Fibrosarcoma

ABSTRACT

Hypericin, a polycyclic quinone obtained from plants of the genus Hypericum, has been shown to be a promising photosensitizer. We investigated the combination of hypericin-photodynamic therapy (PDT) and a bioreductive drug mitomycin C (MMC) in the present study. The radiation-induced fibrosarcoma-1 tumors were exposed to laser light (120 J/cm^sup 2^ at 595 nm) 24 h after an intravenous injection of hypericin (1 mg/kg). Hypericin-PDT alone significantly decreased tumor perfusion and oxygen tension as demonstrated by India ink staining technique and OxyLite pO^sub 2^ measurement, respectively. The in vivo-in vitro cell-survival assay revealed about 60% direct tumor cell killing immediately after PDT. No significant delayed tumor cell death was observed after PDT, which suggests that vascular damage does not contribute significantly to the overall tumor cell death. Injection of a 2.5 mg/kg dose of MMC 20 min before light application significantly decreased tumor cell survival and delayed tumor growth compared with PDT or MMC alone. No greater skin reaction was observed after the combination of MMC and PDT than after PDT alone. Our study demonstrates that combining hypericin-PDT with MMC can be effective in enhancing tumor response with little side effect.

Abbreviations: i.v., intravenous; i.p., intraperitoneal; MEM, minimum essential medium; m-THPC, meso-tetrahydroxyphenylchlorin; MMC, mitomycin C; PBS, phosphate-buffered saline; PDT, photodynamic therapy; RIF, radiation-induced fibrosarcoma.

INTRODUCTION

Photodynamic therapy (PDT) involves the administration of a photosensitizing compound that, upon activation by light, results in tumor destruction via the production of reactive oxygen species, particularly singlet oxygen (1). PDT has become an accepted treatment for certain types of malignant tumors and is showing great promise in the treatment and management of a variety of nonmalignant diseases (1). Studies on the mechanism of tumor responses to PDT in vivo have revealed that the tumor destruction could be due to direct tumor cell killing, tumor vascular damage and immunoreactions (1-3).

Hypericin, isolated from plants of the genus Hypericum, is one of the photosensitizers being investigated as a promising PDT agent because of its potent light-dependent antineoplastic and antiviral activities (4). We (5-7) and others (8,9) have shown that PDT with hypericin significantly inhibits tumor growth in different tumor models. The mechanism of tumor destruction involved in hypericin-PDT could be largely due to vascular targeting or direct tumor cell targeting, which depends on the relative distribution of hypericin in the vasculature and tumor cells. The drug-light interval plays an important role in the drug distribution between the vascular and cellular compartments. For instance, light treatment shortly after injection (e.g. 30 min), when hypericin is mainly in the vasculature, maximizes the tumor vascular damage and leads to a high PDT efficacy (10-12). However, significant normal tissue damage is associated with the short-interval PDT because normal vasculature is also loaded with a high amount of drug at a short time after drug administration. PDT at long intervals (e.g. 24 h), when hypericin is mainly localized in the tumor cells with little in the circulation, causes more selective damage to the tumor cells (7). This selectivity might result from the preferential accumulation of hypericin in the tumor tissue over the surrounding normal tissue at long time points after drug injection (6,7). However, this selectivity in tumor destruction is achieved at the expense of losing efficacy. Hypericin-PDT at long intervals is much less effective in killing tumor cells than the vascular-targeting PDT (7). Several factors are responsible for the low effectiveness of long-interval PDT. Because PDT is an oxygen-dependent process, the existing tumor hypoxia therefore represents a major limitation for PDT efficacy. The depth of light penetration is another limiting factor for PDT treatment. Finally, similar to any therapeutic agent, the morbid tumor microenvironment such as reduced blood How and high interstitial pressure may limit the delivery of a photosensitizer to the tumor (13). Besides ways to diminish these limiting factors, one approach to increase the effectiveness of hypericin-PDT at long intervals is to combine it with other antitumoral modalities. The interaction of two different treatments might potentiate the therapeutic efficacy. Indeed, we have demonstrated that hypericin-PDT sensitizes tumor cells to a subsequent hyperthermic treatment and that combining both treatment modalities leads to enhanced antitumoral effect both in vitro (14) and in vivo (15).

In the present study, we explore the potential of combining hypericin-PDT at a long drug-light interval with mitomycin C (MMC), a bioreductive drug that is especially toxic to hypoxic cells. Our purpose is to determine whether hypericin-PDT using a long drug-light interval can create a tumor microenvironment enabling the activation of MMC and whether the combination of PDT and MMC leads to more tumor cell killing and better tumor control than hypericin-PDT or MMC alone.

