Radiosensitization of head and neck cancer cells by the ...

Original Article

Radiosensitization of Head and Neck Cancer Cells by the Phytochemical Agent Sulforaphane Ulana Kotowski1, Gregor Heiduschka1, Markus Brunner1, Cornelia Czembirek2, Christina Eder-Czembirek2, Rainer Schmidt3, Tammer Fahim1, Dietmar Thurnher1

Background and Purpose: Sulforaphane is a naturally occurring compound found in broccoli and other cruciferous vegetables. Recently it gained attention because of its antiproliferative properties in many cancer cell lines. The aim of this study was to investigate whether sulforaphane could act as a radiosensitizer in head and neck squamous cell carcinoma cell lines. Material and Methods: Four head and neck squamous cell carcinoma cell lines (i.e., (HNSCC) SCC9, SCC25, CAL27, and FADU) were treated with sulforaphane and subsequently irradiated. Then proliferation and clonogenic assays were performed. Apoptosis was detected by flow cytometry. Possible regulation of Akt and Mcl-1 was investigated by western blotting. Results: Sulforaphane and radiation in combination leads to stronger inhibition of cell proliferation and of clonogenic survival than each treatment method alone. Western blot analysis of Akt and Mcl-1 showed no changed expression. Conclusion: Sulforaphane is a promising agent in the treatment of head and neck cancer due to its antiproliferative and radiosensitizing properties. A combination of sulforaphane and radiation decreases clonogenic survival. Apoptosis is not regulated through Akt or the Mcl-1 protein. Key Words: Sulforaphane · Radiation · Radiosensitizer · Head and neck cancer Strahlenther Onkol 2011;187:575–80

DOI 10.1007/s00066-011-2218-6 Strahlensensibilisierung von Kopf- und Halstumorzellen durch den phytochemischen Wirkstoff Sulforaphan Hintergrund und Ziel: Sulforaphan ist ein natürlicher, in Brokkoli und anderen Kreuzblütlern vorkommender Wirkstoff. In letzter Zeit gewann er Beachtung wegen seiner antiproliferativen Wirkung in vielen Zelllinien unterschiedlicher Malignomarten. Ziel dieser Studie war, Sulforaphan auf seine strahlensensibilisierende Wirksamkeit in Kopf- und Halstumorzellen zu erforschen. Material und Methoden: Vier Kopf- und Halstumorzelllinien (SCC9, SCC9, SCC25, CAL27, und FADU) wurden mit Sulforaphan behandelt und anschließend bestrahlt. Dann wurden Proliferations- und Clonogenic Assays durchgeführt. Die Apoptose wurde mittels Durchflusszytometrie gemessen. Eine mögliche Regulation von Akt und Mcl-1 wurde mittels Western Blotting untersucht. Ergebnisse: Sulforaphan und Bestrahlung in Kombination führten zu einer stärker inhibierten Proliferation und Koloniebildung als die einzelnen Behandlungsmethoden allein. Die Western-Blot-Analyse zeigte keine veränderte Expression von Akt und Mcl-1. Schlussfolgerung: Sulforaphan ist aufgrund seiner antiproliferativen und strahlensensibiliserenden Eigenschaften ein vielversprechender Wirkstoff für die Behandlung von Kopf- und Halstumoren. Die Kombination von Sulforaphan und Bestrahlung führt zu einem verminderten klonogenen Überleben. Die Apoptose wird weder durch Akt noch durch Mcl-1 reguliert. Schlüsselwörter: Sulforaphan · Bestrahlung · Radiosensitizer · Kopf- und Halstumoren

Introduction

Cancer is still one of the most frequent causes of death worldwide. Among the different cancer entities, head and neck squamous cell carcinoma is the sixth most common cancer in the world with more than 600,000 deaths every year [17]. Since head and neck tumors cause symptoms very late, they

are often diagnosed at an advanced stage. Survival rates have not increased in recent decades. The currently available treatment options, such as surgery and radiochemotherapy, have the disadvantage of significant side effects [24, 25]. Even with a combination of treatment methods, outcome is often suboptimal for advanced disease [3]. In an on-going process, therapy

1

Department of Otorhinolaryngology, Head and Neck Surgery, Medical University of Vienna, Austria, Department of Cranio-, Maxillofacial and Oral Surgery, Medical University of Vienna, Austria, 3 Department of Radiotherapy and -biology, Medical University of Vienna, Austria. 2

