- Email: [email protected]
Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
Photodiagnosis and Photodynamic Therapy (2014) xxx, xxx—xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/pdpdt
Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro Seong Taek Mun MD, PhD a,∗, Dong Han Bae a, Woong Shick Ahn b a b
Dept. of Obstetrics and Gynecology, SoonChunHyang University, Republic of Korea Dept. of Obstetrics and Gynecology, Catholic University, Republic of Korea
KEYWORDS EGCG; Photodynamic therapy; Anti-tumor effect
Summary Objectives: To evaluate anti-tumor effects of combined photodynamic therapy and epigallocatechin-3-gallate (EGCG). Materials and methods: TC-1 cells were injected into C57BL/6 mice. Mice were grouped by 7 into 4 groups as PDT, EGCG, combined PDT with EGCG, and control group. The photosensitizer Radachlorin was used. The light source was a diode laser with 662 nm wavelength. In vitro TC-1 cells were treated with Radachlorin and irradiated. In vivo, when tumors were 8—10 mm, Radachlorin was injected into mice and irradiated. For in vitro, different doses of EGCG were added to culture dishes. For combination, EGCG was added to the cells. 2.5 or 5 g/ml of Radachlorin was added to the cells. Cells were incubated with EGCG and/or Radachlorin and laser irradiated. In vivo, EGCG were given for 20 days, alone or after PDT treatment. Cell growth inhibition was determined using MTT assay. Tumor growth inhibition assays were done in each group. Tumor growth was measured using caliper. Western blottings were performed with primary antibodies as COX-2, p21, p53, PARP, Bax, P-p38, VEGF, HIF-1␣, MMP9 and actin. Results: The cell growth and the tumor volume in PDT combined with EGCG treatment group was significantly suppressed, compared with control and PDT or EGCG alone treated groups. We have shown that PDT combined with EGCG in vivo increase levels of both p21 and p53. Both Bax and activated PARP genes were significantly expressed. Conclusions: It is suggested that high anti-cancer activity of combined photodynamic therapy with EGCG may be useful for effective cancer therapy. © 2014 Elsevier B.V. All rights reserved.
Introduction ∗ Corresponding author at: Dept. of OBGY, SoonChunHyang University CheonAn Hospital, BongMyung-dong, CheonAn-si, ChungNam 330-721, Republic of Korea. Tel.: +82 41 570 2150; fax: +82 41 571 7887. E-mail addresses: [email protected], [email protected] (S.T. Mun).
Photodynamic therapy (PDT) is a method of treating malignant tumors based on the photodynamic damage of tumor cells resulting in a photochemical reaction [1]. PDT has been successfully used in the treatment of a variety of cancers to induce apoptosis in tumor cells [2—8]. PDT causes
http://dx.doi.org/10.1016/j.pdpdt.2014.03.003 1572-1000/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
2
S.T. Mun et al.
the photochemical generation of cytotoxic reactive oxygen species, such as singlet oxygen, within the target tissue. PDT clinical trials using a photosensitizer, as well as a variety of second-generation photosensitizers, have shown promise in treating malignancies of the esophagus, bronchus, brain, peritoneal cavity, skin, bladder, head and neck, as well as in treating non-oncologic disorders, such as age-related macular degeneration [9,10]. Clinical results of PDT are positive; however, use of this technique requires further improvements, as tumor recurrences can occur [11—13]. The green tea polyphenols have been shown to have a protective effect on prostate cancer in pre-clinical animal models; it has been reported to be effective against several other cancer types, as well [14—18]. Green tea is composed of several catechins, including (−)-epigallocatechin-3-gallate (EGCG), epicatechin (EC), epicatechin-3-gallate (ECG) and epigallocatechin (EGC). Tea polyphenols have been shown to inhibit carcinogenesis in many animal models, and the significance of catechins, the main constituents of green tea, has been shown to be important for cancer prevention. Among them, (−)-epigallocatechin-3-gallate (EGCG), the major catechin found in green tea, has been recognized as a potential therapeutic agent [18—24]. Interest in the study of EGCGs, as anticancer agents, has increased in recent years, due to their both in vitro and in vivo effects on tumor cell signaling pathways regulating growth and apoptosis, including the suppression of COX-2, HIF-1a and VEGF gene expression [24—27]. More recently, it has been found that EGCG was able to enhance growth arrest and apoptosis of cancer cells through many pathways, including activation of p53, p21, inhibition of AKT, ERK1/2 and activation of JNK1/2 [28,29]. It has been shown that drug treatment combined with PDT therapy suppressed potentially harmful genes and enhanced PDT therapy [30—32]. In this study, we evaluated both in vitro and in vivo the effectiveness of the PDT plus EGCG on tumor regression. PDT combined with EGCG demonstrated significant suppression of tumor growth. Using Radachlorin, it was found that PDT exerts its anti-tumor activity through EGCG supplement after PDT, rather than by EGCG supplement prior to PDT.
Materials and methods Cell culture and tumor model TC-1 tumor cells were routinely propagated in complete RPMI 1640 medium (10% fetal bovine serum, 1% Lglutamine, and 1% penicillin/streptomycin), supplemented with 400 g/ml G418. TC-1 is an E7-expressing tumorigenic cell line. It was established from primary lung epithelial cells of C57BL/6 mice immortalized with HPV16 E6 and E7, and then transformed with an activated ras oncogene (1). Female 6-week old C57BL/6 mice were purchased from Daehan Biolink (Daejeon, Korea) and maintained in the specific pathogen free (SPF) animal facility at The Catholic University of Korea. Every 7 mice were divided into 4 groups. Total 28 mice were used. TC-1 cells were washed twice with PBS (phosphate buffered saline) and injected subcutaneously into the right flank of C57BL/6 mice (2 × 105 cells/mouse).
PDT The photosensitizer Radachlorin was purchased from RADAPHARMA group (RADA-PHARMA, Moscow, Russia) and was diluted in PBS buffer to make a 1 mg/ml stock solution. The light source was a diode laser with 662 ± 2 nm wavelength (Won-PDTD662, Won Technology, Daejon, Korea). PDT was carried out as previously described (2). Briefly, in vitro TC1 cells were added with Radachlorin (2.5 or 5 g/ml) for 12 h. Cells were washed with PBS buffer and then irradiated with 6.25 J/cm2 of light (200 mW, 5 min). In vivo, when tumors were approximately 8—10 mm in mean tumor diameter, Radachlorin (10 mg/kg) was injected into the tail vein of mice 3 h before irradiation. The tumor was irradiated at a fluence of 500 J/cm2 with 300 mW power.
