Resveratrol-induced apoptosis in human T-cell acute lymphoblastic leukaemia MOLT-4 cells

Abstract

Resveratrol (RES) is a natural occurring phytoalexin that has been shown to have chemopreventive activity. Resveratrol acts both by suppressing cell proliferation and inducing apoptosis in a variety of cancer cell lines. In this study, we show that RES induces apoptosis in MOLT-4 acute lymphoblastic leukaemia cells by modulating three different pathways that regulate cells survival and cell death. We show for the first time that RES inhibits the survival signalling pathways Notch and their down stream effector and modulates the operation of interacting signalling systems. It induces an increase in the levels of the pro-apoptotic proteins p53, its effector p21waf and Bax. We also show that RES inhibits the PI3K/Akt pathway and activates Gsk-3β. The data presented here demonstrate unequivocally that RES induces apoptosis by inhibiting the Notch pathway and markedly influencing the operation of the interacting apoptosis pathways mediated by p53 and PI3K/Akt. These data support findings from other laboratories that have suggested the use of RES as a chemopreventive agent. Here, we have identified potential signalling pathways influenced by RES and this could lead to the identification of the targets of RES-induced apoptosis and growth control.

Introduction

Current attempts to improve the survival of cancer patients largely depend on strategies to target tumour cell resistance, chemoresistance and late relapses, which are the hallmarks of acute lymphoblastic leukaemia (ALL). Notwithstanding, in recent decades conventional chemotherapy has produced a dramatic improvement in survival of patient with ALL [1]. However, refractory or relapsed disease continues to pose a serious problem. In particular, T-ALL is an aggressive form of cancer that affects children and adolescent and patients whose ALL cells exhibit in vitro resistance to anti-leukaemic agents, who have a substantially worse prognosis than patients whose ALL cells are drug sensitive [2], [3], [4]. It is now widely accepted that the apoptotic capacity of the cancer cells is crucial in determining the response to chemotherapeutic agents and that defects in the core machinery of the apoptotic pathway contribute to chemoresistance and poor outcome in patients with T-ALL [5].

In recent years, many compounds that occur in the diet and beverages have been identified as potential chemopreventive agents. Among them is the hydroxylated stilbene RES (3,4′,5-trihydoxystilbene), which belongs to a class of defense molecules called phytoalexins. These are produced by several plants in response to stress, injury, UV irradiation and fungal infections. RES is normally found in many dietary products such as grapes, peanuts, berries and wine [6]. Jang et al. [7] described the cancer chemopreventive activity of RES, which inhibits cellular events associated with tumour initiation, promotion and progression. These authors demonstrated that RES can inhibit free-radical formation and reduce oxidative and mutagenic stress. Recently many other studies have confirmed the ability of RES to suppress the proliferation of a variety of human cancer cell lines and to induce apoptosis in in vitro assays [8]. It has been shown that RES acts by modulating the activity of many different signalling pathways involved in cell cycle regulation and survival, including the PI3K (phosphoinositide-2 kinase)/Akt pathway.

Akt acts down stream to PI3K to regulate many biological processes, such as proliferation, apoptosis and growth. A relevant target for Akt is glycogen synthase kinase-3 (Gsk-3), which acts as a positive modulator of apoptosis. In unstimulated cells, constitutively active Gsk-3 phosphorylates different proteins, such as several transcription factors necessary for cell survival, promoting their inactivation or degradation. Akt is responsible for mediating the inhibition of Gsk-3β by catalysing the phosphorylation of the Ser9 residue in the amino terminus of Gsk-3β[9]. Recently, it has been shown that Gsk-3β can phosphorylate Notch and regulate its activity [10].

Notch is a transmembrane receptor that mediates intracellular signalling involved in cell differentiation and cell survival. Notch signalling is initiated through receptor–ligand interaction, which leads to the proteolytic cleavage of the intracellular domain of the receptor (NIC) and to its translocation to the nucleus. NIC binds to Cbl and modulates the transcription of genes involved in cell differentiation and cell survival. Notch signalling plays an important role in hematopoiesis and constitutively active intracellular forms of Notch have been shown to have oncogenic activity in T-ALL [11], [12]. Activated Notch signalling confers chemoresistance in a wild-type p53-dependent manner. Notch1 appears to inhibit p53 through PI3K/Akt pathway and its inhibition reverses chemoresistance [13].

P53 is a powerful regulator of the cell cycle and is essential in preventing inappropriate cell proliferation and in maintaining genome integrity following genotoxic stress [14]. Loss of p53 function is commonly encountered in human cancer and restoration of p53 function leads to apoptosis in lymphomas [15]. In the context of the impact of apoptosis on therapeutic outcome, novel strategies to restore the apoptotic p53 pathway have been vigorously pursued [16], [17]. Here, we have explored RES-induced apoptosis in T-cell acute lymphoblastic leukaemia and have attempted to elucidate the signalling pathways mediating the apoptotic effects of RES.

In order to investigate the cellular pathways involved in RES-induced apoptosis we analysed the effects of RES on Notch, p53, and Akt pathways. We found that RES reduces cell proliferation and induces apoptosis in MOLT-4 cells in a dose and time-dependent manner. We also found that RES activates the p53 pathway and inhibits the PI3K/Akt pathway and therefore activates Gsk-3β. RES is also able to reduce the level of Notch, possibly acting on Gsk.