1. DNA-dependent protein kinase modulates the anti-cancer properties of silver nanoparticles in human cancer cells
Authors: Hui Kheng Lima, Resham Lal Gurunga, M. Prakash Handea
Journal: Mutation Research - Genetic Toxicology and Environmental Mutagenesis
Camaryn Hess
29 November 2017

2. Mutation Research - Genetic Toxicology and Environmental Mutagenesis
Publishes papers advancing knowledge in the field of genetic toxicology.
Areas of research include, but are not limited to:
•New developments in genotoxicity testing of chemical agents
The isolation and chemical characterization of novel environmental mutagens.
Nano-genotoxicology, the study of genotoxicity hazards and risks related to novel man-made nanomaterials.
Authors of this paper all appear to be students in Singapore and India

3. Introduction to DNA-dependent protein kinase modulates the anti-cancer properties of silver nanoparticles in human cancer cells
Silver nanoparticles (Ag-np) are toxic to eukaryotic cells.
Effects of Ag-np can be advantageous in experimental therapeutics.
This study demonstrates that Ag-np treatment triggers the activation of DNA- PKcs and JNK pathway at selected doses, as a physiologic response to DNA damage and repair in normal and malignant cells.
1) Sliver nanoparticles are cytotoxic and genotoxic to cells and they may induce cellular apoptosis.
2) They are currently being used to enhance the therapeutic effects of cancer drugs because they can induce cancer cell killing similar to other chemotherapeutics.
JNKs have been reported to regulate DNA repair following genotoxic stress
DNA-PKcs facilitates recruitment of downstream DNA repair factors to mediate repair

4. … what?
DNA-PKCs, or DNA-dependent protein kinase, are essential for the DNA repair and telomere capping.
AKA Telomeres protect chromosome ends by interacting with telomerase
JNKs regulate DNA repair following genotoxic stress.
Therefore, activation of these mechanisms would results in DNA repair and chromosome maintenance.
If Ag-np activate these mechanisms, then DNA repair is activated.
They repair double-strand breaks via non-homologous end joining repair. Cells lacking DNA-PKcs are shown to be highly sensitive to DNA-damaging agents and display impaired DNA repair capacity.
.. Which is a process we learned about in class
via the shelterin complex which is made up of telomere specific proteins that mask the linear ends of telomeres.

5. Methods and Materials GOAL: investigate the role of DNA-PKcs and JNK in the anticancer activity of Ag-np on telomere homeostasis and DNA repair in human brain and breast cancer cells.
Materials
Synthesis of polyvinyl alcohol (PVA) capped Ag-np
Determine cell types and cell culture
normal human fibroblasts
human breast adenocarcinoma cells
human glioblastoma cells
Drug treatments
DNA-PKcs inhibitor
Ag-np (varying doses)
Methods
Trypan blue exclusion assay
Scratch wound healing cell migration assay
Western blot analysis
Cell viability assay
Alkaline single-cell gel electrophoresis (comet assay)
Terminal restriction fragment length analysis
Immunofluorescence staining
The main role of the PVA capping agent is to stabilize the silver nanoparticle
5) To determine the number of viable cells (unstained) vs the nonviable cells (stained blue).
6) To measure the speed and efficiency with which cells in a monolayer migrate to close a scratch wound.
7) Which we have learned about in class, but as a refresher is used to detect protein
8) A viability assay is an assay to determine the ability of organs, cells or tissues to maintain or recover viability. Viability can be distinguished from the all-or-nothing states of life and death by use of a quantifiable index between 0 and 1 (or 0% and 100%).
9) To detect Ag-np induces DNA damage
10) Whole genomic DNA except the subtelomeric and telomeric regions are digested by the restriction enzymes in this analysis allowing the researchers to determine telomere length

6. Results
Ag-np alter morphology and impair migration of human cancer cells
Ag-np treated cells displayed clustering with cellular extensions and showed restricted cell-spreading (A)
Floating and rounded cells were observed upon Ag-np treatment (A)
Cell migration in all the Ag-np treated human cancer cells were inhibited (B)
Exposure to Ag-np for 48 h resulted in altered cell shape in cancer cells compared to their respective controls
a) This could be due to disturbances in cytoskeletal functions
b) indicative of dying cells, in the human breast and brain cancer cells. In contrast, there were minimal changes in the morphology of the normal human lung fibroblasts after the Ag-np treatment
c) Determined by the scratch wound-healing assay – closure of the wound was delayed in the AG-np treated cells
B - These are images of the cell monolayer
Key:
MDA-MB-231 = Breast cancer cells
U251 = Gliobastoma cells
IMR-90 = Normal fibroblasts

