In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered and then activated by exposure to a light source of applicable wavelength. Multidrug resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the anticancer therapy. This study deals with photodynamic therapy with Photofrin II (Ph II) and hypericin (Hyp) on sensitive and doxorubicin-resistant colon cancer cell lines. Changes in cytosolic superoxide dismutase (SOD1) activity after PDT and the intracellular accumulation of photosensitizers in sensitive and resistant colon cancer cell lines were examined. The photosensitizers' distributions indicate that Ph II could be a potential substrate for P-gp, in contrast to Hyp. We observed an increase in SOD1 activity after PDT for both photosensitizing agents. The changes in SOD1 activity show that photodynamic action generates oxidative stress in the treated cells. P-gp appears to play a role in the intracellular accumulation of Ph II. Therefore the efficacy of PDT on multidrug-resistant cells depends on the affinity of P-gp to the photosensitizer used. The weaker accumulation of photosensitizing agents enhances the antioxidant response, and this could influence the efficacy of PDT

Agnieszka Chwilkowsa

Andrzej Pola

Anna Malarska

Anna Marcinkowska

Jolanta Saczko

Julita Kulbacka

Mateusz Lugowski

Teresa Banas

Folia Histochemica et Cytobiologica

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IntroductionThe major drawback of cancer therapy is the emer-gence of multidrug-resistant (MDR) tumor cells,which are cross-resistant to a broad range of struc-turally and functionally unrelated agents, making itdifficult to treat these tumors. Therefore, one of themost important points is to develop new strategies inthe treatment of multidrug-resistant carcinomas. Pho-tosensitizers preferentially accumulate in the regionof a tumor and provide the possibility of killing MDRcells or modulating the MDR phenotype [2]. PDT canthus be promoted as an alternative treatment forMDR tumors. In the last decade, investigations ofphotodynamic effects in MDR cells in culture havegiven promising results [4,5,12]. This relatively newtherapeutic modality for both neoplastic and non-neoplastic diseases involves a dye (photosensitizer)that has been selectively taken up by the target tissue,the presence of molecular oxygen, and light activa-tion. This combined treatment leads to the generationof reactive oxygen species (ROS), such as singletmolecular oxygen, hydroxyl radicals, and/or superox-ide anions [18]. Cells are endowed with enzymaticantioxidant defense systems to prevent cell deathcaused by reactive oxygen species. One of the mostimportant antioxidant enzymes is superoxide dismu-tase, which catalyzes the dismutation of O2- to O2 andH2O2 [9]. In this study, two human-origin colon adenocarci-noma cell lines, LoVo (sensitive) and LoVo DX (dox-orubicin-resistant), were treated with two photosensi-tizers, Photofrin II and hypericin, and irradiated withlaser light. The intracellular accumulation of bothFOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 45, No. 2, 2007pp. 93-97Cytosolic superoxide dismutase activity afterphotodynamic therapy, intracellular distribution of Photofrin II and hypericin, and P-glycoprotein localization in human colon adenocarcinomaJolanta Saczko1, Julita Kulbacka1, Agnieszka Chwilkowska1, Andrzej Pola2, Mateusz Lugowski1, Anna Marcinkowska1, Anna Malarska1 and Teresa Banas11Department of Medical Biochemistry, 2Department of Biophysics, Wroc³aw Medical University, Wroc³aw, PolandAbstract: In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered and then activated by expo-sure to a light source of applicable wavelength. Multidrug resistance (MDR) is largely caused by the efflux of therapeuticsfrom the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the anticancer therapy. This studydeals with photodynamic therapy with Photofrin II (Ph II) and hypericin (Hyp) on sensitive and doxorubicin-resistant coloncancer cell lines. Changes in cytosolic superoxide dismutase (SOD1) activity after PDT and the intracellular accumulationof photosensitizers in sensitive and resistant colon cancer cell lines were examined. The photosensitizers' distributions indi-cate that Ph II could be a potential substrate for P-gp, in contrast to Hyp. We observed an increase in SOD1 activity afterPDT for both photosensitizing agents. The changes in SOD1 activity show that photodynamic action generates oxidativestress in the treated cells. P-gp appears to play a role in the intracellular accumulation of Ph II. Therefore the efficacy ofPDT on multidrug-resistant cells depends on the affinity