MCT1 and MCT4 are overexpressed in many cancers. MCT4 in particular, very efficiently transports lactate in environments with high lactate levels [54] and is well adapted to this transport in highly glycolytic cells [55]. Malignant cells use glycolytic metabolism even when there is sufficient oxygen for an oxidative metabolism (Warburg effect). This type of metabolism is frequently found not only in malignant cells but also in normal cells that are intensely proliferating. Evidently, this type of metabolism represents a growth advantage that took many years to be understood. Now, it is known that glycolytic metabolism allows intense proliferation in a medium depleted of oxygen. It reduces the generation of reactive oxygen species (ROS) and provides building blocks for the synthesis of macromolecules and molecules for antioxidant compounds. Furthermore, glycolytic metabolism can provide energy in a much faster way than oxidative metabolism, albeit at the cost of catabolizing ten times more glucose than usual oxidative metabolism for energy production [56–58]. The harsh and hypoxic environment in which cancers develop exerts a selective pressure on glycolytic cells, which are better adapted for survival [59, 60].

MCTs are over-expressed in many tumors, such as breast [61–63], colorectal [64–66], prostate [67–69], lung [70, 71], esophageal [72, 73], small cell lung [74], ovarian [75], gastric [76–78], endometrial [79], renal [80–82], hepatic [83, 84], pancreatic [85–87] cancers, leukemia [88, 89], lymphoma [90–92], head and neck squamous cell cancer [93–95], glioma [96], soft tissue sarcoma [97], and testicular germ cell tumors [98]. In summary, all kinds of tumors express MCT1–4, and in all of them, they play a role in malignant progression [99, 100]. Hypoxia is a strong driver for the increased expression of MTCs [101] and most tumors have large hypoxic areas.

A meta-analysis showed that MCT4 and its chaperone basigin (CD147) expression were correlated with poor clinical prognosis, including shorter disease-free survival and shorter overall survival across many different cancer types. There was no clear correlation with MCT1 [102]. This last finding does not have a clear explanation however fewer studies with high-quality data were available for this analysis.

Basigin 2 (one of the four isoforms of basigin, see Figure 6) has been found to play an important role in the migration and invasion of ovarian carcinoma cells [103]. Similar findings occurred with melanoma cells [104]. Basigin knock-out blocked lactate export in non-small cell lung cancer (NSCLC) cell lines [105]. Basigin N-glycosylation by N-acetylglucosaminyl transferase V promotes metastasis in hepatocarcinoma [106]. MicroRNA Let-7-b is an endogenous suppressor of basigin expression, and it was shown to reduce invasion and metastasis in mouse melanoma cells [107].

Quercetin is a flavonoid found in many foods in the usual human diet. Its widespread distribution in the vegetal kingdom may give the wrong impression that quercetin is only a nutritional supplement, which is how it is classified by the Food and Drug Administration (FDA). Quercetin is a flavonol, a subclass of flavonoids found in many plants, including fruits and vegetables as well as in seeds, nuts, and bark [118]. More than 5,000 flavonoids have been identified [119]. The normal human diet contains a small amount of quercetin, usually attached to a glycoside [120]. Flavonoids are a group of natural substances abundant in foods like fruits, plants, and some beverages. They have a three-ring polyphenolic structure in common: 2-phenylchromem-4-one (upper panel of Figure 8) [121]. Flavonoids can be modified to form many natural compounds, such as flavones, flavonols, isoflavones, and anthocyanins. Most authors also include chalcones in the group, despite it missing the B ring. The general structure of flavonols is shown in Figure 8.

Quercetin is a flavonol, one of the six subclasses of flavonoid compounds. Most flavonoids usually have glycosides (one or more sugars) attached. When they lack glycosides, they are known as aglycone (without sugars). The type of flavonol depends on the molecule attached in the R1 and R2 positions:

(A)

Quercetin has an -OH at R1 and -H at R2 both in ring C.

Aglycones have very low water solubility, but they are soluble in lipids and alcohol. Adding sugar, whether glucose, rutinose, or rhamnose increases water solubility.

The structural formula of quercetin is shown in Figure 8. Most flavonoids have anti-tumoral effects, and they are all antioxidants. It is of note, that quercetin is the only one that has five hydroxyl groups, and this characteristic influences its effects.

