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The localization of photosensitizers in the subcellular compartments during photodynamic therapy (PDT) plays a major role in the cell destruction; therefore, the aim of this study was to investigate the intracellular localization of Chlorin e6-PVP (Photolon™) in malignant and normal cells. Our study involves the characterization of the structural determinants of subcellular localization of Photolon, and how subcellular localization affects the selective toxicity of Photolon towards tumor cells. Using confocal laser scanning microscopy (CLSM) and fluorescent organelle probes; we examined the subcellular localization of Photolon™ in the murine colon carcinoma CT-26 and normal fibroblast (NHLC) cells. Our results demonstrated that after 30 min of incubation, the distribution of Photolon was localized mainly in the cytoplasmic organelles including the mitochondria, lysosomes, Golgi apparatus, around the nuclear envelope and also in the nucleus but not in the endo-plasmic reticulum whereas in NHLC cells, Photolon was found to be localized minimally only in the nucleus not in other organelles studied. The relationship between subcellular localization of Photolon and PDT-induced apoptosis was investigated. Apoptotic cell death was judged by the formation of known apoptotic hallmarks including, the phosphatidylserine externalization (PS), PARP cleavage, a substrate for caspase-3 and the formation of apoptotic nuclei. At the irradiation dose of 1 J/cm2, the percentage of apoptotic cells was 80%, respectively. This study provided substantial evidence that Photolon preferentially localized in the subcellular organelles in the following order: nucleus, mitochondria, lysosomes and the Golgi apparatus and subsequent photodamage of the mitochondria and lyso-somes played an important role in PDT-mediated apoptosis CT-26 cells. Our results based on the cytoplasmic organelles and the intranuclear localization extensively enhance the efficacy of PDT with appropriate photosensitizer and light dose and support the idea that PDT can contribute to elimination of malignant cells by inducing apoptosis, which is of physiological significance.

Photodynamic therapy (PDT) is currently being used as an alternative treatment modality for various types of cancers. PDT involves the selective uptake and retention of a photosensitizer in the tumor followed by light irradiation of an appropriate wavelength to cause the destruction of tumor cells by the formation of cytotoxic reactive oxygen species. The photosensitizer, hypericin, has shown great potential due to its light-dependent tumor destructive properties. However, as hypericin-mediated PDT primarily targets tumor vasculature, it induces certain pro-angiogenic factors such as vascular endothelial growth factor (VEGF) in the tumor tissue as a result of hypoxia. This study examines the role of hypericin-mediated photodynamic therapy in stimulating the expression of key angiogenesis growth factor VEGF in order to elucidate the process of tumor angiogenesis in nasopharyngeal carcinoma xenografts. We also investigated the effect of angiogenesis inhibitor celebrex on human VEGF levels when combined with hypericin-PDT. These studies were conducted on an in vivo human nasopharyngeal xenograft model. VEGF was measured in the control and hypericin-PDT treated tumors. VEGF levels were found to be higher when the tumors were treated at a 1-h drug-light interval compared to a 6-h interval, due to extensive vascular damage. At 72 h post hypericin-PDT, VEGF levels were upregulated indicating the initiation of regrowth in tumors. The use of angiogenesis inhibitor, celebrex, along with hypericin-PDT downregulated the human VEGF levels suggesting that angiogenesis inhibitors can be used to improve the outcome of hypericin-PDT in nasopharyngeal carcinomas.

Bladder cancer is the fourth most common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate in bladder cancer can be reduced by early treatment following pre-cancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimens, which is both invasive and time-consuming. In this study, ex vivo urine fluorescence cytology was investigated to offer an alternative timely and biopsy-free means for detecting bladder cancers. Sediments in patient urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cancer cells from the other cells based on the cellular size. Intensity histograms of each targeted cell and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cancer cells were observed. A diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this report. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder cancer diagnosis.

Photodynamic therapy (PDT) is a promising treatment that is approved by the US FDA for the treatment of oesophageal and lung cancer as well as for age-related macular degeneration. In this study, using standard tissue culture techniques, the photo cytotoxicity and apoptotic mechanisms of Calphostin C (Cal C), a perylenequinone microbial compound in combination with visible light dose was examined in different tumor cell lines. Our results demonstrated both a time and drug-light dose dependence in Cal-C-PDT induced photo toxicity and apoptotic cell death. The induction of apoptosis by Cal C-PDT was found to transit to necrotic cell death at higher drug and light doses. The detection of apoptosis in irradiated tumor cells was performed using various approaches including cell morphology analysis, flow cytometry [DNA fragmentation and phosphatidylserine (PS) externalization] and biochemical assays (activation of caspases). Time-course analysis of Cal C cellular uptake and distribution showed a rapid increase within the cellular compartments. The activation of caspases and nuclear fragmentation was evidenced at a maximum time point of 3 h after irradiation. By the use of specific caspase substrates, significant activation of caspase-8 and -3 was found. Mitochondrial involvement during Cal C-PDT-induced apoptosis was proven by a rapid reduction of the mitochondrial membrane potential. Furthermore, Cal C-PDT also enhanced FasL expression, which then induced Fas signalling-dependent cell death in NPC and colon cancer cell lines tested. Our results contribute to a deeper understanding of the processes involved in apoptotic cell death following photodynamic treatment with Cal C.

