Role of pH in Regulating Cancer Pyrimidine Synthesis

Replication is a fundamental aspect of cancer, and replication is about reproducing all the elements and structures that form a cell. Among them are DNA, RNA, enzymes, and coenzymes. All the DNA is doubled during each S (synthesis) cell cycle phase. This means that six billion nucleic acids must be synthesized in each cycle. Tumor growth, proliferation, and mutations all depend on this synthesis. Cancer cells require a constant supply of nucleotides and other macromolecules. For this reason, they must stimulate de novo nucleotide synthesis to support nucleic acid provision. When deregulated, de novo nucleic acid synthesis is controlled by oncogenes and tumor suppressor genes that enable increased synthesis and cell proliferation. Furthermore, cell duplication must be achieved swiftly (in a few hours) and in the midst of a nutrient-depleted and hypoxic environment. This also means that the enzymes participating in nucleic acid synthesis must work efficiently. pH is a critical factor in enzymatic efficiency and speed. This review will show that the enzymatic machinery working in nucleic acid synthesis requires a pH on the alkaline side in most cases. This coincides with many other pro-tumoral factors, such as the glycolytic phenotype, benefiting from an increased intracellular pH. An increased intracellular pH is a perfect milieu for high de novo nucleic acid production through optimal enzymatic performance.


Introduction
Folic acid was synthesized in 1937, and it was identified as a necessary factor for bone marrow function. Interestingly, folate-depleted feeding produced bone marrow damage similar to mustard gas. Sidney Farber, a pediatric pathologist working in Boston, used folic acid to treat anemia in children with acute leukemia. The results were devastating. Leukemia lit up and progressed very quickly. Farber concluded that it was necessary to find a substance that could do the opposite: deplete folate. He found the right chemist, Yellapragada Subbarao, who developed aminopterin, a folate antagonist that induced 1.
The de novo pathway starts with the precursor molecules, such as amino acids, CO 2 , NH 3 , and the sugar ribose-5-phosphate. Usually, proliferating and non-proliferating cells preferentially use the de novo pathway rather than the salvage mechanism [15,16].

2.
The salvage pathway is a process of recycling existing nucleotides and bases that originated in the breakdown of existing nucleic acids.
In this review, we focus only on the de novo synthesis of the pyrimidine structure. The de novo synthesis of pyrimidines and purines is similar in all living beings.
A few basic but essential concepts: In purine and pyrimidine biosynthetic pathways, an amino acid is a precursor of each path: Glycine for purines. Aspartate for pyrimidines.
Glutamine is the source of amino groups. In purine and pyrimidine de novo pathways, many enzymes are organized as large multi-enzyme complexes.
In both purine and pyrimidine de novo synthesis, a negative feedback loop regulates the number of molecules to be synthesized. The cellular pools of nucleotides are generally very small compared to the amount needed for DNA or RNA synthesis. Thus, nucleotide synthesis is an essential process for cell replication and growth. This becomes even more evident in highly proliferating cells, such as those found in tumors. Limiting nucleotide synthesis decreases proliferation and growth. Drugs that can inhibit nucleotide synthesis can impede, delay, or decrease malignant proliferation. Pyrimidines are mainly produced by tumor cells, but stromal cells, such as macrophages and cancer-associated fibroblasts, can also produce them. In pancreatic cancer, it has been found that pyrimidines produced by macrophages were able to create resistance to gemcitabine treatment [17]. Glutamine transporters that provide glutamine to the cell (glutamine is a nitrogen donor for pyrimidines) alkalinize the intracellular milieu by simultaneously exporting protons [18]. In this review, we focus only on the de novo synthesis of the pyrimidine structure. The de novo synthesis of pyrimidines and purines is similar in all living beings.
A few basic but essential concepts: ► In purine and pyrimidine biosynthetic pathways, an amino acid is a precursor of each path: ■ Glycine for purines. ■ Aspartate for pyrimidines. ► Glutamine is the source of amino groups. ► In purine and pyrimidine de novo pathways, many enzymes are organized as large multi-enzyme complexes. ► In both purine and pyrimidine de novo synthesis, a negative feedback loop regulates the number of molecules to be synthesized. ► The cellular pools of nucleotides are generally very small compared to the amount

