Photofrin-mediated Photodynamic Therapy Research Articles

Hyperoxygenation enhances the tumor cell killing of photofrin-mediated photodynamic therapy.

Tumor hypoxia, either preexisting or as a result of oxygen depletion during photodynamic therapy (PDT) light irradiation, can significantly reduce the effectiveness of PDT-induced cell killing. To overcome tumor hypoxia and improve tumor cell killing, we propose using supplemental hyperoxygenation during Photofrin-PDT. The mechanism for the tumor cure enhancement of the hyperoxygenation-PDT combination is investigated using an in vivo-in vitro technique. A hypoxic tumor model was established by implanting mammary adenocarcinoma in the hind legs of mice. Light irradiation (200 J/cm2 at either 75 or 150 mW/cm2), under various oxygen supplemental conditions (room air, carbogen, 100% normobaric or hyperbaric oxygen), was delivered to animals that received 12.5 mg/kg Photofrin 24 h before light irradiation. Tumors were harvested at various time points after PDT and grown in vitro for colony formation analysis. Treated tumors were also analyzed histologically. The results show that when PDT is combined with hyperoxygenation, the hypoxic condition could be improved and the cell killing rate at various time points after PDT could be significantly enhanced over that without hyperoxygenation, suggesting an enhanced direct and indirect cell killing associated with high-concentration oxygen breathing. This study further confirms our earlier observation that when a PDT treatment is combined with hyperoxygenation it can be more effective in controlling hypoxic tumors.

Up-regulation of Hsp27 Plays a Role in the Resistance of Human Colon Carcinoma HT29 Cells to Photooxidative Stress¶

The Photofrin-resistant cell line (HT29-P14) was used in the present study to investigate the mechanism(s) involved in Photofrin-mediated photodynamic therapy (PDT). We compared gene expression profiles between the resistant cell line and its parental cell line (HT29) using DNA microarray analysis. A significant up-regulation of small heat shock protein 27 (Hsp27) was found in HT29-P14 cells. The elevated Hsp27 level may play an important role in the resistance of HT29-P14 to Photofrin-PDT. To test this hypothesis, we stably transfected HT29 cells with human Hsp27 complementary DNA. The potential role of Hsp27 in the resistance to PDT was examined in Hsp27-overexpressing cells. Stable trasnfected cells (H13) showed an increased survival after Photofrin-PDT, suggesting that the up-regulation of Hsp27 is related to the induced resistance to Photofrin-PDT. Phosphorylation of Hsp27 has been suggested to play an important role in cytoprotection. We have examined the phosphorylation activity of Hsp27 among the parental and resistant cells, as well as the overexpression cells. An elevated level of Hsp27 resulted in an increased ability of phosphorylation in both resistant and overexpressing cells after PDT. The activation of the phosphorylation of Hsp27 induced by PDT was not mediated by the p38 mitogen-activated protein kinase. These data suggest that Hsp27 may play an important role in mediating the adaptive response to Photofrin-PDT-induced oxidative stress and that the pathways leading to Hsp27 phosphorylation may contribute to the resistance of the cells to photooxidative damage.

Induction of Hsp60 by Photofrin-mediated photodynamic therapy

Photodynamic therapy (PDT) invokes a number of cellular responses. Other studies have shown that PDT induces transcription and translation of heat shock proteins (Hsps). The expression of mitochondrial heat shock protein, Hsp60, was measured following in vitro Photofrin-mediated PDT in the colon cancer cell line HT29 and its PDT-induced resistant variant HT29-P14 as well as the radiation-induced fibrosarcoma cells RIF-1 and its PDT-induced resistant variant, RIF-8A. Basal levels of Hsp60 were found to be similar in the two murine cell lines. In the human model, the resistant HT29-P14 cell line showed a small increase in basal levels relative to its parental population. Incubation with Photofrin ® (PII) alone or photosensitization caused a significant increase in Hsp60 levels in all cell lines as determined by flow cytometry. A dose-dependent and temporal relationship for PDT response was observed, maximum levels were detected 6–8 h post PDT, at which time, Hsp60 induction was found to be significantly greater in the two resistant variants. Induction in the RIF cells was also found to be greater after incubation with PII alone at the highest doses tested. These results indicate that the presence of PII and the subsequent oxidative stress of PDT can induce Hsp60 and implicated it as a common factor that may contribute to the resistance observed in the induced resistant cells.

PHOTOFRIN-mediated photodynamic therapy for treatment of early stage oral cavity and laryngeal malignancies.

