Despite current progress in cancer treatment, breast cancer death rates continue to increase each year in women worldwide [1]. The severe toxicity and multidrug resistance (MDR) of conventional chemotherapeutics remain significant challenges in the treatment of this disease. Hence, efforts need to focus on finding effective treatment strategies with minimized side effects on healthy cells and tissues. A number of investigations regarding the use of natural products and herbal extracts as anti-tumor therapeutics have increased in the last years due to favorable safety profiles and the possibility for long-term patient treatment.

The common butterbur (Petasites hybridus L.) is a medicinal plant of the Asteraceae family and has been used in traditional medicine for the treatment of a wide variety of medical conditions, including migraine, hypertension, bronchial asthma, and allergic rhinitis. This herb is primarily distributed in Europe, as well as in some regions of North America and Asia [2, 3, 4]. Different parts of the plant contain bioactive compounds with anti-inflammatory, spasmolytic, and antioxidant properties. The therapeutic effects of butterbur are principally due to secondary metabolites, primarily sesquiterpenes known as petasins, which are esters of petasol and angelic acid [5, 6]. The concentration of petasins and their derivatives, specifically isopetasin, neopetasin, S-petasin, iso-S-petasin, neo-S-petasin, varies but is generally higher in the plant’s rhizomes and roots. In addition to sesquiterpene esters, the rhizome and roots also contain toxic pyrrolizidine alkaloids (PAs). For this reason, extracts prepared from the plant for medicinal use must be free of PAs [2, 6]. At present, the petasins are commercially available as dietary supplements for the prevention and treatment of migraine and allergic rhinitis [7, 8, 9]. It has been proposed that the mechanisms by which petasins exert anti-migraine and antiallergic effects are mediated by their anti-inflammatory activity and inhibition of leukotriene synthesis. However, calcium channel blockage by petasins stems from their spasmolytic potential and may also explain their anti-migraine action [10, 11]. Although the exact modes of action of butterbur’s compounds remain elusive, their efficacy and favorable tolerance have been extensively documented [7, 8, 10]. In addition, petasins have shown efficacy against cardiovascular, gastrointestinal, and urogenital disorders and might be beneficial for the prevention of neurodegenerative diseases as well [2, 11].

Recently, it was demonstrated that petasins and their derivates, isolated from different Petasites species, also exert anti-tumor activity. In vitro and some in vivo studies have shown strong antiproliferative activities of these bioactive substances towards various types of human cancers [12, 13, 14, 15, 16]. A new benzofuran derivative isolated from P. hybridus roots showed moderate inhibitory activity on human breast cancer MCF-7 cell proliferation [17, 18]. Guo et al. [12] found that S-petasin from Petasites japonicus inhibited proliferation and migration of the human melanoma cell line A375, and induced apoptosis activating the tumor suppressor p53. Similarly, significant anti-tumor effects of petasin have been reported when different colorectal cancer cell lines were used. Furthermore, the authors of this study have also confirmed these effects in a murine colorectal tumor model showing reduced tumor growth. Moreover, findings suggested that petasin inhibited the Akt/mTOR signaling axis [13]. Another study revealed that exposure to S-petasin and iso-S-petasin isolated from common butterbur elicits high cytotoxicity and apoptotic cell death responses through caspase activation and cytochrome c release in prostate cancer cells [14]. Further, isopetasin and S-isopetasin extracted from Petasites formosanus have been shown to act as MDR inhibitors by targeting P-glycoprotein (P-gp) and suppressing the proliferation of multidrug-resistant cancer cells [15]. Our previous findings indicate that standardized Petasites hybridus L. root extract, with the active ingredient petasin and its derivatives, showed a selective cytotoxic effect towards non-invasive MCF-7 and highly invasive MDA-MB-231 breast cancer cells. These studies documented that MDA-MB-231 cells display an approximate two-fold higher sensitivity to this root extract [16]. However, the mechanisms that underlie the anti-breast cancer activities of sesquiterpene esters remain to be elucidated.

The aim of this study was to reveal the role of Bulgarian Petasites hybridus L. root extract on the induction of apoptosis, necrosis, oxidative stress, and NF-$\kappa$B signaling when the highly invasive breast cancer MDA-MB-231 and non-cancerous MCF-10A cells were treated with this extract. The results indicated that standardized Petasites extract exhibited a selective pro-oxidant effect, increased NF-$\kappa$B levels, and induced apoptosis in breast cancer cells whereas non-cancerous MCF10A cells remained only slightly affected. These findings suggest that the active compounds derived from the roots of Petasites hybridus L. may represent a promising anti-breast cancer therapy and that warrants further in vivo and clinical investigation.

