Journal of Photo+mistry

ELSEVIER Journal of Photochemistry and Photobiology 3: Biology 45 ( 1998) 25-27

Tumor versus vascular photodamage in a rat tumor model

David Kessel a,* , James Hampton b, Victor Fingar ‘, Alan Morgan ’ a Wayne State University, School of Medicine, Detroit, MI 48021, USA

‘Medical College of Ohio, Toledo, OH 43699, USA ’ UniversiQ of Louisville, Lmtisville, KY 40208,USA

Received 24 April 1998; accepted 8 July 1998

Abstract

The [ 4-( 5-nitro-Z-furyl)-Z-tbiazoyl] formamide (FANIT)-induced urotbelial tumor in the rat is found to express the mdr gene. The resulting multidrug resistance (MDR) phenotype results in the expression of an outward transport system that prevents cellular accumulation of certain weakly cationic agents. Among the latter is a photosensitizer with known efficacy for the FANFT tumor, the copper benzochlorin iminium salt. FANFI cells are protected from direct cell kill mediated by this drug, suggesting that the substantial delay in tumor regrowth from this tumor/sensitizer combination can be attributed to vascular effects. 0 1998 Elsevier Science S.A. All rights reserved.

Keywords: Photodynamic therapy; Photosensitization; Multidrug resistance; Vasculature

1. Introduction 2. Materials and methods

While PDT of neoplastic tissues in vivo can eradicate tumor cells through direct photodamage, this generally results in less than the 6-8 log reduction required for tumor eradi- cation [ l-51. Photodamage to the tumor vasculature appears to be an important factor in tumor eradication [ 6-9 1. Adverse effects on the normal vasculature also appear to be involved, since the incidence of cures is greatly diminished when nor- mal tissue adjacent to a tumor is shielded from light during the irradiation process [ lo]. In this study, we describeresults which delineate the anti-tumor effects of a PDT-associated vascular effect under conditions where direct tumor cell kill is expected to be minimal.

The preparation of CuBI has been described elsewhere [ 111; a radioactive preparation was carried out by using [ 14C] -N,N’-dimethylformamide in the synthesis [ 121. The carcinogen-induced FANFT tumor was maintained in cell culture using RPMI, 1640 medium supplemented with 10% fetal calf serum, 1% glutamine and antibiotics. Plates were trypsinized and replaced when confluence was reached.

The copper benzochlorin iminium salt (CuBI) is a weakly cationic sensitizer with strong absorption in the near IR [ 111. We have shown that CuBI is excluded from cells that express the multidrug resistance phenotype [ 121. Although the pres- ence of copper decreases the triplet lifetime to less than 20 ns [ 111, studies with the transplantable FANFI urothelial tumor show substantial phototoxicity in culture [ 131 and indicate that this agent can mediate sufficient tumor photo- damage to cause a substantial increase in lifespan of tumor- bearing rats [ 141. In this study, we characterized the expression of MDR on CuBI transport, accumulation and PDT efficacy in the FANFT tumor.

For transport studies, FANFT cells (AY-27) were plated on 24 mm diameter glass cover slips. These were incubated in 1 ml of medium containing 0.1 pCi of i4C-CuBI (0.3 p.M) dissolved in 15% Cremophor EL, for l-30 min at 37°C. The resulting Cremophor dilution was 1:3000. The cover slips were washed in isotonic NaCl and radioactivity was deter- mined by liquid scintillation counting. Where specified, the outward transport system associated with multidrug resis- tance was inhibited by addition of 1 FM DMDP [ 151 during the CuBI-loading incubation. Numbers of cells per slip were determined by typsinization, using replicate coverslips; a Coulter electronic particle counter was used to assess cell numbers.

* Corresponding author. E-mail: dhkessel@med.wayne.edu

Effects of CuBI photodamage on cell viability were deter- mined by treating FANFT cells (non-confluent cultures on cover slips) in vitro with a 1 pM concentration of non- radioactive CuBI for 30 min at 37°C. In some experiments, 1 pM DMDP was also present. The cells were then irradiated at 725 f 5 nm ( 1.5 J cm-‘). The cells were then washed free

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26 D. Kessel et al. /Journal of Photochemistry and Photobiology B: Biology 45 (1998) 25-27

from extracellular drug, incubated in fresh medium for 48 h, and the adhering cells removed by typsinization. Cell num- bers were determined with a Coulter counter. All such exper- iments were carried out in triplicate.

