The initial step in glycolysis involves the transport of glucose molecules from the extracellular environment into the cytoplasmic matrix. This crucial transport action is primarily mediated by the GLUT family members, characterized by their high affinity for glucose and efficient import of glucose into cells to sustain the survival, proliferation, and growth of cancer cells (43, 44). In GBM, the expression of GLUT-1 and GLUT-3 is particularly prominent, and their levels correlate with reduced patient survival (45). Extensive evidence suggests that GLUT1 expression is upregulated in various malignancies and is associated with poor prognosis (45, 46). Studies have demonstrated that silencing GLUT1 expression effectively reduces glioma stem cell (GSC) tumor sphere formation, decreases self-renewal and proliferation in vitro, indicating its potential as a therapeutic target in GBM (46). Zhang Z et al. showed that mutating the CYS207 residue of GLUT1 to serine abolished its palmitoylation and membrane localization, resulting in decreased glycolysis, reduced cancer cell proliferation, and suppression of GBM tumorigenic potential (47). The research by Y. Li et al. found that HSP90B1 is significantly upregulated in radioresistant GBM cell lines, and that HSP90B1 promotes the localization of GLUT1 on the plasma membrane, enhances glycolytic activity, and thereby enhances tumor growth and radioresistance in GBM cells. In vivo experiments showed that HSP90B1 knockdown combined with radiotherapy can improve the survival rate of mice with GBM (48). Furthermore, natural products from plants and fungi, such as quercetin, can specifically block the function of GLUT-1 transporters. Both in vivo and in vitro experiments have confirmed that quercetin can significantly inhibit the growth of GBM and extend the survival time of mice (49). These results indicate that quercetin is a potential therapeutic drug for GBM.

Similar to GLUT-1, GLUT-3, also known as the neuronal glucose transporter, is highly expressed in brain tumor-initiating cells (BTICs) and exhibits a higher affinity for glucose (44). The expression of GLUT-3 is significantly higher in Grade 3 and 4 gliomas compared to lower-grade gliomas, suggesting its crucial role in glucose transport in high-grade gliomas (50). Libby et al. further demonstrated that overexpression of GLUT-3 in GBM is associated with increased invasiveness (51). Indeed, experiments have shown that knocking down GLUT-3 can more effectively regulate anaerobic pyruvate utilization and significantly slow down cancer cell proliferation (52). GLUTs enhance cancer cell glucose uptake, driving glycolytic metabolism and providing the necessary energy support for cancer cell survival, proliferation, and growth. Therefore, targeting GLUT1/GLUT3 is considered an ideal strategy to delay GBM tumor cell proliferation and overcome treatment resistance. Kwak S et al. confirmed the efficacy of GBM cell death induction by HDAC2 knockdown, targeting GLUT3 expression, using an in vivo orthotopic xenograft tumor model (53). Pucci G et al. demonstrated that GLUT-3 gene knockdown provides a better opportunity to control anaerobic pyruvate utilization and significantly reduces GBM tumor proliferation, suggesting GLUT-3 as a suitable silencing target for overcoming radiation resistance (52).

Excessive lactate generated during glycolysis accumulates, leading to the accumulation of MCTs. MCTs utilize a proton symport mechanism to extrude lactate from cells, which is crucial for maintaining high glycolytic rates in cancer cells and promoting extracellular TME acidification (54–56). Among MCTs, MCT1 is the primary plasma membrane transporter responsible for lactate efflux, particularly in Isocitrate Dehydrogenase (IDH)-wildtype gliomas (57). Consequently, MCT1 is considered a reliable target for targeting glycolytic metabolic reprogramming. Miranda-Gonçalves V et al. showed that inhibiting MCT1 significantly improved the survival rate of brain tumor-bearing mice, reduced lactate efflux and cell invasiveness, and enhanced the survival rate of mice treated with orthotopic gliomas, especially when combined with TMZ therapy, MCT1 knockdown significantly increased the sensitivity of GBM cells to TMZ, thereby significantly prolonging the survival of treated mice (58). Based on these findings, MCT1 inhibitors like AZD3965 have entered Phase I/II clinical trials for the treatment of small cell lung cancer (59) and lymphoma (60).