EGCG EGCG was provided by Yukihiko Hara (Tea Solution, Hara Office Inc., Sumida-ku, Tokyo, Japan). EGCG was stored at −20 ◦ C in powder. EGCG was dissolved in sterilized PBS buffer before use. Fresh stocks were made each time. For in vitro experiments, different doses of EGCG (25, 50 and 100 M) were added to culture dishes. For combination, 50 M of EGCG was added to the cells. At the same time, 2.5 or 5 g/ml of Radachlorin was added to the cells. Cells were incubated with drugs (EGCG and/or Radachlorin) for 12 h under environment of 5% CO2 , 37 ◦ C. Incubated cell dishes were warmed at 20 ◦ C before use as with other experiments. Laser irradiated with 6.25 J/cm2 of light. In vivo, EGCG were given by gavages (400 mg/kg per day) for 20 days, alone or after PDT treatment.
MTT assay Cell growth inhibition was determined using the methyl thiazolyl tetrazolium (MTT) assay. MTT assay was performed every 24 h from irradiation time point until the 5th day. 20 l of MTT solution (5 mg/ml) was added to each well. Four hours later, DMSO (dimethyl sulfoxide) was added and then absorbance was measured at 570 nm in the automated spectrophotometric microtitre plate reader (Spectra Max 340, Molecular Devices, USA).
Tumor growth inhibition assay After TC-1 tumor cells had grown to a tumor size of 8—10 mm in mean diameter, the mice were randomly divided into four groups: the control group, the EGCG only group (400 mg/kg per day for 20 days), the PDT only group (10 mg/kg Radachlorin, 300 mW power, 500 J/cm2 dose) and the PDT + EGCG (400 mg/kg per day for 20 days) group. After mice were given treatment, tumor growth was measured twice or three times a week using caliper, and recorded as tumor volume (longest surface length [a], width [b] and height [c], tumor volume (mm3 ) = [a] × [b] × [c]).
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
Epigallocatechin gallate with photodynamic therapy
3
Figure 2 Tumor suppression by PDT, EGCG alone or PDT combination with EGCG in vivo. Mice were injected subcutaneously with 5 × 105 cells per mouse. When the mean tumor size reached 8—10 mm, the EGCG group was fed with 400 mg/kg EGCG for 20 days, whereas the PDT group was given 500 J single treatment; PDT plus EGCG group was treated with Radachlorin/PDT, then EGCG was administered for 20 days. Each group included seven animals (see ‘‘Materials and Methods’’ for details). Error bars indicate the SD. Statistically significant inhibition of tumor growth was evaluated by the Student’s t-test. *P < 0.05, compared with the control; # P < 0.05, compared with EGCG treated group; $ P < 0.05, compared with PDT treated group.
sample underwent electrophoresis for 2.5 h with SDS-PAGE at 10 mA, and Western blotting was performed with a Hybond-ECL membrane (Amersham, Uppsala, Sweden) at 100 V. The blotted membrane was blocked with 5% skimmed milk and reacted with primary antibodies (COX-2, p21, p53, PARP, Bax, P-p38, VEGF, HIF-1a, MMP9 and actin; Santa Cruz Biotechnology Inc., Santa Cruz, CA). After washing with Tris-buffered saline containing 0.1% Tween 20, the membrane was incubated with the horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Protein bands were visualized using the ECL Kit (Amersham, Arlington Heights, IL). Figure 1 In vitro cell growth inhibition by PDT, EGCG alone or PDT combination with EGCG. TC-1 cells (3 × 103) were treated with (A) EGCG, (B) Radachlorin/PDT or (C) PDT combination with EGCG. The control group represents cells only. Samples were assayed in triplicate and MTT assay was performed for five days. Vertical bars indicate SD (n = 3). Statistically significant inhibition of cell growth was measured by the Student’s t-test. *P < 0.05, **P < 0.01 compared with indicated group.
Statistical analysis Statistical analysis included ANOVA and the Student’s t-test. The values for the different groups were compared. P values of <0.05 were considered statistically significant.
Results Western blotting After tumor bearing mice were given treatment at the indicated time, the tumor tissues were harvested and lyzed for protein preparation. The protein was evaluated using the BioRad Protein-Assay kit (Bio-Rad, Hercules, CA), and adjusted for a final concentration of 2 mg/ml. After addition of 2-mercaptoethanol (2%), samples were boiled for 5 min and used for the experiments. 40 g of each protein
In vitro cell growth inhibition by EGCG, PDT alone or PDT combined with EGCG First, we tested the effects of EGCG on the TC-1 cell line. The cell growth inhibition effect was evaluated using MTT assays with increasing amounts of EGCG, and showed significant increase in cell growth inhibition over time (Fig. 1A). Next, we investigated the effects of the PDT on growth
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
4
S.T. Mun et al.
Figure 3 Pictures of mice on the 21st day by PDT, EGCG alone or PDT combination with EGCG in vivo. Every three mice belong to each group. (A) Control group (on the 21st day). (B) EGCG only (on the 21st day). (C) PDT only (on the 21st day). (D) PDT + EGCG (on the 21st day)
inhibition in the TC-1 cell line. A significant decrease in cell survival was detected with PDT, compared to the control group (Fig. 1B). The cell growth was also significantly suppressed in the EGCG plus PDT group (Fig. 1C).
Tumor suppression by PDT, EGCG alone or PDT combined with EGCG in vivo To further enhance the anti-cancer effect with PDT, we investigated the effects of the combination of PDT and EGCG in vivo. After PDT alone, the tumor volume was suppressed, compared with controls, as shown in Fig. 2. Next, the tumor volume in the PDT combined with EGCG treatment group was significantly suppressed, compared with not only control, but also the PDT or EGCG alone treated groups (Fig. 3). Additionally, we investigated the effects of EGCG supplement prior to PDT in vivo. As shown in Fig. 2, the tumor volume after the EGCG supplements were relatively suppressed, but not significantly compared to the PDT alone treatment. The results showed that EGCG lowered the uptake of Radachlorin; this suggested that an EGCG supplement prior to photodynamic irradiation is not desirable. Therefore, EGCG supplementation prior to photodynamic irradiation may play a role as an interference factor against uptake of Radachlorin.
Radachlorin uptake We tested in vivo whether intravenous Radachlorin injection plus EGCG supplement affected Radachlorin accumulation in serum or tumor. As Radachlorin was injected intravenously, it was natural that Radachlorin was highly accumulated in serum in the first time periods (Fig. 4A). We confirmed that Radachlorin was accumulated in sera, and then excreted in each Radachlorin and/or EGCG group after 48 h. For tumors, the highest accumulation was at 3 h after intravenous injection (Fig. 4B). In addition, there was a significant difference with the levels of Radachlorin in either treatment. This was consistent with the anti-tumor effect of PDT, as shown in Fig. 2. Under the same conditions, Radachlorin accumulation in tumors was lower in the combination group (EGCG plus Radachlorin), compared to Radachlorin alone. The results showed that EGCG supplement prior to Radachlorin injection could inhibit Radachlorin uptake in vivo. The time lag of Radachlorin tumor accumulation post injection is also very important for controlling the effect of PDT and/or EGCG combination.
