A Decade of Research on Coffee as an Anticarcinogenic Beverage

Coffee consumption has been investigated as a protective factor against cancer. Coffee is a complex beverage that contains more than 1000 described phytochemicals, which are responsible for its pleasant taste, aroma, and health-promoting properties. Many of these compounds have a potential therapeutic effect due to their antioxidant, anti-inflammatory, antifibrotic, and anticancer properties. The roasting process affects the phytochemical content, and undesirable compounds may be formed. In recent years, there have been contradictory publications regarding the effect of coffee drinking and cancer. Therefore, this study is aimed at evaluating the association of coffee consumption with the development of cancer. In PubMed, until July 2021, the terms “Coffee and cancer” resulted in about 2150 publications, and almost 50% of them have been published in the last 10 years. In general, studies published in recent years have shown negative associations between coffee consumption and the risk or development of different types of cancer, including breast, prostate, oral, oral and pharyngeal, melanoma, skin and skin nonmelanoma, kidney, gastric, colorectal, endometrial, liver, leukemic and hepatocellular carcinoma, brain, and thyroid cancer, among others. In contrast, only a few publications demonstrated a double association between coffee consumption and bladder, pancreatic, and lung cancer. In this review, we summarize the in vitro and in vivo studies that accumulate epidemiological evidence showing a consistent inverse association between coffee consumption and cancer.


Introduction
Drinking coffee has often been discouraged, due to its association with the description of negative effects, mainly attributed to caffeine. Consumers' beliefs about the effects of coffee are strongly influenced by this idea: only 25%, a relevant minority of consumers, think that drinking coffee could have beneficial effects on health [1]. A survey performed by the World Cancer Research Fund (WCRF) revealed that as many as 36% of health professionals thought drinking coffee increases the risk of cancer [2]. Despite the "bad reputation" that has been conferred on coffee for many years, it has been increasingly demonstrated that it offers numerous health benefits, ranging from reduced risk of several cancers to reduced risk of developing depression.
Coffee is widely consumed, ranking as one of the four most commonly consumed beverages in the world [3]. The generic name coffee covers about one hundred species of plants, cultivated in multiple countries with different types of weather conditions: Hawaii, Colombia, Jamaica, Ethiopia, Kenya, Brazil, Vietnam, and some African countries [4]. The coffee tree appertains to the Rubiaceae family, and there are more than 80 different species of the genus Coffea L. Globally, only two species are traded: Coffea arabica, accounting for 63% of the world production, and Coffea canephora, accounting for the rest of the world production [5]. As de Mejia et al. highlight, the species and the origin are only two of the factors that determine that we never drink two cups of coffee with the same chemical composition. Additionally, the final composition of coffee will depend on cultivation practices (traditional or organic), postharvest techniques (wet or dry), duration and conditions of storage, roasting degree (light, medium, or dark), kind of roasting process (standard or torrefaction), type of commercial coffee (roasted ground or instant), and grinding and brewing method (boiled, filtered, or espresso) [6].
The consumption of coffee may have a substantial effect on public health. In 2017, two large reviews based on metaanalyzes were published, which established that coffee consumption was more often associated with benefit than harm, a probable decrease in the risk of breast, colorectal, colon, endometrial, and prostate cancer; cardiovascular disease and mortality; Parkinson's disease; and type 2 diabetes [7,8]. Then, it is no surprise that coffee motivates the interest of researchers and clinicians [9]. In PubMed, until April 2021, the search for the term "coffee" resulted in around 17,200 publications, including 1,430 reviews, 800 clinical trials, and 260 meta-analyses. However, the impact of coffee intake on chronic diseases, including cancer, has been a matter of debate in the last two decades. Accordingly, the search performed by combining both terms, "coffee and cancer", resulted in about 2,150 publications, and almost 50% of them have been published in the last 10 years.
In this work, we present an update on the last decade of research related to coffee and its anticancer activity. For this purpose, a broad approach was used, as follows: (1) analysis of the main coffee compounds and their modifications by the roasting process, (2) review of the bioactivity of coffee extracts by in vitro assays, (3) analysis of the antioxidant activity of coffee and its components, and (4) data collection of large observational human studies.

