PDT Treatment Research Articles

12 HIGH-TECH LASER ACUPUNCTURE RESEARCH

Cerebral metastases occur in 15–40% of cancers and their incidence is increasing. We have studied the use of fluorescence image-guided surgery and repetitive photodynamic therapy in 14 metastatic brain cancers.Case note review of prospectively collected data on patients who were treated with PDT at the time of surgery for brain metastases. Patients were consented for the surgery and PDT. Patients were given 2 mg/kg body weight of Photofrin® IV 48 h before the surgery and 20 mg/kg 5-aminolevulenic acid orally 3 h before surgery. Following resection of the tumor using fluorescence, microsurgical and image guidance techniques, the post-excision cavity is filled with a balloon using 0.32% intralipid solution and up to five consecutive PDT treatments were given using 100 J/cm2 Diode Laser 630 nm. Patients were followed up clinically and by brain imaging every 3 months till their death.Seven were lung in origin and seven of variable sources. One patient with lung metastases died of unrelated cause while the remaining six had remained free from brain disease till their death. Two of the remaining seven patients died of local brain recurrence, one bowel after 4 weeks and one of unknown primary after 70 weeks.Adjuvant repetitive PDT seems to offer an excellent local control of metastatic brain carcinomas with about 79% of patients succumb to the primary and only two out of fourteen died of brain recurrence with the best results obtained in lung cancer.

16 THE METHODOLOGICAL ASPECTS OF PHOTODYNAMIC THERAPY WITH NEW PHOTOSENSITIZER OF CHLORIN LINE IN EXPERIMENT

2.0 2.5mg/kg. Cervix was irradiated superficially with light dose 50 J/cm2 (5 patients) or 100 J/cm2 in other 54 cases. Quality of ectocervix irradiation was monitored by the fluorescence imaging device. Endocervix was irradiated by fiberoptic light delivery system with cylindrical diffuser with active diffusive lengths of 2 3 cm and outer diameters of 1mm with light dose 100 300 J/cm of diffuser length. The PDT outcome was estimated every 3 month after treatment by the expanded colposcopy, proven by morphological and cytological study. HPV presence was detected by PCR (polymerase chain reaction) test. Results: After undergoing the PDT treatment 21 patients have been observed between 6 months and 1 year, 13 patients between 1 and 2 years and 25 patients for 2 years and more. Complete response to PDT, proven by cytological and histological study was observed in case of 53 patients (93%). Moderate skin photosensitivity was observed first 2 days after PDT as systematic side effect. No cervix deformation or scarring occurred due to PDT. HPV was detected in 52 out of 59 observed patients (88%). Followup at three months after PDT revealed that HPV was eradicated in 46 patients (80%). Conclusion: CIN-PDT with chlorin e6-polyvinylpyrrolidone formulation is an effective modality for the treatment of cervical intraepithelial neoplasia. CIN-PDT is effective not only in treating CIN but also for eradicating associated cervical HPV.

Photodynamic therapy outcome for oral verrucous hyperplasia depends on the clinical appearance, size, color, epithelial dysplasia, and surface keratin thickness of the lesion

Our previous studies showed that oral verrucous hyperplasia (OVH) lesions can be successfully treated with a topical 5-aminolevulinic acid-mediated photodynamic therapy (topical ALA-PDT) protocol using a 635-nm light-emitting diode light source. In this study, we report the clinical outcomes of 36 OVH lesions treated by this protocol and assess what clinicopathological parameters of OVH lesions could influence PDT treatment outcomes. We found that all the 36 OVH lesions showed complete response (CR) after an average of 3.8 (range, 1-6) treatments of topical ALA-PDT. OVH lesions with an clinical appearance of a mass, with the greatest diameter <1.5 cm, with the pink color, with epithelial dysplasia, or with the surface keratin layer < or =40 microm needed significantly less mean treatment numbers of PDT to achieve a CR than OVH lesions with an outer appearance of a plaque or a combination type of peripheral plaque and central mass (p=0.000), with the greatest diameter > or =1.5 cm (p=0.011), with the white color (p=0.000), without epithelial dysplasia (p=0.043), or with the surface keratin layer > 40 microm(p=0.003), respectively. Multivariate analysis showed that only the clinical appearance of OVH lesions was the independent factor (p=0.0069). We conclude that complete regression of OVH lesions can be achieved by less than seven treatments of topical ALA-PDT once a week. The PDT treatment outcome for OVH depends on the outer appearance, size, color, epithelial dysplasia, and surface keratin thickness of the lesion.

