I. Introduction to photodynamic therapy and mechanism of action in nasopharyngeal cancer cell

Nasopharyngeal carcinoma is an endemic tumor in southern China and Southeast Asia (Chan et al, 2004). Three main etiologic factors include genetic susceptibility, chemical carcinogens, and association with Epstein-Barr virus (EBV) infection (Chan et al, 2004). Generally, this carcinoma is highly radiosensitive and chemosensitive (Chan et al, 2004). Attempts have been made to improve treatment results by integrating radiotherapy with some form of chemotherapy (Chan et al, 2004). Presently, radiotherapy remains a common treatment for early disease, while concurrent chemoradiotherapy is being increasingly accepted as the standard treatment for advanced disease (Chang et al, 2004). Intensity modulation radiotherapy (IMRT) delivers a more conformal radiation dose to the target area and may spare normal organs such as the parotid gland for patients with early stage disease (Chang et al, 2004). IMRT in conjunction with concurrent chemotherapy offers excellent tumor control with fewer complications (Chang et al, 2004). The outcome of nasopharyngeal carcinoma treatment has greatly improved as advances in imaging techniques increase the accuracy of tumor mapping (Chang et al, 2004).

However, adequate or effective treatments are not always available for most recurrent or residual nasopharyngeal cancers (Kulapaditharom and Boonkitticharoen, 1999). In those cases, photodynamic therapy (PDT) is a new alternative.

PDT is a medical therapy making used of a non-toxic dye termed a photosensitizer together with low intensity visible light, which, in the presence of oxygen, produce cytotoxic species (Demidova and Hamblin, 2004). PS can be targeted to its destination cell or tissue and, in addition, the irradiation can be spatially confined to the lesion giving PDT the advantage of dual selectivity (Demidova and Hamblin, 2004). In therapy, PDT leads to photochemical tissue destruction or immunomodulation (Dragieva et al, 2004). Considering the mechanism of PDT, light-activation of the photosensitizer in the presence of molecular oxygen, which accumulates in cancer tissues, leads to the local production of reactive oxygen species that kill the tumor cells (Agostinis et al, 2004). Hendrickx et al, (2003) reported that hypericin-mediated PDT of human cancer cells led to up-regulation of the inducible cyclooxygenase-2 (COX-2) enzyme and the subsequent release of PGE2. They noted that the combination of PDT with pyridinyl imidazole inhibitors of p38 MAPK might improve the therapeutic efficacy of PDT by blocking COX-2 up-regulation, which contributed to tumor growth by the release of growth- and pro-angiogenic factors, as well as by sensitizing cancer cells to apoptosis (Hendrickx et al, 2003). Agostinis et al, (2004) noted that mitochondria were central coordinators of the mechanisms by which PDT induces apoptosis in the target cells. They proposed that signaling pathways regulated by members of mitogen activated protein kinases and their downstream targets, such as cyclooxygenase-2, appeared to critically modulate cancer cell sensitivity to PDT (Agostinis et al, 2004). Almeida et al (2004) proposed that activation of phospholipases, changes in ceramide metabolism, a rise in the cytosolic free Ca²⁺concentration, stimulation of nitric oxide synthase (NOS), changes in protein phosphorylation and alterations in the activity of transcription factors and levels of gene expression had all been observed in PDT-treated cells (Almeida et al, 2004). Ali et al, (2002) also noted that an important role of PDT in cancer therapy is induction of cellular apoptosis.

PDT has been used as an integrative therapy for many cancers including nasopharyngeal carcinoma (Zhao et al, 1988). Concerning the mechanism of tumor therapy, the general principle of induction of cellular apoptosis is mentioned in PDT for nasopharyngeal cancer (Zhao et al, 1988). In 2001, Lai et al, performed a study to investigate the effect of PDT on expression of the pro-apoptotic gene Bak in nasopharyngeal carcinoma (NPC). In this study, apoptosis and expression of the pro-apoptotic gene Bak on the tumor tissues from both pre- and post-PDT were determined using the in situ end labeling (ISEL), standard immunohistochemistry technique and western blot, respectively, in 24 patients with either persistent or recurrent NPC after radiotherapy (Lai et al, 2001). According to this study, immunohistochemical assay indicated that 75% of the patients had an upgrade of the expression of Bak protein in their tumor tissues after PDT and increases in expression of Bak from PDT were also confirmed by western blot analysis (Lai et al, 2001). Lai et al, 2001 concluded that PDT probably caused NPC cell apoptosis through an upregulation of the pro-apoptotic protein Bak expression. In 2001, Ali et al examined the photodynamic effects of hypocrellin A and B compounds in poorly differentiated (CNE2) and moderately differentiated (TW0-1) human nasopharyngeal carcinoma cells. According to this study, a loss of membrane phospholipid asymmetry associated with apoptosis was induced by both tumor cell lines as evidenced by the externalization of phosphatidylserine and a dose-dependent increase in caspases-3 protease activity inhibitable by the tetrapeptide inhibitor DEVD-CHO was also observed in both cell lines (Ali et al, 2001). Ali et al, (2001) noted that tumor cell death induced by Hypocrellin A and B was mediated by caspase proteases. Ali et al, (2001) proposed that both hypocrellins (A and B) were potent and promising photosensitizers for the treatment of nasopharyngeal carcinoma.

