group members: foo chuan hui joshua (4s2-05) wong tuck wing ryan (4s2-31)

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 cureCurrent methods demonstrate variable effectivenessMay cause harm to other body parts

RationaleSurvived two mass extinction events over the past 400 million years Have been known to benefit cancer research

RationaleLimulus Amebocyte Lysate (LAL)

Detects endotoxins, forms clot

Innate immune systemRich network of proteins Respond to a variety of Pathogen-associated molecular patterns (PAMPs)

RationaleInfection studies on the Singapore horseshoe crab, demonstrated that 106 cfu of Pseudomonas aeruginosa was rapidly suppressedLethal to miceHorseshoe crabs completely cleared the infection within 3 days

RationaleProteins found in the blood of horseshoe crabs potentially provides a more effective way of treating cancer

No damage and irradiation to adjacent cellsChemotherapy – toxicityRadiation therapy – damage from radiation

PurposeElucidate specific proteins in Horseshoe crab blood that recognize and bind surface antigens or PAMPs of cancer cellsTo propose potential peptide-based drugs for cancer detection & treatment.

HypothesisProteins present in horseshoe crab blood recognize and bind to PAMPs of cancer cells.

Variables

MaterialsHorseshoe crab bloodHuman colorectal cancer cell lysateHydrophobic columnSDS-PAGE

Sodium Dodecyl Sulphate Polyscrylamide GelBuffer solutionsUrea solution

MicropipettesCentrifuge15mL centrifuge tubes70% ethanolAutoclaveRefrigerator

Methodology

Method – Collection of blood

Horseshoe crabs were collected from the estuary of the Kranji River

MethodWashed to remove mud and debrisAcclimatized

Stress might affect composition of blood

Washed the carapace around the vicinity of the cardiac chamber with water and swabbed with 70 % ethanol

Removes bacteriaPrevent clotting of blood

MethodThe 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

MethodHemolymph was collected into pre-chilled, pyrogen-free centrifuge tubesClarified from hemocytes

Centrifugation 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

ApplicationIdentified 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

AcknowledgementsMentorSRC lab technicians