Received: September 1, 2010: accepted: February 4, 2011 Published Online: August 16, 2011

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Figure 1. Dose-response cuves after treatment with sulforaphane alone or in combination with radiation. SCC9, SCC25, CAL27, and FADU cells were treated with increasing doses of sulforaphane, subsequently irradiated with 2, 4, or 8 Gy and incubated for 72 hours. Abbildung 1. Dosis-Wirkungs-Kurven nach Behandlung mit Sulforaphan allein oder in Kombination mit Strahlung. SCC9-, SCC25-, CAL27- und FADU-Zellen wurden mit steigenden Dosen Sulforaphan behandelt, anschließend mit 2, 4 oder 8 Gy bestrahlt und 72 Stunden lang inkubiert. SCC9

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protocols are reevaluted and optimized [2, 21, 22]. The aim is, therefore, to find new strategies with fewer side effects for the treatment of malignant tumors. The naturally occurring compound sulforaphane (SFN) is an isothiocyanate isolated from broccoli and other cruciferous vegetables such as cabbage, cauliflower, and Brussels sprouts. Recently it has gained attention because of its multiple biological activities. Several studies showed its chemoprotective properties [15]. In some epidemiological studies, the intake of cruciferous vegetables was associated with lower risk for lung and colorectal cancer [12]. At the same time, other studies have shown that this phytochemical compound posses strong antitumor activities [9]. It inhibits cell growth via induction of cell cycle arrest and apoptosis in several human cancer cell lines, e.g., breast, colon and prostate cancer [10, 13, 16]. In vitro studies have shown that sulforaphane inhibits phase I biotransformation enzymes which are involved in the activation of carcinogenesis and induces phase II detoxification enzymes including UDPglucuronosyl transferase, gluthatione Stransferase, and quinine reductase [1, 4, 14]. Recently, a placebo-controlled study showed that oral intake of SFN increases phase II antioxidant enzymes in the nasal mucosa [19]. In view of these findings and in search for new options to treat cancer, this study was performed. The fact that this compound is naturally occurring and associated with low toxicity was particularly important for us. Lately our group demonstrated that betulinic acid, a plant polyphenol, acts additively in combination with irradiation [8]. The aim of this study was to examine the effect of sulforaphane in the four head and neck cell carcinoma cell lines (HNSCC) SCC9, SCC25, CAL27, and FADU and to investigate whether this agent has a radiosensitizing effect in vitro. Therefore, proliferation and clonogenic assays were performed and apoptosis was measured by flow cytometry. The regulation of the anti-apoptotic proteins Akt and Mcl-1 was examined by western blotting.

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The head and neck squamous cell carcinoma cell lines SCC9, SCC25, and FADU were obtained from the American Type Culture Collection (Manassas, VA, USA). CAL27 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Tumor cells were cultured in RPMI medium (Cambrex, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (PAA Laboratories, Linz, Austria) and 1% penicillin–streptomycin (Gibco BRL, Gaithersburg, MD, USA) at 37°C in a humidified atmosphere of 5% CO2. Cytotoxicity Assay

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Material and Methods Study Drug

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A CCK-8 cell proliferation assay (DojinAbbildung 3. Kombinationsindex-Plot (fa-CI Plot) für die Kombination Sulforaphan und Strahlung. do Molecular Technologies, GaithersCI-Werte wurden mit Hilfe von CalcuSyn®-Software berechnet. CI = 1bedeutet einen addidiven Efburg, MD, USA) was used to determine fekt, CI < 1 bedeutet einen synergistischen Effekt und CI > 1 bedeutet Antagonismus. cytotoxic effects of sulforaphane on tumor cells in vitro. Cells were seeded at Table 1. IC50 values and standard errors of the mean (SEM) after 72 hours 3 × 103 cells per well into 96-well plates and incubated for 24 of treatment with sulforaphane alone or in combination with radiation. hours. Then cells were treated with increasing concentrations of Tabelle 1. IC50-Werte und Standardabweichung 72 Stunden nach alleinisulforaphane (0–20 µM). After 72 hours of incubation, cell proger Behandlung mit Sulforaphan oder in Kombination mit Bestrahlung. liferation was measured by CCK-8 according to manufacturer’s 0 Gy 2 Gy 4 Gy 8 Gy protocol. Experiments were carried out at least three indepenSCC 9 dent times and were performed in triplicates. Irradiation

