NF-κB mediates the antiproliferative and proapoptotic effects of bergamot juice in HepG2 cells
Introduction
Cancer is the second leading cause of death worldwide [1], and liver cancer is one of the most prevalent. Hepatocellular carcinoma (HCC) is the seventh most common cancer and the third leading cause of cancer death worldwide [2]. It accounts for 85–90% of primary liver cancer and, generally, it is lethal at 3–6 months after diagnosis. Globally, over half a million people develop HCC each year and for an equivalent number of people it is the cause of death.
Over the past few decades, significant progress has been made in the diagnosis and treatment of HCC. Surgery offers the best chance of successful outcomes, but the majority of patients with HCC are not good candidates for it, thus chemotherapy represents the adjuvant treatment for them. Unluckily, no single agent or combination of them really improve survival rates of HCC patients, and their use is no longer recommended [3]. Therefore, development of novel preventive strategies or co-adjuvant therapy for HCC are warranted.
Growing evidence indicate that regular assumption of fruits and vegetables may protect against major chronic and degenerative diseases such as cancer, although studies have cast some doubts on whether their use is really producing cancer risk reduction [4]. Numerous epidemiological studies demonstrated that regular intake of Citrus fruits may be associated with reduced incidence of various types of cancers [5], [6], [7] and meta-analyses have confirmed the relation with decreased risk for stomach [8], pancreas [9], breast [10] and bladder cancers [11], although some studies have reported their ineffectiveness in the cancer prevention [8], [12], [13].
Citrus bergamia Risso et Poiteau (bergamot) is an endemic tree of the Calabria region (Italy). Bergamot fruit is employed to extract the essential oil obtained from the peel, largely exploited by fragrance industries, but also used in aromatherapy [14], and recently studied for its neuroprotective [15] and anti-cancer potential [16], [17]. Bergamot juice (BJ) instead, is considered just a secondary or even waste product. Previously our studies have demonstrated the ability of BJ to reduce growth rate and migration of SHSY-5Y neuroblastoma cells by interacting with specific intracellular targets linked to the cell cycle, adhesion and migratory machinery [18]. The impairment of both cell adhesiveness and motility by BJ may underlying the slight inhibitory effects on lung metastasis colonization observed in a spontaneous metastatic neuroblastoma severe combined immunodeficiency disease (SCID) mouse model [19]. In addition, we demonstrated that the anti-cancer effect of BJ is due to its flavonoid content, because the flavonoid fraction of BJ (BJe) reduced growth rate in colon cancer cells [20]. Moreover, we have shown that low concentrations of BJe possess antioxidant activities [21] and decreased inflammatory response induced by lipopolysaccharide (LPS) in THP-1 monocytes, through a mechanism involving Sirtuin 1-mediated NF-κB inhibition [22]. Furthermore, it exerted anti-inflammatory effect in an in vivo model of bowel disease [23], suggesting a potential role in rheumatoid arthritis [24]. Finally, very recently we demonstrated the anti-Helicobacter pylori of BJe alone or in combination with antibiotics [25].
In this study, we evaluated the antiproliferative activity of BJ on the HepG2 human hepatocellular carcinoma cells, focusing on its effect on cell cycle distribution and apoptosis. Furthermore, we attempted to investigate the effect of BJ on NF-κB signaling pathways.
Section snippets
Chemical characterization of coumarins and furocoumarins in bergamot juice
Citrus bergamia Risso & Poiteau fruits were collected from trees crop growing in Ionian coast of the province of Reggio Calabria in Southern Italy (Italy). Prof. Antonio Rapisarda (pharmaceutical botany expert, University of Messina) has provided the identification of Citrus bergamia fruits used to obtain the bergamot juice employed in this experimental study. A voucher specimen of the plant, identified according to the botanical literature, was deposited in the herbarium (H.N. 3263–3266 #
Content of oxygen heterocyclic components in bergamot juice
Since in this study we used the same BJ already employed in other investigations where we showed its flavonoids composition [18], [19], in this research we focused on coumarins and furocoumarins determination. As shown in Fig. 1, BJ used in this study contains all the oxygen heterocyclic components detected in bergamot essential oil, except citropten. Although present in very low concentrations, bergamottin and bergapten are the major compounds measured in BJ.
Inhibition of the HepG2 cell growth by bergamot juice
Treatment of HepG2 cells with
Discussion
Very recently, Tomasetti and Vogelstein [36] suggested that a third of the variation in cancer risk among tissues is attributable to environmental factors or inherited predispositions, and that changes in lifestyle can have a huge impact on certain types of cancer, including liver cancer. In this line, several bodies of evidence have demonstrated that the two most important ways to reduce cancer risk are the avoidance of cancer-causing agents and the habitual consumption of foods that may
Conclusions
Our experimental research demonstrate that BJ strongly inhibits the growth of HepG2 cells by inducing cell cycle arrest and apoptosis through the activation of both intrinsic and extrinsic pathways and the involvement of p53 and NF-κB ones. These findings, together with our previous reports, strengthen the hypothesis that BJ exerts antiproliferative effects in vitro by different mechanisms depending on the cell lines. This study enhances the potential of BJ as anticancer agent, emphasizing that
Conflict of interest statement
The authors declare they have no conflicts of interest.
Authors' contributions
Ferlazzo N. and Cirmi S.: performed the experiments and assisted with drafting the manuscript;
Trapasso E., Ursino M. R. and Lombardo G. E.: performed the experiments;
Russo M.: performed the HPLC analyses;
Gangemi S.: analyzed the data;
Calapai G.: assisted in the interpretation of the data;
Navarra M.: conceived and designed the experiments and drafted the manuscript.
Acknowledgments
This research was supported by grants from Calabria Region (PSR Calabria 2007/2013 Misura 124, project “ABSIB”). We are grateful to Drs. Irene Bonaccorsi and Gregorio Costa (University of Messina, Italy) for their help during the cytofluorimetric analysis.
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These two authors contributed equally to this work and share the first authorship.