Access Type

Open Access Dissertation

Date of Award

January 2012

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

David Kessel

Abstract

Photodynamic therapy (PDT) is based on the ability of certain photosensitizing agents to selectively localize in neoplastic cells and their vasculature. Subsequent irradiation at a wavelength corresponding to a photosensitizer absorbance band excites the photosensitizer molecules, leading to energy transfer reactions and fluorescence. It was initially concluded that the phototoxic effect occurred when energy from the excited state of the photosensitizer was transferred to dissolved oxygen to form singlet oxygen. This product has a very brief half-life and will cause cellular damage only in the immediate vicinity of its formation. But an excited-state photosensitizer can also interact with oxygen to form superoxide anion radical, which in turn (through the action of superoxide dismutase) is converted to hydrogen peroxide and hydroxyl radical via the Fenton reaction. These reactive oxygen species (ROS) all posses properties (i.e. lifetime, reactivity and diffusion distances) that make them at least as cytotoxic as singlet oxygen. The hypothesis examined in this dissertation was that the particular reactive oxygen species being formed might be an important determinant of photodynamic therapy efficacy.

Initial studies were designed to assess specificity of several commercially available fluorogenic probes that could used for identification of ROS generated during PDT. APF (for hydroxyl radical) and DADB (for singlet oxygen) were found to be useful for cell culture studies in the context of PDT while other probes were found to have less specificity than advertised.

Using an inhibitor of catalase (3-AT) and an endogenous source of catalase that localizes to peroxisomes (termed CATSKL) it was feasible to alter the level of hydrogen peroxide produced by PDT. Promoting persistence of hydrogen peroxide resulted in enhanced photokilling and vice versa. This indicates a role for hydrogen peroxide and its downstream product in the photokilling process.

The efficacy of singlet oxygen generation vs. oxygen radicals was then compared. The photosensitizer NPe6 localizes in lysosomes and generates singlet oxygen in a high yield. The photosensitizer WST11 also localized in lysosomes, and has been reported to produce only oxygen radicals upon irradiation under aqueous conditions. This was confirmed by studies using the singlet oxygen probe DADB. The efficacy of these photosensitizers was compared under conditions where the oxygenation level was varied. In cell culture, lowering the oxygenation levels from 20% to 1% did not alter the phototoxicity of NPe6. WST11 phototoxicity was reduced in the 1% oxygen.

In a cell-free system, the initial rate of formation of singlet oxygen from NPe6 was independent of the oxygen content. For both NPe6 and WST11, formation of hydroxyl was, however, highly correlated with the level of oxygenation.

These data indicate important roles for several different ROS that can be formed during PDT. With most common photosensitizing agents, effects of singlet oxygen generally predominate, especially under hypoxic conditions, but in a well-oxygenated system, formation of hydrogen peroxide and hydroxyl radical can also promote lethal photodamage. The photosensitizing agent WST11 represents a possibly unique situation, where only hydroxyl radical is involved in photobleaching and tumor eradication.

Download

Share

COinS