Pyrimidine De Novo Synthesis
Pyrimidine nucleotides are essential building blocks not only for nucleic acid synthesis but DNA repair and other cell functions as well [19]. Pyrimidine biosynthesis is initiated and regulated by a multienzymatic complex, CAD (CPS-II, aspartate transcarbamoylase, and dihydroorotase), that harbors the enzymes required for the first three steps of biosynthesis. This association of enzymes in one big polypeptide is an unusual finding in superior eukaryotes. This explains the difficulties found in characterizing and defining the properties of each of the enzymatic domains in a separate manner. In addition, CAD Pyrimidine nucleotides are essential building blocks not only for nucleic acid synthesis but DNA repair and other cell functions as well [19]. Pyrimidine biosynthesis is initiated and regulated by a multienzymatic complex, CAD (CPS-II, aspartate transcarbamoylase, and dihydroorotase), that harbors the enzymes required for the first three steps of biosynthesis. This association of enzymes in one big polypeptide is an unusual finding in superior eukaryotes. This explains the difficulties found in characterizing and defining the properties of each of the enzymatic domains in a separate manner. In addition, CAD self-assembles in hexameres. CAD, which controls the pathway, is in turn, controlled by pro-proliferation and pro-biosynthesis pathways ( Figure 2).

Figure 2.
Signaling pathways that control CAD activity. This diagram is based on references [20][21][22][23]. CAD initiates de novo pyrimidine synthesis. CAD is activated by the activation of growth factors binding growth factor receptors and triggering the MAP kinases pathway. This activation takes place at the beginning of the S phase in the cell cycle. After the S phase is over, CAD is deactivated by protein kinase A (PKA) phosphorylation. The lower panel shows that phosphorylation of the Thr456 residue activates the enzyme, while phosphorylation of Ser1406 acts in the opposite way. The diagram also shows the three enzymes that form CAD. In this regard, the activation and deactivation residues form part of the first enzyme that initiates synthesis, namely, CPS (carbamoyl phosphate synthetase). DHO: dihydroorotase; ATC: aspartate transcarbamoylase. Signaling pathways that control CAD activity. This diagram is based on references [20][21][22][23]. CAD initiates de novo pyrimidine synthesis. CAD is activated by the activation of growth factors binding growth factor receptors and triggering the MAP kinases pathway. This activation takes place at the beginning of the S phase in the cell cycle. After the S phase is over, CAD is deactivated by protein kinase A (PKA) phosphorylation. The lower panel shows that phosphorylation of the Thr456 residue activates the enzyme, while phosphorylation of Ser1406 acts in the opposite way. The diagram also shows the three enzymes that form CAD. In this regard, the activation and deactivation residues form part of the first enzyme that initiates synthesis, namely, CPS (carbamoyl phosphate synthetase). DHO: dihydroorotase; ATC: aspartate transcarbamoylase. Figure 3 shows a full view of the de novo synthesis of pyrimidines from their origin in glutamine up to the pyrimidine uridine monophosphate. The diagram shows the origin of the different parts of a uracil molecule, which receives contributions from aspartic acid, glutamine, and CO 2 (converted into HCO 3− by cytoplasmic carbonic anhydrases). The major amino acid contributors are aspartic acid and glutamine. Each of them also donates an amino group. Glutamine contributes to an NH 2 group (see right panel of Figure 3).

The Pyrimidine Synthesis Pathway
CPS II: carbamoyl phosphate synthetase II; ATCase: aspartate transcarbamoylase; OPRT: orotate phosphoribosyltransferase; PRPP: phosphoribosyl diphosphate (PRPP). This whole synthetic process consumes seven ATP molecules. Figure 3 shows a full view of the de novo synthesis of pyrimidines from their origin in glutamine up to the pyrimidine uridine monophosphate. The diagram shows the origin of the different parts of a uracil molecule, which receives contributions from aspartic acid, glutamine, and CO2 (converted into HCO 3− by cytoplasmic carbonic anhydrases). The major amino acid contributors are aspartic acid and glutamine. Each of them also donates an amino group. Glutamine contributes to an NH2 group (see right panel of Figure 3). CPS II: carbamoyl phosphate synthetase II; ATCase: aspartate transcarbamoylase; OPRT: orotate phosphoribosyltransferase; PRPP: phosphoribosyl diphosphate (PRPP). This whole synthetic process consumes seven ATP molecules.

