Download Global DNA methylation status of colorectal cancer cells exposed to

Global DNA methylation status of
colorectal cancer cells exposed
to photodynamic therapy
Photodynamic Therapy
• 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
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 (CH3) 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
DNA Methylation
• 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, Cancer and PDT
• 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
* 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
5-aza-dC
Problem Statement
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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
IRRADIATION AND
PHOTOSENSITIZER
5 J/cm2 at 680 nm
ZnPC
Assays at
3 h & 24 h post LILI
Colorectal: CaCo-2
GROUP 1:
Control
Cancer
cells only
GROUP 2:
Cancer cells + LI
GROUP 3:
Cancer cells + PS + LI
GROUP 4:
Cancer cells + 5-aza-dC
GROUP 5:
Cancer cells + 5aza-dC
+ PS + LI
Add PS, Incubate, Irradiate,
Incubate
Conduct assays
Cell Viability and
Proliferation:
Trypan Blue and alamarBlue
DNA Configuration:
Agarose gel electrophoresis
Live fluorescent imaging
Hoechst 3328 stain
Global Methylation Status:
ELISA
Modified Comet Assay
RT-PCR
Results: Morphology
Results: Viability (AlamarBlue)
3 hour incubation
n=4
* = P < 0.05
** = P < 0.01
*** = P < 0.001
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