Lonidamine

Targeting glucose metabolism to suppress cancer progression:prospective of anti-glycolytic cancer therapy

Ali F. Abdel-Wahab, Waheed Mahmoud, Randa M. Al-Harizy

PII: S1043-6618(19)31673-1
DOI: https://doi.org/10.1016/j.phrs.2019.104511
Reference: YPHRS 104511

To appear in: Pharmacological Research

Received Date: 11 August 2019
Revised Date: 19 October 2019
Accepted Date: 23 October 2019

Please cite this article as: Abdel-Wahab AF, Mahmoud W, Al-Harizy RM, Targeting glucose metabolism to suppress cancer progression: prospective of anti-glycolytic cancer therapy, Pharmacological Research (2019), doi: https://doi.org/10.1016/j.phrs.2019.104511

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© 2019 Published by Elsevier.

Targeting glucose metabolism to suppress cancer progression: prospective of anti-glycolytic cancer therapy

Ali F. Abdel-Wahaba,b,⁎, Waheed Mahmoudc, Randa M. Al-Harizyd,e

aDepartments of Clinical Pharmacology, Faculty of Medicine, Cairo University, Egypt
bDepartments of Pharmacology and Toxicology, Faculty of Medicine, Umm Al-Qura University, Saudi Arabia
cDepartments of Medical Biochemistry, Faculty of Medicine, Cairo University, Egypt
dDepartments of Internal Medicine, Faculty of Medicine, Cairo University, Egypt
eDepartments of Internal Medicine, Ibn Sina National College for Medical Sciences, Saudi Arabia

Corresponding author:

Ali F. Abdel-Wahab, MD
Department of Pharmacology, Faculty of Medicine, Cairo University, Cairo, Egypt; and Department of Pharmacology, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia

E-mail: [email protected] [email protected]

Tel: 00966548085598
00201024035540
0020225329227
Fax: 0096625352834

Graphical Abstract:

Highlights:

⦁ Cancer is the second major cause of mortality worldwide despite current chemotherapy
⦁ Targeting aerobic glycolysis has become a research focus for developing anticancer drugs
⦁ These antiglycolytic agents might also sensitize tumor cells to other cytotoxic therapies
⦁ Although preclinical studies are promising, this antiglycolytic therapy has not yet been successfully translated into clinical practice
⦁ This review presents the current state of this emerging glycolytic inhibitors discussing the challenges and the opportunities for this antiglycolytic cancer therapy

Abstract
Most solid tumor cells adapt to their heterogeneous microenvironment by depending largely on aerobic glycolysis for energy production, a phenomenon called the Warburg effect, which is a hallmark of cancer. The altered energy metabolism not only provides cancer cell with ATP for cellular energy, but also generate essential metabolic intermediates that play a pivotal role in the biosynthesis of macromolecules, to support cell proliferation, invasiveness, and chemoresistance. The cellular metabolic reprogramming in cancer is regulated by several oncogenic proteins and tumor suppressors such as hypoxia-inducible factor (HIF-1), Myc, p53, and PI3K/Akt/mTOR pathway. A better understanding of the mechanisms involved in the regulation of aerobic glycolysis can help in developing glycolytic inhibitors as anticancer agents. These metabolic antiglycolytic agents could be more effective if used in drug combinations to combat cancer.

Several preclinical and early clinical studies have shown the effectiveness of targeting the glycolytic pathway as a therapeutic approach to suppress cancer progression. This review aimed to present the most recent data on the emerging drug candidate targeting enzymes and intermediates involved in glucose metabolism to provide therapeutic opportunities and challenges for antiglycolytic cancer therapy.

Chemical compounds:
Chemical compounds studied in this article;
3-Bromopyruvate (PubChem CID: 70684); Dichloroacetate (PubChem CID: 517326); Fasentin (PubChem CID: 879520); Koningic acid (PubChem CID: 124361); Lonidamine (PubChem CID: 39562); Metformin (PubChem CID: 4091); Omeprazole (PubChem CID:4549); Oxamate (PubChem CID: 974); Phloretin (PubChem CID: 4788); Rapamycin (PubChem CID: 5284616).

Abbreviations:
3-BrPA, 3-bromopyruvate; 2-DG, 2-deoxy-D-glucose; 3PO, 3-(3-pyridinyl)-1-(4-pyridinyl)-2- propen-1-one; ALDH, aldehyde dehydrogenase; AMP, adenosine monophosphate; AMPK, AMP- activated protein kinase; ATP, adenosine triphosphate; BET, bromodomain and extra-terminal motif; β-CD, β-cyclodextrin; ß-CD–3BrPA, micro-encapsulated formulation of 3BrPA; CAFs, cancer associated fibroblasts; DCA, dichloroacetate; DNA, deoxyribonucleic acid; EGF, endothelial growth factor; EGFR, EGF receptor; EMT, epithelial-mesenchymal transition; ENO1, enolase 1; ERRα: estrogen-related receptor alpha; ETC, electron transport chain; GBM, glioblastoma multiforme; Glo1, glyoxalase1; FDG, 18fluoro-2-deoxy-D-glucose; F-2,6-BP, fructose-2,6-bisphosphate; GAPDH, glycerladehyde-3-phosphate dehydrogenase; G-6-P, glucose- 6-phosphate; GLUTs, glucose transporters; GPx, glutathione peroxidase; GSH, glutathione; H2O2, hydrogen peroxide; HCC; hepatocellular carcinoma; HF, halofuginone; HK2, hexokinase 2; HIF-1 α, hypoxia-inducible factor-1 alpha; LDHA, lactate dehydrogenase A; LN, lonidamine; MCT, monocarboxylate transporters; mtDNA, mitochondrial DNA; mTOR, mammalian target of rapamycin; mTORC1, mTOR kinase1; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; OxPhos, oxidative phosphorylation; OA, oleanolic acid; PDAC, pancreatic ductal adenocarcinoma; PDH, pyruvate dehydrogenase, PDK1, pyruvate

dehydrogenase kinase1; PEL, primary effusion lymphoma; PEP, phosphoenolpyruvate; PET, positron emission tomography; PFK1, phosphofructokinase1; PFKFB, 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatases; PFK15, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PHD, prolylhydroxylase; PHGDH, phosphoglycerate dehydrogenase; PI3K, phosphoinositide 3-kinase; PK, pyruvate kinase; PK-M2, pyruvate kinase M2; PPP, pentose phosphate pathway; RCC, renal cell carcinoma; ROS, reactive oxygen species; SGLT1, sodium-glucose linked transporter 1, siRNA, small interfering RNA; TCA, tricarboxylic acid; TKTL1, transketolase1; TNBC, triple negative breast cancer, VEGF, vascular endothelial growth factor.

Keywords: Aerobic glycolysis; antiglycolytic agents; cancer therapy; Warburg effect

⦁ Introduction
Cancer incidence is rapidly growing with an expected increase of 70% over the next two decades. Cancer is ranked as a leading cause of death, accounting for 13% of all mortality worldwide (GLOBOCAN 2018 database) [1]. Metabolism of cancer cells is different from that of normal cells, which allows them to sustain a high rate of proliferation and resist signals of apoptosis [2]. Normally, cells utilize multiple metabolic pathways to produce energy depending on the availability of metabolites and biosynthetic requirements for cellular function. Cells typically use glycolysis to convert glucose into pyruvate in the cytosol. The pyruvate is further metabolized in the mitochondria by oxidative phosphorylation (OxPhos) through the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), to produce the energy-storing adenosine triphosphate (ATP). Under hypoxic conditions, cells can utilize anaerobic glycolysis, and converts pyruvate into lactate, producing much less amount of ATP, but at a faster rate [3]. Mitochondrial oxidation of one glucose molecule yields 36 molecules of ATP, while its metabolism to lactate by glycolysis produces only 2 ATP molecules. Under aerobic conditions, cells can also utilize fatty-acid oxidation (called beta-oxidation) or glutamine oxidation, if these metabolites are available [3].
Tumor cells, unlike normal cells, depend largely on glycolysis for producing energy even in the presence of adequate levels of oxygen, a process termed aerobic glycolysis [4]. Thus, inhibition of glycolytic pathways has the potential to provide an effective approach to cancer research aiming to develop new targeted anticancer agents. This approach has been proven effective in suppressing

tumor progression, and several of these glycolytic inhibitors are currently under investigation in preclinical and clinical studies with promising results [5-7]. This review will present the most recent data on the emerging candidate agents targeting glycolytic enzymes and intermediates to be useful in cancer therapy.

