Effects of sevoflurane on metalloproteinase and natural killer group 2, member D (NKG2D) ligand expression and natural killer cell-mediated cytotoxicity in breast cancer: an in vitro study

Abstract

Background: We investigated the effects of sevoflurane exposure on the expression of matrix metalloproteinase (MMP), expression and ablation of natural killer group 2, member D (NKG2D) ligands (UL16-binding proteins 1-3 and major histocompatibility complex class I chain-related molecules A/B), and natural killer (NK) cell-mediated cytotoxicity in breast cancer cells.

Methods: Three human breast cancer cell lines (MCF-7, MDA-MB-453, and HCC-70) were incubated with 0 (control), 600 (S6), or 1200 μM (S12) sevoflurane for 4 h. The gene expression of NKG2D ligands and their protein expression on cancer cell surfaces were measured using multiplex polymerase chain reaction (PCR) and flow cytometry, respectively. Protein expression of MMP-1 and -2 and the concentration of soluble NKG2D ligands were analyzed using western blotting and enzyme-linked immunosorbent assays, respectively.

Results: Sevoflurane downregulated the mRNA and protein expression of the NKG2D ligand in a dose-dependent manner in MCF-7, MDA-MB-453, and HCC-70 cells but did not affect the expression of MMP-1 or -2 or the concentration of soluble NKG2D ligands in the MCF-7, MDA-MB-453, and HCC-70 cells. Sevoflurane attenuated NK cell-mediated cancer cell lysis in a dose-dependent manner in MCF-7, MDA-MB-453, and HCC-70 cells (P = 0.040, P = 0.040, and P = 0.040, respectively).

Conclusions: Our results demonstrate that sevoflurane exposure attenuates NK cell-mediated cytotoxicity in breast cancer cells in a dose-dependent manner. This could be attributed to a sevoflurane-induced decrease in the transcription of NKG2D ligands rather than sevoflurane-induced changes in MMP expression and their proteolytic activity.

Keywords: Breast neoplasms; Inhalation anesthetics; Matrix metalloproteinases; Natural killer cells; Sevoflurane; Tumor escape..

Fig. 1.

Experimental protocol used in the study. Treatment (1200 [S12], 600 [S6], and 0 [control, C] µM) was administered for 4 h, and each sevoflurane and control group solution was replaced on an hourly basis. mRNA expression analysis was performed 18 h after completion of the 4-h treatment. The other tests were performed 24 h after the 4-h treatment.

Fig. 2.

Effect of sevoflurane on the mRNA expression of NKG2D ligands. Results were analyzed using Kruskal-Wallis tests with post-hoc Conover comparisons. Variables are presented as the median with the first and third quartiles (n = 6 per group). C: control group, S6: sevoflurane 600 µM group, S12: sevoflurane 1200 µM group, ULBP: UL16-binding proteins, MICA/B: major histocompatibility complex class I chain-related molecules A/B, RPL19: ribosomal protein L19. ^*P < 0.05 compared to C.

Fig. 3.

Effect of sevoflurane on surface expressions of NKG2D ligands assessed by flow cytometry. Results were analyzed using Kruskal-Wallis tests with post-hoc Conover comparisons. Variables are presented as the median with the first and third quartiles (n = 6 per group). C: control group, S6: sevoflurane 600 µM group, S12: sevoflurane 1200 µM group, ULBP: UL16-binding proteins, MICA/B: major histocompatibility complex class I chain-related molecules A/B, PE: phycoerythrin. ^* and ^†P < 0.05, compared with C and S6, respectively.

Fig. 4.

Effect of sevoflurane on MMP expression and concentration of soluble NKG2D ligands. Variables are presented as medians with the first and third quartiles (n = 6 per group). (A) Western blot analysis was performed to evaluate MMP-1 and -2 expression (Supplementary Fig. 1). The protein expression levels of MMP-1 and -2 between the control and sevoflurane-treated groups in MCF-7, MDA-MB-453, and HCC-70 cells remained unchanged. (B) Enzyme-linked immunosorbent assay (ELISA) tests were performed to evaluate the concentration of soluble NKG2D ligands. No differences in the levels of soluble NKG2D ligands were observed between the control and sevoflurane treatment groups in the MCF-7, MDA-MB-453, and HCC-70 cells. MMP: matrix metalloproteinase, MICA/B: major histocompatibility complex class I chain-related molecules (MIC) A/B, C: control group, S6: sevoflurane 600 µM group, S12: sevoflurane 1200 µM group.

Fig. 5.

Effect of sevoflurane on NK cell-mediated cytotoxicity assessed by flow cytometry. Results were analyzed using Kruskal-Wallis tests with post-hoc Conover comparisons. Variables are presented as the median with the first and third quartiles (n = 4 per group). Target cancer cells (T; MCF-7, MDA-MB-453, and HCC-70) were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) and co-cultured with NK92-MI cells (effector cells [E]) at a 1:1 ratio (e.g., E:T = 1 × 10⁵ : 1 × 10⁵) or 10:1 (e.g., E:T = 1 × 10⁶ : 1 × 10⁵). PI: propidium iodide, C: control group, S6: sevoflurane 600 µM group, S12: sevoflurane 1200 µM group. ^*P < 0.05 compared to C.