Natalie Rucker

Properties and applications of photodynamic therapy.

Photodynamic therapy (PDT) is the treatment of malignant lesions with visible light following the systemic administration of a tumor-localizing photosensitizer. Pharmacological and photochemical properties of the photosensitizer are combined with precise delivery of laser-generated light to produce a treatment which can offer selective tumoricidal action. Hematoporphyrin derivative (HD) and a purified component called Photofrin II are currently being used in clinical PDT. Initial patient results have been encouraging, and considerable interest has developed in the synthesis and evaluation of new photosensitizers with improved photochemical and pharmacological characteristics. In addition, there has been a gradual increase in knowledge related to in vitro and in vivo mechanisms of action of PDT. This report provides an overview of the properties and applications of PDT. Information and data related to drug development, photochemistry, subcellular targets, in vivo responses, and clinical trials of PDT are presented.

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.

INCREASED TRANSCRIPTION AND TRANSLATION OF HEME OXYGENASE IN CHINESE HAMSTER FIBROBLASTS FOLLOWING PHOTODYNAMIC STRESS OR PHOTOFRIN II INCUBATION

Abstract— Porphyrin mediated photosensitization can enhance the transcription and translation of several oxidative stress genes. In this study, we report on the enhanced expression of the gene encoding for heme oxygenase in Chinese hamster fibroblasts by; (1) incubation in Photofrin II; (2) Photofrin II mediated photosensitization; and (3) photosensitization induced by Rose Bengal. Increased expression of heme oxygenase mRNA was accompanied by a concomitant increase in the synthesis of the 34 kDa heme oxygenase protein. Western blot analysis using antibody to heme oxygenase confirmed the immunoreactivity of the 34 kDa protein induced by Photofrin II and PDT. These results demonstrate that heme oxygenase can be activated by non‐metalloporphyrins as well as by photosensitization associated with singlet oxygen mediated subcellular injury.

The Matrix Metalloproteinase Inhibitor Prinomastat Enhances Photodynamic Therapy Responsiveness in a Mouse Tumor Model

Photodynamic therapy (PDT) clinical results are promising; however, tumor recurrences can occur and, therefore, methods for improving treatment efficacy are needed. PDT elicits direct tumor cell death and microvascular injury as well as expression of angiogenic, inflammatory, and prosurvival molecules. Preclinical studies combining antiangiogenic drugs or cyclooxygenase-2 inhibitors with PDT show improved treatment responsiveness (A. Ferrario et al., Cancer Res 2000;60:4066–9; A. Ferrario et al., Cancer Res 2002;62:3956–61). In the present study, we evaluated the role of Photofrin-mediated PDT in eliciting expression of matrix metalloproteinases (MMPs) and modulators of MMP activity. We also examined the efficacy of a synthetic MMP inhibitor, Prinomastat, to enhance tumoricidal activity after PDT, using a mouse mammary tumor model. Immunoblot analysis of extracts from PDT-treated tumors demonstrated strong expression of MMPs and extracellular MMP inducer along with a concomitant decrease in expression of tissue inhibitor of metalloproteinase-1. Gelatin zymography and enzyme activity assays performed on protein extracts from treated tumors confirmed the induction of both latent and enzymatically active forms of MMP-9. Immunohistochemical analysis indicated that infiltrating inflammatory cells and endothelial cells were primary sources of MMP-9 expression after PDT, whereas negligible expression was observed in tumor cells. Administration of Prinomastat significantly improved PDT-mediated tumor response (P = 0.02) without affecting normal skin photosensitization. Our results indicate that PDT induces MMPs and that the adjunctive use of an MMP inhibitor can improve PDT tumor responsiveness.

Survivin, a Member of the Inhibitor of Apoptosis Family, Is Induced by Photodynamic Therapy and Is a Target for Improving Treatment Response

We observed that photodynamic therapy (PDT) induces the expression and phosphorylation of the inhibitor of apoptosis (IAP) protein survivin in murine and human cancer cells and tumors. Survivin inhibits caspase-9, blocks apoptosis, and is associated with resistance to chemotherapy and radiation. Survivin is a client protein for the 90-kDa heat shock protein (Hsp-90), and the binding of survivin to Hsp-90 assists in the maturation, proper folding, assembly, and transport of this IAP protein. A derivative of the antibiotic geldanamycin, 17-allylamino-17-demethoxygeldanamycin (17-AAG), interferes with proper binding of client proteins, such as survivin, to Hsp-90 and leads to misfolding of client proteins, ubiquination, and proteasome degradation. We hypothesized that PDT efficacy may be reduced by treatment-mediated expression and phosphorylation of survivin, and therefore, targeting the survivin pathway could increase PDT responsiveness. To address this hypothesis, we examined cellular and molecular responses following exposure to PDT, 17-AAG, and the combination of PDT plus 17-AAG in human BT-474 breast cancer cells using Photofrin and NPe6 as photosensitizers. Cells treated with the combination of PDT and 17-AAG exhibited decreased expression of the Hsp-90 client proteins phosphorylated survivin, phosphorylated Akt, and Bcl-2. The decreased expression of these client proteins was accompanied by higher apoptotic indexes and increased cytotoxicity. To confirm a specific role for survivin in modulating PDT, we used a human melanoma cell line, YUSAC2/T34A-C4, stably transfected with an inducible dominant-negative survivin gene under the control of a tetracycline-regulated (tet-off) promoter. PDT treatment of melanoma cells expressing the dominant-negative survivin resulted in increased cleavage of the caspase substrate poly(ADP-ribose) polymerase, apoptosis, and cytotoxicity when compared with results following PDT of the same melanoma cell line expressing wild-type survivin. These results show for the first time that targeting survivin and possibly other Hsp-90 client proteins improves in vitro PDT responsiveness and suggest that manipulation of the antiapoptotic pathway maintained by survivin may enhance PDT-mediated cancer therapy.