MATERIALS AND METHODS

Drugs. Hypericin was synthesized and purified with silica and Sephadex LH-20 column chromatography as described previously (16). A stock solution of hypericin (1 mg/mL) was prepared in polyethylene glycol 400 and stored at -20[degrees]C in the dark. Immediately before intravenous (i.v.) injection into the animals, it was diluted five times in phosphate-buffered saline (PBS) (GIBCO BRL, Paisley, UK) to obtain a final concentration of 0.2 mg/mL.

MMC (Kyowa, Japan) was dissolved in PBS to a concentration of 0.5 mg/mL and injected intraperitoneally (i.p.) with a dose of 2.5 mg/kg. This dose has been shown to be effective with no observed side effect (17). For the combination of PDT and MMC, MMC was injected 20 min before light treatment to allow optimal drug distribution (17).

Animals and tumor system. Female C3H/Km mice (10-14 weeks old, weight range 21-25 g) obtained from the Katholieke Universiteit Leuven Animal Facility were used throughout this study. The radiation-induced fibrosarcoma (RIF)-1 murine tumor line (kindly provided by Dr. F. Stewart, The Netherlands Cancer Institute) was maintained and passaged according to established in vivo-in vitro procedures (18). Approximately 1 x 10^sup 5^ cells were inoculated subcutaneously on the depilated lower dorsum of the mice. Tumor growth was documented regularly by caliper measurements in three orthogonal dimensions. Tumors were used for experimentation 7 to 15 days after inoculation on reaching a surface diameter of 4 to 6 mm and a thickness of 2 to 3 mm. All aspects of the animal experiment and husbandry were carried out in compliance with national and European regulations and were approved by the Animal Care and Use Committee of the Katholieke Universiteit Leuven.

PDT treatment of the tumor. Tumor-bearing mice were given an i.v. injection of 1 mg/kg hypericin. At 24 h after administration, the animals were anesthetized by i.p. injection of 60 mg/kg sodium pentobarbital. Tumors were then given external 595 nm laser light treatment as described previously (6). The light (120 J/cm^sup 2^) was delivered at a fluence rate of 100 mW/cm^sup 2^ measured by an IL 1400 A photometer (International Light, Newburyport, MA). The irradiation spot, centered on the tumor mass, was adjusted to 1 cm diameter.

Tumor oxygenation determination. Tumor oxygen partial pressure (pO^sub 2^) was profiled by the OxyLite pO^sub 2^ system (Oxford Optronics, Oxford, UK) as described (19). In brief, the 220 [mu]m diameter fiber optical probe was inserted into the tumor tissue through a pinpoint hole made with a 26 gauge needle immediately before probe insertion. The measurements started when the pO^sub 2^ value became stable. Three measurements at different tumor areas were taken along a straight track made by moving the fiber probe forward in the tumor, and two such tracks were made for each tumor. The pO^sub 2^ signals were recorded using a data-acquisition system (MacLab ADI Instruments, Sydney, Australia). Tumor pO^sub 2^ histograms were generated by pooling all the measurements from animals in each group (including at least five animals) and expressed as percentages within different pO^sub 2^ ranges.

Functional vasculature assay and histology. To examine the effect of hypericin-PDT on tumor functional vasculature, animals were sacrificed within 2 min after i.v. injection of India ink (0.5 mL/mouse) at different time points after PDT (20). Tumor tissues were harvested, immediately mounted in embedding medium (Tissue Tek, Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen-precooled isopentane. Cryostat cross sections (5 [mu]m) were taken from the center of the tumor, stained with hematoxylin and eosin and examined under a light microscope. The black India ink is visible only within the lumen of functional blood vessels. The number of India ink-positive vessels in each field (with an objective lens of 10x) was determined. Five to seven fields were counted randomly for each section. The data of various groups (three tumors in each group) were compared by the nonparametric Wilcoxon test using GraphPad software (GraphPad, San Diego, CA).

In vivo-in vitro cell survival assay. To assess tumor cell killing after PDT alone, MMC alone or the combination of PDT and MMC, the in vivo-in vitro cell survival assay was performed as described elsewhere (21). In brief, at various times after treatment, tumors were removed from the surrounding normal tissues, weighed and minced under sterile and subdued light conditions. The minced tumor was disaggregated in 5 mL sterile PBS containing 0.05% protease (Sigma Type IX), 0.02% deoxyribonuclease (Sigma Type I) and 0.02% collagenase (Sigma Type IV) under continuous stirring at 37[degrees]C for 40 min. The resulting suspension was passed through a cell strainer to eliminate any remaining tissue clumps and cell aggregates. The cells were washed with PBS, pelleted and then resuspended in complete minimum essential medium (MEM) containing antibiotics and 10% fetal bovine serum. Cell numbers were determined with a Coulter Particle Counter. Known numbers of cells were plated for colony formation in complete MEM. The resulting colonies were fixed and stained with 1% methylene blue in methanol after incubation at 37[degrees]C for 7-10 days. Colonies containing more than 50 cells were counted. The number of viable cells (clonogenic cells) per gram of tumor tissue was calculated as the product of cell number recovered per gram of tumor tissue and the plating efficiency obtained from the clonogenic assay. Each data point represents the mean of three experiments (performed in duplicate) and is expressed relative to untreated control tumors.