The Main Bioactive Compounds of Coffee and Their Modifications by the Roasting Process
The traditional recommendation to reduce the consumption of coffee as a beverage or not to drink it at all because of a global risk profile has progressively given rise to a less negative view due to its better-known phytochemistry [10]. The knowledge that coffee and caffeine are not equivalent has increased the interest in discovering whether other components of coffee might contribute to the protective action in the human body [9]. Coffee is a complex beverage containing more than 1000 described phytochemicals responsible for its pleasant flavor, aroma, and health promoters [1,6,10]. Many of these compounds have therapeutic potential antioxidant, anti-inflammatory, antifibrotic, and anticancer effects. As a consequence of roasting, profound changes occur in the chemical composition, which leads to the transformation of natural substances present in green beans into compounds derived from the Maillard reaction [4]. Table 1 shows the main compounds of green coffee, i.e., coffee beans before roasting, and the appearance of or increase in specific components in black coffee beans. This well-known roasting process involves the caramelization of carbohydrates and the pyrolysis of organic compounds. Here, we detail the main phytochemicals belonging to groups of sugars and sugar metabolites, protein and amino acids, fatty acids, chlorogenic acids (CGAs), organic acids, and other compounds.
"Bioactive compounds" are extra nutritional constituents that typically occur in small quantities in foods and have a positive effect on human health. These are also referred to as nutraceuticals, a term that reflects their existence in the human diet and their biological activity. They consist of a wide range of chemical compounds with different structures, physiological activities, and molecular mass between 200 and 1000 Da [11]. Green coffee beans have been shown to have high levels of bioactive compounds, but even after the roasting process and exposure to hot water, dark coffee maintains and develops numerous new phytochemicals that are beneficial to consumers' health [12].
As highlighted in Table 1, during the roasting process, the concentration of some nutraceutical compounds, such as 5-CQA, 3,4-diCQA, 3,5-diCQA, phenolic acid, trigonelline, polyphenolic, cafestol, and kahweol, decreases while others are formed, such as melanoidins, chlorogenic lactones, acid gallic, acid nicotinic, caffeic acid, flavonoids, and N-methylpyridinium (all of them marked in bold). This delicate balance between the formation and decomposition of bioactive compounds allows the biological effects of dark roasted coffee to be found in experimental models. Sometimes, these effects are higher in dark than lighter roasted coffee, despite the antioxidant content in dark coffee is lower [1]. In fact, Priftis et al. reported that in eight out of 13 coffee varieties, toasted coffee increased the free radical scavenging activity [13].
In summary, although in vitro and in vivo studies using individual components of coffee revealed multiple biological activities, the physiological properties of whole coffee will likely differ because coffee is a complex, nonstandardized beverage. Therefore, it is a variable mixture of hundreds of compounds, and its bioactivity may be influenced by possible matrix, synergistic, and/or antagonist effects. Additionally, only a small percentage of the ingested compounds may enter the circulatory system and reach the tissues, and very little of the absorbed material may retain the original structure present in the beverage [4]. For these reasons, it is worth pointing out that the prevention of various diseases derived from coffee consumption is usually the joint action of multiple components, and sometimes the synergistic effect of various types of compounds is much better than the activity of single compounds [29].
In the last decade, numerous research groups have focused on the effect of coffee on tumors by conducting in vitro studies, to understand the bioactivities of this beverage in greater depth. Table 2 presents many of these in vitro studies, which have addressed a great diversity of cancer types, coffee varieties, degrees of roasting [5,19,30,31], and diverse effects including cell-cycle arrest, antiproliferative effects, and antiapoptotic and high antioxidant activities.
Remarkably, most of the investigations report a wide range of coffee effects on cancer cells. For example, studies on breast cancer cells focused on antiproliferative and anti-oxidant effects, cell-cycle arrest, induced apoptosis, regulation of gene expression, modulation of enzyme activity, and enhanced efficiency of cancer treatments [30,[32][33][34]. Studies on prostate cancer cells reported antiproliferative effects, cell-cycle arrest, apoptosis, and high antioxidant capacity [5,19,31,32]. Research on kidney cancer cells found antiproliferative and antimigratory effects, EMT downregulation, and gene downregulation [35]. Furthermore, antiproliferative effects were reported in esophageal, urinary, bladder, lung, colon, oral, osteosarcoma, and glioblastoma cancer cells [12,18,30,36].