Photoactivation of hypericin is known to generate reactive oxygen species and induce phototoxic effects. However, modulation of the cellular antioxidant defense would influence the extent and severity of the photodynamic effects. We have previously shown that hypericin-mediated photodynamic therapy (PDT) induced a significant reduction of Glutathione S-transferase activity. In this study, we investigated the phototoxic effects of hypericin-mediated PDT in nasopharyngeal cancer (NPC) in vitro and analyzed the expression of metallothionein (MT), a family of potential free radical scavengers. HK1 NPC cells were subjected to PDT treatment in vitro, and the effects on cell death were analyzed by flow cytometry (using propidium iodide and Annexin V staining) and transmission electron microscopy. The expression profile of MT-1E and MT-2A isoforms (the only functional MT isoforms expressed in HK1 NPC cells) were determined by quantitative real-time RT-PCR. The results showed that hypericin PDT induced necrotic cell death as evidenced by the absence of a subdiploid peak and decreased Annexin-V fluorescence. Ultrastructural examination verified the presence of cell necrosis. There was a significant up-regulation of MT-1E and MT-2A isoforms six hours following PDT, with an approximately 50-fold rise in the expression level of MT-1E and a 15-fold increase of MT-2A. Hence, despite the up-regulation of MT, cells still succumbed to PDT-induced necrosis. It appears that the oxidative stress induced by PDT overwhelmed the antioxidant defense mechanism such as the alteration of MT levels in tumor cells.

5-Aminolevulinic acid (5-ALA) and its esters have been under intense investigation to enhance the endogenous production of protoporphyrin IX (PpIX) in tumour cells for the purpose of photodynamic diagnosis. In this study we have investigated the use of exogenous PpIX and its dimethyl ester (PME) and compared the results with endogenous PpIX produced via 5-ALA and ALA methyl ester (AME) in poorly differentiated NPC/CNE-2 nasopharyngeal carcinoma cells in both in vivo and in vitro systems. All prodrugs and photosensitizers were administered to tumour bearing balb/c nude mice either intravenously or topically. in vitro results show that 5-ALA induced more PpIX fluorescence when compared with AME in NPC/CNE-2 cells and PME showed better uptake than PpIX. in vivo results show that exogenous PpIX and PME show promise as good candidates as photosensitizers for photodynamic diagnosis as they exhibit significant selectivity between tumour tissue and normal tissue at 3 h. Modification of delivery vehicle used for application of exogenous PpIX and PME could allow for rapid uptake; better selectivity and localization of the photosensitizer.

Photodynamic therapy (PDT) is being investigated as an alternative treatment modality in cancer treatment. It has been shown to induce tumor hypoxia and upregulation of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF). The objective of this study was to improve in vivo tumor growth control of nasopharyngeal carcinoma (NPC), treated at a subcurative dosage by using a combination of Hypericin-PDT and COX-2 inhibitor, Celebrex (CX). The effect of an initial CX dose at 6- and 24-h post-PDT was investigated simultaneously. It was observed that hypoxic NPC/CNE2 cells upregulate both COX-2 and VEGF A genes in vitro. In vivo studies, down-regulation of COX-2 and hypoxia inducible factor-1alpha (HIF-1alpha) genes at 24-h post-PDT and bulk tumor ablation at 48-h post-PDT was observed. However, 24-28 days later regrowth was observed. In a combination treatment, 1st CX dose at 6-h post-PDT had the highest tumor control in which tumors were

The Y-Box binding protein, YB-1, belongs to a group in the DNA and RNA binding protein family. YB-1 is known to be expressed in a number of different cancers and reported to protect cells against DNA-damaging agents such as ultraviolet light. The purpose of this study was to determine if YB-1 levels are altered in photodynamic therapy (PDT)-treated nasopharyngeal cancer (NPC) cells. In this report, we show for the first time that YB-1 is expressed at both the mRNA and protein levels in well-differentiated NPC in vivo. YB-1 mRNA expression in tumor tissues from a murine well-differentiated HK1/NPC model showed no significant difference in mRNA levels following hypericin-PDT. Localization of the YB-1 protein by immuno-histochemistry revealed the presence of cytoplasmic YB-1 in the more mature and better differentiated cells of the untreated group, as well as cells that survived PDT treatment. Expression of the YB-1 protein may possibly influence the cellular stress response to hypericin-PDT and protect the cells from phototoxicity.