Synthesis and biological investigations of tetrakis(p-carboranylthio-tetrafluorophenyl)chlorin (TPFC)

We describe the total synthesis and biological properties of a new carboranyl-containing chlorin (TPFC) that might find application as a dual sensitizer in the PDT and BNCT treatment of cancer. TPFC was found to be non-toxic in the dark but showed extensive photosensitizing ability both in vitro and in vivo despite its relatively low singlet oxygen quantum yield. In particular, TPFC exhibited significant photosensitizing activity against highly pigmented melanotic melanoma tumors in mice.

Monitoring Pc 4 photodynamic therapy in clinical trials of cutaneous T-cell lymphoma using noninvasive spectroscopy

Silicon phthalocyanine Pc 4 photodynamic therapy (Pc 4-PDT) has emerged as a potentially effective treatment for cutaneous T-cell lymphoma (CTCL). Noninvasive reflectance and fluorescence spectroscopy before, during, and after PDT may provide useful dose metrics and enable therapy to be tailored to individual lesions. We present the design and implementation of a portable bedside spectroscopy system for initial clinical trials of Pc 4-PDT of CTCL. Reflectance and fluorescence spectra were obtained from an early stage CTCL patient throughout the course of the PDT treatment. Preliminary patient data show a significant effect of Pc 4 on the tissue absorption, modest Pc 4 photobleaching, and heterogeneity of Pc 4 within and between the lesions.

Clinical trial of verteporfin photodynamic therapy in Chinese age-related macular degeneration with subfoveal predominant choroidal neovascularization patients

To determine the safety and efficacy of verteporfin (visudyne) photodynamic therapy in age-related macular degeneration patients with subfoveal predominant choroidal neovascularization in China. Multicenter, open-label, non-controlled clinical study. The included patients are diagnosed AMD patients with predominant classic CNV. The included patients received verteporfin intravenously followed by 689 nm laser light administration 15 minutes after the infusion start. The patients were be followed up for 24 weeks (+/-12 days) after initial verteporfin PDT treatment. Clinical follow-up was done at the end of week 12 (+/-12 days) and week 24 (+/-12 days) after the initial treatment. Additional treatment was given after 12 weeks from initial treatment if leakage from CNV was observed on fundus fluorescein angiogram. The visual acuity with ETDRS visual chart and the retinal lesion changes were documented and compared with baseline. The adverse events both in the process of treatment and in the follow-up were recorded throughout the entire study period. Thirty-one patients (31 eyes) were included and completed the trial with verteporfin PDT treatment. During the 24 weeks of the trial, 38.7% of the treated eyes had a vision gain more than 5 letters, 83.9% of the treated eyes had less than 15 letters vision loss. At week 12 after the initial treatment, 12.9% of the treated eyes had no leakage; 61.3% of the treated eyes had leakages, but limited to the former lesion, 25.8% of the treated eyes had increased leakage. The results at week 24 after the initial treatment were similar to those at week 12. At week 24 after the initial treatment, there were only slight enlargements in the lesion size, area of retinal lesion, the lesion surrounding area, and greatest linear dimension (GLD) of the lesion, but no statistical significant difference was found between baseline and week 12 after initial treatment (P = 0.65, 0.31, 0.12, respectively). No obvious progress of the fibrosis was detected in most of the PDT treated eyes. Eleven cases of adverse events (AE) occurred in our trial and the incidence was 34.4%. Among the 11 patients with reported adverse event, 7 (21.9%) had mild adverse event; 3 (9.4%) had moderate adverse events; 1 (3.1%) had a serious adverse event. During the study period, no abnormal changes were found in most of the laboratory tests including serum and urine biochemistry, hematology and Electrocardiogram. The results of this trial showed positive efficacy of PDT with verteporfin in the treatment of predominantly subfoveal CNV secondary to AMD by reducing the risk of vision loss. The incidence of serious adverse events was only 3.1%. It is highly safe to use PDT with verteporfin in Chinese AMD patients with subfoveal predominant classical CNV.