Protein expression changes in treated tumor tissue In addition, we determined the molecules responsible for the enhanced anticancer effects following PDT combination with EGCG in vivo. We have shown that PDT combined
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
Epigallocatechin gallate with photodynamic therapy
5
Figure 5 Changes in tumor protein expression by combined PDT and EGCG treatment. Tumor bearing mice were injected intravenously with Radachlorin (10 mg/kg). Three hours after injection, the animals were anesthetized and the tumors were given external light treatment (100 J/cm2 ); mice in the EGCG treatment group were fed with 400 mg/kg for 20 days, whereas mice in the combination therapy group were fed with EGCG for 20 days after PDT were performed. At 6 and 20 days after PDT treatment, tumor tissues were collected and homogenized with lysis buffer. Tissue extracts (30 g of protein) were run and immunoblot analysis was performed with each specific antibody.
Figure 4 Radachlorin uptake in mice. Radachlorin uptake was expressed as g/g of wet tissue in the tumor, and expressed as g/ml of sera in vivo, as a function of the time after intravenous injection at a dose of 10 mg/kg to the C57BL/6 mice with TC-1 tumors. The bars represent the mean SD of three animals.
with EGCG in vivo led to significant increase in levels of both p21 and p53, at 6 days post PDT plus EGCG combination treatment (Fig. 5). These changes disappeared 20 days after treatment and both Bax and activated PARP genes were significantly expressed.
Discussion The main finding of this study was that the combination therapy approach using PDT and one of the green tea constituents, (−)-epigallocatechin-3-gallate (EGCG), was effective for reducing tumor growth. Moan and Berg [33] reported that photodynamic therapy (PDT) is a promising cancer treatment modality, based on
its selective killing of malignant cells by singlet oxygen 1 O2 , and other reactive products generated by photoactivated photosensitizer (PS) molecules that accumulate in tumor tissue. However, further development is needed to improve outcomes, as tumor recurrences occur, and tumor size and depth affect the efficiency of PDT irradiation. This maybe due to the increase in survival of molecules expressing COX2, HIF-1a and the VEGF gene. Photodynamic therapy (PDT) is a method of treating malignant tumors that produces photodynamic damage of tumor cells by photochemical reactions. At present, there are many chemicals used as photosensitizers of PDT for clinical trials [34]. We used Radachlorin, a promising sensitizer for photodynamic therapy. Radachlorin is a second generation photosensitizer, with promising physicochemical properties and high photodynamic efficiency. A tumor treated by PDT is reabsorbed and is gradually replaced by connective tissue [34]. The location of photodynamic damage of a tumor is determined by the photosensitizer’s ability to accumulate in tumor tissues and by the direction and localization of precise laser irradiation [34]. In our study, the influence of PDT time after Radachlorin injection on anti-cancer effect was investigated. We evaluated tumor suppression at 3, 6, 9, 12, and 15 days after PDT treatment. A significant difference was confirmed on day 15, and a tendency for the tumor to grow larger was observed. In contrast, in the control group, the tumor sizes increased almost linearly with time, until the end of the observation period. The results showed that tumor suppression was most effective at 3 h post Radachlorin injection (data not shown). As shown in Fig. 2, as Radachlorin was injected intravenously, it is natural that Radachlorin was highly accumulated in serum in the first time periods. For tumors, the
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
6
S.T. Mun et al.
highest accumulation was at 3 h after intravenous injection. Additionally, Radachlorin accumulation in tumors was lower in the combination group (EGCG plus Radachlorin), compared to Radachlorin alone. Therefore, we performed our PDT treatment at 3 h after Radachlorin injection in all experiments. EGCG, the major catechin found in green tea, has been recognized as a potential therapeutic agent, as previously described [22—24]. The study of EGCGs as anticancer agents has increased in recent years, due to their in vitro and in vivo effects on tumor cell signaling pathways growth and apoptosis, including the suppression of COX-2, HIF-1a and VEGF gene expression [24—27]. In this study, EGCG suppressed cancer efficiently and showed enhanced anti-tumor growth activity in combination with PDT therapy. We confirmed that the tumor growth was significantly delayed in groups treated with Radachlorin/PDT and EGCG, compared to either the Radachlorin/PDT or EGCG alone groups. In vivo, PDT combination with EGCG led to a significant increase in the levels of both p21 and p53, at 6 days after PDT plus EGCG combination treatment. Recently, it has been reported that EGCG can directly lead to apoptosis, without requiring Bax (as is the case in response to agents that induce DNA damage) and can also induce p21-mediated growth arrest in HCT116 cells [28]. Interestingly, in our study, these changes disappeared 20 days after treatment and both Bax and activated PARP genes were significantly expressed. Also, we evaluated the roles of the molecules responsible for angiogenesis, such as VEGF, HIF-1a, and MMP9. The changes in the levels of proteins were not seen after the PDT combination with EGCG (data not shown). In vitro, we found significant decrease in the levels of COX-2 expression at 48 h post PDT plus EGCG combination treatment, but we did not observe similar trends in vivo (data not shown). These findings demonstrated a high anti-cancer activity of photodynamic therapy with the green tea constituent EGCG, on TC-1 tumor cell implanted mice. In this study, EGCG lowered the uptake of Radachlorin, a promising sensitizer for photodynamic therapy; this suggested that an EGCG supplement prior to photodynamic irradiation is not desirable, and that the supplement administered only after photodynamic irradiation enhances the effect. Therefore, we suggest that EGCG supplementation prior to photodynamic irradiation may decrease Radachlorin uptake. In conclusion, the present experiments showed that the combination of photodynamic therapy with the green tea constituent EGCG was very useful in cancer therapy by regulation of tumor cell signaling pathways, growth and induction of apoptosis, together with increased levels of p21 and p53 expression at the beginning of combined treatment, and other molecular changes in longer time. Therefore, it is strongly suggested that high anti-cancer activity of combined photodynamic therapy with green tea constituent EGCG may be useful for effective cancer therapy; additional studies are needed for further consideration of clinical applications.
Conflict of interest None declared.