The current study investigates the potential of the photosensitizer Fotolon as a photodynamic diagnostic agent in an in vivo system of human nasopharyngeal carcinoma (NPC) cells. Fotolon (formerly known as Photolon) represents a complex of trisodium salt Chlorin e6 and polyvinylpyrrolidone (PVP). It is a photosensitizing agent that selectively accumulates in tumour tissues. A poorly differentiated human nasopharyngeal carcinoma cell line CNE-2 was xenografted on 6-8 weeks old male balb/c nude mice for our photodynamic diagnostic (PDD) studies. A fluorescence endoscope system was used to perform the in vivo macroscopic fluorescence digital imaging of tumours on the mice. The macroscopic images were further analyzed for distribution of fluorescence intensity. Laser fluorescence confocal microscopy was used to capture microscopic fluorescence images of the tumour tissue. In our PDD studies, we observed intense fluorescence in the tumour and tumour vasculature of human NPC xenografts on nude mice as early as 15-min post administration of Fotolon. High fluorescence selectivity in the tumour tissue was observed between 3-h and 6-h time points. The tumour to normal tissue selectivity ratio was highest at 6 h. The microfluorescence tumour imaging shows similar trends confirming the macroscopic fluorescence data. Fotolon shows much promise as a good photodiagnostic agent.

The chorioallantoic membrane (CAM) assay is a widely used bioassay in early in vivo cancer research. The CAM allows non-invasive study of in vivo microvasculature and blood circulation. This report describes the first topical application investigation of photodynamic response in the CAM model using Hypericin (HY) and Hypocrellin B (HB) that belongs to the perylenequinone family. Briefly, cultivated carcinoma of the human bladder cell line (MGH), were inoculated on the CAM of fertilized eggs of embryo age (EA) 9. Tumor growth was evaluated by digital stereomicroscopy. Photodynamic therapy (PDT) was performed following topical application of the photosensitizers. We were able to demonstrate that these perylenequinones localized selectively in the xenografted bladder tumor and in the vasculature of the CAM. Photodynamic treatments were performed using a custom-made non-laser light source coupled into a flexible fiber bundle to selectively excite the photosensitizers in order to induce photodamage to the tumor and vasculature. The vascular damage induced was quantitatively measured following topical application of the photosensitizers. Both photosensitizers exhibited very similar degrees of photodamage to the CAM. The CAM model offers an exciting avenue for the study of PDT induced effect on the vasculature. Our preliminary results support that the CAM model could potentially serve as a customized model to study photodynamic therapy effects of various photosensitizers on specific tumor models.

Hypocrellin B (HB), a monomeric perylenequinone pigment, is a promising second-generation photosensitizer for photodynamic therapy. We have evaluated the efficacy of HB mediated PDT by experimenting with various drug-light intervals, based on the biodistribution analysis in human bladder tumor (MGH cell line) models. Tumor growth rates were assessed at 10-day post treatment followed by morphometric analysis. Biodistribution of HB was evaluated using spectrofluorophotometry analysis (Ex: 480 nm, Em: 620-630 nm). The level of HB peaked at 6 h postinjection in tumor, peritumoral skin and normal muscle followed by a decline over the next 42 h. Concurrently, the ratio of drug in tumor versus skin was relatively low at all times in comparison to tumor to muscle ratio. In serum, concentration of HB peaked at 1 h. Almost 88% of its original uptake level was cleared at 48 h. The level of PDT response revealed a strong dependence on the drug-light intervals (DLI) and light dose. For both high and low fluence/fluence rate, comparable tumor response was observed at 1 h DLI; treated tumors exhibited significant tumor regression compared to 6 and 24 h DLI. The absence of tumor response was observed at 24 h DLI even at high light dose (100 J/cm(2); 100 mW/cm(2)). Tumor response detected at low light dose (12 J/cm(2); 12 mW/cm(2)) at short DLI suggests that the tumor vasculature is a more sensitive target compared to the cellular compartment of the tumor, correlating significantly with the bioavailability of the drug in serum. Therefore, HB mediated PDT effect is characteristics of a predominantly vascular mediated effect. This study confirms that for short drug-light intervals, PDT seems to target tumor vasculature, which contributes to tumor destruction.