The Steps in De Novo Pyrimidine Synthesis
Step 1

The Steps in De Novo Pyrimidine Synthesis
Step 1: In the presence of ATP, L-glutamine and bicarbonate are converted to carbamoyl phosphate (CAP) by carbamoyl phosphate synthetase II (CPS-II) in the cytoplasm (Figure 4). ture. Uracil is being used as an example.

The Steps in De Novo Pyrimidine Synthesis
Step 1 In the presence of ATP, L-glutamine and bicarbonate are converted to carbamoyl phosphate (CAP) by carbamoyl phosphate synthetase II (CPS-II) in the cytoplasm ( Figure  4). There are two types of CPS: (1) CPS-I is an intra-mitochondrial enzyme; (2) CPS-II is cytosolic and is the enzyme that participates in de novo pyrimidine biosynthesis. This is the rate-limiting enzyme in pyrimidine biosynthesis.
CPS-II is upregulated in many cancers [24], and mainly in hepatoma [25][26][27] The optimal pH for CPS-II activity is 7.4 [28]. Normal cells have an intracellular pH between 7.1 and 7.2. This means that to achieve an efficient function of this rate-limiting enzyme, the cytoplasm requires a higher pH. Interestingly, tumors raise their intracellular There are two types of CPS: (1) CPS-I is an intra-mitochondrial enzyme; (2) CPS-II is cytosolic and is the enzyme that participates in de novo pyrimidine biosynthesis. This is the rate-limiting enzyme in pyrimidine biosynthesis.
CPS-II is upregulated in many cancers [24], and mainly in hepatoma [25][26][27] The optimal pH for CPS-II activity is 7.4 [28]. Normal cells have an intracellular pH between 7.1 and 7.2. This means that to achieve an efficient function of this rate-limiting enzyme, the cytoplasm requires a higher pH. Interestingly, tumors raise their intracellular pH to 7.4-7.5 [29]. The difference seems small, but it must be remembered that pH is a logarithmic function, thus the difference represents an important increase in proton concentration. When the pH reaches the optimal point for an enzyme, this means that the enzyme is working at its maximum efficiency and speed.
The end product of the events leading to pyrimidine synthesis is uridine monophosphate (UMP), which inhibits CPS-II via a negative feedback loop (not shown in Figure 3).
CPS-II activators. Many activators, such as ATP and phosphoribosyl diphosphate (PRPP), stimulate the expression of this enzyme [30]. mTORC1 is a key modulator that upregulates the whole pathway [31] and, through its downstream protein S6K1, induces the expression of all the enzymes involved in de novo pyrimidine synthesis, including CPS-II [32] and the other two enzymes that participate in steps 2 and 3. Actually, the three enzymes that catalyze the first three steps of pyrimidine synthesis, namely, CPS-II, aspartate transcarbamoylase, and dihydroorotase, form an enzymatic complex that is known by the acronym CAD, as mentioned above.
CPS-II Inhibitors. CPSII is inhibited by acivicin, an analog of glutamine [33]. In this respect, a glutamine-restricted diet might limit pyrimidine biosynthesis, interrupting the nucleic acid synthesis.
The impact of pH on ATCase (aspartate transcarbamoylase) activity depends on the substrate concentration: the optimal action occurs at an alkaline pH of 8.5 in bacteria [34] and at around 9 in rat liver [35]. Here again, we find that a high intracellular pH favors the synthesizing steps. respect, a glutamine-restricted diet might limit pyrimidine biosynthesis, interrupting the nucleic acid synthesis.
The effect of pH on dihydroorotase in cancer cell lines is a controversial and debated issue. However, the current concept is that dihydroorotase is driven forward by an acidic pH, while the reversible reaction (e.g., the formation of CAA) reaches its optimal efficiency at an alkaline pH [43][44][45][46][47][48].
At this point, the three enzymes forming CAD have catalyzed the ring structure that will form pyrimidines in the next steps from glutamine, CO 2 , and aspartate. The optimal pH for the first two enzymes is above 7.4, while the third enzyme requires a lower pH (around 7 or lower).
Step 3: Carbamoyl aspartic acid (CAA) is converted to dihydroorotic acid (DHOA droorotase (EC 3.5.2.3), which is also known as dihydroorotate hydrolase, bamoylaspartic dehydrase. Dihydroorotase is a metalloenzyme that catalyzes th ble conversion of carbamoyl aspartic acid into dihydroorotic acid and closes the ure 6). The effect of pH on dihydroorotase in cancer cell lines is a controversial an issue. However, the current concept is that dihydroorotase is driven forward by pH, while the reversible reaction (e.g., the formation of CAA) reaches its op ciency at an alkaline pH [43][44][45][46][47][48].
At this point, the three enzymes forming CAD have catalyzed the ring stru will form pyrimidines in the next steps from glutamine, CO2, and aspartate. Th pH for the first two enzymes is above 7.4, while the third enzyme requires a (around 7 or lower).
Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and deferoxamine, metal c ducing HIF-1α expression, show inhibitory effects on the CAD multienzymatic com This seems paradoxical because HIF-1 alpha is a strong tumor driver.
Therefore, although the dihydroorotase appears to be an enzyme that do hance the pyrimidine biosynthetic pathway, some data suggest that the same supports tumorigenesis [50]. However, it was also found that CAD downreg Wnt/Beta catenin pathway in colon cancer cells, decreasing migration [51]. Therefore, although the dihydroorotase appears to be an enzyme that does not enhance the pyrimidine biosynthetic pathway, some data suggest that the same CAD also supports tumorigenesis [50]. However, it was also found that CAD downregulates the Wnt/Beta catenin pathway in colon cancer cells, decreasing migration [51].