⦁ Tumor glucose metabolism and Warburg effect
Cancer progression involves an inappropriate proliferation of cells, which have enhanced abilities for energy production to resist metabolic stresses [8]. Tumor cell metabolism is reprogrammed in favor of aerobic glycolysis despite the presence of plentiful oxygen [9]. This observation was first reported many decades ago by the German scientist Otto Warburg and is thus referred to as the “Warburg effect” [10]. This metabolic alteration to a high glycolysis rate has been observed in a variety of malignant tumors using positron emission tomography (PET) [11]. Warburg first hypothesized that the high rate of glycolysis is a result of mitochondrial injury and this can convert differentiated cells into proliferating malignant cells [10]. However, this primary defect in mitochondrial function is not supported by later studies in cancer [12], and many observations have suggested that mitochondrial OxPhos is the main source of ATP in most cancer tissues [13]. The switch in the metabolism of some cancer cells appears to happen because of the altered conversion of phosphoenolpyruvate to pyruvate, which is catalyzed by the enzyme pyruvate kinase M2 overexpressed in cancer cells [14]. The generation of pyruvate through this unique enzymatic mechanism in cancer cells, is uncoupled with ATP production but is converted primarily into lactic acid, rather than acetyl-CoA to enter the TCA cycle [7].
Cancer cells adapt to the low energy yield of glycolysis by increasing uptake of glucose to support a higher glycolytic rate. This increased glucose uptake has been exploited clinically in diagnosis and follows up of cancer via the use of 18Fluoro-2-deoxy-D-glucose (FDG), a radiolabeled glucose analog, in PET [11,15]. It is likely that, the high rate of glycolysis benefit cancer cells by providing a high rate of ATP production, in addition to providing many intermediates, that are used in subsidiary metabolic pathways for de novo synthesis of nucleotides, amino acids, lipids and NADPH, that are required for rapid cell proliferation [16]. Besides, recent evidence indicated a critical role for mitochondria in cancer cell metabolism [17]. Inhibition of mitochondrial OxPhos has been shown to inhibit tumor invasion in hepatocellular and breast cancer [18], and to reduce the multidrug resistance of melanoma cells [19]. Indeed, it has been observed that mitochondrial

OxPhos and glutaminolysis contributes to cancer progression and metastasis [20]. However, the presence of aerobic glycolysis under the normoxic condition and functionally efficient mitochondria is a very interesting fingerprint of cancer cells. Thus, the use of tumor glycolysis as a potential target for cancer therapy is the most intriguing.

⦁ Dual metabolic phenotype and hybrid state
The glycolytic phenotype is known to be expressed by many cancers [21], but the dependence of cancer cells on such phenotype remains unclear. It has been demonstrated cancer cell death can be induced by reversing the glycolytic state to OxPhos [22]. The glycolytic cancer cells can exhibit a non-glycolytic phenotype under acidic conditions by intracellular lactic acid accumulation. Lactic acidosis is a common consequence of the Warburg effect in most solid tumors [23]. Tumor cells cultured with sufficient glucose are initially glycolytic and then lactate generation leads to acidification of the medium. The lowered cellular pH decreases glycolytic flux and inhibits the activities of glycolytic enzymes, leading to the suppression of glycolysis. The limited supply of glucose can lead to the rapid death of tumor cells, but under lactate acidosis, the cells switch from Warburg effect to a non-glycolytic oxidative phenotype and metabolize glucose at a slower rate to support cell survival [24]. During hypoxia or mitochondrial dysfunction, the cells can switch energy metabolism from mitochondrial respiration to glycolysis, thus maintaining cancer growth [25]. It has been demonstrated that breast cancer cells can resist radiation injury by shifting from glycolytic phenotype to OxPhos, thus producing more ATP to enhance survival [26].
In addition to the concept of a central role of the Warburg effect in cancer metabolism, some studies suggested the concept of dual metabolic nature or hybrid state (Warburg effect and oxidative phenotype) in cancer cells under the stressful microenvironment [27]. It is proposed that normal cells can exhibit either a glycolytic or oxidative state, but tumor cells can have a hybrid metabolism, where both states coexist, enhanced by increased reactive oxygen species (ROS) and/or activation of oncogenes such as RAS, MYC, and c-SRC [28,29]. The hybrid metabolic state, with coexisting oxidative respiration and glycolysis, has been demonstrated in some aggressive tumor cell lines, such as SiHa and HeLa, due to strong activation of hypoxia-inducible factor-1 (HIF-1) from lactate accumulation [30]. The hybrid state is also verified by in vitro experiments with aggressive triple-negative breast cancer (TNBC) cells [31]. These data support the hypothesis of a hybrid (glycolytic and oxidative) model and its critical role in tumorigenesis.

The hybrid state model could explain the phenomenon of oxygen shock [32] when glycolytic cells reach the blood vessel and exposed to a large amount of oxygen. These cells could switch to the hybrid metabolic phenotype, which further induces metastasis [33]. Moreover, the hybrid phenotype enhances the metabolic plasticity and enables malignant cells to switch their phenotypes between glycolysis and OxPhos. This metabolic plasticity facilitates cancer cell adaptation to the various tumor microenvironment, such as hypoxia and acidic conditions, enhancing invasion and metastasis [34]. Therefore, targeting the hybrid state to eliminate metabolic plasticity could be a new therapeutic strategy against cancer progression.
Recent evidence has demonstrated that tumor cells can acquire a hybrid metabolic state, thereby using both glycolysis and OxPhos for the production of energy and formation of the required macromolecules [35]. The hybrid glycolysis/OxPhos phenotype enhances the metabolic plasticity of tumor cells supporting cancer invasion, metastasis, and chemoresistance [20]. Also, it is noted that the metabolic phenotype is heterogeneous in different tumors, even in the same type of tumor [36]. Detection of the metabolic plasticity of cancer could be helpful in targeting specific metabolic pathways to prevent the hybrid state and suppress tumor growth. Moreover, metastasis of cancer cells is facilitated by epithelial-mesenchymal transition (EMT), where epithelial tumor cells lose adhesion and acquire the mesenchymal characteristics of migration and invasiveness [37]. Rapid growth and expansion of solid tumors usually outpace angiogenesis leading to a progressive hypoxic condition, inducing HIF-1α, which induces both EMT and aerobic glycolysis [38]. This hybrid glycolysis/OxPhos metabolic state indicates that dual blockade of both metabolic processes, glycolysis and OxPhos, could be more effective in suppressing tumor and preventing metabolic plasticity [39]. This combination treatment could drive tumor cells away from the hybrid phenotype leading to more effective anticancer response [36].

⦁ Reverse Warburg effect or metabolic coupling
Another type of tumor metabolism has been recently observed in certain types of cancers called the “reverse Warburg effect” or “metabolic coupling” which have high mitochondrial respiration and low glycolysis rate [40]. In this type, the tumor cells and adjacent stromal fibroblasts form a two-compartment model of cancer metabolism, where aerobic glycolysis occurs in fibroblasts, and the generated metabolites are transferred to malignant cells, to fuel the TCA cycle and maintain ATP generation [41]. This metabolic coupling reveals a parasite-host relationship between tumor

cells and the cancer-associated fibroblasts, as observed in some forms of breast cancer [42]. The two-compartment model of metabolism may contribute to certain forms of drug resistance and therapeutic failure in some types of cancers [43]. It has been demonstrated that tamoxifen-sensitive MCF7 cancer cells become tamoxifen-resistant when co-cultured with fibroblasts. The drug combination of tamoxifen and the tyrosine kinase inhibitor dasatinib has been shown to decrease this fibroblast-induced tamoxifen-resistance and reprogram tumor cells to the glycolytic phenotype, leading to the death of MCF7 cancer cells [44]. However, although recent advances have shed more light on the biological significance of tumor metabolism, the underlying mechanistic details of regulation and consequences of such metabolic types remain unclear.

⦁ Warburg effect and tumor acidosis
The microenvironment of solid tumors is markedly heterogenous, so tumor cells tend to increase their uptake of glucose to maintain energy production [13]. Increased glycolysis and decreased mitochondrial oxidation lead to increased formation of lactic acid, increased glutaminolysis, increased beta-oxidation of fatty acids and activation of the pentose phosphate pathway [45]. To maintain the intracellular pH (pHi), tumor cells promptly export the excess intracellular acid load to the extracellular compartment. The rapid turnover of cancer cells, glucose fermentation, and hypoxia, all can result in the generation and release of excessive amounts of protons into the extracellular space [46]. In contrast to normal cells, cancer cells show a reversed pH gradient with increased pHi and decreased extracellular pH (pHe) [47]. To survive in this hostile microenvironment, cancer cells develop prompt ATP-requiring mechanisms to extrude protons. These mechanisms include the vacuolar H+-ATPase, Na+/H+ exchanger, Na-bicarbonate cotransport via carbonic anhydrase (CA) 9 or 12, and monocarboxylate transporters (MCTs) [46]. The alkaline pHi and acidic pHe, enhance metabolic adaptation, proliferation, invasiveness and metastatic behavior of tumor cells [48]. Moreover, many chemotherapeutic drugs are weak bases that are trapped and neutralized in the acidic microenvironment of tumors contributing to chemoresistance. It has been demonstrated that restoration of mitochondrial function could suppress tumor growth and restore chemosensitivity [49].

