2.2.1 Promoting the angiogenesis in TME

Angiogenesis mainly encompasses degrading the vascular basement membrane, proliferating and migrating vascular endothelial cells, and forming new vessels [46], which involves multiple regulatory molecules, for instance, fibroblast growth factor (FGF), VEGF, and interleukin-8 (IL-8) [47]. Numerous studies found that the upgraded glycolysis of tumor cells activated the expression of angiogenesis-associated proteins, which in turn transported more nutrients for tumor cells to promote cell growth and tumor metastasis [48-50].

Both enzymes and products involved in glycolytic process can promote tumor angiogenesis. In a study of mouse transplantation models of breast and colon cancers, intracellular lactate was observed to stimulate angiogenesis of tumors through the NF-κB/IL-8 pathway [51]. In addition, by inducing endothelial cell proliferation and adhesion, the dimer of glycolytic enzyme PKM2 initiated and promoted tumor angiogenesis [52]. Furthermore, under hypoxic conditions, PKM2 translocated to the nucleus and induced VEGF secretion by upregulating HIF-1α expression through the NF-κB/p56 pathway, thereby facilitating tumor angiogenesis [53]. In tumor cells, the activity of VEGF was correspondingly enhanced due to the upregulation of glycolytic enzyme PFKFB3, and VEGF expression was decreased by the inhibition of PFKFB3 [54], suggesting that glycolytic enzyme PFKFB3 promotes tumor angiogenesis by modulating VEGF activity.

Lactate and H⁺ produced during glycolysis could acidify TME and promote the secretion of angiogenic factors like VEGF and IL-8 [55], which increase angiogenesis in HCC. Pyruvate, one of the products in glycolytic process was found to promote HIF-1α expression, which accelerated angiogenesis in the TME of HCC by upregulating the expression of plasminogen activator inhibitor-1 (PAI-1) and VEGF [56]. Elevated plasma levels of PKM2 in patients with pancreatic and colon cancers were found to promote tumor angiogenesis in a mouse xenograft model [52]. Further study on colon cancer showed that PKM2 promoted angiogenesis through PI3K/Akt and Wnt/β-catenin signaling pathways [57]. Knockdown of PKM2 reduced the expression of angiogenesis-related proteins (including N-cadherin, Vimentin, and VEGFA), thereby inhibiting angiogenesis in thyroid tumor [58]. Additionally, PKM2 knockout caused impaired angiogenesis and inhibited the growth of pancreatic cancer cells [53]. Besides, breast cancer with the silenced ENO1 gene exhibited a reduction in neovascularization and tumor size [59], indicating that ENO1 has an essential function in promoting tumor angiogenesis and proliferation. The above findings suggested that glycolytic enzymes and their products could promote tumor angiogenesis and tumor progression, which provided new ideas for targeting angiogenesis in tumor treatment.

2.2.2 Promoting the immune escape of tumor cells

The extracellular environment that is excessively acidic and hypoxic has been shown to dampen immune cell activity and aid in the immune evasion of tumor cells [60]. Most antitumor immune cells could be inhibited directly by lactate according to several studies. For example, lactate could inhibit the growth and cytotoxicity of cytotoxic T lymphocytes (CTLs) [61], diminish the secretion of cytokines from CTLs [61], and ultimately repress the antitumor capacity of T cells [61]. Recent research demonstrated that lactate inhibited the antitumor function of CTLs by inhibiting JNK/c-Jun and MAPK-p38 signaling pathway [62].

Additionally, there has been extensive research into the modulation of macrophages by lactate. Tumor-associated macrophages (TAMs) consist of two subtypes: M1 macrophages have been mainly involved in the immune response against tumors, while M2 macrophages repress the inflammatory reaction and facilitate the evasion of immune surveillance. Macrophages in an acidic environment are more inclined to polarize into M2 macrophages to exert immunosuppressive effects [63]. Furthermore, LDHA expression was also found to correlate positively with CD163, a marker of M2-like macrophages, in extramammary Paget's disease [64] and head and neck cancer [65]. The above evidence suggested that glycolysis regulated the polarization of macrophages within the TME, opening up new avenues for tackling tumor cell immune evasion.

