits complex I and multiple pro-death signaling pathways associated with mitochondrial permeability transition in CRC (70). Both ME-143 and ME-344 disrupt NADH oxidation at complex I, blocking electron flow through the ETC. ME-344 further induces Bax translocation to the mitochondrial outer membrane, triggering mitochondrial permeability transition and releasing pro-apoptotic molecules (71).

The natural product Rhein, known for its mitochondrial-targeting properties, has been conjugated with dichloroacetate (DCA) to create Rhein-DCA, a dual glycolysis and OXPHOS inhibitor. Rhein-DCA accumulates in mitochondria, inhibits glycolysis via the PDK-PDH axis, and disrupts the respiratory chain. In CRC models, it induces oxidative stress, decreases lactate levels, and promotes immunogenic cell death (72). Frondoside A, a triterpene glycoside derived from marine organisms, induces mitochondrial apoptosis by decreasing antiapoptotic proteins Bcl-2 and survivin, increasing ROS production, and promoting cytochrome c release (73). Similarly, ginsenoside compound K, a natural derivative of ginseng, activates ROS-mediated mitochondrial apoptosis, leading to cytochrome c release and caspase-9/-3 activation (99). These natural compounds offer promising avenues for CRC therapy by targeting mitochondrial function and enhancing oxidative stress-induced cell death.

Fatty acid synthase (FASN), a pivotal enzyme in de novo lipogenesis, is inversely correlated with CRC prognosis (102, 121). Several FASN-specific inhibitors—such as cerulenin, C75, Orlistat, and TVB-series compounds—have demonstrated pro-apoptotic activity and therapeutic potential across various cancer types (103).

Cerulenin induces apoptosis in CRC cell lines by activating the caspase cascade and inhibiting DNA replication and S-phase progression (122). In HT-29 and LoVo cells, cerulenin also disrupts energy metabolism and inhibits mTOR signaling, thereby suppressing the malignant phenotype of CRC (123). Combination therapy with cerulenin and oxaliplatin may attenuate oxaliplatin-induced neurotoxicity, reduce required dosages, and improve long-term chemotherapeutic tolerance in clinical trials (124). C75 and C93 structurally related to cerulenin, also target FASN (105). Both compounds, including cerulenin, influence carnitine palmitoyltransferase 1 (CPT1), thereby enhancing fatty acid oxidation (104, 105). Notably, treatment with C75 and cerulenin significantly reduces food intake and body weight in murine models (105). Orlistat, another FASN inhibitor, activates caspase-3 in a dose-dependent manner, induces G1 cell cycle arrest, and reduces both proliferation and lipid synthesis in HT-29 cells (106). Second-generation FASN inhibitors—TVB-3664, TVB-3166, and TVB-2640—exhibit potent antitumor activity in vitro and in vivo and are currently undergoing clinical evaluation in CRC patients (107).

In addition to synthetic compounds, natural products such as luteolin and resveratrol also exhibit FASN-inhibitory and anticancer properties. In HT-29 cells, luteolin downregulates anti-apoptotic proteins and induces cell cycle arrest (108). Resveratrol inhibits proliferation in Caco-2 cells, suppresses aberrant crypt foci formation (109), and induces apoptosis by modulating the IGF1R/AKT/Wnt pathway and activating p53 (125).

ATP citrate lyase (ACLY), which catalyzes the conversion of citrate to acetyl-CoA, acts as a rate-limiting enzyme in early lipid biosynthesis. ACLY has been implicated in promoting CRC progression in both in vitro and in vivo models (110). ETC-1002, a potent ACLY inhibitor, activates the AMPK pathway and suppresses lipid and cholesterol synthesis, although its clinical efficacy has been predominantly observed in cholesterol regulation (126). Nonetheless, co-administration of ETC-1002 with the IGF1R inhibitor linsitinib has demonstrated significant synergistic effects in inhibiting CRC metastasis (111). Curcumin, a natural compound, also inhibits ACLY activity, lowering acetyl-CoA levels and disrupting lipid synthesis essential for tumor growth (112). Other natural agents, such as berberine, similarly suppress ACLY activity, thereby attenuating tumor progression and metastatic potential (127).