⦁ Tumor glycolysis and its clinical relevance to cancer progression

The glycolysis process takes place in the cytoplasm by converting glucose into pyruvate through nine reaction steps, involving several glycolytic enzymes. First, glucose is transported into tumor cells at a high rate by glucose transporters (GLUTs), GLUT1 and sodium-glucose linked transporter 1 (SGLT1) which are overexpressed in most cancers [50]. Glucose is phosphorylated into glucose-6-phosphate by hexokinase (HK), a rate-limiting step that provides direct feedback inhibition to preserve energy. The HK is bound to the mitochondrial membrane and has a high affinity for glucose, facilitating initiation of glycolysis with low glucose levels [51]. There are four HK isoforms (HK 1-4), HK1 is ubiquitously expressed, whereas HK2 is expressed in insulin- sensitive adipose tissue and muscles [52] and is overexpressed in many cancer cells [53]. Glucose- 6-phosphate isomerase then converts glucose-6-phosphate into fructose-6-phosphate, which is further phosphorylated to form fructose-1,6-biphosphate and fructose-2,6-biphosphate, under the effect of phosphofructokinase-1 (PFK1) and PFK2, respectively, and consuming one ATP molecule. The PFK1 is a crucial driver of glycolysis and is inhibited by high ATP levels. The PFK2 is overexpressed in tumor cells, generating excess fructose-2,6-bisphosphate, which activates PFK1, leading to maintenance of high glycolytic rate irrespective of ATP level [54]. Next, fructose-1,6-bisphosphate is transformed by aldolase enzyme into glyceraldehyde-3- phosphate and dihydroxyacetone phosphate. Glyceraldehyde-3-phosphate is transformed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into glycerate-1,3-diphosphate, which is further transformed by phosphoglycerate kinase (PGK) into 3-phosphoglycerate and producing two ATP molecules. The phosphoglycerate mutase (PGM) isomerizes 3-phosphoglycerate into 2- phosphoglycerate, followed by the formation of phosphoenolpyruvate (PEP). Lastly, PEP is converted into pyruvate by pyruvate kinase (PK), with the generation of one ATP molecule. The PK catalyzes a rate-limiting step of glycolysis and its activity is affected by the cellular pH and ATP/AMP ratio [55]. The PK exists in four isoforms; PKL, PKR, PKM1, and PKM2 [56]. The PKM2 is overexpressed in tumor cells and tumor-associated fibroblasts [57]. Also, PKM2 can be translocated into the nucleus, acting as a protein kinase to regulate gene transcription [58]. Unlike PKM2, PKM1 seems to have little effect on cell proliferation [59]. The ratio of PKM1/PKM2 has been shown to decrease during malignant progression [60] and switching PKM2 to PKM1 can have therapeutic implications. Replacing PKM2 with PKM1 has been demonstrated to inhibit glycolysis in lung cancer cells and suppress tumor xenograft formation in nude mice [14,61]. The pyruvate resulting from glycolysis can be converted into acetyl-CoA to enter the TCA cycle for

OxPhos, but during hypoxia, pyruvate is transformed into lactic acid by lactate dehydrogenase (LDH). The LDH is overexpressed in tumor cells thus maintaining high glycolytic flux [62].
Although glycolysis yields less amount of ATP (18 times lower) compared to mitochondrial oxidation, it can provide many benefits for cancer cells competing for shared energy sources [63]. The accelerated rate of glycolysis leads to faster and greater ATP production, which reaches 100 times faster than oxidative phosphorylation [64]. Besides ATP generation, glycolysis provides tumor cells with metabolic intermediates and precursors that fuel pathways for biosynthesis of macromolecules required for cell proliferation and tumor progression [65]. The accumulated glycolytic intermediates enhance the pentose phosphate pathway (PPP) with the formation of NADPH and ribose-5-phosphate, which are needed for the synthesis of phospholipids and nucleic acids. Also, NADPH enables cancer cells to maintain supplies of the antioxidant glutathione (GSH), required for maintaining the intracellular redox status and protecting cancer cells against the damaging effects of chemotherapeutic agents [66]. The glycolytic pathway also produces NADH and serine, which can be used in the formation of signaling molecules and important amino acids such as glycine and cysteine [67] (figure 1).
Several lines of evidence have established a link between cancer progression and overexpression of GLUTs and glycolytic enzymes including HK2, GAPDH, LDH, and PFK-2 [68,69]. A biochemical link has also been demonstrated between cancer glycolysis and resistance to chemotherapy and radiotherapy [70,71]. Moreover, the transketolase1 (TKTL1) enzyme, involved in PPP, plays an important role in cell survival under starvation and stresses [72]. The TKTL1 has been shown to affect cancer cell sensitivity to drugs such as imatinib [73] and cetuximab [74]. Also, the pyruvate dehydrogenase kinase (PDK) isoforms, PDK1 and PDK3, have been found to confer chemotherapeutic resistance in the cervical cancer cell line, HeLa [75]. Similarly, in colon carcinoma cell line, LoVo, drug resistance correlated with increased lactate production by aerobic glycolysis [76]. This key signature of cancer metabolism, providing energy and supporting uninterrupted growth, could be an attractive target for cancer therapy and sensitization to chemotherapeutics [77].
A high lactate level, indicating the prevalence of glycolytic phenotype, correlated significantly with tumor growth, spread, and recurrence [78]. Lactate was originally thought to be an acidifying molecule that can cause a dangerous lowering of intracellular pH if not exported from tumor cells. However, different roles of lactate efflux and influx have been proposed that contribute to cancer

cell survival through some type of “metabolic symbiosis” between normoxic and hypoxic tumor cells co-existing in the heterogeneous microenvironment of most solid tumors [79]. The lactate is produced and exported by hypoxic glycolytic tumor cells, to be imported and utilized by normoxic tumor cells for energy production by mitochondrial OxPhos [80]. This process of lactate transfer is achieved through the monocarboxylate transporters (MCTs), mainly MCT4 for lactate release and MCT1 for lactate uptake, which is overexpressed in most tumors [81]. The MCT1 is abundant in normoxic non-glycolytic cancer cells of vascularized area whereas MCT4 is expressed in hypoxic glycolytic cancer cells. A significant correlation has recently been demonstrated, in breast cancer cell lines (MCF-7 and MDA-MB-231), between the distribution of MCT isoforms (1 and 4) and the expression profile of LDH isoforms (A and B) [82]. In the MDA-MB-231cell line, LDHA is abundant to convert pyruvate into lactate and MCT4 is overexpressed to release lactate. While in the MCF-7 cell line, MCT1 is overexpressed to uptake lactate and LDHB is abundant to convert lactate back into pyruvate to fuel the TCA cycle [83]. Thus, cancer cells organize their glycolytic phenotype to achieve efficient energy supply.

⦁ Non-glycolytic functions of glycolytic enzymes and metabolic intermediates
Many glycolytic enzymes have also important roles in several non-glycolytic processes involved in cellular functions that support cancer cell survival and growth [84]. For instance, the mitochondrial membrane-bound HK2 can antagonize the proapoptotic pathway in cancer cells [85]. Also, HK2 acting as a nuclear enzyme is involved in transcriptional regulation of some nuclear proteins [84]. Similarly, GAPDH has a critical role in maintaining the cellular redox balance by catalyzing the production of NADH, and protection against free radical-induced injury [86]. Also, GAPDH has some nuclear functions contributing to the pro-apoptosis and the oncogenic process by affecting nucleic acid binding properties of hepatitis viruses [87]. Several non-glycolytic functions have been demonstrated for PKM2, including phosphorylation of histone H3 to favor tumorigenesis [88], transactivation of β-catenin [89], and binding phosphotyrosine to interact with other proteins [90].
Results revealed that GAPDH can bind directly to telomeric DNA preventing its degradation by chemotherapy [91]. In prostate cancer, GAPDH has been shown to enhance the transcriptional activity of androgen receptors [92]. The LDH has been found to cooperate with the transcriptional factor Oct-4, and LDH gene silencing leads to the downregulation of Oct-4 and suppression of

gastric tumorigenesis. Also, the nuclear translocation of LDH can affect the functions of DNA polymerases [93]. Moreover, the influx and efflux of lactate achieved through the MCTs, are involved in the regulation of the CD147, a matrix metalloproteinase inducer, that increases cancer cell invasion and metastasis [94]. Also, fructose-1,6 biphosphate has an anti-apoptotic effect in tumor cells by reducing cytochrome C [95]. Besides, pyruvate has been involved in resistance to chemotherapy by over-expression of p-glycoprotein [96]. Therefore, evidence indicates that many glycolytic enzymes and intermediates participate in non-glycolytic processes at various subcellular locations such as mitochondria, nucleus, and cytosol, to support cancer progression.

