pH Deregulation as the Eleventh Hallmark of Cancer

By Tomas Koltai, Larry Fliegel, Stephan J. Reshkin and 4 more

pH Deregulation as the Eleventh Hallmark of Cancer presents key concepts about pH deregulation in a concise and straight-forward manner. The book discusses topics such as pH regulation and metabolism, sodium hydrogen exchanger, monocarboxylate transporter, V-ATPase proton pump, carbonic anhydrases, and voltage gated sodium channels. In addition, it covers clinical and therapeutic implications and future perspectives. This is a valuable resource for researchers, oncologists, students and members of the biomedical and medical fields who want to learn more about the role of pH deregulation in cancer treatment.

pH deregulation can improve the outcome of classical treatments without adding toxicity to them, and the book shows that treating the pH peculiarities of cancer is simple and can be performed with existing drugs. Based on the classification of tumor malignancy in ten hallmarks, the authors put pH deregulation at the spotlight and separated from metabolic reprogramming due to its impact on all other hallmarks, proposing it as an additional characteristic to evaluate and fight cancer.

• Proposes that pH deregulation should be considered as an independent hallmark of cancer from metabolic reprogramming due to its impact on all other hallmarks (based on seminal work of Hanahan and Weinberg)
• Explains basic issues of cancer pH deregulation and its consequences in a simple and concise manner
• Discusses the subject from the start with very elementary concepts on pH and pH regulation to help readers understand key concepts without proper background
• Presents key concepts through original illustrations and table for easy comprehension

• Language: English
• Publisher: Academic Press
• Release date: Jul 3, 2023
• ISBN: 9780443154621

Tomas Koltai

Tomas Koltai is Board Certified Specialist in Medical Oncology (1985), PhD in Chemistry (1997) and Master’s in Sciences in Molecular Oncology, University of Buenos Aires (2011). He has vast experience on cancer treatment at several positions: staff oncologist, Sanatorio Mater Dei, Argentina (1985-1990); Head of Oncology, Central Hospital of the Centro Gallego de Buenos Aires (1990-2000) and Medical Director, at the same institution (2000-2002); Head of the Department of Chemotherapy, National Social Services for Retirees, Argentina (2002-2014); Head of the Department of Oncology, Social Services of the National Food Workers Union, Argentina (2010-2014) and Medical Director at the same institution (2014-2016). He has authored several seminal publications on cancer and created the new “triple-edged cancer treatment”.

Book preview

Chapter 1: pH Regulation and metabolism: Basic concepts

Abstract

pH is a strictly

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Differences between aminopterin and folic acid arrows show the differences.

controlled parameter in cell homeostasis. Buffer systems, proton extruders, membrane transporters and enzymes are the main tools that maintain a tight range of compartmentalized pH which is an invisible participant in all cellular events. pH plays an important role from the beginning of malignant transformation to cell death,. Malignant cells deregulate the intricate mechanisms that govern pH homeostasis and create a new paradigm, inversion of the pH gradient, in which intracellular pH is higher than the extracellular. This gives a growth and progression advantage to malignant cells. This chapter discusses the basic concepts of pH chemistry, the redox process, metabolism, and ion, glucose and lactate transport through the cell membrane.

Keywords

pH; pH regulation; Buffers; Proton extruders; pH gradient inversion

pH regulation

Water is the molecular compound that represents the core component of all living beings. This means that there is no life without water. Water is the universal solvent inside all cells and extracellular fluids.

Therefore, studying the properties of this molecular compound is essential to understanding how organisms function in health and disease.

At first glance, water would seem to be an inert compound, however, this is not so, because it is a very weak electrolyte that participates in almost all the biochemical processes that take place in living organisms. What makes it a weak electrolyte is that water molecules collide and this produces a transfer of a hydrogen ion from one molecule to the other. As a consequence, there will be positively charged ions (H3O+) and negatively charged hydroxide ions (OH−).

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An aqueous solution, whether in the cell or in the intercellular space, can be characterized by the concentration of charged ions: positively with hydrogen ions and negatively with hydroxide ions. Many chemical reactions in living creatures involve exchanges of hydrogen ions. Acidity or alkalinity can affect the structure and chemical reactivity of molecules, therefore, cells must constantly maintain an acid-base balance that is appropriate for these reactions.