HK is a key enzyme in the first step of glycolysis, catalyzing the conversion of glucose to Glucose-6-Phosphate (G-6-P). HK2 is the most well-characterized isoform of the HK family and is significantly overexpressed in GBM tumor cells compared to adjacent normal tissues, correlating with GBM prognosis (61). Wolf A et al. demonstrated that transient inhibition of HK2 in GBM cells suppresses tumor cell proliferation and enhances sensitivity to apoptosis-inducing stimuli such as radiotherapy and chemotherapeutic alkylating agent TMZ. Furthermore, stable HK2 depletion inhibits aerobic glycolysis and promotes normal oxidative glucose metabolism, including decreased extracellular lactate, increased OXPHOS protein expression, and increased oxygen consumption. Interestingly, they found that supplementing HK1 in HK2-deficient cells restored overall HK activity but failed to rescue the aerobic glycolysis phenotype, strongly indicating the unique role of HK2 in GBM progression beyond that of HK1 (62). The role of HK2 in inhibiting tumor cell growth has been confirmed in breast cancer (63), oral cancer (64), and cervical cancer (65). The main therapeutic approaches targeting HK2 include reducing its catalytic activity by inhibiting its expression, directly inhibiting its enzymatic activity to block glucose phosphorylation, and disrupting its interaction with mitochondria to cut off an important energy pathway for tumor cells. Natural products like resveratrol and ginsenosides have been shown to effectively inhibit HK2 activity and exhibit potential in suppressing glycolysis and inducing apoptosis in various cancer experimental models (66, 67). Resveratrol has been shown to significantly inhibit the proliferation of U87 glioma cells and multiple patient-derived GSC cell lines in GBM (68). Additionally, antifungal azole drugs have been identified as a class of potential tumor metabolism inhibitors. Studies have revealed that ketoconazole and posaconazole can effectively inhibit the growth of GBM in vitro and in vivo by regulating HK2-related genes and signaling pathways. These drugs improve survival rates in GBM mouse models, reduce tumor cell proliferation, and decrease overall tumor metabolism levels (69).

The development of small-molecule HK2 inhibitors is also actively pursued and shows promising prospects. 2-DG is a small-molecule HK2 inhibitor that competitively inhibits HK2-mediated glucose phosphorylation, weakens the immunosuppressive network and macrophage polarization, enhances anti-tumor immunity, and contributes to local tumor control in mouse models (69). The research by Pająk et al. confirms that novel 2-DG analogs, such as WP1122, and HDAC inhibitors (like sodium butyrate and sodium valproate) exhibit significant synergistic anticancer effects, inhibiting the proliferation of GBM cells and inducing apoptosis. In vivo experimental results demonstrate that, in mouse models, the combination therapy significantly suppressed tumor growth, improved the survival rate of mice, and no significant toxicity was observed (70). This suggests that 2-DG, as a glycolysis inhibitor, can enhance the sensitivity of GBM cells to radiotherapy and other chemotherapeutic drugs, providing a new strategy for the treatment of GBM. In addition to combination with chemotherapy and radiotherapy, the combination of HK inhibitors and anti-PD-1 antibodies has also shown promising efficacy. HK2 can act as a protein kinase and phosphorylate IκBα at T291, leading to NF-κB-dependent upregulation of PD-L1 expression, which suppresses CD8 T cell activation and infiltration into tumor tissues, accelerating brain tumor growth and increasing resistance to immune checkpoint blockade therapy. Therefore, the combination of HK inhibitors and anti-PD-1 antibodies exhibits superior anti-tumor effects compared to monotherapy with either drug (71).

