International Journal of Radiation Biology, 2014; Early Online: 1–5 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.906765
Effect of photodynamic therapy with hypocrellin B on apoptosis, adhesion, and migration of cancer cells Yuan Jiang1,3, Albert Wingnang Leung2, Xinna Wang2, Hongwei Zhang2 & Chuanshan Xu2,4 1Department of Rehabilitation Medicine, First Affiliated Hospital of Chengdu Medical College, Sichuan, 2School of Chinese
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Medicine, The Chinese University of Hong Kong, Hong Kong, 3Department of Sonodynamic and Photodynamic Therapy, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, and 4Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China
used therapeutic modalities (Hogberg 2010, Hu et al. 2010, Eckstein 2011). Therefore, there is an urgent need to develop novel safe and effective therapeutic strategies. Photodynamic therapy (PDT) is an alternative strategy for managing malignancies via reactive oxygen species (ROS) produced by laser-activated photosensitizer (Dougherty et al. 1998). Hypocrellin B is an effective photosensitizer, which is isolated from a traditional Chinese herb Hypocrella bambuase (Ali et al. 2001, Yu et al. 2002, Xu et al. 2004, Wang P et al. 2010). Hypocrellin B-mediated photodynamic action has been confirmed to effectively kill tumor cells and pathogenic microbes (Hudson et al. 1994, Ali et al. 2001, Ma et al. 2003, Ma et al. 2004, Jiang et al. 2013). Emerging evidences have shown that hypocrellin B exhibits great physicochemical advantages over hematoporphyrin derivative (HpD), such as a single compound, definite chemical structure, low dark toxicity, high singlet oxygen quantum yield (Φ(1O2) ⫽ 0.76) and dual photochemical mechanisms of Type I and Type II (Yu et al. 2002, Liu et al. 2008, Zhao et al. 2010). Comparative study showed that hypocrellin B had stronger photodynamic killing effect on tumor cells than HpD (Zhou et al. 2004, Shang et al. 2005). However, the maximum absorption spectrum of hypocrellin B in visible wavelength range locates in the wavelength range of 460 ∼ 470 nm (Zhao et al. 2010). To effectively activate hypocrellin B, we have set up a novel blue light source from lightemitting diodes (LED) with the wavelength of 470 nm and our previous studies showed that blue light from LED source could activate hypocrellin B, causing cell death of cancer cells (Jiang et al. 2012a). In the present study we focused on investigating the effect of photodynamic therapy with hypocrellin B on apoptosis, adhesion, and migration of cancer cells in vitro.
Abstract Purpose: In the present study, we investigated effects of photodynamic therapy with hypocrellin B on apoptosis, adhesion, and migration of cancer cells in vitro. Materials and methods: Human ovarian cancer HO-8910 cell as a cancer model cell was incubated with hypocrellin B at a concentration of 2.5 mM for 5 h and irradiated by light from a light-emitting diodes (LED) source. Cell apoptosis was analyzed by flow cytometry with annexin V/propidium iodide (PI) staining and nuclear staining 6 h after hypocrellin B photoirradiation. Cell adhesion was assessed using the 3-(4, 5-dimthylthiazol-2-yl)-2, 5 diphenyl-tetrazolium bromide (MTT) assay 4 h after photodynamic treatment. Cell migration was measured 48 h after photodynamic treatment. Results: Flow cytometry with annexin V/PI staining showed that early apoptotic and late apoptotic (necrotic) rates following photodynamic therapy with hypocrellin B markedly increased to 16.40% and 24.67%, respectively. Nuclear staining found nuclear condensation and typical apoptotic body in the treated cells. The number of cell migration was significantly decreased to 183 ⴞ 28 after photodynamic therapy with hypocrellin B (p ⬍ 0.01). Light irradiation alone and hypocrellin B alone had no significant effect on cell migration. The cell adhesion inhibitory rate due to photodynamic action of hypocrellin B was 53.2 ⴞ 1.8%, significantly higher than 2.7 ⴞ 2.1% of light treatment alone and 1.0 ⴞ 0.4% of hypocrellin B treatment alone (p ⬍ 0.01). Conclusion: The findings demonstrated that photodynamic therapy with hypocrellin B remarkably induced apoptosis and inhibited adhesion and migration of cancer cells in vitro. Keywords: Hypocrellin B, photodynamic therapy, apoptosis, adhesion, migration, ovarian cancer
Introduction
Materials and methods
Cancer remains one of the most lethal diseases threatening the life and health of human beings. Serious side-effects and drug resistance limit clinical application of currently
Sensitizer Hypocrellin B was provided by the Institute of Chemistry, Chinese Academy of Sciences, China. A stock solution of
Correspondence: Dr. Chuanshan Xu, School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China. Tel: ⫹ 852 3943 3769. Fax: ⫹ 852 3943 1278. E-mail: [email protected] (Received 3 April 2013; revised 8 March 2014; accepted 13 March 2014)
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hypocrellin B (100 mM) was made in dimethyl sulfoxide (DMSO) (Sigma, St Louis, MO, USA) and kept in the dark at ⫺ 20°C until used. The working solution of hypocrellin B was diluted in the Roswell Park Memorial Institute (RPMI-1640) medium without 10% fetal calf serum (FCS).
