Dickinson State University INBRE Program April 2006

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Dickinson State UniversityINBRE ProgramApril
2006

• Lynn C. Burgess, Ph.D.
• Dickinson State University, Dickinson, ND

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Projects

• The anticarcinogenic mechanism of lycopene
• The pathology of exuberant granulation tissue in
  equine

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Lycopene Hypothesis

• Our hypothesis is that lycopenes
  anticarcinogenic properties is due to its ability
  to increase or renew gap junctional connexin
  protein expression. Therefore, lycopene
  maintains or creates gap junctional communication
  from cell to cell, which can control
  proliferation of neoplastic cells from
  surrounding normal cells.

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Lycopene

• To test the hypothesis that lycopenes
  anticarcinogenic property was due to suppression
  of the cancer cells proliferation.
• We tested the following human cell lines
• DU-145, prostate adenocarcinoma cells
• HS-68, foreskin fibroblast cells
• A549, lung carcinoma cells
• HS-578T, breast carcinoma cells
• RWPE-1, prostate cells
• IMR-90, lung cells

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Cell Proliferation Results

• None of the cell lines showed any significant
  changes to their proliferation at any of their
  sample times contrary to other published reports.
  All the cells grew normally and continued to
  proliferate through the 72 hours of incubation.
• Treated cells were compared to controls using the
  Mann-Whitney U Test

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Lycopene and Connexin mRNAs

• RNAs of treated cells were extracted and
  converted to cDNAs. With the use of standard
  PCR, 17 connexins mRNAs were tested for their
  expression in the cell lines
• Connexins 26, 29, 30, 30.3, 31, 31.1, 31.9, 32
  36, 37, 40, 43, 45, 50, and 59 were expressed in
  the cells and there was no expressions from 46
  and 47 in any of the cell lines.

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Connexin Protein Expression

• ELISA and Western Blots are being used to test
  the changes to the connexin proteins after
  lycopene treatment

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Experiments for Next Year

• Three more cell lines will be added
• QPCR will be used to measure any changes in
  connexin mRNA expression due to the lycopene
  treatments
• The effect of lycopene on cell migration will be
  examined

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Exuberant Granulation TissueHypothesis

• The capillary endothelial cells from equine
  exuberant granulation tissue are self simulating
  due to the expression of VEGF and causing the
  overgrowth of the tissue
• We also needed to culture and characterized
  capillary endothelial cells from the disorder.

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Exuberant Granulation TissueProud Flesh
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Endothelial Cell Culturing

• Tissue was minced and treated with typsin then
  sparely seeded on plates. The cells were
  generally attached after 24 h.
• The cells had a fusiform to polygonal appearance.
  They grew to a monolayer by 2 weeks and formed
  round colonies of cells that had a cobblestone
  appearance.
• The EC appeared to be uniform in type without
  contaminating fibroblasts.
• Growth rates of the cells increased as the
  initial seeding was cultured for the 2 weeks.
  The cells retained their appearance and continued
  to after multiple passages.
• A immunofluorescence study confirmed the
  phenotype with an antibody to von Willebrand
  factor (factor VIII). Confluence cultures of
  showed strong cytoplasmic staining in all the
  cells

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Growth Factors Expression in Endothelial Cells

• mRNAs from cultured endothelial cells were tested
  with PCR for angiogenic and inflammation growth
  factor expression
• VEGF, midkine, fibroblast growth factors a and
  ß, somastatin, platelet factor-4, thrombospondin,
  leukemia inhibitory factor, interferon ?,
  transforming growth factor-ß, and insulin-like
  growth factor-1.

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Growth Factor Expression

• The EC expressed two isoforms of vascular
  endothelial growth factor (VEGF) these were 121
  and 165
• There was no expression of other angiogenic and
  inflammation growth factors

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Exuberant Granulation TissueConclusions

• Exuberant granulation tissue provides an easily
  cultured source of capillary EC, which should be
  valuable in studies of the disease
• The expression of VEGF by the EC shows a
  self-simulation that may be the pathology for
  this disease
• VEGF was the angiogenic growth factor expressed
  and could be a possible target for treatment

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The DSU INBRE Research Group