Author:

Bernardino Fantini

Abstract

In the early 1920s Otto Warburg, one of the leading biochemists of the time, studying cellular energetics, discovered a surprising tumour phenotype, characterized by a very low energy yield, in the form of very high rates of lactic acid fermentation, not only in anoxia, but also in the presence of oxygen levels that do not compromise cellular respiration. After an eclipse lasting several decades, in recent years the Warburg Effect has been rediscovered. And alterations in cellular metabolism once again emerge as a hallmark of tumour development and a possible tool for diagnosis and treatment of cancer.

Introduction

In the early decades of the twentieth century, biochemistry was the leading science in biology and medicine. The dominant paradigm sought to explain every biological phenomenon - from fermentation to development and physiological or pathological processes - through cycles of chemical reactions catalyzed by enzymes. A central research objective was the study of metabolic networks that provide cells with usable forms of energy and precursors for biosynthesis, particularly of proteins. The model for this type of scientific explanation is the “Krebs cycle,” a complex set of chemical reactions found in all cells, through which cellular respiration provides the energy necessary for living organisms to synthesize lipids, carbohydrates, and proteins.

Hans Krebs, a German biochemist who emigrated to England after the Nazis came to power, was awarded the Nobel Prize in Physiology or Medicine in 1953 for his discoveries made in the 1930s, after training under Professor Otto Warburg at the Kaiser Wilhelm Institut in Berlin-Dahlem. Krebs would later dedicate a biographical book to his mentor, titled Otto Warburg: Cell Physiologist, Biochemist, and Eccentric (1981), expressing his deep admiration for Warburg’s “penetrating intelligence and original and imaginative approach to any situation” and for his “independence from beliefs and prejudices.”

Indeed, Warburg played a central role in the history of biochemistry and oncology due to his ability to discover surprising facts and propose original explanatory theories. Between 1908 and 1914, Otto Warburg spent several research periods at the Zoological Station in Naples - then considered the “Mecca” of biological research - where he conducted studies on the oxygen consumption of sea urchin eggs after fertilization, thus initiating a new research program on cellular respiration. Warburg precisely measured oxygen consumption, observing that it increased six- to seven-fold after fertilization.

The “Warburg apparatus” and the study of cellular respiration

To conduct these studies, Warburg developed laboratory techniques and introduced new instruments that would become classics in all cell physiology and biochemistry laboratories for studying fine biological samples. Using a microtome, thin histological sections of animal or plant tissue samples were produced, while a complex experimental setup - called the “Warburg apparatus” - enabled the measurement of gas pressure at a constant volume and temperature, so that pressure changes reflected the production or absorption of gas. It was then sufficient to take measurements with a manometer to determine the variations in the amount of gas produced or absorbed by a sample maintained at a constant temperature.

The greatest difficulty in this type of research is keeping the tissue alive throughout the entire duration - sometimes lasting hours - of the manometric observations. Thin slices of tissue are cut with a razor, and these must be thin enough to allow light to pass through, yet thick enough to reduce the percentage of cells killed by the cuts. Warburg developed a mathematical theory to calculate the “limit thickness” of the slices, that is, the maximum thickness compatible with tissue survival. In the case of liver tissue, for example, the calculated limit thickness was 0.5 mm, corresponding to about 150 layers of cells. This allowed Warburg to obtain quantitative and consistent measurements of lactic acid fermentation and cellular respiration for at least 30–60 minutes using only a few milligrams of tissue, whereas traditional chemical methods required 50 times more material and yielded much more variable results.

By rigorously applying, and for the first time, enzymology techniques to the study of cellular physiology, Warburg isolated the coenzymes responsible for hydrogen transport, such as nicotinamide, and a “yellow enzyme,” which would later be identified as a flavoprotein. In this way, Warburg provided a description of oxidation and reduction reactions within the cell, opening new directions for research on metabolism and cellular respiration. This is a vital process through which substances supplied directly to cells, or stored within them, are broken down into simpler components with the consumption of oxygen. It is through this process that the energy needed for other vital functions is made available to the cells in a readily usable form. For this body of research, Warburg was awarded the Nobel Prize in Physiology or Medicine in 1931, “for his discovery of the nature and mode of action of the respiratory enzyme,” now known as cytochrome oxidase.

A particularly suitable experimental system for studying cellular metabolism, which became a model at the Zoological Station in Naples, is the developing sea urchin embryo. This model is characterized by rapid cell division accompanied by high metabolic activity and a significant increase in oxygen consumption. Since tumour tissues are also characterized by rapid and uncontrolled cell division, Warburg hypothesized that a similar increase in oxygen consumption should also be observable in these tissues.

The biochemical study of tumour growth

Starting in the 1920s, Warburg devoted himself to the biochemical study of tumours and chose as his experimental cancer model the Flexner-Jobling rat carcinoma, discovered at the Rockefeller Institute in 1906 in the seminal vesicle. This tumour could be transplanted into other rats, thereby facilitating research.

According to the typical ‘biochemical paradigm’ dominant at the time, Warburg argued that in order to explain tumour growth, attention should be focused on the reactions that generate the energy required for growth, particularly respiration. Having observed that respiration in sea urchin eggs increases sixfold at the moment of fertilization, Warburg expected to find much higher respiration rates in cancerous tissue compared to normal epithelium. However, the first measurements showed that the respiration of the carcinoma, when placed in solution at body temperature, was significantly lower than that of liver and kidney tissue under the same conditions.