A study on in vitro model of tumor-associated dendritic cells (TADCs) showed that the presence of lactate reduced the production of multiple antitumor cytokines in DCs, such as IL-6 and tumor necrosis factor-alpha (TNF-α) [66], suggesting that lactate restrains the antitumor effects of DCs and encourages immune escape of tumor cells. Elevated expression of LDH was found to induce lactate accumulation in melanoma, which suppressed the immune function of natural killer (NK) cells and T cells, and promoted immune escape [67]. Elevated lactate levels in TME inhibited glycolysis in monocytes, which decreased the secretion of TNF-α [68], thus suppressing antitumor immunity. Lactate and the glycolytic enzyme ENO1 were essential for the enhancement and maintenance of the immunosuppressive capacity of regulatory T cells (Tregs) [69, 70], demonstrating that glycolysis can promote immune escape by regulating Treg cells in multiple dimensions. The aforementioned results suggest that glycolysis products and enzymes can interfere the antitumor effect of immune cells, providing a fresh angle for the therapy of tumors by targeting glycolysis.

Since neutrophils have few mitochondria, they mainly rely on aerobic glycolysis for their biological functions [71]. Tumor-associated neutrophils (TANs) are classified into two phenotypes, anti-tumorigenic (N1) and pro-tumorigenic (N2) [72]. Elevated levels of glycolysis in tumor cells led to increased levels of lactate in TME, which promoted N2 polarization of neutrophils, exerting their pro-tumorigenic effects and leading to immune escape of tumor cells [73]. Metabolomic analysis of neutrophil lysates from pancreatic ductal adenocarcinoma (PDCA) showed that several glycolytic metabolites, such as glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate, and lactate, were significantly up-regulated in TANs, indicating enhanced neutrophil glycolysis in the PDCA TME [74]. Further, when LDHA-overexpressed neutrophil-like differentiated HL-60 (dHL-60) cells were co-cultured with PDCA cells, enhanced proliferation of tumor was found, revealing that glycolysis exerts its pro-tumor effects by regulating neutrophils [74]. Moreover, lactate drove the expression of programmed death-ligand 1 (PD-L1) in infiltrating neutrophils through the monocarboxylate transporter 1 (MCT1)/NF-κB/Cyclooxygenase 2 (Cox2) pathway, which reduced the sensitivity of HCC cells to immunotherapy and led to the progression of tumor [75]. The above results implied that the tumor-promoting effects of neutrophils could be suppressed by inhibiting the glycolytic function, which provides a new direction for clinical treatment of tumors.

2.2.3 Promoting the invasion and metastasis of tumor cells

The metastasis of tumor, one of the characteristics of tumor aggressiveness [76], affects the patient's outcome and contributes to poor prognosis, which is a challenge that we need to address urgently at present. Recent reports unveiled that glycolytic enzymes and products serve a significant function in promoting tumor metastasis, which has attracted attention [77, 78]. The discovery indicated that lactate concentration was markedly increased in areas with tumor cell metastasis compared to those without tumor cell metastasis [79, 80], suggesting that lactate might promote the migration of tumor cells. Several pieces of research have demonstrated that ENO1 expression was positively correlated with tumor cell invasion and metastasis [77, 81]. ENO1 could function as a receptor for plasminogen and accelerate the activation of the fibrinolytic system, resulting in fibrinolysis and the decomposition of extracellular matrix (ECM), which provides conditions for tumor cell invasion and metastasis [82, 83]. Through downregulating the expression of ENO1, retinoic acid (RA) reduced the migration of follicular thyroid cancer cells [84], suggesting that ENO1 promoted the migration of tumor cells.

Transcription factors including c-Myc, forkhead box protein M1 (FOXM1), and ETS proto-oncogene 1 (ETS1) promoted the glycolysis process by activating the promoters of glycolytic genes (for example LDHA, PFKFB3, and hexokinase 1 [HK1]), thus increasing the capability of pancreatic cancer to invade and metastasize [85, 86]. Moreover, pancreatic cancer could promote glycolysis through post-translational modification and the alteration of glycolytic enzyme activity, which further strengthened the viability of tumor cells [87]. In addition, studies focused on HCC showed that more aggressive cell lines (such as HCC-LM3) exhibited stronger glycolytic activity than less aggressive cell lines (such as HepG2) [88, 89], indicating that the degree of glycolytic activity might influence the level of tumor invasiveness.

