Elsevier

Biochemical Pharmacology

Volume 68, Issue 12, 15 December 2004, Pages 2387-2396
Biochemical Pharmacology

Involvement of both endoplasmic reticulum and mitochondria in photokilling of nasopharyngeal carcinoma cells by the photosensitizer Zn–BC–AM

https://doi.org/10.1016/j.bcp.2004.08.024Get rights and content

Abstract

Photodynamic therapy (PDT) is recently developed as an effective treatment for malignant disease. In PDT, the photosensitizer eradicates tumour by induction of apoptosis. In this study, we investigated the mechanistic actions of a recently developed second generation photosensitizer, Zn–BC–AM, on nasopharyngeal carcinoma (NPC) cells. Zn–BC–AM was found to localize in the mitochondria, endoplasmic reticulum (ER), and golgi body. Photoactivation of Zn–BC–AM loaded NPC cells resulted in a rapid collapse of mitochondrial membrane potential (Δψm) (15 min), followed by the release of cytochrome c (1 h), and activation of caspases-9 and -3 (4 h). Expression of ER chaperones Bip/Grp78 and Grp94, and ER resident lectin-like chaperone calnexin (CNX) was also enhanced in PDT-stressed NPC cells. Caspase-12, an important caspase involved in ER stress-induced apoptosis, was also activated. Inhibition of Ca2+ uptake into mitochondria by ruthenium red (RR) or loading the cells with EGTA–AM, an agent that buffers intracellular Ca2+ released from ER, resulted in a significant reduction of Zn–BC–AM PDT-induced cell death. These observations suggest that both ER and mitochondria are the subcellular targets of Zn–BC–AM. Effective activation of ER- and mitochondria-mediated apoptotic pathways is responsible for Zn–BC–AM PDT-induced NPC cell death.

Introduction

Nasopharyngeal carcinoma (NPC) is one of the major causes of death in Hong Kong and Southern China. The disease is rare in Europe and North America, and very few oncologists in the Western world have extensive experience in treating this neoplasm [1]. NPC is a malignant squamous cell carcinoma and radiotherapy is the primary form of treatment. The combination of radiotherapy and chemotherapy are used for the treatment of advanced stage NPC. Photodynamic therapy (PDT) is a cancer treatment modality applicable for a wide variety of tumours. These include the use of ALA for skin cancer [2] and porfimer sodium (Photofrin) for endobronchial and oesophageal tumours [3]. PDT involves the sensitization of tumour cells with a photosensitizing compound followed by exposure of the sensitized tissue to visible light [4]. Photoactivation of the sensitizer results in the liberation of highly reactive oxygen species and cell death. Both necrotic and apoptotic cell death have been reported in PDT-induced tumour cell death [5]. Among those recently developed photosensitizers, long-wavelength-absorbing photosensitizers, such as benzochlorins received much interest. Zn(II) benzochlorin analog was found to be quite effective in tumour eradication in vivo [6].

Earlier studies showed the unique subcellular localization of PDT compounds. Cationic photosensitizer triaryl methane dye Victoria Blue BO (VBBO) was found to localize in mitochondria [7]. In human epidermoid carcinoma A431 cells, the plasma membranes and golgi complex were the main target sites of photofrin [8]. The initial oxidative reactions lead to damage to organelles in which the dye is bound, culminating in cell death and destruction of the tumour or abnormal tissue [9]. Both the plasma membrane [8] and mitochondria [10] have been implicated in PDT-induced apoptotic cell death.

Several distinct apoptotic pathways have recently been established [11]. These include the ligand binding of death receptor expressed on the tumour cell surface, oxidative stress on the mitochondria, and the induction of ER stress. Other intracellular signaling pathways have also been implicated in modulating PDT-induced tumour cell death. PDT-induced tumour cell apoptosis was increased by blocking the p38MAP kinase cascade pathway [12]. In this study, the induction of ER stress during photodynamic treatment of nasopharyngeal carcinoma cells (NPC) was investigated. We herein show the subcellular localization of a Zn(II) benzochlorin analog Zn–BC–AM and the involvement of both mitochondria and ER in apoptosis of the NPC cells.

Section snippets

NPC cell lines and chemicals

In Hong Kong, most of the nasopharyngeal carcinomas are undifferentiated carcinomas or poorly differentiated squamous cell carcinomas. In this study, poorly differentiated CNE-2 and undifferentiated HONE-1 squamous cell carcinoma cell lines were grown in 10% heat-inactivated fetal calf serum (FCS, Gibco), 2 mM glutamine, and antibiotics (50 μg/ml penicillin and 50 μg/ml streptomycin, Gibco) supplemented RPMI-1640 and DMEM medium, respectively. Ruthenium red (RR) was obtained from Calbiochem.

Synthesis of photosensitizer Zn–BC–AM

The

Synthesis and absorption characteristics of Zn–BC–AM

When 2,3,8,8,12,13,17,18-octaethylbenzochlorin was treated with thionyl bromide, followed by firstly a substitution reaction with copper(II) cyanide and then zinc metal replacement, of the desired cyano precursor (4) could be obtained with an overall yield of about 42% from the starting material. The Zn–BC–AM (5) was then synthesized from the cyano compound by reacting (4) with methyl aluminium(III) chloroamide. The yield of this last step was 57%. During PDT, photosensitizers are activated by

Discussion

Recent studies reveal two distinct apoptotic mechanisms leading to cell death, namely the extrinsic cell surface receptor-mediated (e.g. tumour necrosis factor receptor) and the intrinsic mitochondria- or ER-mediated cell death pathway [20], [21], [22], [23], [24], [25], [26]. The ER stress-induced apoptotic cell death represents a novel and mitochondrial-independent intrinsic apoptotic pathway [27]. ER is essential to the functions and survival of a cell. Any perturbation of ER functions may

Acknowledgement

This work was supported by the Research Grants Council of Hong Kong (Project nos. CA99/00SC2 and HKBU 2052/02M).

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