Photodynamic therapy mediates the oxygen-independent activation of hypoxia-inducible factor 1α

Photodynamic therapy (PDT) induces the expression of the hypoxia-inducible factor 1alpha (HIF-1alpha) subunit of the HIF-1 transcription factor and its target genes in vitro and in vivo. PDT also induces the expression of the enzyme cyclooxygenase-2 and its metabolite, prostaglandin E2 (PGE2). PGE2 and hypoxia act independently and synergistically to increase HIF-1alpha accumulation and nuclear translocation. To examine the expression of HIF-1 target genes in response to PDT-mediated oxidative stress and PGE2 under normoxic conditions, we established EMT6 cells transfected with a plasmid consisting of a hypoxia response element promoter and a downstream gene encoding for green fluorescent protein (GFP). To examine the temporal kinetics of HIF-1alpha nuclear translocation in response to PDT, we transfected a second line of EMT6 cells with a GFP-tagged HIF-1alpha fusion vector. Cell monolayers were incubated with 1 microg mL(-1) Photofrin for 24 h and irradiated with fluences of 1, 2.5, and 5 J cm(-2). Direct measurement of oxygen concentration during irradiation confirmed that cells remained well oxygenated. Cells were also exposed to 1 and 10 micromol/L PGE2 for 3 h. In normoxic conditions, Photofrin, PDT, and PGE2 treatment activated HIF-1alpha and induced its nuclear translocation. Maximal Photofrin-PDT-mediated HIF-1alpha activation was intermediate in magnitude between that induced by PGE2 and that by the hypoxia mimic cobalt chloride. This work establishes that PDT induces significant activation of the HIF-1alpha pathway in the absence of hypoxia and supports the interpretation that the induction of HIF-1 target genes by PDT may be mediated, at least in part, by the prostaglandin pathway.

Evidence for a bystander role of neutrophils in the response to systemic 5‐aminolevulinic acid‐based photodynamic therapy

A significant increase in the number of circulating and tumour neutrophils immediately after therapy was observed while investigating the increase in response of tissues to aminolevulinic acid-based photodynamic therapy (ALA-PDT) using a twofold illumination scheme with a prolonged dark interval. The action of (tumour) neutrophils is an important therapeutic adjunct to the deposition of singlet oxygen within the treatment volume, for many photosensitizers. It is not known if those phagocytes contribute to the improved outcome of ALA-PDT. In this study we investigated the role of neutrophils in the response to PDT using systemic ALA with and without light fractionation. Rhabdomyosarcoma, transplanted in the thigh of female WAG/Rij rats were illuminated transdermally using 633 nm light following i.v. administration of 200 mg/kg ALA. The pharmacokinetics of protoporphyrin IX (PpIX) within the tumour tissue during therapy were determined to compare with that observed in other models for topical administration of ALA. PDT was performed under immunologically normal or neutropenic conditions using various illumination schemes. The number of neutrophils in tumour and in the circulation were determined as a function of time after treatment and compared with growth delay of each scheme. Fluorescence spectroscopy revealed similar pharmacokinetics of PpIX to those observed during and after topical ALA-PDT. The number of neutrophils within the illuminated tumour and in the circulation increased significantly following therapy. This increase in the number of neutrophils was associated with an increase in the efficacy of therapy: the more effective the therapy the greater the increase in tumour and blood neutrophils. Administration of anti-granulocyte serum treatment prevented the influx of neutrophils after ALA-PDT, but did not lead to a significant decrease in the efficacy of the PDT treatment on the growth of the tumour for any illumination scheme investigated. These results indicate that the magnitude of damage inflicted on the tumour by ALA-PDT does not depend on the presence of neutrophils in the tumour or circulation and that the role of neutrophils in ALA-PDT is much less important than in PDT using other photosensitizers. These data contribute to the understanding of the mechanism of response of tissue to systemic ALA-PDT.

Synthesis and cellular studies of a carboranylchlorin for the PDT and BNCT of tumors

The syntheses of closo- and nido-carboranylchlorins 4 and 5 from a known carboranylporphyrin are described. Water-soluble nido-carboranylporphyrin 5 was found to have very low dark cytotoxicity (IC 50 > 500 μM using a MTT-based assay) but to be toxic in the presence of red light (IC 50 = 80 μM at 0.55 J/cm 2 light dose). Under the same experimental conditions, carboranylchlorin 5 was taken up by human glioma T98G cells to a significantly higher extent than chlorin e 6, a chlorophyll degradation product. The preferred sites of subcellular localization of carboranylchlorin 5 were found to be the cell lysosomes. Our results suggest that carboranylchlorin 5 is a promising new dual sensitizer for the PDT and BNCT treatment of tumors.