Additionally, as Samoggia and Riedel and Montenegro et al. pointed out, intake of green coffee-based beverages has become popular in recent years due to the belief in their beneficial antioxidant properties [1,31]. Some researchers have observed that green coffee treatments have antiproliferative activity, though not as powerful as that of roasted coffee treatments [12]. However, further cell experiments Oxidative Medicine and Cellular Longevity should be conducted to evaluate the deep molecular mechanisms and pharmacokinetics involved in the effects observed. The bioavailability and bioaccessibility of extracts should be investigated to determine the quantity of coffee required to achieve such effects, due to possible losses during digestion, absorption, and metabolization by the gut microbiota. Toxicity assays should be performed to ensure safety. In addition, in vivo and clinical tests would be required to recommend the consumption of different coffee types to help protect against cancer.

Antioxidant Properties of Coffee
As highlighted in the previous sections, among the main biological activities of the beverage and its components, the antioxidant activity was made evident in numerous investigations, both in chemical tests and different tumor cell cultures.
When, in aerobic life and our metabolism, the electron flow becomes uncoupled (transfer of unpaired single electrons), free radicals are continuously produced by the body's normal use of oxygen such as respiration. Oxygen-centered free radicals or ROS radicals are superoxide, hydroxyl, peroxyl, alkoxyl, hydroxyl radical, nitric oxide, and lipid hydroperoxides, while ROS nonradicals are singlet oxygen, hydrogen peroxide, and hypochlorous acid. Therefore, these intermediates, also called oxidants or prooxidants, can easily initiate the peroxidation of membrane lipids [37]. The imbalance towards high ROS concentration could result in oxidative damage to critical cellular biopolymers (proteins, lipids, and nucleic acids), especially if the free radicals are produced and accumulate unchecked for a prolonged period. The sustained oxidative damage of these biological macromolecules is linked to the development of chronic diseases like cancer. [38]. The reason why antioxidants are thought to be beneficial to our health is that ROS have been shown to be involved in many disease processes, including cancer [39].
In terms of food, an antioxidant has been defined as any substance that, when present at lower concentrations than those of an oxidizable substrate, significantly delays or inhibits the oxidation of the substrate. In recent years, there has been great interest in identifying alternative natural and safe sources of food antioxidants, especially those of plant origin. Nowadays, there is a growing interest in substances exhibiting antioxidant properties, which are supplied to human organisms as food components or as specific preventive pharmaceuticals [37]. The possibility of complementing the body's natural antioxidant defense system with exogenous antioxidants has continued to receive significant research attention as a result of their potential for wide applications [38].
In this sense, the antioxidant activity of coffee and of many of its components has been demonstrated [29,[40][41][42][43]. Additionally, recent studies have shown that coffee components can trigger tissue antioxidant gene expression and protect against gastrointestinal oxidative stress [44].
Recent studies conclude that coffee infusions significantly extend the chronological lifespan of the Saccharomy-ces cerevisiae yeast cells by protecting cells against reactive oxygen species, double DNA-strand break, and the decrease in metabolic activity [45]. However, the bibliography currently gives contradictory information as to whether slightly roasted or dark coffee generates greater antioxidant activity. Water extracts from green coffee are characterized by significant antioxidant properties and a high capacity to reduce transition metal ions because it contains many polyphenolic compounds that oxidize in the potential range tested [19,46]. Jung et al. found that the cellular antioxidant activity of coffee extracts (in AML-12 and RAW 264.7 cells) has physiological antioxidant and anti-inflammatory activities, and that these effects are negatively correlated with roasting levels in the cell models [47]. Bobková et al. also support this theory, since they established that total antioxidant capacity reached the highest values in light roasted coffee, and the roasting process affected both the oxidative activity and the polyphenolic content [48]. Schouten et al. confirmed that acrylamide levels and antioxidant activity reached a maximum in the first coffee roasting degrees and then decreased as the heating process continued, both in Arabica and Robusta samples [49]. Wolska et al. showed that the method of brewing Arabica coffee and green coffee (simple infusion, espresso maker, French press, overflow espresso, or Turkish coffee) significantly affected the antioxidant potential of infusions [42]. In other research, antioxidant activity significantly rose with the degree of roasting, where strongly roasted coffee had higher activity than lightly roasted coffee [50].