Quercetin’s anticancer properties have been shown to act on different molecular targets and there is a great diversity of effects [122, 123]. These studies demonstrated preventive and therapeutic characteristics which will not be discussed here. For a review in this regard see Rauf et al. [124] and Ezzati et al. [125].

Various reports, including earlier studies, have characterized quercetin’s cellular activities. In 1969, Carpenedo et al. [126] discovered that quercetin inhibited mitochondrial ATPase activity. They concluded that quercetin “shows an affinity for membrane-dependent cellular activities”. The first papers showing that quercetin was able to rewire metabolism and inhibit glycolysis in malignant cells appeared in 1974 [127, 128]. The initial conclusion was that quercetin’s metabolic effects were due to inhibition of mitochondrial ATPase. In 1975, Suolinna et al. [129] worked with Ehrlich ascites tumor cells and discovered that hydroxyl-rich flavonoids (quercetin) can inhibit glycolysis. Furthermore, concentrations of quercetin between 5 μg/mL and 20 μg/mL induced growth inhibition. The explanation they gave was the “interfering with the generation of adenosine diphosphate and inorganic phosphate which are required for glycolysis”. However, it must be remembered that in 1975 the MCTs were not known. They also did not measure intracellular pH. Additionally, they found that ion pumps worked with greater efficiency. This is logical because now it is known that quercetin acidifies intracellular pH and the cells tried to survive by increasing the activity of sodium hydrogen exchanger 1 (NHE1) that exports protons. Therefore, unknowingly, Suolinna et al. [129] found that quercetin reduced the glycolytic behavior due to the accumulation of lactate.

Confirming our re-interpretation of the experiment, they found that the inhibitory effect of quercetin on glycolysis was hampered when the culture medium was enriched with bicarbonate. This is also logical, because the cell incorporates bicarbonate through sodium bicarbonate cotransporter 1 (NBC1), thus increasing intracellular pH. All these ion transporters were not identified at the time of the experiments.

Of all the flavonoids tested by Suolinna et al. [129], quercetin (at a concentration of 8 µg/mL) had the highest percent inhibition of lactate production (78%), meaning that it was the most efficient flavonoid to inhibit glycolysis. It was followed by luteolin with 75% inhibition (8 µg/mL) and kaempferol (16 µg/mL) with 52% inhibition. At a concentration of 40 µg/mL, quercetin completely inhibited lactate production. With 5 days of incubation, 5 µg/mL of quercetin reduced P388 leukemia cell growth to zero, while 2.5 µg/mL had no effect at all. Details of the mechanism of action of flavonoids were still missing at this time, though later, in 1979, Belt et al. [130] found that flavonoids, and in particular quercetin were potent inhibitors of lactate transport, and glycolysis, in Ehrlich ascites tumor cells.

More details were elucidated in the studies of Volk et al. [131] and Albatany et al. [132]. They induced intracellular acidification with quercetin through the inhibition of the MCT1 and MCT4 in glioma cells. This inhibition prevents lactate extrusion and increases intracellular lactate in malignant cells. Quercetin can inhibit lactate extrusion to the extent of increasing intracellular lactate levels 3-fold to 4-fold. At the same time, it decreases intracellular pH to 6.9. Importantly, Volk et al. [131] showed that these effects are limited to malignant cells. Unfortunately, the concentrations used to achieve lactate inhibition were in the order of 50 μmol/L which is higher than what can be achieved by oral administration. That does not mean that lower concentrations cannot achieve some positive results, but this possibility has not been fully tested [133].

Oral administration of quercetin can hardly achieve a level of 1 μmol/L in tissues, according to our study of the published literature. The highest concentration achieved was recorded by Graefe et al. [134]. Administering 100 mg oral quercetin to normal volunteers gave a peak plasma concentration of 2.3 μg/mL ± 1.5 μg/mL and a mean level of 2.1 μg/mL ± 1.6 μg/mL (approximately 7 μmol/L).

