Chemotherapeutic treatment is associated with severe toxic effects that vary with the dose and the patient, and lead sometimes to chemotherapy discontinuation which adversely affects the prognosis and eventually reduces the survival rates of patients. The therapies target specifically not only cancer cells but also normal proliferating cells, causing inhibition of cell proliferation, genomic instability, overproduction of different reactive toxic products and mediators and necrosis, leading to severe toxic effects and complications. 5-Fluorouracil (5-FU), a pyrimidine analogue, is among the most widely used drugs in the treatment of many types of cancer. Like most chemotherapeutic agents, 5-FU does not have a selective action targeting cancer cells and this leads to a number of serious side effects [1
]. Different complications are associated with 5-FU therapy and among the most common complications are gastrointestinal effects and dermatitis [2
]. In particular, dermatitis is a frequent side effect of chemotherapy and radiotherapy treatment in cancer patients. Dermatitis arises as the beginning of erythema, swelling, an acneiform rash, severe pruritus, xerosis cutis, skin wounds and inflammatory ulcers, which can lead to chronic inflammation, necrosis, fibrosis and increased risk of infections in patients [9
]. 5-FU may rapidly harm dividing immature keratinocytes, as well as dividing stem cells [12
] and the basal cell layer of epithelium can be directly damaged leading to the loss of the renewal capacity of the epithelium, which may result in ulceration [15
Among the etiological factors that can determine the pathogenesis of toxic side effects induced by chemotherapy treatments, oxidative stress and inflammation are important [16
]. In particular, chemotherapy drugs are responsible for an excessive production of radical oxygen species (ROS) and inflammatory mediators that trigger pathological mechanisms of cell damage, altering the normal physiological parameters of the cells [18
]. In particular, ROS levels are also increased after 5-FU treatment and its induction is also critical to promote both inflammation and drug-mediated apoptotic cell death [20
]. Therefore, inhibiting or limiting ROS production and inflammation may help in the treatment of dermatitis in cancer patients.
Although various types of therapies have been introduced for the prevention and treatment of chemotherapy-induced dermatitis, the efficacy of these treatments remains limited, and thus researchers have a great interest in finding new therapeutic strategies also in the field of natural products.
Pomegranate (Punica granatum
L.) was one of the first domesticated fruits to be cultivated and has also gained considerable recognition as a functional food in the modern era. A large body of literature has highlighted the health-promoting activities of pomegranate juice and fruit extracts. Pomegranate has been used to expel parasites [21
], its seeds and fruit peels to treat diarrhea [22
], its plant flowers to manage diabetes [24
], its tree barks and roots to end bleeding and heal ulcers, and its leaves to manage inflammation and handle digestive system disorders [25
]. The ingredients of pomegranate also show a chemopreventive role through the inactivation and activation of different cell signaling pathways [26
] and their effect in counteracting some chemotherapy-induced side effects, such as nephrotoxicity, hepatotoxicity and intestinal mucositis, have also been reported [28
]. Considering the properties of the polyphenols present in pomegranate juice extract (PPJE), in this study we evaluated its effect on oxidative stress, apoptosis and cellular migration in a human keratinocyte cell line (HaCaT) treated with 5-FU.
Chemotherapy is the most widely used therapeutic approach for cancer treatment but its results are also associated with, sometimes, substantial adverse effects. The mechanism of action of many of these drugs, as well as radiotherapy, is the induction of tumor cells’ necrosis or apoptosis, with consequent ROS overproduction. Although oxidative stress, inflammation and apoptosis play a pivotal role as cellular mechanisms related to therapies for cancer treatment, to cause cancer cells elimination, they can also affect healthy cells. These side effects are particularly evident in tissue with a rapid turnover, such as skin, thus inducing effects that can significantly impair the quality of life in these patients. Therefore, control of oxidative stress and of the inflammatory response is of primary importance to avoid deleterious pathological conditions and to counteract anticancer treatment side effects.
Evidence has shown that many foods rich in polyphenols could be used for cancer prevention as adjuvants in chemotherapy treatments, but they could be used to also reduce cancer therapy side effects [41
]. Pomegranate is a fruit rich in punicalagin, cyanidin 3,5-diglucoside and pelargonidin 3-glucoside [29
], which have potent free-radical scavenging properties [46
] and pomegranate has the potential to be an agent for cancer prevention related to the anti-inflammatory, pro-apoptotic and anti-proliferative activities of its constituents. In this study we focused our attention on the whole PPJE, reporting its protective effect on 5-FU-treated keratinocytes by: (i) reducing oxidative stress, (ii) reducing the inflammatory response, (iii) reducing apoptosis and (iv) promoting cell migration rate.