⦁ Molecular regulation of tumor glycolysis
It is increasingly evident that coordinated networks of signaling pathways regulate reprogramming of cancer cells to balance their metabolic state, supporting tumor growth and stress resistance. Several studies have demonstrated the affection of cancer cell metabolism by many regulators including protooncogenes (e.g. Myc), transcription factors (e.g. HIF-1), signaling pathways (e.g. PI3K/Akt/mTOR), and tumor suppressors (e.g. p53) [97]. The c-Myc is a transcription factor, encoded by Myc oncogene, that control cellular growth, and metabolism, and its expression is upregulated in many cancers, such as breast, colon, prostate, and bladder cancers [98]. Experimental studies have demonstrated that overexpression of c-Myc in the liver of transgenic mice can increase the activity of the glycolytic enzymes with the overproduction of lactic acid [99]. Activation of the c-Myc has been found to upregulate many genes of the glycolytic enzyme, such as GLUTs, HK2, PFK, enolase1 (ENO1), LDHA, and pyruvate dehydrogenase kinase1 (PDK1) [62,100]. Also, c-Myc is linked to increased mitochondrial ROS, leading to mitochondrial dysfunction and switching cancer cells to glycolysis for energy production.
The PI3K/Akt pathway contributes to several cellular processes including inflammation, autophagy, and tumorigenesis. The Akt oncogene has been shown to stimulate the metabolism of glucose and production of lactate without increasing oxygen consumption, in glioblastoma and hematopoietic cancer cells [101]. Activation of Akt, the serine/threonine kinase, has been found to mobilize GLUTs and activate HK2, thus enhancing glycolysis and promoting cancer growth [102]. The mammalian Target of Rapamycin (mTOR) kinase exists in two forms, mTORC1 and mTORC2, both are involved in the regulation of metabolism and cell proliferation. The mTORC1, as a downstream effector of PI3K/Akt signaling, can enhance protein translation, lipogenesis, and

glycolysis [103]. Activation of mTORC1 induces GLUT1 and HK2, leading to increased glucose uptake and a high rate of glycolysis [104]. The mTORC2 acts primarily through phosphorylating Akt on serine 473, to enhance the expression of GLUT1, and activate HK2 and PFK-1, increasing glycolysis rate [105]. Also, mTORC2 was shown to upregulate intracellular c-Myc and enhance glycolysis in glioblastoma [106].
Hypoxia-inducible factor-1 (HIF-1) consists of oxygen-labile α subunit and constitutive β subunit, and control gene transcriptions, regulating many processes such as angiogenesis, erythropoiesis, inflammation and energy production [107]. The level of HIF-1α is sensitive to oxygen and is regulated by HIF prolyl hydroxylase (PHD), which enhances its degradation in normoxic conditions [108]. During hypoxia, the HIF-1α protein increases due to lower degradation by PHD [109]. Thus, under hypoxic condition, cancer cell accumulates the HIF-1α, leading to upregulation of GLUTs and glycolytic enzymes, thereby increasing the rate of glycolysis. Studies in human glioblastoma multiforme (GBM) cells demonstrated overexpression of HIF-1α, PDK1 and EGF receptor (EGFR) under hypoxic conditions [110]. Also, HIF-1 activates PDK1 to inhibit the PDH activity and suppress the conversion of pyruvate to acetyl-CoA, thereby impairing mitochondrial function [111]. Furthermore, activation of EGFR was found to enhance nuclear translocation of PKM2 in cells of glioblastoma, breast and prostate cancers, leading to increased expression of cyclin D1 and glycolytic enzymes, thus promoting aerobic glycolysis and cellular proliferation [88,89].
The tumor suppressor p53 can negatively regulate cell growth by inhibiting mTOR via inducing the transcription of several genes. The p53 can inhibit glycolysis by decreasing GLUTs and increasing fructose-2,6-bisphosphatase activity [112]. Also, p53 can enhance mitochondrial oxidation through activation of the SCO2 gene of the respiratory chain [113]. Also, AMP-activated protein kinase (AMPK), the main sensor of cellular energy, is often associated with p53 mutation in human cancers, leading to enhanced aerobic glycolysis [43].

Glucose

GLUTs
Phloretin, fasentin, WZB117, ritonavir

Glucose
HK
2-DG, LN, 3-BrPA,
genistein-27, benserazide

Glucose-6-P
ribose-5-phosphate
NADPH

13

Nucleotide synthesis

G-6-PI

ATP PFK [PFKFB3]

Glyceraldehyde-3-P
Aldolase

NADH GAPDH

Serine
2ATP PGK
PGM

ATP

Figure 1: The glycolysis process with catalytic enzymes, metabolic targets (red) and glycolytic inhibitors (green)

⦁ Targeting tumor metabolism and Glycolytic inhibitors
Recent cancer research focuses on selective inhibition of metabolic pathways to deprive cancer cells of essential metabolic needs and interfere with tumor growth. The improved understanding of aerobic glycolysis as a hallmark of cancer and underlying mechanisms may pave the way for the development of targeted metabolic agents for antiglycolytic cancer therapy [114]. There are several approaches to disrupt energy production and prevent glucose utilization by cancer cells.

Indeed, carbohydrate-restricted diets have been reported to have therapeutic benefits in cancer patients [115]. Targeting glycolytic pathways to achieve cancer treatment seems appealing since the involved enzymes are attractive molecular targets. However, to be useful targets, these enzymes must have a significant difference in the activity or expression between cancer cells and normally proliferating cells.
Currently, several agents inhibiting glycolysis are under intensive investigation in preclinical and clinical studies exploiting the glycolytic activity of tumor cells [116]. These antiglycolytic agents can target glucose transporters (e.g. phloretin, WZB117) or glycolytic enzymes such as HK2 (e.g. 2-deoxy-D-glucose, lonidamine), GAPDH (e.g. 3-bromopyruvate, koningic acid), LDH-A (e.g. oxamate) or PDK (e.g. dichloroacetate) (Table 1). Although preliminary results of tumor growth inhibition are promising, there are concerns of significant toxicity related to the wide expression of these target enzymes in normally proliferating cells [117]. This review will present the available preclinical and clinical results with recent drug candidates to provide a future perspective of therapeutic opportunities for antiglycolytic cancer therapy.

9.1- Targeting glucose uptake (GLUTs inhibitors)
A direct antiglycolytic approach would be to block the uptake of glucose in malignant cells via GLUTs, leading to a total disruption of energy production pathways. Several small molecules can selectively inhibit GLUTs including phloretin, fasentin, genistein, ritonavir, STF-31, WZB117, and cytochalasin B. These agents have demonstrated anticancer effects in preclinical models by inhibiting glucose uptake, thus leading to cell death through glucose and energy deprivation [118]. The polyphenol phloretin was recently shown to inhibit GLUT2 in TNBC leading to suppression of tumor growth and metastasis [119]. Also, phloretin can inhibit GLUT1 that is overexpressed in the hypoxic area of resistant colon cancer cell lines and induce apoptosis by activating p53- mediated signaling, leading to suppression of growth in resistant cancer cells [120]. Fasentin and its analogs have shown to inhibit glucose uptake and decrease the resistance of caspase activation, which is involved in chemoresistance of tumor cells [121]. Other studies have demonstrated that STF-31 is a selective GLUT1 inhibiting agent that can kill the VHL-deficient renal cell carcinoma (RCC), suppressing tumor growth with lower toxicity to normal cells [122]. The WZB117 is a bis- hydroxybenzoate compound that produces fast selective irreversible blocking of glucose uptake by GLUT1, leading to inhibition of tumor glycolysis and reduction of cellular ATP levels with the

arrest of cell-cycle [123]. This drug also produced a synergistic effect when combined with other anticancer drugs, such as paclitaxel or cisplatin, against lung and breast cancer cell lines in vitro and in vivo [118]. Moreover, the HIV-protease inhibitor, ritonavir was shown to inhibit GLUT4 leading to suppression of multiple myeloma cells, with synergistic effects with other drugs as metformin [124]. However, since GLUTs are expressed ubiquitously in all cells, selective inhibition of cancer glucose uptake is an important challenge. For instance, several glucose transport inhibitors, tested in phase I clinical trials for hepatocellular and prostate cancer, are associated with significant side effects [125].

9.2- Targeting glucose phosphorylation (HK inhibitors)
Another antiglycolytic approach is to target the HK enzymes responsible for the first, rate-limiting step of glycolysis. The HK2 is overexpressed in many cancer cells, and its inhibition produced effective anticancer activity in preclinical studies [53]. Several HK inhibitors have been exploited for the anticancer effect such as 2-deoxy-D-glucose (2-DG), lonidamine, 3-Bromopyruvate (3- BrPA), genistein-27 (GEN-27) and benserazide. Many natural products such as resveratrol, astragalin, chrysin, have also been shown to inhibit HK2, suppressing growth and inducing apoptosis in hepatocellular carcinoma cells [126-128]. The genistein derivative, GEN-27, is a synthetic flavonoid that was shown to suppress breast cancer cells via inhibition of HK2 and induction of cell apoptosis [129]. Also, benserazide was found to specifically inhibit HK2 and induce apoptosis in colorectal cancer cells, suppressing tumor growth in xenograft cancer model [130].
9.2.1- 2-deoxy-D-glucose (2-DG)
The glucose analog 2-deoxy-D-glucose (2-DG) is one of the well-studied antiglycolytic agents, with promising anticancer effects in preclinical models [131]. The 2-DG enters the cell via GLUTs competitively inhibiting glucose uptake. Inside the cell, 2-DG competes with glucose-6-phosphate interfering with HK activity. 2-DG is phosphorylated to 2-DG-P which is an end substance that cannot be metabolized and accumulate intracellularly, thereby blocking the glycolysis process [132]. Subsequently, therapeutic doses of 2-DG caused ATP depletion and oxidative stress, and eventually facilitated cell death [133]. Although 2-DG has been shown to inhibit the proliferation of several cancer cell lines in vitro, animal studies revealed heterogeneous results with 2-DG [134- 136]. So, contrary to its expected anti-cancer effects, 2-DG may enhance some pro-survival

