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

and incubated in crystal violet (0.01% in 10% buffered and CP-724714 (1 µM) in DMEM medium containing 1%
formalin; Sigma-Aldrich, St. Louis, MO, USA) for 30 min, FBS for 24 h, respectively. Cell lysates were electrophoresed
followed by washing with tap water and air-drying at room by means of sodium dodecyl-sulfate polyacrylamide gel
temperature. The number of colonies was photographed electrophoresis (SDS-PAGE), and afterward, the protein
and quantified using ImageJ software. bands were transferred to polyvinylidene difluoride
(PVDF) membrane. The PVDF membrane was blocked
2.5. Melanoma animal model with 5% skim milk in TBS-T for 1 h and then incubated
Four-week-old C57BL/6J male mice were purchased from with the indicated primary antibodies (1:1000 dilutions
the National Laboratory Animal Center (Taipei, Taiwan, in TBS-T containing 5% FBS) and horseradish peroxidase
Republic of China). All animal procedures in this study were (HRP)-conjugated secondary antibodies (1:10000 dilutions
approved by Animal Care and Use Committee (IACUC) in 5% skim milk) for 1 h, respectively. The signals on the
of National Sun Yat-sen University (approval ID: 11128). membrane were detected using chemiluminescent HRP
To achieve primary melanoma induction, 5 × 10 B16-F10 substrate (Millipore Corporation; Billerica, MA, USA),
5
melanoma cells in 0.1 mL of medium were inoculating into and then, the membrane was exposed to X-ray film for
the back of C57BL/6J mice. After implantation for 7 days, autoradiography.
tumor mass started to develop in the mice. The tumor- 2.8. Statistical analysis
bearing mice were then randomly divided into control,
excipient, polysaccharide, and Me-EGF groups (n = 6 per Data were analyzed using GraphPad Prism 8.0 software
group). Each melanoma in mice was treated daily with (GraphPad Software, San Diego, CA) and expressed
topical Me-EGF or other adjuvant (0.1 mL) for 14 days. as mean ± standard deviation (SD). One-way analysis
The tumor volumes were measured with a dial-caliper of variance (ANOVA), coupled with post hoc multiple
according to the following formula : comparison test using the Tukey procedure, was used to
[18]
analyze comparisons involving more than two groups.
Tumor volume (mm ) = width × length × 0.52 (I) A p-value of less than 0.05 was considered statistically
2
3
significant.
2.6. Histological and immunohistochemical analyses 3. Results
To examine histological profiles of melanoma tissues
treated with Me-EGF and other adjuvants, the paraffin 3.1. Administration of Me-EGF did not affect the
sections were deparaffinized, rehydrated, and stained viability and anchorage-independent growth of
with modified Mayer’s hematoxylin (ab220365; Abcam; B16-F10 melanoma cells in vitro
Cambridge, MA, USA) and eosin solutions (SI-E6003; To investigate whether topical application of Me-EGF
Sigma-Aldrich; St. Louis, MO, USA) (H & E). For the increased the risk of skin cancers, B16-10 melanoma cells
analysis of proliferative index in melanoma tissues, were treated with Me-EGF (10 EGF ng/mL) for 24 h and
the paraffin sections were deparaffinized, blocked with 48 h, respectively. At the end of experiment, the viability
3% hydrogen peroxide for 10 min, and subjected to of B16-F10 melanoma cells was determined by MTT
antigen retrieval with microwave in 0.01 M citrate buffer assay. The results showed that Me-EGF and its ingredients
for 30 min. The slides were washed three times with (excipient, EGF, and polysaccharide) had no significant
phosphate-buffered saline (PBS) and then incubated with effect on the proliferation of B16-F10 melanoma cells after
Ki-67 antibody (1:500 dilutions in PBS) for 1 h, followed by 24-h and 48-h incubation (Figure 1). The colony formation
incubation with secondary antibody for 30 min. The signal assay results showed that Me-EGF had no significant effect
was detected using a polymer detection system (Zymed on the anchorage-independent growth of B16-F10 cells
Laboratories, San Francisco, CA). Finally, the slides were (Figure 2). Interestingly, polysaccharide exhibited some
counterstained with modified Mayer’s hematoxylin, cytotoxicity to the colony-forming capability of melanoma
dehydrated, and mounted before microscopic viewing. cells. These findings suggest that the application of Me-EGF
The percentage of nuclear Ki-67-positive cells was did not stimulate the oncogenic behaviors of melanoma
counted from five random images at 200× magnification cells.
per melanoma tissue and expressed as mean ± standard
deviation (n = 6 each group). 3.2. Me-EGF topical application did not enhance
the progression of primary B16-F10 melanoma in
2.7. Western blot analysis C57BL/6J mice
B16-F10 cells were treated with excipient, polysaccharide Further, we evaluated the effect of Me-EGF topical
(0.002%), EGF (100 ng/mL), Me-EGF (100 EGF ng/mL), application on the tumor growth of melanoma in

Volume 2 Issue 4 (2023) 3 https://doi.org/10.36922/gpd.1848