rhEGF Treatment Improves EGFR Inhibitor-Induced Skin Barrier and Immune Defects

Abstract

The mechanisms of epidermal growth factor (EGF) affecting EGF receptor inhibitor (EGFRI)-related skin toxicities are as yet unknown. We investigated which mechanisms are involved in EGF's positive effects. Two types of EGFRIs, cetuximab and gefitinib, were used to treat the cells or 3d-cultured human skin tissue with recombinant human EGF (rhEGF). As a result, rhEGF increased EGFR and pEGFR expression. Furthermore, rhEGF induces EGFR signaling by pAKT and pPI3K expression in gefitinib and rhEGF co-treated cells. In addition, rhEGF bound to EGFR after than cetuximab, but cetuximab bound to EGFR more strongly than rhEGF. Moreover, expressions of proliferation and differentiation proteins, both ki-67 and filaggrin, were decreased in EGFRI-treated tissue. However, in rhEGF and EGFRI co-treated tissue, those expressions were increased. Expression of IL-1α, IL-8, and TNF-α was increased by EGFRIs and down-regulated by rhEGF. Furthermore, hBD-2 and hBD-3 protein expressions were inhibited by cetuximab or gefitinib treatment, and those decrements were increased by rhEGF treatment. In patients' tissue evaluation, compared with controls, patients' Ki-67 and EGFR expression were decreased (p = 0.015, p = 0.001). Patients' IL-17 and TNF-α expression intensity was higher than that of the control group (p = 0.038, p = 0.037). After treatment with EGF ointment, average values of Ki-67, EGFR, and Melan-A were changed to normal values. Oppositely, patients' proportions of IL-17 and TNF-α were decreased to low stain level. In conclusion, treatment of rhEGF improved EGFRI-induced skin eruption via normalizing the proliferation and differentiation of keratinocytes, reducing inflammatory cytokines by the affected EGFRIs.

Keywords: cetuximab; epidermal growth factor; epidermal growth factor inhibitor related skin rash; gefitinib.

Figure 1

The recombinant human epidermal growth factor (rhEGF) increased the epidermal growth factor receptor inhibitor (EGFR) and EGFR signaling was interrupted by EGFR inhibitors. The expression of the EGFR of human epidermal keratinocytes was decreased by cetuximab treatment. However, EGFR and phosphorylated EGFR expression was induced by rhEGF treatment (a). Similarly, in gefitinib-treated keratinocytes, it was observed that phosphorylated AKT and PI3K were slightly decreased. On the other hand, rhEGF and gefitnib co-treatment induced AKT and PI3K phosphorylation (b). Representative Western blot images and relative densitometric bar graphs of EGFR and phosphorelated EGFR (c) β-actin was used as protein loading control. rhEGF treatment induced pEGFR expression decreased by cetuximab (c). Representative Western blot images and relative densitometric bar graphs of AKT, PI3K, phosphorylated AKT, and phosphorylated PI3K (d) β-actin was used as protein loading control. Gefirinib treatment inhibited the expression of EGFR signaling related molecules. That decrement was increased by rhEGF (d).

Figure 2

rhEGF normalized proliferation and differentiation of epidermis-erupted EGFRIs. Expression of ki67 in the nucleus of keratinocytes in the epidermal basal, para-basal cell layer (white arrow) (a). Expression of filaggrin in upper epidermis (b). K5 expression was seen in the epidermal basal layer (white arrow) (c). K10 expression was observed in upper epidermis, especially in the stratum corneum (d). (Magification, ×20, Scale bar: 20 μM).

Figure 3

Tight junction expression was erupted by EGFRIs, which was weekly normalized by rhEGF. Claudin-1 expression was seen in epidermal layers, especially the suprabasal layer (a). Expression of claudin-3 in stratum corneum and stratum granulosum junction (b). Expression of occludin was observed in all epidermal layers (c). (Magification, ×20, Scale bar: 20 μM).

Figure 4

EGFRI-induced inflammatory cytokine expression was inhibited by rhEGF treatment. IL-1α, IL-8, and TNF-α mRNA expression was increased in keratinocytes treated with cetuximab or gefitinib (a,b). However, that increment was decreased by rhEGF and cetuximab (a) or gefitinib (b) co-treatment. The protein expression of IL-1α, IL-8, and TNF-α also induced cetuximab (c) or gefitinib (d) treated keratinocytes (c,d), and that was inhibited by rhEGF and EGFRI co-treatment (c,d). IL-1α expression was observed in the epidermal layer (white arrow) (e). The expression of IL-8 was slightly observed in the epidermis (white arrow) (f). TNF-α expression was seen in the epidermal layer (white arrow) (g). Results are presented as mean ± standard deviation (S.D.), representative of three separated experiments. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001). (Magification, ×20, Scale bar: 20 μM).

Figure 5

EGFRI-inhibited AMP expression was increased by rhEGF treatment. LPS increased mRNA and protein expression of antimicrobial peptides, including hBD-2, -3, LL37, and RNase 7, in human epidermal keratinocytes (a–f). It was observed that mRNA and protein expression of hBD-2 and -3 was decreased in LPS- and EGFRI-treated cells. Also, rhEGF increased hBD-2 and -3 mRNA and protein expression respectably (a,c,e,f). However, in rhEGF- and cetuximab-treated cells, rhEGF only increased RNase 7 but not LL37 mRNA expression (b). Unlike the mRNA expression results, rhEGF did not affect the protein expression of LL37 and RNase 7 (e). Similarly, in rhEGF and gefitinib treated cells, rhEGF affected only RNase 7 mRNA expression (d) but not the protein expression of both LL37 and RNase 7 (f). Results are presented as mean ± standard deviation (S.D.), representative of three separate experiments. Asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

Pathologic comparison between normal control and patient who had EGFRI-related side effects (ERSE). (x 200) Expression of Ki-67 in the nucleus of keratinocytes in the epidermal basal, para-basal cell layer (a: control, b: ERSE). Expression of EGFR in the membrane of keratinocytes in the epidermal basal cell layer (c: control, d: ERSE). Cytoplasmic Melan-A expression was seen in the basal melanocytes of the epidermis (e: control, f: ERSE). Cytoplasmic IL-17 expression in the dermis (g: control, h: ERSE) and Nuclear with or without cytoplasmic TNF-α expression in the dermis and epidermis (i: control, j: ERSE).

Figure 7

ERSE skin pathologic change pre- and post-EGF ointment treatment. (x 200) H&E stain (a: pre-, b: post-EGF treatment). The expression of Ki-67 in the nucleus of keratinocytes in the epidermal basal, para-basal cell layer (c: pre-, d: post-EGF treatment). The expression of EGFR in the membrane of keratinocytes in the epidermal basal cell layer (e: pre-, f: post-EGF treatment). Cytoplasmic Melan-A expression was seen in the basal melanocytes of the epidermis (g: pre-, h: post-EGF treatment). Cytoplasmic IL-17 expression in the dermis (i: pre-, j: post-EGF treatment) and Nuclear with or without cytoplasmic TNF-α expression in the dermis and epidermis (k: pre-, l: post-EGF treatment).