Cell culture Ovarian cancer HO-8910 cells were stored at the Institute of Ultrasound and Medicine Engineering, Chongqing. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS, Gibco, Grand Island, NY, USA), 50 μg/ml penicillin, 50 μg/ml streptomycin and 10 μg/ml neomycin. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Int J Radiat Biol Downloaded from informahealthcare.com by Oakland University on 06/08/14 For personal use only.
Photodynamic treatment HO-8910 cells were incubated with hypocrellin B (2.5 μM) at 37°C for 5 h in the dark. Unbound hypocrellin B was washed away and the cells were exposed (except for the dark controls) to blue light emitted from LED system (Integrated LED multi-wavelength special light source, type II, Southwest University, Chongqing, China) with the wavelength of 470 ⫾ 10 nm and power density of 60 mW/cm2 distributing uniformly over an area of 78.5 cm2. All experiments were randomly divided into four groups: Photodynamic therapy with hypocrellin B, sham irradiation, hypocrellin B treatment alone and light irradiation alone. After light irradiation, the cells were incubated for analysis.
Apoptotic analysis After the photosensitization of hypocrellin B irradiated by blue light from LED with the wavelength of 470 nm and the energy density of 1 J/cm2, HO-8910 cells were incubated in culture flasks at 37°C for 6 h, and then treated using an annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime, Jiangsu, China) following our previous protocol (Wang P et al. 2010, Jiang et al. 2012b, Wang et al. 2012). Cell apoptosis was analyzed using flow cytometric method (FCM) (SE, Becton Dickinson, USA) according to the manufacture’s instruction; 10,000 cells were acquired and data processed by CellQuest software.
bated for 4 h, and then were washed using PBS buffer to remove the non-adhesive cells. After the washing, the MTT reagent was added to each well for staining the adhered cells. MTT reagent was removed after incubation of 4 h, and 150 μl DMSO was added into each well. After shaking for 10 min, the optical density (OD) was measured using an iEMS Analyzer (Lab-system, Type1401, Helsinki, Finland) at 570 nm. The inhibitory rate was calculated using the following equation: Inhibitory rate (%) ⫽ (OD control group ⫺ OD treatment group) / OD control group ⫻ 100%
Migration assay Cell migration after photodynamic therapy with hypocrellin B was assessed using the Boyden Transwell system (8.0 μm, Neuro Probe, Inc. Gaithersburg, USA). The bottom chamber was filled with RPMI 1640 medium enriched with 10% fetal calf serum (FCS). The medium in the top chamber contained serum-free RPMI1640. The cells (1 ⫻ 105) were seeded into the top chamber. The cells on the lower surface of the filter were fixed with 70% ethanol for 30 min after an incubation of 48 h, and stained with hematoxylin for 15 min, and counted under a light microscopy. Five fields were randomly selected, and the counts were averaged.
Statistical analysis The statistical analysis was conducted using SPSS 13.0 for Windows. Differences between groups were analyzed by ANOVA (analysis of variance). A p-value of less than 0.05 was considered as significant difference.
Results Apoptosis induction Flow cytometry showed that photodynamic therapy with hypocrellin B increased the early and late apoptotic
Nuclear staining After photodynamic therapy with hypocrellin B, HO-8910 cells (1 ⫻ 105 cells/well) were incubated in a 24-well microplate at 37°C for 6 h. Cells were stained with Hoechst 33258 (5 μg/ml) for 5 min at 37°C. The stained cells were washed twice with phosphate buffered saline (PBS) and immediately observed under a fluorescence microscope with an Excitation (ex) / emission (em) BP330-380/LP420 nm filter set with images recorded by a colorful charge-coupled device camera (Panasonic, WV-CL320, Japan).
Adhesion Cell adhesion was measured after photodynamic therapy with hypocrellin B using the MTT assay (Yu et al. 2007). Briefly, The treated or control cells (2 ⫻ 104) were implanted on Matrigel-precoated (100 μg/ml) 96-well plate and incu-
Figure 1. Apoptosis of HO-8910 cells was analyzed 6 h after photodynamic therapy with hypocrellin B (2.5 μM) irradiated by light from LED source with the wavelength of 470 nm and the energy density of 1 J/cm2. This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
Hypocrellin B-mediated PDT on cancer cells
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(necrotic) rates of HO-8910 cells. Figure 1 shows that the early apoptotic and late apoptotic (necrotic) rates after photodynamic therapy with hypocrellin B markedly increased to 16.40% and 24.67%, respectively. However, no remarkable change in early apoptotic and late apoptotic rates was found in cells treated by hypocrellin B alone or light irradiation alone (Supplementary Figure 1 to be found online at http://informahealthcare.com/abs/doi/10.3109/09553002. 2014.906765).
