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Vol. 6, 2021–2027, May 2000 Clinical Cancer Research 2021

Photodynamic Therapy with the Phthalocyanine Photosensitizer Pc 4 of SW480 Human Colon Cancer Xenografts in Athymic Mice1

Cecilia M. Whitacre,2 Denise K. Feyes, showed that tumor growth resumed after a delay of 9–15 Taroh Satoh, Johannes Grossmann,3 days. Our results suggest that: (a) Pc 4-PDT is effective in John W. Mulvihill, Hasan Mukhtar, and the treatment of SW480 human colon cancer xenografts independent of p53 status; (b) PARP cleavage may be me- Nancy L. Oleinick diated by caspase-9 and caspase-3 activation in the Pc Departments of Medicine [C. M. W., T. S., J. G.], Radiation Oncology 4-PDT-treated tumors; and (c) p38 phosphorylation may be [J. W. M., N. L. O.], and Dermatology [D. K. F., H. M.], Case a trigger of apoptosis in response to PDT in vivo in this Western Reserve University School of Medicine, Cleveland, Ohio 44106-4937 tumor model.

INTRODUCTION ABSTRACT PDT,4 recently approved by the U.S. Food and Drug Ad- Photodynamic therapy (PDT) using the silicon phtha- ministration for the treatment of esophageal and lung cancer, is locyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N- undergoing clinical trials for the treatment of a variety of solid (CH3)2] is an oxidative stress associated with induction of cancers as well as numerous noncancerous conditions (1). PDT apoptosis in various cell types. We assessed the effectiveness is a novel mode of cancer treatment in which visible light of an of Pc 4-PDT on SW480 colon cancer xenografts grown in appropriate wavelength activates a tumor-associated photosen- athymic nude mice. Animals bearing xenografts were sitizer to produce reactive oxygen (2–5). Although the primary treated with 1 mg/kg body weight Pc 4 and 48 h later were mechanism of tumor regression by PDT is still not completely 2 irradiated with 150 J/cm 672-nm light from a diode laser understood (6, 7), it is known to induce damage to the tumor 2 delivered at 150 mW/cm . Biochemical studies were per- vasculature and elicits an immune and inflammatory response in formed in xenografts resected at various time points up to the treated tumors (1). PDT causes lipid peroxidation and dam- 26 h after Pc 4-PDT treatment, whereas tumor size was age to multiple cellular sites, including membranes, DNA, and evaluated over a 4-week period in parallel experiments. In cytoskeleton (8, 9), followed by apoptosis in some cell lines (8, the tumors resected for biochemical studies, apoptosis was 10–14) and apoptosis and rapid regression of solid tumors (1, 5, visualized by activation of caspase-9 and caspase-3 and a 15). Apoptosis is characterized by a pattern of PARP cleavage gradual increase in the cleavage of the nuclear enzyme (10, 16–18) distinctive from the pattern observed during necro- poly(ADP-ribose) polymerase (PARP) to a maximum of sis (19). PARP is one of many regulatory and structural proteins ϳ60% of the total PARP present at ϳ26 h. At that time all that are cleaved during apoptosis after activation of a cascade of Pc 4-PDT-treated tumors had regressed significantly. Two caspases (20, 21). This proteolytic cleavage by caspases divides signaling responses that have previously been shown to be the NH2-terminal DNA binding domain of PARP from its associated with Pc 4-PDT-induced apoptosis in cultured COOH-terminal catalytic domain (16, 22), generating two frag- cells, p38 mitogen-activated protein kinase and p21/WAF1/ ϳ ments of Mr 90,000 and 26,000. It has been previously shown Cip1, were examined. A marked increase in phosphorylation that PDT induces cleavage of PARP in murine lymphoma cells of p38 was observed within 1 h after Pc 4-PDT without (10) and in other cell lines (23, 24). changes in levels of the p38 protein. Levels of p21 were not In cell culture, the rapid oxidative stress induced by PDT altered in the xenografts in correspondence with the pres- results in a complex series of events, including activation of ence of mutant p53 in SW480 cells. Evaluation of tumor size phospholipases and sphingomyelinase and release of the lipid second messengers inositol-1,4,5-trisphosphate and ceramide (25), induction of p21/WAF1/Cip1 (26), activation of nitric oxide synthase and release of nitric oxide (27), activation of Received 12/28/99; revised 2/11/00; accepted 2/16/00. stress kinases (8, 28–30) and nuclear factor ␬B (31), and in- The costs of publication of this article were defrayed in part by the creased expression of early response genes such as c-fos,c-jun, payment of page charges. This article must therefore be hereby marked c-myc, and egr-1 and cellular stress proteins such as GRP-78 advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and heme oxygenase (4, 32, 33). p38 MAP kinase, also named 1 This work was supported by NIH Grants KO1 CA-77065, P01 CA- 48735, and CA-51802. 2 To whom requests for reprints should be addressed, at Hematology Oncology Division BRB347B, School of Medicine, 10900 Euclid Av- enue, Cleveland, OH 44106-4937. Phone: (216) 368-5344; Fax: 4 The abbreviations used are: PDT, photodynamic therapy; bw, body (216) 368-1166; E-mail: [email protected]. weight; Pc, phthalocyanine; PARP, poly(ADP-ribose) polymerase; 3 Present address: University of Regensburg, Department of Internal MAP, mitogen-activated protein; SAPK, stress-activated protein kinase; Medicine I, Regensburg, 93053 Germany. JNK, c-Jun N-terminal kinase; HOG, hyperosmotic glucose.