Confocal endomicroscopy is a novel and non-invasive microscopic technique that enables surface and subsurface imaging of living tissues or cells in vivo. The purpose of this study was to assess the possibility of utilizing a rigid confocal endomicroscope (RCE) system developed for detecting morphological changes in living normal and neoplastic human tongue tissue in combination with 5-aminolevulinic acid (ALA)-induced endogenous protoporphyrin IX (PPIX) fluorescence. Three patients with squamous cell carcinoma (SCC) of the tongue were examined using the novel RCE system with the excitation wavelength at 488 nm from an argon-ion laser and the detection wavelengths of the tissue fluorescence above 515 nm. Patients were topically applied with 0.4% of 5-ALA rinsing solution to the oral mucosa for approximately 15 min, and then the confocal endomicroscopic imaging of tissue PPIX fluorescence was performed on the lesion sites of the tongue after an optimal incubation period of 90-120 min. For comparison purposes, ALA-PPIX fluorescence confocal endomicroscopic imaging was also carried out on the normal sites of the tongue in vivo from two healthy volunteers. Image distortions due to tissue motion can be minimized using a specially designed tissue stabilizer attached to the RCE probe. Good quality ALA-mediated confocal fluorescence images of the tongue can be acquired in real-time, providing well-defined micro-morphological structures (e.g., filiform papillae, keratinized epithelium and fungiform papillae) of the tongue in vivo. Changes of tissue structures in oral tissue associated with cancer transformation can also be clearly identified using the RCE imaging. Preliminary results obtained in this study suggest that ALA-mediated rigid confocal endomicroscopy may have a significant potential for the rapid, non-invasive diagnosis and evaluation of early oral cancers in vivo.

We have mapped the microscopic distribution of aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) in normal rabbit brain and in brain tumour at various time intervals after i.v. injection of ALA. In normal brain, regions without a blood-brain barrier (BBB) such as the choroid plexus and meninges and also structures associated with the CSF synthesized the highest amounts of PPIX. We also detected ALA in the CSF within 2 h after ALA administration when the serum concentration was high. Our fluorescence studies have shown that ALA is able to cross an intact BBB and be metabolized in regions with a BBB. This is evident in regions of the brain such as the hippocampus, thalamus, cerebellar folia and brain stem that are remote from the meninges or choroid plexus. However, the relative peak PPIX fluorescence in these regions are much lower than in regions without a BBB. Our imaging studies have shown that neurons in different regions of normal brain with an intact BBB exhibit varying capacities to metabolize ALA. The grey matter regions of the cerebrum and cerebellum also showed higher PPIX fluorescence compared to white matter regions in these same structures. Our study showed that tumour tissue had the highest PPIX fluorescence compared to normal contralateral cerebral cortex. Maximum selectivity was achieved between tumour and normal tissue at 24 h. PPIX fluorescence in normal blood vessels appear to be mainly due to metabolism of internalized ALA from serum and not due to high PPIX levels. In conclusion, ALA does cross the BBB and all regions of the brain with the exception of the hypothalamus, exhibit a dynamic synthesis of PPIX from ALA. Increased PPIX accumulation in tumour and inflamed brain tissue in comparison to normal brain tissue suggest the feasibility of selective photosensitization of such tissue.

Singlet oxygen (1O2) generated by ultraviolet A irradiation has been reported to induce cytokine-dependent Matrix metalloproteinase-1 (MMP-1) expression in dermal fibroblasts. In the present study, we analyzed the effect of hypericin-based photodynamic therapy (PDT) on MMP-1 expression in two nasopharyngeal cancer (NPC) cell lines and an animal tumor model. MMP-1 protein and mRNA expression were evaluated by Western blot analysis and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) respectively. 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 NPC cells in vitro. Similarly, there was up-regulation of MMP1 mRNA expression in hypericin-PDT-treated NPC/HK1-tumors. To our knowledge, this is the first time that modulation of MMP-1 expression has been demonstrated as a photodynamic effect of hypericin in NPC cells. This has clinical implications as MMP-1 is known to play an essential role in the biological process of matrix remodeling.

In this study we have evaluated the use of hypericin ex vivo urine fluorescence cytology as a non-invasive method for detecting early bladder cancers. To date this is the first study reported using this technique with hypericin. Urine samples from patients with early bladder cancers were processed for fluorescence cytology by incubation with hypericin, a novel photosensitizer. Normal urine samples incubated with hypericin served as normal controls. Laser confocal microscopy and spectroscopy was used to detect the fluorescence in the exfoliated low-grade urothelial tumor cells. Fluorescence cytology was considered positive if hypericin fluorescence of the low-grade urothelial tumor cells was detected to be stronger (>8.5 times) compared to the baseline fluorescence established for normal urine samples. Automated analysis for an objective reproducible outcome appears possible. The possibility of detection of malignant urothelial cells in early cancer makes ex vivo fluorescence cytology promising for routine diagnostic screening.