pathways in tumor cells [137]. Also, hypoxic tumor cells may resist the therapeutic effect of 2-DG by overexpression of HIF-1 leading to the upregulation of GLUT and glycolytic enzymes [138]. These disappointing results challenge the use of 2-DG as a single agent for antiglycolytic cancer therapy. Experimental studies demonstrated that 2-DG combined with the PI3K/mTOR inhibitor, PF-04691502, can convert aerobic glycolysis into mitochondrial OxPhos in the cells of primary effusion lymphoma (PEL). This drug combination was cytotoxic to PEL cells with less toxicity to normal lymphocytes [139]. Also, it is suggested that 2-DG can be used with radiotherapy to prevent the recovery of tumor cells from radiation damage, acting as a radiosensitizer [140]. Enhanced killing of malignant cells has also been observed in several preclinical studies using 2- DG in combination with irradiation [141,142].
Despite the promising results of 2-DG in preclinical studies, results of clinical trials are inconsistent. Recently, 2-DG activity has been tested in phase II clinical trial in patients with pancreatic cancer, but the trial was terminated due to the slow accrual [143]. The anticancer activity of 2-DG is tested in two other clinical trials, but there are real concerns about significant inhibition of the glycolytic metabolism in the heart and brain [135,144]. Daily administrations of 2-DG at therapeutic doses were associated with hypoglycemia-like symptoms that can limit its use as a single-agent therapy in vivo [145,146]. The safety of 2-DG is evaluated in phase I dose- escalation trial in prostatic cancer and identified 45 mg/kg as the recommended oral dose, before stoppage due to insignificant effects on tumor growth [147]. Overall, the preliminary results from clinical trials of 2-DG as a monotherapy are inconclusive and ambiguous.
Currently, 2-DG is reintroduced for use in combination approach, as reported in more recent preclinical and clinical studies, using 2-DG at lower doses to produce synergistic anticancer effects with other chemotherapeutic agents [136,148]. These synergistic effects of 2-DG have been observed in combination with cytotoxic agents in the in vitro studies and in phase I clinical trial with docetaxel [149,150]. Also, a combination of 2-DG with cisplatin has been shown enhanced cytotoxicity in head and neck cancer by increasing the ROS levels [136]. A recent phase I clinical trials have shown that the use of 2-DG is well tolerated and has low toxicity [151,152]. An early clinical trial examined the safety and effectiveness of orally administered 2-DG and showed favorable results in patients with cerebral glioma [153]. Another clinical trial in glioblastoma using 2-DG 250 mg/kg, in combination with radiotherapy, showing a favorable toxicity profile, but significant side effects occurred at higher doses in two of six patients [154]. Histological

examination of tumors in patients treated with the recommended dosage schedule of 2-DG revealed extensive necrosis of tumor tissue with preserved surrounding tissue [155]. Similar good tumor control, in addition to improved quality of life, was achieved in patients with brain tumors using 2-DG combination regimens [156]. Moreover, combining 2-DG with metformin produced a synergistic inhibitory effect on energy metabolism with depleting ATP levels in the cancer cell lines [157]. This combination therapy was more effective in the resistant poorly glycolytic pancreatic cancer cells, indicating that metformin sensitizes cancer cells to 2-DG [158]. Thus, combination treatments using 2-DG may have encouraging outcomes providing a new opportunity for cancer combination therapy.
9.2.2- Lonidamine (LN)
Lonidamine (LN) is an indole derivative, was used as an anti-spermatogenic agent, but now recognized with anti-tumor and pro-apoptotic activity [159]. The mechanism of action is not clear, but studies indicated that LN acts as an inhibitor of HK-2 suppressing glycolysis and/or mitochondrial respiration. LN is an ANT ligand inducing mitochondrial channel formation and causing inhibition of complex II and complex I [160]. This leads to decreased mitochondrial uptake of pyruvate and decreased formation of fumarate and malate in treated cells, together with the accumulation of succinate [161]. It is also suggested that LN can inhibit the MCTs responsible for lactate efflux, leading to decreased extracellular lactate [160].
LN is an emerging anticancer agent alone and in combination with other anticancer therapies. This agent has completed pre-clinical studies and entered phase II clinical trials for cancer treatment [162]. Inhibition of glycolytic enzyme HK2, with LN, has also been tested in several types of cancers, such as lung, breast and ovarian cancer [163-166], but elevated toxicity recorded with no significant survival benefit for patients. Also, early clinical testing of this drug showed no benefits for patients and the trial was terminated [167]. Results of combination therapy studies showed that LN combined with other chemotherapeutics, such as doxorubicin, produced better anticancer effects for the treatment of breast, prostate and ovarian tumors [168,169]. LN was found to enhance the apoptotic response to many anticancer agents and γ irradiation both in vivo and in vitro [162]. However, the clinical success of LN has been impaired by significant pancreatic and hepatic toxicities [170]. Therefore, current research focused on developing alternative dosage forms or local targeted delivery of LN to reduce its toxicity.

9.3- Targeting fructose phosphorylation by PFK (PFKFB3 inhibitors)
Another glycolytic enzyme, PFK, that catalyzes the rate-limiting step of fructose phosphorylation, is a potential target for anticancer agents. PFK is allosterically activated by AMP and fructose 2,6- bisphosphate (F-2,6-BP). The PFK activity is liable to feed-back inhibition by excess ATP, but this inhibition can be overcome by the elevated F-2,6-BP level to maintain uninterrupted glycolytic flux in tumor cells [171]. The PFK is also controlled by a family of bi-functional enzymes including PFKFBs, which is overexpressed in many cancers, providing an interesting target for cancer therapy. The PFKFB3 is specifically inhibited by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen- 1-one (3PO), and its derivative, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15). Administration of 3PO was shown to produce a rapid reduction of glucose uptake, lactate production and ATP generation in the cells of Jurkat T-cell leukemia [172]. Also, PFK15 has been reported to exhibit significant anti-tumor activity by reducing 18FDG uptake and F-2,6-BP level in xenografted tumors. Moreover, PFK15 exhibits a pro-apoptotic effect in the transformed tumor cells in vivo and in vitro [173]. Thus, preliminary studies with PFKFB3 inhibitors revealed potentially useful anticancer effects.

9.4- Targeting glucotrioses metabolism (GAPDH inhibitors)
One of the most promising therapeutic approaches in antiglycolytic cancer therapy has been targeting the enzyme GAPDH, which catalyzes the unique reaction for producing energy in the form of NADH with the conversion of 3-phosphoglycerate into serine. The NADH molecule plays a critical role in the regulation of intracellular ROS levels, and the biosynthesis of macromolecules. Recent studies demonstrated that the growth of some subsets of melanoma and breast cancers are dependent on the expression of high levels of PHGDH enzyme to enhance tumorigenesis and chemoresistance [174,175]. Therefore, inhibition of this multifunctional enzyme, GAPDH, would result in multipronged effects within cancer cells, including reduced NADH and ATP production with disturbed cellular redox balance, leading eventually to cell death [176]. Metabolism of the glucotrioses, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate is inhibited, and their partial degradation leads to the formation of methylglyoxal. This cytotoxic metabolite is usually detoxified by the glyoxalase system (Glo 1 and 2). In presence of excess ROS and depletion of GSH, the glyoxalase (Glo1) activity diminishes leading to the accumulation of the toxic

methylglyoxal [177]. Thus, inhibiting GAPDH represents an attractive therapeutic strategy, not only affecting glycolysis but also exploiting other cytotoxic mechanisms in cancer cells.
A reversed pH gradient, manifested by extracellular acidosis and intracellular alkalization, with metabolic reprogramming is a hallmark of cancer metabolism. By integrating the pH-dependent enzyme activities, Persi et al., [178], developed a computational approach to explore how pHi can modulate metabolic adaptation. They showed that, alkaline pHi enhances tumor cell proliferation associated with increased glycolysis (more Warburgness). On the other hand, acidic pHi inhibits tumor growth and prevents metabolic adaptation of cancer cells to be more oxidative (less Warburgness). Their analysis explored GAPDH as a main metabolic modulator of cancer cells and its inhibition at acidic pHi increases the anti-Warburg effect of low pHi on tumor growth [178]. Several GAPDH inhibitors, such as 3-BrPA, arsenate, iodoacetate, together with the natural compound Koningic acid, have been studied in cell cultures and animal models, with evident anticancer efficacy [179, 180].
9.4.1- Koningic acid (KA)
Koningic acid (KA), also called heptelidic acid, is a sesquiterpene metabolite isolated from a fungus, that inhibits GAPDH by covalent binding to its active site [181]. The KA is cytotoxic to highly glycolytic tumor cells due to ATP depletion associated with the progression of glucose phosphorylation. Thus, KA may be effective in disrupting metabolic pathways in aggressively glycolytic tumors with little effect on healthy cells [182]. Furthermore, KA produced a marked toxic effect on tumor cells with minimal systemic toxicity after its intraperitoneal administration [183]. These data reinvigorate the hope to exploit this selective targeting of GAPDH by KA in the treatment of cancer.
9.4.2- 3-Bromopyruvate (3-BrPA)
The pyruvate analog, 3-Bromopyruvate (3-BrPA), is one of the most effective antiglycolytic drugs currently under evaluation. This small molecule analog of pyruvate has demonstrated an interesting ability to inhibit GAPDH and HK enzymes by alkylation of the active site. Administration of 3-BrPA causes profound inhibition of tumor glycolysis and mitochondrial OxPhos, leading to profound depletion of intracellular ATP, depriving cancer cells of energy [184,185]. This alkylating agent can react by covalent binding with thiol groups in cysteine of many targets involved in the glycolytic pathway including mt-HNK, SERCA-1, MCT1, GAPDG, and others [186]. However, the predominant mechanism of action of 3-BrPA is selective inhibition