Dihydroorotase Inhibitors
Dihydroorotase seems to interact with the thymidylate synthase inhibitor 5-fluorouracil [52]. However, this needs further research. Plumbagin, a natural product obtained from the carnivorous plant Nepenthes miranda, has shown strong competitive inhibition, causing apoptosis in tumors [52]. Analogs of carbamoyl aspartate have also shown inhibitory effects [53].
Step 4: Dihydroorotic acid (DHOA) is converted to orotic acid (OA) via dihydroorotate dehydrogenase. This is the only redox reaction in the de novo biosynthesis pathway of pyrimidine nucleotides.
The optimal pH for this is around 8 [64,65]. This is logical because this enzyme carries out its work in the mitochondria [66], which have a higher pH than the cytoplasm.
Leflunomide is a drug used to treat rheumatoid arthritis that has shown significant efficacy against dihydroorotate dehydrogenase [67], and it could be considered as an anticancer drug candidate [68][69][70].
Other inhibitors, such as brequinar sodium, atovaquone, and ML390. have also been identified (See Box 1).
Phosphoribosyl pyrophosphate originates in the first part of the oxidative phase of the pentose phosphate pathway [85]. Therefore, this step of the pentose phosphate pathway (PPP) branches to merge and/or amalgamate with de novo pyrimidine biosynthesis. Interestingly, there is a shared regulation of the glycolytic and PPP pathways by intracellular pH pHi. Indeed, as with glycolysis, the optimal activity of the enzymes driving the PPP occurs at an alkaline pHi, which is compatible with the cytoplasmic pH of cancer cells (Figures 8 and 9).
Step 4: Dihydroorotic acid (DHOA) is converted to orotic acid (OA) via dihydroorotat hydrogenase. This is the only redox reaction in the de novo biosynthesis pathway o rimidine nucleotides.

Figure 7.
Step 4. Chemical reaction catalyzed by dihydroorotate dehydrogenase. The pyrim ring is initially formed as orotate. Then, in the next step (step 5), it is attached to ribose phos (which is generated in the pentose phosphate pathway) and finally converted to the pyrim nucleotides that will be used for DNA and RNA synthesis.
The optimal pH for this is around 8 [64,65]. This is logical because this enzyme ca out its work in the mitochondria [66], which have a higher pH than the cytoplasm.
Leflunomide is a drug used to treat rheumatoid arthritis that has shown signif efficacy against dihydroorotate dehydrogenase [67], and it could be considered as a ticancer drug candidate [68][69][70].