It is noteworthy that disrupting the interaction between HK2 and mitochondria is another promising strategy. Li R et al. found that Gomisin J, a lignan derivative, can inhibit the proliferation of glioma cells, induce apoptosis, and suppress glycolysis regulated by HK2, thereby inhibiting the progression of gliomas. Gomisin J may represent a potential therapeutic strategy for the treatment of gliomas with relatively low toxicity (72). Similarly, Shteinfer-Kuzmine A et al. focused on cell-penetrating peptides based on Voltage-Dependent Anion Channel 1 (VDAC1), which can perturb the energy and metabolic homeostasis of GBM cells, leading to apoptosis of GBM cells and derived stem cell lines. These peptides also demonstrated inhibitory

effects on tumor growth, invasion, metabolism, stemness, and promoted apoptosis in subcutaneous and intracranial orthotopic GBM xenograft mouse models, suggesting that peptide compounds based on VDAC1 may become a novel candidate for GBM treatment (73). Additionally, the use of VDAC1-specific short interfering RNA (si-VDAC1) to treat GBM cell lines and subcutaneous or intracranial orthotopic GBM xenograft mouse models has been shown to significantly inhibit tumor growth. The remaining tumor tissue exhibited reversed oncogenic characteristics, including metabolic reprogramming, proliferation inhibition, reduced angiogenesis, weakened EMT, decreased invasiveness, and loss of stemness, with some tumor cells differentiating into neuron-like and astrocyte-like cells (74).

Phosphofructokinase-1 (PFK1) is a key regulatory and rate-limiting step in glycolysis, catalyzing the conversion of fructose-6-phosphate and ATP to fructose-1,6-bisphosphate (F-1,6-BP) and ADP (75). PFKFB3 is one of the isoforms of PFK-2 and has been confirmed to be the most highly expressed enzyme in GBM (76). Inhibition of PFKFB3 expression or activity can decrease the level of F-2,6-BP, thereby inhibiting the activity of PFK1 and the rate of glycolysis, ultimately suppressing cell proliferation (77, 78). Clem BF et al. demonstrated that chemical inhibitors targeting PFKFB3, a key enzyme in glycolysis, can effectively reduce glycolysis rate and inhibit tumor cell proliferation (79). This key finding not only confirms the crucial role of PFKFB3 in tumor metabolism regulation but also opens up new strategies for cancer treatment. Li FL et al. further demonstrated the impact of PFKFB3 acetylation status on its activity, showing that acetylated PFKFB3 can significantly promote glycolysis and protect cells from cisplatin-induced apoptosis, revealing the potential role of PFKFB3 modification status in tumor resistance mechanisms (80). Current research trends also lean towards combining PFKFB3 inhibitors with other existing therapies to achieve more comprehensive and effective treatment outcomes. Zhang J’s research team found that simultaneous inhibition of VEGF and glycolysis activator PFKFB3 can significantly prolong the survival of GBM patients and slow down tumor growth. This dual therapy not only inhibited cell proliferation but also promoted apoptosis and enhanced tumor vessel normalization, improving tumor hypoxia, reducing lactate accumulation, and increasing the efficacy and delivery efficiency of chemotherapeutic drugs like doxorubicin (81). This indicates that by integrating the synergistic effects of VEGF and PFKFB3 blockade, it is possible to overcome the current limitations of bevacizumab monotherapy. Nakada M et al. explored the possibility of using PFKFB3 inhibitors to address TKI (tyrosine kinase inhibitor) resistance in GBM, suggesting that the combination of PFKFB3 inhibitors and TKIs may bring breakthroughs in clinical practice (82). Additionally, a series of small-molecule inhibitors targeting PFKFB3 have been extensively investigated, including KAN0438757, developed by Gustafsson NMS et al. Experimental data show that KAN0438757 significantly reduces glioblastoma cell viability and migration ability, with a time- and dose-dependent effect (83). Other inhibitors like 3-PO, PFK15, PFK158, YN1, and N4A have also shown strong antitumor proliferation efficacy in preclinical studies (84). 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) is a novel compound widely reported to inhibit glycolysis flux by competitively inhibiting PFKFB3 (85). Li J’s research team compared the differences in glycolysis flux (analyzed by glucose uptake and lactate secretion) and cell viability (assessed by CCK8 assay) between experimental groups with and without 3-PO addition, providing strong evidence for 3-PO’s role in significantly inhibiting tumor cell glycolytic activity and proliferation (86). The combined application of 3-PO and bevacizumab (a monoclonal antibody targeting VEGF) has been confirmed to reduce GBM cell proliferation and increase apoptosis, thereby delaying tumor growth and improving patient survival (81). PFK15, as a derivative of 3-PO, has demonstrated approximately 100 times higher PFKFB3 inhibitory activity than 3-PO in preclinical studies. Although its anti-GBM growth activity is slightly inferior to that of TMZ, preclinical studies still indicate that PFK15 and its closely related structural derivatives have the potential to exhibit potent cytotoxic activity against various human cancers in future clinical trials. However, it is noteworthy that these compounds have not yet entered the clinical trial phase (79). The combination of PFK158 with CTLA-4 antibody has demonstrated synergistic inhibition of cancer growth, providing a promising perspective for the integration of immunotherapy and targeted glycolytic metabolism therapy (88).