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Nuclear staining After photodynamic therapy, HO-8910 cells were stained using Hoechst 33258. The untreated HO-8910 cells showed with weak fluorescence. Figure 2 shows that nuclear condensation and typical apoptotic body with high fluorescence were observed in the treated cells 6 h after photodynamic therapy with hypocrellin B. However, no nuclear condensation and typical apoptotic body were observed in the cells treated by hypocrellin treatment alone or light irradiation alone (Supplementary Figure 2 to be found online at http://informahealthcare.com/abs/doi/10.3109/09553002.2014.906765).
Cell adhesion The inhibitory rate of photodynamic therapy with hypocrellin B on cell adhesion of HO-8910 cells is shown in Figure 3. The inhibitory rate of hypocrellin B treatment alone on cell adhesion was 1.0 ⫾ 0.4%, 2.7 ⫾ 2.1% after light treatment alone. The inhibitory rate of photodynamic therapy with hypocrellin B on cell adhesion was 53.2 ⫾ 1.8%. The inhibitory rate of photodynamic action of hypocrellin B on cell adhesion was significantly higher than that of light treatment alone and hypocrellin B treatment alone (p ⬍ 0.01), demonstrating that photodynamic therapy with hypocrellin B had significant inhibition on cell adhesion of HO-8910 cells.
Figure 2. Nuclear features in HO-8910 cells 6 h after photodynamic therapy with hypocrellin B (2.5 μM) irradiated by light from LED source with the wavelength of 470 nm and the energy density of 1 J/ cm2 (⫻ 400). This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
Figure 3. Cell adhesion of HO-8910 cells measured after photodynamic therapy with hypocrellin B (2.5 μM) irradiated by light from LED source with the wavelength of 470 nm and the energy density of 0.4 J/cm2. (n ⫽ 3).
Cell migration The treated or control cells were incubated for 48 h for measuring cell migration. The number of cell migration after sham treatment was 325 ⫾ 30, 320 ⫾ 23 after hypocrellin B treatment alone, and 322 ⫾ 27 after light treatment alone. The number of cell migration after photodynamic therapy with hypocrellin B was significantly decreased to 183 ⫾ 28 (p ⬍ 0.01), demonstrating that photodynamic therapy with hypocrellin B remarkably inhibited cell migration of HO-8910 cells (Figure 4).
Discussion Apoptosis is an active mode of cell death characterized by specific nuclear and cytoplasmic features (Jäckel et al. 1999, LaMuraglia et al. 2000). Apoptotic induction has become an
Figure 4. Cell migration of HO-8910 cells was measured after photodynamic therapy with hypocrellin B (2.5 μM) irradiated by light from LED source with the wavelength of 470 nm and the energy density of 0.4 J/cm2 (n ⫽ 3).
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important strategy in the management of malignant tumor (Varfolomeev and Vucic 2011). In our past study, we found microvillin disappearance and topical apoptotic body in the cells at 6 h after photodynamic treatment of hypocrellin B under transmission electron microscopy (TEM), indicating apoptosis might be an important mode of cell death induced by photodynamic therapy with hypocrellin B (Jiang et al. 2012a). To reinforce the observation, we used nuclear staining with Hoechst 33258 and flow cytometric method with annexin V-FITC and PI staining to investigate cell death of ovarian cancer HO-8910 cells at 6 h after photodynamic therapy with hypocrellin B. Nuclear staining observed nuclear condensation and typical apoptotic body in the cells treated by photodynamic action of hypocrellin B. Flow cytometry also showed that the apoptotic and late apoptotic (necrotic) rates markedly increased after photodynamic action of hypocrellin B. These findings demonstrated that photodynamic therapy with hypocrellin B increased apoptotic and late apoptotic (necrotic) rates of HO-8910 cells, and also underlined that apoptosis was an important cell death mode of ovarian cancer HO-8910 cells after photodynamic therapy with hypocrellin B. Metastasis, often involving cell adhesion, migration and invasion, is a major cause of therapeutic failure in the clinical management of malignancies (Hwang and Park 2010, Yang et al. 2010, Yodkeeree et al. 2010). Inhibiting cell adhesion, migration or invasion has become a promising strategy for preventing tumor metastasis (Hwang and Park 2010, Yodkeeree et al. 2010). Our previous study showed that hypocrellin B has significant photocytoxicity of cancer cells in a dose-dependent manner (Jiang et al. 2012a). To investigate the effect of photodynamic action of hypocrellin B on cell adhesion and migration of cancer cells, in our present study we used a lower energy-density