Figure 7.
Step 4. Chemical reaction catalyzed by dihydroorotate dehydrogenase. The pyrimidine ring is initially formed as orotate. Then, in the next step (step 5), it is attached to ribose phosphate (which is generated in the pentose phosphate pathway) and finally converted to the pyrimidine nucleotides that will be used for DNA and RNA synthesis.
Brequinar sodium (a quinoline-carboxylic acid sodium salt) is a small molecule that acts as a specific inhibitor of dihydroorotate dehydrogenase, the fourth enzyme of the de novo pyrimidine biosynthetic pathway. Brequinar sodium inhibits dihydroorotate dehydrogenase that has ubiquinone as a cofactor but not when NAD is the cofactor [71].
Brequinar has shown a dose-dependent antineoplastic and immunosuppressant activity against different tumor models [72] and went through phase I and II clinical trials during the 1990s [73][74][75][76]. A narrow therapeutic window and high myelotoxicity led this drug to oblivion. In 2018, new studies confirmed that brequinar was not active at lower doses in solid tumors but was quite effective in leukemia [77]. This different behavior in non-solid tumors seems to confirm two concepts: Solid tumors have a higher level of pyrimidines that require toxic doses of brequinar, while leukemias respond to lower doses due to a decreased pool of uridine; The pyrimidine synthetic pathway is somehow related to the inhibition of myeloid differentiation because the inhibition of dihydroorotate dehydrogenase overcame a differentiation blockade in acute myeloid leukemia in vivo [78].
Interest in brequinar as a complementary drug for cancer treatment in both non-solid and solid tumors has been revived after 20 years [79][80][81][82][83]. The fact that brequinar has shown the ability to induce ferroptosis and to increase the ferroptotic effect of drugs such as sulfasalazine [84] is particularly interesting. Importantly, OPRT is an enzyme that contributes to the conversion of 5-FU into fdUMP, which is the active form of 5-FU [86]. Resistance to 5-FU seems to be associated with low OPRT expression [87,88]. OPRT enhances the chemotherapeutic response to 5-fluorouracil (5-FU) [89,90].
OPRT is overexpressed in many tumors, such as bladder cancer [91], and its expression is correlated with recurrence. Further, OPRT is useful as a marker of prognosis and for predicting chemosensitivity [90,92,93].
The optimal pH of the OPRT for the forward reaction is 8, while for the reverse reaction it ranges between 6.5 and 7.5 [94].  The optimal pH of the OPRT for the forward reaction is 8, while for the reverse reaction it ranges between 6.5 and 7.5 [94].
There is not much information about the expression of ODCase in tumors. How pyrazofurin and 6-Aza uridine 5′-monophosphate are examples of ODCase inhi with antitumor properties [99,100].

Step 7 (Uracil Formation):
Two consecutive phosphorylations take place: steps 7.1 and 7.2 ( Figure 11). There is not much information about the expression of ODCase in tumors. However, pyrazofurin and 6-Aza uridine 5 -monophosphate are examples of ODCase inhibitors with antitumor properties [99,100].
Step 7.1 is a crossroad of de novo pyrimidine biosynthesis, and it can follow two main branches.
Here, the optimal pH is 8 [113]. NM23 (nucleoside-diphosphate kinase), which is known as a metastasis suppressor [114], produces nucleoside-diphosphate kinase, although in some tumors (e.g., melanoma, breast, and colon), its expression is associated with low metastatic potential. In others (neuroblastoma and osteosarcoma), it is related to a decrease in survival rate [115]. Therefore, its role is unclear. One of the possible explanations is that, while NM23 protein suppresses metastasis, it does not alter the growth of primary tumors [116].
Step 7.1 is a crossroad of de novo pyrimidine biosynthesis, and it can follow two main branches.
Here, the optimal pH is 8 [113]. NM23 (nucleoside-diphosphate kinase), which is known as a metastasis suppressor [114], produces nucleoside-diphosphate kinase, although in some tumors (e.g., melanoma, breast, and colon), its expression is associated with low metastatic potential. In others (neuroblastoma and osteosarcoma), it is related to a decrease in survival rate [115]. Therefore, its role is unclear. One of the possible explanations is that, while NM23 protein suppresses metastasis, it does not alter the growth of primary tumors [116].