In 2002, Du et al, investigated the endogenous production of interleukin (IL)-8 and IL-10 in vitro by two EBV-positive nasopharyngeal carcinoma cell lines, HK1 and CNE-2. According to this study, Du et al noted that PDT which was known to upregulate IL-8 transcription via reactive oxygen species and activate the IL-10 promoter did not alter IL-8 levels in either of the NPC cell lines nor induced the production of IL-10 (Du et al, 2002). In 2003, Du et al showed by electron microscopy that subcutaneously implanted HK1 NPC cells from Balb/c nude mice perished by cell necrosis with hypericin-PDT treatment. They said that there was evidence of cytoplasmic swelling accompanied by loss of cell membrane integrity and autophagic vacuolization of cytoplasm but no nuclear changes and there was also no significant difference in the apoptotic index of control and PDT-treated tumors, when analyzed by in situ end labeling of DNA strand breakage to detect apoptosis (Du et al, 2003b). Du et al (2003b) said that these further finding supported the observation that cell death in PDT-treated nasopharyngeal cell /HK1 tumors was by necrosis. They noted that lipid peroxidative stress analyzed by the malondialdehyde assay was significantly elevated in PDT-treated cells, however, PDT had no effect on the activity of superoxide dismutase, an intracellular antioxidant enzyme (Du et al, 2003b). Finally, they concluded that hypericin-PDT of nasopharyngeal tumors in vivo induced tumor necrosis with accompanying lipid peroxidation (Du et al, 2003b). In 2003, Du et al also investigated the effect of hypericin-mediated PDT on subcutaneously implanted NPC/HK1 tumor cells and the relationship between the biodistribution of hypercin and photodynamic effects (Du et al, 2003a). They concluded that hypericin-mediated PDT induces both vascular damage and direct tumor cell killing, thereby bringing about tumor necrosis and shrinkage (Du et al, 2003a). In 2004, Du et al analyzed the effect of hypericin-based PDT on matrix metalloproteinase-1 (MMP-1) expression in two NPC cell lines and an animal tumor model. MMP-1 protein. In addition, mRNA expression were evaluated by Western blot analysis and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) respectively (Du et al, 2004a). According to this study, photoactivation of hypericin, a polycyclic phenanthroperylenedione, elicited an increase in MMP-1 protein and mRNA expression in well differentiated HK1 and poorly differentiated CNE-2 nasopharyngeal cell cells in vitro and there was similar up-regulation of MMP1 mRNA expression in hypericin-PDT-treated nasopharyngeal cell /HK1-tumors (Du et al, 2004a). Du et al proposed that this was the first time that modulation of MMP-1 expression had been demonstrated as a photodynamic effect of hypericin in nasopharyngeal carcinoma cells (Du et al, 2004a). In 2004, they observed that photoactivated hypericin induced the generation of reactive oxygen intermediates in nasopharyngeal cancer cells in vitro (Du et al, 2004a). They noted that there was also significant reduction of glutathione S-transferase (GST) activity in HK1 and CNE-2 NPC cells and in tumor tissues from the nasopharyngeal cell/HK1 murine tumor model by hypericin-mediated PDT (Du et al, 2004b). Du et al (2004b) concluded that down-regulation of GST activity would potentiate the efficacy of hypericin-PDT treatment as antioxidants protect cells against phototoxicity.