Topical methyl aminolaevulinate photodynamic therapy for treatment of facial acne vulgaris: results of a randomized, controlled study

There is a need for alternative treatments for moderate to severe acne vulgaris. Preliminary experience suggests that topical methyl aminolaevulinate photodynamic therapy (MAL-PDT) may have potential. To investigate the efficacy and tolerability of MAL-PDT for treatment of moderate inflammatory facial acne. Thirty patients aged 15-28 years with moderate to severe acne were included in a blinded, prospective, randomized, placebo-controlled multicentre study. Each side of each patient's face was randomly assigned to treatment with MAL (160 mg g1) or placebo cream, applied for 3 h prior to illumination. A second treatment was given 2 weeks later. On each occasion, patients assessed the intensity of pain using a 10-cm visual analogue scale. Inflammatory and noninflammatory acne lesions were counted at baseline and 4 and 10 weeks after the last PDT treatment. The investigator assessed the global severity of acne at baseline (seven patients had severe acne on at least one side of the face) and each study visit using a six-point rating scale. Data were analysed on an intention-to-treat basis, including all 30 patients. There was a statistically significant greater reduction in the total inflammatory lesion count with MAL-PDT compared with placebo PDT at week 12; median reduction 54% [95% confidence interval (CI) 35-64%] vs. 20% (95% CI 8-50%), P = 0.0006. MAL-PDT was associated with more pain than placebo PDT, although intensity varied across centres and was reduced with repeated treatment. Local adverse events were consistent with this treatment modality. MAL-PDT is effective in the treatment of moderate to severe inflammatory facial acne. Further studies are warranted to optimize this promising procedure.

Radiofrequency Ablation and Photodynamic Therapies: What Are the In-Vivo EUS and Histologic Correlates?

Tissue vaporization or coagulation from PDT and balloon-based radiofrequency (RF) ablation has been gaining acceptance as a potential treatment for Barrett esophagus and local esophageal adenocarcinoma. However, there is a paucity of data regarding the extent of epithelial changes secondary to these forms of ablative therapy. Aim: To determine EUS and histologic changes in human esophageal epithelium subsequent to PDT and RF treatments. Methods: Prospective EUS examinations and systematic biopsies in four quadrants, each cm, and at set time intervals were performed following treatment of Barrett esophagus with PDT or RF. Energy was delivered at 200 J/cm for PDT, whereas the RF energy density was pre-set at 12 J/cm2. All patients were placed on PPI's. Results: 55 patients (51 men, 4 women), mean age 68 (38-81), underwent PDT or RF from 11-2003 to 12-2005. The length of Barrett segments ranged from 1 cm to 10 cm. Eleven patients had histologically proven and verified high grade and were treated with RF. A total of 44 EUS examinations with mechanical radial, electronic radial, electronic linear echoendoscopes and through-the-scope probes were performed in 6 PDT and 4 RF patients. Biopsies were obtained in 4 quadrants every cm with jumbo forceps. Post PDT changes affected the mucosa, submucosa, and muscularis propria with thickness up to 8 mm, which decreased to 3.4 mm @ month 19. RT changes included the muscularis propria with thickness of 4 mm @ month 9. One patient in each group developed adenocarcinoma at 8 and 9 months. The longest post ablative EUS follow-up was 19 months. Conclusions: Ablative therapy with PDT and RF can cause epithelial changes through the muscularis propria. The predominant alterations are seen as hypoechoic thickening of the mucosal and submucosal layers. The increased thickness lessens with time. EUS hypoechoic thickening of the mucosa, submucosa, and muscularis propria without endoscopically visible nodule/mass is likely non-malignant in this clinical setting.