References [1] Gomer CJ, Rucker N, Ferrario A, et al. Properties and applications of photodynamic therapy. Radiat Res 1989;20:1—18. [2] Corti L, Mazzarotto R, Belfontali S, et al. Photodynamic therapy in gynaecological neoplastic diseases. J Photochem Photobiol B 1996;36:193—7. [3] Ji HT, Chien LT, Lin YH, et al. 5-ALA mediated photodynamic therapy induces autophagic cell death via AMP-activated protein kinase. Mol Cancer 2010;9:91. [4] Moan J, Berg K. Photochemotherapy of cancer: experimental research. Photochem Photobiol 1992;55:931—48. [5] Liu T, Wu LY, Choi JK, et al. Targeted photodynamic therapy for prostate cancer: inducing apoptosis via activation of the caspase-8/-3 cascade pathway. Int J Oncol 2010;36:777—84. [6] Lim DS, Ko SH, Lee CH, et al. DH-I-180-3-mediated photodynamic therapy: biodistribution and tumor vascular damage. Photochem Photobiol 2006;82:600—5. [7] Lim DS, Bae SM, Kwak SY, et al. Adenovirus-mediated p53 treatment enhances photodynamic antitumor response. Hum Gene Ther 2006;17:347—52. [8] Ahn WS, Bae SM, Huh SW, et al. Necrosis-like death with plasma membrane damage against cervical cancer cells by photodynamic therapy. Int J Gynecol Cancer 2004;14:475—82. [9] Mataix J, Desco MC, Palacios E, et al. Photodynamic therapy for age-related macular degeneration treatment: epidemiological and clinical analysis of a long-term study. Ophthalmic Surg Lasers Imaging 2009;40:277—84. [10] Tsuchihashi T, Mori K, Peyman G, et al. Photodynamic effects on retinal oxygen saturation, blood flow, and electrophysiological function in patients with neovascular age-related macular degeneration. Retina 2009;29:1450—6. [11] Laukka MA, Wang KK, Bonner JA. Apoptosis occurs in lymphoma cells but not in hepatoma cells following ionizing radiation and photodynamic therapy. Dig Dis Sci 1994;39:2467—75. [12] Luo Y, Chang CK, Kessel D. Rapid initiation of apoptosis by photodynamic therapy. Photochem Photobiol 1996;63:528—34. [13] Gomer CJ, Ferrario A, Luna M, et al. Photodynamic therapy: combined modality approaches targeting the tumor microenvironment. Lasers Surg Med 2006;38:516—21. [14] Paschka AG, Butler R, Young CY. Induction of apoptosis in prostate cancer cell lines by the green tea component, (−)epigallocatechin-3-gallate. Cancer Lett 1998;130:1—7. [15] Suganuma M, Ohkura Y, Okabe S, et al. Combination cancer chemoprevention with green tea extract and sulindac shown in intestinal timor formation in Min mice. J Cancer Res Clin Oncol 2004;127:69—72. [16] Fujiki H, Suganuma M, Okabe S, et al. Cancer prevention with green tea and monitoring by a new biomarker, hnRNPB1. Mutat Res 2001;480—481:299—304. [17] Jin YM, Nam SL, Ahn WS. Growth inhibition and induction of apoptosis in cervical cancer cell line by green tea polyphenols. Kor J Obstet Gynecol 2002;45:560—8. [18] Ji BT, Chow WH, Hsing AW, et al. Green tea consumption and the risk of pancreatic and colorectal cancers. Int J Cancer 1997;70:255—8. [19] Ahmad N, Feyes DK, Nieminen AL, et al. Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. J Natl Cancer Inst 1997;89:1881—6. [20] Pronix S, Liederer BM, Blanchard J. Preformulation study of epigallo-catechin, a promising antioxidant for topical skin cancer prevention. J Pharm Sci 2002;92:111—6. [21] Ahn WS, Huh SW, Bae SM, et al. A major constituent of green tea, EGCG, inhibits the growth of human cervical cancer cell lines, CaSki cells, through apoptosis, G1 arrest, and regulation of gene expression. DNA Cell Biol 2003;22:217—24.
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
Epigallocatechin gallate with photodynamic therapy [22] Otsuka T, Ogo T, Eto T, et al. Growth inhibition of leukemic cells by (−)-apigallocatechin-3-gallate, the main constituent of green tea. Life Sci 1998;63:1397—403. [23] Yang GY, Liao J, Kim K, et al. Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenols. Carcinogenesis 1998;19:611—6. [24] Hussain T, Gupta S, Adhami VM, et al. Green tea constituent epigallocatechin-3-gallate selectively inhibits COX-2 without affecting COX-1 expression in human prostate carcinoma cells. Int J Cancer 2005;113:660—9. [25] Kundu JK, Na HK, Chun KS, et al. Inhibition of phorbolesterinduced COX-2 expression by epigallocatechin gallate in mouse skin and cultured human mammary epithelial cells. J Nutr 2003;133, 3805S—10S. [26] Tang FY, Nguyen N, Meydani M. Green tea catechins inhibit VEGF-induced angiogenesis in vitro through suppression of VEcadherin phosphorylation and inactivation of Akt molecule. Int J Cancer 2003;106:871—8. [27] Fassina G, Vene R, Morini M, et al. Mechanisms of inhibition of tumor angiogenesis and vascular tumor growth by epigallocatechin-3-gallate. Clin Cancer Res 2004;10:4865—73. [28] Thakur VS, Ruhul Amin AR, Paul RK, et al. p53-Dependent p21mediated growth arrest pre-empts and protects HCT116 cells
7
[29]
[30]
[31]
[32]
[33]
[34]
from PUMA-mediated apoptosis induced by EGCG. Cancer Lett 2010;296:225—32. Zhang T, Yang D, Fan Y, et al. Epigallocatechin-3-gallate enhances ischemia/reperfusion-induced apoptosis in human umbilical vein endothelial cells via AKT and MAPK pathways. Apoptosis 2009;14:1245—54. Ferrario A, Fisher AM, Rucker N, et al. Celecoxib and NS-398 enhance photodynamic therapy by increasing in vitro apoptosis and decreasing in vivo inflammatory and angiogenic factors. Cancer Res 2005;65:9473—8. Zhou Q, Olivo M, Lye KY, et al. Enhancing the therapeutic responsiveness of photodynamic therapy with the antiangiogenic agents SU5416 and SU6668 in murine nasopharyngeal carcinoma models. Cancer Chemother Pharmacol 2005;56:569—77. Zuluaga MF, Lange N. Combination of photodynamic therapy with anti-cancer agents. Curr Med Chem 2008;15: 1655—73. Moan J, Berg K. The photodegradation of porphyrins in cells can be used to estimate the life time if singlet oxygen. Photochem Photobiol 1991;53:549—53. Solban N, Rizvi I, Hasan T. Targeted photodynamic therapy. Lasers Surg Med 2006;38:522—31.
