Elucidating Acute-phase cancer responsive proteins from Horseshoe crabs (Carsinocorpius rotundicauda) Group Members: Foo Chuan Hui Joshua (4s2-05) Wong Tuck Wing Ryan (4s2-31) Anu Venkatachalam (AOS) Estelle Gong (AOS)
Jan 04, 2016
Elucidating Acute-phase cancer responsive proteins from Horseshoe crabs (Carsinocorpius rotundicauda)
Group Members:Foo Chuan Hui Joshua (4s2-05)Wong Tuck Wing Ryan (4s2-31)Anu Venkatachalam (AOS)Estelle Gong (AOS)
Carcinoscorpius rotundicauda
Background
“Cancer” refers to a class of diseases with no single cure
Current methods demonstrate variable effectiveness
May cause harm to other body parts
Rationale
Survived two mass extinction events over the past 400 million years
Have been known to benefit cancer research
Rationale
Limulus Amebocyte Lysate (LAL)Detects endotoxins, forms clot
Innate immune systemRich network of proteins
Respond to a variety of Pathogen-associated molecular patterns (PAMPs)
Rationale
Infection studies on the Singapore horseshoe crab, demonstrated that 106 cfu of Pseudomonas aeruginosa was rapidly suppressed
Lethal to mice
Horseshoe crabs completely cleared the infection within 3 days
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
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.
Hypothesis
Proteins present in horseshoe crab blood recognize and bind to PAMPs of cancer cells.
Variables
MaterialsHorseshoe crab blood
Human colorectal cancer cell lysate
Hydrophobic column
SDS-PAGE
Sodium Dodecyl Sulphate Polyscrylamide Gel
Buffer solutions
Urea solution
Micropipettes
Centrifuge
15mL centrifuge tubes
70% ethanol
Autoclave
Refrigerator
Methodology
Method – Collection of blood
Horseshoe crabs were collected from the estuary of the Kranji River
Method
Washed to remove mud and debris
AcclimatizedStress 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
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
Method
Prosoma
Opisthosoma
Method
Needle inserted at hinge
Method
Hemolymph was collected into pre-chilled, pyrogen-free centrifuge tubes
Clarified from hemocytesCentrifugation at 150 x g for 15 min at 4 ºC
Cell debris, contaminants and excess hemocyanin were removed
Further centrifugation at 9,000 x g for 10 min at 4°C
The hemolymph was then quick-frozen in liquid nitrogen and stored at -80 °C.
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.
Method – Separation of proteins
Collected proteins will be analysed by Sodium Dodecyl Sulphate Polyscrylamide Gel Electrophoresis (SDS-PAGE).
Proteins from the SDS-PAGE profile will then be extracted and digested by trypsin.
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
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
Application
Identified proteins can serve as an alternative method of curing cancer, without harmful side effects on the patient.
ReferencesNg 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. Curr Opin Immunol. 14: 87-95
Stormer L. 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
Acknowledgements
Mentor
SRC lab technicians