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Gene & Protein in Disease Topical Me-EGF application in melanoma tumor growth

1. Introduction VA, USA). Cells were cultured in Dulbecco’s Modified
Eagle Medium (DMEM; Invitrogen, Carlsbad, CA, USA)
Epidermal growth factor (EGF) is an important mitogen containing 10% fetal bovine serum (FBS; Hyclone, Logan,
and an excellent wound-healing agent, given its biological UT, USA), 2 mM glutamine, 100 mg/mL streptomycin
functions including regulation of skin cell growth, (Invitrogen; Carlsbad, CA, USA), and 100 U/mL penicillin
proliferation, and differentiation . EGF ligand binds to at 37°C in 5% CO atmosphere. The antibodies used in this
[1]
2
its receptor, EGF receptor (EGFR), and induces EGFR work include p-EGFR antibody (sc-101668; Santa Cruz
homodimerization or heterodimerization with other Biotechnology, Santa Cruz, CA, USA), p-HER2 antibody
member of ErbB family, including ErbB2/HER2, ErbB3/ (6B12; Cell Signaling Technology, Danvers, MA, USA),
HER3, or ErbB4/HER4, thereby triggering downstream EGFR antibody (ab52894; Abcam, Cambridge, MA, USA),
PI3K/AKT or RAS/MEK signaling activation . EGF Ki-67 antibody (MA5-14520; Thermo Fisher Scientific,
[2]
stimulates re-epithelialization by promoting keratinocyte Waltham, MA, USA), HER-2 antibody (06-562; Merck
proliferation and migration to accelerate the healing process KGaA, Darmstadt, Germany), and β-actin antibody
of damaged tissues . Despite the numerous benefits in (A5441; Sigma-Aldrich, St. Louis, MO, USA). HER2
[3]
improving chronic wounds and surgical incisions [4,5] , the inhibitor (CP-724714) was purchased from Selleckchem
native EGF protein is easily degradable, especially when in (Houston, TX, USA).
liquid form, due to poor biological stability.
Through modification with polysaccharide 2.2. Preparation of Me-EGF
microencapsulation techniques, the microencapsulated The human EGF (hEGF) is a small polypeptide containing
recombinant human EGF (Me-EGF) exhibits improvements 53 amino acid residues. NewEpi (Me-EGF), which
®
in protein stability, integrity, and effectiveness even contained microencapsulated EGF (10 µg/mL EGF with
after long-term storage [6,7] . Accumulating studies have 0.002% chitosan polysaccharide [Sigma] in excipient
demonstrated that Me-EGF has excellent efficacy to [NaCl, 0.9%]), was provided by JoyCom Bio-chem Co. Ltd.,
promote healing and reduce scar formation in several Kaohsiung, Taiwan region. Me-EGF was made by JoyCom
types of wounds, including cesarean section and burning. with a patented technology that microencapsulates EGF
Moreover, Me-EGF also contributes to the rapid repair of with polysaccharide to maintain a complete structure
osteoarthritis injury [8-10] . through static electricity stabilized with the negatively
The oncogenic potential of EGF remains controversial. charged EGF to form nanoparticles [16,17] . In addition to
Previous studies indicate that high serum levels of EGF being able to coat EGF and maintain the integrity of the
were found in patient with melanoma . The elevated structure, the polysaccharide has good biocompatibility
[11]
EGF levels are associated with melanoma lymph node and biodegradability, can promote wound healing, and is
metastasis; contrarily, EGF knockdown leads to a endowed with antibacterial properties.
significant reduction of lymph node metastasis and 2.3. Cell proliferation assay
primary tumor lymphangiogenesis in vivo . However,
[12]
some studies showed that EGF possesses anti-tumor B16-F10 melanoma cells were cultured in a 96-well
4
properties. Multiplex cytokines assay reveals that patients plate at a density of 1 × 10 cells/mL. After being treated
with unresectable in-transit melanoma have a lower EGF with excipient, polysaccharide, EGF (10 ng/mL), and
[13]
secretion compared with normal healthy controls . Me-EGF (10 ng EGF/mL), respectively, for 24 h and 48 h,
Besides, EGF administration attenuates the growth of the cells were supplemented with medium containing
xenograft tumors derived from A431 human squamous cell 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
carcinoma cells . Interestingly, the EGF-induced apoptosis bromide (MTT; 0.2 mg/mL) and incubated for 2 h at 37°C.
[14]
is dependent on the expression status of EGFR and HER2 . The formazan in viable cells was dissolved with 100 µL
[15]
In the present study, we aim to elucidate the influence of of dimethyl sulfoxide (DMSO), and the absorbance was
microencapsulated recombinant EGF (Me-EGF; brand determined using ELISA reader (Dynex Technologies,
®
name NewEpi ) and its ingredients on the tumorigenicity of Inc., Chantilly, VA, USA) at the wavelength of 570 nm.
skin cancer cells using murine B16-F10 melanoma model. 2.4. Colony formation assay
2. Materials and methods Cells were cultured in 6-well plates (1 × 10 cells per
3
well) and treated with excipient, polysaccharide, EGF
2.1. Cell cultures and reagents
(10 ng/ml), and Me-EGF (10 EGF ng/mL), respectively,
The B16-F10 mouse melanoma cells were purchased once per 2 days for a period of 7 days. At the end of the
from the American Type Culture Collection (Manassas, experiment, the cells were fixed in 4% paraformaldehyde

Volume 2 Issue 4 (2023) 2 https://doi.org/10.36922/gpd.1848