Group Members: Foo Chuan Hui Joshua (4s2-05) Wong Tuck Wing Ryan (4s2-31)

1. Elucidating Acute-phase cancer responsive proteins from Horseshoe crabs (Carsinocorpiusrotundicauda) Group Members: Foo Chuan Hui Joshua (4s2-05) Wong Tuck Wing Ryan (4s2-31) AnuVenkatachalam (AOS) Estelle Gong (AOS)

2. Carcinoscorpiusrotundicauda

3. Background • “Cancer” refers to a class of diseases with no single cure • Current methods demonstrate variable effectiveness • May cause harm to other body parts

4. Rationale • Survived two mass extinction events over the past 400 million years • Have been known to benefit cancer research

5. Rationale • Limulus AmebocyteLysate (LAL) • Detects endotoxins, forms clot • Innate immune system • Rich network of proteins • Respond to a variety of Pathogen-associated molecular patterns (PAMPs)

6. Rationale • Infection studies on the Singapore horseshoe crab, demonstrated that 106cfu of Pseudomonas aeruginosa was rapidly suppressed • Lethal to mice • Horseshoe crabs completely cleared the infection within 3 days

7. Rationale • Proteins found in the blood of horseshoe crabs potentially provides a more effective way of treating cancer • No damage and irradiation to adjacent cells • Chemotherapy – toxicity • Radiation therapy – damage from radiation

8. Purpose • Elucidate specific proteins in Horseshoe crab blood that recognize and bind surface antigens or PAMPs of cancer cells • To propose potential peptide-based drugs for cancer detection & treatment.

9. Hypothesis • Proteins present in horseshoe crab blood recognize and bind to PAMPs of cancer cells.

10. Variables

11. Materials • Micropipettes • Centrifuge • 15mL centrifuge tubes • 70% ethanol • Autoclave • Refrigerator • Horseshoe crab blood • Human colorectal cancer cell lysate • Hydrophobic column • SDS-PAGE • Sodium DodecylSulphatePolyscrylamide Gel • Buffer solutions • Urea solution

12. Methodology

13. Method – Collection of blood • Horseshoe crabs were collected from the estuary of the Kranji River

14. Method • Washed to remove mud and debris • Acclimatized • Stress might affect composition of blood • Washed the carapace around the vicinity of the cardiac chamber with water and swabbed with 70 % ethanol • Removes bacteria • Prevent clotting of blood

15. Method • The crabs partially bled by inserting a sterile needle (18 gauge; Becton Dickinson™), puncturing the cardiac chamber • Pressure differences caused blood to be ejected • About 10 mL collected for each crab

16. Method Prosoma Opisthosoma

17. Method Needle inserted at hinge

18. Method • Hemolymph was collected into pre-chilled, pyrogen-free centrifuge tubes • Clarified from hemocytes • Centrifugation at 150 xg for 15 min at 4 ºC • Cell debris, contaminants and excess hemocyanin were removed • Further centrifugation at 9,000 xg for 10 min at 4°C • The hemolymph was then quick-frozen in liquid nitrogen and stored at -80 °C. 

19. Method – Hydrophobic Column • Hemolymph will be passed through an hydrophobic column pre-loaded with the membrane extract of human colorectal cancer cell membranes. • Proteins that recognise PAMPs associated with these cancer cells will bind to the column. • These proteins will be eluted with increasing concentrations of urea solution.

21. Method – Separation of proteins • Collected proteins will be analysed by Sodium DodecylSulphatePolyscrylamide Gel Electrophoresis (SDS-PAGE). • Proteins from the SDS-PAGE profile will then be extracted and digested by trypsin.

22. Method • SDS-PAGE • An electric field is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the anode • Proteins are separated according to electrophoretic mobility • Molecular mass

23. Method – Identification via mass spectrometry • Lastly, Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) analysis will be conducted to identify proteins or peptides of interest

24. Application • Identified proteins can serve as an alternative method of curing cancer, without harmful side effects on the patient.

25. References • Ng P M L, Jin Z, Tan S S H, Ho B & Ding J L. 2004.C-reactive protein: a predominant LPS-binding protein responsive to Pseudomonas infection. J Endotoxin Res. 10 (3): 163-74. • Medzhitov R & Janeway C Jr. 2000. Innate Immune Recognition: mechanisms and pathways. Immunol Rev. 173: 89-97. • Iwanaga S .2002. The molecular basis of innate immunity in the horseshoe crab. CurrOpinImmunol. 14: 87-95 • StormerL. 1952. Phylogeny and taxomony of fossil horseshoe crabs. J Paleontol. 26: 630-39. • ERDG (2003-2009). The Horseshoe Crab. Available online at: http://horseshoecrab.org/med/med.html • Sharon Rorem (2001). Horseshoe Crabs: True Blue Bloods. Available online at: http://www.suite101.com/article.cfm/aquatic_animals/79177 • Maryland Horseshoe Crabs. Available online at: http://www.dnr.state.md.us/fisheries/general/hscpix/hscbiol.html • Maryland Department of Natural Resources (2005). Medical Uses. Available online at: http://www.dnr.state.md.us/education/horseshoecrab/other.html • Radiation Therapy. Available online at: http://en.wikipedia.org/wiki/Cancer#Radiation_therapy

26. Acknowledgements • Mentor • SRC lab technicians