Photochemistry and Photobiology, 1997, 65(3): 387-388

Symposium-in-Print

Subcellular Localization of Photosensitizing Agents Introduction

David Kessel* Departments of Pharmacology and Medicine, Wayne State University, Detroit, MI, USA

BACKGROUND tumor stroma, macrophages and the vasculature. While stud-

Photodynamic therapy (PDT)? was initially described in 1990 (1) and has periodically been rediscovered during the past 70 years. The modem era began with a series of ex- periments by T. J. Dougherty and his associates at the Ros- well Park Cancer Center, which demonstrated the usefulness of the technique for cancer control in animal models and in the clinic (2).

The photosensitizer used in the Roswell trials was HPD (hematoporphyrin derivative), a complex mixture of porphy- rin monomers, dimers and oligomers with the porphyrin units joined by ester and ether linkages. Attempting to cor- relate subcellular localization with efficacy using such a mixture was difficult because the drug components varied widely in tumor selectivity, photodynamic efficacy and sta- bility. As HPD was gradually refined into the present prod- uct, Photofine, and second-generation sensitizers of known structure began to appear, it became possible to compare sites of cellular and subcellular binding of these agents with sites of photodamage and to assess the role of sensitizer biodistribution as a factor in PDT efficacy. It was soon ap- preciated that an additional consideration was the contribu- tion of vascular photodamage to the PDT-induced eradica- tion of photosensitized tissues (3).

ies on the role of these diverse factors can often be examined in animal models, there are substantial differences between such models and the clinical situation. An example is the distribution of lipoprotein components of the blood. The common experimental animals (rodents, dogs, cats) exhibit high high-density lipoprotein and low low-density lipopro- tein levels, as do equine and bovine sera, common compo- nents of tissue culture media. The opposite is true in man (5 ) .

It is not generally feasible to carry out extensive studies of sensitizer biodistribution, sites of photodamage and ultra- structural alterations in clinical trials. The major clinical ad- vantage of PDT is that the procedure is either noninvasive or minimally invasive, and enthusiasm for extended series of biopsies to evaluate mechanistic factors is generally lim- ited. As a result, what we know regarding subcellular effects of PDT mainly relates to studies in cell culture, along with a limited series of animal studies and very few systematic clinical studies. The design of noninvasive techniques for assessing PDT mechanisms in man will doubtless improve the situation, but for the immediate future, experiments in culture or in animal models continue to provide the infor- mation that may not necessarily be relevant to the human situation.

MODES AND SITES OF BlODlSTRlBUTlON APPROACHES TO THE PROBLEM Studies carried out in cell culture can reveal the affinity of different sensitizers for subcellular structures. The sensitizers are usually fluorescent, permitting identification of binding sites via fluorescence microscopy. A wide variety of fluo- rescent probes is now available, facilitating assessment of sites of photodamage. We now appreciate that these subcel- ''Iar regions do not to sites Of sen- sitizer binding (4). The situation in vivo is even more com- plex: binding of sensitizers to lipoprotein components of plasma can affect biodistribution patterns, and there are ad- ditional considerations, e.g. photodamage to other tissues,

In a recent review, Peng et al. (4) concluded that little is of localization patterns of sensitizers in neoplastic

and normal tissues or of the correlation between these pat- terns and the targets of PDT. The current state of the prob- lem can be compared with initial efforts to gain an under- standing of the modes of action the more conventional anti- tumor agents. Even here, a relatively complete understanding of anti-tumor mechanisms is known for a relatively few agents, e.g. methotrexate, fluorouracil and some alkylating agents, The picture concerning PDT is clearly very complex, and continued interest in solutions must necessarily be driv- en by evidence of clinical successes.

Because the bulk of the current PDT studies are associated with new drug development and improved light sources, it is perhaps not surprising that data concerning ultrastructural responses to PDT have been sparse. A recent topic Of interest has been the discovery that PDT-induced cell death can in- volve an apoptotic response (6). Because resistance to drug-

*To whom correspondence should be addressed at: Department of Pharmacology, WSU School of Medicine, 540 East Canfield Street, Detroit, MI 48201, USA. Fax: 3 13-577-6739; e-mail: dhkessel @med.wayne.edu.

namic therapy. TAbbreviafions: HPD, hematoporphyrin derivative; PDT, photody-

8 1997 American Society for Photobiology 0031-8655/97 $5.00+0.00

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388 David Kessel

induced apoptosis has been cited as a mechanism of drug resistance (7,8), perhaps the wide range of responses pro- duced by PDT may relate to the bypassing of steps in the process that are blocked and impede or prevent initiation of apoptosis by conventional chemotherapy.

