Global DNA methylation status of colorectal cancer cells exposed to photodynamic therapy L. Vorster, N. Houreld, H. Abrahamse Laser Research Centre Faculty of Health Sciences University of Johannesburg

Photodynamic Therapy (PDT) is the use of Low Intensity Laser Irradiation (LILI) in conjunction with a photosensitizer (PS) to initiate cell death (apoptosis) The PS, when activated, by LILI, forms singlet state oxygen molecules. Oxygen molecules in an excited state are called reactive oxygen species (ROS) ROS reacts with surrounding cellular structures causing cell death PDT is used to treat cancer due to the PS’s high affinity for cancer cells Photodynamic Therapy Robertson C A, Hawkins Evans D, Abrahamse H (2009) Photodynamic therapy (PDT): a short review on cellular mechanisms and cancer research applications.Journal of Photochemistry and Photobiology B: Biology 96: 1-8

DNA Methylation It is a chemical change to the DNA without changing the sequence A methyl group (CH 3 ) is covalently added to the 5’ position of a cytosine that is part of a Cytosine-p-Guanine di-nucleotide (CpG) DNA methylation is an epigenetic regulator and contributes to the tertiary structure of DNA

CpG rich areas in the genome are called CpG islands These CpG islands are mostly found in promotors of genes Hypomethylation (under-methylated) usually leads to over expression of a gene Hypermethylation (over-methylated) usually leads to silencing of a gene DNA Methylation

DNA methylation is associated with Cancer due to the silencing and over expression of genes caused by a mutation 5’-aza-decibine is a hypomethylating agent Recent studies have shown that 5-aza-dc upregulates the tumour rejection antigen P1A By opening up the chromatin structure, more ROS can penetrate, thus leading to more oxidative damage The use of PDT in conjunction with a demethylating agent can have a beneficial effect DNA Methylation, Cancer and PDT 5-aza-dC * Mroz P, Hamblin M (2009) Combination of PDT and a DNA demethylating agent produces anti-tumour immune response in a mouse tumour model. Proc SPIE 7380: 73800H-1 – 73800H-9

Problem Statement The treatments used for cancer are chemotherapy and radiationtherapy Side effects are : Anemia, nausea, memory changes, constipation, appetite changes, bleeding problems and diarrhea to name but a few Recent studies have shown that 5-aza-dC works well with cisplatin (a chemotherapeutic drug) Previous studies done on PDT by the Laser Research Centre have shown that metallophthalocyanine (MPc) are highly effective PSs Zincphthalocyanine (ZnPc) has shown to be an effective PS with cancer cell lines colorectal (CaCo 2), osophageal (SNO), breast (MCF-7), lung (A549) and skin (A375) * Manoto S, Sekhejane P, Houreld N, Abrahamse H. Localization and phototoxic effect of zinc sulfophthalocyanine photosensitizer in human colon (DLD-1) and lung (A549) carcinoma cells. (2011) Photodiagnosis and Photodynamic therapy in press. * Patties I, et al. (2009) Additive Effects of 5-Aza-2'-deoxycytidine and Irradiation on Clonogenic Survival of Human Medulloblastoma Cell Lines.Strahlentherapie und Onkologie 185:331–338 * Robertson C A, Abrahamse H (2010) The In Vitro PDT Efficacy of a Novel Metallophthalocyanine (MPc) Derivative and Established 5-ALA Photosensitizing Dyes Against Human Metastatic Melanoma Cells. Lasers in Surgery and Medicine 42:766–776

Methodology Colorectal: CaCo-2 IRRADIATION AND PHOTOSENSITIZER 5 J/cm 2 at 680 nm ZnPC Assays at 3 h & 24 h post LILI GROUP 1: Control Cancer cells only GROUP 5: Cancer cells + 5aza-dC + PS + LI GROUP 4: Cancer cells + 5-aza-dC GROUP 3: Cancer cells + PS + LI GROUP 2: Cancer cells + LI Add PS, Incubate, Irradiate, Incubate Conduct assays Cell Viability and Proliferation: Trypan Blue and alamarBlue Global Methylation Status: ELISA Modified Comet Assay RT-PCR DNA Configuration: Agarose gel electrophoresis Live fluorescent imaging Hoechst 3328 stain

Results: Morphology

3 hour incubation n = 4 * = P < 0.05 ** = P < 0.01 *** = P < Results: Viability (AlamarBlue)

24 hour incubation n = 4 * = P < 0.05 ** = P < 0.01 *** = P < 0.001

Results: DNA Damage (Hoechst)

Conclusion: The global DNA methylation status of cells does have an effect on PDT efficiency Demethylation prior to PDT treatment enhances cancer cell death induction Future work: Investigate effect of combination therapy - demethylation (5’-aza-dc) and PDT on DNA integrity Determine demethylation + PDT on variety of different cancer types and using different PSs

ACKNOWLEDGEMENTS