7. Results (cont.)
Ag-np induce cell death and reduce proliferative potential in human brain and breast cancer cells
Ag-np induced apoptotic cell death in cancer cells (C)
Reduced proliferative potential only in human cancer cells upon Ag-np treatment (D)
cell cycle analysis revealed that.. normal human cells displayed minimal effects.
significant increase in sub-G1 population in MDA-MB-231 cells while minimal effect was observed in IMR-90 cells
trypan blue exclusion assay showed significantly
The 48 h-treatment with Ag-np decreased the viable proliferative breast cancer cells by 57% and glioblastoma cells by 42%
Key:
MDA-MB-231 = Breast cancer cells
U251 = Gliobastoma cells
IMR-90 = Normal fibroblasts

8. Results (cont.)
Inhibition of DNA-PKcs increases cell death in Ag-np treated cells
Treatment with Ag-np resulted in 74.8% reduction in survival in MDA-MB-231 and U251 and cells as compared to their untreated control (F).
Cancer cells pre-treated with NU7026, however, showed further reduction in cell viability (F).
This validated the importance of DNA-PKcs in governing the survival of Ag-np treated cancer cells as well as demonstrated the effects of Ag-np treatment
Key:
MDA-MB-231 = Breast cancer cells
U251 = Gliobastoma cells
IMR-90 = Normal fibroblasts
NU7026 = DNA-PKcs inhibitor

9. Results (cont.)
Ag-np treatment produces DNA damage in human brain and breast cancer cells
IMR-90 cells appear to be less susceptible to Ag-np as indicated by a minimal increase in DNA damage (A).
There was 4–8 fold increase in DNA damage in cancer cells following treatment with Ag-np.
Comet assay was used to measure DNA damage - Nuclei with undamaged DNA appear round; nuclei with damaged DNA result in DNA fragments that migrate faster during gel electrophoresis and give rise to a ‘tail’. The tail moment (product of tail length and fraction of DNA) was used as a measure of DNA damage.
Key:
MDA-MB-231 = Breast cancer cells
U251 = Gliobastoma cells
IMR-90 = Normal fibroblasts

10. Results (cont.)
Ag-np cause telomere dysfunction in human brain cancer cells and breast cancer cells
Telomere length was decreased by 19% in breast cancer cells (B)
Followed by 12% reduction in glioblastoma cells (B)
No detectable change was observed in normal cells (B)
monitored changes in telomere length following Ag-np treatment using terminal restriction fragment analysis to determine whether Ag-np-induced genotoxicity influences telomere dynamics
Key:
MDA-MB-231 = Breast cancer cells
U251 = Gliobastoma cells
IMR-90 = Normal fibroblasts

11. Results (cont. – last one I promise!)
Ag-np exposure activates DNA-PKcs and JNK pathway in human brain and breast cancer cells
There was a small increase in p-DNAPKcs level Ag-np treatment in normal cells (C).
All human cancer cell types showed significant activation of DNAPKcs (C).
Increase in p-JNK1 level was observed in cancer cells after being exposed to Ag-np (D).
In contrast, minimal activation of JNK pathway seen in normal cells (decrease in p-JNK1 level) (D).
Western Blot Analysis
Examined the activation status of DNA-PKcs and JNK in cancer and normal cells after Ag-np treatment.
To test for the activation of JNK pathway following Ag-np treatment, the protein expression of main components of JNK pathway were analyzed (p-JNK1)
These are images of the proteins bands that were visualized on X-ray film.
Key:
MDA-MB-231 = Breast cancer cells
U251 = Gliobastoma cells
IMR-90 = Normal fibroblasts
P-JNK1 = JNK pathway protein

12. Discussion KEY FINDINGS
Ag-np exerts toxicity in human breast cancer cells and glioblastoma cells but has little effect on normal human cells.
Activation of DNA-PKcs mediates repair of Ag-np induced DNA damage in human cancer cells by NHEJ and JNK1 pathways.
DNA-PKcs activity is essential in the repair of Ag-np mediated DNA damage
Pharmacological inhibition of DNA-PKcs in cancer cells potentially enhanced the anticancer effect of Ag-np.