MATERIALS AND METHODS Cell Lines. SW480 colon cancer cells were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in DMEM containing 10% fetal bovine

serum at 37°C in an atmosphere of 95% air and 5% CO2 in a humidified incubator. Pc 4 Formulation. The compound used in this study was obtained from the National Cancer Institute (NSC 676418). The silicon Pc 4 stock solution (1 mg/ml) was prepared as described previously (15, 46) by dissolving Pc 4 in 50% Cremophor EL and 50% absolute ethanol (National Cancer Institute diluent 12), followed by addition of 9 volumes of normal saline while vortexing at low speed. The solution was passed through a 0.22-␮m syringe filter, and the precise concentration was determined by absorption spectrophotometry and then aliquoted for storage at Ϫ20°C. For injection, this solution was further diluted with an equal volume of 5% Cremophor EL, 5% ethanol, and 90% saline solution. Fig. 1 Effect of Pc 4-PDT on SW480 xenografts. SW480 xenografts Xenograft Generation and Animal Treatment. Four- were either untreated or treated with Pc 4 alone, light alone, or Pc week-old female athymic mice (athymic nu/nuϪ, Case Western 4-PDT, and tumor volumes were measured as described in “Materials ϳ and Methods.” The data represent the means Ϯ SEM of the volumes of Reserve University Animal Facility; 20–22 g bw) were in- four xenografts in each arm. jected s.c. on the flank with 5 ϫ 106 SW480 colon cancer cells in 0.2 ml of serum-free MEM, as described previously (43, 44). Animals were maintained under pathogen-free conditions. After xenografts reached ϳ50–100 mm3 in size, the animals were RK (34) and CSBP (35), participates in a signal transduction randomized into four groups of four animals (four xenografts) network that regulates cellular responses to cytokines and stress per group to determine tumor growth rate after the following triggered by osmotic shock, inflammatory cytokines, lipopo- treatments: (a) control, no treatment; (b) 1 mg/kg bw Pc 4 alone; lysaccharides, UV light, and growth factors (34–38). In cell (c) light alone; and (d) PDT, 1 mg/kg bw Pc 4 and light culture, PDT strongly activates this stress kinase (28, 30, 39). exposure. Pc 4 was delivered via tail vein injection, and 48 h Mechanisms of PDT may be markedly different when later a 1-cm-diameter area encompassing the tumor was irradiated (power density, 150 mW/cm2; ␭, 670 Ϯ 1 nm; fluence, 150 assessed in vitro and in vivo (1) and, further, may differ in J/cm2) with light from a 250-mW diode laser (Applied Optron- rodent tumor models compared with human tumors. Some ics Corp., South Plainfield, NJ) coupled to a 400-␮m quartz photosensitizers that are porphyrins or porphyrin derivatives fiber-optic cable terminating in a microlens to distribute light tend to accumulate in cells of the tumor vasculature and upon uniformly throughout the treatment field. Growth curves repre- photoillumination cause various types of damage including senting tumor regrowth for the control and treated groups were stasis, vessel collapse, and vessel leakage (1, 5) in the tumor. estimated by measuring tumors in three dimensions using a In contrast, Pc 4 [HOSiPcOSi(CH ) (CH ) N(CH ) ] seems 3 2 2 3 3 2 caliper. Tumor volume (V) was determined by the following to preferentially localize into the tumor parenchyma where equation: V ϭ (L ϫ W ϫ H ϫ 0.5236, where L is length, W is the damage occurs after light activation (40). Although Pc the width, and H is the height of the xenograft (44). 4-PDT has been shown to be a strong inducer of apoptosis in For biochemical studies, animals were sacrificed at the several tumor models (15, 41, 42), the mechanism for the in following times after treatment: 0, 0.08, 0.25, 0.5, 1, 3, 6, 10, 18, vivo induction of apoptosis by Pc 4-PDT is not known. and 26 h. Xenografts were resected and frozen instantly in liquid Therefore, the goals of this study were: (a) to evaluate the nitrogen and stored at Ϫ80°C for further biochemical studies. effectiveness of PDT in a human colon cancer model in vivo; Western Blotting. Xenografts were pulverized using a (b) to further evaluate in vivo the participation of signal mortar and pestle on dry ice and then lysed by sonication in a transduction pathways shown to operate during Pc 4-PDT- solution comprising 0.5% sodium deoxycholate, 0.2% SDS, 1% induced apoptosis in vitro; and (c) to test the utility of PARP Triton X-100, 1% NP40, 5 mM EDTA, 10 ␮g/ml leupeptin, 10 cleavage and other previously observed responses as poten- ␮g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride in tial indicators of apoptosis in human tumors treated with Pc ice-cold PBS (all reagents were from Sigma Chemical Co., St. 4-PDT. We chose the human SW480 colon cancer model for Louis, MO). Samples (35 ␮g of protein determined by Dc this study because its growth as a xenograft in athymic nude Bio-Rad protein assay from Bio-Rad, Hercules, CA) were sep- mice has been well characterized, and it is known to undergo arated by PAGE consisting of a 5% (w/v) acrylamide stacking apoptosis in response to topoisomerase inhibitors (43, 44). gel and a 12.5% (w/v) acrylamide separating gel containing Furthermore, SW480 colon cancer cells are known to un- 0.1% SDS (47). The running buffer comprised 0.1% SDS, 25 dergo apoptosis in response to PDT with merocyanine-540 mM Tris, and 250 mM glycine (pH 8.3). Electrophoretic frac- and chemotherapeutic agents (44, 45). tionation was carried out at a constant current of 15 mA until