Pyruvate kinase (PK) has two main isoforms: PKM1 and PKM2. It catalyzes the transfer of inorganic phosphate from Phosphoenolpyruvate (PEP) to ADP, generating pyruvate and the energy-rich molecule ATP (82). PKM2 is minimally expressed in healthy brains but highly expressed in GBM cells. Studies have shown that GBM cells undergo a switch from PKM1 to PKM2 isoforms (87). Further experiments have revealed that replacing PKM2 with PKM1 variants in GBM cells leads to restricted biosynthetic pathways and inhibits tumor growth (88). These studies collectively indicate that PKM2 is an important biomarker of glycolytic reprogramming. PKM2 primarily exists in the forms of tetramers and dimers. Dimeric PKM2 is predominantly located in the nuclei of tumor cells and plays a pivotal role in tumor cell proliferation, invasion, and metastasis. Specifically, when the expression of dimeric PKM2 is preferred, it can regulate aerobic glycolysis through metabolic reprogramming of cancer cell metabolic pathways, leading to increased macromolecular biosynthesis in cancer cells and accelerated cancer cell growth. Conversely, activating PKM2 can promote the formation of tetramers, which can inhibit the nuclear import of PKM2 and subsequently suppress the STAT3 signaling pathway, thereby inhibiting the proliferation and metastasis of GBM cells (88, 89). This dynamic equilibrium mechanism between the dimeric and tetrameric forms of PKM2 enables proliferating cancer cells to regulate their anabolic and catabolic demands (90). Liang J et al. demonstrated that activation of the epidermal growth factor receptor (EGFR) induces PKM2 nuclear translocation, upregulating cyclin D1 expression in GBM U87 cell lines and other human cancer cells, accelerating tumor formation (91). On the other hand, the naphthoquinone compound Shikonin, extracted from the traditional Chinese herb Zicao, has been found to inhibit PKM2 activity. In vitro experiments have demonstrated its inhibitory effect on the proliferation, migration, and invasion of glioblastoma cell lines U87 and U251. Furthermore, studies have shown that Shikonin can also inhibit the migration and invasion of GBM cells by targeting phosphorylated β-Catenin and phosphorylated PI3K/Akt signaling pathways (92). Ding Y et al. extensively investigated the effects of a series of parthenolide dimers as PKM2 activators and found that compound 5 exhibited high activity. It promotes PKM2 tetramer formation in GBM cells, reduces PKM2 nuclear translocation without affecting total PKM2 expression, and thereby inhibits the STAT3 signaling pathway in vitro and in vivo, as a result, it suppresses the proliferation and metastasis of GBM cells and induces apoptosis (93). These findings provide potential drug targets and therapeutic approaches for the development of new GBM treatment strategies. Metformin and vitamin K, as PKM2 inhibitors, have shown inhibitory effects on glycolysis, cell proliferation, and drug resistance in various cancers such as osteosarcoma (94), non-small cell lung cancer (95), and hepatocellular carcinoma (96). Metformin has been evaluated for its potential therapeutic effect on glioblastoma in several clinical trials. For example, a cohort study by Adeberg et al. involving 276 patients with primary GBM suggested that the use of metformin in diabetic GBM patients may help slow disease progression and improve patient survival (97),This study provides evidence for metformin as a potential therapeutic agent and lays the foundation for future clinical trials. Additionally, research by Qiao X et al. suggests that metformin can penetrate the blood-brain barrier (BBB) and enter brain tissue, inhibiting the malignant progression of gliomas. The study showed that metformin alone can induce apoptosis in glioma cells (98). However, the specific role of vitamin K in gliomas has not been reported as of now, and further research is needed to elucidate it in the future. Notably, a small molecule compound called dimethylaminochlorocycline (DMAMCL) can modulate glycolysis pathways by activating PKM2, reducing GBM cell proliferation. DMAMCL has been applied in clinical trials for recurrent GBM. This compound selectively binds to the monomeric form of PKM2, promoting PKM2 tetramerization and increasing its pyruvate kinase activity. The inhibitory effect of DMAMCL in GBM cells is weakened when PKM2 is depleted, further demonstrating the critical role of PKM2 in mediating the sensitivity of GBM cells to DMAMCL (99). Furthermore, research by Gao M et al. has shown that Trametinib inhibits the growth and aerobic glycolysis of glioma cells by targeting the PKM2/c-Myc axis. Trametinib suppresses the expression of PKM2 in glioma cells and inhibits the translocation of PKM2 into the nucleus, thereby affecting the expression of c-Myc (100). These findings not only reveal the potential mechanism of Trametinib in antitumor activity but also provide new insights into the clinical drug treatment of gliomas.