II. Summary on previous reports of photodynamic therapy for nasopharyngeal carcinoma

A. General reports on photodynamic therapy for nasopharyngeal cancer

As previously mentioned, PDT can be applied in nasopharyngeal carcinoma. In 1990, Sun reported a case series, consisting of 57 nasopharyngeal carcinoma cases treated with PDT with hematoporphyrin derivative in China (Sun, 1990). In this report, 25 (43.9%) achieved complete response, 25 (43.9%) marked response and 7 (12.2%) mild response (Sun, 1990). In 1992, Sun et al, (1992) reported another case series of 137 cases of nasopharyngeal carcinoma treated by PDT with hematoporphyrin derivative. In this report, the results were: complete response 76 cases (55.47%) and marked response 47 cases (34.31%), with an over-all marked response rate of 89.78% (123/137) (Sun, 1992). In 1997, Lai et al studied the effect of PDT on selected laboratory values of patients with nasopharyngeal carcinoma. According to this study, the results showed that the post-PDT serum level of soluble interleukin-2 receptor (SIL-2R) had significantly declined, while that of IL-2 and the NK cell activity had significantly increased, compared with pre-PDT values, suggesting an immunoenhancing (Lai et al, 1997). The apparent benefit of PDT in nasopharyngeal carcinoma on survival and quality of life must be confirmed. Here, the reports on these outcomes of PDT in nasopharyngeal carcinoma are summarized and present.

B. Reports on photodynamic therapy for recurrent nasopharyngeal cancer

There are some reports on PDT for recurrent nasopharyngeal cancer. In 1996, Tong et al reported their initial experience in using PDT for 12 patients with recurrent nasopharyngeal carcinoma. In this report, all patients were treated with an infusion of hematoporphyrin derivative (5 mg/kg) 48-72 h before exposure to 200 J/cm² light (wavelength, 630 nm) delivered from a gold vapor laser (Tong et al, 1996). According to this study, all 12 patients showed a dramatic response as judged by computed tomography or magnetic resonance imaging at 6 months post-PDT (Tong et al, 1996). Concerning the side-effects of therapy, skin hypersensitivity occurred in two patients and was the only significant complication encountered (Tong et al, 1996). Tong et al concluded that PDT could be an encouraging palliative or definitive management for recurrent superficial nasopharyngeal carcinoma (Tong et al, 1996). In 1995, Lofgren et al performed another similar pilot study to determine if PDT could be a safe and efficacious treatment for recurrent or persistent nasopharyngeal cancer (Lofgren et al, 1995). In this study, 4 patients were injected intravenously with hematoporphyrin derivative (2.5 mg/kg) and one patient with porfimer sodium (2 mg/kg) 48 hours before treatment by a 630-nm laser light passed down a 1-mm core quartz fiber to a miniaturized convex mirror positioned in the nasopharynx via the contralateral nasal cavity (Lofgren et al, 1995). According to this study, all patients survived at 5-years, indicating good outcome of treatment (Lofgren et al, 1995). Lofgren et al, (1995) found that long-term tumor control could be achieved by photodynamic therapy in cases where very high doses of ionizing radiation have failed. They proposed that the entire treatment could be accomplished in 30 minutes under topical anesthesia and the technique carried no serious side effects (Lofgren et al, 1995). In 1999, Kulapaditharom and Boonkitticharoen evaluated the PDT using hematoporphyrin derivative for its effectiveness in treating patients with recurrent nasopharyngeal carcinoma, who failed conventional therapy, with curative or palliative intent in 13 patients. They demonstrated that hematoporphyrin derivative -PDT could effectively control locally recurrent or residual NPC at T1-T2 stages and offered good palliation for more advanced diseases (Kulapaditharom and Boonkitticharoen, 1999). They also proposed that combined PDT and chemotherapy seemed to prolong survival time for a period longer than 2 years in T3-T4 tumors.

