NUGC-4

Secretion of IL-1α and VEGF Protein by Gastric Cancer Cell Lines

Interleukin-1 alpha (IL-1α) plays an important role in tumorigenesis and angiogenesis of gastric cancer. The interleukin-1 receptor antagonist (IL-1RA) inhibits IL-1 selectively, specifically through IL-1R type I (IL-1RI). The results of the reverse transcription (RT)-PCR analysis revealed that IL-1α mRNA was expressed only in the MKN45 cell line; no expression of IL-1α mRNA was detected in the NUGC-4 and AGS cell lines (Fig. 1a). Relative expression of IL-1α mRNA was determined by semi-quantitative RT-PCR, with the results agreeing with those of the RT-PCR experiment (Fig. 1b).

IL-1α protein was detected in the supernatants of cultured MKN45 and HUVEC cells (7.922 ± 0.525 and 5.231 ± 0.367 pg/mL/2 × 105cells, respectively), but not in those of cultured NUGC-4 and AGS cells (Fig. 2a). IL-1α significantly enhanced VEGF secretion by HUVECs in a dose-dependent manner (*P < 0.01, **P < 0.05), while VEGF secretion by HUVECs was blocked by rIL-1RA (*P < 0.01; Fig. 2c). The secretion of VEGF protein by MKN45 cells was higher than that by NUGC-4 and AGS cells (*P < 0.01). RIL-1RA blocked VEGF secretion by MKN45 cells in a dose-dependent manner (compared with control, *P < 0.01, **P < 0.05), but that by NUGC-4 and AGS was not affected (Fig. 2b).

Fig. 1 Expression levels of interleukin-1 alpha (IL-1α), interleukin 1 receptor type I (IL-1RI), and vascular endothelial growth factor (VEGF) mRNA in gastric cancer cell lines MKN45, NUGC-4, and AGS. (Gong Z, et al., 2018)

Fig. 2 (a) Secreted IL-1α levels in human umbilical vein endothelial cells (HUVECs) and gastric cancer cell lines MKN45, NUGC-4, and AGS. (b) Effect of IL-1α and IL-1RA on the level of VEGF secreted by HUVECs. (c) Interleukin-1 receptor antagonist (IL-1RA) influences the secretion of VEGF in gastric cancer cell lines. (Gong Z, et al., 2018)

Anti-cancer Activity of iPS-ML Expressing Anti-HER2 scFv in Vitro

iPS-ML (iPS-cell-derived myeloid/macrophage line), generated by introducing proliferation and anti-senescence factors into iPS-cell-derived myeloid cells, grew continuously in an M-CSF-dependent manner. A cancer-related antigen, HER2/neu, is expressed in various human cancers, including breast and gastric cancers. The anti-cancer effect of iPS-ML expressing anti-HER2 single chain variable region fragment (scFv) was examined against a HER2-expressing gastric cancer cell line, NUGC-4 (Fig. 3A). iPS-ML stably expressing anti-HER2 scFv (iPS-ML/anti-HER2) was generated (Fig. 3B). iPS-ML/anti-HER2 were generated from iPS-MC derived from an iPS cell clone introduced with an expression vector for anti-HER2 scFv by a previously described method. The expression of the scFv by iPS-ML/anti-HER2 was examined and confirmed.

At first, the effect of iPS-ML/anti-HER2 against NUGC-4 cells was evaluated in vitro. Firefly luciferase-introduced NUGC-4 cells were co-cultured with iPS-ML with or without anti-HER2 scFv expression. iPS-ML reduced live NUGC-4 cells, and the expression of anti-HER2 scFv in iPS-ML enhanced the inhibitory effect against the growth of NUGC-4 cells (Fig. 3C).

Fig. 3 Effect of trypsin treatment on adhesion of MKN-1 cells to fibronectin. (Miyata S, et al., 2000)