As mentioned above, many coffee micronutrients are considered bioactive due to their high antioxidant capacity. Investigations have revealed that crude caffeine possesses hydrophilic antioxidant activity (145 ?mol Trolox equivalent (TE)/g) and lipophilic antioxidant activity (66 ?mol TE/g) [51], and its administration has led to the inhibition of the cyclooxygenase-2 enzyme [40]. The antioxidant activities of CGA exhibited protection against oxidative damage of macromolecules such as DNA, lipids, and proteins. When administered to mice under scopolamine-induced amnesia, CGA showed a neuroprotective function via the inhibition of acetylcholinesterase [40]. Coffee melanoidins, compounds formed during coffee roasting, have also been demonstrated as potential food ingredients due to their antioxidant properties. The antioxidant activity of these compounds, evaluated in vitro, was 725-750 μmol Trolox/g, and it was tripled by the addition of sugar during coffee roasting, namely, torrefaction, known to increase the content of melanoidins [41]. Czachor et al. provide strong evidence that coffee flavonoids are responsible for scavenging free radicals and leading to longevity in yeast lacking Sod1, Sod2, and Rad52 proteins [45].

Have We Been Drinking a Medicine Daily for
Hundreds of Years without Knowing It?
In recent years, the number of consistent epidemiological evidence of coffee and cancer has accumulated but due to similar methodological limitations, it is sometimes unsatisfactory. Therefore, interest has focused on meta-analyses or 5 Oxidative Medicine and Cellular Longevity Table 3: Coffee consumption and cancer risk. 78 meta-analyses, cohort, or prospective studies, published between 2010 and 2021, which reported some type of significant association between coffee consumption and different cancer types, were analyzed. Italic rows: studies that report a negative association between coffee and cancer risk; bold rows: studies that report a positive association between coffee and cancer risk.  6 Oxidative Medicine and Cellular Longevity   The study found a significant inverse linear trend between consumption and distal colon cancer and a tendency toward a lower risk of rectal cancer.
Micek, 2019 [43] 14 prospective studies (1,381,085 Pt and 28,404 Cc) August 2018 Restriction to decaffeinated coffee revealed a 15% lower risk of cancer for the highest category consumption. Coffee consumption was related with a decreased risk of cancer in a subgroup of never-smokers and in Asian countries.

Sartini, 2019 [56] 26 prospective studies Uninformed
Regarding colorectal cancer, a protective effect emerged in US subjects. Concerning colon cancer, a significant protective effect was noted only in European men and only in Asian women. Decaffeinated coffee exhibited a protective effect against colorectal cancer in men and women combined. 9 Oxidative Medicine and Cellular Longevity  10 Oxidative Medicine and Cellular Longevity Highest consumption was significantly associated with an increased risk of cancer.
Wang, 2012 [135] 9 publications (3,008 Cc) 2005 A significantly positive association was found between coffee consumption and the risk of cancer. 11 Oxidative Medicine and Cellular Longevity pooled analyses to bypass the shortage of individual studies [10].
The database of human meta-analysis studies is more abundant today than ever before. Among them, there is a great diversity and due to contradictions between them or the limited scope of their conclusions, they are often not considered, or their interpretation is difficult. For this reason, in this review, we endeavored to make an exhaustive summary of meta-analysis studies (78 out of 115 published in PubMed until April 2021), which found a significant association (positive or negative) between coffee consumption and different types of cancer development, treatment, or advancement. Table 3 shows meta-analyses, cohort, or prospective studies published between 2010 and 2021. Importantly, the large number of patients involved in these studies provides evidence on the effect of years of coffee consumption on humans. We collected and analyzed more than 75 studies (as shown in Table 3), the vast majority of which (63) reported beneficial effects of coffee consumption, such as less development, metastasis, or mortality from cancer of different origins.
Most publications showed a negative association between coffee consumption and the risk or development of different types of cancer (Table 3, italic rows); these include breast, oral, oral, and pharyngeal, melanoma, skin and skin nonmelanoma, prostate, colorectal, endometrial, liver, leukemic and hepatocellular carcinoma, brain, and thyroid cancer among others (18 cancer types). Instead, a dual association was observed in bladder, gastric, pancreatic, and lung cancer, although only a few publications demonstrated this association. Surprisingly, we found that only 12 publications showed positive associations between cancer and coffee consumption (bold rows), as observed in a few types of cancer, including lung, bladder, pancreatic, laryngeal, and gastric cancer.