of P-gp to the photosensitizer used. The weaker accumulation ofphotosensitizing agents enhances the antioxidant response, and this could influence the efficacy of PDT.Key words: Photodynamic therapy - Colon adenocarcinoma - Superoxide dismutase - P-glycoprotein - Photofrin II -HypericinCorrespondence: J. Saczko, Dept. Medical Biochemistry, Wroc³aw Medical University, Cha³ubiñskiego 10, 50-368Wroc³aw, Poland; e-mail: michal@bioch.am.wroc.plphotosensitizers and the influence of the photodynam-ic therapy on cytosolic superoxide dismutase activity,one of the crucial antioxidant enzymes, were exam-ined. Materials and methods Cell lines. Two human colon adenocarcinoma cell lines were used(doxorubicin-sensitive LoVo and doxorubicin-resistant LoVo DX).The cell lines were purchased from Institute of Immunology andExperimental Therapy, Polish Academy of Sciences. The cell lineswere grown in F-12 Ham medium nutrient mixture (Sigma) withaddition of 10% fetal bovine serum in 25 cm3 Falcon flasks. Thecells were maintained in a humidified atmosphere at 37°C and 5%CO2. For the experiments the cells were removed by trypsinizingand washed with phosphate-buffered saline. Photodynamic treatment. The cells were treated with 30 µg/mlPhotofrin II and 2.5 µg/ml hypericin in complete media for 4 h inthe dark. Then they were irradiated with a light dose of 2.5 J/cm2using red laser light (λ= 632.8 nm). After irradiation, the cells wereincubated in a humidified atmosphere at 37°C and 5% CO2 for 30min.Photosensitizer distribution. Microcultures were derived fromthe culture dishes and harvested on cover glasses for 24 hours.The cells were subsequently incubated with two photosensitizers:Photofrin II (Ph II) (QLT Phototherapeutics, Inc., Vancouver,Canada), at a concentration of 30 µg/ml, and hypericin (Sigma), ata concentration of 2.5 µg/ml, for 4 hours. After incubation, thecells were fixed in 4% formalin buffer, washed in PBS, and exam-ined under a fluorescence microscope (Nikon Eclipse TE 2000-E)using a filter with an excitation wavelength of 528-553 nm andemission wavelength of 578-633 nm. Immunofluorescence - P-gp expression. The cells were plated ingrowth medium into 8-dip glass (Nunc) and grown for two days.Then the cells were fixed in 4% formalin buffer and washed inPBS. The intracellular localization of P-gp (1:40, Novoacstra) waslabeled with FITC (1:60, Sigma, excitation wavelength: 470 ± 20nm and emission wavelength: λ=525 ± 20 nm). DAPI (0.2 µl/ml,Molecular Probes, excitation wavelength: 541 nm and emissionwavelength: 445 ± 50 nm) was used for nuclei staining. Fluores-cence was monitored using a microscope with LED illumination(Zeiss Axio Imager.A1). SOD1 activity. After photodynamic treatment in vitro, the cellswere removed by trypsinizing and washed twice in PBS. Then thecells were suspended in 50 mM phosphate buffer with a proteaseinhibitor mixture (Complete Mini EDTA-free, Roche). SOD1activity was measured using the method describe by Segura-Aguilar [16]. The substrate for cytosolic superoxide dismutase isprovided by the reduction of oxygen during the autoxidation ofriboflavin in the presence of UV light. Hydrogen peroxide is quan-titated using a coupled reaction where horseradish peroxidase cat-alyzes the formation of a fluorescent product, 6,6'-diOH-(1,1'-biphenyl)-3,3'-diacetic acid, from 4-OH-phenylacetic acid andhydrogen peroxide. SOD1 activity was measured after incubationwith Ph II and Hyp without irradiation immediately following irra-diation and 30 min after irradiation. Statistical analysis. The significance of the differences betweenmean values of different groups of cells was assessed by Stu-dent's t-test with values of p≤0.05 taken to imply statistical sig-nificance.Results Distribution of photosensitizersThe intracellular distribution of Ph II and Hyp in theLoVo and LoVo DX cell lines was monitored after 4hours of incubation with the photosensitizers. Weobserved different distributions of these photosensitiz-ers in the two investigated cell lines. The fluorescenceemitted by Photofrin II in LoVo was stronger than inLoVo DX (Fig. 1C and D). In contrast, the accumula-tion of Hyp was stronger in LoVo DX then in LoVocells (Fig. 1A and B). The present study shows thatboth photosensitizers are diffused after 4 hours in thecytoplasm predominantly in intracellular membranes(Fig. 1A, B, C and D).P-gp expressionThe P-gp expression allowed evaluation of the level ofmultidrug resistance in LoVo and LoVoDX cells (Fig.2). In LoVo cells, P-glycoprotein is mainly accumulat-ed in the cytoplasm and around the nuclear envelope(Fig. 2A). Cytoplasmatic localization was also identi-fied in LoVoDX