Assessment of tumor response. Tumors were measured regularly after treatments by a caliper, and tumor volume was calculated using the formula ([pi]/6 abc), where a, b and c are the three-dimensional diameters of the tumor. Tumor responses to various treatments were presented as Kaplan-Meier curves in which the percentages of animals with tumor size less than four times the initial tumor volume were plotted against the number of days after treatment. The curves were compared by log rank analysis (Graph-Pad). The control group consisted of tumor-bearing mice without treatment. At least six animals were included in each group.

RESULTS AND DISCUSSION

The potential of using MMC to enhance the antitumoral effect of long drug-light interval PDT with hypericin was examined in the present study. The RIF-1 tumors were irradiated with 120 J/cm^sup 2^ light dose at 24 h after the administration of 1 mg/kg hypericin (i.v.). We selected this PDT treatment because it resulted in a similar modest antitumoral effect in the tumor regrowth assay as observed in the case of MMC (2.5 mg/kg, i.p.) (see later), anticipating that the combination effect could be assessed easily.

PDT at 24 h after administration of hypericin (1 mg/kg) switches the major target from the vasculature to the tumor cells because it has been shown that hypericin is almost exclusively localized in the tumor cells with little amount in the vasculature (7). Under these conditions PDT had little effect on the surrounding normal tissue, and no apparent skin edema or necrosis was observed in our study. The in vivo-in vitro tumor cell survival assay revealed about 60% tumor cell death immediately after PDT (Fig. 1). However, tumor cell survival recovered to more than 80% by 2 h after treatment and remained at this level thereafter. A delayed tumor cell death, which is indicative of a secondary vascular effect (22), was absent in this PDT treatment. Our data therefore indicate that, at least after long-interval hypericin-PDT, a direct tumor cell killing is primarily responsible for the tumor cell death, whereas the contribution of a secondary vascular effect is minor. This outcome is completely different from hypericin-PDT at a short interval, where opposite results were observed (12).

Surprisingly, both tumor oxygenation and functional vasculature measurements demonstrated that long-interval PDT with hypericin did induce a significant vascular effect. As shown in Fig. 2, hypericin-PDT leads to a significant increase in the percentage of low pO^sub ^2 values, especially in the values less than 2.5 mm Hg. The percentage of pO^sub ^2 measurements less than 2.5 mm Hg increased abruptly from 28.5% in control tumors to 70.3% in tumors immediately after PDT. Tumor hypoxia was maintained for at least 24 h after treatment. Similarly, PDT with hypericin was found to decrease the number of functional blood vessels per low-power field (10x), as examined by India ink labeling (Fig. 3). The decrease in functional vessels was observed immediately after PDT and was not reversed throughout the 24 h period.

It is not yet clear why PDT at a long drug-light interval still possesses an apparent vascular effect. It is possible that a slight amount of hypericin that lingered in the vasculature accounts for the vascular photosensitizing effect, or it can be that the release of vasoactive substances from PDT-damaged tumor cells triggers the secondary vascular effect. Even though long-interval PDT with hypericin caused a significant decrease in tumor oxygenation and perfusion, this PDT-induced tumor hypoxia did not translate into an extra tumor cell killing (as shown in Fig. 1), which suggests that the tumor ischemia induced by PDT under these conditions is not strong enough to kill tumor cells. Ideally, PDT aiming at targeting tumor cells should exclude the vascular effect during the PDT process because reduction in tumor perfusion as a result of vascular damage aggravates tumor hypoxia, making PDT less effective. SInce laser light interferes with the signal from the OxyLite probe, if is technically impossible to measure tumor pO^sub ^2 during light treatment. However, on the basis of the fact that reduced oxygen tension and tumor perfusion were already present immediately after PDT, it can be assumed that the vascular damage already occurs during the PDT process. Thus, tumor hypoxia induced by long-interval PDT with hypericin might render a self-limitation for PDT efficacy.

One approach for improving the effectiveness of hypericin-PDT using long intervals is to exploit PDT-induced tumor hypoxia by combining it with bioreductive drugs. Because bioreductive drugs are activated to form highly effective cytotoxins under hypoxic conditions (23), they can be used to inactivate hypoxic tumor cells that are refractory to the direct cytotoxicity of hypericin-PDT. MMC is the first bioreductive drug and has been widely used for the treatment of various types of cancer (24). Studies have demonstrated that PDT using other photosensitizers in combination with MMC gives enhanced antitumoral effect (17,21,25,26). In the present study, MMC was injected 20 min before light application to allow an optimal intratumoral accumulation (17).