Treatment frequency and visual outcome in subfoveal choroidal neovascularization related to pathologic myopia treated with photodynamic therapy

The purpose of this study was to examine the influence of treatment frequency on visual acuity of patients with PDT treatment for subfoveal predominantly classic CNV related to pathological myopia. Retrospective case series. Thirty-seven patients with subfoveal predominantly classic CNV caused by pathologic myopia and treated with PDT were included. All patients received a full ophthalmic examination, including best-corrected visual acuity, slit-lamp biomicroscopy, fundus photography and fluorescein angiography, before first treatment and every 3 months thereafter. Photodynamic therapy was performed according to standard protocol. Main outcome measurements were visual acuity and treatment frequency. The number of treatments received was 3.35+/-1.83 (average: 1-7). In 12 eyes (32.43%); the BCVA was stable or increased during the entire follow-up period. In eight eyes (21.62%), the BCVA decreased and did not return to the baseline values. A transient loss of visual acuity (over 3-9 months) with subsequent improvement in visual function was found in 68% (17 eyes). A gain of three or more lines compared with lowest BCVA was found in 56% (14 eyes). The number of treatments did not correlate with baseline BCVA, greatest linear dimension of CNV at baseline or with the change of BCVA from baseline. In cases with transient worsening of BCVA, the recovery of visual acuity correlated significantly with the number of treatments (r=-0.522, P<0.05; Spearman rank correlation) received. Visual acuity recovery correlates with the number of PDT re-treatments; in many cases, an improvement in visual function after temporary decrease of BCVA can be observed after re-treatment according to current treatment guidelines. The number of PDT treatments has no negative effect on the visual outcome in subfoveal CNVs caused by pathological myopia.

Choroidal vein and artery occlusion following photodynamic therapy in eyes with pathologic myopia

To describe highly myopic patients in which either the large choroidal veins or arteries were occluded following PDT treatment. Demographic features of two highly myopic patients in which large choroidal vessels were occluded at 1 week following PDT, among a total of 23 patients who received PDT due to choroidal neovascularization (CNV) caused by high myopia, were demonstrated. In case 1, ICG angiography demonstrated the complete occlusion of several large choroidal veins within the laser-applied area at 1 week after PDT. In case 2, ICG angiography demonstrated an occlusion of choroidal artery emanating from beneath the CNV. One month after PDT, re-perfusion of the occluded choroidal vessels was noted in both patients. Occlusion of choroidal veins has never been reported in eyes with CNV treated by verteporfin therapy. Although further studies are necessary, occlusion of large choroidal vessels might happen in highly myopic eyes more commonly than expected. ICG angiography at 1 week after PDT was useful to evaluate the impact of PDT on large choroidal vessels.

ASCORBIC ACID SUPPRESSES CELL DEATH IN RAT DS‐SARCOMA CANCER CELLS AFTER ALA‐BASED PHOTODYNAMIC THERAPY

Especially in the public, vitamin C is considered supportive for the treatment of cancer and supplementation is common. However, the underlying mechanism that most chemotherapeutic agents, ionizing radiation and photodynamic therapy exert on tumor cell kill is an increased production of reactive oxygen species (ROS) leading to irreversible tissue injury. Therefore, antioxidants like ascorbic acid (AA) may prevent cancer cells of cellular free radical damage and may therefore be contraindicated in patients undergoing tumor treatment. We report on the effects of AA on markers of oxidative stress and apoptosis in rat DS-sarcoma cells upon 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT). AA dose-dependently protected cancer cells against lipid- and protein oxidation caused by ALA-PDT treatment. By real-time RT-PCR analysis an impressive increase of FasL (124-fold) and TNF-alpha (121-fold) mRNA was detected after PDT treatment. In addition, a decrease in mitochondrial transmembrane potential followed by the mitochondrial release of apoptosis inducing factor (AIF) was observed. All these early signs of apoptosis were significantly reduced by AA, resulting in a 2.1 fold increased cell survival rate upon ALA-PDT treatment. In conclusion, AA functions as a potent antioxidant, protecting mitochondria and other cell structures of oxidative cell injury induced by ALA-PDT.