Many non-steroidal anti-inflammatory drugs (NSAIDs) have shown anti-tumoral effects such as decreased proliferation and growth, which was attributed to cyclooxygenase 2 (COX2) inhibitory abilities. In addition, non-COX2 dependent effects were found that modified glucose metabolism by decreasing GLUT1, lactate dehydrogenase A, and MCT1 expression in tumor cells [139].

Many NSAIDs have a monocarboxylic acid as part of their structure. It is attractive to speculate that NSAIDs may compete with lactate for MCTs, but this idea has not been fully confirmed experimentally. However, supportive evidence for the concept is that the NSAID diclofenac (Figure 9), interferes with the cellular uptake of gamma-hydroxybutyric acid, which enters the cell through the action of MCT1 [140]. Lactic acid and diclofenac also seem to compete in transplacental transport and may share the same transplacental transfer system [141]. There is additional evidence showing that MCTs play a role in monocarboxylic NSAID uptake [142]. A report by Sasaki et al. [143] showed that diclofenac exerted a powerful inhibitory effect on the lactate uptake of Caco-2 cells while Renner et al. [144] have shown that diclofenac can inhibit MCTs.

Interestingly, diclofenac has the opposite effect in retinal cells HRPE and ARPE-19 where it increases proton-coupled and sodium-coupled MCT activity [145]. This may be related to the high expression of basigin 1 in retinal cells, although there is no experimental evidence to sustain this idea.

Syrosingopine (Figure 10) is a reserpine derivative used for treating hypertension. This is a weak antihypertensive medication that was developed by CIBA Pharmaceuticals (now Novartis) and first used in 1958. Due to poor sales, it was discontinued in 1968. Importantly, it is also a dual MCT inhibitor that targets MCT1 and MCT4. Evidence for its physiological actions comes from several studies. In a study by Buyse et al. [146], syrosingopine decreased extracellular acidity, glucose consumption, and lactate secretion, and importantly decreased tumor cell proliferation in vitro. However, it showed no effects in vivo. In contrast, in a different study at least at the cellular level, the co-administration of syrosingopine with metformin showed important anti-tumor effects. However, this work did not test the compound in vivo. Similarly, Benjamin et al. [147] found that syrosingopine sensitized cancer cells to the administration of far lower doses of metformin or phenformin than were necessary for anti-cancer effects with metformin or phenformin alone. We think that what happens is something complex with many contributing factors. Metformin or phenformin increases the production of lactate by inhibiting complex I in the electron transport chain. This additional load of lactate is added to that produced by the increased glycolytic metabolism, plus the load from the inhibition of its extrusion by syrosingopine. This leads the cell to acidic stress and eventual apoptosis. Supporting this theory is that the only confirmed effect of metformin in normal or tumor cells is inhibition of complex I. All the supposed antitumoral effects are secondary to this inhibition. Several authors also suggest the main effect of syrosingopine is due to intracellular acidification [148–150]. In another later publication, Benjamin [151] suggested that metformin potentiates syrosingopine through intracellular acidic stress-mediated effects.

Statins have shown an ability to inhibit lactic efflux from muscle cells. In this regard atorvastatin seemed the most potent: it can cause a 2.5-fold increase in intracellular lactate. Simvastatin and rosuvastatin had no effects [152]. Regarding cancer cells, similar findings were observed with atorvastatin earlier, as were much lower effects of fluvastatin. Lovastatin showed no inhibitory effects [153]. Kobayashi et al. [154] found that lipophilic statins inhibited MCT4. On the other hand, hydrophilic statins had no such effects. In a recent publication, all statins were found to exert inhibitory effects on MCT1, MCT2, and MCT4. Atorvastatin showed selective and potent inhibition of MCT2 [155]. This is not an unimportant observation, because MCT2 is a high affinity pyruvate transporter [156].

Statin treatments are known to cause myotoxicity, and this is fully explained by their inhibition of MCTs [157]. However, in clinical practice, statin-related myotoxicity is a multifactorial development in which many other facts play a role, such as dose, concentration, interaction with fibrates, statin-induced interruption of glycoprotein synthesis in the muscle membrane, increased intracellular calcium concentrations leading to impaired membrane function and decreased membrane fluidity.