This surprising result was followed by another: shortly after placing slices of the tumour into a solution containing physiological concentrations of glucose, the solution became acidic. Since acidification was not observed in the absence of glucose, Warburg attributed it to the generation of lactic acid from the sugar. The high rate of lactic acid production exhibited by the Flexner-Jobling rat carcinoma resulted from an unusually elevated fermentative capacity- or, in Warburg’s terminology, from a high glycolytic activity (glykolytische Fähigkeit) or"Aerobic glycolysis." Normal cells primarily release energy through glycolysis followed by the mitochondrial citric acid cycle and oxidative phosphorylation. In contrast, most cancer cells release energy predominantly through a high rate of glycolysis followed by lactic acid fermentation, even in the presence of abundant oxygen.

Starting in 1924, after making modifications to his manometric measurement protocols to more accurately reproduce physiological conditions, Warburg conducted a long series of in vitro experiments, extending his investigations to a variety of other tumours, both benign and malignant, including human tumours of various origins, while simultaneously studying tissues from 3- to 5-day-old chicken embryos, whose growth rate at this stage of development is comparable to that of young rat carcinomas.

Metabolic changes as the primary cause of tumours

Based on his laboratory observations and manometric measurements on tissues, Warburg also developed a metabolic theory to explain the cause of tumours, hypothesizing that the transition from orderly to disordered growth occurred through a disturbance in the ratio between fermentation and respiration. Aerobic fermentation is a distinctive characteristic of cancer cells, not shared by normal tissues. While all growing tissues, whether normal or malignant, exhibit a high fermentative capacity, only in proliferating normal tissues is respiration sufficient to abolish aerobic fermentation. In other words, the difference between orderly and disordered growth lies in the ability of respiration to stop fermentation in the presence of oxygen. Consequently, the aerobic metabolism of normal cells, both growing and mature, consists solely of respiration, whereas that of cancer cells, whether malignant or benign, is a mix of fermentation and respiration. The differences between malignant and benign tumours are differences in degree rather than in nature, as suggested by pathology.

If oxygen deficiency occurs in a resting tissue, only those few cells with high fermentative capacity will survive, while all other cells will die. In the case of chronic oxygen deficiency, a tissue will form that possesses the fermentative capacity of embryonic tissue, while its respiration, impaired by the lack of oxygen, is abnormally low, hence typical of tumour tissue.

Throughout his life, Warburg continued to argue that aerobic glycolysis is 'the primary cause of cancer,' but the biochemical paradigm to which this theory refers has now been replaced by a new paradigm, based on the function of genes and in some cases the role of viruses (which are nothing more than 'free genes') in the origin of tumours. The new type of explanation proposed by molecular oncology focuses on the genetic bases of the proliferation, differentiation, and cell death that characterize tumours. It is currently believed that malignant transformation occurs through successive mutations in specific cellular genes, leading to the activation of oncogenes and the inactivation of tumour suppressor genes. Oncogenes and tumour suppressor genes normally function as key regulators of physiological processes such as proliferation, cell death or apoptosis, differentiation, and senescence, as well as how these cellular programs become deregulated in cancer due to mutations. Consequently, the effect discovered by Warburg is considered a consequence of these mutations rather than a cause of cellular transformation.

The ‘Warburg Effect’

After a long period of obscurity, the phenomenon discovered by Warburg would only be reconsidered decades later and termed the 'Warburg Effect' by Efraim Racker in 1972, in an article on the bioenergetics of tumour growth, to designate a metabolic phenotype typical of many cancer cells, characterized by high anaerobic glycolytic activity, that is, the production of lactic acid, even in the presence of a high concentration of oxygen. And in recent decades, the number of publications using the term 'Warburg Effect' in cancer research has increased almost exponentially, a sign of the resurgence of the study of tumour metabolism as a diagnostic indicator and potential therapeutic target.

A new research field is also being discussed, known as oncometabolism, which focuses on the metabolic changes occurring in the cells that make up the tumour microenvironment (Tumour Microenvironment, or TME) and accompany oncogenesis and neoplastic progression. A new scientific society, The International Society of Cancer Metabolism (ISCaM), has also been established.

The increase in glucose consumption by cancer cells resulting from the Warburg effect is used as a diagnostic tool for a wide variety of tumours in combination with imaging techniques such as positron emission tomography, using a radioactive isotope of glucose (18F-fluorodeoxyglucose FDG-PET). This technique allows for the diagnosis of a tumour, as glucose is detected at higher concentrations in malignant tumours compared to other tissues. A clear correlation has also been established between the degree of FDG uptake and the grade of the tumour.

The causes of the Warburg effect remain largely unclear. The most widespread hypothesis is that it could simply be a consequence of damage caused by mutations that block the mitochondria (the organelles responsible for aerobic cellular respiration). It may also be an adaptation to low-oxygen environments within tumours.

A hallmark of cancer

In recent years, the importance of changes in cellular metabolism accompanying oncogenesis has been highlighted, going beyond the energetic metabolism revealed by the Warburg effect to include many cellular biosynthetic cycles. This has led to a reevaluation of the role played by 'bioenergetic disorder' in carcinogenesis. Douglas Hanahan and Robert A. Weinberg, in a famous 2000 article in the journal Cell, defined the six distinctive capabilities (Hallmarks) of tumours (self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, the ability to avoid apoptosis, the ability to replicate indefinitely, induction of angiogenesis, and the ability to form metastases). In a subsequent article in 2011, also in Cell, titled 'Hallmarks of cancer: the next generation,' the same authors identified the two emerging distinctive characteristics of tumour forms: the deregulation of cellular energetic metabolism and the ability to evade destruction by the immune system. Thus, alterations in cellular metabolism or ‘bioenergetic reprogramming ‘, particularly the Warburg effect, once again emerge as a hallmark of tumour development.

Although the energetic theory of the origin of tumours proposed by Warburg has proven to be a 'false lead,' linked to a scientific paradigm that has now been abandoned, the phenomena he discovered continue to play an important role in diagnosis and also hint at the possibility of some therapeutic indications.

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