To determine the efficacy of PHOTOFRIN-mediated photodynamic therapy (PDT) for treatment of diffuse "field cancerization" of the oral cavity and Cis-T2N0M0 squamous cell carcinoma (SqCCa) of the larynx in patients not amenable or that have failed conventional head and neck treatment. Over the past 15 years 10 patients with early stage Tis-T2N0M0 SqCCa of the oral cavity and oropharynx and 10 patients with Tis-T2N0M0 SqCCa of the larynx were treated. Intravenous PHOTOFRIN (porfimer sodium) (dose 2.0 mg/kg) was administered outpatient, followed 48-60 hours later by intraoperative light photoactivation at 630 nm via fiberoptic microlens (ML) delivery (surgical light dose, 50-100 J/cm(2)) and/or cylindrical diffuser (CD) delivery (80-100 J/cm). Complete responses (CRs) (follow up 6 months-9 years) were achieved in eight of 10 patients with diffuse field cancerization of the oral cavity. CRs (follow up 6 months-8 years) were achieved in eight of 10 patients with superficial laryngeal cancer obviating the need for total laryngectomy in previously treated radiation therapy patients. PHOTOFRIN-mediated PDT provides a surgical oncologic modality for potentially curative treatment of early stage oral cavity and laryngeal malignancies with minimal side effects, absence of systemic toxicity, preservation of oral function and voice quality, with multiple drug administration, and laser light retreatment capability.

Analysis of photosensitivity in Japanese cancer-bearing patients receiving photodynamic therapy with porfimer sodium (Photofrin).

A major disadvantage of a new cancer treatment, porfimer sodium (Photofrin)-mediated photodynamic therapy (PF-PDT), is photosensitivity for several weeks after cessation of the treatment. To characterize persistent sensitivity to visible light following PF-PDT, phototestings were performed in 59 Japanese cancer-bearing patients with a slide projector lamp 3 weeks or more after the treatment. The duration of photosensitivity was analyzed in relation to the patients' sex, skin phototype (SPT), site of tumor and liver function. There was no correlation of the photosensitivity persistency with the site of cancers and the function of liver. However, female subjects needed significantly longer recovery periods than male subjects from potential photosensitivity after PF-PDT. Patients with SPT2 were significantly more sensitive than patients with SPT3 and 4. These results suggest that the prolonged photosensitivity occurs after PF-PDT especially in female patients and in cases with a lighter SPT. Such patients should be carefully followed up for post-PDT photosensitivity.

The Role of the p53 Tumor Suppressor in the Response of Human Cells to Photofrin-mediated Photodynamic Therapy

The Role of the p53 Tumor Suppressor in the Response of Human Cells to Photofrin-mediated Photodynamic Therapy

The role of the p53 tumor suppressor in the response of human cells to photofrin-mediated photodynamic therapy.

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li-Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 +/- 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 +/- 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.

In vivo resistance to photofrin-mediated photodynamic therapy in radiation-induced fibrosarcoma cells resistant to in vitro Photofrin-mediated photodynamic therapy.

The effects of Photofrin-mediated photodynamic therapy (PDT) on the in vitro cell survival and in vivo tumor growth of murine radiation-induced fibrosarcoma (RIF) cell tumors have been examined following in vivo PDT treatment of tumors. The response to in vivo PDT is examined in tumors derived from RIF-1 mouse fibrosarcoma cells and in tumors derived from RIF-8A cells, which show in vitro resistance to PDT. A significant reduction in tumor volume is observed over the first three days following in vivo PDT treatment of either 5 or 10 mg/ kg. The reduction in tumor volume is greater for a 10 compared to a 5 mg/ml dose and occurs to a similar extent for both RIF-1 and RIF-8A tumors. The re-growth is significantly delayed for RIF-1 compared to RIF-8A tumors, indicating a greater response for RIF-1 tumors compared to RIF-8A tumors following PDT. A reduced response of the RIF-8A compared to the RIF-1 tumor cells is also observed in the clonogenic survival of cells from tumors that were excised and explanted in vitro immediately following in vivo PDT treatment. These data indicate that the intrinsic cell sensitivity to PDT is an important component in the mechanism that leads to tumor response following in vivo photodynamic therapy.

Photofrin-mediated photodynamic therapy of chemically-induced premalignant lesions and squamous cell carcinoma of the palatal mucosa in rats

Photodynamic therapy (PDT), an experimental cancer therapy, was studied in an animal model of chemically-induced epithelial dysplasia and squamous cell carcinoma. PDT was performed 24 hours after i.v. injection of 2.5 mg/kg bw Photofrin, and using 100 J/cm 2 incident light at two activation wavelengths (514.5 nm or 625 nm). Two days after PDT, the majority of rats macroscopically showed a marked erythema of the entire palatal region. Microscopically all the rats showed oedema, haemorrhage, and necrosis of the epithelium of the intermolar area. The long-term results were not so favourable. No evidence of disease was found in 6 out of 20 rats in the 514.5 nm group and in 2 out of 20 rats in the 625 nm treated group. Epithelial dysplasia was found in 14 out of 20 rats in the 514.5 nm group, and in 18 out of 20 rats of the 625 nm treated group. Squamous cell carcinomas were found in 4 out of 20 rats treated with 514.5 nm and in 7 out of 20 rats in the 625 nm treated groups. Comparing both treatment wavelengths, better results were obtained in the 514.5 nm groups as this wavelength gave less normal tissue damage. Based on the results of this study the application of PDT for the treatment of field cancerization and squamous cell carcinoma of the oral cavity, is discussed.

