elucidating acute phase cancer responsive proteins from horseshoe crabs carsinocorpius rotundicauda n.
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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.

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elucidating acute phase cancer responsive proteins from horseshoe crabs carsinocorpius rotundicauda

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)

background
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
Rationale
  • Survived two mass extinction events over the past 400 million years
  • Have been known to benefit cancer research
rationale1
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)
rationale2
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
rationale3
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
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
Hypothesis
  • Proteins present in horseshoe crab blood recognize and bind to PAMPs of cancer cells.
materials
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
method collection of blood
Method – Collection of blood
  • Horseshoe crabs were collected from the estuary of the Kranji River
method
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
method1
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
method2
Method

Prosoma

Opisthosoma

method3
Method

Needle inserted at hinge

method4
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. 
method hydrophobic column
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
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.
method5
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
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
Application
  • Identified proteins can serve as an alternative method of curing cancer, without harmful side effects on the patient.
references
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
acknowledgements
Acknowledgements
  • Mentor
  • SRC lab technicians