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