LED light (0.4 J/cm2) to activate hypocrellin B in ovarian cancer cells. We found that the number of cell migration more significantly decreased after photodynamic therapy with hypocrellin B than that of sham treatment, hypocrellin B treatment alone, and light irradiation alone, demonstrating that photodynamic therapy with hypocrellin B had a remarkable inhibitory effect on cell migration of HO-8910 cells. Cell adhesion is an initial step of tumor cell metastasis (Wang L et al. 2010). Our cell adhesion assay showed that the inhibitory rate of photodynamic with hypocrellin B on cell adhesion was significantly higher than that of hypocrellin B treatment alone or light irradiation alone. These findings demonstrated that photodynamic therapy with hypocrellin B had significant inhibition on adhesion and migration of HO-8910 cells. However, tumor metastasis is a complex multi-step process involving many cellular and molecular events (Hwang and Park 2010, Wang et al. 2010, Yodkeeree et al. 2010). Therefore, the exact inhibitory mechanisms of photodynamic therapy with hypocrellin B on the adhesion and migration of HO-8910 cells needs to be determined in our future investigations. In summary, our findings demonstrated that photodynamic therapy with hypocrellin B had significant induction of cell apoptosis and decrease of cell adhesion and migration of HO-8910 cells, indicating that photodynamic therapy with hypocrellin B might not only induce cell death, but it
also inhibited cell adhesion and migration of cancer cells. However, the maximum absorption spectrum of hypocrellin B mainly locates in the blue light range (460 ∼ 470 nm) and in the red light range (580 ∼ 600 nm); one-photon photodynamic therapy with hypocrellin B was suggested for use in the treatment of mucosal cancer cells (Ali et al. 2001). The interesting fact is that the hypocrellin molecule with a large conjugated p-electron system has the capability of twophoton absorption. Liu’s study found hypocrellin B could be activated by the infrared light with the wavelength of 800 nm to effectively damage cancer cells, confirming that hypocrellin B might be a potential two-photon photodynamic agent (Liu et al. 2002). Two-photon photodynamic therapy can overcome the disadvantages of one-photon photodynamic therapy in clinically therapy on solid tumor. Thus, hypocrellin B might be a very promising agent for two-photon photodynamic therapy on mucosal cancer and solid tumor.
Acknowledgements This work was supported by the general research fund (GRF) from Hong Kong Research Grants Council (RGC) (476912), the Direct Grant from the Chinese University of Hong Kong (4053026), and Innovation and Technology Fund of Shenzhen (CXZZ20120619150627260). We express our sincere thanks to Professor Faqi Li, Mr Jianyong Wu, Mr Jingchuan Fan, and Mr Kejian Wang for their helpful assistance.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References Ali SM, Chee SK , Yuen GY, Olivo M. 2001. Hypericin and hypocrellin induced apoptosis in human mucosal carcinoma cells. J Photochem Photobiol B 65:59–73. Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J, Peng Q. 1998. Photodynamic therapy. J Natl Cancer Inst 90:889–905. Eckstein N. 2011. Platinum resistance in breast and ovarian cancer cell lines. J Exp Clin Cancer Res 30:91. Hogberg T. 2010. Chemotherapy: Current drugs still have potential in advanced ovarian cancer. Nat Rev Clin Oncol 7:191–193. Hudson JB, Zhou J, Chen J, Harris L, Yip L, Towers GH. 1994. Hypocrellin, from Hypocrella bambuase, is phototoxic to human immunodeficiency virus. Photochem Photobiol 60:253–255. Hu L, McArthur C, Jaffe RB. 2010. Ovarian cancer stem-like sidepopulation cells are tumourigenic and chemoresistant. Br J Cancer 102:1276–1283. Hwang ES, Park KK. 2010. Magnolol suppresses metastasis via inhibition of invasion, migration, and matrix metalloproteinase-2/-9 activities in PC-3 human prostate carcinoma cells. Biosci Biotechnol Biochem 74:961–967. Jäckel MC, Dorudian MA, Marx D, Brinck U, Schauer A, Steiner W. 1999. Spontaneous apoptosis in laryngeal squamous cell carcinoma is independent of bcl-2 and bax protein expression. Cancer 85:591–599. Jiang Y, Leung AWN, Xiang JY, Xu CS. 2012a. Blue light-activated hypocrellin B damages ovarian cancer cells. Laser Phys 22:300–305. Jiang Y, Xia X, Leung AW, Xiang J, Xu C. 2012b. Apoptosis of breast cancer cells induced by hypocrellin B under light-emitting diode irradiation. Photodiagnosis Photodyn Ther 9:337–343. Jiang Y, Leung AW, Wang X, Zhang H, Xu C. 2013. Inactivation of Staphylococcus aureus by photodynamic action of hypocrellin B. Photodiagnosis Photodyn Ther 10:600–606.