of GAPDH, which has been demonstrated in vitro in many cancer cell lines and, also in vivo in animal studies [187,188]. Inhibition of GAPDH disrupts the metabolism of glucotriosis leading to marked energy depletion, redox imbalance and eventually cancer cell death [189,190]. The anticancer effects of 3-BrPA included suppression of tumor growth, invasion, metastasis, angiogenesis, together with increased oxidative stress, and regulation of apoptotic pathways [191]. Moreover, 3-BrPA has been shown to enhance the cytotoxic effect and decrease resistance to other anticancer drugs by inhibiting the ATP-dependent MDR transporter, providing a promising candidate in combination therapy [192-194].
Interestingly, 3-BrPA is more stable in the acidic microenvironment of tumors with a longer half- life, thus producing more marked cytotoxic effects in cancer cells without serious organ toxicity [191]. Also, the cellular uptake of 3-BrPA is mediated through MCTs that is upregulated in most cancers, thereby providing a preferential uptake of 3-BrPA in tumor cells. The selective uptake of this potentially toxic agent by cancer cells has recently been demonstrated [195]. However, concern remains about the rather non-specific alkylating properties of 3-BrPA that may be associated with significant toxicity limiting the use of this potent anticancer agent. Thus, many studies have focused on the local regional catheter-based delivery of 3-BrPA for the treatment of solid tumors, such as hepatic VX2 tumor model in rabbits [195,196], murine orthotopic pancreatic and breast cancer models [197,198]. In the liver VX2 tumor model, histopathological assessment of liver samples 4 days after treatment, revealed complete necrosis of tumor cells with no evident damage to surrounding tissue or other organs [199]. Subsequent dose-escalation study in the VX2 tumor-bearing rabbits defined 1.75 mM 3-BrPA as a therapeutic concentration, to be injected intra- arterially or given as continuous intra-arterial infusion for 1 h duration is equally effective. Higher concentrations of 2.5 mM 3-BrPA caused peripheral liver necrosis [200]. Treatment with 3-BrPA was found to achieve survival benefits in treated cancer animal models [201-204].
The systemic delivery of 3-BrPA has been investigated in an orthotopic xenograft mouse model of pancreatic cancer, after micro-encapsulation of 3-BrPA using β-cyclodextrin (β-CD), as a molecular carrier, to achieve stability of 3-BrPA molecule for systemic administration and reduce its nontarget toxicity. This micro-encapsulated formulation (ß-CD–3BrPA) showed similar anticancer efficacy as that observed with the free drug in vitro [184]. A recent in vivo experiment using daily intraperitoneal injections of free or micro-encapsulated 3-BrPA (up to 5 mg/kg) in tumor-bearing mice showed similar efficacy with tumor eradication, confirmed by

histopathological analysis, under both formulations of systemic 3-BrPA therapy. However, the micro-encapsulated formulation was associated with lower toxicity as compared with the free 3- BrPA [205].
Early clinical trials showed promising results for efficacy and safety of 3-BrPA in the local regional delivery, but drug toxicity is considered the main obstacle for its systemic delivery. In a clinical trial, two patients diagnosed with end-stage liver cancer (HCC and cholangiocarcinoma, respectively) were treated with intra-arterial 3-BrPA, showing favorable results with no significant toxicity [5]. Also, 3-BrPA has been accepted by the US FDA as an investigational new drug application for use in Phase I clinical trial in patients with liver cancer [206]. However, most scheduled trials regarding 3-BrPA are suspended after the reports of three deaths, in 2016. All these patients died within a few days after receiving 3-BrPA as alternative medicine by a nonmedical practitioner in Germany and the condition is under investigation by the prosecutor about the possible improper use of 3-BrPA [207]. Currently, no approved clinical trials on 3-BrPA are available in the database website of approved clinical studies; https: //www.clinicaltrials.gov/. There are only two reported case studies on using 3-BrPA for the treatment of patients with resistant cancers. The first is reported by Ko et al., [208], on voluntary treating a boy with advanced fibrolamellar hepatocellular carcinoma using transcatheter arterial administration of formulated 3- BrPA over several months. The boy survived with a higher quality of life for a much longer period than expected without major toxicity. Unfortunately, the patient died after two years of diagnosis, due to overload liver function [208]. The second is reported by El Sayed et al., [209], using an intravenous infusion of 3-BrPA (1–2.2 mg/kg) to treat a man with stage IV metastatic melanoma and response was monitored by serum LDH level. Repeated 6 infusion doses of 3-BrPA over 10 days led to a partial reduction of serum LDH level. The patient was also given paracetamol as a GSH scavenger, to avoid tumor resistance by the high GSH level. This is followed by a sharp drop of LDH level, and no significant cytotoxicity observed except burning sensation at the infusion site. However, the man died due to respiratory distress and hypoxemia [209]. Currently, it is suggested that unformulated 3-BrPA should not be used and only formulated forms can be used in clinical oncology studies [191].

9.5- Targeting pyruvate formation (PKM2 inhibitors)

Pyruvate is regarded as a “hub” metabolite, regulating metabolic reprogramming of cancer cells, and linking glycolysis in the cytosol to mitochondrial oxidation. The enzyme PK catalyzes the formation of pyruvate from phosphoenolpyruvate (PEP) with the generation of ATP. This essential enzyme for aerobic glycolysis is liable to regulation by allosteric effectors as fructose-1,6- bisphosphate and by phosphorylation [210]. The isoform PKM2 is overexpressed in tumors enhancing cellular proliferation, and thus could be an attractive target for anticancer therapy [61,211]. Interestingly, data are suggesting that either inhibition or activation of PKM2 could diminish the growth of cancer cells. The PKM2 inhibitors could allow glycolytic intermediates to accumulate and feed biosynthetic pathways, promoting tumor growth. For instance, Anastasiou et al. [212], have demonstrated that inhibition of PKM2 by oxidation on cysteine 358, leads to diversion of glycolytic intermediates into the PPP, with NADPH production and promotion of redox balance, supporting tumor growth. Thus, the anticancer effect can be achieved by the expression of a non-oxidizable PKM2 mutant or by PKM2 activators that suppress the PPP and increase oxidative stress. On the contrary, Goldberg and Sharp [213], found that using small interfering RNA (siRNA) to inhibit PKM2, could inhibit tumor growth and increase cell apoptosis in vitro. Also in vivo delivery of siRNA to inhibit PKM2 in mouse xenograft model can induce tumor regression.
These contradictory results could be explained by variable responses to various degrees of hypoxia. Under moderate hypoxia hydrogen peroxide (H2O2) formation is increased, activating signaling pathways involved in the cellular response to hypoxia. Thus, PKM2 activity is suppressed by its oxidation leading to increased flux through the PPP and enhanced redox balance, preventing oxidative cellular damage [212]. On the other hand, in severe hypoxia mitochondrial production of ATP and generation of H2O2 stops. Thus, cells dependent on PK activity for energy production and can be affected by PKM2 inhibitors. This implies that the anticancer effects of PKM2 inhibitors or activators could be tumor-dependent according to tumor size and vascularization. So, PKM2 activators can enhance oxidative damage in moderately hypoxic cells, while PKM2 inhibitors can prevent energy production in severely hypoxic cells. Also, PKM2 inhibition can induce caspase-dependent cell death, increasing apoptosis [213]. Recent studies revealed that PKM2 can increase the transcriptional activity of HIF-1, which mediates cellular adaptation to hypoxic stress via transcription of diverse targets such as GLUTs, HK2 and LDH-A

[214]. Thus, it would be also important to clarify the effects of PKM2 inhibition or activation on the non-glycolytic functions involved in tumorigenesis.
Several compounds such as TT-232, VK3, VK5 and Compound 3, can inhibit PKM2 leading to suppression of glycolysis in tumor cells [211]. The natural compound oleanolic acid (OA), has been recently found to exert its anticancer effect by shifting PKM2 to PKM1, thus inhibiting aerobic glycolysis [215]. However, it has been recently observed that knockdown of PKM2 in the breast cancer model could enhance tumor formation, indicating that PKM2 inhibition may not be efficient alone in the treatment of cancer [216]. These results highlighted the importance of understanding the tumor metabolic needs to target metabolic pathways for cancer therapy.