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
ARTICLE IN PRESS
Photodiagnosis and Photodynamic Therapy (2014) xxx, xxx—xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/pdpdt
Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro Seong Taek Mun MD, PhD a,∗, Dong Han Bae a, Woong Shick Ahn b a b
Dept. of Obstetrics and Gynecology, SoonChunHyang University, Republic of Korea Dept. of Obstetrics and Gynecology, Catholic University, Republic of Korea
KEYWORDS EGCG; Photodynamic therapy; Anti-tumor effect
Summary Objectives: To evaluate anti-tumor effects of combined photodynamic therapy and epigallocatechin-3-gallate (EGCG). Materials and methods: TC-1 cells were injected into C57BL/6 mice. Mice were grouped by 7 into 4 groups as PDT, EGCG, combined PDT with EGCG, and control group. The photosensitizer Radachlorin was used. The light source was a diode laser with 662 nm wavelength. In vitro TC-1 cells were treated with Radachlorin and irradiated. In vivo, when tumors were 8—10 mm, Radachlorin was injected into mice and irradiated. For in vitro, different doses of EGCG were added to culture dishes. For combination, EGCG was added to the cells. 2.5 or 5 g/ml of Radachlorin was added to the cells. Cells were incubated with EGCG and/or Radachlorin and laser irradiated. In vivo, EGCG were given for 20 days, alone or after PDT treatment. Cell growth inhibition was determined using MTT assay. Tumor growth inhibition assays were done in each group. Tumor growth was measured using caliper. Western blottings were performed with primary antibodies as COX-2, p21, p53, PARP, Bax, P-p38, VEGF, HIF-1␣, MMP9 and actin. Results: The cell growth and the tumor volume in PDT combined with EGCG treatment group was significantly suppressed, compared with control and PDT or EGCG alone treated groups. We have shown that PDT combined with EGCG in vivo increase levels of both p21 and p53. Both Bax and activated PARP genes were significantly expressed. Conclusions: It is suggested that high anti-cancer activity of combined photodynamic therapy with EGCG may be useful for effective cancer therapy. © 2014 Elsevier B.V. All rights reserved.
Introduction ∗ Corresponding author at: Dept. of OBGY, SoonChunHyang University CheonAn Hospital, BongMyung-dong, CheonAn-si, ChungNam 330-721, Republic of Korea. Tel.: +82 41 570 2150; fax: +82 41 571 7887. E-mail addresses: [email protected], [email protected] (S.T. Mun).
Photodynamic therapy (PDT) is a method of treating malignant tumors based on the photodynamic damage of tumor cells resulting in a photochemical reaction [1]. PDT has been successfully used in the treatment of a variety of cancers to induce apoptosis in tumor cells [2—8]. PDT causes
http://dx.doi.org/10.1016/j.pdpdt.2014.03.003 1572-1000/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
2
S.T. Mun et al.
the photochemical generation of cytotoxic reactive oxygen species, such as singlet oxygen, within the target tissue. PDT clinical trials using a photosensitizer, as well as a variety of second-generation photosensitizers, have shown promise in treating malignancies of the esophagus, bronchus, brain, peritoneal cavity, skin, bladder, head and neck, as well as in treating non-oncologic disorders, such as age-related macular degeneration [9,10]. Clinical results of PDT are positive; however, use of this technique requires further improvements, as tumor recurrences can occur [11—13]. The green tea polyphenols have been shown to have a protective effect on prostate cancer in pre-clinical animal models; it has been reported to be effective against several other cancer types, as well [14—18]. Green tea is composed of several catechins, including (−)-epigallocatechin-3-gallate (EGCG), epicatechin (EC), epicatechin-3-gallate (ECG) and epigallocatechin (EGC). Tea polyphenols have been shown to inhibit carcinogenesis in many animal models, and the significance of catechins, the main constituents of green tea, has been shown to be important for cancer prevention. Among them, (−)-epigallocatechin-3-gallate (EGCG), the major catechin found in green tea, has been recognized as a potential therapeutic agent [18—24]. Interest in the study of EGCGs, as anticancer agents, has increased in recent years, due to their both in vitro and in vivo effects on tumor cell signaling pathways regulating growth and apoptosis, including the suppression of COX-2, HIF-1a and VEGF gene expression [24—27]. More recently, it has been found that EGCG was able to enhance growth arrest and apoptosis of cancer cells through many pathways, including activation of p53, p21, inhibition of AKT, ERK1/2 and activation of JNK1/2 [28,29]. It has been shown that drug treatment combined with PDT therapy suppressed potentially harmful genes and enhanced PDT therapy [30—32]. In this study, we evaluated both in vitro and in vivo the effectiveness of the PDT plus EGCG on tumor regression. PDT combined with EGCG demonstrated significant suppression of tumor growth. Using Radachlorin, it was found that PDT exerts its anti-tumor activity through EGCG supplement after PDT, rather than by EGCG supplement prior to PDT.
Materials and methods Cell culture and tumor model TC-1 tumor cells were routinely propagated in complete RPMI 1640 medium (10% fetal bovine serum, 1% Lglutamine, and 1% penicillin/streptomycin), supplemented with 400 g/ml G418. TC-1 is an E7-expressing tumorigenic cell line. It was established from primary lung epithelial cells of C57BL/6 mice immortalized with HPV16 E6 and E7, and then transformed with an activated ras oncogene (1). Female 6-week old C57BL/6 mice were purchased from Daehan Biolink (Daejeon, Korea) and maintained in the specific pathogen free (SPF) animal facility at The Catholic University of Korea. Every 7 mice were divided into 4 groups. Total 28 mice were used. TC-1 cells were washed twice with PBS (phosphate buffered saline) and injected subcutaneously into the right flank of C57BL/6 mice (2 × 105 cells/mouse).
PDT The photosensitizer Radachlorin was purchased from RADAPHARMA group (RADA-PHARMA, Moscow, Russia) and was diluted in PBS buffer to make a 1 mg/ml stock solution. The light source was a diode laser with 662 ± 2 nm wavelength (Won-PDTD662, Won Technology, Daejon, Korea). PDT was carried out as previously described (2). Briefly, in vitro TC1 cells were added with Radachlorin (2.5 or 5 g/ml) for 12 h. Cells were washed with PBS buffer and then irradiated with 6.25 J/cm2 of light (200 mW, 5 min). In vivo, when tumors were approximately 8—10 mm in mean tumor diameter, Radachlorin (10 mg/kg) was injected into the tail vein of mice 3 h before irradiation. The tumor was irradiated at a fluence of 500 J/cm2 with 300 mW power.