Comparison of reduced and standard light application in photodynamic therapy of the eye in two rabbit models

Current PDT treatment for age-related macular degeneration uses a standard radiant exposure of 50 J/cm(2) at an irradiance of 600 mW/cm(2). However; there is a general problem with the unusually high irradiance; in fact, the rate of photochemical production of singlet oxygen may be limited by insufficiently oxygenized neovascular tissue. It was the aim of this study to evaluate the efficacy of verteporfin (Visudyne) photoactivation to induce thrombosis of choriocapillaries and in experimentally induced corneal neovascularizations in rabbits by varying irradiance and retinal radiant exposure. The light-dose threshold to induce micro-thrombosis in the choriocapillaries (seven eyes) and in corneal neovascularizations (eight eyes) of Chinchilla-Bastard rabbits using different retinal irradiances (100 and 600 mW/cm(2)) at different radiant exposures (20, 10, 5, 2.5, 1.25, 0.62, and 0.3 J/cm(2)) was evaluated. Induction of neovascularizations was performed 7 days prior to PDT treatment using intracorneal silk sutures. A dose of 2 mg/kg verteporfin was intravenously infused 10 min before standard PDT. The criterion for vascular thrombosis was vessel closure as determined by fluorescein angiography 1 h and 1 day post exposure. Experiments on the choroid revealed vessel closure 1 h after irradiation at ED(50) = 10.8 J/cm(2) (both 600 and 100 mW/cm(2)) and after 24 h at ED(50) = 2.4 J/cm(2) (600 mW/cm(2)) versus 1.8 J/cm(2) (100 mW/cm(2)). Vessel closure was enhanced at irradiation with 100 mW/cm(2). Regarding corneal neovascularizations, vessel thrombosis was observable by dark appearance of irradiated clotted neovascular tissue and angiographically by a lack of leakage at ED(50) thresholds of 0.62 J/cm(2) (1 h) and 0.41 J/cm(2) (1 day) for 100 mW/cm(2) and of 0.99 J/cm(2) (1 h), and 0.67 J/cm(2) (1 day) for 600 mW/cm(2). Thus in both experiments thresholds for vessel closure were reduced by a factor of 1.5 for the lower intensity. Histology revealed more selective vessel occlusion without RPE and photoreceptor damage for 100 mW/cm(2) rather than 600 mW/cm(2) intensity at threshold irradiation. Low-intensity PDT with verteporfin for neovascular tissue seems to be more effective than regular high-intensity PDT. Future preclinical trials should address the issue of proper dosimetry for effective PDT in age-related macular degeneration.

The temperature dependence of porphyrin production in Propionibacterium acnes after incubation with 5-aminolevulinic acid (ALA) and its methyl ester (m-ALA)

Topical PDT treatment of the common skin disease acne vulgaris is now in clinical use. Propionibacterium acnes (P. acnes) is known to play an important role in acne. 5-Aminolevulinic acid (ALA) supplementation leads to an enhanced porphyrin production in the bacteria. Subsequent illumination with light of the proper wavelengths can reduce the number of bacteria and this might at least partly explain the PDT effect on acne. We have assessed the effects of temperature on P. acnes washed cell suspensions incubated for 4 h with ALA or ALA methyl ester (m-ALA). The effect on porphyrin production of both the cell suspension incubation temperature as well as the initial growth temperature of the cultivated cells prior to harvesting and use in suspension experiments was investigated. The bacterial porphyrin content was estimated from fluorescence emission spectra. It was found that incubation with ALA or m-ALA at a temperature 42 degrees C resulted in an approx. 100% and 33% increase in the total amount of PDT-relevant porphyrins produced as compared to incubation at 37 degrees C. These results support increasing the skin temperature during incubation with ALA or m-ALA in the clinic. The initial growth temperature, prior to the incubation, had no apparent effect on the ALA or m-ALA induced porphyrins. Activation energy studies indicate slightly higher temperature dependence in the case of ALA produced porphyrins as compared to m-ALA produced porphyrins (77 and 65 kJ mol(-1), respectively).

Extramammary Paget's disease treated with topical and systemic photodynamic therapy

Surgical excision and radiotherapy for extramammary Paget's disease (EMPD) at specific sites such as the groin and genitalia is often inappropriate. An 80-year-old man with histologically proven EMPD of the left groin and scrotum was referred for PDT. The lesion and normal skin thickness were measured by a 20MHz, two-dimensional 'B' ultrasound scanner (US). δ-5-Aminolaevulinic acid (ALA), was applied followed 6h later by irradiation using a filtered xenon-arc lamp. Clinical improvement was apparent four weeks after first PDT session with a reduction in ulceration and lesion size and moderate thickness reduction on US. Superficial ulceration recurred 9 months after the fifth treatment. This recurrence was treated with intravenously administered porfimer sodium. This, followed by one topical PDT treatment resulted in significant clinical and US cure and the patient remains disease free at 1-year follow-up.