9.6- Targeting pyruvate oxidation (PDK inhibitor)
Pyruvate in the cytosol is converted into mitochondrial acetyl-CoA to enter the Krebs cycle by pyruvate dehydrogenase (PDH) enzymes. The PDH is negatively regulated by the enzyme pyruvate dehydrogenase kinase (PDK), leading to a shift of glucose from oxidative phosphorylation to glycolytic metabolism [217]. Thus, targeting PDK may be an attractive approach to inhibit cellular proliferation and cancer growth by inducing tumor cell reprogramming from the glycolytic to oxidative phenotypes. This reversing of the Warburg effect has been demonstrated in glioblastoma multiforme (GBM) cells using the PDK inhibitor, dichloroacetate (DCA), that lowered PDK1-EGFR activation and shifted glycolytic phenotype to oxidative metabolism [110].
9.6.1- Dichloroacetate (DCA)
Dichloroacetate (DCA) is a small molecule recently introduced for antiglycolytic therapy. This drug has been used for more than 25 years in the treatment of congenital mitochondrial dysfunctions [218]. The mechanism of DCA action involves its ability to activate mitochondrial PDH by inhibiting its regulator PDK. Thus, DCA enhances the reprogramming of energy production from the glycolytic state toward mitochondrial OxPhos with the consumption of pyruvate and decreasing lactate accumulation [219]. So, cancer cells become more susceptible to apoptotic signals and their proliferation is suppressed [220]. During hypoxia, HIF-1-alpha activates gene transcription of some glycolytic regulators such as GLUTs, PDK and LDHA. This leads to redirection of cells from oxidative to glycolytic state with accumulation of pyruvate [111].

The compelling concept of the ability to reverse ‘Warburg effect’ in tumor cells prompted several oncologic studies to investigate the potential use of DCA in antiglycolytic cancer therapy. A study conducted in rats having subcutaneous cancer cells showed that orally administered DCA can induce apoptosis and decrease cell proliferation [221]. Similar promising anticancer effects were demonstrated for DCA administration in tumor xenograft studies in animals and humans with glioblastoma [222]. However, it has been recently observed that DCA treatment could not suppress or even enhance cell proliferation in cancer cell lines of human breast cancer, and cell lines of human and murine neuroblastoma [223]. On the contrary, it has been reported that treatment with DCA can overcome the resistance to sorafenib in a mouse model of sorafenib-resistant xenograft HCC cells [224]. Also, combining oral DCA with adriamycin leads to enhanced cytotoxic effects in treated hepatoma cells in vitro and in mice with subcutaneous xenografts in vivo [225]. Thus, preclinical data of DCA use in cancer therapy are not conclusive and need further confirmation. Several phase I/ II clinical trials are ongoing for testing DCA activity and safety as an anticancer agent. A single-arm prospective study examined the safety of oral DCA in patients with brain tumors, revealed no acute dose-limiting toxicities after at least one 4-week cycle [222]. Some of the patients treated with DCA for about 75 days remained with stable disease, but two patients reported grade 0–1 paresthesia. Another dose-escalation trial included 24 patients with solid malignancies to receive oral treatment with DCA at a dose of 6.25 mg/kg. Dose-limiting fatigue, vomiting, and diarrhea are reported by three patients at a dose of 12.5 mg/kg. Tumor response assessed by FDG-PET revealed stable disease in eight patients but no response in others [226]. Overall, the current clinical results of DCA use in antiglycolytic therapy are preliminary, supporting a favorable toxicity profile rather than prominent anticancer efficacy.

9.7- Targeting lactate dehydrogenase (LDHA inhibitors)
The LDH is a major glycolytic enzyme, that has five isozymes catalyzing the interconversion of pyruvate and lactate coupled with NAD+ recycling. Accumulation of lactate can drastically lower pH inside the cells. Thus, it is necessary for cells to actively efflux lactate into the outside, through specific transporters (MCTs), as the final port of the lactate shuttle. The MCT1 and MCT4 isoforms play a crucial role in cancer metabolism through the efflux and influx of lactate between adjacent cells [81]. Experimental studies demonstrated the overexpression of LDHA in several tumor cells and its inhibition can markedly delay tumor progression [227]. LDHA is directly

regulated by the c-myc oncogene, and so the genetic or pharmacologic inhibition can diminish myc-dependent tumors [62].
Inhibitors of LDHA, such as FX11 and oxamate, have demonstrated encouraging results in preclinical studies and remain to show similar promising results in clinical trials. FXII, a catechol- containing small compound that inhibits LDHA, was shown to inhibit tumor growth in xenografts [228]. Oxamate, another LDHA inhibitor, can sensitize resistant tumor cells to the cytotoxic effect of chemotherapeutic agents [229]. The LDHA inhibitors probably act by a mechanism similar to that of DCA, involving increased delivery of pyruvate into the mitochondria to undergo decarboxylation to acetyl-CoA, with enhanced mitochondrial activity, thereby increasing oxidative stress leading to cancer cell death [228].

9.8- Targeting tumor acidosis:
Targeting tumor acidosis and decreasing the intracellular alkalosis can be a useful addition to metabolic anticancer therapy [230]. Tumor acidity can be targeted by inhibition of proton release using proton pump inhibitors (PPIs) such as omeprazole, either alone or in combination with chemotherapeutic agents [47]. It has been demonstrated that PPIs can inhibit vacuolar-type ATPase to increase the lysosomal pH and increase lysosomal-endosomal turnover in tumor cells [231]. In a case-control study, it was found that breast cancer patients were 25% less likely to have prior use of PPIs compared with control women [232]. Also, a strong association was detected between PPIs use and improved survival in patients with head and neck cancer [233]. Several other reports revealed survival benefits from the combined use of PPIs with conventional chemotherapeutics in many types of cancers including osteosarcoma [234], breast cancer [235] or gut cancers [236]. The safety profile of PPIs on long-term use makes these drugs ideal candidates for more wide clinical anticancer studies.
Targeting the lactate shuttle through MCTs, as important regulators of pH status, could also be exploited for anti-tumor effects. The specific MCT1 inhibitor, AZD3965, has shown promising anticancer effects in preclinical studies [237]. The mechanism of the anticancer effect involves intracellular trapping of lactate, decreasing pHi, with the death of tumor cells. AZD3965 is currently under testing in phase I clinical trials in patients with B-cell lymphoma and prostate cancer [238,239]. Inhibition of MCTs using α-cyano-4-hydroxycinnamic acid has also, been demonstrated to produce a significant effect on tumor growth of malignant glioma [240].

Cinnamate is another small molecule inhibitor of MCTs, that was shown to decrease pHi and inhibit tumor growth [241]. Further investigations are required to confirm the target specificity and effectiveness of these candidate drugs before translation into anticancer clinical studies.
Moreover, Carbonic anhydrase 9 (CA9) enzyme is active at low pH and is involved in tumor acidosis. CA9 is an important prognostic indicator in many tumors, associated with invasion and metastasis [242]. Inhibition of CA9 was shown to suppress growth and metastasis in breast cancer [243]. The CA9/CA12 inhibitor SLC0111 is investigated in phase I clinical trial in solid tumors showing favorable results (NCT02215850).

9.9- Targeting other regulatory aspects of energy metabolism
The molecular regulators of glycolysis could also be targets for potential anticancer activity. For instance, KRAS mutation is found in most cases (>90%) of pancreatic cancer [244], where it facilitates glycolysis and drive the PPP for the synthesis of nucleic acids [245], to enhance tumorigenesis [246]. Inhibition of KRAS or its downstream pathways revealed promising anticancer effects in preclinical studies [247,248]. However, clinical trials in pancreatic cancer patients reported no effect on overall survival from these KRAS inhibitors [249-251]. The c-MYC oncogene is also a driver of glycolysis and tumorigenesis in many cancers [252]. Agents targeting MYC showed encouraging anticancer effects, and many of these agents are currently under investigation in clinical trials. For example, inhibition of BET proteins downregulates MYC expression and inhibits tumor growth [253,254]. Moreover, MYC inhibition can prevent the development of resistance to mitochondrial inhibitors [255], making combination therapy with these agents a potentially useful anticancer strategy.
The components of PI3K/Akt/mTOR signaling pathways are extensively investigated for potential anticancer effects. Many inhibitor agents for the PI3K/Akt pathway are under testing in preclinical and early clinical studies as targeted anticancer therapies, including Afuresertib (GSK2110183), Uprosertib (GSK2141795), Ipatasertib (GDC-0068), MK-2206, GDC‑ 0068, TCN and TCN-P [256]. Most of these clinical trials with Akt inhibitors showed modest activity as single anticancer agents, being most effective in cancers with PIK3 mutations and PTEN deficiency [257]. They produce more promising effects when combined with other chemotherapeutic agents or with radiotherapy [258].