Cells were treated with sulforaphane and subsequently irradiated with a single boost of 2, 4, or 8 Gy using a conventional radiation source with 250-kV X-rays. Analysis of Combination Effects

To determine the concentrations of the drug to be investigated in the combination study, dose–response curves were generated with the GraphPad 4.0 software from PRISM® (GraphPad Software Inc., San Diego, CA, USA). Interactions with radiation were calculated with the CalcuSyn® software (Version 2.0, Biosoft, GB) based on the Chou-Talalay equation [7]. Colony-Forming Assays

Aliquots of 5 × 105 cells were seeded in 10 cm culture dishes and incubated for 24 hours. Then cells were treated with 2.5 µM, 5 µM, and 10 µM sulforaphane and/or irradiated Strahlenther Onkol 2011 · No. 9

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with a single boost of 4 Gy. After 72 hours of incubation, cells were trypsinized and counted using the automated CASY® Cell Counting System (Schärfe System GmbH, Reutlingen, Germany). Thereafter, 4 × 102 of the analyzed, surviving cells were plated in 6-well plates in drug-free medium. After 2 weeks of incubation, cells were washed three times with PBS,

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A total of 1 × 10 cells were seeded in 6-well plates. After 24 hours, cells were treated with sulforaphane and/or radiation. Apoptosis was measured after 48 hours using the Annexin-V Apoptosis Detection Kit (Bender MedSystems, Vienna, Austria). Apoptosis was defined as Ann+/PI–. Ann–/PI+, and Ann+/PI+ were both defined as necrotic as late apoptosis and necrosis cannot be differentiated with this assay. 5

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Aliquots of 5 × 105 cells were seeded in Figure 4. Clonogenic survival of treatment with sulforaphane in combination with radiation. SCC9, 10 cm culture dishes and incubated for SCC25, CAL27, and FADU cells where treated with 2.5, 5, and 10 µM sulforaphane and irradiated with 24 hours. Then cells were treated with 4 Gy. After 72 hours, 400 viable cells where transferred into drug-free medium and incubated for 2 10 µM sulforaphane and/or irradiated weeks. Then surviving colonies where counted. The results are shown in a semi-logarithmic scale. with 4 Gy. After 0, 12, and 24 hours, cell Abbildung 4. Klonogenes Überleben nach Behandlung mit Sulforaphan in Kombination mit Bemonolayers were washed twice with strahlung. SCC9-, SCC25-, CAL27- und FADU-Zellen wurden mit 2,5, 5 und 10 µM Sulforaphan und cold PBS, frozen with liquid nitrogen, einer Strahlendosis von 4 Gy behandelt. Nach 72 Stunden wurden 400 Zellen in frisches Medium transferiert und für 2 Wochen inkubiert. Danach wurden die überlebenden Kolonien gezählt. Die and lysed with lysis buffer as described Ergebnisse sind in einer semi-logarithmischen Skala dargestellt. previously [11]. The lysates were centrifuged at 14,000 rpm at 4°C for 20 minutes and the supernatants were collected. Protein concengrowth inhibition after treatment with sulforaphane was found trations were determined using Micro BCA protein countat 20 µM. Dose–response curves after 72 hours of treatment ing kit from Pierce (Rockford, IL, USA). Thereafter, 20 µg of are shown in Figure 1. The IC50 values for sulforaphane ranged protein was separated by SDS-PAGE (10%) and electroblotted from 5.03–7.36µM (Table 1). Dose–response curves for survivonto nitrocellulose membranes (Schleicher & Schuell, Dassel, al after irradiation alone are shown in Figure 2. Radiation with Germany). After blocking with 5% BSA in TBS-Tween over4 Gy inhibited cell viability up to 71%. night, membranes were incubated with the appropriate diluted primary antibody. Bound antigen was visualized with the ImCombined Effect of Sulforaphane and Radiation mun-Star Western C Kit (Bio-Rad Laboratories, CA, USA) and on Cell Proliferation detected by ChemiDoc-It Imaging System (UVP, CA, USA). The combined effect of sulforaphane and radiation was investigated thereafter. Cells were first treated with different concentrations of sulforaphane (0–20 µM) and subsequently irradiStatistical Analysis ated (0–8 Gy). As shown in Figure 1, the combined treatment Statistical analysis was performed using Graph Pad 4.0 software led at low doses of sulforaphane to a stronger growth inhibition by PRISM® (GraphPad Software Inc., San Diego, CA, USA). All than treatment with sulforaphane or radiation alone. A posexperiments were repeated at least three times. sible synergistic effect was calculated with CalcuSyn® software. A synergism could be demonstrated in all cell lines (Figure 3). Results Effect of Sulforaphane and Radiation on Cell Proliferation

