Sanguinarine induces apoptosis of human osteosarcoma cells through the extrinsic and intrinsic pathways

https://doi.org/10.1016/j.bbrc.2010.07.114Get rights and content

Abstract

The quaternary benzo[c]phenanthridine alkaloid sanguinarine inhibits the proliferation of cancerous cells from different origins, including lung, breast, pancreatic and colon, but nothing is known of its effects on osteosarcoma, a primary malignant bone tumour. We have found that sanguinarine alters the morphology and reduces the viability of MG-63 and SaOS-2 human osteosarcoma cell lines in concentration- and time-dependent manner. Incubation with 1 μmol/L sanguinarine for 4 and 24 h killed more efficiently MG-63 cells than SaOS-2 cells, while incubation with 5 μmol/L sanguinarine killed almost 100% of both cell populations within 24 h. This treatment also changed the mitochondrial membrane potential in both MG-63 and SaOS-2 cells within 1 h, caused chromatin condensation and the formation of apoptotic bodies. It activated multicaspases, and increased the activities of caspase-8 and caspase-9 in both MG-63 and SaOS-2 cells. These data highlight sanguinarine as a novel potential agent for bone cancer therapy.

Research highlights

► We show for the first time the effect of sanguinarine (SA) on MG63 and SaOS-2 cells. ► SA altered osteosarcoma cell viability in a concentration and time dependent manner. ► SA induced osteosarcoma cell apoptosis and increased caspase-8 and -9 activities. ► SA decreased dose dependently the Bcl-2 protein level only in MG63 cells. ► SaOS-2 which are osteoblast-derived, seemed more resistant to SA than MG63.

Introduction

Osteosarcoma is a primary malignant tumour of the skeleton that frequently metastasizes to the lungs [1]. This very aggressive cancer is the second most common childhood cancer. Around 75% of all osteosarcoma patients are aged between 10 and 25 [1], [2]. The main treatment for osteosarcoma is presently surgery and chemotherapy. But surgery does not prevent metastases, which can cause the death of 75% of patients within 2 years of diagnosis [3]. Chemotherapy with high doses of agents like methotrexate, cisplatin and doxorubicin helps to eradicate metastases and attacks primary tumour cells, but it can have side effects on the heart and kidneys, and may cause infertility [4]. A combination of surgery and chemotherapy produces a small but constant improvement in patient survival, but 30–40% of patients suffer a relapse within 3 years of diagnosis [3]. The grim prognosis for osteosarcoma patients makes it imperative to develop new drugs that can produce cell-cycle arrest and apoptosis, the lack of which are the hallmarks of cancerous cells [3], [5], [6]. Particularly promising are plant-derived alkaloids such as sanguinarine, a quaternary benzo[c]phenanthridine alkaloid extracted from members of the Papaveraceae [7], [8], [9]. Sanguinarine is a phytoalexin with anti-microbial, anti-oxidant, anti-inflammatory and pro-apoptotic effects [9], [10], [11], [12], [13]. Apoptosis is characterized by distinct morphological changes that include cell rounding, membrane blebbing, cytoskeletal disassembly, and chromatin condensation, with or without DNA fragmentation [14]. This cellular suicide can be induced by extrinsic and intrinsic apoptotic pathways [15], [16]. The extrinsic apoptotic pathway is triggered by the ligand-induced activation of receptors at the cell surface, which promotes the activation of caspase-8 [16]. The intrinsic apoptotic pathway leads to the release of cytochrome c from the intermembrane space of the mitochondria into the cytoplasm and the activation of caspase-9 [16]. Apoptosis can be regulated by the Bcl-2 family of anti-apoptotic (Bcl-2, Bcl-xL) and pro-apoptotic (Bax, Bad, Bak) proteins [17]. Sanguinarine acts on both apoptotic pathways in human promyelocytic leukemia HL-60 cells [18].

However, the impact of sanguinarine on osteosarcoma cells is unknown. We therefore investigated the effects of sanguinarine (1 and 5 μmol/L) on two human osteosarcoma cell lines, MG-63 and SaOS-2. We determined its concentration- and time-dependent impact on cell morphology and viability. We also examined the capacity of sanguinarine to activate apoptosis in osteosarcoma cells by measuring changes in mitochondrial membrane potential, multicaspase activation, and caspase-8 and caspase-9 activities. Lastly, we verified the effect of sanguinarine on the expression of Bcl-2 and Bax proteins.

Section snippets

Chemicals

Sanguinarine chloride hydrate, 98% pure, was purchased from Fluka (Sigma–Aldrich, Oakville, ON, Canada) and dissolved in methanol.

Cell culture and treatments

MG-63 (CRL-1427, American Type Culture Collection (ATCC), Manassas, VA, USA) human osteosarcoma cells were incubated at 37 °C in alpha minimum essential medium without ascorbic acid (α-MEM, Gibco®, Grand Island, NY, USA), while SaOS-2 (HTB-85, ATCC) human osteosarcoma cells were grown in McCoy’s 5A medium (ATCC). The growth media (GM) contained heat-inactivated foetal

Effects of sanguinarine on the morphology of osteosarcoma cells

We examined the morphology of osteosarcoma cells that had been incubated with sanguinarine for 4 h (Fig. 1A) and 24 h (Fig. 1B). After 4 h in 1 μmol/L sanguinarine, many of the MG-63 cells had rounded up and started to detach from the PS surface, while most of the SaOS-2 cells remained well-spread (Fig. 1A). Both osteosarcoma cells became rounded up after 4 h with 5 μmol/L sanguinarine. Incubation for 24 h with 1 μmol/L sanguinarine produced morphological changes in both osteosarcoma cell lines (Fig. 1

Discussion

Current multidisciplinary therapies use bone resection and multi-agent chemotherapy to control the proliferation and survival of osteosarcoma cells [3], [15], [20]. Osteosarcoma cells can be treated with chemotherapeutic drugs that cause them to undergo apoptosis [21]. Micromolar concentrations of sanguinarine promoted apoptosis in several human cancerous cell lines, such as A549 lung cells [11], AsPC-1 and BxPC-3 pancreatic cells [22] and HCT116 and SW480 colon cells [12]. But the effect of

Conflict of interest statement

All the co-authors have no conflict of interest.

Acknowledgments

We thank Dr. Owen Parkes for editing the English text. This research was supported through a Canada Research Chair in Cell-Biomaterial Biohybrid Systems, a Natural Sciences and Engineering Research Council of Canada program, and Université de Sherbrooke through “Équipe de recherche en production et immobilisation de principes actifs” research group. Eric Bergeron was supported by a Fonds de la recherche en santé du Québec fellowship.

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