ROS are one of the principal mediators in tissue and cellular damage triggered by chemotherapy. In accordance with this, our data show that 5-FU induced a significant ROS release with respect to control cells and PPJE significantly inhibited ROS release in 5-FU-treated cells, at the three highest tested concentrations. This antioxidant effect of PPJE is in accordance with other evidence reporting the antioxidant effect of pomegranate on keratinocytes exposed to UV radiation [47
] and in keratinocytes against H2
-induced oxidative stress and cytotoxicity in human keratinocytes HaCaT [48
]. If in normal conditions ROS production can be controlled by antioxidant mechanisms, including small-molecular-weight antioxidants and antioxidant enzyme systems protecting the organism from the damaging effects of oxidative stress, under conditions of severe oxidative stress, for example during the administration of certain drugs as chemotherapeutic agents, cellular endogenous antioxidant defense mechanisms may fail to control the excessive oxidative stress due to ROS overproduction. An oxidative stress condition is due to an imbalance between oxidant factors, such as ROS, and antioxidant factors, such as nuclear factor erythroid 2-like 2 (Nrf2). Nrf2 is a key regulator of the antioxidant response also in keratinocytes, being a redox-sensitive transcriptional regulator that controls the expression of antioxidant and detoxification enzymes such as HO-1 and NQO1 [49
]. Our results indicated that PPJE significantly increased these Nrf-2-related antioxidant enzymes, in accordance with other experimental models reporting the protective effect of Nrf-2 pathway activation in keratinocytes during oxidative stress [50
]. Evidence has shown that ROS activate NF-κB, thus amplifying the pro-inflammatory response [53
]. Nitrotyrosine formation, due to the reaction between ROS and nitric oxide (NO), a pro-inflammatory mediator, is the stable product of the action of peroxynitrite on tyrosine-containing proteins. Nitrotyrosine residues can significantly impair the protein structure and alter its function, leading to potentially pathological results also in keratinocytes [55
]. Our data indicated that PPJE was also able to significantly reduce nitrotyrosine formation thus also reducing the nitrosative stress response.
Proinflammatory cytokine production by epidermal keratinocytes plays a key role in skin inflammation [56
]. Among these pro inflammatory cytokines, TNF-α has been implicated in the promotion of inflammatory reactions via the activation of cytokines IL-6 and IL-1β [56
]. TNF-α released from keratinocytes would facilitate the subsequent influx of inflammatory cells on neighboring endothelial cells and it has been shown to promote the immune/inflammatory reactions via the activation and induction of other cytokines, such as IL-6 and IL-1β [58
]. Therefore, inhibiting or limiting the production of these pro-inflammatory cytokines may help in the treatment of inflammation and dermatitis in cancer patients. PPJE treatment significantly inhibited TNF-α, IL-6 and IL-1β release, thus strongly contributing to the anti-inflammatory response. These data are in accordance with previous studies reporting the anti-inflammatory effect of pomegranate in different experimental models [60
NF-κB is the most important transcriptional factor regulating inflammatory pathways. It also directly controls pro-inflammatory cytokine cellular production in keratinocytes suffering from 5-FU-induced dermatitis [15
]. Our findings revealed that PPJE attenuated 5-FU-induced p65 NF-κB nuclear translocation, thereby reducing the expression of pro-inflammatory cytokines. Previous studies have reported that other agents, such as caffeic acid, saikosaponin A and palmitic acid, can reduce keratinocyte inflammation via NF-κB, TNF-α, IL-1β and IL-6 inhibition [55
] thus highlighting the modulation of these factors as crucial in regulating 5-FU-induced skin side effects.
As a result of oxidative stress and inflammatory response onset, apoptosis occurs in 5-FU-treated keratinocytes relative to non-damaged control cells and PPJE significantly inhibited apoptosis in 5-FU-treated cells. The normal balance between pro-apoptotic members of the family (e.g., Bax) and anti-apoptotic components (e.g., Bcl-2) might also be disrupted by the administration of chemotherapy in healthy cells. PPJE was able to reduce Bax expression and to increase the anti-apoptotic protein Bcl-2, thus contributing to protecting keratinocytes from cell death. Our results are in accordance with other studies reporting the protective effect of pomegranate in protecting normal cells from various stimuli-induced toxicities and apoptosis [65
Keratinocytes play a fundamental role in maintaining the integrity of the epidermal barrier [66
], also taking part in skin repair processes and for these reasons their homeostasis is of primary importance. 5-FU treatment induced a significant reduction in cell migration rate, as assessed in wound healing assay, but PPJE induced a significant increase in cell migration speed, thus promoting the skin damage resolution. The obtained results highlight the potential use of PPJE in counteracting chemotherapy-induced side effects, also because this potential can be exerted with topical use. Thus, PPJE would not interfere in reducing the pro-oxidant, pro-inflammatory and pro-apoptotic effect of the therapy on the cancer cells.