In earlier days, the major mystery concerning PDT was the nature and composition of HPD. A large number of early papers dealt with approaches to solving this problem, for which advances in HPLC, mass spectrometry and controlled synthetic approaches were needed. As we begin to consider the much more complex problem of PDT mechanisms, an initial period of examining diverse systems and procedures will be involved. A variety of these approaches are illus- trated by this Symposium-in-Print. Drs. Malik, Amit and Rothmann (pp. 389-396) describe localization and PDT-in- duced relocalization of sulfonated tetraphenylporphines in colon carcinoma cells in vitro using a spectral imaging tech- nique. The paper also describes potential applications of this procedure. Wood, Holroyd and Brown (pp. 397402) report on the use of fluorescence microscopy to examine initial sites of localization and relocalization after PDT, and pro- vide evidence to suggest that the latter may be the more important determinant of PDT efficacy. Berg and Moan (pp. 403409) discuss the consequences of PDT-induced photo- damage to lysosomes and microtubules. Woodburn et al. (pp. 410415) provide information on lysosomal localization of a lutetium texaphyrin, a phenomenon that leads to an apoptotic cell death in the mouse mammary EMT-6 tumor in vivo. Gibson et al. (pp. 416421) describe the enzymatic determinants of conversion of exogenous aminolevulinic acid to protoporphyrin, in four different monolayer cultures. Kessel et al. (pp. 422426) have examined some of the de- terminants of PDT-induced apoptosis that involve sites of photodamage. Because failure of conventional chemotherapy often derives from intrinsic resistance of tumor cells to un- dergo apoptotic death, the ability of PDT to elicit an apop- totic response could explain, at least in part, the general ef- ficacy of this modality.

At this stage, we can begin to design further explorations

into the role of sensitizer localization as a factor in PDT successes. To provide a useful framework for further re- search, both initial localization and sites of photodamage need to be taken into account, along with the role of tissue and subcellular biodistxibution. All of these variables will be affected by drug structure, hydrophobicity, charge distribu- tion, along with properties of tumor cell types, time between photosensitization and irradiation, light dose and (where per- tinent) drug-delivery vehicle (9). The suggestion made in the latter reference Concerning the need for establishment of common screening protocols becomes especially relevant because the PDT involves all of the uncertainties found in conventional drug chemotherapy plus additional considera- tions relating to photoprocesses.

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Raab, 0. (1900) Uber die wirkung fluorescirender Stoffe auf In- fosorium. Z. Biol. 39, 524-546. Dougherty, T. J., B. W. Henderson, S. Schwartz, J . W. Winkel- man and R. L. Lipson (1992) Historical perspective. In Phoro- dynamic Therapy (Edited by B. W. Henderson and T. J. Dough- erty), pp. 1-15. Marcel Dekker, New York. Henderson, B. W. and V. H. Fingar (1987) Relationship of tumor hypoxia and response to photodynamic therapy in an experimen- tal mouse tumor. Cancer Res. 47, 31 10-3 1 14. Peng, Q., J. Moan and J. M. Nesland (1996) Correlation of sub- cellular and intratumoral photosensitizer localization with ultra- structural features after photodynamic therapy. Ulrrasrrucr. Path-

Chapman, M. J. (1986) Comparative analysis of mammalian plas- ma lipoproteins. Methods Enzymol. 128, 70-143. Agarwal, M. L. M. E. Clay, E. J. Harvey, H. H. Evans, A. R. Antunez and N. L. Oleinick (1991) Photodynamic therapy in- duces rapid cell death by apoptosis in L5 178Y mouse lymphoma cells. Cancer Res. 51, 5993-5996. Hickman, J. A., C. S . Potten, A. J. Merritt and T. C. Fisher (1994) Apoptosis and cancer chemotherapy. Phil. Trans. R. SOC. Lond.

Powis, G. (1994) Recent advances in the development of anti- cancer drugs that act against signaling pathways. Tumori 80.68- 87. Boyle, R. W. and D. Dolphin (1996) Structure and biodistribution relationships of photodynamic sensitizers. Phorochem. Photobiol. 64, 469485.

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