ACTION SPECTRUM (620–640 nm) FOR HEMATOPORPHYRIN DERIVATIVE INDUCED CELL KILLING

Abstract— Monochromatic red light generated by a tunable dye laser is currently being utilized for the treatment of solid tumors with hematoporphyrin derivative (HpD) photoradiation therapy (PRT). Experiments were performed using mammalian cells to determine the most efficient wavelength of red light (620 to 640 nm range) for HpD induced cellular photoinactivation. Decrease in the clonogenic potential of Chinese hamster ovary (CHO) cells was examined following both short (I h) and extended (12 h) HpD incubation times. Maximal photosensitization was observed with wavelengths ranging from 630 to 632.5 nm and the action spectra for cell killing matched the absorption spectra for HpD bound to cells. Similar observations were obtained following both short and extended HpD‐cell incubation times. The potential relevance of these results as they relate to clinical HpD PRT are discussed.

Differential photosensitivity in wild-type and mutant p53 human colon carcinoma cell lines

Tumor sensitivity to cancer therapies may be modulated by the p53 status of the malignant cells. Generally, tumors retaining wild-type p53 are more sensitive to radiotherapy and some chemotherapeutic agents than are tumors with either a mutated or deleted p53 phenotype. The role of p53 in the responsiveness to PDT as a cancer treatment is clinically unknown. In the current study, we evaluated the photosensitivity of two human colon carcinoma cell lines, one expressing wild-type p53 protein and the other expressing mutant p53. Wild-type p53 cells were found to be significantly more sensitive to Photofrin-mediated photodynamic treatment measured by clonogenic assay. Uptake of the photosensitizer was equivalent for both cell lines. Interestingly, sensitivity of the colon carcinoma cell lines to ionizing radiation was similar. These two cell lines represent a useful model for examining p53 involvement in the cellular response to PDT-mediated oxidative stress.

Transformation and mutagenic potential of porphyrin photodynamic therapy in mammalian cells.

The transformation and mutagenic potential of porphyrin photodynamic therapy has been examined in mammalian cells. The mutagenic frequency in Chinese hamster cells at the Na+/K+ ATPase locus was measured by resistance to ouabain following treatment with either photodynamic therapy (PDT) or UV irradiation. The C3H 10T 1/2 mouse embryo cell system was used to document the transformation frequency following PDT, UV irradiation, gamma irradiation or exposure to 3-methylcholanthrene (MCA). Treatments with UV irradiation were effective in producing mutants resistant to ouabain, and treatments with UV irradiation, gamma irradiation and MCA generated transformants at frequencies comparable to those which are reported in the literature. However, PDT treatment conditions (which produced a full range of cytotoxicity) did not induce any mutagenic or transformation activity above background levels.

Multiple components of photodynamic therapy can phosphorylate akt

A growing number of clinically relevant molecular and cellular responses are observed following photodynamic therapy (PDT). PDT‐mediated oxidative stress and PDT‐induced tissue hypoxia can elicit the transcriptional and/or translational expression of genes associated with cellular stress, inflammation, angiogenesis, immuno‐modulation, apoptosis and signal transduction. One of the signaling molecules activated by oxidative stress is Akt/protein kinase B. Phosphorylation of Akt/protein kinase B activates this signaling molecule and induces a survival response in effected cells and tissue. We hypothesized that PDT using Photofrin (PH) as the photosensitizer could also induce increased levels of Akt phosphorylation. Results from our initial set of experiments demonstrated that in vitro and in vivo PDT treatments induced Akt phosphorylation. Interestingly, incubation of mouse and human breast cancer cells with the porphyrin‐based photosensitizer, PH, increased the expression of Akt phosphorylation in the absence of light. Exposure of the corresponding mouse and human‐derived breast cancer tumors growing in mice to 630 nm light in the absence of PH administration also induced Akt phosphorylation. These results demonstrate that individual components of the PDT process, photosensitizer alone and light alone, as well as the complete PDT procedure can activate the Akt signaling pathway.

Pro-apoptotic and anti-inflammatory properties of the green tea constituent epigallocatechin gallate increase photodynamic therapy responsiveness

A polyphenol constituent of green tea, epigallocatechin gallate (EGCG), has anti‐carcinogenic properties. A growing number of studies document EGCG‐mediated induction of apoptotic pathways and inhibition of pro‐survival factors when combined with chemotherapy or radiation. We evaluated the efficacy of EGCG in modulating photofrin (PH)‐mediated photodynamic therapy (PDT) responses.