C. Reports on photodynamic therapy for nasopharyngeal cancer with different photosensitizers

In additional to general photosensitizer, hematoporphyrin derivative, there are also some reports on PDT for nasopharyngeal cancer with other photosensitizers. Yee et al found that protoporphyrin IX dimethyl ester (PME), a dimethyl esterification of protoporphyrin IX (PPIX), exhibited higher intracellular uptake into NPC/CNE2 cells, a poorly differentiated human nasopharyngeal carcinoma, than did PPIX (Yee et al, 2002). They noted that phototoxicity studies revealed PME to be a more potent photosensitizer than was PPIX, at the early and late incubation time points (Yee et al, 2002). According to this study, correlating phototoxicity with subcellular localization indicated that PME was a more potent photosensitizer when its primary target of photodamage was mitochondria (Yee et al, 2002). In 2002, Betz et al investigated the potential use of 5-aminolevulinic acid (5-ALA, 5-amino-4-oxovaleric acid) induced PPIX for PDT of nasopharyngeal carcinoma and its related mechanisms of inducing cell death. PPIX biosynthesis via fluorescence analysis (Betz et al, 2002). In this study, mechanisms of PDT-induced cell death were investigated via anncxin-V/propidium iodide staining and DNA electrophoresis (Betz et al, 2002). According to this study, more than 80% of induced cell deaths thereby occurred via apoptosis within the first 12 h following irradiation; necrosis was accountable for less than 20% and high level induction of apoptosis by 5-ALA-PDT was confirmed by DNA electrophoresis (Betz et al, 2002). Betz et al reported that their investigations showed promising results of 5-ALA based PDT of nasopharyngeal carcinoma in vitro (Betz et al, 2002).

In 2003, Mak et al studied two sulfonamide derivatives of porphycene, namely PS6 and PS6A, were synthesized, and their photodynamic efficacies on the nasopharyngeal carcinoma cell line NPC/CNE-2. They found that over 99% of CNE-2 cells were sensitized by PS6A 24 h after drug treatment and collapse of the mitochondrial membrane potential was also observed in PS6A PDT-treated CNE-2 cells 1.5 h after PDT (Mak et al, 2003). In addition, confocal microscopy revealed that PS6A was predominantly localized in the mitochondria, lysosomes and Golgi bodies of NPC cells. Significant genotoxicity was not observed in CNE-2 cells (Mak et al, 2003). Mak et al concluded that PS6A mediated both in vitro antitumor and antiangiogenic activities and PS6A might be a candidate for photodynamic treatment of nasopharyngeal carcinoma (Mak et al, 2003). In 2000, Yow et al studied two clinical photosensitizers, Temoporfin (meta-tetra-hydroxyl-phenyl-chlorin; mTHPC) and merocyanine 540 (MC540) for their photocytotoxic and genotoxic effects on nasopharyngeal carcinoma cells (Yow et al, 2000). According to this study, mTHPC-mediated PDT exerted a more potent effect than MC540-mediated PDT, even though the molar extinction coefficient of the main absorption peak for MC540 was much higher than that of mTHPC (Yow et al, 2000). In addition, confocal laser scanning microscopy showed that mTHPC and MC540 localized in the cytoplasm but not in the nucleus of the tumor cells, which provided evidence for undetectable DNA damage under dark and low photodynamic dose (Yow et al, 2000).

References

Agostinis P, Buytaert E, Breyssens H, Hendrickx N (2004) Regulatory pathways in photodynamic therapy induced apoptosis. Photochem Photobiol Sci 3, 721-9.

Ali SM, Chee SK, Yuen GY, Olivo M (2002) Photodynamic therapy induced Fas-mediated apoptosis in human carcinoma cells. Int J Mol Med 9, 257-70.

Ali SM, Olivo M, Yuen GY, Chee SK (2001) Photodynamic-induced apoptosis of human nasopharyngeal carcinoma cells using Hypocrellins. Int J Oncol 19, 633-43.

Almeida RD, Manadas BJ, Carvalho AP, Duarte CB (2004) Intracellular signaling mechanisms in photodynamic therapy. Biochim Biophys Acta 1704, 59-86.

Betz CS, Lai JP, Xiang W, Janda P, Heinrich P, Stepp H, Baumgartner R, Leunig A (2002) In vitro photodynamic therapy of nasopharyngeal carcinoma using 5-aminolevulinic acid. Photochem Photobiol Sci 1, 315-9.

Chan AT, Teo PM, Huang DP (2004) Pathogenesis and treatment of nasopharyngeal carcinoma. Semin Oncol 31, 794-801.

Chang JT, Ko JY, Hong RL (2004) Recent advances in the treatment of nasopharyngeal carcinoma. J Formos Med Assoc 103, 496-510.

Demidova TN, Hamblin MR (2004) Photodynamic therapy targeted to pathogens. Int J Immunopathol Pharmacol 17, 245-54.

Dragieva G, Scharer L, Dummer R, Kempf W (2004) Photodynamic therapy--a new treatment option for epithelial malignancies of the skin. Onkologie 27, 407-11.

