Improved Therapeutic Effectiveness of Photodynamic Therapy Combined with Antiangiogenic Therapy in a Mouse Tumor Model
January 1, 2001 7 minutes read
Improved Therapeutic Effectiveness of Photodynamic Therapy Combined with Antiangiogenic Therapy in a Mouse Tumor Model
By Angela Ferrario, PhD and Charles J. Gomer, PhD
Photodynamic therapy (pdt) is an effective clinical cancer treatment that utilizes tumor-localizing photosensitizers activated by tissue-penetrating laser light to mediate cytotoxic events that lead to the eradication of solid malignancies.1-3 The porphyrin photosensitizer, Photofrin porfimer sodium (PH), recently received Food and Drug Administration approval for PDT treatment of esophageal and endobronchial carcinomas.1 PDT applications continue to be encouraging for bladder, head and neck, brain, intrathoracic, and skin malignancies, as well as for non-oncological disorders such as age-related macular degeneration.1,4 Nevertheless, recurrences are observed after PDT, and methods to improve the therapeutic efficacy of this procedure are needed.
Introduction
PDT elicits direct tumor cell damage via the photochemical generation of cytotoxic singlet oxygen as well as microvascular injury within exposed tumors. Vascular effects induced by PH-mediated PDT include perfusion changes, vessel constriction, macromolecular vessel leakage, leukocyte adhesion, and thrombus formation.1,5 Reduction in vascular perfusion occurring during and following PDT leads to a significant blood flow disruption, which can lead to tumor tissue hypoxia.6,7
Tissue hypoxia can serve as a catalyst for an inducible response associated with gene activation.8 An initial step in hypoxia-mediated gene activation is the formation of the hypoxia-inducible transcription factor-1 (HIF-1) transcription factor complex.8,9 HIF-1 is a heterodimeric complex of two helix-loop-helix proteins: HIF-1b, which is expressed consti-tutively; and HIF-1a, which is degraded rapidly by the ubiquitin-proteasome system under normoxic condition.8-11 Hypoxia induces the stabilization of the HIF-1a subunit, allowing the formation of the transcriptionally active protein complex.10,12 Several HIF-1-responsive genes have been identified, including vascular endothelial growth factor (VEGF), erythropoietin, and glucose transporter-1.10
VEGF is an endothelial cell-specific mitogen involved in the induction and maintenance of the neovasculature in solid tumors.10,12 VEGF expression in areas of tumor necrosis originally led to the suggestion that hypoxia is a major regulator of tumor angiogenesis.12,13 VEGF expression in hypoxic tumor tissue increases as a result of both transcriptional activation and increased stabilization.10,13 Exposure of rat endothelial cells to hydrogen peroxide-mediated oxidative stress and exposure of various mouse and human tumor cells to ionizing radiation also have been shown to up-regulate VEGF expression.14,15
In the current study using a murine BA mammary carcinoma, we examined whether PDT-mediated cytotoxicity could serve as an activator of molecular events leading to increased VEGF expression within treated tumor tissue.16 Interestingly, a growing number of reports have indicated that antiangiogenic agents can enhance the tumoricidal effectiveness of chemotherapy and radiation treatments.14,17,18 Therefore, we also examined whether antiangiogenic compounds that counter the actions of VEGF would improve PDT responsiveness.16
Results
Since hypoxia induces expression and stabilization of the HIF-1a subunit and activates the HIF-1 transcription complex, we initially investigated whether PDT-induced microvascular damage and resulting tumor tissue hypoxia also would stabilize HIF-1a and initiate HIF-1-mediated transcription. BA mammary carcinoma tumors growing in C3H mice were collected immediately after PDT treatment and evaluated for HIF-1a expression by Western immunoblot analysis. HIF-1a was not detectable in control tumors; however, both PDT (5 mg/kg PH; 200 J/cm2) and 45 minutes of tumor clamping (used as a positive control for hypoxia) resulted in induced expression of HIF-1a. The response was rapid, being observed within the first five minutes following treatments.