EGCG EGCG was provided by Yukihiko Hara (Tea Solution, Hara Office Inc., Sumida-ku, Tokyo, Japan). EGCG was stored at −20 ◦ C in powder. EGCG was dissolved in sterilized PBS buffer before use. Fresh stocks were made each time. For in vitro experiments, different doses of EGCG (25, 50 and 100 M) were added to culture dishes. For combination, 50 M of EGCG was added to the cells. At the same time, 2.5 or 5 g/ml of Radachlorin was added to the cells. Cells were incubated with drugs (EGCG and/or Radachlorin) for 12 h under environment of 5% CO2 , 37 ◦ C. Incubated cell dishes were warmed at 20 ◦ C before use as with other experiments. Laser irradiated with 6.25 J/cm2 of light. In vivo, EGCG were given by gavages (400 mg/kg per day) for 20 days, alone or after PDT treatment.
MTT assay Cell growth inhibition was determined using the methyl thiazolyl tetrazolium (MTT) assay. MTT assay was performed every 24 h from irradiation time point until the 5th day. 20 l of MTT solution (5 mg/ml) was added to each well. Four hours later, DMSO (dimethyl sulfoxide) was added and then absorbance was measured at 570 nm in the automated spectrophotometric microtitre plate reader (Spectra Max 340, Molecular Devices, USA).
Tumor growth inhibition assay After TC-1 tumor cells had grown to a tumor size of 8—10 mm in mean diameter, the mice were randomly divided into four groups: the control group, the EGCG only group (400 mg/kg per day for 20 days), the PDT only group (10 mg/kg Radachlorin, 300 mW power, 500 J/cm2 dose) and the PDT + EGCG (400 mg/kg per day for 20 days) group. After mice were given treatment, tumor growth was measured twice or three times a week using caliper, and recorded as tumor volume (longest surface length [a], width [b] and height [c], tumor volume (mm3 ) = [a] × [b] × [c]).
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
Epigallocatechin gallate with photodynamic therapy
3
Figure 2 Tumor suppression by PDT, EGCG alone or PDT combination with EGCG in vivo. Mice were injected subcutaneously with 5 × 105 cells per mouse. When the mean tumor size reached 8—10 mm, the EGCG group was fed with 400 mg/kg EGCG for 20 days, whereas the PDT group was given 500 J single treatment; PDT plus EGCG group was treated with Radachlorin/PDT, then EGCG was administered for 20 days. Each group included seven animals (see ‘‘Materials and Methods’’ for details). Error bars indicate the SD. Statistically significant inhibition of tumor growth was evaluated by the Student’s t-test. *P < 0.05, compared with the control; # P < 0.05, compared with EGCG treated group; $ P < 0.05, compared with PDT treated group.
sample underwent electrophoresis for 2.5 h with SDS-PAGE at 10 mA, and Western blotting was performed with a Hybond-ECL membrane (Amersham, Uppsala, Sweden) at 100 V. The blotted membrane was blocked with 5% skimmed milk and reacted with primary antibodies (COX-2, p21, p53, PARP, Bax, P-p38, VEGF, HIF-1a, MMP9 and actin; Santa Cruz Biotechnology Inc., Santa Cruz, CA). After washing with Tris-buffered saline containing 0.1% Tween 20, the membrane was incubated with the horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Protein bands were visualized using the ECL Kit (Amersham, Arlington Heights, IL). Figure 1 In vitro cell growth inhibition by PDT, EGCG alone or PDT combination with EGCG. TC-1 cells (3 × 103) were treated with (A) EGCG, (B) Radachlorin/PDT or (C) PDT combination with EGCG. The control group represents cells only. Samples were assayed in triplicate and MTT assay was performed for five days. Vertical bars indicate SD (n = 3). Statistically significant inhibition of cell growth was measured by the Student’s t-test. *P < 0.05, **P < 0.01 compared with indicated group.
Statistical analysis Statistical analysis included ANOVA and the Student’s t-test. The values for the different groups were compared. P values of <0.05 were considered statistically significant.
Results Western blotting After tumor bearing mice were given treatment at the indicated time, the tumor tissues were harvested and lyzed for protein preparation. The protein was evaluated using the BioRad Protein-Assay kit (Bio-Rad, Hercules, CA), and adjusted for a final concentration of 2 mg/ml. After addition of 2-mercaptoethanol (2%), samples were boiled for 5 min and used for the experiments. 40 g of each protein
In vitro cell growth inhibition by EGCG, PDT alone or PDT combined with EGCG First, we tested the effects of EGCG on the TC-1 cell line. The cell growth inhibition effect was evaluated using MTT assays with increasing amounts of EGCG, and showed significant increase in cell growth inhibition over time (Fig. 1A). Next, we investigated the effects of the PDT on growth
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
4
S.T. Mun et al.
Figure 3 Pictures of mice on the 21st day by PDT, EGCG alone or PDT combination with EGCG in vivo. Every three mice belong to each group. (A) Control group (on the 21st day). (B) EGCG only (on the 21st day). (C) PDT only (on the 21st day). (D) PDT + EGCG (on the 21st day)
inhibition in the TC-1 cell line. A significant decrease in cell survival was detected with PDT, compared to the control group (Fig. 1B). The cell growth was also significantly suppressed in the EGCG plus PDT group (Fig. 1C).
Tumor suppression by PDT, EGCG alone or PDT combined with EGCG in vivo To further enhance the anti-cancer effect with PDT, we investigated the effects of the combination of PDT and EGCG in vivo. After PDT alone, the tumor volume was suppressed, compared with controls, as shown in Fig. 2. Next, the tumor volume in the PDT combined with EGCG treatment group was significantly suppressed, compared with not only control, but also the PDT or EGCG alone treated groups (Fig. 3). Additionally, we investigated the effects of EGCG supplement prior to PDT in vivo. As shown in Fig. 2, the tumor volume after the EGCG supplements were relatively suppressed, but not significantly compared to the PDT alone treatment. The results showed that EGCG lowered the uptake of Radachlorin; this suggested that an EGCG supplement prior to photodynamic irradiation is not desirable. Therefore, EGCG supplementation prior to photodynamic irradiation may play a role as an interference factor against uptake of Radachlorin.
Radachlorin uptake We tested in vivo whether intravenous Radachlorin injection plus EGCG supplement affected Radachlorin accumulation in serum or tumor. As Radachlorin was injected intravenously, it was natural that Radachlorin was highly accumulated in serum in the first time periods (Fig. 4A). We confirmed that Radachlorin was accumulated in sera, and then excreted in each Radachlorin and/or EGCG group after 48 h. For tumors, the highest accumulation was at 3 h after intravenous injection (Fig. 4B). In addition, there was a significant difference with the levels of Radachlorin in either treatment. This was consistent with the anti-tumor effect of PDT, as shown in Fig. 2. Under the same conditions, Radachlorin accumulation in tumors was lower in the combination group (EGCG plus Radachlorin), compared to Radachlorin alone. The results showed that EGCG supplement prior to Radachlorin injection could inhibit Radachlorin uptake in vivo. The time lag of Radachlorin tumor accumulation post injection is also very important for controlling the effect of PDT and/or EGCG combination.