α-cyano-4-hydroxycinnamate, an MCT4 inhibitor also produced the same type of lesions induced by atorvastatin in embrional rabdomyosarcoma cells [158]. This seems bad news for the systematic use of statins for lowering cholesterol, but it is the opposite regarding their clinical utility in cancer.

Statins can achieve inhibition of MCTs. Atorvastatin, the most potent in this regard, was found to be a non-competitive inhibitor of MCT1 with a constant concentration of 40 μmol/L [159]. The problem is that the maximum achievable concentration of atorvastatin after a normal high dose does not go beyond nmol/L levels [160]. The unanswered question that remains here is: can clinically acceptable doses of atorvastatin inhibit MCTs? One study improved statin delivery to tumors by using cell-derived microparticules loaded with fluvastatin. These attenuated lung adenocarcinoma cells growth in vivo [161]. Improving pharmaceutical delivery methods through nanoparticles may be a useful avenue to pursue to increase the amount of statins deliverable to the tumor.

Lonidamine is a derivative of indazole-3-carboxylic acid (Figure 11). It is an old and almost forgotten drug with low toxicity, that interferes with glucose metabolism in different ways. Research is now in a period of rediscovery of what lonidamine can do in cancer cells and how it inhibits glycolysis.

Lonidamine was originally developed in the 1970s as a spermicide. Discovery of its anti-tumoral effects came shortly after [162, 163]. After more than 30 years of research, the exact mechanisms of action of lonidamine are not fully known. Lonidamine inhibits glycolytic and oxidative metabolism of glucose, decreasing the production of ATP whether mitochondrial or glycolytic. Furthermore, it also inhibits lactate extrusion, thus producing an important intracellular acidification.

Six mechanisms of action have been identified (Figure 12):

(A)

Inhibition of the mitochondrial pyruvate carrier which impedes mitochondrial uptake of pyruvate [164].

The main effect of lonidamine seems to be the targeting of the mitochondrial pyruvate transporter which it accomplishes with low concentrations. For MCT inhibition, much higher concentrations are required (above 150 μmol/L) [172]. However, all the effects mentioned above produce an important acidification of the cytoplasm and an energy restriction [173, 174]. Adjudin, a derivative of lonidamine showed similar antitumor effects [175].

The lack of positive results in cancer treatment with lonidamine is produced by its use as a stand-alone drug, which is not how it should be utilized. Its minimal toxicity permits it to be added to more toxic treatments without adding adverse effects, which may be a more productive use of the compound. To date, the clinical experience with lonidamine as a stand-alone drug has been poor. This may be because the drug has reversible, and short-duration effects and as noted above, because the drug was not administered with other anticancer drugs. When lonidamine was co-administered with other anticancer drugs, it was shown to potentiate their effects [176, 177]. In one example, when administered with metformin, lonidamine showed increased apoptotic effects in vitro and in vivo [178]. Pharmokinetic studies have demonstrated that lonidamine administered to humans can achieve concentrations high enough to block mitochondrial pyruvate transporter and MCTs [179–181].

The drugs described above are weak MCT inhibitors and usually require high concentrations to achieve inhibition. On the other hand, the drugs that will be considered below are direct MCT blockers and achieve inhibition at single-digit nanomolar concentrations, therefore they are much more potent than most of the above-named compounds.

Cyano cinnamic acid (Figure 13) is the structural base from which two powerful MCT1 and MCT4 inhibitors have been developed [182]. Although the exact mechanism by which they inhibit MCTs is unclear, it was found that one of the derivatives, α-cyano-4-hydroxycinnamate bound strongly to the MCT without being translocated. However, it could be displaced by the addition of lactate [183].

Many of these derivatives have shown antitumoral effects in vivo [184]. They are still undergoing pre-clinical experimentation.

BAY-8002 (Figure 14) is an orally available potent dual MCT1/2 inhibitor that suppresses bidirectional lactate transport. Laboratory and pre-clinical studies showed that large B cell lymphoma cells were particularly sensitive to MCT1 inhibition by BAY-8002 (these cells lack MCT4 expression). BAY-8002 also increased intracellular lactate. Resistant cells developed increased MCT4 expression [185].

