Differential effect of recombinant human epidermal growth factorrhEGF on the proliferation and radia

1
Differential effect of recombinant human
epidermal growth factor(rhEGF) on the
proliferation and radiation survival in normal
fibroblast cell lines and cancer cell lines
Implication for use in patients with
radiation-induced oral mucositis
Eun Kyoung Kwon1, Seung Hee Lee1 ,Hong-Gyun Wu1,
Sang-Wook Lee2 1.Department of Radiation
Oncology, Seoul National University College of
Medicine Institute of Radiation Medicine, Medical
Research Center, Seoul National University Cancer
Research Center, Seoul National University
College of Medicine Department of Radiation
Oncology, 2. Asan Medical Center, College of
Medicine, University of Ulsan
Introduction
Results
Results
Keratinocyte growth factor is approved in North
America, Australia, and Europe to prevent
development of severe oral mucositis in the
patients with hematologic malignancies receiving
myelotoxic therapy and hematopoietic stem cell
transplantation. But application for
radiation-induced oral mucositis in solid tumors
is still under investigation. This study was to
evaluate effects of rhEGF on proliferation and
radiation survival on both normal fibroblast cell
lines and cancer cell lines, so as to apply for
radiation-induced oral mucositis in solid
tumors. And, we found a potent cytoprotective
effect of EGF for human epitherium in vitro .

• EGF- Receptor Expression
• The effect of rhEGF in cancer cell proliferation

Normal Fibroblast Cell Clonogenic Assay

HN3 HN9 H460 H1299 A549 EMT-6 Hela HEL299
Cervix-Fb
EGFR(170KDa)
a-Tubulin
HN3 A549 EMT-6 H M L
a-Tubulin
EGFR
Material Methods
The effect of combination Radiation with rhEGF

• Cell culture
• Cancer cell AMC-HN3 , AMC-HN9 ,NCI- H460,
  NCI-H1299, A549, EMT-6, Hela
• Normal cell F65, HEL299, Cervix Fibroblast
• Cultured at 37?, 5 CO2 in RPMI 1640 medium,
  DMEM medium, MEM medium
  supplemented with 10FBS, 50 U/ml penicillin and
  50 mg/ml streptomycin
• Passage twice a week using 0.05 trypsin-EDTA
• In vitro clonogenic assay
• Mid-log phase cells seeding plating at a
  density of 100 cells per well in six-well culture
  plate.
• The cell were then incubated for 24 hours prior
  to treatment.
• Adding rhEGF(01000nM)
• Without medium change, the cell underwent an
  additional 10 days of incubation at 37?,
  resulting in the formation of colonies
• Fixing with 100 methanol, and stained with
  0.5crystal violet in methanol.
• Colonies counting (defined as more than 50 cells
• Cell proliferation
• Trypan Blue Staining

Conclusion

• Both normal fibroblast and cancer cell lines
  showed varying degree of EGFR expression.
• rhEGF alone stimulated proliferation of normal
  fibroblast cell lines, but had no ef- fect on
  cancer cell lines with medium or low expression
  of EGFR and inhibited proliferation of cancer
  cell lines with high expression of EGFR.
• Compared to radiation alone, addition of rhEGF
  attenuated cell killing effect of radiation in
  normal fibroblast cell lines, but it had no
  effect or augmented radiation effect in cancer
  cell lines.
• Further animal study and clinical study is
  necessary, but results of this study suggest
  apotential approach for prevention or treatment
  of radiation- induced oral mucositis.
• The mechanisms of the action of rhEGF remain
  unclear and it should be elucidated.