The role and its mechanism of intermittent fasting in tumors: friend or foe?

Intermittent fasting (IF) is becoming a prevailing topic worldwide, as it can cause changes in the body’s energy metabolism processes, improve health, and affect the progression of many diseases, particularly in the circumstance of oncology. Recent research has shown that IF can alter the energy metabolism of tumor cells, thereby inhibiting tumor growth and improving antitumor immune responses. Furthermore, IF can increase cancer sensitivity to chemotherapy and radiotherapy and reduce the side effects of these traditional anticancer treatments. IF is therefore emerging as a promising approach to clinical cancer treatment. However, the balance between long-term benefits of IF compared with the harm from insufficient caloric intake is not well understood. In this article, we review the role of IF in tumorigenesis and tumor therapy, and discuss some scientific problems that remain to be clarified, which might provide some assistance in the application of IF in clinical tumor therapy.


Introduction
Intermittent fasting (IF) is a diet-based therapy that alternates between fasting and free feeding/eating for a period of time. This practice was developed by people seeking practical and relatively safe fasting methods to achieve daily caloric restriction. IF regimens generally include short-term intermittent fasting [such as [16][17][18] h of daily fasting, alternate-day fasting or 5:2 IF (2 days a week)], long-term intermittent fasting, and so on 1-8 ( Table 1). More than a century ago, Moreschi 9 first described the beneficial effects of fasting and restricting caloric intake on tumors in animals. In 1997, Weindruch et al. 10 found that reducing food availability over a lifetime (caloric restriction, CR) had significant effects on aging and the lifespan of animals (Figure 1). Follow-up studies have subsequently showed that IF over a period of 12 h to several weeks can prevent diseases and delay aging in many organisms (such as bacteria, yeasts, worms, and mice). Specifically, IF can improve the function of the entire body, including tissues and organs. Moreover, after conducting an IF regimen, some tissues and organs are more resistant to a variety of harmful stimuli that involve metabolic, oxidative, ionic, traumatic, and proteotoxic stresses 21 . Animal model studies of experimental diseases have further shown that IF can alleviate the development of many chronic diseases, including obesity, diabetes, vascular diseases, cancer, and neurodegenerative diseases 22 .
From a mechanistic perspective, studies have demonstrated that the effect of IF is related to the adaptive energy metabolic response of organs, tissues, and cells triggered by IF (mainly the metabolic conversion from glucose to ketone bodies as an energy source), which manifests as increased ketone body production, autophagy, DNA repair, and anti-stress abilities, and antioxidant defense is enhanced in the early stage of IF. During periods of recovery (including eating and sleeping), the conversion from ketones to glucose as the main energy source of cells results in an enhanced ability to produce glucose and synthesize intracellular proteins and increased mTOR expression and mitochondrial biogenesis. During the long-term adaptation period, the insulin sensitivity of cells and body resistance to insulin are increased, blood glucose homeostasis and lipid metabolism are further improved, and abdominal fat and inflammation are reduced [23][24][25][26] (Figure 2). These processes are accompanied by alterations in the insulin-like growth factor-1 (IGF-1)/mTOR signaling pathway, a decrease in leptin levels, an increase in adiponectin levels [27][28][29] , an improvement in antistress ability, a decrease in free radical production, growth and functional remodeling, and an increase in body resistance to stress 30,31 .
A recent study reported that the miRNA machinery, particularly the miRNA-processing enzyme Drosha/drsh-1, is involved in fasting-induced changes in gene expression and IF-induced longevity. This study found that miRISC components (alg-1, alg-2, ain-1, and ain-2) and the miRNA-processing enzyme, drsh-1, are upregulated by fasting, suggesting that the miRNA IF significantly reduces the incidence of lymphoma in aged mice and exerts a beneficial antioxidant effect and modulates oxidative stress associated with aging.

Moreschi 9
Weindruch et al. 10 Prokopenko et al. 11 Descamps et al. 12 Meynet et al. 15 Mattison et al. 16 O'Flanagan et al. 17 Nencioni et al. 18 IF reduces the occurrence of spontaneous tumors during normal aging in rodents and suppresses the growth of many types of induced tumors while increasing their sensitivity to chemotherapy and irradiation.

Kim et al. 19
More than a century ago machinery is activated in response to fasting (Figure 2). The expression of miRNA machinery proteins (Argonaute, Dicer, and Drosha) in mouse adipose tissues has been reported to decrease with aging, and these decreases are suppressed by CR. Moreover, miRNA array analyses revealed that the expression levels of numerous miRNAs changed after 2 days of fasting 32 . These results indicate that components of the miRNA machinery, especially the miRNA-processing enzyme, drsh-1, and miRNA molecules, might play important roles in mediating IF-induced longevity via the regulation of fasting-induced changes in gene expression.