7-aminocarboxycoumarin 2 (7ACC2, Figure 15) is a potent inhibitor of lactate influx into the cell. It has minimal or no effects on efflux, thus it is an inhibitor of MCT1 but not of MCT4. However, the laboratories that sell the product suggest that it inhibits both proteins (Merck KGaA, Darmstad, Germany).

7ACC2 was tested on cervical carcinoma (SiHa cell) tumors, colorectal HCT116 tumors, and xenografted orthotopic MCF-7 breast tumors, and delayed growth in all of them [186].

Several studies support the potential use of 7ACC2 in cancer treatment. Corbet et al. [187] mention the possibility that inhibiting lactate uptake via MCT inhibitors may increase glucose utilization in oxidative malignant cells. Importantly, they found that 7ACC2 also inhibited mitochondrial pyruvate transport. This leads to intracellular pyruvate accumulation which inhibits lactate uptake. They also found that 7ACC2 had an important radiosensitizing effect. Sandforth et al. [188] also showed that 7ACC2 reduced stemness in pancreatic cancer cells expressing MCT1. Unfortunately, its multiple off-target effects interrupted further development.

AZD3965 (Figure 16) is an updated variant of AR-C155858, its predecessor, which was an MCT1/2 inhibitor [189]. AZD3965 is probably the most promising MCT1 inhibitor for cancer treatment. Some clinical trials have already been completed. There was a phase I clinical trial concluded with 35 patients with solid tumors. The 10 mg twice-a-day dose was shown to be the safest. Some adverse effects on the heart (troponin elevation) and retina (reversible electroretinographic changes) were also suggested [190]. In a phase I expansion study with the drug, a twice-daily dose of 10 mg was found to be safe. Of the 11 patients with diffuse large B cell lymphoma, 1 patient had a complete remission and 1 achieved stable disease [191]. A multicenter phase I trial of dose escalation in 40 patients with advanced solid tumors (mainly colorectal adenocarcinoma and mesothelioma) or lymphoma and no standard therapy options showed similar adverse effects on the retina and heart and grade 3 acidosis. The dose of 10 mg twice a day was confirmed as the most convenient. From 39 evaluable patients, 9 achieved stable disease as the best response [192].

In vitro, other studies have suggested that AZD3965 is more useful in tumors with high MCT1 and low MCT4 expression [193]. This may be explained by other results that showed that elevated MCT4 expression is a mechanism of resistance to MCT1 and MCT2 inhibition [194, 195].

In conclusion, the drugs’ utility is restricted to tumors with high MCT1 and low MCT4 expression. As a stand-alone drug, the performance of AZD3965 is poor.

According to the preliminary results with AZD3695, it is evident that some further co-administered drug is needed if a better outcome is desired. It may be possible to improve the effect of AZD3965 by co-administration with simvastatin as this showed additive effects in tumor growth delay in mouse-bearing head and neck squamous cell carcinoma (HNSCC) xenografts [196]. Metformin deserves to be tested in this regard. In 2016, one of us (Koltai T) proposed the double approach of increasing intracellular lactate production by inhibiting complex I in the electron transport chain, with metformin and simultaneously inhibiting lactate extrusion [197]. This theoretical idea was further developed by other authors [198] and was proposed by Benjamin and Hall [199] to be used with AZD3965 acting as a lactic acid extrusion inhibitor. By itself, to have a significant effect AZD3965 may require doses that are toxic, and results are poor. Co-administered metformin may allow the use of a lower dose of AZD3965 and may achieve higher toxicity in cancer glycolytic cells. However, it will likely have no effect on cancer oxidative cells.

CYT-851 is an inhibitor of RAD51, the protein that participates in homologous recombination repair of DNA. It was discovered that CYT-851 is also an MCT inhibitor. It has been tested in phase I clinical trials in combination with capecitabine or gemcitabine [200]. In a cohort of 8 patients with heavily pre-treated solid tumors receiving capecitabine, there was 1 partial response and 7 cases of stable disease. In 6 patients of the gemcitabine cohort, there was one partial response, and 4 achieved stable disease (ClinicalTrials.gov identifier: NCT03997968). This drug also showed important activity in hematological tumors such as non-Hodgkin lymphoma [201]. Unfortunately, the company developing the drug has stopped operations due to financial problems.