Multiple investigations have demonstrated that glycolysis could promote epithelial-mesenchymal transition (EMT) [90-92]. EMT is a biological process that involves the transformation of epithelial cells to cells with a mesenchymal phenotype, with distinct features including reduced levels of cell adhesion proteins (for instance E-cadherin) and alteration of the cytokeratin cytoskeleton to a cytoskeleton based on vimentin [93]. The occurrence of EMT makes tumor cells more motile and invasive, and therefore, more prone to metastasis [94, 95]. ALDOA, one of the isoenzymes of glycolytic enzyme ALDO, has been found to be positively correlated with EMT [96]. The researchers found that E-cadherin expression was down-regulated and metastasis was more likely to occur in pancreatic cancer tissues with high levels of ALDOA relative to those with low levels [96]. Moreover, the silencing of ALDOA was associated with increased E-cadherin expression levels and decreased vimentin levels [96], indicating that ALDOA might accelerate tumor progression through promoting EMT and metastasis. Additionally, ALDOA might contribute to bladder cancer invasion and metastasis through the E-cadherin-epidermal growth factor receptor (EGFR) signaling pathway [78]. Furthermore, ENO1 promoted EMT through interacting with macrophages and hepatocyte growth factor receptor (HGFR, also called c-Met), thereby facilitating the metastasis of oral squamous cell carcinoma (OSCC) [97] and lung cancer [98], respectively. Moreover, by inducing the upregulation and nuclear relocation of STAT3, PKM2 could induce the metastasis of colon cancer cells [99].

The GLUT family contains 14 members, which are key gating proteins responsible for the entry and exit of glucose into and out of the cell, and play an important role in maintaining homeostasis of intracellular and extracellular glucose [100]. Impaired membrane transport of GLUT is one of the most important reasons for the disruption of glucose levels in the body and the development of hyperglycemia and diabetes [101]. GLUT1 has a high affinity with glucose, and its expression is regulated by HIF-1α [102, 103]. Studies have shown that GLUT1 was highly expressed in a variety of tumor cells, such as lymphoma, kidney, lung, and prostate cancers, which enhanced the uptake of glucose by cancer cells [104-106]. In addition, GLUT2 and GLUT3 have been found to be highly expressed in certain tumors, including HCC, breast cancer, gastric cancer and so on [103]. A recent study has demonstrated that colorectal cancer (CRC) patients with high GLUT3 expression had worse prognosis, and further mechanistic research revealed that GLUT3 promoted EMT in tumor cells through the transforming growth factor-β (TGF-β)/c-Jun N-terminal kinase (JNK)/activating transcription factor-2 (ATF2) signaling pathway, thereby promoting tumor invasion and metastasis [107]. In summary, glycolysis can affect EMT and promote tumor cell invasion and migration, suggesting that it is possible to delay tumor progression by antagonizing glycolytic enzymes, which provides a new idea for the therapy of malignant tumors in the clinic.

3.3.1 Inhibitors of GLUT1

High expression of GLUT1 provides tumor cells with more glucose to participate in glycolysis, thereby promoting tumor progression [151]. Currently, a series of GLUT1 inhibitors have been identified, such as cytochalasin B, CG-5, BAY-876, STF-31, and WZB117. Cytochalasin B, a cell-permeable fungal toxin, reduced glucose uptake and inhibited proliferation of breast cancer by inhibiting GLUT1 [152]. In addition, STF-31 was found to inhibit the growth of renal cancer cells by directly binding to GLUT1 and reducing glucose uptake [153]. WZB117 inhibited glucose transport by binding to the glucose-binding site of GLUT1, thereby inhibiting glycolytic function in tumor cells and inducing cell cycle arrest [154-156]. Furthermore, WZB117 could be used in combination with cisplatin, paclitaxel and other anti-tumor drugs for the treatment of lung cancer and breast cancer [154]. BAY-876 could inhibit the activity of GLUT1 by targeting it for the treatment of tumors, which has been demonstrated in a variety of tumors such as breast cancer [157] and ovarian cancer [158]. In recent years, a variety of GLUT1 inhibitors have been discovered, such as rapaglutin A [159, 160], EF24 [161], quinazolines [162], and phenylalanine amides [163]. Additionally, some natural products including trehalose [164], melatonin [165], bezielle [166], resveratrol [167], and caffeine [168] have been found to inhibit GLUT1. It was found that natural product compounds generally had a better safety and less toxicity than synthetic compounds in inhibiting tumor progression [169]. The above studies have shown that targeting GLUT might be an effective strategy for the treatment of tumors.