Intermittent fasting and tumorigenesis
Recent studies have shown that IF can affect the energy metabolism of tumor cells and inhibit tumor cell growth, and can also improve the function of immune cells and promote antitumor immune responses, which indicates that IF has potential value in tumor immunotherapy 33 .

IF and tumor growth
The molecular mechanism by which IF inhibits tumor cell growth is as follows. CR induced by IF inhibits the IGF-1/AKT and mTORC1 pathways in tumor cells, while adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), which is dependent on the nicotinamide adenine dinucleotide coenzyme deacetylase-1 (Sirtuin-1, SIRT1) and SIRT3 pathways, is activated, thus hindering tumor cell growth. Notably, AMPK and SIRTs depend on each other in IF-associated metabolic adaptation 34,35 . AMPK can induce activation of SIRT1 through nicotinamide phosphoribosyltransferase (NAMPT), while SIRT1 can activate AMPK through liver kinase B1 (LKB1) regulation. In addition, FOXO3a (a downstream molecule of SIRT1 and SIRT3) and AMPK can each enhance the other's transcriptional activities 36 . SIRT3 inhibits tumor growth by activating FOXO3a and the expression of superoxide dismutase 2 (SOD2), thereby reducing the level of reactive oxygen species (ROS) 37-39 and negatively regulating the expression of hypoxia inducible factor-1α (HIF-1α) 40,41 . SIRT3 activates SOD2 via upregulation of FOXO3a [42][43][44][45] . Furthermore, IF can selectively inhibit tumor growth by upregulating leptin receptor (LEPR) and its downstream signaling pathway protein, PR/SET domain gene family 1 (PRDM1) 46 (Figure 3). However, the major decreases in glucose, insulin, and IGF-1 caused by fasting, which is accompanied by cell death and/ or atrophy in a wide range of tissues and organs, including the liver and kidneys, is followed by a period of abnormally high cellular proliferation in these tissues, driven in part by Periods of caloric intake restriction Figure 2 The effect of different periods of intermittent fasting (IF). In the early stage of IF, the effect of IF mainly occurs through ketogenesis, which manifests as increased ketone body production, autophagy, and DNA repair, as well as enhanced anti-stress abilities and antioxidant defense. During periods of recovery (including eating and sleeping), the main energy sources of cells are converted from ketones to glucose, which leads to increased glucose, mTOR expression, and mitochondrial biogenesis, enhanced ability to synthesize intracellular proteins, and remodeled growth and functions. During the long-term adaptation period, the insulin sensitivity of cells and body resistance to stress are increased, blood glucose homeostasis and lipid metabolism are further improved, and abdominal fat and inflammation are reduced.
the replenishment of growth factors during refeeding. When combined with carcinogens during refeeding, this increased proliferative activity can increase carcinogenesis and/or precancerous lesions in tissues, including the liver and colon 47 . In addition, some previous studies have shown that IF fails to inhibit the formation of carcinomas in mice [48][49][50] . Together with these studies, we therefore hypothesize that excessive use of IF may have the opposite effect in inhibiting the occurrence of tumors. To summarize, it is suggested that the mechanism by which IF inhibits tumor growth is complex, and the specific molecular mechanisms that contribute to specific tumors may be different when different IF or CR regimens are implemented.