Du H, Olivo M, Mahendran R, Bay BH (2004a) Modulation of Matrix metalloproteinase-1 in nasopharyngeal cancer cells by photoactivation of hypericin. Int J Oncol 24, 657-62.

Du HY, Bay BH, Mahendran R, Olivo M (2002) Endogenous expression of interleukin-8 and interleukin-10 in nasopharyngeal carcinoma cells and the effect of photodynamic therapy. Int J Mol Med 10, 73-6.

Du HY, Bay BH, Olivo M (2003a) Biodistribution and photodynamic therapy with hypericin in a human NPC murine tumor model. Int J Oncol 22, 1019-24.

Du HY, Olivo M, Tan BK, Bay BH (2003b) Hypericin-mediated photodynamic therapy induces lipid peroxidation and necrosis in nasopharyngeal cancer. Int J Oncol 23, 1401-5.

Du HY, Olivo M, Tan BK, Bay BH (2004b) Photoactivation of hypericin down-regulates glutathione S-transferase activity in nasopharyngeal cancer cells. Cancer Lett 207, 175-81.

Hendrickx N, Volanti C, Moens U, Seternes OM, de Witte P, Vandenheede JR, Piette J, Agostinis P (2003) Up-regulation of cyclooxygenase-2 and apoptosis resistance by p38 MAPK in hypericin-mediated photodynamic therapy of human cancer cells. J Biol Chem 278, 52231-9.

Kulapaditharom B, Boonkitticharoen V (1999) Photodynamic therapy for residual or recurrent cancer of the nasopharynx. J Med Assoc Thai 82, 1111-7.

Lai J, Tao Z, Xiao J, Yan Y, Wang X, Wang C, Zhou S, Tian Y (2001) Effect of photodynamic therapy (PDT) on the expression of pro-apoptotic protein Bak in nasopharyngeal carcinoma (NPC). Lasers Surg Med 29, 27-32.

Lai JP, Tao ZD, Xiao JY, Zhao SP, Tian YQ (1997) Effect of photodynamic therapy on selected laboratory values of patients with nasopharyngeal carcinoma. Ann Otol Rhinol Laryngol 106, 680-2.

Lofgren LA, Hallgren S, Nilsson E, Westerborn A, Nilsson C, Reizenstein J (1995) Photodynamic therapy for recurrent nasopharyngeal cancer. Arch Otolaryngol Head Neck Surg 121, 997-1002.

Mak NK, Kok TW, Wong RN, Lam SW, Lau YK, Leung WN, Cheung NH, Huang DP, Yeung LL, Chang CK (2003) Photodynamic activities of sulfonamide derivatives of porphycene on nasopharyngeal carcinoma cells. J Biomed Sci 10, 418-29.

Sun ZQ (1990) Hematoporphyrin derivative (HPD) plus laser photodynamic therapy for nasopharyngeal carcinoma--analysis of 57 cases. Zhonghua Zhong Liu Za Zhi 12, 120-2.

Sun ZQ (1992) Photodynamic therapy of nasopharyngeal carcinoma by argon or dye laser--an analysis of 137 cases. Zhonghua Zhong Liu Za Zhi 14, 290-2.

Tong MC, van Hasselt CA, Woo JK (1996) Preliminary results of photodynamic therapy for recurrent nasopharyngeal carcinoma. Eur Arch Otorhinolaryngol 253, 189-92.

Yee KK, Soo KC, Bay BH, Olivo M (2002) A comparison of protoporphyrin IX and protoporphyrin IX dimethyl ester as a photosensitizer in poorly differentiated human nasopharyngeal carcinoma cells. Photochem Photobiol 76, 678-82.

Yow CM, Mak NK, Szeto S, Chen JY, Lee YL, Cheung NH, Huang DP, Leung AW (2000) Photocytotoxic and DNA damaging effect of temoporfin (mTHPC) and merocyanine 540 (MC540) on nasopharyngeal carcinoma cell. Toxicol Lett 115, 53-61.

Zhao SP, Tao ZD, Xiao JY, Peng YY, Yang YH, Zeng QS, Liu ZW (1988) Clinical use of hematoporphyrin derivative and photoradiation therapy in nasopharyngeal carcinoma. Chin Med J (Engl) 101, 86-91.

Review Article

Received: 22 February 2005; Revised: 2 March 2005

Accepted: 4 March 2005; electronically published: May 2005

I. Introduction to photodynamic therapy and mechanism of action in nasopharyngeal cancer cell

References