Breast cancer is the second leading cause of death among women worldwide and, despite recent advances in breast cancer therapy, the mortality rate continues to rise [1]. Thus, new alternative approaches, with non or low-toxic treatment regimens, need to be developed. One principal therapeutic strategy is targeting apoptosis specifically within cancer cells and without harming normal cell function [35]. Apoptosis is a tightly coordinated process which is essential for the development and homeostasis of multicellular organisms. This type of regulated cell death efficiently eliminates harmful, damaged and malignantly-transformed cells [20, 21]. In contrast to necrotic cell death, apoptosis does not cause inflammation in surrounding tissues, this feature makes induction of apoptosis in tumor cells a preferential cell response in cancer therapy. However, most currently used chemotherapeutics are highly toxic and induce apoptosis in both tumor and healthy cells causing systemic cellular injury.

Many plants and their bioactive compounds have long been the subject of study due to their multiple therapeutic properties. Such properties may make these compounds useful for the prevention and treatment of various diseases. Excluding toxic plant alkaloids that are currently used in cancer chemotherapy, the interest in herbal plant products with anti-proliferative activity and tolerability to normal cells has dramatically increased in recent years. A growing body of evidence indicates that different plant-derived compounds such as polyphenols, flavonoids, phytosterols, and terpenoids (sesquiterpenes), exert anti-tumor effects through modulating diverse signaling pathways associated with cancer cell growth and invasiveness and, primarily, selectively inducing apoptosis [36, 37, 38, 39, 40, 41]. We previously demonstrated selective cytotoxic effects and morphological changes associates with apoptosis in MCF-7 and MDA-MB-231 breast cancer cells after exposure to a standardized root extract of the medicinal plant Petasites hybridus L. with active sesquiterpenes termed petasins [16]. Common butterbur and its bioactive components are well-studied for treatment of migraine and other disorders, but their potential anti-tumor activity remains poorly investigated and available literature on this topic are quite limited [9].

Following the results of our previous work [16] which determined that the triple negative breast cancer cell line MDA-MB-231 displayed almost twice the sensitivity to common butterbur extract when compared to the MCF-7 cell line, in the present study we confirmed that standardized Bulgarian Petasites hybridus L. root extract triggered apoptosis in MDA-MB-231 cells after 72 hours of treatment. Moreover, Annexin V/PI flow cytometry showed that breast cancer cells were committed to late stages of apoptosis, whereas parallel treatment of non-cancerous MCF-10A cells displayed an early apoptotic response. Late stages of apoptosis are characterized by the formation of apoptotic bodies through a process dependent on activation of a class of proteases termed caspases. Caspase activation, in turn, commits the cell to a process of irreversible programmed cell death. In contrast, early apoptosis is a reversible process as studies have determined that early apoptotic cells can fully recover after the elimination of early apoptotic inducers [42, 43]. Moreover, the phagocytosis of early apoptotic cells trigger the processes of regeneration and angiogenesis at sites of tissue injury suggesting that early apoptotic cell uptake is anti-inflammatory in nature and induces a tolerogenic response [44]. Meanwhile, neither MDA-MB-231 nor MCF-10A cells underwent necrosis after exposure to various doses of the extract, indicating that common butterbur selectively kills breast cancer cells through activation of programmed cell death. Other studies showed induction of late stages of apoptosis via different biological mechanisms when various cancer cells were exposed to purified petasin or its derivates [12, 13, 14, 15]. Based on the findings outlined in this study, we propose that the pronounced pro-apoptotic effect of Petasites hybridus L. root extract on metastatic MDA-MB-231 is due to the presence of petasins.

A possible mechanism by which Petasites hybridus L. root extract induces apoptosis in cancer cells could be through increased production of reactive oxygen species (ROS). Under normal physiological conditions, ROS play an essential role in cellular metabolism and homeostasis. ROS levels are strictly regulated by the endogenous antioxidant defense system which principally comprised of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). Excessive ROS formation impairs redox homeostasis and leads to the occurrence of oxidative stress which is harmful to cellular macromolecules such as proteins, lipids, and DNA [45, 46]. Typically, cancer cells display increased levels of ROS and, in order to maintain the redox homeostasis, they upregulate antioxidant defense systems. Several studies have demonstrated that ROS can induce cell proliferation [47], resistance to apoptosis [48], increased angiogenesis [49], and invasion and metastasis [50]. However, a further increase in ROS above certain thresholds stemming from either endogenous or exogenous sources disturbs redox balance in tumor cells resulting in oxidative stress [51, 52]. Oxidative stress may trigger apoptosis by activating the apoptosome protein complex which initiates the caspase cascade resulting in activation of the intrinsic apoptotic pathway [46]. It should be noted that pro-oxidant agents can increase cellular levels of ROS to cytotoxic levels, and thus this type of agent may induce selective killing of cancer cells and therefore be therapeutically useful [23]. Abdelfatah et al. [15] previously documented that sesquiterpenes from Petasites formosanus act as ROS generators and can trigger a predominantly late apoptotic response in multidrug-resistant cancer cells. Additionally, several other lines of evidence demonstrated that different plant-derived compounds selectively induce apoptosis in cancer cells by driving ROS accumulation and further promoting oxidative stress [53]. Beyond their antioxidant capacities, in certain circumstances, various plant-derived products can exhibit a pro-oxidant role [54, 55]. Accordingly, we showed that the standardized Petasites hybridus L. root extract disturbs the redox balance in MDA-MB-231 and promotes a state of oxidative stress as marked by enhanced MDA levels and depleted GSH levels. MDA, a common marker of oxidative stress, is an end-product of ROS and fatty acid interaction and results in lipid peroxidation and cell membrane damage [26]. GSH depletion is also indicative of oxidative stress [25] as this is the most abundant non-protein intracellular thiol and is commonly present in high (1–10 mM) concentrations. Reduced GSH abundance can have potential application as a marker for various human diseases [56].