Besides, inhibition of mTOR kinase was proposed as an effective approach for tumor suppression. Recently, mTOR inhibitors have demonstrated significant activity in the treatment of several types of tumors such as neuroendocrine, endometrial and breast cancers, medulloblastoma, glioblastoma, osteosarcoma, leukemia and lymphoma [259-265]. The mTOR inhibitors have been classified into three generations; including rapamycin and other rapalogs, temsirolimus, everolimus, and ridaforolimus, as first-generation; the ATP-competitive inhibitor of mTOR kinase (inhibit both mTORC1 and mTORC2), MLN0128, AZD2014, AZD8055 and CC223, as second- generation inhibitors; and the dual PI3K/mTOR inhibitors, PP242, MLN0128, KU-0063794 and BEZ235, as third-generation inhibitors [266]. Rapalogs, are mTORC1 inhibitors, showed limited activity in clinical trials, as single anticancer agents, probably related to the various cross-talks of the complicated mTOR pathway with other signaling pathways [267]. The newer generations of dual mTOR kinase inhibitors are less liable to induce tumor resistance than the rapalogs. These agents are tested in preclinical studies and recently entered some clinical trials [268]. Also, halofuginone (HF) treatment have demonstrated potent anticancer activity in human colorectal cancer cells. Halofuginone can reduce the Akt/mTORC1 signaling pathway, decreasing HK2 and GlU1, leading to glycolysis inhibition [269]. Combination therapy targeting multiple pathways may produce a better therapeutic outcome and overcome tumor resistance [266].
Currently, the concept of reverse Warburg effect provides a new approach to anticancer strategies by targeting glycolysis and oxidative phosphorylation [27]. Thus, it could be relevant for cancer therapy to suppress glycolysis by targeting autophagy in the stroma decreasing the fuel supplied to mitochondria of cancer cells, thereby inhibiting the two-compartment tumor metabolism. Several such drugs have been approved including N-acetylcysteine, metformin, hydroxychloroquine, and rapamycin [43]. Metformin is a biguanide oral antidiabetic drug, that activates AMPK, inhibits mitochondrial ETC Complex-1 and inhibits mTOR, which further inhibits HIF1. It can reduce the activity of mitochondrial complex I, to inhibit OxPhos and reduce ATP production, suppressing cancer progression [270]. Recent epidemiological studies revealed that metformin-treated diabetic patients have lower cancer risk. Later the drug has been proven to exhibit significant anti-proliferative activity in many clinical trials in cancer patients with or without diabetes [271]. This antitumor activity involved several mechanisms including inhibition of the respiratory complex I, and reduction of mTOR activity, in addition to the reduction of blood glucose and insulin levels, decreasing its mitogen effect [272]. However, despite the promising

results in preclinical studies, the results of some clinical trials are inconclusive [273]. Inhibition of both the main metabolic pathway and its main escape mechanism can completely suppress malignant cells. Thus, the use of dual metabolic inhibitors such as metformin (inhibits mitochondrial ETC complex 1) and 2-DG, (inhibits of glycolysis), was shown to produce profound metabolic inhibition with more effective anticancer activity in preclinical studies [39]. The synergetic effect has also been demonstrated for metformin combination with either the bromodomain and extra-terminal motif (BET) inhibitor JQ-1 in pancreatic cancer [255] or PI3K inhibition for ovarian cancer [274], which produces simultaneous inhibition of OxPhos and glycolysis. Similarly, glycolysis inhibitor can be combined with other DNA damaging chemotherapeutic agents could provide an effective approach to overcome anticancer resistance through increasing cytotoxicity and decreasing repair capacity.
The hypoxic condition prevailing in rapidly growing tumors can switch the balance between the angiogenic activators and inhibitors, in favor of the angiogenesis activators such as vascular endothelial growth factor (VEGF), transforming growth factor-beta, fibroblast growth factor-2, platelet-derived growth factor, interleukin-8, and angiopoietins [275]. The newly formed blood vessels in tumors are structurally and functionally abnormal with tortuosity, leakiness and poor covering by vascular supportive cells. These poorly functioning vessels lead to chaotic blood flow with the incorporation of tumor cells into the endothelial wall, enhancing blood dissemination of tumors [276]. Agents inhibiting tumor angiogenesis have been tested for anticancer therapy, including agents targeting the angiogenic factor VEGF by the monoclonal antibody, bevacizumab [277], or by the fusion protein, aflibercept [278]. Several other agents are tyrosine kinase inhibitors (sorafenib, sunitinib, regorafenib) that target the angiogenic receptors [279]. These agents have shown promising results in the treatment of various types of cancers but, with little survival benefit [280]. However, complete vascular suppression can lead to marked hypoxia with decreased perfusion and decreased the supply of tumors with oxygen, nutrients and also drugs. This may result in a more aggressive tumor with a worse prognosis [281]. Moreover, anti-VEGF drugs can decrease the vascular density in normal tissues, with reduced function of healthy organs [282]. Thus, further studies are required to support the beneficial role of antiangiogenic agents in cancer therapy.

Table 1: Glycolytic targets, and main antiglycolytic agents in preclinical and clinical development for anticancer therapy

Target metabolic pathway Glycolytic inhibitors State of development References
Glucose transporters (GLUTs) Phloretin Fasentin STF-31 WZB117
Ritonavir Silybin Preclinical Preclinical Preclinical Preclinical
Preclinical Phase I [119,120]
[121]
[122]
[118,123]
[124]
[125]
Hexokinase2 (HK2) 2-Deoxy-D-glucose (2-DG) lonidamine (LN)
Genistein-27 Benserazide Resveratrol Astragalin
Chrysin Phase II Phase II Preclinical Preclinical Preclinical Preclinical
Preclinical [142,143,147,150,153]
[163-168]
[129]
[130]
[126]
[127]
[128]
Glyceraldehyde-3- phosphate dehydrogenase
(GAPDH) 3-Bromopyruvate (3-BrPA) Koningic acid
Iodoacetate Phase I Preclinical
Preclinical [204-209]
[181-182]
[179]
Pyruvate dehydrogenase
kinase (PDK) Dichloroacetate (DCA) Phase I [222-226]
Lactate dehydrogenase
(LDHA) Oxamate
FX11 Preclinical
Preclinical [289]
[288]
Monocarboxylate transporte (MCTs) α-Cyano-4-hydroxycinnamic acid Cinnamate
AZD3965 Preclinical
Preclinical Phase I [240]
[241]
[238,239]
Carbonic anhydrase (CA)
9/12 SLC0111 Phase I [243]
Proton pump
(H+/K+-ATPase) Omeprazole, Esomeprazole,
Lansoprazole, Pantoprazole Phase I [232-236]
Mitochondrial AMPK Metformin Phase III [270-273]
Akt/mTORC1 Halofuginone Phase I [269]
mTORC1

mTORC1/mTORC2 PI3K/mTOR Rapamycin, temsirolimus, everolimus, ridaforolimus MLN0128, OSI-027
PP242, NVP-BEZ235 Phase I

Phase I Phase I [259-265]

[267]
[268]
PI3K/Akt Afuresertib
Uprosertib Ipatasertib Phase I
Phase I Phase I [256-258]
KRAS mutation Sorafenib Trametinib
Selumetinib Phase 1 Phase I
Phase 1 [249]
[250]
[251]
Angiogenesis factors (e.g. VEGF) and receptors Bevacizumab
Aflibercept Regorafenib Phase II
Phase II Phase II [277]
[278]
[279, 280]

⦁ Conclusion and future perspective for metabolic cancer therapy

Although aerobic glycolysis is a known signature of cancer cells, targeting this pathway for therapy has not yet been successfully translated into clinical practice. Recently, this hallmark of cancer metabolism has become a focus of cancer research and drug discovery, aiming for the introduction of effective metabolic agents as a promising strategy to combat cancer. These candidate drugs might also sensitize tumor cells to other more effective cytotoxic therapies. Many emerging drugs have been tested in preclinical studies with promising results and some are under investigation in clinical trials with mixed favorable and disappointing results. For example, 2-DG is recently resurrected in clinical trials in combination therapy with other chemotherapeutics or with irradiation, after disappointing results as monotherapy. Other new antiglycolytic agents such as DCA and 3-BrPA have been tested for monotherapy in solid glycolytic tumors such as HCC with encouraging results and improved survival in animal models.
However, a great concern arises about the significant toxicities of inhibiting metabolic enzymes in normal cells especially in the immune system and stem cells that also display aerobic glycolysis. Novel solutions to circumvent this problem of systemic toxicity include selective targeted delivery using recent imaging technology to deliver the drug directly into the tumor or intraarterially near the tumor. Other approaches included micro-encapsulation and developing antiglycolytic agents with high specificity for the molecular target, such as 3-BrPA that enters cells through MCTs which is overexpressed in cancer.
Moreover, cancer cells display metabolic plasticity and can overcome the inhibition of a specific metabolic pathway via the expression or up-regulation of alternative pathways. Also, adjacent cells such as fibroblasts and adipocytes can offer metabolic intermediates for the synthetic needs of cancer cells. The great metabolic heterogeneity and cellular plasticity observed in solid tumors made metabolic inhibitors unlikely to become effective as monotherapy for cancer. For instance, combination treatment with two or more antimetabolic gents inhibiting different metabolic pathways simultaneously would decrease resistance and prevent relapse. Also, tumor glycolysis has an important role in cancer resistance to chemotherapeutic agents, therefore antiglycolytic agents could be helpful in sensitization of tumor cells and improving the therapeutic outcome for treatment of resistant cancers as shown in preclinical models. Therefore, it is worth to explore the outcome of combining conventional drugs with antiglycolytic agents to provide novel therapeutic or preventive strategies for fighting against cancer.

Conflict of interest
The authors declare that there is no conflict of interests.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgments
We thank Dr. Abdul-Salam Noor Waly, Dean Faculty of Medicine, Umm Al-Qura University, Makkah, for providing access to the Saudi Digital Library.

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