Photoactivation of hypericin is known to generate singlet oxygen and superoxide anion radicals. Reactive oxygen species (ROS) produced by photodynamic therapy (PDT) has the capacity to induce oxidative damage and tumor destruction. We have previously shown that hypericin-PDT induces tumor shrinkage and regression in the human nasopharyngeal cancer (NPC)/HK1 murine tumor model. In this extended study, we show by electron microscopy that subcutaneously implanted HK1 NPC cells from Balb/c nude mice perished by cell necrosis with hypericin-PDT treatment. There was evidence of cytoplasmic swelling accompanied by loss of cell membrane integrity and autophagic vacuolization of cytoplasm but no nuclear changes. 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. This further supports the observation that cell death in PDT-treated NPC/HK1 tumors was by necrosis. Lipid peroxidative stress analyzed by the malonaldehyde assay was significantly elevated in PDT-treated cells. However, PDT had no effect on the activity of superoxide dismutase, an intracellular antioxidant enzyme. The findings show that hypericin-PDT of nasopharyngeal tumors in vivo induces tumor necrosis with accompanying lipid peroxidation.

The early detection of carcinoma is very essential for the diagnosis and prognosis of a bladder cancer patient. In this study we have investigated the use of hypericin as a fluorescent tumour marker and laser confocal microscopy as a diagnostic tool to aid the diagnosis of such cancers. Both cellular and clinical studies have been conducted. In the cellular studies, we have compared two bladder cell lines for the uptake and sub-cellular localization of hypericin. It was found that there was a rapid uptake and clearance of hypericin and significant localization in mitochondria and lysosomes. The study also revealed that there was a time-dependent increase in fluorescence intensity in bladder cells. The optimum localization was found to be 2-4 h post drug incubation. In the clinical study, consisting of 30 patients, both white light and fluorescence cystoscopy were performed after hypericin instillation. Biopsies taken from fluorescing regions were imaged using the confocal microscope. The order of fluorescence was detected to be as follows: normal < inflammation < grade 1 < grade 2 < CIS < grade 3. It was also found that the fluorescence intensity increased with the stage of the disease thereby enabling the determination of the degree of invasiveness of cancer. This enables the use of hypericin as a prognostic marker and laser confocal microscopy as a tool to aid in diagnosis of bladder cancer.

Despite the age-old belief that most anti-cancer agents kill tumor cells by necrosis, recent findings have demonstrated that photosensitizers could also kill tumor cells by triggering genetically programmed series of events termed apoptosis. Cell death by apoptosis is a very neat way to eliminate unwanted cells: no traces are left and the cell contents are never released or accessible to the immune system. Hence there is no inflammation. This is in contrast to death by necrosis. Under these conditions, normally the cell swells and then, when membrane integrity comes under attack, the cell collapses like a balloon and the contents spill out into the extracellular milieu. This may result in an inflammatory response. Because of the relatively clean nature of the apoptotic process, it is desirable to identify compounds that effectively activate the apoptotic pathway. Photodynamic therapy (PDT), a new mode of treatment, is based on the combined use of light-absorbing compounds and light irradiation. Recent developments in understanding the mechanisms of the PDT effect of photosensitizers indicate that a critical factor in the success of the agent is the ability to induce apoptosis in the malignant cell population. Hypericin and Hypocrellins are perylquinones, which are novel natural photosensitizers characterized by high absorption around 470 nm and high singlet oxygen yield. To study the signaling mechanism in vitro we have investigated uptake kinetics, intracellular localization, mode of cell death and mechanisms involved in the photodynamic action following PDT in human cell lines of poorly differentiated (CNE2) and moderately differentiated (TW0-1) nasopharyngeal carcinoma (NPC) and also poorly differentiated colon (CCL-220.1) and bladder (SD) cells.

The use of photodynamic therapy (PDT) for the treatment of recurrent and residual nasopharyngeal carcinoma (NPC) has been encouraging. To determine the potential of hypericin as a PDT tool in the treatment of NPC, we 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. The plasma hypericin level increased rapidly and reached its peak concentration at 1 h after injection. The uptake of hypercin in tumor tissue was maximal 6 h after hypericin administration, at which time the drug concentration in the circulation was low. The efficacy of hypericin-mediated PDT was maximal when light irradiation was performed at 6 h after hypericin administration. Tumor relative regression percentage (RRP) induced by PDT at 1-h interval was comparable to that at 6-h interval, whereas light treatment performed at other time intervals induced less tumor RRP, albeit significant when compared to the control group. Hypericin appears to be an effective photosensitizer for the treatment of NPC. It is likely that hypericin-mediated PDT induces both vascular damage and direct tumor cell killing, thereby bringing about tumor necrosis and shrinkage.