Concentration of protons ([H+]) and pH

The concentration of protons (or H+ or hydrogen ions) in a solution is usually a very small number such as 0.0000001 M and this creates some conceptual difficulties for understanding its meaning at a glance. To simplify the concept, Søder P. L. Sørensen created a new way of expressing a solution’s acidity or alkalinity: the pH (pondus hydronium or hydrogen power) [1–3].

pH is the negative of the logarithm of H+ concentration ([H+]).

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Water dissociates weakly into H+ and OH− ions. There will always be a balance between H+ and OH− ions concentration in pure water.

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The amount of water molecules in 1 L of pure water at 25°C is always constant: 55.5 mol. This is because 1 L of pure water at 25°C weighs 1000 g. The molarity is the number of grams per liter divided by the molecular weight (18) which gives us 55.5 mol/L. And the dissociation of water molecules is also a constant. When we measure the amount of H+ions in distilled water it is 10-7 per mol, which matches perfectly the amount of OH−ions which is also 10-7 per mol.

Therefore, when we say that the pH of pure water is 7 it means that we will find 0.0000007 mol of ionized hydrogen in 1 mol of water. This is not a theoretical number but an empirically proven one. Handling the concept of a number such as 0.0000007 is complex. Therefore, we use pH and the pH of pure water becomes 7 (negative logarithmic value of [H+]). This number represents a state in which the [H+] and [OH−] are exactly the same. Therefore, pure water is neither acidic nor alkaline: it is neutral.

If we add an acid substance such as H2SO4 (sulfuric acid) to water the concentration of H+ will increase, for example to 10-6 and the pH of the solution will be 6. A pH below 7 indicates acidity and over 7 alkalinity. Therefore, pH is a counterintuitive virtual creation: the lower the pH the greater the acidity, and vice versa, the higher the pH the lesser the acidity.

pH may sometimes be misleading in the field of molecular biology. If we analyze Fig. 1 we can see that extracellular pH is 7.3–7.35 while cytoplasmic pH is 7.15–7.25. The difference seems negligible. Actually, it is not. It represents a big difference in [H+]. It is the nature of the logarithmic scale used for pH that makes it look negligible.

Fig. 1

Fig. 1 Compartments of a complex multicellular organism. The numbers shown in each compartment represent the approximate pH usually found in the compartment under normal physiological conditions [4]. Endosomes increase their acidity on the basis of their proximity to the cell surface. Each organelle has a pH that is the most appropriate for its functions.

Let’s examine this apparent negligibility: if the acidity of a solution goes up 10-fold its pH goes down by only 1 unit. Therefore, the difference of 0.1–0.3 units of pH between extracellular substance and cytoplasm represents a much more important difference than what it apparently seems. For example, a pH of 7, is a concentration of 1 × 10−7 mol/L of H+. A pH of 7.3 is 5.0 × 10−8 mol/L of H+, or only half as much H+’s in solution with a change in pH of only 0.3.

In summary pH indicates the concentration of hydrogen ions (protons, [H+]) dissolved in a solution. A solution with low pH has a higher concentration of protons than a solution with a higher pH and vice versa.

Some basic definitions

An acid is a substance that has the ability to give away protons or hydrogen ions (H+) and/or accept an electron pair. Acids have a bonded hydrogen atom which, upon dissociation, yields a cation and anion in water. A high concentration of hydrogen ions produced by an acid, lowers the solution’s pH. Acids are proton donors. Strong acids completely dissociate in water, while weak acids dissociate only partially.

pH homeostasis

pH is strictly regulated in complex multicellular organisms. There is a pH difference among the different compartments of such an organism [5,6].

(Figs. 1 and 2).

Fig. 2

Fig. 2 The two compartments of mitochondria, namely the matrix and the intermembrane space have different pH levels that create an intra-mitochondrial pH gradient essential for ATP (adenosine triphosphate) production through the electron transfer chain and the maintenance of the mitochondrial membrane potential [ 7– 9].

What is the need for these different pH compartments?

The evolutionary process of compartmentalization seems to be related to the need to have an optimum environment for the various metabolic pathways. These pathways have specific requirements, including different pH levels in order to perform their tasks in the best possible manner [10].

