Apigenin, by activating p53 and inhibiting STAT3, modulates the balance between pro-apoptotic and pro-survival pathways to induce PEL cell death

Apigenin is one of the major flavonoids being present in a variety of natural sources such as parsley, chamomile, celery, artichokes, and oregano [1]. Several studies have reported that apigenin can be used to prevent or successfully control tumor progression in vivo, in several animal models [2]. These effects have been shown to occur either through the activation of the p53 oncosuppressor gene [2, 3] or the inhibition of oncogenic pathways, such as NFkB [4], HIF1alfa [5] and AKT [6]. The latter has been reported to be a target of apigenin in Primary Effusion Lymphoma (PEL) cells [7]. PEL is a Kaposi Sarcoma Associated Herpesvirus (KSHV)-associated malignant B cell lymphoma highly refractory to conventional chemotherapies [8], but displaying a good susceptibility to treatment with natural products such as capsaicin [9, 10]. These molecules share the characteristic to concomitantly inhibit multiple oncogenic pathways such as AKT and STAT3, strongly involved in PEL cell survival [11‐13]. The latter indeed positively regulates the expression of pro-survival molecules such as survivin, cyclinD1 and c-myc [14, 15] or anti-apoptotic proteins such as c-FLIP [16, 17].

Similarly to other flavonoids, apigenin has been reported to target and to inhibit STAT3 in several cancers [18, 19] sometimes concomitantly with the activation of p53 tumor suppressor functions [18], leading to reduction of cancer cell survival. Interestingly, it has been described that activated STAT3 may inhibit p53, repressing its pro-apoptotic activity [20] and that, on the other hand, wtp53 activation may reduce STAT3 tyrosine phosphorylation and interfere with its DNA binding activity in prostate cancer cell lines displaying constitutive STAT3 activation [21]. Furthermore, it has been reported that the loss of p53 function activates JAK2-STAT3 signaling to promote pancreatic tumor growth and gemcitabine resistance [22]. All these studies suggest a reciprocal influence between p53 and STAT3 in cancer. Based on this knowledge and on the reports showing that apigenin can target both STAT3 and p53 in cancer cells, in this study we investigated the effect of apigenin in PEL cells that display constitutive STAT3 activation and wtp53, and the underlying mechanisms [21]. ROS, highly produced in cancer cells including PEL, are essential for their survival since sustain the activation of STAT3 as well as of other oncogenic pathways. Intracellular ROS can be regulated by wtp53 [23] that, among its numerous activities, may reduce the intracellular ROS by up-regulating catalase, one of the most important ROS scavenger [24] or other anti-oxidant enzymes [25]. Thus, we next investigated whether ROS reduction by apigenin could lead to STAT3 de-phosphorylation and whether STAT3 inhibition could down-regulate the expression of downstream molecules such as c-myc and cyclin D1 that sustain cancer cell proliferation and/or affect cellular or viral anti-apoptotic molecules. Finally, we evaluated whether apigenin, previously reported to interfere with AKT activation, could also affect autophagy in PEL cells.

PEL, the KSHV-associated B cell lymphoma, is highly malignant and characterized by poor response to chemotherapies. In this study, we show that apigenin could represent a valid therapeutic strategy against PEL, also confirming a previous reported study [7]. Apigenin is a flavonoid widely distributed in plant kingdom displaying several beneficial effects also against cancer [3, 35‐37]. We found that, among the underlying mechanisms leading to PEL cell death, apigenin induced a cross-talk between p53 and STAT3. Indeed apigenin induced the activation of p53, as revealed by p21 up-regulation, and induced catalase expression, reducing ROS, dephosphorylating STAT3 and preventing STAT3 inhibitory influence on p53 (Fig. 6). Although the influence of p53 on STAT3 [21] and of STAT3 on p53 [20], have been previously described, this study shows, for the first time, a cross-talk between the two pathways mediated by the reduction of intracellular ROS. p53 is strongly involved in the control of ROS level, since it can have both pro-oxidant and anti-oxidant properties that can be mediated by the inhibition or the activation of catalase activity [38]. The finding that apigenin, by activating the anti-oxidant response, reduced PEL cell survival is quite surprising, since it usually elevated oxidant species to induce cancer cell death [39]. However, it is well known that ROS levels need to be finely regulated since either their increase or their reduction reduce cell survival [23]. In one hand, high levels of ROS sustain oncogenic signaling, indeed PEL cells die when ROS decrease following treatment with metformin [26] or with NAC, while on the other hand ROS increase may also lead to cancer cell death, although cancer cells are equipped with high expression of molecules, such NRF2, that allow them to rapidly adapt to ROS increase.

STAT3, one of the pathway strongly sustained by ROS, is constitutively activated in PEL cells and plays a fundamental role for the survival of these cells, as well as of several other cancer cell types [13, 40]. It indeed may promote the transcription of a variety of pro-survival molecules such as survivin, c-myc and cyclin D1. Interestingly, STAT3 also interacts with AKT/mTOR pathway in PEL cells, indeed they can positively influence each other to promote cell survival and both are sustained by high levels of intracellular ROS [9, 10, 33]. On the other hand, ROS production is influenced by the expression of KHSV proteins such as v-FLIP [41], strongly down-regulated by apigenin, similarly to its cellular homologue c-FLIP. C-FLIP and v-FLIP are molecules involved either in the inhibition of apoptosis and of autophagy [42‐44], therefore their down-regulation may contribute to the induction of both processes by apigenin in PEL cells. However, the inhibition of STAT3 is likely one of the main stimuli leading to autophagy induction by apigenin and indeed its activation, by regulating the release of cytokines by cancer cells [9] as well as by immune cells [45], may result in autophagy inhibition [46]. STAT3 activation and autophagy inhibition also correlated with an impairment of DC phenotypic and functional properties [45], besides interfering with other aspects of the immune response for which the autophagic process is also required [47]. Regarding cancer cells, autophagy activated by chemotherapies before or concomitantly with apoptosis can influence cell survival either positively, by helping cells to cope with basal or chemotherapy-induced stress, or negatively, by promoting the degradation of oncogenic molecules such as mutant p53 [48] or c-myc [10]. Furthermore, the effect of anti-cancer drugs on autophagy is worth to investigate, also beacause autophagy can promote the release of ATP [49] and activate the immune system and influence the overall survival of anti-cancer treatments [50]. Regarding this point, there are increasing evidences that the chemotherapy must not only be not toxic for the cells of the immune system, but it should also promote the exposure, or the release, of hidden molecules called damage-associated molecular patterns (DAMPs) that stimulate the immune response against cancer [51].

In conclusion this study, showing the activation of a cross-talk between p53 and STAT3 by apigenin, unveils new molecular mechanisms through which this flavonoid leads to apoptosis and autophagy induction in PEL, an aggressive lymphoma harboring wt p53 and constitutive STAT3 activation, that is very difficult to successfully treat.