cells, but we observed very intensivefluorescence in the nuclear envelope (Fig. 2B).SOD1 activitySOD1 activity was seen to increase after incubationwith hypericin with and without irradiation comparedwith all control cells without PDT (Fig. 3A). Howev-er, in the cells treated with Ph II without irradiation,the level of SOD1 activity was not significantlydecreased. An increase in enzyme activity was alsoobserved in the cells incubated with Ph II after irradi-ation. (Fig. 3B). The activity was lower in both celllines treated with Ph II than in cells treated with hyper-icin. SOD1 activity was observed to increase more inLoVo cells treated hypericin compared with the LoVoDX cell line. On the other hand, the level of SOD1activity in the cells after Ph II PDT was not signifi-cantly higher in LoVoDX cells compared with LoVocells. DiscussionThe resistance to chemotherapeutic drugs is a frequentphenomenon in which tumor cells develop cross-resistance to chemically unrelated cytotoxic agents.The overexpression of multidrug-resistant transporterproteins in cell membranes is one of the reasons for theresistance and can affect the efficacy of photodynamictherapy by reducing the intracellular accumulation ofthe photosensitizer [4,13].The mechanism of cell response to PDT was inves-tigated through the effects of two different photosensi-94 J. Saczko et al.95SOD1 activity and P-gp in colon adenocarcinoma cells after PDTFig. 1. Fluorescence distribution of (a) hypericin (cHyp=2.5 µg/ml) and (b) Photofrin II (cPhII=30 µg/ml) in LoVo and LoVoDX cell linesafter 4-h incubation; left: in LoVo, right: in LoVoDX cells. Fig. 2. LoVo (a) and LoVoDX (b) cells, right: DAPI fluorescence (blue) showing nuclear localization; left: P-glycoprotein labelled FITC(green). tizing agents in cell lines sensitive to (LoVo) andresistant to (LoVo DX) doxorubicin. Other investiga-tions of MDR in PDT have focused primarily on P-gp-mediated resistance, and differing results for specificcells lines and photosensitizers have led to variousconclusions [14]. Cells overexpressing P-gp display areduced intracellular concentration of drugs that aresubstrates for this transporter. In our previous studies,some chemical features of the uptake of both photo-sensitizers, Ph II and Hyp, were examined [3,10]. Thepresent study demonstrates the different sensitivities ofLoVo and LoVoDX cells to the two photosensitizers. Several authors have claimed that there was no dif-ference in Photofrin II accumulation and photosensiti-zation between sensitive and MDR Friend leukemiacell lines. But the accumulation of the hematopor-phyrin derivative was weaker in multicellular sphe-roids derived from a human colorectal carcinoma cellline, a kind of tumor that usually overexpresses P-gp[2,11]. Doxorubicin-resistant and parental cancer cellswere treated with mTHPC (m-tetrahydroxyphenyl-chlorin)-mediated PDT by Teiten et al. [17]. Theyobserved that the effect of photodynamic action wassignificantly greater in the resistant than in the sensi-tive, parental cell line. The highest applied concentra-tion of 7.5 mM of mTHPC resulted in a significantlygreater accumulation in MDR cells. We observed thesame correlation for LoVoDX cells incubated withhypericin compared with the LoVo cell line. Interest-ingly, we obtained conflicting results after incubationwith Photofrin II. These differences in the accumula-tion of the two photosensitizers in sensitive and resist-ant colon cancer cells suggest that Ph II could be asubstrate for P-gp in MDR cells. However, Merlin etal. [12] showed that the cellular accumulation of Ce6in MCF-7 and MCF-7/DXR induced no significantalteration after 3 h of incubation. These data were con-sistent with the fact that Ce6 should not be a substratefor P-glycoprotein. The authors demonstrated thenuclear localization of P-gp in MCF-7/DX. We alsoobserved accumulation of P-gp in the nuclear environ-ment and the cytoplasm, but in the sensitive cell lineLoVo (Fig. 2B). P-gp accumulated in the nuclei can beinvolved in active cytotoxic drug removal [1,13]. Inturn, the LoVoDX cell line revealed intensive P-gpaccumulation in the cell membrane. Calcabrini et al. [1] demonstrated that earlier andhigher depolarization after treatment with BSAO andspermine was found in the membrane of LoVoDX thanin the membrane of the drug-sensitive cells. In addi-tion, basal ROS production was higher in LoVoDXcells. Other results comparing Rh 123 (Rhodamine123) and Photofrin® showed that there was lowerPhotofrin® accumulation in resistant than in wild-typecells. This seems to be mainly related to intracellulardrug turnover [4]. The results obtained in this studysuggest