Protoporphyrinogen oxidase (PPOX) targeted gene silencing based photodynamic therapy (PDT) in combination with photofrin-PDT
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Photodynamic therapy ( PDT ) has proved to be an effective approach for the treatment of solid tumors. Tumors are treated with an exogenous photosensitizer and the photosensitizer is excited using light. The excited photosensitizer converts oxygen to singlet oxygen, a form of reactive oxygen species, which causes cell death resulting in tumor response. Photofrin, an FDA approved photosensitizer, is administered intravenously and tumors are treated with light after 2-3 days when the drug selectively localizes in the tumor with residual localization in normal tissue and skin. The major drawback of photofrin is its prolonged accumulation in the skin, because of this accumulation patients are advised to protect themselves from external light to avoid cutaneous photosensitive reactions for about 4-6 weeks thereby restricting the dose that could be used for enhanced tumor response. Although, PDT has proven to be very effective, the molecular mechanism underlying PDT still remains unclear. To identify genes involved in the mechanism of PDT, which includes genes that confer resistance and sensitivity to PDT, 466 mitochondrial dysfunction mutants in the yeast model system Saccharomyces Cerevisiae were screened using the photosensitive dye Rose Bengal. 53 genes were found to confer resistance and 1 gene was found to confer sensitivity to Rose Bengal mediated PDT. HEM 14 was one among the 53 genes which conferred resistance. The mammalian homolog of HEM 14 is Protoporphyrinogen Oxidase (PPOX). PPOX is an enzyme in the heme biosynthesis pathway, catalyzing the conversion of protoporphyrinogen IX to protoporphyrin IX ( PpIX ), an endogenous photosensitizer. The heme biosynthesis pathway is exploited in Aminolevulenic acid (ALA) based PDT, which involves the generation of endogenous photosensitive porphyrins. Administration of ALA, a precursor in the heme biosynthesis pathway leads to excessive accumulation of PpIX , which when treated with light elicits a tumor response. Loss of function mutation in PPOX is associated with a disease termed variegate porphyria (1-5). Patients with this disease are light sensitive. Accumulation of porphyrin precursors have been shown in the plasma, feces and urine of these patients (6,7). Blocking of PPOX using inhibitors results in increased PpIX which accumulates in the mitochondria and diffuses into the cytoplasm, where it spontaneously oxidizes to form PpIX (8). PPOX inhibitors have been identified as potential compounds useful in the generation of endogenous PpIX for photodynamic therapy of solid tumors. Inhibiting PPOX activity in mice using PPOX inhibitor FP486 has been shown to elicit tumor response using PDT with significant levels of toxicity and mice death at higher doses(9). Silencing the expression of PPOX using siRNA could serve as a novel strategy leading to excessive accumulation of PpIX which when treated with light could result in tumor response. This treatment methodology can be termed "PPOX targeted gene silencing based PDT" and when it is used in combination with Photofrin-PDT could serve as a novel combination scheme providing an enhancement in treatment efficacy. Also, the cutaneous photosensitivity associated with patients treated with Photofrin can be reduced by utilizing the combination scheme which could help in lowering the photofrin dose used for treatment thereby reducing the levels of accumulation and patient discomfort. Consequently, I hypothesize that Protoporphyrinogen oxidase (PPOX) is a novel target for gene silencing based photodynamic therapy in combination with Photofrin-PDT for enhancement in treatment efficacy and simultaneous reduction in cutaneous photosensitivity.