At 24 hours following treatment, both PDT and tumor clamping induced significant increases in VEGF expression within exposed lesions compared to control levels as documented by Western and ELISA analysis.
In vitro PDT of BA mammary carcinoma cells growing in culture dishes was not as effective as in vivo tumor treatments in inducing VEGF expression. The in vitro PDT conditions used in our study would be expected to involve singlet oxygen-mediated oxidative stress but not induced hypoxia. A 210 J/m2 PDT dose resulted only in a small increase in VEGF levels when measured 24 hours after treatment. These results suggest that the increased VEGF expression observed in tumors after in vivo PDT may be associated with treatment-induced hypoxia and to a lesser extent with treatment-induced oxidative stress.
Next we examined whether antiangiogenic treatments, using IM862 or endothelial monocyte-activating polypeptide-II (EMAP-II), could enhance the tumoricidal action of PDT. IM862, obtained from Cytran Inc. (Kirkland,WA), is a synthetic dipeptide of L-glutamyl-L-tryptophan that initially was isolated from the thymus.18 Preclinical studies have shown that the dipeptide inhibits angiogenesis in chorioallantoic membrane assays and VEGF production in monocytic lineage cells. IM862 also inhibits tumor growth in xenograft models not by direct cytotoxic effect on tumor cells, but by inhibiting VEGF production and activating natural killer cells. Intranasal administration of IM862 exhibits anti-tumor activity in patients with AIDS-associated Kaposi’s sarcoma.19 EMAP-II is a single chain polypeptide that inhibits tumor growth and has antiangiogenic properties.20 EMAP-II induces apoptosis in growing capillary endothelial cells in both a time- and dose-dependent manner. EMAP-II also prevents vessel ingrowth in experimental angiogenesis models and in primary tumors.
Both compounds appear to have minimal systemic toxicity. Cytotoxic effects have not been observed following administration of IM862 to patients or in experimental animals exposed to EMAP-II. BA mammary carcinomas growing in C3H mice were treated with PDT only, antiangiogenic therapy alone, or with PDT combined with an antiangiogenic agent. A PDT dose (5 mg/kg PH; 200 J/cm2) that produced a moderate (39%) cure rate by itself was used to measure changes in tumor response when the PDT treatment was combined with 10 daily injections of IM862 (25 mg/kg) or EMAP-II (50 mcg/kg).21 Antiangiogenic treatments significantly enhanced the tumoricidal action of PDT as measured by increased tumor cures rate from 39%, for PDT alone, to 78% and 89% when PDT was combined with IM862 or EMAP-II, respectively. Interestingly, these antiangiogenic compounds were observed to decrease PDT-induced VEGF levels in tumor samples indicating that potentiated PDT responsiveness may involve attenuating the angiogenic actions of VEGF.
Significance
The results of this study suggest that an adjunctive antiangiogenic approach for improving PDT responsiveness may have, because of minimal systemic toxicity, a positive clinical impact.16 Optimization of antiangiogenic parameters and an examination of various methods to block angiogenesis currently are being performed. Combination procedures using antiangiogenic therapy may provide an efficient strategy for selectively enhancing PDT tumor responsiveness and possibly may improve PDT procedures for pathologies marked by neovascularization, including age-related macular degeneration.4 (Dr. Ferrario is a Research Specialist, Clayton Center for Ocular Oncology, Childrens Hospital Los Angeles; and Dr. Gomer is a Professor, Childrens Hospital Los Angeles, and Department of Pediatrics and Radiation Oncology, Keck School of Medicine, University of Southern California in Los Angeles.)
Acknowledgments
This investigation was performed in conjunction with the Clayton Foundation for Research and was supported in part by USPHS Grants CA-31230; United States Army Medical Research Grant BC981102 from the Department of Defense; Neil Bogart Memorial Fund of the T.J. Martell Foundation for Leukemia, Cancer and AIDS Research; and Las Madrinas Endowment for Experimental Therapeutics in Ophthalmology.
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