Photodynamic therapy (PDT) is a therapeutic modality used for the treatment of a variety of solid neoplasms. The principle of PDT is based on the selective uptake of a photosensitizing chemical in tumor tissue/cell followed by irradiation of tumors with visible light. The treatment results in a cascade of oxidative events causing cell death both in vitro and in vivo. Nitric oxide (NO) is a gaseous free radical, which is an important modulator of immune, endocrine and neuronal functions and plays an important role in the induction of apoptosis. Hypericin (HY) is a photosensitizing pigment from Hypericum perforatum that displays phototoxic effects in neoplastic cell lines. Our previous studies have shown HY induced apoptotic cell death in nasopharyngeal carcinoma and other tumor cells. To better understand the oxidative mechanism of apoptosis induced by HY, we hypothesized the role of NO in PDT, which is considered to be involved in a variety of physiological and pathological processes. We first demonstrated the presence of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) reactivity, a potential marker of NO synthesizing (NOS) enzyme both at light (LM) and electron microscopic (EM) level. Immunocytochemistry, using specific antibodies for NOS subtypes (constitutive, NOS I and inducible, NOS II), we observed that both NOS I and NOS II was present in all cell lines. The expression of both NOS I and NOS II was further verified using Western blot analysis as early as 15 min post PDT compared to that of drug-treated non-irradiated and light alone treated control cells. Our observation of NO production and distribution using the DAF-2 method is direct evidence of NO production in PDT-treated cells.

5-aminolevulinic acid mediated changes in tissue specific fluorescence were studied in bladder cancer. Bladders of normal patients and also patients diagnosed with cancer were instilled with 5-aminolevulinic acid and the resultant protoporphyrin IX mediated fluorescence intensity was imaged and quantified with confocal laser microscopy and fluorescence image analysis. Urothelial tumour cells were observed to fluoresce more intensely than normal urothelial cells. Submucosa and muscle tissues exhibited minimal fluorescence compared to urothelial cells of malignant origin and also normal urothelial cells. Degree of fluorescence intensity was in the order of malignant urothelium > normal urothelium > normal submucosa > normal muscles. Fluorescence intensity was also found to increase with duration of ALA instillation. Grade 3 malignant cells produced more fluorescence compared to grade 2 and grade 1. Similarly, T1 transitional cell carcinoma (TCC) showed increased fluorescence intensity than that of Ta TCC. Also, tumour blood vessels fluoresced more intensely compared to blood vessels found in normal bladder tissue. Tissue specific ALA mediated PpIX micro fluorescence can be used as a diagnostic technique for early detection of neoplasms and confocal laser microscopy and fluorescence image analysis are advantageous diagnostic tools for the photodynamic diagnosis of bladder neoplasms in vivo.

The use of 5-aminolevulinic acid and its esters to induce endogenous porphyrins for the purpose of detection of epithelial cancers is being studied extensively in many centres around the world. The challenge is to prepare an efficacious formulation for the purpose of cancer detection. Photodynamic diagnosis of cancer using 5-aminolevulinic acid (ALA) and its ester derivatives is being actively investigated. In this study, we compared ALA with ALA methyl ester (AME) derivative in terms of PpIX fluorescence intensity in in vitro and in vivo systems of bladder carcinoma. For the in vivo system consisting of RT112 xenografts, the modes of drug administration compared were intravenous administration and topical application. The Karl Storz fluorescence endoscopy system was used to obtain macroscopic fluorescence images. The macroscopic images were further analysed for fluorescence intensity distribution. For the intravenous administration, over all time points studied (1, 3, 6 h), AME-PpIX fluorescence was lower than ALA-PpIX fluorescence and was cleared at a faster rate than the ALA-PpIX when administered intravenously. Topical application with two different polymers, Gantrez and Polyvinyl pyrrolidone (PVP) which are fast releasing polymers was found to be comparable in inducing PpIX fluorescence. Topical AME-PpIX fluorescence was found to be comparable with ALA-PpIX fluorescence. The results of this study suggest that the AME can also be used as a good diagnostic agent.

Hypocrellins A and B are pigments which are isolated from the parasitic fungi Hypocrella bambuase sacc and Shiraia bambusicola P. Heen found in the People's Republic of China and other parts of Asia including Sri Lanka. These agents, which belong to the general class of perylene quinonoid pigments, have a long history of traditional medicinal agents especially in Asia. Hypocrellins are under extensive investigation as photosensitizing agents for photodynamic therapy (PDT). Hypocrellin compounds were selected as potential photosensitizers for PDT owing to their high quantum yields of singlet oxygen (1O2), and facility for site-directed chemical modification to enhance phototoxicity, pharmacokinetics, solubility, and light absorption in the red spectral region, among other properties. The cellular uptake, evaluated by spectrofluorimetry and confocal laser scanning microscopy (CLSM) demonstrated that both HA and HB exhibited high and fast uptake and rapid internalization as revealed by their bio-distribution pattern. In addition, the present study employed both immunocytochemical and Western blot techniques to explore the photo-induced expression of apoptosis related proteins in NPC as well as other human carcinoma cells. Using spectrofluorimetry and CLSM we have determined the cellular fluorescence as a marker for the uptake of HA and HB. Co-staining with either HA or HB and fluorescent dyes specific for cell organelles revealed an intracellular localization of HA and HB in lysosomes other than mitochondria.