The findings outlined in this study are consistent with previous literature regarding the selective pro-oxidant properties of other terpenoids and their ability to activate oxidative stress-mediated apoptosis in cultured human cancer cells [21, 22, 57]. In addition, the pro-apoptotic transcription factor NF-$\kappa$B has been characterized as a cellular “sensor” for oxidative stress [58]. Thus, it is not surprising that we found elevated levels of the NF-$\kappa$B (p105/p50) in breast cancer cells treated with Petasites hybridus L. root extract. Similarly, a previous study by Ricca et al. [59] demonstrated that overexpression of NF-$\kappa$B promotes apoptotic cell death accompanied by ROS generation in multidrug-resistant MCF-7 breast cancer cells. Another in vitro study determined that cisplatin, a well-characterized inducer of oxidative stress [60] and a commonly used first-line chemotherapeutic for various malignancies [61], promotes upregulation of NF-$\kappa$B and induction of apoptosis in head and neck squamous cell carcinoma [62]. Cisplatin has been found to promote severe oxidative stress, associated with decreased antioxidant abundance and increased lipid peroxidation both of which can provoke injury to normal tissues as well [63, 64]. In contrast, our finding determined that Petasites hybridus L. root extract blunts oxidative stress in healthy MCF-10A cells. Moreover, the present work confirmed that NF-$\kappa$B activation is cell-specific [65], and that the protective effect of NF-kB can result in suppression of oxidative stress through increasing antioxidant abundance thus reducing cellular damage in non-cancerous cells.

Studies conducted in rats have indicated increased NF-$\kappa$B activity and activation of pro-apoptotic proteins as a result of exogenously induced oxidative stress [66]. Although cancer is not the direct subject of this particular investigation, this study demonstrates the relationship between oxidative stress, NF-$\kappa$B activation, and its pro-apoptotic role an in vivo setting. Several in vitro studies using human cancer cell lines have also confirmed the pro-apoptotic function of NF-$\kappa$B following exposure to various stimuli. Stark et al. [67] observed that the nonsteroidal anti-inflammatory drug aspirin mediates apoptosis in colon cancer cells through activation of NF-$\kappa$B. A similar report observed induction of apoptosis, that was NF-$\kappa$B-dependent, after treatment of melanoma cells with a biotransformed soybean extract [68]. Moreover, a recent study suggested the involvement of NF-$\kappa$B in activation of apoptotic cell death after exposure of MCF-7 breast cancer cells to anti-tumor tetrahydroquinolines [69]. However, further studies are required to clarify whether NF-$\kappa$B is responsible for the initiation of the apoptotic pathway in MCF-7 cells.

Based on all these findings, which are in line with our study and that a pro-apoptotic role for NF-$\kappa$B is observed in both in vitro and in vivo models, evidence suggests the potential use of Petasites hybridus L. root extract in anti-tumor therapy. However, further studies are needed to confirm NF-$\kappa$B activation by documenting its translocation to the nucleus as well as elucidating the pathways that regulate this transcription factor and determining the signaling pathways that lie upstream of NF-$\kappa$B.

In conclusion, our findings indicate that the Bulgarian standardized Petasites hybridus L. root extract induces oxidative stress and promotes a strong apoptotic response most likely, at least in part, as a result of elevated NF-$\kappa$B levels in MDA-MB-231 metastatic breast cancer cells. The administration of parallel doses of extract affected non-cancerous MCF-10A cells to a lesser extent. Taken together, these results suggest that Petasites hybridus L. root extract might be useful in treatment of invasive breast cancers; however, further studies using in vivo tumor models and clinical trials are necessary to confirm these effects.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conceptualization — RT and LM; Methodology — VU; Software — IG; Validation — SA, TO and IG; Formal analysis — TO; Investigation — SA; Resources — TO; Data curation — IG; Writing—original draft preparation — SA; writing—review and editing — RT; Visualization — IG; Supervision — RT; Project administration — LM; Funding acquisition — LM. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

This research was funded by the National Science Fund of Bulgaria, grant number KII-06-H21/12/2018.

This research was funded by BSF, grant number KII-06-H21/12/2018 and The APC was funded by KII-06-H21/12/2018.

Given her role as Guest Editor, Rumiana Tzoneva had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Amedeo Amedei. The authors declare no conflict of interest.