IF and tumor immunity
Immune escape is a key factor in tumorigenesis. Studies have shown that IF can affect the development and function of a variety of immune cells, thereby regulating antitumor immune responses and affecting tumorigenesis. IF affects antitumor immunity mainly by increasing the self-renewal ability of hematopoietic stem cells and improving immunosuppression. Moreover, the conversion of energy metabolism results in dramatic downregulation of IGF-1 and upregulation of insulin-like growth factor binding protein-1 51 and increased tumor cell autophagy and programmed cell death. These effects are accompanied by a decrease in extracellular nucleoside triphosphate diphosphohydrolase expression and ATP accumulation, thus inhibiting regulatory T cells (Tregs) and stimulating cytotoxic T lymphocyte (CTL) functions, which enhances antitumor immune responses 52 . Moreover, high expression of heme oxygenase-1 (HO-1) in tumors can inhibit tumor cell apoptosis and immunostimulatory effects 53 . Notably, IF can decrease the expression of HO-1 in tumor cells and increase tumor cell apoptosis, as well as enhance stimulation of CD8 + T cells by reducing the caloric supply, which forms a positive feedback loop of IF-induced CD8 + T cell-medicated killing of tumor cells (Figure 4) 54 .
Furthermore, IF can also regulate antitumor immune responses by affecting the function and polarization of innate immune cells, such as natural killer (NK) cells and tumorassociated macrophages (TAMs) 55 . Lactic acid derived from tumor cells can directly inhibit NK cell-mediated killing and also increase the number of myeloid-derived suppressor cells (MDSCs) to indirectly inhibit NK cell functions. IF can reduce the production of lactic acid by tumor glycolysis, thereby restoring the function of NK cells, reducing the enrichment of MDSCs, and inhibiting tumor growth 56 . In addition, the occurrence of tumors is also closely related to polarization of local TAMs. Recent studies have shown that IF can reduce the metabolism and inflammatory activity of monocytes due to the activation of peroxisome proliferator-activated receptor-α (PPAR-α) during fasting, and also enhance repair and immune surveillance in the body via activation of AMPK. Furthermore, IF inhibits the production of chemokine C-C motif ligand 2 (CCL2) to reduce the number of inflammation-related monocytes in the blood and tissues 57 . Moreover, IF can also inactivate the JAK1/STAT3 signaling pathway and reduce the expression of CD73 and adenosine in the tumor microenvironment, which in turn affects the M2 polarization of TAMs and inhibits tumor growth 58 (Figure 4).
The above studies suggest that IF can cause favorable changes in the immune cell population, but there are results that contradict these studies. For example, IF in a lupus mouse model leads to the expansion of Tregs 59 . Moreover, IF can exacerbate acute immunity and behavioral sickness in response to stimulation with the viral mimic, poly(I:C) 60 . These findings suggest that the effect of a specific IF regimen on the development and function of immune cells is complex and remains to be elucidated.

IF and tumor therapy IF, chemotherapy, and radiotherapy
Previous studies have shown that fasting cycles combined with chemotherapy (CT) are viable and might slow tumor progression and reduce CT-induced side effects in some patients with different types of cancer. The beneficial therapeutic effect of IF combined with CT on tumors indicate that IF enhances the therapeutic effect of CT on tumors 61,62 and also significantly ameliorates the inflammation caused by CT [63][64][65][66][67][68][69] . Early studies suggested that IF can produce effective immunity and reduce the incidence of infection and febrile neutropenia. Moreover, in animal models of systemic bacterial infection, IF can inhibit inflammation and protect cognitive function 70,71 . Further analysis has shown that IF can inhibit the expression of proinflammatory cytokines, including RANTES/CCL5, TNF-α, IL-1β, IL-6, and IL-18, and the inflammatory body proteins NLRP1 and NLRP3 by activating NF-κB, thereby reducing the incidence of inflammation 72,73 .
However, the exact mechanism involving the effects of IF combined with CT might also be complex due to the varied influence of IF on gene expression. IF before CT has been shown to protect the host from treatment toxicity by reducing the expression of some oncogenes, such as RAS and the AKT signaling pathway. This reduction is mediated by decreases in circulating IGF-1 and glucose. Moreover, IF-induced CR activates other oncogenes in cancer cells, induces autophagy, and decreases cellular growth rates while increasing the sensitivity of tumor cells to antimitotic drugs. In addition, the effect of IF combined with CT on tumor growth may depend entirely on the cellular immune system, because CT combined with IF cannot control tumor growth in nonthymic mice due to the lack of T lymphocytes 74 .
Similarly, some studies have shown that IF increases cancer sensitization to radiotherapy (RT) and extends the survival of starved experimental animals after different IF regimens. For example, IF improves the survival of mice with orthotopic pancreatic tumors subjected to lethal abdominal radiation compared with controls with free access to food. Moreover, IF does not affect radiation therapy-mediated tumor cell killing and enhances γ-H2AX staining after radiation therapy, suggesting an additional mild radiosensitizing effect 75 .
Importantly, preclinical and some preliminary clinical data support the hypothesis that IF could be utilized as a complementary treatment to improve the outcome after RT, both in terms of improved tumor control and a reduced probability of normal tissue complications 76,77 .
Although IF combined with these traditional anticancer therapies has shown promising results in these basic studies, more preclinical experimental data are still needed because clinical relevance remains low due to insufficient human data, including few clinical outcome studies, a lack of safety data, and almost no randomized controlled trials 78 . Future studies therefore need to focus on safety, and ultimately  89 increase benefits to current therapies associated with IF. Moreover, the potential of IF to enhance the response to lower doses of CT and radiation therapy also should be further investigated.