3.3.2 Inhibitors of HK2

As HK2 has been discovered to be upregulated in tumor cells and is prominent in promoting tumor development [6, 170], targeting HK2 can safely and effectively inhibit tumor cell growth [171]. Now, many small molecule inhibitors have been extensively identified for the purpose of inhibiting HK. Preclinical studies showed that the glucose analogue 2-DG inhibited glycolysis in tumor cells by competitively inhibiting HK2 [172]. The anti-tumor effects of 2-DG can be enhanced when combined with other drugs such as metformin [173], sorafenib [174], and 2-aminophenoxazine-3-one (Phx-3) [175] in the treatment of HCC. As 3-bromopyruvate (3-BrPA), a brominated derivative of pyruvate, directly inhibits the activity of HK2 and displays a strong glycolytic inhibitory effect [176], Rai et al. [177] discovered that the sensitivity of leukemic cells to daunorubicin was heightened by 3-BrPA through the inhibition of HK2 efficiency. Lonidamine (LN) is an indazole derivative targeting glycolytic enzyme HK2, which has undergone clinical trials for the treatment of a variety of tumors, like breast [178] and ovarian cancers [179]. In studies involving the treatment of ovarian cancers, prostate, and breast, the combination of LN with doxorubicin was found to exhibit enhanced efficacy [180, 181]. Moreover, metformin, the commonly prescribed drug for managing type 2 diabetes was found to inhibit the activity of HK1 and HK2, and reduce glycolytic function, ultimately attenuating cell proliferation in lung cancer [182]. In recent years, many flavonoid derivatives including amentofavone (AF) [183], GL-V9 [183], Gen-27 [20] and natural compounds such as shikonin [184] and astragalin [185] have been discovered to inhibit HK2 and exert anti-tumor effects in multiple solid tumors. In addition, numerous natural compounds also exhibited HK2 inhibitory effect, such as arsenic trioxide (ATO), the main active ingredient of arsenic, which had the capability to reduce the proliferation of gastric cancer cells through down-regulating HK2 expression [186]. Furthermore, the combination with vitamin C might enhance sensitivity of osteosarcoma cells to ATO by strengthening the inhibition of glycolysis [187]. Additionally, an in vivo mouse experiment showed that metformin attenuated the toxic effects of ATO on the liver [188]. However, the inhibitory effect of ATO in combination with metformin on HK2 has not been investigated, which requires further exploration. By inhibiting the expression of HK2, resveratrol induced apoptosis in HCC cells [189]. In summary, various HK2 inhibitors have been demonstrated to serve as effective antitumor drugs in preclinical studies, and accelerated clinical studies are expected to promote the application of HK2 inhibitors in the early treatment of tumor patients.

3.3.3 Inhibitors of PKM2

Several novel inhibitors targeting PKM2 have been discovered, including shikonin, metformin, and vitamin K (VK). Among them, the most specific and effective PKM2 inhibitor is shikonin, and its analogue alkannin has potential therapeutic value by targeting PKM2 [190]. A previous study found that shikonin could down-regulate PKM2 expression through the AKT/mTOR/SREBP-1c signaling pathway, thus inhibiting bladder cancer cell growth [191]. Besides, metformin can enhance osteosarcoma stem cell responsiveness to cisplatin by down-regulating PKM2 expression, thereby exerting an anti-tumor effect [192]. In addition, metformin reduced kidney cancer cell intrusion and metastasis by activating AMPK and down-regulating PKM2 [193]. Moreover, VK is a lipid-soluble substance, with VK3 and VK5 exhibiting more potent inhibitory effect on PKM2 compared to PKM1 [194]. Chen et al. [195] demonstrated that VK3 and VK5 could reduce glycolysis by targeting PKM2, which inhibited the proliferation of cervical cancer cells. Furthermore, C3k [196], tannic acid (TA) [197], and some natural compounds, such as MC-4 [198], proanthocyanidin B2 (PB2) [199], and wogonin [200] were also demonstrated to display the inhibitory effect on PKM2. Altogether, a variety of PKM2 inhibitors can restrict the reproduction, invasion and spread of tumor cells, thereby delaying the progression of tumor and prolonging patient survival.