First, the growth inhibitory effects of sulforaphane and radiation in the HNSCC cell lines SCC9, SCC25, CAL27, and FADU were examined. Cells were treated with sulforaphane in a dose ranging from 0–20 µM or irradiated with doses from 0–8 Gy. Both sulforaphane and irradiation showed a dose-dependent growth inhibition in all tested cell lines. The maximal cell

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Effect of Combined Treatment on Clonogenic Survival

To determine the long-term effect of treatment on cells, clonogenic assays were performed. Cells were treated with 2.5, 5, or 10 µM and then irradiated with 4 Gy. It was observed in all tested cell lines that treatment with sulforaphane and/or radiation led to a reduced clonogenic survival (Figure 4). Combined



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Figure 5. Evaluation of apoptosis by flow cytometry. The head and neck cancer cell lines SCC25, CAL27, and FADU where treated with 5, 10, or 20 µM sulforaphane and subsequently irradiated. Apoptosis was measured after 48 hours by flow cytometry using the Annexin-V Apoptosis Detection Kit. Abbildung 5. Evaluation von Apoptose mittels Durchflusszytometrie. Die Kopf- und Halstumorzelllinien SCC25, CAL27 und FADU wurden mit 5, 10 und 20 µM Sulforaphan behandelt und anschließend bestrahlt. Apoptose wurde nach 48 Stunden unter Anwendung des Annexin-V Apoptosis Detection Kit mittels Durchflusszytometrie gemessen.

treatment resulted in a synergistic inhibition of colony formation. Sulforaphane and Irradiation Induces Apoptosis in HNSCC

Because treatment with sulforaphane and radiation led to reduced proliferation of cancer cells, flow cytometry was performed to evaluate the induction of apoptosis. SCC25, CAL27, and FADU were treated with 5, 10, and 20 µM sulforaphane, subsequently irradiated with 4 Gy, and incubated for 48 hours. Treatment with sulforaphane resulted in an increased apoptosis at 10 µM (Figure 5). In the cell lines SCC25 und FADU, doubling of the treatment dose to 20 µM did not lead to a further increase of apoptosis rates. In addition, there was no significant difference of apoptosis rates between treatment with the single substance and treatment in combination with radiation. Regulation of Akt and Mcl-1

Regulation of the anti-apoptotic proteins Akt and Mcl-1 was analyzed before and after 12 and 24 hours of treatment with 10 µM sulforaphane and/or 4 Gy. In all tested cell lines, no altered expression of the two enzymes could be demonstrated (data not shown). Discussion

In this study, we demonstrated the radiosensitizing effect of the phytochemical agent sulforaphane in head and neck cancer cells. Our experiments showed that treatment with sulforaphane in combination with radiation leads to a stronger inhibited proliferation than with sulforaphane or radiation alone as demonstrated with proliferation and clonogenic assays. To our knowledge, this work shows for the first time the synergistic effect of radiation and sulforaphane in HNSCC in vitro. Sulforaphane is known as a naturally occurring compound with chemopreventive as well as antiproliferative properties. This effect has been proven in many tumor entities [9]; however, little


