Circulating Natural Autoantibodies to HER2-Derived Peptides Performed Antitumor Effects on Oral Squamous Cell Carcinoma

Abstract

Natural autoantibodies play a crucial role in destruction of malignant tumors due to immune surveillance function. Epidermal growth factor receptor 2 (HER2) has been found to be highly expressed in a variety of epithelial tumors including oral squamous cell carcinoma (OSCC). The present study was thus undertaken to investigate the effect of anti-HER2 natural autoantibodies on OSCC. Compared with cancer-adjacent tissues, cancer tissues from OSCC patients exhibited higher HER2 expression especially in those with middle & advanced stage OSCC. Plasma anti-HER2 IgG levels examined with an enzyme-linked immunosorbent assay (ELISA) developed in-house showed differences between control subjects, individuals with oral benign tumor and patients with OSCC. In addition, anti-HER2 IgG-abundant plasma was screened from healthy donors to treat OSCC cells and to prepare for anti-HER2 intravenous immunoglobulin (IVIg). Both anti-HER2 IgG-abundant plasma and anti-HER2 IVIg could significantly inhibit proliferation and invasion of OSCC cells by inducing the apoptosis, and also regulate apoptosis-associated factors and epithelial-mesenchymal transition (EMT), respectively. Besides, the complement-dependent cytotoxicity (CDC) pathway was likely to contribute to the anti-HER2 IgG mediated inhibition of OSCC cells. After the HER2 gene was knocked down with HER2-specific siRNAs, the inhibitory effects on OSCC cell proliferation and apoptotic induction faded away. In conclusion, human plasma IgG, or IVIg against HER2 may be a promising agent for anti-OSCC therapy.

Keywords: HER2; circulating natural autoantibodies; intravenous immunoglobulin; oral squamous cell carcinoma; trastuzumab.

FIGURE 1

The expression of HER2 in OSCC tissues. (A) H&E and IHC staining of OSCC tissues and cancer-adjacent tissues in early (n = 5), middle & advanced stages (n = 5). (B) The average optical density (AOD) data of IHC staining were presented as mean ± standard deviation (SD). *p < 0.05; **p < 0.01.

FIGURE 2

Effects of anti-HER2 IgG-abundant plasma on the proliferation of OSCC cells. (A–C) Proliferation of three OSCC cell lines treated with anti-HER2 IgG-abundant plasma for 48 h. Plasma A and plasma B obtained from two individual healthy donors were abundant in anti-HER2 IgG. The data of proliferation were expressed as mean ± SD in cell viability (%). Plasma A showed an inhibitory effect on CAL27 and plasma B on both SCC15 and SCC25 cells. (D) Apoptosis of three OSCC cell lines treated with anti-HER2 IgG-abundant and -deficient plasma for 24 and 48 h, respectively. The data were expressed as mean ± SD in proportion of apoptotic cells. (E–F) Three OSCC cell lines treated with either 20% anti-HER2 IgG-abundant or -deficient plasma for 48 h were photographed and bar chart showed the proportion of invasive cells relative to total number of seeding cells. (G) Three OSCC cell lines were cultured in 20% anti-HER2 IgG-abundant or -deficient plasma and the expression of EMT-related factors, E-cadherin, Vimentin, or Snail was then determined by Western blot with β-actin as reference. D: anti-HER2 IgG-deficient plasma; A: anti-HER2 IgG-abundant plasma; *p < 0.05; **p < 0.01.

FIGURE 3

Possible involvement of complement-dependent cytotoxicity. (A–C) Inhibitory effects of trastuzumab (TZ) on proliferation of CAL27, SCC15, and SCC25 cells. A range of the TZ concentrations was used to treat OSCC cells and the concentration of 200 μg/ml TZ was considered as the optimum concentration for subsequent experiments. (D–F) Viability of CAL27, SCC15, and SCC25 cells treated with 200 μg/ml TZ in medium containing either 20% inactivated FBS or 20% activated FBS. (G–I) Viability of CAL27, SCC15, and SCC25 cells treated with inactivated 200 μg/ml TZ in medium containing either 20% inactivated FBS or 20% activated FBS. (J–L) Viability of CAL27, SCC15, and SCC25 cells treated with 20% anti-HER2 IgG-deficient plasma, 20% anti-HER2 IgG-abundant plasma and 20% inactivated anti-HER2 IgG-abundant plasma. The data of proliferation were expressed as mean ± SD in cell viability (%). Deficient, anti-HER2 IgG-deficient plasma; Abundant, anti-HER2 IgG-abundant plasma; TZ, trastuzumab; *p < 0.05; **p < 0.01.