IF and tumor immunotherapy
Presently, there are no reports about IF and tumor immunotherapy. In the tumor microenvironment, IF can reduce glucose uptake and glycolysis in tumor cells. However, effector T cells of antitumor immune responses also rely on glycolysis to maintain their clonal expansion and function. Importantly, studies have shown that immune checkpoint blockade (ICB) therapy with PD-L1 antibodies selectively protects T cells from decreased glucose utilization in the tumor microenvironment 79 , which suggests that ICB combined with IF may become a promising clinical tumor treatment strategy. However, IF can reduce the level of circulating glucose and slow the growth of tumor cells 80 . ICB is used to improve the energy metabolism of T cells and maintain their function 81 to achieve an improved effect of clinical tumor immunotherapy. However, considering the complexity of the energy metabolism mechanism triggered by IF and the differences in ICB immunotherapy methods, more prospective studies are needed to characterize the regimens and effects of IF combined with tumor immunotherapy.

IF and other therapies
One recent study showed that the combination of IF and vitamin C was a promising intervention with low toxicity, which could be tested in randomized clinical trials against colorectal cancer and possibly other tumors with KRAS mutations. Specifically, the anticancer activity of vitamin C is limited by the upregulation of HO-1. However, IF selectivity reverses vitamin C-induced upregulation of HO-1 and ferritin in KRAS-mutant cancer cells, consequently increasing reactive iron, oxygen species, and cell death, an effect that is further potentiated by CT 82 . These findings indicate that IF combined with noncytotoxic compounds could be novel and promising treatments of tumors. In addition, the related progression on IF combined with tumor therapy was listed in the Table 2.

Conclusions
Current studies have shown that IF possesses a wide range of effects that improve energy metabolism and the occurrence of many diseases; IF has a crucial effect on tumor immune responses, suggesting that IF is emerging as a promising strategy in clinical tumor therapy. However, because of the complexity of the effects of IF on tissues and cell energy metabolism, there are still many scientific challenges to be further addressed. In the present review, we summarized three aspects of these scientific problems ( Figure 5).
(1) Regarding the mechanism, what are the direct effects and mechanisms of IF on the development and function of different tissue-specific cells and immune cells? For example, one recent study showed that 30-day IF was associated with an anticancer serum proteomic signature, upregulated key regulatory proteins associated with glucose and lipid metabolism, and affected the circadian clock, DNA repair, cytoskeletal remodeling, the immune system, and cognitive function, and resulted in a serum proteome that was protective against cancer, metabolic syndrome, and inflammation. Furthermore, it is important that there are some different nutritional interventions (Table 1), which may lead to different effects on physiological indices including blood glucose, triglycerides, growth hormone, insulin, and insulin-like growth factor 1 (IGF-I). Different clinical treatment strategies also might affect the ultimate effects of IF in cancer patients. Further research is therefore needed before the use of IF as an intervention can be recommended to increase the quality of life for tumor patients.
(2) Regarding side effects, the different influences of distinct IF regimens on the energy metabolism of tumor patients remain largely unknown. To better monitor the side effects of IF, is it possible to standardize IF regimens for different cancer treatments? One study reported that it will be necessary to test and standardize IF regimens by methodologies that are similar to those performed for the approval of drugs by the FDA, to allow these interventions to be implemented by a large portion of the population.
(3) Regarding application, does IF exacerbate malnutrition or other adverse reactions in tumor patients? Some studies report that malnutrition and sarcopenia frequently occur in cancer patients and have a negative effect on clinical outcomes. The reason may be driven by inadequate food intake, decreased physical activity, and catabolic metabolic derangements. The process regarding the application of IF in cancer patients should therefore be more rigorous, and examination of major adverse clinical events (including malnourishment, cachexia, possibly a weakened immune system, and increased susceptibility to certain infections, which need to be carefully monitored) are a vital step in determining whether nutritional intervention is actually beneficial. In addition, because existing research has shown limited safety outcomes, the findings are only useful in developing longer-term trials. Therefore, future studies will have to take into consideration the risk of malnutrition and sarcopenia, and the immunological and metabolic state of the enrolled patients.
In summary, IF has emerged as a promising and powerful tool in clinical tumor therapy. However, IF is a double-edged sword that can affect tumorigenesis, increase immune responses, and alter the energy metabolism of tumor patients. Additionally, there is a notable lack of research on the effects of IF combined with tumor immunotherapy and gene therapy. Thus, further research is needed before the use of IF as an intervention can be recommended to increase the quality of life for tumor patients. We believe that with further elucidation of the mechanism of IF accompanied by the development of molecular biology, systems biology, and mega data, specifically regarding the relationship between IF and tumor immune responses, IF will result in new strategies for clinical tumor immune prevention and treatment in the near future, with wide application prospects.