Cross-resistance to photofrin-mediated photodynamic therapy and UV light and recovery from photodynamic therapy damage in Rif-8A mouse fibrosarcoma cells measured using viral capacity

Photodynamic therapy (PDT) utilizes the localized delivery of light to activate a photosensitizing drug (such as Photofrin) which is selectively retained by the tumuor tissues. The intrinsic in vitro sensitivity of tumour cells to PDT is thought to be an important determinant of clinical tumour response to PDT. In this work we show the feasibility of using a viral capacity assay for adenovirus (Ad) DNA synthesis as an indicator of cellular sensitivity to and recovery from Photofrin-mediated PDT. Rif-1 mouse fibrosarcoma cells and a PDT resistant derivative, Rif-8A, as well as Chinese hamster ovary (CHO) cells and CHO-MDR multi-drug resistant mutant cells were studied. Consistent with the clonogenic survival of these cells, the capacity of PDT-treated cells for Ad DNA synthesis was greater for Rif-8A compared to Rif-1 cells and for CHO-MDR compared to CHO-N cells. Delaying infection of the Rif cells from immediately after, to 6 hours after PDT, resulted in an increased capacity for Ad DNA synthesis, which was greater for Rif-8A compared to Rif-1 cells, suggesting that the increased resistance of Rif-8A cells to PDT results from an elevated recovery and/or repair of PDT damage. The capacity of UV-irradiated cells for Ad DNA synthesis was also greater for Rif-8A compared to Rif-1 cells indicating a cross-resistance of Rif-8A cells to UV. These results suggest some overlap in the types of cellular damage induced by UV and PDT and/or an overlap in the pathways for the repair of UV and PDT damage in Rif cells.

Subcellular Localization of Photofrin and Aminolevulinic Acid and Photodynamic Cross‐Resistance in Vitro in Radiation‐Induced Fibrosarcoma Cells Sensitive or Resistant to Photofrin‐Mediated Photodynamic Therapy

The subcellular and, specifically, mitochondrial localization of the photodynamic sensitizers Photofrin and aminolevulinic acid (ALA)-induced protoporphyrin-IX (PpIX) has been investigated in vitro in radiation-induced fibrosarcoma (RIF) tumor cells. Comparisons were made of parental RIF-1 cells and cells (RIF-8A) in which resistance to Photofrin-mediated photodynamic therapy (PDT) had been induced. The effect on the uptake kinetics of Photofrin of coincubation with one of the mitochondria-specific probes 10N-Nonyl acridine orange (NAO) or rhodamine-123 (Rh-123) and vice versa was examined. The subcellular colocalization of Photofrin and PpIX with Rh-123 was determined by double-label confocal fluorescence microscopy. Clonogenic cell survival after ALA-mediated PDT was determined in RIF-1 and RIF-8A cells to investigate cross-resistance with Photofrin-mediated PDT. At long (18 h) Photofrin incubation times, stronger colocalization of Photofrin and Rh-123 was seen in RIF-1 than in RIF-8A cells. Differences between RIF-1 and RIF-8A in the competitive mitochondrial binding of NAO or Rh-123 with Photofrin suggest that the inner mitochondrial membrane is a significant Photofrin binding site. The differences in this binding may account for the PDT resistance in RIF-8A cells. With ALA, the peak accumulations of PpIX occurred at 5 h for both cells, and followed a diffuse cytoplasmic distribution compared to mitochondrial localization at 1 h ALA incubation. There was rapid efflux of PpIX from both RIF-1 and RIF-8A. As with Photofrin, ALA-induced PpIX exhibited weaker mitochondrial localization in RIF-8A than in RIF-1 cells. Clonogenic survival demonstrated cross-resistance to incubation in PpIX but not to ALA-induced PpIX, implying differences in mitochondrial localization and/or binding, depending on the source of the PpIX within the cells.