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Hypocrellin B-mediated PDT on cancer cells LaMuraglia GM, Schiereck J, Heckenkamp J, Nigri G, Waterman P, Leszczynski D, Kossodo S. 2000. Photodynamic therapy induces apoptosis in intimal hyperplastic arteries. Am J Pathol 157:867–875. Liu J, Zhao YW, Zhao JQ, Xia AD, Jiang LJ, Wu S, Ma L, Dong YQ, Gu YH. 2002. Two-photon excitation studies of hypocrellins for photodynamic therapy. J Photochem Photobiol B: Biol 68:156–164. Liu Y, Wang X, Zhang B. 2008. Hypocrellin-based photodynamic sensitizers. Progress in Chem 20:1345–1352. Ma G, Khan SI, Jacob MR, Tekwani BL , Li Z, Pasco DS, Walker LA , Khan IA . 2004. Antimicrobial and antileishmanial activities of hypocrellins A and B. Antimicrob Agents Chemother 48:4450–4452. Ma L, Ta Hi, Li C, Zhang Y, Wang ZH, Ji WZ. 2003. Photodynamic inhibitory effects of three perylenequinones on human colorectal carcinoma cell line and primate embryonic stem cell line. World J Gastroenterol 9:485–490. Shang L, Zhou N, Gu Y, Liu F, Zeng J. 2005. Comparative study on killing effect of esophageal cancer cell line between hypocrellin B-photodynamic therapy and hematoporphyrin derivative-photodynamic therapy. Chin J Cancer Prev Treat 12:1139–1142. Varfolomeev E, Vucic D. 2011. Inhibitor of apoptosis proteins: Fascinating biology leads to attractive tumor therapeutic targets. Future Oncol 7:633–648. Wang L, Ling Y, Chen Y, Li CL, Feng F, You QD, Lu N, Guo QL. 2010. Flavonoid baicalein suppresses adhesion migration and invasion of MDA-MB-231 human breast cancer cells. Cancer Lett 297:42–48. Wang P, Xu CS, Xu J, Wang X, Leung AW. 2010. Hypocrellin B enhances ultrasound-induced cell death of nasopharyngeal carcinoma cells. Ultrasound Med Biol 36:336–342.
Supplementary material available online Supplementary Figures 1 and 2.
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Wang X, Leung AW, Jiang Y, Yu H, Li X, Xu C. 2012. Hypocrellin B-mediated sonodynamic induces apoptosis of hepatocellular carcinoma cells. Ultrasonics 52:543–546. Xu S, Chen S, Zhang M, Shen T. 2004. Hypocrellin derivative with improvements of red absorption and active oxygen species generation. Bioorg Med Chem Lett 14:1499–1501. Yodkeeree S, Ampasavate C, Sung B, Aggarwal BB, Limtrakul P. 2010. Demethoxycurcumin suppresses migration and invasion of MDA-MB-231 human breast cancer cell line. Eur J Pharmacol 627:8–15. Yu C , Xu S, Chen S, Zhang M, Shen T. 2002. Investigation of photobleaching of hypocrellin B in non-polar organic solvent and in liposome suspension. J Photochem Photobiol B 68: 73–78. Yu J, Qian H, Li Y, Wang Y, Zhang X, Liang X, Fu M, Lin C. 2007. Arsenic trioxide (As2O3) reduces the invasive and metastatic properties of cervical cancer cells in vitro and in vivo. Gynecolog Oncol 106:400–406. Yang CM, Liu YZ, Liao JW, Hu ML. 2010. The in vitro and in vivo antimetastatic efficacy of oxythiamine and the possible mechanisms of action. Clin Exp Metastasis 27:341–349. Zhao J, Deng H, Xie J, Huang N, Gu Y. 2010. Hypocrellin photosensitizers and photodynamic therapy to microvascular diseases. Acta Biophysica Sinica 26:1005–1014. Zhou N, Shang L, Liu X. 2004. Comparative study on the killing effect between hypocrellin B-photodynamic therapy and hematoporphyrin derivative-photodynamic therapy on human lung cancer cells. Med J Chin PLA 29:1076–1078.
Effect of photodynamic therapy with hypocrellin B on apoptosis, adhesion, and migration of cancer cells Yuan Jiang1,3, Albert Wingnang Leung2, Xinna Wang2, Hongwei Zhang2 & Chuanshan Xu2,4 1Department of Rehabilitation Medicine, First Affiliated Hospital of Chengdu Medical College, Sichuan, 2School of Chinese
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Medicine, The Chinese University of Hong Kong, Hong Kong, 3Department of Sonodynamic and Photodynamic Therapy, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, and 4Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China
used therapeutic modalities (Hogberg 2010, Hu et al. 2010, Eckstein 2011). Therefore, there is an urgent need to develop novel safe and effective therapeutic strategies. Photodynamic therapy (PDT) is an alternative strategy for managing malignancies via reactive oxygen species (ROS) produced by laser-activated photosensitizer (Dougherty et al. 1998). Hypocrellin B is an effective photosensitizer, which is isolated from a traditional Chinese herb Hypocrella bambuase (Ali et al. 2001, Yu et al. 2002, Xu et al. 2004, Wang P et al. 2010). Hypocrellin B-mediated photodynamic action has been confirmed to effectively kill tumor cells and pathogenic microbes (Hudson et al. 1994, Ali et al. 2001, Ma et al. 2003, Ma et al. 2004, Jiang et al. 2013). Emerging evidences have shown that hypocrellin B exhibits great physicochemical advantages over hematoporphyrin derivative (HpD), such as a single compound, definite chemical structure, low dark toxicity, high singlet oxygen quantum yield (Φ(1O2) ⫽ 0.76) and dual photochemical mechanisms of Type I and Type II (Yu et al. 2002, Liu et al. 2008, Zhao et al. 2010). Comparative study showed that hypocrellin B had stronger photodynamic killing effect on tumor cells than HpD (Zhou et al. 2004, Shang et al. 2005). However, the maximum absorption spectrum of hypocrellin B in visible wavelength range locates in the wavelength range of 460 ∼ 470 nm (Zhao et al. 2010). To effectively activate hypocrellin B, we have set up a novel blue light source from lightemitting diodes (LED) with the wavelength of 470 nm and our previous studies showed that blue light from LED source could activate hypocrellin B, causing cell death of cancer cells (Jiang et al. 2012a). In the present study we focused on investigating the effect of photodynamic therapy with hypocrellin B on apoptosis, adhesion, and migration of cancer cells in vitro.