Fig. 2 InductionofPARPclea- vage by Pc 4-PDT in SW480 xenografts. Western blotting to PARP shows increased PARP cleavage after treatment with Pc 4-PDT. Animals were sacrificed at the times indicated, and tumors were excised and pro- cessed as described in “Materi- als and Methods.” Maximum PARP cleavage is observed at 26 h after Pc 4-PDT.

bromphenol blue migrated ϳ10 cm. Proteins were electrotrans- in the Pc 4-PDT-treated tumors. Treatment with Pc 4 alone or ferred onto an Immobilon p15 membrane (Millipore, Bedford, light alone did not exert any detectable effect on tumor growth. MA). The filters were blocked with 5% nonfat dry milk in 0.1% Apoptosis in Pc 4-PDT-treated SW480 Xenografts. To Tween 20 in PBS and then incubated overnight at 4°C with 1 determine whether evidence of apoptosis could be observed in ␮g/ml primary antibody (directed to PARP, caspase-9, the SW480 xenografts after Pc 4-PDT, animals were sacrificed caspase-3, p38, phospho-p38, p21/WAF1/Cip1, or actin). The at various time points after treatment. The xenografts were secondary antibody was horseradish peroxidase-conjugated an- excised, and lysates were prepared as described in “Materials ti-mouse or anti-rabbit immunoglobulin (1:1000 in blocking and Methods.” The time course of the cleavage of PARP, as solution). Bands were visualized with enhanced chemilumines- measured by Western blot analysis, is shown in Fig. 2. Trace cence reagent and subsequent exposure to Hyperfilm-enhanced amounts of the Mr 90,000 PARP cleavage product were detected chemiluminescence (Amersham, Arlington Heights, IL). The in the control untreated samples and those treated with light intensity of the bands was quantified by densitometric scanning alone or photosensitizer (Pc 4) alone. In contrast, a gradual (SCIscan 5000 USB densitometer; United States Biochemicals, increase in levels of PARP cleavage was observed in the first 6 h Cleveland, OH) and normalized with respect to actin. The after PDT with a significant increase of ϳ60% PARP cleavage percentage of PARP cleavage was estimated as described pre- at 26 h after treatment. Interestingly, Pc 4-PDT-induced PARP ϭ viously (44, 45): percentage of PARP cleaved intensity of Mr cleavage appeared to be mediated by caspase 9 and caspase-3, ϫ 90,000 PARP band 100/(intensity of Mr 90,000 PARP band which were evaluated in the same blots. Activation of caspase-9 ϩ intensity of Mr 116,000 PARP band). and caspase-3 was shown by proteolytic cleavage of the respec- Origin of Antibodies. The monoclonal antibody to pu- tive proenzymes after Pc 4-PDT (Fig. 3A). The intensity of the ␬ rified human PARP (4C10–5) is an IgG1 that shows specific- bands corrected to actin as determined by densitometric scan- ity for the NAD binding domain of PARP and reacts