mediated photodynamic therapy on MCF-7 human breast cancer cell lines Combination treatment effects of doxorubicin and sulfonated zinc phthalocyanine mediated photodynamic therapy on MCF-7 human breast cancer cell lines Eric Chekwube Aniogo Blassan P. George and Heidi Abrahamse Laser Research Centre, Faculty of Health Sciences, University of Johannesburg P.O. Box 1701, Doornfontein Campus 2028, South Africa. ABSTRACT This study aimed to investigate cellular responses to treatment with a combination of Doxorubicin and sulfonated zinc phthalocyanine (ZnPcS) mediated phototherapy on breast cancer cells (MCF-7). Doxorubicin is a broad spectrum chemo-therapeutic antibiotic drug used to treat different human malignancies such as breast cancer. However, it has undesired and severe adverse effects in the form of alopecia, cardiotoxicity, hepatotoxicity and bone marrow degeneration etc. which limit its use in chemotherapy applications. Hence, to minimize these adverse side effects and still enhance its chemotherapeutic destructive abilities, current studies are examining its use in combination therapy approaches. Photodynamic therapy (PDT) is a photochemical process of inducing localized tissue damage through administration of a photosensitizer (PS) that is preferentially taken up by tumour cells, followed by light irradiation that initiates tumour damage through the formation of singlet oxygen. MCF-7 cell proliferation were inhibited by both treatments in a dose dependent manner but combined treatment with Doxorubicin and PDT were more effective in inhibiting cell growth than each of the two treatment alone. Cell viability assay was measured using a homogenous ATP quantitation method while qualitative changes in cellular morphology were observed using fluorescent microscopy. Findings from this study suggest that a low dose of Doxorubicin can effectively be combined with photodynamic therapy to enhance breast cancer treatment and minimise side effects. However, further studies to evaluate cell death mechanism as well as in vivo studies using animal models are required. METHODS BACKGROUND Breast cancer is one of the most dangerous form of cancer among women worldwide [1]. Doxorubicin is a chemotherapeutic agent used against breast cancer but its severe dose-limiting toxicity have hindered its use [2]. Photodynamic therapy (PDT) is a photochemical process of inducing localized tissue damage. It involves administration of a photosensitizer that is preferentially taken up by tumor cells, followed by light irradiation that initiates tumor necrosis/damage through formation of reactive oxygen species [3]. PDT with its localized effect can be used in combination with other therapies like conventional chemotherapy (Doxorubicin) to reach a desirable death proportion of malignant cells with lower doses of chemotherapeutic drug [4]. Thus minimizing Doxorubicin side effects and prevents its drug resistance. Cellular quantification: Trypan blue viability assay ATP proliferation assays DNA damage assessment: Fluorescent microscopy and live cell imaging using Hoechst stain MCF-7 Breast cancer cells, (ATCC HTB-22); 5x105 cells Morphological assessment: Inverted light microscopy Dose responses ZnPcSmix (PS): 0.125, 0.25, 0.5, 1 and 2 µM Doxorubicin: 0.3, 0.6, 0.9, 1.2 and 1.5 µM Group 1 Controls Group 2 PS + Light (PDT) Group 3 Doxorubicin treated Group 4 Dox + PS + Light (Combination treatment) Cellular responses after 24 h Fig 2. Methodology flow diagram Name and type Semiconductor (diode) Wavelength 681.5 nm Spectrum Red (visible) Wave emission Continuous Spot size 9.1 cm2 Power output 43 mW Power density 4.74 mW/cm2 Fluence 5 J/cm2 Irradiation time 17 Minutes 36 Seconds Fig 1. Mechanism of photodynamic therapy Fig 3. Laser parameters used Fig 4. 681.5 nm diode laser RESULTS Morphological assessment Cellular viability assessment DNA damage assessment using Hoechst stain p˂0.05* p˂0.01** p˂0.001*** A B C Fig 10. MCF-7 control cells were stained with Hoechst stain. The cells showed perfectly round nucleus without any damage Fig 11. MCF-7 cells treated with Doxorubicin alone showed irregular small nuclear shrinking and fragmentation Fig 5. MCF-7 control cells without showed no change in morphology Fig 6. MCF-7 cells treated with Doxorubicin showed some morphological changes as cells became rounded up and detached from the culture dishes p˂0.05* p˂0.01** p˂0.001*** D E F Fig 9. Cell viability was assessed by Trypan blue assay (A,B,C) and cell proliferation was assessed using ATP luminescence assay (D,E,F). Treatment with Doxorubicin alone showed a dose dependent decrease in cell viability (A) and likewise photodynamic treated cell viability (B). But approximately 45% cell viability was observed when treated with 0.3 µM concentration of Doxorubicin with IC50 of PDT treated cells. A progressive decrease in the cell proliferation was observed in Doxorubicin treated cells (D) and PDT treated cells (E) with lowest proliferation when both treatment was combined together (F). Fig 12. MCF-7 cells treated with photodynamic therapy showed nuclear swelling with nucleus larger than the normal Fig 13. MCF-7 cells treated with combination of Doxorubicin and photodynamic therapy showed nuclear destruction as nucleus gradually shrinks and become fragmented hence, could not retain the Hoechst stain Fig 7. MCF-7 cells treated with photodynamic therapy showed crenated irregular cellular structure and gradually rounding up. Fig 8. Breast cancer cells treated with combination therapy of Doxorubicin and photodynamic therapy showed floating rounded detached dead cells. CONCLUSION Combination of Doxorubicin with zinc phthalocyanine mediated photodynamic therapy decreases the viability and proliferation of MCF-7 breast cancer cells. Changes in cell morphology and DNA damage assessment using fluorescence microscopy supports the efficacy of combination treatment. Hence, this study suggests that a low dose of Doxorubicin can effectively be used in combination with PDT to enhance its treatment efficacy, thus minimizing dose-limiting Doxorubicin side effects. References [1] Tobias J, Hochhanser D. Breast cancer in cancer and its management. 7th ed. United Kingdom (UK): John Wiley and Sons; 2015. 237-274. [2] Mitry AM, Edwards GJ. Doxorubicin induced heart failure: Phenotype and molecular mechanisms. Int J Cardiol Heart Vasc. 2016; 10:17-24. [3] Tynga IM, Abrahamse H. Cell death pathways and phthalocyanine as an efficient agent for photodynamic cancer therapy. Int J Mol Sci. 2015; 16: 10228-10241. [4] Diez B, Ernst G, Teijo MJ, et al. Combined chemotherapy and ALA-based photodynamic therapy in leukemic murine cells. Leuk Res. 2012; 36: 87-108. CONTACT DETAILS Heidi Abrahamse (Prof) Director: Laser Research Centre NRF SARChI Chair: Laser Applications in Health Faculty of Health Sciences University of Johannesburg Tel. (+27) 11 559 6550 Email: habrahamse@uj.ac.za ACKNOWLEDGMENTS C