Stearoyl-CoA desaturase (SCD), an endoplasmic reticulum membrane enzyme, catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids, thus facilitating lipid biosynthesis (128). Elevated expression of SCD1, the predominant isoform, is negatively associated with CRC prognosis (113). The novel oral SCD inhibitor T-3764518 has been shown to promote apoptosis by disrupting lipid raft integrity and inhibiting oncogenic signaling in CRC xenograft models (114). Betulinic acid, a natural SCD1 inhibitor derived from birch bark, induces G2/M cell cycle arrest and inhibits CRC growth (129). Furthermore, it reduces clonogenicity and induces apoptosis in CRC stem-like cells, highlighting its potential as a therapeutic agent targeting cancer stemness (130).

Carnitine palmitoyltransferase 1A (CPT1A) is a key rate-limiting enzyme in fatty acid oxidation. CPT1A-mediated β-oxidation supports reactive oxygen species (ROS) detoxification and enhances reduced glutathione synthesis by increasing intracellular NADPH levels (115). Etomoxir, an irreversible CPT1 inhibitor, significantly impairs fatty acid uptake and ATP production without affecting tumor cell stemness or angiogenesis (115, 116). Notably, combining etomoxir with cisplatin enhances cisplatin-induced apoptosis in HCT116 colorectal cancer (CRC) cells in a dose-dependent manner (117). Another CPT1 inhibitor, perhexiline, induces ROS accumulation and apoptosis, thereby suppressing gastrointestinal tumor progression (118). Co-treatment with perhexiline and oxaliplatin further promotes apoptosis and sensitizes HCT116 cells to oxaliplatin (131).

Numerous studies have established a positive correlation between elevated dietary and plasma cholesterol levels and increased CRC risk, whereas statin-mediated inhibition of cholesterol biosynthesis is associated with reduced risk (119, 132). Lovastatin inhibits both canonical Wnt signaling and alternative oncogenic pathways, such as YAP/TAZ, thereby suppressing CRC progression (133). At low doses, lovastatin promotes CRC cell differentiation and significantly increases their sensitivity to 5-fluorouracil (5-FU) (120). Hesperetin, a cholesterol-lowering flavonoid found in citrus juices, reduces foam cell formation, intracellular cholesterol levels, and cholesterol esterification, while enhancing cholesterol efflux in THP-1 macrophages (134). These findings suggest that hesperetin may also regulate cholesterol metabolism and inhibit CRC progression.

Glutaminase 1 (GLS1) catalyzes the conversion of glutamine to glutamate, and its overexpression is strongly associated with poor prognosis in multiple cancers. Inhibiting GLS1 may disrupt glutamine metabolism and hinder tumor progression (136). BPTES (Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide), a potent GLS1 inhibitor, suppresses glutamine utilization and inhibits CRC growth (141), although its clinical development is limited by poor solubility and metabolic instability (137). CB-839, a selective and clinically advanced GLS1 inhibitor, has demonstrated promising results. In a phase II trial, CB-839 combined with 5-FU extended progression-free survival beyond 6 months in 21.8% of patients, potentially through modulation of neutrophil extracellular traps (142). Ongoing clinical trials are evaluating CB-839 in combination with palbociclib for KRAS-mutant CRC and with nivolumab for melanoma and renal cell carcinoma (138). Compound 968, another GLS1 inhibitor with a distinct mechanism of action, has shown broad anticancer efficacy across multiple cell lines (143).

The amino acid transporter ASCT2 has emerged as a critical pro-tumorigenic factor, with elevated expression linked to poor prognosis in various cancers (139). In CRC, ASCT2 overexpression is strongly associated with KRAS mutations. Given the therapeutic resistance commonly observed in KRAS-mutant tumors, ASCT2 represents a promising target for this CRC subset (144, 145).

A monoclonal antibody against ASCT2, Ab3-8, significantly reduced glutamine uptake and inhibited AKT and ERK phosphorylation in SW1116 and HCT116 CRC cells in vitro. In vivo, Ab3–8 treatment markedly suppressed tumor growth in KRAS-mutant CRC xenografts. Additionally, V-9302, a small-molecule ASCT2 inhibitor, competitively blocks glutamine transport. Combined with ASCT2 gene silencing, V-9302 further impairs glutamine uptake and significantly inhibits tumor progression in preclinical models (140).