The use of 5-aminolevulinic acid to induce endogenous porphyrins for the purpose of detection of epithelial cancers is being studied extensively in many centres around the world. The challenge is to prepare an efficacious formulation of 5-ALA for the purpose of cancer detection. In this study, we compared two formulations of topical 5-ALA applications with intravenous administration in NPC/CNE-2 xenografts on balb/c nude mice. One of the formulations was a gantrez muco-adhesive patch and the other was a polyvinyl-pyrolidone muco-adhesive patch. The Karl Storz fluorescence endoscopy system was used to obtain macroscopic fluorescence images. Microscopic fluorescence imaging was done by laser confocal microscopy. The macroscopic images were further analysed for fluorescence intensity distribution. It was found that between the two formulations of topical application of 5-ALA; there was very little difference in the fluorescence biodistribution. When the topical applications were compared with the intravenous administration, the tumour to normal differential in biodistribution was significantly higher with the topical application compared to the intravenous application.

Studies of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PPIX) fluorescence have shown a sensitivity of 95-100% for oral cancer diagnosis, but the specificity is only about 50-60%. To improve the diagnostic specificity, a 5-ALA mediated digitized fluorescence endoscopic imaging system was built in this study to enable the on-line image acquisition, analysis and fluorescence quantification for the early detection of neoplasms in the oral cavity. PPIX fluorescence endoscopy and fluorescence image quantification were performed on 16 patients with known or suspected premalignant or malignant lesions in the oral cavity. Preliminary data from the head and neck clinical trials show that the red-to-blue intensity ratio of malignant tissue is larger than that of benign tissue. By applying the intensity ratio as a diagnostic algorithm, both high specificity and sensitivity can be achieved for discriminating malignant tissue from benign tissue, and the capability of classifying different histopathological stages of oral lesions has also been demonstrated. It was found that the red fluorescence intensity distribution in the lesion area could also be obtained to better understand the situation of PPIX accumulations in the tissues. Our initial results indicate that the digitized endoscopic imaging system combined with the fluorescence image quantification method and the ratio diagnostic algorithm developed in this study has the potential to significantly improve the non-invasive diagnosis of early oral neoplasms in vivo.

The development of new-generation photosensitizers to improve photodynamic therapy (PDT) and photodynamic diagnosis (PDD) is an area of extensive research. One such compound that has been studied in our group is Hypericin (HY). To study the mechanism of action we have investigated uptake, intracellular localization, cell phototoxicity and morphological changes especially to ultrastructures following photodynamic treatment in poorly (CNE2) and moderately (TW0-1) differentiated human nasopharyngeal carcinoma (NPC) cells and also other tumor cells such as colon (CCL-220.1) and bladder (SD) cells in vitro. Following irradiation, phototoxicity was determined by crystal fast violet assay and apoptosis was assessed using annexin-V assay. Using spectrofluorimetry and confocal laser scanning microscopy (CLSM) we have determined cellular fluorescence localization and uptake of HY. Co-labeling with HY and fluorescent dyes specific for cell organelles revealed an intracellular localization of HY predominantly in mitochondria and lysosomes. Since many photosensitizing agents in current clinical use have mitochondrial targets, HY may be a valuable addition to current protocols. In addition, our results also indicate that leakage of lysosomal protease into cytosolic compartment might be involved in the induction of apoptosis. Electron microscopy revealed damage to plasma membrane with high drug dose (>5 microM); indicating a mechanism related to necrosis, whereas sub-lethal lower doses (<2.5 microM) resulted in induction of apoptosis indicated by typical ultrastructural signs of apoptosis. Our results based on mitochondrial and lysosomal localization support the idea that PDT can contribute to elimination of malignant cells by the induction of apoptosis, and can be of physiological significance.

Interleukin-8 (Il-8) and IL-10 are cytokines associated with the Epstein Barr Virus (EBV), which is linked with nasopharyngeal cancer (NPC). In the present study, we investigated the endogenous production of IL-8 and IL-10 in vitro by two EBV-positive NPC cell lines, viz., HK1 and CNE-2. IL-8 was expressed by both cell lines although the level of IL-8 was 2-fold higher in supernatant from HK1 cells. Incubation with hypericin, a natural photosensitizer increased IL-8 significantly but only in HK1 cells. Hypericin-based photodynamic therapy (PDT) did not alter the expression of IL-8 levels. Il-10 was not constitutively expressed in either cell line and could not be induced by PDT. It is interesting that PDT which is 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.