Abstract Purpose: In the present study, we investigated effects of photodynamic therapy with hypocrellin B on apoptosis, adhesion, and migration of cancer cells in vitro. Materials and methods: Human ovarian cancer HO-8910 cell as a cancer model cell was incubated with hypocrellin B at a concentration of 2.5 mM for 5 h and irradiated by light from a light-emitting diodes (LED) source. Cell apoptosis was analyzed by flow cytometry with annexin V/propidium iodide (PI) staining and nuclear staining 6 h after hypocrellin B photoirradiation. Cell adhesion was assessed using the 3-(4, 5-dimthylthiazol-2-yl)-2, 5 diphenyl-tetrazolium bromide (MTT) assay 4 h after photodynamic treatment. Cell migration was measured 48 h after photodynamic treatment. Results: Flow cytometry with annexin V/PI staining showed that early apoptotic and late apoptotic (necrotic) rates following photodynamic therapy with hypocrellin B markedly increased to 16.40% and 24.67%, respectively. Nuclear staining found nuclear condensation and typical apoptotic body in the treated cells. The number of cell migration was significantly decreased to 183 ⴞ 28 after photodynamic therapy with hypocrellin B (p ⬍ 0.01). Light irradiation alone and hypocrellin B alone had no significant effect on cell migration. The cell adhesion inhibitory rate due to photodynamic action of hypocrellin B was 53.2 ⴞ 1.8%, significantly higher than 2.7 ⴞ 2.1% of light treatment alone and 1.0 ⴞ 0.4% of hypocrellin B treatment alone (p ⬍ 0.01). Conclusion: The findings demonstrated that photodynamic therapy with hypocrellin B remarkably induced apoptosis and inhibited adhesion and migration of cancer cells in vitro. Keywords: Hypocrellin B, photodynamic therapy, apoptosis, adhesion, migration, ovarian cancer
Introduction
Materials and methods
Cancer remains one of the most lethal diseases threatening the life and health of human beings. Serious side-effects and drug resistance limit clinical application of currently
Sensitizer Hypocrellin B was provided by the Institute of Chemistry, Chinese Academy of Sciences, China. A stock solution of
Correspondence: Dr. Chuanshan Xu, School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China. Tel: ⫹ 852 3943 3769. Fax: ⫹ 852 3943 1278. E-mail: [email protected] (Received 3 April 2013; revised 8 March 2014; accepted 13 March 2014)
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hypocrellin B (100 mM) was made in dimethyl sulfoxide (DMSO) (Sigma, St Louis, MO, USA) and kept in the dark at ⫺ 20°C until used. The working solution of hypocrellin B was diluted in the Roswell Park Memorial Institute (RPMI-1640) medium without 10% fetal calf serum (FCS).
Cell culture Ovarian cancer HO-8910 cells were stored at the Institute of Ultrasound and Medicine Engineering, Chongqing. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS, Gibco, Grand Island, NY, USA), 50 μg/ml penicillin, 50 μg/ml streptomycin and 10 μg/ml neomycin. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Int J Radiat Biol Downloaded from informahealthcare.com by Oakland University on 06/08/14 For personal use only.
Photodynamic treatment HO-8910 cells were incubated with hypocrellin B (2.5 μM) at 37°C for 5 h in the dark. Unbound hypocrellin B was washed away and the cells were exposed (except for the dark controls) to blue light emitted from LED system (Integrated LED multi-wavelength special light source, type II, Southwest University, Chongqing, China) with the wavelength of 470 ⫾ 10 nm and power density of 60 mW/cm2 distributing uniformly over an area of 78.5 cm2. All experiments were randomly divided into four groups: Photodynamic therapy with hypocrellin B, sham irradiation, hypocrellin B treatment alone and light irradiation alone. After light irradiation, the cells were incubated for analysis.
Apoptotic analysis After the photosensitization of hypocrellin B irradiated by blue light from LED with the wavelength of 470 nm and the energy density of 1 J/cm2, HO-8910 cells were incubated in culture flasks at 37°C for 6 h, and then treated using an annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime, Jiangsu, China) following our previous protocol (Wang P et al. 2010, Jiang et al. 2012b, Wang et al. 2012). Cell apoptosis was analyzed using flow cytometric method (FCM) (SE, Becton Dickinson, USA) according to the manufacture’s instruction; 10,000 cells were acquired and data processed by CellQuest software.