Ascorbic acid suppresses cell death in rat DS-sarcoma cancer cells induced by 5-aminolevulinic acid-based photodynamic therapy

Especially in the public, vitamin C is considered supportive for the treatment of cancer and supplementation is common. However, the underlying mechanism that most chemotherapeutic agents, ionizing radiation, and photodynamic therapy exert on tumor cell kill is an increased production of reactive oxygen species (ROS) leading to irreversible tissue injury. Therefore, antioxidants like ascorbic acid (AA) may prevent cancer cells of cellular free radical damage and may therefore be contraindicated in patients undergoing tumor treatment. We report on the effects of AA on markers of oxidative stress and apoptosis in rat DS-sarcoma cells on 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT). AA dose-dependently protected cancer cells against lipid and protein oxidation caused by ALA-PDT treatment. By real-time RT-PCR analysis an impressive increase of FasL (124-fold) and TNF-α (121-fold) mRNA was detected after PDT treatment. In addition, a decrease in mitochondrial transmembrane potential followed by the mitochondrial release of apoptosis-inducing factor (AIF) was observed. All these early signs of apoptosis were significantly reduced by AA, resulting in a 2.1-fold increased cell survival rate on ALA-PDT treatment. In conclusion, AA functions as a potent antioxidant, protecting mitochondria and other cell structures of oxidative cell injury induced by ALA-PDT and may therefore be contraindicated in patients undergoing tumor treatment.

Celecoxib and NS-398 Enhance Photodynamic Therapy by Increasing In vitro Apoptosis and Decreasing In vivo Inflammatory and Angiogenic Factors

Photodynamic therapy (PDT) elicits both apoptotic and necrotic responses within treated tumors and produces microvascular injury leading to inflammation and hypoxia. PDT also induces expression of angiogenic and survival molecules including vascular endothelial growth factor, cyclooxygenase-2 (COX-2), and matrix metalloproteinases. Adjunctive administration of inhibitors to these molecules improves PDT responsiveness. In the current study, we examined how the combination of PDT and COX-2 inhibitors improve treatment responsiveness. Photofrin-mediated PDT combined with either celecoxib or NS-398 increased cytotoxicity and apoptosis in mouse BA mammary carcinoma cells. Immunoblot analysis of protein extracts from PDT-treated cells also showed poly(ADP-ribose) polymerase cleavage and Bcl-2 degradation, which were further enhanced following combined therapy. Tumor-bearing mice treated with PDT and either celecoxib or NS-398 exhibited significant improvement in long-term tumor-free survival when compared with PDT or COX-2 inhibitor treatments alone. The combined procedures did not increase in vivo tumor-associated apoptosis. Administration of celecoxib or NS-398 attenuated tissue levels of prostaglandin E2 and vascular endothelial growth factor induced by PDT in treated tumors and also decreased the expression of proinflammatory mediators interleukin-1beta and tumor necrosis factor-alpha. Increased tumor levels of the antiinflammatory cytokine, interleukin 10, were also observed following combined treatment. This study documents for the first time that adjunctive use of celecoxib enhances PDT-mediated tumoricidal action in an in vivo tumor model. Our results also show that administration of COX-2 inhibitors enhance in vitro photosensitization by increasing apoptosis and improve in vivo PDT responsiveness by decreasing expression of angiogenic and inflammatory molecules.

Detection of caspase-3 activation in single cells by fluorescence resonance energy transfer during photodynamic therapy induced apoptosis

In many apoptosis pathways, activation of caspase-3 is considered the final stage. In this study, we studied PDT induced caspase-3 activation with fluorescence resonance energy transfer (FRET) technique. A recombinant caspase-3 substrate, SCAT3, was used as the FRET probe. FRET fluorescence images were collected after PDT or TNF-α induced apoptosis. By analyzing the dynamic changes of FRET fluorescence, the results indicate that the caspase-3 activation started immediately after the PDT treatment. In contrast, FRET disruption caused by caspase-3 activation started at 3 h after TNF-α treatment, due to different signaling pathway. The results have proofed, for the first time, that FRET is a sensitive technique that can be used to investigate PDT-induced activation of caspase-3 in real-time and in single cells. By choosing appropriate recombinant substrates as FRET probes, it is likely that FRET technique will provide a new real-time means to study the mechanism of PDT at single cell level.