Nasopharyngeal carcinoma (NPC) is a malignant disease of the head/neck region with a 5-year survival level of approximately 65%. To explore the novel therapeutic strategies in the management of this disease, the potential effects of photodynamic therapy (PDT) in NPC cells were investigated. PDT, a new mode of treatment, is based on the combined use of light-absorbing compounds and light irradiation. Two human NPC cells such as, poorly differentiated (NPC/CNE2) and moderately differentiated (NPC/TW0-1) and other types of tumor cells like colon (CCL-220.1) and bladder (SD) undergo rapid apoptosis when treated with PDT sensitized with hypericin (HY). It has been shown that this compound has a strong photodynamic effect on tumors and viruses. However, the initiating events of PDT sensitized HY-induced apoptosis are not identified completely. In this study, we sought to determine whether Fas/FasL upregulation and involvement of mitochondrial events are an early event in HY-treated PDT induced apoptosis. Loss of mitochondrial transmembrane potential, release of cytochrome c, involvement of caspases 8 and 3 and the status caspase-3 specific substrate PARP, were evaluated in PDT treated tumor cells. Photosensitization of HY enhanced both CD95/CD95L expression and induced CD95-signaling dependent cell death in all tumor cell lines studied. CD95/CD95L expression appeared within 2 h following light irradiation and appeared to be a principal event in PDT induced apoptosis. Furthermore, these results indicate that release of mitochondrial cytochrome c into the cytoplasm within 2-3 h post PDT is a secondary event following the activation of initiator caspase-8 preceding Apaf-1, caspase-9 and caspase-3 activation, cleavage of PARP and DNA fragmentation.

We examined whether generation of H2O2 is a critical event for the apoptotic pathway upstream of mitochondrial involvement and caspase-3 protease activation. Perylquinone photosensitizers such as Hypocrellin A (HA), Hypocrellin B (HB) and Hypericin (HY) induced activation of caspase-3 and apoptosis upon photoactivation. Generation of H2O2 was commonly detected after photoactivation within an hour, and scavenging of H2O2 caused cells to fail to undergo apoptosis. Flow cytometry demonstrated that H2O2 production preceded loss of mitochondrial membrane potential (DeltaPsim) in photoactivated cells treated with HA, HB and HY. Then caspase-3 activity was activated, followed by DNA fragmentation. These findings suggest that HA, HB and HY upon photoactivation induce H2O2 generation, which causes (DeltaPsim) and subsequently caspase-3 activation, resulting in apoptosis. These findings suggest that generation of H2O2 by photoactivation of HA, HB and HY causes activation of caspase-3. Therefore, H2O2 may function as a common mediator for apoptosis induced by HA, HB and HY. The present study also demonstrated that upon photoactivation HA, HB and HY induced a decrease in intracellular acidification, glutathione (GSH) depletion and an array of mitochondrial damage together with apoptotic morphological changes in the irradiated cells.

Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. Human poorly (CNE2) and moderately differentiated (TW0-1) human nasopharyngeal carcinoma (NPC) cells undergo rapid apoptosis when treated with PDT sensitized with Hypocrellin A (HA) and Hypocrellin B (HB). It has been shown that these compounds have a strong photodynamic effect on tumors and viruses. The initiating events of PDT sensitized HA and HB-induced apoptosis are poorly defined. In the current study, we sought to determine whether Fas/FasL upregulation and involvement of mitochondrial events are an early event in HA and HB-treated PDT induced apoptosis. Loss of mitochondrial transmembrane potential, release of cytochrome c, involvement of caspases-8 and -3 and the status caspase-3 specific substrate PARP, were evaluated in PDT treated tumor cells. Photoactivation of HA and HB enhanced both CD95/CD95L expression and induced CD95-signaling dependent cell death in all tumor cell lines studied. CD95/ CD95L expression appeared within 2 h following light activation and appeared to be a primary event in PDT induced apoptosis. Furthermore, these results indicate that release of mitochondrial cytochrome c into the cytoplasm is a secondary event following the activation of initiator caspase-8 preceding caspase-3 activation, cleavage of PARP and DNA fragmentation. Cytochrome c appeared in the cytosol within 2-3 h post PDT. Cleavage of PARP was observed at 3-4 h following PDT and caspase-3 specific inhibitor DEVD-CHO and broad-spectrum caspases inhibitor z-VAD-fmk blocked caspase-3 activation and PARP cleavage suggesting that caspase-3 plays an important role in HA and HB-induced apoptosis.