Protein expression changes in treated tumor tissue In addition, we determined the molecules responsible for the enhanced anticancer effects following PDT combination with EGCG in vivo. We have shown that PDT combined
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
Epigallocatechin gallate with photodynamic therapy
5
Figure 5 Changes in tumor protein expression by combined PDT and EGCG treatment. Tumor bearing mice were injected intravenously with Radachlorin (10 mg/kg). Three hours after injection, the animals were anesthetized and the tumors were given external light treatment (100 J/cm2 ); mice in the EGCG treatment group were fed with 400 mg/kg for 20 days, whereas mice in the combination therapy group were fed with EGCG for 20 days after PDT were performed. At 6 and 20 days after PDT treatment, tumor tissues were collected and homogenized with lysis buffer. Tissue extracts (30 g of protein) were run and immunoblot analysis was performed with each specific antibody.
Figure 4 Radachlorin uptake in mice. Radachlorin uptake was expressed as g/g of wet tissue in the tumor, and expressed as g/ml of sera in vivo, as a function of the time after intravenous injection at a dose of 10 mg/kg to the C57BL/6 mice with TC-1 tumors. The bars represent the mean SD of three animals.
with EGCG in vivo led to significant increase in levels of both p21 and p53, at 6 days post PDT plus EGCG combination treatment (Fig. 5). These changes disappeared 20 days after treatment and both Bax and activated PARP genes were significantly expressed.
Discussion The main finding of this study was that the combination therapy approach using PDT and one of the green tea constituents, (−)-epigallocatechin-3-gallate (EGCG), was effective for reducing tumor growth. Moan and Berg [33] reported that photodynamic therapy (PDT) is a promising cancer treatment modality, based on
its selective killing of malignant cells by singlet oxygen 1 O2 , and other reactive products generated by photoactivated photosensitizer (PS) molecules that accumulate in tumor tissue. However, further development is needed to improve outcomes, as tumor recurrences occur, and tumor size and depth affect the efficiency of PDT irradiation. This maybe due to the increase in survival of molecules expressing COX2, HIF-1a and the VEGF gene. Photodynamic therapy (PDT) is a method of treating malignant tumors that produces photodynamic damage of tumor cells by photochemical reactions. At present, there are many chemicals used as photosensitizers of PDT for clinical trials [34]. We used Radachlorin, a promising sensitizer for photodynamic therapy. Radachlorin is a second generation photosensitizer, with promising physicochemical properties and high photodynamic efficiency. A tumor treated by PDT is reabsorbed and is gradually replaced by connective tissue [34]. The location of photodynamic damage of a tumor is determined by the photosensitizer’s ability to accumulate in tumor tissues and by the direction and localization of precise laser irradiation [34]. In our study, the influence of PDT time after Radachlorin injection on anti-cancer effect was investigated. We evaluated tumor suppression at 3, 6, 9, 12, and 15 days after PDT treatment. A significant difference was confirmed on day 15, and a tendency for the tumor to grow larger was observed. In contrast, in the control group, the tumor sizes increased almost linearly with time, until the end of the observation period. The results showed that tumor suppression was most effective at 3 h post Radachlorin injection (data not shown). As shown in Fig. 2, as Radachlorin was injected intravenously, it is natural that Radachlorin was highly accumulated in serum in the first time periods. For tumors, the
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
6
S.T. Mun et al.
highest accumulation was at 3 h after intravenous injection. Additionally, Radachlorin accumulation in tumors was lower in the combination group (EGCG plus Radachlorin), compared to Radachlorin alone. Therefore, we performed our PDT treatment at 3 h after Radachlorin injection in all experiments. EGCG, the major catechin found in green tea, has been recognized as a potential therapeutic agent, as previously described [22—24]. The study of EGCGs as anticancer agents has increased in recent years, due to their in vitro and in vivo effects on tumor cell signaling pathways growth and apoptosis, including the suppression of COX-2, HIF-1a and VEGF gene expression [24—27]. In this study, EGCG suppressed cancer efficiently and showed enhanced anti-tumor growth activity in combination with PDT therapy. We confirmed that the tumor growth was significantly delayed in groups treated with Radachlorin/PDT and EGCG, compared to either the Radachlorin/PDT or EGCG alone groups. In vivo, PDT combination with EGCG led to a significant increase in the levels of both p21 and p53, at 6 days after PDT plus EGCG combination treatment. Recently, it has been reported that EGCG can directly lead to apoptosis, without requiring Bax (as is the case in response to agents that induce DNA damage) and can also induce p21-mediated growth arrest in HCT116 cells [28]. Interestingly, in our study, these changes disappeared 20 days after treatment and both Bax and activated PARP genes were significantly expressed. Also, we evaluated the roles of the molecules responsible for angiogenesis, such as VEGF, HIF-1a, and MMP9. The changes in the levels of proteins were not seen after the PDT combination with EGCG (data not shown). In vitro, we found significant decrease in the levels of COX-2 expression at 48 h post PDT plus EGCG combination treatment, but we did not observe similar trends in vivo (data not shown). These findings demonstrated a high anti-cancer activity of photodynamic therapy with the green tea constituent EGCG, on TC-1 tumor cell implanted mice. In this study, EGCG lowered the uptake of Radachlorin, a promising sensitizer for photodynamic therapy; this suggested that an EGCG supplement prior to photodynamic irradiation is not desirable, and that the supplement administered only after photodynamic irradiation enhances the effect. Therefore, we suggest that EGCG supplementation prior to photodynamic irradiation may decrease Radachlorin uptake. In conclusion, the present experiments showed that the combination of photodynamic therapy with the green tea constituent EGCG was very useful in cancer therapy by regulation of tumor cell signaling pathways, growth and induction of apoptosis, together with increased levels of p21 and p53 expression at the beginning of combined treatment, and other molecular changes in longer time. Therefore, it is strongly suggested that high anti-cancer activity of combined photodynamic therapy with green tea constituent EGCG may be useful for effective cancer therapy; additional studies are needed for further consideration of clinical applications.
Conflict of interest None declared.