As shown in Fig. 1, MMC administered 20 min before hypericin-PDT resulted in a dramatic decrease in tumor cell viability as compared with PDT or MMC alone. As mentioned, the tumor cell viability was high throughout the 24 h posttreatment after PDT alone. Besides, as expected, no apparent loss of cell viability was detected immediately after MMC alone. Cell survival was found to be about 10-20% at 16-24 h after drug administration. When MMC was followed by hypericin-PDT, the survival fraction was decreased to 0.7% and 1.7% immediately and 24 h after treatment, respectively. Tumor responses to PDT, MMC and the combination of PDT and MMC are shown as Kaplan-Meier curves in Fig. 4. Compared with the control group, PDT or MMC alone had significant antitumoral effect (P

Similar to hypericin-PDT alone, no apparent skin damage was noticed after the combined treatments. In the previous studies with Photofrin or meso-tetrahydroxyphenylchlorin (m-THPC), it was demonstrated that the photosensitizer dose or the light dose could be reduced by a factor of 2 for the equivalent tumor response when PDT was combined with MMC (21,25). The lower doses of photosensitizer and light may lead to reduced skin phototoxicity and short treatment time. Actually, this enhanced therapeutic effect has been confirmed in a small clinical study where four patients with recurrent skin metastasis of a mammary carcinoma were treated with Photofrin-PDT in combination with MMC (25).

The mechanism involved in the observed synergism is still uncertain because it could be multifactorial. First of all, it is very likely that long-interval PDT with hypericin not only consumes oxygen but also cuts the oxygen (blood) supply through its vascular effect during the PDT process. Consequently, this PDT together with the existing tumor hypoxia acts as a shield to protect tumor cells from any PDT effect. Combination of hypericin-PDT with MMC dramatically enhanced the damage to those tumor cells living under hypoxic conditions and, therefore, greatly reduced tumor cell survival, as shown in the present study. As a result, tumor regrowth was significantly delayed by the combined treatments of MMC and hypericin-PDT compared with PDT or MMC treatment alone. Similarly, van Geel et al. (21) also found that PDT with Photofrin or m-THPC significantly decreased tumor perfusion, and PDT in combination with MMC increased the tumor response. However, other factors also might contribute to the enhanced antitumoral effect. For instance, MMC has been found to retard tumor cells in S-phase when they are very sensitive to PDT treatment, which leads to enhanced tumor cell killing (26). Moreover, it has been shown that MMC could increase the uptake of Photofrin in WiDr human colon adenocarcinoma tumors (26). Although a different photosensitizer and tumor model were used in this study, the contribution of these factors to the enhanced antitumoral effect by hypericin-PDT in combination with MMC cannot be ruled out.

In conclusion, we demonstrated in the present study that hypericin-PDT using a 24 h drug-light interval induced significant decreases in tumor perfusion and oxygenation. Even though the resultant tumor ischemia was not effective enough to inactivate tumor cells, it might be able to establish a tumor microenvironment good enough for a bioreductive drug MMC to be effective. The combination of hypericin-PDT with MMC caused a dramatic decrease in tumor cell survival and significantly delayed tumor regrowth compared with PDT or MMC alone. No apparent surrounding tissue damage was observed after the combination of MMC and hypericin-PDT. Our study suggests that hypericin-PDT at long drug-light intervals can be used in combination with MMC to achieve a selective and effective local tumor control.

Acknowledgements-This work was supported by grants awarded by the Onderzoeksfonds of the Katholieke Universiteit Leuven (Onderzoekstoelage) and by a Geconcerteerde Onderzoeksactie (GOA) of the Flemish Government.

[para]Posted on the website on 5 June 2003

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Bin Chen1[dagger], Bissan Ahmed2, Willy Landuyt2, Yicheng Ni3, Robert Gaspar1, Tania Roskams4 and Peter A. M. de Witte*1

1 Laboratory of Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium;

2 Laboratory of Experimental Radiobiology, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium;

3 Division of Radiodiagnosis, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium and

4 Division of Histochemistry and Cytochemistry, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium

Received 28 February 2003; accepted 1 June 2003

* To whom correspondence should be addressed at: Laboratory of Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Van Evenstraat 4, B-3000, Leuven, Belgium. Fax: 32-16-323460; e-mail: peter.dewitte@farm.kuleuven.ac.be

[dagger] Current address: Thayer School of Engineering, Dartmouth College, Hanover, NH, USA.

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