has been published about the combined application of sulforaphane and radiation therapy. Sulforaphane as a single agent showed a dose dependent inhibition of cell proliferation with an IC50 from 5.03–7.36 µM after 72 hours in our four tested cell lines. This indicates that sulforaphane is an effective antiproliferative substance. These values are markedly lower than the results of Cho et al. [5]. In their work, the IC50 was at 22.2 and 25.6 µM for two squamous cells cell lines of the tongue. However, these results cannot be compared directly as their data was collected after 24 and 48 hours. The results of our group and those of Cho et al. coincided insofar as that after treatment with sulforaphane apoptosis rates increased up to fourfold. To our knowledge, there is only one publication that investigated the combined treatment of sulforaphane and radiation so far. That work examined the combined effect in HeLa human cervix carcinoma cells and demonstrated an enhanced radiosensitivity [28]. We demonstrated an increased radiosensitivity for our head and neck cell lines, but combined treatment showed no enhanced apoptosis rates compared with treatment with sulforaphane alone. However, apoptosis was measured after 48 hours and cannot be compared with the clonogenic survival assay that investigates long-term effects. To examine whether there is a regulation of apoptosis on the molecular biological level, we analyzed the expression of the anti-apoptotic proteins Mcl-1 and Akt. Mcl-1 is a member of the Bcl-2 family and highly expressed in head and neck squamous cell carcinoma [23]. After treatment with sulforaphane, Bcl-2 was downregulated in many cancer cell lines, e.g., breast cancer cells [18], hepatoma cells [27], leukemia cells [6], prostate cancer cells [20], and oral squamous carcinoma cells [5]. We did, however, not detect an altered expression of Mcl-1 in our head and neck cancer cell lines or a regulation of Akt, which is in accordance with the work by Yao et al. [26], who also did not observe a change in the expression of Akt.

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Conclusion

The results of this work have shown that sulforaphane is an effective phytochemical compound with antiproliferative and radiosensitizing properties in head and neck cancer cells. Because of its low toxicity, we believe that sulforaphane could perhaps be used as an adjuvant treatment to radiotherapy in the future. Moreover it is not expensive and easily available. References 1. Basten GP, Bao Y, Williamson G. Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP-glucuronosyl transferase (UGT1A1) and glutathione transferase (GSTA1) in cultured cells. Carcinogenesis 2002;23:1399–404. 2. Berger B, Belka C, Weinmann M et al. Reirradiation with alternating docetaxelbased chemotherapy for recurrent head and neck squamous cell carcinoma: update of a single-center prospective phase II protocol. Strahlenther Onkol 2010;186:255–61. 3. Bieri S, Bentzen SM, Huguenin P et al. Early morbidity after radiotherapy with or without chemotherapy in advanced head and neck cancer. Experience from four nonrandomized studies. Strahlenther Onkol 2003;179:390–5. 4. Brooks JD, Paton VG, Vidanes G. Potent induction of phase 2 enzymes in human prostate cells by sulforaphane. Cancer Epidemiol Biomarkers Prev 2001;10:949–54. 5. Cho NP, Han HS, Leem DH et al. Sulforaphane enhances caspase-dependent apoptosis through inhibition of cyclooxygenase-2 expression in human oral squamous carcinoma cells and nude mouse xenograft model. Oral Oncol 2009;45:654–60. 6. Choi WY, Choi BT, Lee WH et al. Sulforaphane generates reactive oxygen species leading to mitochondrial perturbation for apoptosis in human leukemia U937 cells. Biomed Pharmacother 2008;62:637–44. 7. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006;58:621–81. 8. Eder-Czembirek C, Erovic BM, Czembirek C et al. Betulinic acid a radiosensitizer in head and neck squamous cell carcinoma cell lines. Strahlenther Onkol 2010;186:143–8. 9. Fimognari C, Hrelia P. Sulforaphane as a promising molecule for fighting cancer. Mutat Res 2007;635:90–104. 10. Gamet-Payrastre L, Li P, Lumeau S et al. Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res 2000;60:1426–33. 11. Heiduschka G, Erovic BM, Vormittag L et al. 7beta-hydroxycholesterol induces apoptosis and regulates cyclooxygenase 2 in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2009;135:261–7. 12. Higdon JV, Delage B, Williams DE et al. Cruciferous vegetables and human cancer risk: epidemiologic evidence and mechanistic basis. Pharmacol Res 2007;55:224–36. 13. Jackson SJ, Singletary KW. Sulforaphane: a naturally occurring mammary carcinoma mitotic inhibitor, which disrupts tubulin polymerization. Carcinogenesis 2004;25:219–27. 14. Maheo K, Morel F, Langouet S et al. Inhibition of cytochromes P-450 and induction of glutathione S-transferases by sulforaphane in primary human and rat hepatocytes. Cancer Res 1997;57:3649–52.

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Address for Correspondence Dietmar Thurnher, MD Department of Otorhinolaryngology, Head and Neck Surgery Medical University of Vienna Waehringer Guertel 18–20 1090 Vienna Austria Phone (+43/1) 40400-3372, Fax -3355 e-mail: [email protected]