FIGURE 4

Inhibitory effect of anti-HER2 IVIg on the proliferation of OSCC cells. (A,B) Viability of CAL27 cells and SCC25cells treated with anti-HER2 IgG-deficient plasma only, anti-HER2 IgG-deficient plasma containing 200 μg/ml trastuzumab, 2.5 mg/ml regular IVIg, 2.5 mg/ml anti-HER2 IVIg, 5 mg/ml regular IVIg, and 5 mg/ml anti-HER2 IVIg, respectively. (C–E) EdU staining of CAL27 and SCC25 cells treated with anti-HER2 IgG-deficient plasma only, 200 μg/ml trastuzumab, 2.5 mg/ml regular IVIg, 2.5 mg/ml anti-HER2 IVIg, 5 mg/ml regular IVIg, and 5 mg/ml anti-HER2 IVIg, in medium containing 20% anti-HER2 IgG-deficient plasma. The data were expressed as mean ± SD in cell viability (%). Deficient, anti-HER2 IgG-deficient plasma; IVIg, intravenous immunoglobulin; *p < 0.05; **p < 0.01.

FIGURE 5

The effects of anti-HER2 IVIg on OSCC cells apoptosis. (A–C) Apoptosis of CAL27 and SCC25 cells treated for 48 h with anti-HER2 IgG-deficient plasma only, 200 μg/ml trastuzumab, 5 mg/ml regular IVIg, and 5 mg/ml anti-HER2 IVIg, respectively. The data were expressed as mean ± SD in proportion of apoptotic cells. (D) CAL27 cells and SCC25 cells were treated for 48 h with anti-HER2 IgG-deficient plasma only, 200 μg/ml trastuzumab, 5 mg/ml regular IVIg, and 5 mg/ml anti-HER2 IVIg, respectively; the expression of apoptosis-related factors, including Bcl-2, Bax, Bak, cytochrome c, caspase-9, caspase-3, and cleaved caspase-3, was then determined by Western blot with β-actin as a reference. Deficient, anti-HER2 IgG-deficient plasma; IVIg, intravenous immunoglobulin; *p < 0.05; **p < 0.01.

FIGURE 6

Inhibitory effects of anti-HER2 IVIg on OSCC cells migration. (A–C) CAL27 and SCC25 cells treated for 48 h with anti-HER2 IgG-deficient plasma only, 200 μg/ml trastuzumab, 5 mg/ml regular IVIg, and 5 mg/ml anti-HER2 IVIg, respectively, were photographed; bar chart showed the proportion of invasive cells relative to the total number of seeding cells. (D) CAL27 and SCC25 cells were cultured for 48 h with anti-HER2 IgG-deficient plasma only, 200 μg/ml trastuzumab, 5 mg/ml regular IVIg, and 5 mg/ml anti-HER2 IVIg, respectively; the expression of EMT-related factors, E-cadherin, Vimentin, or Snail was then determined by Western blot with H3 as a reference. Deficient: anti-HER2 IgG-deficient plasma; IVIg: intravenous immunoglobulin; *p < 0.05; **p < 0.01.

FIGURE 7

Inhibitory effects of anti-HER2 IVIg on HER2 knockdown OSCC cells. (A–D) Three HER2-specific siRNAs were transfected into CAL27 and SCC25 cells. The expression of HER2 mRNA and protein was then examined by qPCR and Western blot assay, respectively. (E–F) CAL27 and SCC25 cells were transfected with either HER2-specific siRNAs or control siRNAs; OSCC cells were treated for 48 h with anti-HER2 IgG-deficient plasma containing 200 μg/ml trastuzumab, 5 mg/ml regular IVIg, and 5 mg/ml anti-HER2 IVIg, respectively. The data were expressed as mean ± SD in cell viability (%). (G) CAL27 and SCC25 cells were transfected with either HER2-specific siRNAs or control siRNAs, and then treated for 48 h with anti-HER2 IgG-deficient plasma containing 200 μg/ml trastuzumab, 5 mg/ml regular IVIg, and 5 mg/ml anti-HER2 IVIg, respectively. The data were expressed as mean ± SD in proportion of apoptotic cells. IVIg, intravenous immunoglobulin; si-Ctrl, control siRNA; si-HER2, HER2-specific siRNAs; *p < 0.05; **p < 0.01.