3.3.4 Inhibitors of LDHA

As LDHA is one of the important tumor biomarkers and lactate (produced from pyruvate catalyzed by LDH) significantly contributes to promoting tumor proliferation and migration, the development of drugs targeting LDHA is a promising anti-tumor strategy. Oxamate impeded the proliferation and metastasis of tumor cells by competitively inhibiting LDHA activity in leukemia [201] and prostate cancer [43]. Besides, oxamate increased the drug responsiveness of glioblastoma (GBM) cells to temozolomide [202]. Moreover, it was found that FX11, a small molecule inhibitor of LDHA, inhibited lactate production by targeting LDHA, thereby restraining the progression of pancreatic cancer and lymphoma [203]. Galloflavin inhibited breast cancer cell proliferation by blocking glycolysis and reduced ATP production through targeting LDHA [204]. In addition, gossypol, a natural compound targeting LDHA, has exhibited anti-tumor effects in diverse types of tumors, for example breast cancer [205], glioma [206] and adrenal cancer [206], but the non-specific toxicity of gossypol limits its clinical application [207].

3.3.5 Inhibitors of PFKFB3

Many inhibitors targeting PFKFB3 have been discovered, such as 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) [24, 208], 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) [209], and PFK158 [210]. 3PO was found to rapidly decrease glucose absorption in leukemic cells, thereby reducing the production of lactate and ATP, which caused leukemia proliferation inhibition [208]. PFK15, a derivative of 3PO, has higher PFKFB3 inhibitory activity, which was found to improve the efficacy of imatinib in chronic granulocytic leukemia [211], and it could be used in combination with oxaliplatin for better treatment in CRC [120]. In addition, benzoindoles [212], BrAcNHEtOP [213], and YZ9 [213], and other compounds have been identified to hinder PFK function, and delay the progression of tumor cells.

3.3.6 Inhibitors of glycolytic non-rate-limiting enzymes

Glycolytic non-rate-limiting enzymes also contribute significantly to tumor progression, so it is necessary to explore their inhibitors for tumor treatment. TDZD-8, specifically targeting ALDOA, inhibited the glycolytic function in cancer cells and acted as an anti-breast cancer agent [214]. Raltegravir prolonged the patient's survival by inhibiting the metastasis of lung cancer cells through targeting ALDOA [215]. PGK1 inhibitors and their molecular mechanisms in tumors have not been elucidated [216], and the function of its potential suppressors, like CBR-470-1, terazosin, and bisphosphonates, in tumor therapy needs to be further explored [217]. MJE3 inhibited breast cancer cell growth by targeting phosphoglycerate mutase 1 (PGAM1), which is one of the isoenzymes of the glycolytic enzyme PGAM [218]. Furthermore, phosphonoacetohydroxamicacid (PhAH) was found to decrease ENO's enzymatic function, thus inhibiting the growth of pancreatic cancer and GBM [219, 220].

Despite the current widespread development of anti-tumor drugs, the incidence of tumor drug resistance has increased, leading to poor therapeutic efficacy in tumor patients. Therefore, it is an urgent problem to diminish the resistance to anti-tumor drugs, and many studies have shown that metabolic reprogramming may be related to the chemoresistance of tumors [221]. Several studies illustrated that enhanced glycolysis was associated with insensitivity to sorafenib in HCC cells [88, 199]. The upregulation of HK2 in breast cancer cells could cause tumor cell resistance to paclitaxel [222] and tamoxifen [223]. Moreover, PKM2 overexpression decreased the sensitivity of acute promyelocytic leukemia cells to RA, whereas inhibiting PKM2 attenuated RA resistance in APL cells [224], indicating that PKM2 could serve as a target to ameliorate drug resistance in leukemia. In tumors with cisplatin resistance, such as cervical, colorectal, and advanced bladder cancers, shikonin reduced the resistance to platinum by inhibiting PKM2 activity [190]. In addition, the overexpression of PFKFB3 has been linked to resistance to sorafenib in HCC [88] patients and to the tyrosine kinase inhibitor imatinib in chronic myeloid leukemia (CML) patients [211]. The above studies demonstrated that overexpressing glycolytic enzymes can lead to tumor cell resistance to antitumor drugs, ultimately resulting in poor therapeutic outcomes and even reduced survival time in tumor patients. Furthermore, some of the glycolysis inhibitors do not specifically target a particular glycolytic enzyme, which is not conducive to further drug discovery and clinical drug selection. Therefore, it is urgent to explore more effective, safe and specific inhibitors of glycolytic enzymes to improve the therapeutic efficiency of tumors.