with an Mr ning is shown in Fig. 3B. There was continued processing of 90,000 degradation product in mitogen-stimulated human lym- both proenzymes over the first 26 h after PDT. phocytes (PharMingen, San Diego, CA). The polyclonal anti- PDT Induces Phosphorylation of p38 in SW480 Xe- body to phospho-p38 (Thr-180 and Tyr-182) MAP kinase was nografts. Several laboratories have reported that treatment of from New England Biolabs (Beverly, MA); monoclonal anti- cultured cells with PDT promotes the phosphorylation and ac- human p38 MAP kinase, mouse anti-human p21 monoclonal, tivation of SAPK/JNK and p38/HOG (28, 30, 39). We have and anti-human caspase-9 monoclonal antibodies were from demonstrated that the p38 inhibitor SB202190 can interfere with PharMingen. Monoclonal anti-human caspase-3 antibody was PDT-induced apoptosis, suggesting that the activation of p38 is from Transduction Laboratories (Lexington, KY). Peroxidase- necessary for apoptosis in PDT-treated cells in culture (30). linked anti-rabbit and anti-mouse immunoglobulin were from Therefore, we have evaluated the possible involvement of p38 in Amersham. apoptosis by Pc 4-PDT in the SW480 human colon cancer xenografts. Treatment of the xenografts with Pc 4-PDT induced RESULTS rapid phosphorylation of p38 at Thr-180 and Tyr-182, which Growth Inhibition of SW480 Xenografts by Pc 4-PDT. could be observed by Western blot analysis in samples taken Treatments were performed as described in “Materials and within 5 min after Pc 4-PDT. Levels of phospho-p38 remained Methods.” Untreated tumors or those receiving Pc 4 alone or elevated for ϳ1 h and then gradually decreased to baseline light alone grew with a doubling time of ϳ6 days. Fig. 1 shows levels within the next 25 h. Control untreated samples or sam- the growth rate of untreated xenografts, xenografts treated with ples treated with light alone or Pc 4 alone showed little or no Pc 4 alone or light alone, and xenografts exposed to Pc 4-PDT. p38 phosphorylation. Levels of p38 protein remained constant in Treatment with Pc 4-PDT resulted in almost complete tumor all samples analyzed (Fig. 4). ablation in all four xenografts within 26 h after treatment. The Effect of PDT on p21/WAF1/Cip1 Levels. Activation curves revealed a growth delay of ϳ9–15 days in tumor growth of p21 has been observed in human A431 skin carcinoma in

Fig. 3 Pc 4-PDT induces caspase-9 and caspase-3 activation. A, a time-dependent decrease in the levels of pro- caspase-9 and pro-caspase-3 was detected by Western blotting after Pc 4-PDT treatment. B, densitometric scanning of the Hyperfilm ECL indicates gradual decrease of pro-caspase-9 and pro-caspase-3 over the ini- tial 26 h after PDT. Values were corrected to actin as internal standard for protein loading.

