Elsevier

Biochemical Pharmacology

Volume 79, Issue 3, 1 February 2010, Pages 361-372
Biochemical Pharmacology

Apoptotic effects of mahanine on human leukemic cells are mediated through crosstalk between Apo-1/Fas signaling and the Bid protein and via mitochondrial pathways

https://doi.org/10.1016/j.bcp.2009.09.007Get rights and content

Abstract

Apo-1 (Fas/CD95), a cell surface receptor, triggers apoptosis after binding to its physiological ligand, Apo-1L (FasL/CD95L). This study reports that mahanine, purified from the leaves of Murraya koenigii, has a dose- and time-dependent anti-proliferative activity in acute lymphoid (MOLT-3) and chronic myeloid (K562) leukemic cell lines and in the primary cells of leukemic and myeloid patients, with minimal effect on normal immune cells including CD34+ cells. Leukemic cells underwent phosphatidylserine externalization and DNA fragmentation, indicating mahanine-induced apoptosis. An increase in reactive oxygen species suggests that the mahanine-induced apoptosis was mediated by oxidative stress. A significant drop in the Bcl2/Bax ratio, the loss of mitochondrial transmembrane potential as well as cytochrome c release from the mitochondria to the cytosol suggested involvement of the mitochondrial pathway of apoptosis. Cytochrome c release was followed by the activation of caspase-9, caspase-3 and caspase-7, and cleavage of PARP in both MOLT-3 and K562 cells. In MOLT-3 cells, formation of the Fas-FasL-FADD-caspase-8 heterotetramer occurred, leading to the cleavage of Bid to its truncated form, which consequently resulted in formation of the mitochondrial transmembrane pore. The incubation of MOLT-3 cells with mahanine in the presence of caspase-8 inhibitor or FasL-neutralizing NOK-2 antibody resulted in the decrease of mahanine-induced cell death. Mahanine was also a potent inhibitor of K562 xenograft growth, which was evident in an athymic nude mice model. In summary, these results provide evidence for involvement of the death receptor-mediated extrinsic pathway of apoptosis in the mahanine-induced anticancer activity in MOLT-3 cells, but not in K562 cells, which are deficient in Fas/FasL.

Introduction

The death receptor-mediated extrinsic pathway is one of the primary apoptotic pathways operative in different cell types. The cell surface Fas receptor (Apo-1 or CD95) belongs to the family of tumor necrosis factor (TNF) and nerve growth factor (NGF) [1], [2], [3]. The mechanism of cell death induced by the Fas/Fas ligand (FasL) involves binding of FasL to Fas receptors on the cell surface that eventually results in caspase-8 activation [3], [4]. Death receptor-mediated activation of caspase-8 could lead to the processing of Bid to form tBid, which mediates crosstalk between cell surface receptor-mediated apoptotic signals and the mitochondria, facilitating the release of cytochrome c into the cytosol and further amplifying relevant apoptotic signaling [5], [6]. The apoptosis induced by Fas is known to regulate tumor progression and chemotherapeutic drug-induced death [7], [8]. The importance of the Fas/FasL pathway is evident from the aggressive disease presentation and decreased survival of patients with several cancer types in which the loss of function of Fas is reported. In addition, the disruption of Fas has been shown to result in enhanced tumor development in experimental animal models [9], [10], while Fas restoration delays outgrowth of the primary tumor. Fas and FasL interactions are known to play a role in the control of distant metastases [11], as well as in the development of chemotherapeutic resistance in some cell types [12]. These observations suggest that Fas is a frequent target for inactivation during oncogenesis and that Fas-induced apoptosis plays a crucial role in the biology and response of malignant diseases.

Leukemia is the most common hemato-oncological disorder that affects different age groups. Acute type leukemia is seen in children and adults, but the chronic phase of leukemia mainly affects adults [13], [14]. Due to the presence of minimal residual disease, relapse and drug resistance are the major problems in these malignancies [15], [16], [17], [18]. Different combinational chemotherapy is available, but to improve the subset of cancer therapeutics and to upgrade the cancer prevention policy, the establishment of a novel drug is still in demand. Various cell lines derived from leukemia patients have generally been used to study the apoptotic pathways. Understanding the influence of a given drug on these pathways provides opportunities for manipulation of the pathways for better drug efficacy [19], [20].

New drugs, particularly those from herbal sources, are promising modalities for the treatment of a variety of cancers [21], [22]. Mahanine, a carbazole alkaloid occurring in Micromelum minutum, Murraya koenigii and related species, has been shown to exhibit antimutagenicity, antimicrobial activity and cytotoxicity [23], [24], [25], [26]. Mahanine is also known to induce apoptosis in histiocytic lymphoma and promyelocytic leukemia [27], [28], and in prostate cancer cells [29], [30]. However, the involvement of various apoptotic pathways in mahanine-induced cell death is not known. Here, we demonstrate the involvement of the Fas/FasL pathway during mahanine-induced cell death in Fas/FasL-positive acute lymphoid (MOLT-3) leukemic cell line, but not in Fas/FasL-deficient chronic myeloid (K562) leukemic cell line. To our knowledge, no study has thus far shown the involvement of the Fas/FasL system specifically in lymphoid and myeloid leukemic cells as a result of mahanine treatment.

Section snippets

Reagents

The primary antibodies (Bcl2, Bax, Fas, caspase-9, caspase-3, caspase-7, caspase-8, cytochrome c and PARP), secondary HRP-conjugated antibodies, all flow cytometry compatible fluorescence-conjugated antibodies, annexinV–FITC, the ApoDitect TUNEL assay kit, FasL-neutralizing NOK-2 antibody and growth factors (SCF, G-CSF, GM-CSF) were purchased from BD Bioscience (USA). The primary antibodies (FasL, FADD, Bid and β-actin) and growth factor (IL-2) were obtained from Cell Signaling Technology

Mahanine inhibits the proliferation of leukemic cells of myeloid (K562) and lymphoid (MOLT-3) origin

Leukemic cells (K562 and MOLT-3) were subjected to mahanine treatment to evaluate its cytotoxic effect. Mahanine showed significant anti-proliferative activity in both a dose- and time-dependent manner as assessed by the trypan blue (Fig. 1B) and thymidine uptake assays. The IC50 values of mahanine after 48 h of treatment were 10.6 μM and 13.0 μM for MOLT-3 and K562 cells, respectively. The radioactive thymidine uptake assay also showed the same trend of viability. Complete inhibition of

Discussion

The development of novel herbal remedies for the treatment of leukemia is important because of the side effects of existing synthetic pharmaceutical medications [44], [45], which target a variety of target molecules [46], [47], [48]. We have recently demonstrated two key enzymes responsible for induction of leukemia-associated antigens, which may also be considered as drug targets in leukemia [49], [50]. Treatments can be further improved if the mechanisms of induction of cell death associated

Acknowledgements

This work received financial support from the Department of Biotechnology (GAP 235) and the Council of Scientific and Industrial Research (IICB), Govt. of India, New Delhi.

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