Lactate dehydrogenase (LDH) is a core component of the Warburg effect. In hypoxic or oxidative stress conditions, tumor cells adaptively upregulate LDH activity to promote the rapid conversion of pyruvate to lactate during glycolysis, rather than entering the tricarboxylic acid cycle (TCA cycle) for complete oxidation (101, 102). In GBM, the Lactate Dehydrogenase A (LDHA) isoform is typically overexpressed, promoting the flux of substrates towards lactate production during glycolysis, enhancing tumor cell survival and proliferation (103). Studies on various cell lines have shown that inhibiting LDHA function leads to decreased cancer cell proliferation, increased apoptosis, and weakened migration and invasion, further confirming the importance of LDHA as a potential therapeutic target. Galloflavin, a synthesized LDHA and LDHB inhibitor, exhibits high levels of cell penetration (104, 105). Research has shown that Galloflavin inhibits the conversion of pyruvate to lactate, directing pyruvate into the TCA cycle, prompting tumor cells that rely on glycolysis to switch to oxidative phosphoryation (OXPHOS) for energy production, this metabolic shift is detrimental to tumor cells as it increases oxygen consumption and reactive oxygen species (ROS) generation, leading to mitochondrial oxidative damage (106). Gossypol, a derivative of cottonseed oil, has been found to inhibit LDHA. Research by T. Coyle et al. has demonstrated that gossypol exhibits significant in vitro and in vivo cytotoxicity against central nervous system tumor cells. In vitro, gossypol displays concentration- and time-dependent cytotoxicity against various glioma cell lines. In vivo, gossypol significantly inhibits tumor growth in a xenograft model in nude mice, suggesting its potential as a therapeutic agent for the treatment of primary malignancies of the central nervous system (107). Gossypol has been shown to be well-tolerated in clinical trials and has shown promise in trials for recurrent malignant gliomas (NCT00540722 and NCT00390403). FX11, a novel gossypol-derived selective LDHA small-molecule inhibitor, has been developed and has shown promise in preclinical studies. It inhibits LDHA activity, decreases intracellular ATP levels, and induces oxidative stress and cell death, which can be partially reversed by the antioxidant N-acetylcysteine (108). Recently, FX11 has been commercialized, and studies in human lymphoma and pancreatic cancer xenografts have shown that it inhibits tumor progression and induces significant oxidative stress and necrosis (108) Daniele S et al. found that NHI-1 and NHI-2 (LDH-A inhibitors) can reduce GBM cell proliferation, trigger apoptosis, and block the cell cycle (103). Additionally, oxalate has been considered to have LDH-inhibitory effects (109). The study of SunT et al. confirmed that Oxamate can reprogram glucose metabolism of cancer stem cells, and can also alleviate immunosuppression of TME and reduce tumor-infiltrating CAR-Treg cells, which may be a potential strategy to enhance CAR-T function in glioblastoma therapy (110). Moorhouse AD et al. designed a novel dual-functional ligand that inhibits LDHA, with activity 9 times higher than sodium oxalate,however, subsequent reports on detailed molecular modeling studies and biological tests targeting cancer cell lines have not been forthcoming (111). The specific application of targeting glycolytic metabolic reprogramming pathways in GBM is demonstrated in Table 1.