vitro (26) and in human OVCAR-3 xenografts after Pc 4-PDT mice (42), and in a human ovarian tumor xenograft (OVCAR-3) treatment (15). Therefore, levels of p21 were evaluated by in athymic nude mice (15). In a search for molecular markers of immunoblotting of the same membranes used to detect p38 and human tumor response to Pc 4-PDT, both PARP cleavage and phospho-p38. PDT appeared not to affect levels of p21 in induction of p21/WAF1/CIP1 were found to be correlated with SW480 xenografts. Fig. 5 shows that control and treated sam- response to Pc 4-PDT treatment in the OVCAR-3 cancer model, ples express similar levels of p21. a tumor that is wild type for p53 (15). Among the response markers that have been studied in vitro, both p38 phosphoryla- DISCUSSION tion (30) and release of ceramide (25) have been strongly The mechanisms by which PDT induces cell death have correlated with PDT-induced cell death. In the present report, been extensively evaluated with a variety of photosensitizers in we have examined the response of the SW480 human colon tissue culture and to a lesser extent in animal models (1, 8). cancer xenograft, a tumor that expresses mutant p53 (48). The Interestingly, apoptosis has been observed in all animal tumors possible role of p53 in PDT response has been examined by in which it has been sought, arguing that this is an important Fisher et al. (49) in three cell systems. With either Photofrin or process contributing to the rapid responses of the tumors to SnET2 as photosensitizer, PDT-induced cell killing was greater PDT. For Pc 4-PDT, apoptosis has been demonstrated in the for HL60 cells that expressed a transfected wild-type p53 than in early post-treatment hours in a murine transplantable (RIF-1) HL60 cells with either deleted or mutated p53; however apo- tumor in C3H mice (41), in an induced skin tumor in SENCAR ptosis was prominent in all three cell lines (49). Comparison of

Fig. 4 Pc 4-PDT induces phosphorylation of p38-MAP kinase in SW480 xenografts. Pc 4-PDT induced phosphorylation of p38 within 5 min after treatment. Levels of phospho- rylated p38 (p38P) remained elevated for ϳ1 h and gradually decreased thereafter. Levels of p38 protein were not altered by Pc 4-PDT as measured by Western blotting.

Fig. 5 Pc 4-PDT does not al- ter levels of p21/WAF1/CIP1 in SW480 xenografts. Levels of p21 protein remained constant throughout the experiment at all times analyzed as determined by Western blotting.

two human colon carcinoma cell lines with wild-type (LS513) caspase-9 and caspase-3 activation were observed in association or mutant (LS1034) p53 for their sensitivity to PDT revealed with tumor regression (Figs. 1–3). Although we have not spe- greater sensitivity of the LS513 line; however, differences be- cifically addressed the contribution of necrosis in the present tween the two lines other than their p53 status may have ex- study, PDT-treated tumors often contain regions of necrosis in plained the differential photosensitivities (50). In a recent study addition to apoptosis. Some of the necrosis may represent late (51), abrogation of p53 function by transfection of the HPV16 stages of apoptosis. However, within the time course of the

E6 oncoprotein into the wild-type p53-expressing LS513 colon present study, there was little evidence of PARP fragments at Mr carcinoma cell line or MCF-7 human breast cancer cell line had 35,000–50,000 that are characteristic of necrosis (19, 44). In little effect on the photosensitivity of the cells. Thus, PDT addition, despite the fact that the majority of cells constituting a response appears to be independent of p53 status. Because we tumor are the transformed cells, the presence of host cells, such have shown tumor regression in the p53 mutant SW480 colon as endothelial cells, stromal fibroblasts, and peripheral blood cancer xenografts (Fig. 1) as well as the p53 wild-type cells, and the responses of those cells are always a concern in OVCAR-3 ovarian cancer xenografts (15), the in vivo tumor measuring apoptosis, because any or all of these cell types may response appears to be independent of tumor origin and p53 undergo apoptosis. In the present case, although we cannot be status as well. Furthermore, Pc 4-PDT increased p21 levels in certain that only the SW480 cells are undergoing apoptosis, the the OVCAR-3 xenografts (15) but not in the SW480 xenografts high percentage of PARP cleavage (Ͼ60%) and the marked (Fig. 5), suggesting that induction of cell death in these tumors reduction in tumor size over 26 h after treatment suggest that the may be triggered by a p53- and p21-independent mechanism. bulk of the PARP cleavage derives from the malignant cells. However, because we have not compared SW480 xenografts Although the principal mechanism of tumor regression by Pc that differ only in their p53 expression, it is possible that 4-PDT is not completely understood, certain mechanisms are being introduction of wild-type p53 to SW480 cells might confer elucidated in cells in vitro. Pc 4 localizes in mitochondria, as well additional sensitivity to PDT with respect to induction of apo- as in other intracellular organelles, of cells in culture (52), and ptosis or overall tumor response. Our results further indicate that mitochondrial damage appears to play a central role in cell killing Pc 4-PDT caused tumor cells to die by apoptosis, because by Pc 4-PDT (8, 53), as it does for PDT with other mitochondria- generation of the Mr 90,000 PARP cleavage fragment and localizing photosensitizers (24). Immediately upon photodamage,

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