bated for 4 h, and then were washed using PBS buffer to remove the non-adhesive cells. After the washing, the MTT reagent was added to each well for staining the adhered cells. MTT reagent was removed after incubation of 4 h, and 150 μl DMSO was added into each well. After shaking for 10 min, the optical density (OD) was measured using an iEMS Analyzer (Lab-system, Type1401, Helsinki, Finland) at 570 nm. The inhibitory rate was calculated using the following equation: Inhibitory rate (%) ⫽ (OD control group ⫺ OD treatment group) / OD control group ⫻ 100%
Migration assay Cell migration after photodynamic therapy with hypocrellin B was assessed using the Boyden Transwell system (8.0 μm, Neuro Probe, Inc. Gaithersburg, USA). The bottom chamber was filled with RPMI 1640 medium enriched with 10% fetal calf serum (FCS). The medium in the top chamber contained serum-free RPMI1640. The cells (1 ⫻ 105) were seeded into the top chamber. The cells on the lower surface of the filter were fixed with 70% ethanol for 30 min after an incubation of 48 h, and stained with hematoxylin for 15 min, and counted under a light microscopy. Five fields were randomly selected, and the counts were averaged.
Statistical analysis The statistical analysis was conducted using SPSS 13.0 for Windows. Differences between groups were analyzed by ANOVA (analysis of variance). A p-value of less than 0.05 was considered as significant difference.
Results Apoptosis induction Flow cytometry showed that photodynamic therapy with hypocrellin B increased the early and late apoptotic
Nuclear staining After photodynamic therapy with hypocrellin B, HO-8910 cells (1 ⫻ 105 cells/well) were incubated in a 24-well microplate at 37°C for 6 h. Cells were stained with Hoechst 33258 (5 μg/ml) for 5 min at 37°C. The stained cells were washed twice with phosphate buffered saline (PBS) and immediately observed under a fluorescence microscope with an Excitation (ex) / emission (em) BP330-380/LP420 nm filter set with images recorded by a colorful charge-coupled device camera (Panasonic, WV-CL320, Japan).
Adhesion Cell adhesion was measured after photodynamic therapy with hypocrellin B using the MTT assay (Yu et al. 2007). Briefly, The treated or control cells (2 ⫻ 104) were implanted on Matrigel-precoated (100 μg/ml) 96-well plate and incu-
Figure 1. Apoptosis of HO-8910 cells was analyzed 6 h after photodynamic therapy with hypocrellin B (2.5 μM) irradiated by light from LED source with the wavelength of 470 nm and the energy density of 1 J/cm2. This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
Hypocrellin B-mediated PDT on cancer cells
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(necrotic) rates of HO-8910 cells. Figure 1 shows that the early apoptotic and late apoptotic (necrotic) rates after photodynamic therapy with hypocrellin B markedly increased to 16.40% and 24.67%, respectively. However, no remarkable change in early apoptotic and late apoptotic rates was found in cells treated by hypocrellin B alone or light irradiation alone (Supplementary Figure 1 to be found online at http://informahealthcare.com/abs/doi/10.3109/09553002. 2014.906765).
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Nuclear staining After photodynamic therapy, HO-8910 cells were stained using Hoechst 33258. The untreated HO-8910 cells showed with weak fluorescence. Figure 2 shows that nuclear condensation and typical apoptotic body with high fluorescence were observed in the treated cells 6 h after photodynamic therapy with hypocrellin B. However, no nuclear condensation and typical apoptotic body were observed in the cells treated by hypocrellin treatment alone or light irradiation alone (Supplementary Figure 2 to be found online at http://informahealthcare.com/abs/doi/10.3109/09553002.2014.906765).
Cell adhesion The inhibitory rate of photodynamic therapy with hypocrellin B on cell adhesion of HO-8910 cells is shown in Figure 3. The inhibitory rate of hypocrellin B treatment alone on cell adhesion was 1.0 ⫾ 0.4%, 2.7 ⫾ 2.1% after light treatment alone. The inhibitory rate of photodynamic therapy with hypocrellin B on cell adhesion was 53.2 ⫾ 1.8%. The inhibitory rate of photodynamic action of hypocrellin B on cell adhesion was significantly higher than that of light treatment alone and hypocrellin B treatment alone (p ⬍ 0.01), demonstrating that photodynamic therapy with hypocrellin B had significant inhibition on cell adhesion of HO-8910 cells.
Figure 2. Nuclear features in HO-8910 cells 6 h after photodynamic therapy with hypocrellin B (2.5 μM) irradiated by light from LED source with the wavelength of 470 nm and the energy density of 1 J/ cm2 (⫻ 400). This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
Figure 3. Cell adhesion of HO-8910 cells measured after photodynamic therapy with hypocrellin B (2.5 μM) irradiated by light from LED source with the wavelength of 470 nm and the energy density of 0.4 J/cm2. (n ⫽ 3).
Cell migration The treated or control cells were incubated for 48 h for measuring cell migration. The number of cell migration after sham treatment was 325 ⫾ 30, 320 ⫾ 23 after hypocrellin B treatment alone, and 322 ⫾ 27 after light treatment alone. The number of cell migration after photodynamic therapy with hypocrellin B was significantly decreased to 183 ⫾ 28 (p ⬍ 0.01), demonstrating that photodynamic therapy with hypocrellin B remarkably inhibited cell migration of HO-8910 cells (Figure 4).