Photofrin-mediated photodynamic therapy of rat palatal mucosa: Normal tissue effects and light dosimetry

Photodynamic therapy (PDT) is a treatment modality with potential application for premalignant lesions and squamous cell carcinoma of the oral mucosa. PDT in principle has dual selectivity. This may result from a ‘preferential’ retention of the photosensitizer in target tissue. In addition, the photodynamic activity will be limited to the irradiated area because PDT will not affect tissues in the absence of excitation light. The specificity of PDT is limited by the fact that normal tissues also retain the photosensitizer to some degree, which makes these tissues susceptible to PDT damage. To optimize PDT for oral malignancies, a study was undertaken on normal tissue to investigate the responses in rat palatal mucosa and surrounding anatomical structures. Eighty male Wistar rats were used in the study. Photofrin was administered i.v. at four doses (0, 2.5, 5 or 10 mg kg−1 body weight). Irradiation for PDT was performed 24 h later. An argon pumped dye laser system was used to produce light of two different treatment wavelengths (514.5 and 625 nm), and various energy density levels (0, 25, 50, 100 or 200 J cm−2). Early effects of PDT were studied at 2 days and late effects at 2 months after treatment. Twenty-four hours after i.v. administration of Photofrin, it was found that PDT affects normal tissues of the oral cavity both macroscopically and microscopically. Combinations of photosensitizer doses ≥5 mg kg−1 and light doses≥100 J cm−2 caused severe and permanent damage to the palatal mucosa and adjacent normal structures such as palatal bone and dentition. Light scattering and internal reflection usually raise the fluence rate in tissue above the irradiance of the incident beam. In an additional study using six male Wistar rats, the energy fluence rate at two treatment wavelengths (514.5 and 625 nm) was measured ex vivo in the palatal mucosa and adjacent anatomical structures. As expected, the energy fluence rates were wavelength, tissue and depth dependent. At the air-mucosa boundary, light of 625 nm was found to have a three-times higher fluence rate than the primary incident beam. Under similar conditions, the fluence rate of 514.5 nm was found to be less, but still twice as high as the primary incident beam. At deeper levels of the rat maxilla, fluence rates were still elevated compared with the incident beam. For 625 nm light, this phenomenon was observed up to the level of the nasal cavity. These increased fluence rates could largely explain the pattern of damage to normal mucosa and surrounding anatomical structures.

Vascular perfusion and hypoxic areas in RIF-1 tumours after photodynamic therapy.

The influence of photodynamic therapy (PDT) on vascular perfusion and the development of hypoxia was investigated in the murine RIF-1 tumour. Image analysis was used to quantify changes in perfusion and hypoxia at 5 min after interstitial Photofrin-mediated PDT. The fluorescent stain Hoechst 33342 was used as an in vivo marker of functional vascular perfusion and the antibody anti-collagen type IV as a marker of the tumour vasculature. The percentage of total tumour vasculature that was perfused decreased to less than 30% of control values after PDT. For the lower light doses this decrease was more pronounced in the centre of the tumour. The observed reduction in vascular perfusion showed a good linear correlation (r = 0.98) with previously published tumour perfusion data obtained with the 86Rb extraction technique. The image analysis technique provides extra information concerning the localisation of (non)-perfused vessels. To detect hypoxic tumour areas in vivo, an immunohistochemical method was used employing NITP [7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline]. A large increase in hypoxic areas was found for PDT-treated tumours. More than half the total tumour area was hypoxic after PDT, compared with < 4% for control tumours. Our studies illustrate the potential of image analysis systems for monitoring the functional consequences of PDT-mediated vascular damage early after treatment. This provides direct confirmation that the perfusion changes lead to tissue hypoxia, which has implications for the combined treatment of PDT with bioreductive drugs.

Therapy of superficial bladder cancer

Intravesical therapy is used in the management of superficial transitional cell carcinoma of the urinary bladder (i.e. Ta, T1, and carcinoma in situ) with specific objectives, including treating existing or residual tumor, preventing recurrence of tumor, preventing progression of disease, and prolonging survival. The initial clinical stage and grade remain the main determinant factors in survival, irrespective of the treatment. Involvement of prostatic urethral mucosa with bladder cancer can be effectively treated with bacillus Calmette-Guerin intravesical immunotherapy. Results of current investigations suggest that biologic and molecular markers might be useful in monitoring the outcome of intravesical therapy. Intravesical chemotherapy has demonstrated a reduction in short-term tumor recurrence rates, but has had no positive impact on disease progression or prolongation of survival. Bacillus Calmette-Guerin immunotherapy remains the most effective treatment and prophylaxis for transitional cell carcinoma, with a positive impact on tumor recurrence rate, disease progression, and prolongation of survival. Interferons, keyhole limpet hemocyanin and Photofrin-mediated photodynamic therapy are under investigation in the management of transitional cell carcinoma and the efficacy results are encouraging. This review summarizes the recent advances in intravesical therapy and prophylaxis of transitional cell carcinoma and the important role of intravesical immunotherapy.