Elsevier

Biochemical Pharmacology

Volume 67, Issue 1, 1 January 2004, Pages 41-51
Biochemical Pharmacology

Mechanism of mahanine-induced apoptosis in human leukemia cells (HL-60)

https://doi.org/10.1016/j.bcp.2003.07.021Get rights and content

Abstract

Mahanine, a carbazole alkaloid occurs in the edible part of Micromelum minutum, Murraya koenigii and related species has been found to induce apoptosis in human myeloid cancer cell (HL-60). Concentration of 10 μM mahanine caused a complete inhibition of cell proliferation and the induction of apoptosis in a time dependent manner. Mahanine-induced cell death was characterized with the changes in nuclear morphology, DNA fragmentation, activation of caspase like activities, poly(ADP-ribose) polymerase cleavage, release of cytochrome c into cytosol and stimulation of reactive oxygen species generation. The cell death was completely prevented by a pancaspase inhibitor benzyloxycarbonyl-l-aspart-1-yl-[(2,6-dichlorobenzoyl)oxy]methane (Z-Asp-CH2-DCB). Mahanine activated various caspases such as caspase-3, -6, -8 and -9 (like) activities but not caspase-1 like activity. More than 70% cell survival was observed in the presence of a caspase-3 inhibitor. In addition, co-treatment of cyclosporin A markedly increased the survival of mahanine-treated HL-60 cells. Flow cytometric analysis revealed that mahanine decreased the mitochondrial membrane potential of intact cells, and disrupted cell cycle progression by increasing the number of cells in sub-diploid region, concomitantly with the decrease of cells in diploid phases, particularly at late hours of apoptosis. The overall results suggest that mahanine down regulates cell survival factors by activation of caspase-3 through mitochondrial dependent pathway, and disrupts cell cycle progression.

Introduction

Identifying mode of cell death has been given central importance in the study of various diseases, such as cancer and neurodegenerative diseases (e.g. Alzheimer’s and Parkinson’s diseases) [1], [2]. Cells undergo death by two major mechanisms: necrosis, in which primary damage to the metabolic or membrane integrity of the cell occurs, or apoptosis, which is an internal suicide program contained in all cells [3]. Programmed cell death (apoptosis) plays an important role in the regulation of tissue turnover in most normal and cancerous tissues.

The present trends in the management of cancer development include increasing awareness and chemoprevention that suggest using natural or synthetic substances to prevent initiational and promotional events associated with cancer development and this strategy has been considered to be the most direct way to counteract malignancy development [4]. Epidemiological studies suggest that dietary intake of fruits and vegetables, is effective against many diseases including cancer [5], [6]. Mahanine (3,11-dihydro-3,5-dimethyl-3-(4-methyl-3-pentenyl)-pyrano[3,2-a]carbazol-9-ol), a carbazole alkaloid has been reported to be present in the edible parts of some plants such as Micromelum minutum and Murraya koenigii, that are consumed in some parts of Southeast Asia, particularly Thailand, and in South Asia [7], [8]. The compound showed various bioactivities such as antimutagenicity against heterocyclic amines, antimicrobial activity against gram positives, anti-inflammatory effect, and appeared to be cytotoxic against several cancer cell lines [7], [8], [9], [10] although the mechanism of action is elusive.

It is becoming clear that caspases (cysteine aspartic acid-specific protease) play central roles in the execution of apoptosis [11], [12]. They are found in cells as inactive precursors, being converted into active form dismantle key cellular structure by generating proteolytic torrent [13], [14]. There are evidences that show caspase activation occurs in cell death pathway through the initiation of certain member of tumor necrosis factors [15]. However, more recently an alternative route to caspase activation involving mitochondria has emerged [16]. To investigate the possible mechanism(s) that involved in mahanine-induced apoptosis we examined the effect of this compound on cell morphology, DNA fragmentation, DNA ladder formation, activation of various caspases, release of cytochrome c, ROS production, mitochondrial membrane potential and cell cycle progression. In some experiments we used various inhibitors to conclude the observed results. In this study, HL-60 cell line was selected as a model system since it is broadly used in the study related to the evaluation of antiproliferative activity, differentiation, and apoptotic effect of potential active molecules.

This study revealed that mahanine actively induces apoptosis in HL-60 cells and reduces proliferation of HL-60 cells. The pathways that involved in the induction of apoptosis were activation pathways in the expression of caspase-3 activity related with the accumulation of cytochrome c in cytosol. Our data show that mahanine can induce ROS production in HL-60 cells and affects normal cell cycle progression.

Section snippets

Reagents

Fluorogenic caspase substrates and specific caspase inhibitors were obtained from Peptide Institute Inc. Z-Asp-CH2-DCB general caspase inhibitor, cyclosporin A (cs A), and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbo-cyanine iodide (JC-1) were obtained from Wako Pure Chemicals. For Western blotting, anti-PARP (poly(ADP-ribose) polymerase) IgG was obtained from Santa Cruz Biotechnology, anti-cytochrome c IgG was obtained form R&D System, Inc. All other chemicals were purchased

Effect of mahanine on cell proliferation, morphological changes, and cellular DNA of HL-60 cells

The doubling time of HL-60 was about 24 hr. To assess the effect of mahanine on cell proliferation alamar blue reduction and trypan blue exclusion methods were used. Mahanine inhibited proliferation of HL-60 cell in a concentration- and time-dependent manner. Proliferation inhibition was observed as early as 8 hr, and a complete inhibition was detected when cells were treated with mahanine at a concentration higher than 10 μM for 72 hr. About 8.5 μM of mahanine inhibited 50% of cell growth after 48 

Discussion

There are reports that indicate mahanine possess a wide variety of physiological activities [7], [8], [20]. It has also been shown that the vegetable species M. minutum, M. koenigii, M. zeylanicum, and M. euchrestiolia have significant pharmacological values, the edible parts of which contain many cabazole alkaloids [7], [8], [9], [10], [20], [21] including mahanine. Very recently, identifying active components from foods with apoptosis inducing activity against cancer cell lines has been

Acknowledgements

We thank Dr. Tadashi Yoshihashi, Dr. Eizo Tasumi, and Dr. Masayoshi Saito for their expert technical assistant. We are also indebted to Dr. Masuko Kobori, Dr. Masao Goto, Dr. Keiko Iwashita (National Food Research Institute, Tsukuba, Japan) for using their facilities in FACS analysis. This work was supported by JIRCAS under Visiting Research Fellowship Program.

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