Discussion Apoptosis is an active mode of cell death characterized by specific nuclear and cytoplasmic features (Jäckel et al. 1999, LaMuraglia et al. 2000). Apoptotic induction has become an
Figure 4. Cell migration of HO-8910 cells was measured after photodynamic therapy with hypocrellin B (2.5 μM) irradiated by light from LED source with the wavelength of 470 nm and the energy density of 0.4 J/cm2 (n ⫽ 3).
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Y. Jiang et al.
important strategy in the management of malignant tumor (Varfolomeev and Vucic 2011). In our past study, we found microvillin disappearance and topical apoptotic body in the cells at 6 h after photodynamic treatment of hypocrellin B under transmission electron microscopy (TEM), indicating apoptosis might be an important mode of cell death induced by photodynamic therapy with hypocrellin B (Jiang et al. 2012a). To reinforce the observation, we used nuclear staining with Hoechst 33258 and flow cytometric method with annexin V-FITC and PI staining to investigate cell death of ovarian cancer HO-8910 cells at 6 h after photodynamic therapy with hypocrellin B. Nuclear staining observed nuclear condensation and typical apoptotic body in the cells treated by photodynamic action of hypocrellin B. Flow cytometry also showed that the apoptotic and late apoptotic (necrotic) rates markedly increased after photodynamic action of hypocrellin B. These findings demonstrated that photodynamic therapy with hypocrellin B increased apoptotic and late apoptotic (necrotic) rates of HO-8910 cells, and also underlined that apoptosis was an important cell death mode of ovarian cancer HO-8910 cells after photodynamic therapy with hypocrellin B. Metastasis, often involving cell adhesion, migration and invasion, is a major cause of therapeutic failure in the clinical management of malignancies (Hwang and Park 2010, Yang et al. 2010, Yodkeeree et al. 2010). Inhibiting cell adhesion, migration or invasion has become a promising strategy for preventing tumor metastasis (Hwang and Park 2010, Yodkeeree et al. 2010). Our previous study showed that hypocrellin B has significant photocytoxicity of cancer cells in a dose-dependent manner (Jiang et al. 2012a). To investigate the effect of photodynamic action of hypocrellin B on cell adhesion and migration of cancer cells, in our present study we used a lower energy-density LED light (0.4 J/cm2) to activate hypocrellin B in ovarian cancer cells. We found that the number of cell migration more significantly decreased after photodynamic therapy with hypocrellin B than that of sham treatment, hypocrellin B treatment alone, and light irradiation alone, demonstrating that photodynamic therapy with hypocrellin B had a remarkable inhibitory effect on cell migration of HO-8910 cells. Cell adhesion is an initial step of tumor cell metastasis (Wang L et al. 2010). Our cell adhesion assay showed that the inhibitory rate of photodynamic with hypocrellin B on cell adhesion was significantly higher than that of hypocrellin B treatment alone or light irradiation alone. These findings demonstrated that photodynamic therapy with hypocrellin B had significant inhibition on adhesion and migration of HO-8910 cells. However, tumor metastasis is a complex multi-step process involving many cellular and molecular events (Hwang and Park 2010, Wang et al. 2010, Yodkeeree et al. 2010). Therefore, the exact inhibitory mechanisms of photodynamic therapy with hypocrellin B on the adhesion and migration of HO-8910 cells needs to be determined in our future investigations. In summary, our findings demonstrated that photodynamic therapy with hypocrellin B had significant induction of cell apoptosis and decrease of cell adhesion and migration of HO-8910 cells, indicating that photodynamic therapy with hypocrellin B might not only induce cell death, but it
also inhibited cell adhesion and migration of cancer cells. However, the maximum absorption spectrum of hypocrellin B mainly locates in the blue light range (460 ∼ 470 nm) and in the red light range (580 ∼ 600 nm); one-photon photodynamic therapy with hypocrellin B was suggested for use in the treatment of mucosal cancer cells (Ali et al. 2001). The interesting fact is that the hypocrellin molecule with a large conjugated p-electron system has the capability of twophoton absorption. Liu’s study found hypocrellin B could be activated by the infrared light with the wavelength of 800 nm to effectively damage cancer cells, confirming that hypocrellin B might be a potential two-photon photodynamic agent (Liu et al. 2002). Two-photon photodynamic therapy can overcome the disadvantages of one-photon photodynamic therapy in clinically therapy on solid tumor. Thus, hypocrellin B might be a very promising agent for two-photon photodynamic therapy on mucosal cancer and solid tumor.
Acknowledgements This work was supported by the general research fund (GRF) from Hong Kong Research Grants Council (RGC) (476912), the Direct Grant from the Chinese University of Hong Kong (4053026), and Innovation and Technology Fund of Shenzhen (CXZZ20120619150627260). We express our sincere thanks to Professor Faqi Li, Mr Jianyong Wu, Mr Jingchuan Fan, and Mr Kejian Wang for their helpful assistance.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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