A simple example will show the importance of the different pH levels in the compartments. Many biological processes are dependent on the binding of proteins or the binding of a protein to DNA. For this to occur, among many other conditions is the spatial conformation of the protein. This in turn depends, among many other conditions on the state of protonationa of the molecules involved, and this protonation depends on the concentration of protons which is the same as the pH [11].

pH compartmentalization is constantly and rigorously maintained in multicellular organisms: this results in pH homeostasis [12].

Cells have many overlapping mechanisms to do this task. Basically, we can divide them into two groups:

(a)intra and extracellular buffering systems;

(b)specialized channels, transporters, pumps and exchangers located in plasma and intracellular membranes.

Quick pH changes are mainly handled by the buffering system. It is the first response to a pH change that may be unfavorable for cell function and even threaten its survival. However, this system is finally depleted if the pH challenge is intensive and extended in time. This is the point where proton extruders (channels, transporters, pumps, and exchangers) take the lead. This scheme applies to both normal and malignant tissues. Actually, the specialized channels, transporters and exchangers start working well before dangerous buffer system depletion occurs, and complement the action of the buffers. However, they work differently in one case and the other.

The intra- and extracellular pH balance required for cancer to develop and progress differs from that of normal cells as will be explained in detail in this chapter. The small difference between the pH of the extracellular space and the cytoplasm (range 0.1–0.3) in normal cells, where extracellular pH (pHe) is higher than intracellular pH (pHi), undergoes striking changes in cancer:

(a)intracellular pH becomes higher (more alkaline) than extracellular pH;

(b)extracellular pH becomes more acidic (usually well below 7);

(c)the difference between them (ΔpH = pHe − pHi), which in a normal cell is in the range of 0.1–0.3 becomes close to −1 in cancer. This means that [H+] in the extracellular space is 10 times higher than in the cytoplasm.

This phenomenon is known as the inversion of the pH gradient.

To achieve this inversion all the proteins engaged in proton export (channels, pumps, transporters, exchangers, enzymes) become over-active and/or over-expressed.

Whether normal or malignant, cells are permanently striving against acids. These acids are the consequence of metabolism. For this reason, cells have developed many, sometimes overlapping, mechanisms to get rid of the excess acids (actually the excess protons). Cancer cells have a metabolism that produces more acids than normal cells, and in spite of this, they have the ability not only to dispose of them, but to increase intracellular pH above normal levels, and to establish an inverted gradient generating extracellular acidity. This extracellular acidity is the direct consequence of the increased amount of protons extruded from the malignant cells, and it shows the failure of extracellular buffers to compensate for it.

In spite of this evident difference of pH in cancer compared with normal cells, they share one common feature: they both constantly have to deal with intracellular production of acids.

However, while normal cells deal with this problem using their buffering systems and proton extruders, cancer cells need to over-express or over-activate their proton extruders in order to avoid excessive intracellular acidification. And they are very successful in this, to the point that they reach a higher intracellular pH while they dump a huge load of acids into the extracellular substance surrounding them. The buffer systems are absolutely overwhelmed in cancer and can do little to prevent the process.

Buffers

A buffer is a substance that when added to water dissociates into either a weak acid and its salt, or a weak base and its salt, and has the ability to moderate changes in pH. They serve to rapidly absorb or release H+ ions, in order to preserve the compartment’s ideal pH when it is challenged by pH-altering compounds. However, this first aid mechanism of buffers is short lived if defiance is prolonged. The reservoir of buffers is limited and ultimately it may be overwhelmed. Other pH regulators are necessary for the long run.

Cells have many different buffering systems. Buffers contribute to maintaining a stable pH in a solution, because they can neutralize small quantities of additional acids or bases. The small amount they can neutralize avoiding a change in pH is called the buffering capacity. When this capacity is overrun the pH will change, unless the second system of the pH homeostasis apparatus, the proton extruders, does its task.

Normal cells handle pH homeostasis mainly with the buffering system and under normal metabolic conditions, they also use some collaboration from the proton extruders. Cancer cells, on the other hand, with a different metabolic profile require a very important activity from the proton extruders and the buffering capacity is always overwhelmed from the very beginning of cancerization. The proton extruding systems are overexpressed or overactive in cancer (Fig. 3).