a more effective PDT action in the sensitivehuman colon adenocarcinoma cell line.Apart from the uptake of photosensitizer, this studyshows the antioxidant response to PDT. The presentexperimental results suggest an influence of hypericinand Photofrin II in combination with irradiation onincreased SOD1 activity caused by the generation ofROS and oxidative stress in both cell lines. Johnsonand Pardini performed photodynamic treatment withhypericin on EMT6 cells. A significant increase inSOD activity was already noticed after 30 minutes [8].Golab et al. [6] showed that Ph II-mediated PDT96 J. Saczko et al.Fig. 3. The effect of hypericin (a) and Photofrin (b), and light on cytosolic superoxide dismutase activity (SOD1). The activity was meas-ured control cells without PDT, after incubation with Ph II and Hyp without irradiation, directly after irradiation and after 30 min. p≤0.05statistically significant compared with the control.induced the expression of MnSOD in murine colon-26(C-26). The inhibition of SOD activity in tumor cellsinduced an increase in the cytotoxic effect of PDT. Incontrast, transient transfection with MnSOD generesulted in decreased effectiveness of PDT. Theseresults suggest that MnSOD plays a leading role in theresponse to PDT and that mitochondria impairmentmay be a critical factor in phototoxicity. The SODsappear to be important antioxidative enzymes that reg-ulate the sensitivity of cancer cells in some therapeuticmethods, such as photodynamic therapy [7]. This study indicates a difference in the uptake ofvarious photosensitizers by two colon adenocarcinomacell lines. We suggest that the stronger accumulation ofPh II in LoVo and Hyp in LoVoDX cells causes theimpairment of SOD1 activity after PDT. On the otherhand, weaker fluorescence was observed in cells withhigher SOD1 activity after PDT. Verification of thedependency between dyes applied in PDT and mul-tidrug-resistant transporters could clarify the problemof the proper photosensitizer. The development andcharacterization of multidrug-resistant cells not onlyhelps to understand the mechanism of PDT in theresistance of tumor cells, but also may contribute tothe application of more effective treatment. The searchfor new antitumor drugs has been directed toward find-ing compounds having a novel mechanism of action,activity against resistant cancers, and a higher thera-peutic index [2,4,15].References[ 1] Calcabrini A, Arancia G, Marra M, Crateri P, Befani O, Mar-tone A, Agostinelli E. 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Photochem Photo-biol Sci, 2002; 1: 71-78[ 5] Dellinger M, Moreno G, Salet C, Tapiero H, Lampidis TJ.Cytotoxic and photodynamic effects of Photofrin on sensitiveand multidrug-resistant Friend leukaemia cells. Int J RadiatBiol, 1992; 62: 735-741[ 6] Golab J, Nowis D, Skrzycki M, Czeczot H, Baranczyk-Kuzma A, Wilczynski GM, Makowski M, Mroz P, Kozar K,Kaminski R, Jalili A, Kopec M, Grzela T, Jakobisiak M. Anti-tumor effects of photodynamic therapy are potentiated by 2-metoxyestradiol, a superoxide dismutase inhibitor. J BiolChem, 2003; 278: 407-414[ 7] Huang P, Feng L, Oldham EA, Keating MJ, Plunkett W.Superoxide dismutase as a target for the selective killing ofcancer cells. Nature, 2000; 407: 390-395[ 8] Johnson SAS, Pardini RS. Antioxidant enzyme response tohypericin in EMT6 mouse mammary carcinoma cells. FreeRad Biol Med, 1998; 24: 817-826[ 9] Kasugai N, Murase T, Ohse T, Nagoka S, Kawakami H, Kub-ota S. 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Chem Biol Interact, 1993; 86: 69-78[17] Teiten MH, Bezdetnaya L, Merlin JL, Bour-Dill C, Pauly ME,Dicatio M, Guillemin F. Effect of meta-tetra(hydroxyphenyl-)chlorin (mTHPC)-mediated photodynamic therapy on sensi-tive and multidrug-resistant human breast cancer. J Pho-tochem Photobiol B, 2001; 62: 146-152[18] Wang HP, Hanlon JG, Rainbow AJ, Espiritu M, Singh G. Up-regulation of Hsp27 plays a role in the resistance of humancolon carcinoma HT29 cells to photooxidative stress. Pho-tochem Photobiol, 2002; 76: 98-104Received: October 24, 2006Accepted after revision: December 11, 200697SOD1 activity and P-gp in colon adenocarcinoma cells after PDT

Cytosolic superoxide dismutase activity after photodynamic therapy, intracellular distribution of Photofrin II and hypericin, and P-glycoprotein localization in human colon adenocarcinoma.

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Cytosolic superoxide dismutase activity after photodynamic therapy, intracellular distribution of Photofrin II and hypericin, and P-glycoprotein localization in human colon adenocarcinoma.

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