Step 8 (Cytosine Formation):
UTP is converted to cytosine triphosphate via cytosine triphosphate synthase (CTP synthase), also known as UTP: ammonia ligase. Glutamine is also crucial in carrying out this step [118,119]. Therefore, the presence of glutamine is essential for starting de novo pyrimidine biosynthesis and forming cytosine ( Figure 12).
The optimal pH of CTP synthase is around 8 [120]. CTP synthase expression is markedly increased in very rapidly growing tumors [121,122]. Moreover, a CTP synthase abnormality may be associated with resistance [123].
The steps discussed above show the synthesis of the two pyrimidine bases that form the nucleotides for RNA, namely, the uracil and cytosine nitrogen bases and the uridine and cytidine nucleotides, respectively.

Step 9 (Thymine Formation):
DNA has thymine instead of uracil. In this step, UDP is reduced to produce dUDP (deoxyuridine diphosphate) via ribonucleotide reductase, which converts NADPH to NADP + (Figure 13).
Steps 9 and 10 depend on NADPH and its possible cellular level; therefore, this reaction might occur in the cytoplasm rather than the nucleus. Furthermore, it might interact with additional pathways, e.g., the pentose phosphate pathway (PPP) and the methylglyoxal pathway [85,125]. Therefore, it raises more critical questions: Does de novo pyrimidine biosynthesis occur in the nucleus or cytoplasm or even in the mitochondrion? Does the nucleus have its own de novo pyrimidine biosynthesis pathway, while the mitochondrion has a separate biosynthetic pathway?
Step 8 (Cytosine Formation): UTP is converted to cytosine triphosphate via cytosine triphosphate synthase (CTP synthase), also known as UTP: ammonia ligase. Glutamine is also crucial in carrying out this step [118,119]. Therefore, the presence of glutamine is essential for starting de novo pyrimidine biosynthesis and forming cytosine (Figure 12).
The optimal pH of CTP synthase is around 8 [120]. CTP synthase expression is markedly increased in very rapidly growing tumors [121,122]. Moreover, a CTP synthase abnormality may be associated with resistance [123].
Cyclopentenyl cytosine is an inhibitor of CTP synthase [121], while GTP is a CTP synthase activator [120,122,124]. The steps discussed above show the synthesis of the two pyrimidine bases that form the nucleotides for RNA, namely, the uracil and cytosine nitrogen bases and the uridine and cytidine nucleotides, respectively.
Step 9 (Thymine Formation): DNA has thymine instead of uracil. In this step, UDP is reduced to produce dUDP (deoxyuridine diphosphate) via ribonucleotide reductase, which converts NADPH to NADP + (Figure 13) Step 9: The sugar, ribose, is reduced to deoxyribose, forming dUDP.
Step 10: dUDP is dephosphorylated, yielding dUMP (deoxyuridylic acid or deoxyuridylic acid or deoxyuridylate) (see Figure 14). Steps 9 and 10 depend on NADPH and its possible cellular level; therefore, this reac tion might occur in the cytoplasm rather than the nucleus. Furthermore, it might interac with additional pathways, e.g., the pentose phosphate pathway (PPP) and the methylgly oxal pathway [85,125]. Therefore, it raises more critical questions: Does de novo pyrim dine biosynthesis occur in the nucleus or cytoplasm or even in the mitochondrion? Doe the nucleus have its own de novo pyrimidine biosynthesis pathway, while the mitochon drion has a separate biosynthetic pathway?
Because this step supports DNA synthesis, NADPH is crucial for DNA synthesis. Ribonucleotide reductase (RR), also known as ribonucleoside diphosphate reductas (rNDP), is a ubiquitous cytosolic enzyme with an optimal pH of around 7.5 [126].
Step 11: dUMP is converted to dTMP via thymidylate synthase (TS), which requires 5,10 methylenetetrahydrofolate as a co-enzyme. Then, dTMP can be phosphorylated to dTD and dTTP, which is the molecule used for DNA synthesis (Figure 15) [146].