Expression of MDR by the FANFT tumor was assessed by a histochemical staining procedure, using a polyclonal anti- body ( Ab- 1) to MDR ( Oncogene Research Products, Cam- bridge, MA). Staining was detected with a biotinylated secondary antibody stain and ABC-alkaline phosphatase. As a control, we used rat proximal renal tubules which express MDR, as contrasted with glomeruli which do not [ 161.

3. Results and discussion

Accumulation of [ 14C] -CuBI did not increase during a 30 min incubation unless DMDP was present (Fig. 1) . These results are consistent with other studies that indicate the exclusion of CuBI [ 121 by cells which express the MDR phenotype [ 161. PDT studies indicate that CuBI had a pho- totoxic effect only when the outward transport system asso- ciated with MDR was circumvented by DMDP (Table 1) . CuBI and DMDP showed no dark toxicity under the condi- tions used in these studies. The final concentration of Cre-

CuBl Transport

l control 0 DMDP

15 , I

“0 lo20 3040 50 60 70

time (minutes) Fig. 1. Accumulation of W-CuBI by FANFT tumor cells in culture. Effect of 1 pM DMDP is also shown. Data are expressed in terms of the distribution ratio (drug concentration in cells/medium).

Table 1 Photosensitization of FANFT cells in vitro. FANFT cells were treated with 1 (LM CuBI and/or 1 pM DMDP, using a light dose of 1.5 J cm-‘at 725 f 5 nm. Effects of these treatments on cell numbers 48 h later are expressed as a percentage of the control

System Viability

Dark Light

Controls CuBI alone DMDP alone CuBI + DMDP

100% 96*2% 93*40/o 91*3% 96+2% 94*3% 87+4% 41*5%

mophor present (a 1:3000 dilution) was insufficient to reverse multidrug resistance, as indicated by the lack of CuBI uptake by FANFT cells unless DMDP was added (Fig. 1) .

Staining patterns of rat kidney tubules and frozen sections of the FANFT tumor were identical, indicating expression of MDR by both cell types. Rat kidney glomeruli do not express MDR, and provide a control. MDR expression in the FANFT tissue is demonstrated by the antibody staining pattern (Fig. 2). This study was carried out on tumor tissue obtained from tumor-bearing rats; a similar result (not shown) was obtained with FANFI cells grown in culture.

These results suggest a more potent anti-tumor effect than can be obtained under conditions where sensitizer accumu- lation by tumor tissues is inhibited. Evidence indicating a substantial vascular shut-down during PDT using CuBI has been reported [ 111: a rapid decrease in tumor blood flow was observed after irradiation of FANFI tumors in the rat sensi- tized with CuBI, along with a substantial decrease in the time required for tumor regrowth. Since MDR is expressed only in cerebral capillary endothelial cells [ 171, the mechanism of tumor regrowth inhibition by PDT with CuBI is consistent with a process in which vascular shut-down is the predomi- nant factor.

It has been reported that the drug-formulation vehicle Cre- mophor EL can sometimes [ 181, but not always [ 191, reverse

Fig. 2. Immunohistochemical staining for MDR in frozen sections of rat tissues. Left, rat kidney tissue (C = glomen$ which show no mR expression; T (mows) = kidney tubules which express MDR). Right, FANFT tumor showing expression of MDR (arrows),

D. Kessel et al. /Journal of Photochemistry and Photobiology B: Biology 45 (1998) 25-27 21

examples of MDR in mouse tumor models in vivo. We have recently determined that solubilization of CuBI via an emul- sification procedure with phospholipids and soy-bean oil does not compromise the photodynamic efficacy of the drug [ 201.

4. Abbreviations

CuBI copper benzochlorin iminium salt DMDP dimethoxyphenyl-2( naphthyl) -2-propylamine FANFT [ 4- ( 5nitro-2-furyl) -Zthiazolyl] formamide MDR multidrng resistance PDT photodynamic therapy

Acknowledgements

This work was partially supported by grant CA 65561 from the National Cancer Institute, NIH.

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