References [1] Gomer CJ, Rucker N, Ferrario A, et al. Properties and applications of photodynamic therapy. Radiat Res 1989;20:1—18. [2] Corti L, Mazzarotto R, Belfontali S, et al. Photodynamic therapy in gynaecological neoplastic diseases. J Photochem Photobiol B 1996;36:193—7. [3] Ji HT, Chien LT, Lin YH, et al. 5-ALA mediated photodynamic therapy induces autophagic cell death via AMP-activated protein kinase. Mol Cancer 2010;9:91. [4] Moan J, Berg K. Photochemotherapy of cancer: experimental research. Photochem Photobiol 1992;55:931—48. [5] Liu T, Wu LY, Choi JK, et al. Targeted photodynamic therapy for prostate cancer: inducing apoptosis via activation of the caspase-8/-3 cascade pathway. Int J Oncol 2010;36:777—84. [6] Lim DS, Ko SH, Lee CH, et al. DH-I-180-3-mediated photodynamic therapy: biodistribution and tumor vascular damage. Photochem Photobiol 2006;82:600—5. [7] Lim DS, Bae SM, Kwak SY, et al. Adenovirus-mediated p53 treatment enhances photodynamic antitumor response. Hum Gene Ther 2006;17:347—52. [8] Ahn WS, Bae SM, Huh SW, et al. Necrosis-like death with plasma membrane damage against cervical cancer cells by photodynamic therapy. Int J Gynecol Cancer 2004;14:475—82. [9] Mataix J, Desco MC, Palacios E, et al. Photodynamic therapy for age-related macular degeneration treatment: epidemiological and clinical analysis of a long-term study. Ophthalmic Surg Lasers Imaging 2009;40:277—84. [10] Tsuchihashi T, Mori K, Peyman G, et al. Photodynamic effects on retinal oxygen saturation, blood flow, and electrophysiological function in patients with neovascular age-related macular degeneration. Retina 2009;29:1450—6. [11] Laukka MA, Wang KK, Bonner JA. Apoptosis occurs in lymphoma cells but not in hepatoma cells following ionizing radiation and photodynamic therapy. Dig Dis Sci 1994;39:2467—75. [12] Luo Y, Chang CK, Kessel D. Rapid initiation of apoptosis by photodynamic therapy. Photochem Photobiol 1996;63:528—34. [13] Gomer CJ, Ferrario A, Luna M, et al. Photodynamic therapy: combined modality approaches targeting the tumor microenvironment. Lasers Surg Med 2006;38:516—21. [14] Paschka AG, Butler R, Young CY. Induction of apoptosis in prostate cancer cell lines by the green tea component, (−)epigallocatechin-3-gallate. Cancer Lett 1998;130:1—7. [15] Suganuma M, Ohkura Y, Okabe S, et al. Combination cancer chemoprevention with green tea extract and sulindac shown in intestinal timor formation in Min mice. J Cancer Res Clin Oncol 2004;127:69—72. [16] Fujiki H, Suganuma M, Okabe S, et al. Cancer prevention with green tea and monitoring by a new biomarker, hnRNPB1. Mutat Res 2001;480—481:299—304. [17] Jin YM, Nam SL, Ahn WS. Growth inhibition and induction of apoptosis in cervical cancer cell line by green tea polyphenols. Kor J Obstet Gynecol 2002;45:560—8. [18] Ji BT, Chow WH, Hsing AW, et al. Green tea consumption and the risk of pancreatic and colorectal cancers. Int J Cancer 1997;70:255—8. [19] Ahmad N, Feyes DK, Nieminen AL, et al. Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. J Natl Cancer Inst 1997;89:1881—6. [20] Pronix S, Liederer BM, Blanchard J. Preformulation study of epigallo-catechin, a promising antioxidant for topical skin cancer prevention. J Pharm Sci 2002;92:111—6. [21] Ahn WS, Huh SW, Bae SM, et al. A major constituent of green tea, EGCG, inhibits the growth of human cervical cancer cell lines, CaSki cells, through apoptosis, G1 arrest, and regulation of gene expression. DNA Cell Biol 2003;22:217—24.
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003
+Model PDPDT-545; No. of Pages 7
ARTICLE IN PRESS
Epigallocatechin gallate with photodynamic therapy [22] Otsuka T, Ogo T, Eto T, et al. Growth inhibition of leukemic cells by (−)-apigallocatechin-3-gallate, the main constituent of green tea. Life Sci 1998;63:1397—403. [23] Yang GY, Liao J, Kim K, et al. Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenols. Carcinogenesis 1998;19:611—6. [24] Hussain T, Gupta S, Adhami VM, et al. Green tea constituent epigallocatechin-3-gallate selectively inhibits COX-2 without affecting COX-1 expression in human prostate carcinoma cells. Int J Cancer 2005;113:660—9. [25] Kundu JK, Na HK, Chun KS, et al. Inhibition of phorbolesterinduced COX-2 expression by epigallocatechin gallate in mouse skin and cultured human mammary epithelial cells. J Nutr 2003;133, 3805S—10S. [26] Tang FY, Nguyen N, Meydani M. Green tea catechins inhibit VEGF-induced angiogenesis in vitro through suppression of VEcadherin phosphorylation and inactivation of Akt molecule. Int J Cancer 2003;106:871—8. [27] Fassina G, Vene R, Morini M, et al. Mechanisms of inhibition of tumor angiogenesis and vascular tumor growth by epigallocatechin-3-gallate. Clin Cancer Res 2004;10:4865—73. [28] Thakur VS, Ruhul Amin AR, Paul RK, et al. p53-Dependent p21mediated growth arrest pre-empts and protects HCT116 cells
7
[29]
[30]
[31]
[32]
[33]
[34]
from PUMA-mediated apoptosis induced by EGCG. Cancer Lett 2010;296:225—32. Zhang T, Yang D, Fan Y, et al. Epigallocatechin-3-gallate enhances ischemia/reperfusion-induced apoptosis in human umbilical vein endothelial cells via AKT and MAPK pathways. Apoptosis 2009;14:1245—54. Ferrario A, Fisher AM, Rucker N, et al. Celecoxib and NS-398 enhance photodynamic therapy by increasing in vitro apoptosis and decreasing in vivo inflammatory and angiogenic factors. Cancer Res 2005;65:9473—8. Zhou Q, Olivo M, Lye KY, et al. Enhancing the therapeutic responsiveness of photodynamic therapy with the antiangiogenic agents SU5416 and SU6668 in murine nasopharyngeal carcinoma models. Cancer Chemother Pharmacol 2005;56:569—77. Zuluaga MF, Lange N. Combination of photodynamic therapy with anti-cancer agents. Curr Med Chem 2008;15: 1655—73. Moan J, Berg K. The photodegradation of porphyrins in cells can be used to estimate the life time if singlet oxygen. Photochem Photobiol 1991;53:549—53. Solban N, Rizvi I, Hasan T. Targeted photodynamic therapy. Lasers Surg Med 2006;38:522—31.
Please cite this article in press as: Mun ST, et al. Epigallocatechin gallate with photodynamic therapy enhances anti-tumor effects in vivo and in vitro. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.03.003