Although caffeine has been shown to have anticancer activity both in vivo and in vitro, many epidemiological studies have published evidence indicating that the risk of cancer decreased even in people who ingested decaffeinated coffee [43,[52][53][54][55][56][57][58]. In this sense, Hall et al. discovered that the amounts of many of the bioactive components, such as caffeic acid, chlorogenic acid, ferulic acid, pyrogallic acid, and trigonelline, did not change when decaffeinated coffee was compared to caffeinated coffee [59].
This update reveals a growing body of statistically significant evidence from epidemiological studies, suggesting that coffee drinking in most people (including different sexes, ethnicity, and ages) is beneficial and inversely associated with cancer risk [6,60]. The fact that we found few publications on the positive association of coffee consumption and cancer does not imply that it is less important but rather suggests that more research should be carried out, as this would make it possible to formulate a clearer hypothesis.
Undoubtedly, moderate coffee consumption of up to 4 cups/day can be enjoyed as part of a healthy, balanced diet and an active lifestyle [9], since health claims associated with its consumption are broad enough to recommend it as a protective beverage. Probing for the detrimental effects of coffee should be focused on further drawing a conclusive approach for end-users to eliminate the ambiguities [29].
Higher doses of coffee have higher benefits in terms of risk reduction. However, further biological and epidemiological studies are required to determine the exact mechanism and analyze the specific subgroups [61]. In addition, it is important to recommend caution to avoid the concomitant use of coffee with drugs that have a significant interaction with coffee. There should be an adequate time interval between the intake of drugs and coffee based on the properties of the drugs. Pharmacists and physicians must be aware of the potential risks of drug-coffee interaction and counsel patients appropriately. Further in vitro and in vivo studies should be performed on frequently prescribed drugs to obtain robust evidence of the pharmacokinetic interaction with coffee [62].

Conclusions
In the last decade of research, the overall view on the impact of coffee on health has shifted from mostly detrimental outcomes towards a beneficial profile. In fact, the data on cancer disease are mostly balanced towards beneficial effects. The subsequent production of more clinical data with a higher number of cases, together with a better understanding of the components of coffee, has contributed to changing this perspective. It can be concluded according to the current knowledge that the labeling of coffee as a mostly unhealthy beverage lacks scientific support.
Throughout this review, we have detailed the main components of coffee, their variation in the roasting process, and the bioactivity of many of them. We provide a wealth of up- Cancer risk is significantly increased by 47% in the population with the highest category intake of coffee compared with that with the lowest category intake.
Zhu, 2020 [137] 17 prospective cohort studies (1.1 million Pt and 20,280 Cc) Uninformed Higher consumption of coffee is associated with increased cancer risk. 12 Oxidative Medicine and Cellular Longevity to-date information on the existing experimental evidence for the anticarcinogenic effects of this beverage and a comprehensive review of the important significant evidence for these effects on humans. We have summarized the accumulating epidemiological evidence pointing towards a consistently inverse association between coffee consumption and the risk of breast, oral, pharyngeal, melanoma, endometrial, hepatocellular, prostate, colorectal, liver, and brain cancers, among others. The prospects for new bioactive compounds and the development of new drugs are focused on natural products. Considering the wide range and depth of evidence already collected, coffee, a beverage that has been widely included in the human diet for years, could be used not only for its well-known taste and stimulant, pleasurable effects, but also for social, pharmaceutical, and clinical purposes, as it provides health benefits and significant effects on cancer treatments.
More research is needed to find the right dosage and balance between the beneficial health effects of coffee (such as anticancer and antioxidant activities) and those traditionally considered negative. However, many of the "harmful" effects noted in antiquity can be attributed to excessive consumption or specific compounds (such as caffeine), which can be regulated or even eliminated from the beverage, thus minimizing the associated risks, without necessarily affecting the important beneficial effects described by the numerous research studies included in this review.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest
The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper.

Authors' Contributions
All authors contributed equally to this work. Ayelén D. Nigra performed the conceptualization and acquisition of data, methodology, validation, formal analysis, investigation, and writing of the original draft, writing and editing of the review, and final approval of the manuscript. Anderson Junger Teodoro performed the conceptualization and interpretation of data, investigation, methodology, resources, writing and editing of the review, supervision, project administration, funding acquisition, and final approval of the manuscript. German A. Gil performed the conceptualization and design of the study, formal analysis and interpretation of data, investigation, methodology, resources, writing of the original draft, writing and editing of the review, supervision, project administration, funding acquisition, and final approval of the manuscript.