Fig. 3

Fig. 3 The bicarbonate/carbonic acid buffer system, the most important in complex organism, modulating pH. The bicarbonate anion can bind one H + increasing the pH while the carbonic acid, a weak acid can release H + acidifying the solution and decreasing pH. In blood, where pH is maintained around 7.4, the bicarbonate and carbonic acid ratio is 20:1, and is the most suitable for the buffering function.

Maintaining a physiological range of pH in the different compartments (Fig. 1) is of capital importance for complex multicellular organisms to function. A very low or very high pH will substantially modify the enzymes’ activity. Furthermore, enzymes are exquisitely pH. When the pH shifts beyond a very narrow range, the cell will eventually die, due to the enzymes’ inability to function. Therefore, in addition to the buffering system, organisms have developed a sophisticated system of channels, transporters and exchangers to prevent these risks.

pH and enzymatic activity

There is an optimum pH for every enzyme. That is why pH homeostasis is so important for cells. The rate (speed and amount of catalyzed substrate) of a chemical reaction is influenced by the structure/shape of the enzyme. pH changes can modify the enzymatic (and/or the substrate) structure/shape affecting the rate of reaction or even reducing it to zero (Figs. 4 and 5).

Fig. 4

Fig. 4 Relation between pH and the rate of a chemical reaction. Most biological reactions occur within a narrow range of pH. Modifications in the pH have an important impact on the biological functions. Protein folding and enzymatic activities are pH-dependent. Since cancer cells proliferation requires enzymatic activity, tumor growth and reproduction are dependent on pH.

Fig. 5

Fig. 5 The maximum activity of two different enzymes is shown according to the pH of their environment. Enzyme activity is at its maximum at the optimum pH. However, there is a working range. For example pepsin has an optimum of 2.5 and a working range between 1 and 4.

Enzymes change their conformation according to pH. At the optimum pH the conformation of the enzyme is the best suited for its substrate. Increasing or decreasing the pH causes changes in the enzyme’s structure that decrease its activity, until finally it becomes fully inactive. However, there is a working pH range within which the enzyme is still active even if the pH is not at the optimum level.

Therefore, an interesting method for manipulating intracellular and extracellular chemical reactions consists of modifying the pH [13,14]. One example can illustrate the issue. Phosphofructokinase is an enzyme that catalyzes the phosphorylation of fructose-6-phosphate to fructose-1-6-bisphosphate and represents the third step of the glycolytic pathway. This cytoplasmic metabolic pathway is essential for tumor metabolism. When intracellular pH is 7.3 the enzyme’s activity is inhibited by small amounts of ATP. When cytoplasmic pH rises to 7.6 much larger amounts of ATP are needed for enzymatic inhibition, thus increasing glycolysis [15]. This difference in pH seems small (0.3), however due to its logarithmic nature the difference in proton concentration is significant. This shows that the increased intracellular pH of tumors favors the metabolic switch to glycolytic metabolism instead of oxidative. Acidifying the intracellular milieu reduces phosphofructokinase activity, thus reducing fermentative glycolysis.

pH determines the rate (speed) of a chemical reaction through the alteration of enzymatic activity. This enzymatic activity makes the modifications we know as metabolism. Therefore, a specific pH is required to set the metabolic cascade in motion. If this pH is not achieved, the metabolism will be substantially reduced or, in some cases certain types of metabolic processes cannot take place (Box 1).

Box 1

How pH influences metabolism

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We have called this the pH fundamental biologic effect equation. No matter that later metabolism in turn can modify pH, the equation remains unchanged. This is probably the reason why initiation of the malignant transformation starts with an intracellular pH modification which will be explained in detail in Chapter 2. Apoptosis also begins with an initial stage of pH acidification [16].

Transport of substances and ions in and out of cells

pH homeostasis is dependent on compartmentalization, buffering and movement of ions through the cell’s bilayer lipid membrane. Cells use both passive and active processes for this transport. While passive transport requires no energy expenditure and is dependent on the physical-chemical properties of the substances themselves, active transport is energy-dependent and it is usually independent of the substances’