Discussion
The de novo biosynthesis of pyrimidine is a complex process. Each step of the bio synthetic pathway is influenced by many factors. Part of these factors includes the redo state of the cell NADPH/NADP + , ATP, GTP levels, the optimal pH for the related enzyme the availability of glutamine and aspartate among others. The synthesis of cytosine an Ribonucleotide reductase (RR), also known as ribonucleoside diphosphate reductase (rNDP), is a ubiquitous cytosolic enzyme with an optimal pH of around 7.5 [126].
Step 11: dUMP is converted to dTMP via thymidylate synthase (TS), which requires 5,10methylenetetrahydrofolate as a co-enzyme. Then, dTMP can be phosphorylated to dTDP and dTTP, which is the molecule used for DNA synthesis (Figure 15) [146].

Discussion
The de novo biosynthesis of pyrimidine is a complex process. Each step of the biosynthetic pathway is influenced by many factors. Part of these factors includes the redox state of the cell NADPH/NADP + , ATP, GTP levels, the optimal pH for the related enzymes, the availability of glutamine and aspartate among others. The synthesis of cytosine and uracil might occur at a higher pH compared to thiamine.
Pyrimidine is not limited to the biosynthesis of nucleic acid. It also plays crucial roles in tumor metabolism. Therefore, can pyrimidine act as an oncometabolite?
Interestingly, the optimal pH of most enzymes participating in this pathway is slightly higher than that of normal cells. An increased intracellular pH is usual in malignancies. Table 1 shows these optimal pH levels and in all cases they are above 7.3, except for thymidylate synthase. The protumoral effects of an alkaline or hyperalkaline intracellular milieu has been extensively investigated and confirmed [6,158,159].  [160] For example, in the case of ribonucleotide reductase (RR), the key enzyme to produce nucleotides for DNA, indirect evidence shows that the optimal pH is between 7.5 and 8. These data come from the fact that at this pH range less iron is needed for the maximum activity of the enzyme [160]. However, with high levels of iron, the optimum pH is in the range of 6.5-7 [161]. RR requires iron and folic acid for its adequate functioning. This case shows the difficulties in correctly determining the optimal pH of many of the enzymes involved in nucleotide synthesis. Furthermore, tumors are high iron consumers.
Hormesis is a biphasic pharmacological dose-dependent response characterized by stimulation by a low dose and inhibition by a higher dose [162,163]. Moreover, hormesis is a redox-dependent process that might support cell death (apoptosis), such as with reactive oxygen species (ROS), an example of a stressful condition; however, the release of ROS at different concentrations supports genetic mutations.
Heat shock proteins (HSPs) are proteins (molecular chaperones) synthesized in response to stressful conditions. They play a crucial role in protein maturation and folding, so they are defense proteins that support cellular survival in stressful conditions. Therefore, it will not be surprising if many tumor cells are found to be HSP-dependent. It has been shown there is an association between HSPs, cancer, and pyrimidine synthesis. Hence, one of the future directions in managing cancer is further studying the de novo pyrimidine biosynthesis-HSP interactions and the potential opportunity that offers a new therapeutic window in managing cancer [164,165].
This review shows another face of pH deregulation and its link with increased proliferation. Hypothetically, we may assume that by lowering intracellular pH the activity of these enzymes may be slowed down, thus decreasing pyrimidine synthesis. Table 1 also shows the possible inhibitors for each enzyme.

1.
Cancer cells have an increased rate of pyrimidine synthesis, as expected in a cell that must duplicate all its DNA and RNA at an accelerated pace.

2.
The enzymes participating in pyrimidine synthesis have an optimal efficiency at a pH that is higher than the intracellular pH of normal cells.

3.
This increased intracellular pH is constantly found in malignant cells as part of the pH paradigm.

4.
On a theoretical basis, we may assume that lowering the intracellular pH will hamper the efficiency of pyrimidine synthesis and decrease tumor proliferation. There is direct and indirect evidence that intracellular acidification is a valid method for complementing standard treatment schemes.