Volatile anaesthetics enhance the metastasis related cellular signalling including CXCR2 of ovarian cancer cells

Abstract

The majority of ovarian cancer patients relapse after surgical resection. Evidence is accumulating regarding the role of surgery in disseminating cancer cells; in particular anaesthesia may have an impact on cancer re-occurrence. Here, we have investigated the metastatic potential of volatile anaesthetics isoflurane, sevoflurane and desflurane on ovarian cancer cells. Human ovarian carcinoma cells (SKOV3) were exposed to isoflurane (2%), sevoflurane (3.6%) or desflurane (10.3%) for 2 hours. Metastatic related gene expression profiles were measured using the Tumour Metastasis PCR Array and qRT-PCR. Subsequently vascular endothelial growth factor A (VEGF-A), matrix metalloproteinase 11 (MMP11), transforming growth factor beta-1 (TGF-β1) and chemokine (C-X-C motif) receptor 2 (CXCR2) proteins expression were determined using immunofluorescent staining. The migratory capacities of SK-OV3 cells were assessed with a scratch assay and the potential role of CXCR2 in mediating the effects of volatile anaesthetics on cancer cell biology were further investigated with CXCR2 knockdown by siRNA. All three volatile anaesthetics altered expression of 70 out of 81 metastasic related genes with significant increases in VEGF-A, MMP-11, CXCR2 and TGF-β genes and protein expression with a magnitude order of desflurane (greatest), sevoflurane and isoflurane. Scratch analysis revealed that exposure to these anesthetics increased migration, which was abolished by CXCR2 knockdown. Volatile anaesthetics at clinically relevant concentrations have strong effects on cancer cell biology which in turn could enhance ovarian cancer metastatic potential. This work raises the urgency for further in vivo studies and clinical trials before any conclusions can be made in term of the alteration of clinical practice.

Keywords: CXCR2; isoflurane; ovarian cancer; tumour metastasis.

Figure 1. Isoflurane, Sevoflurane and Desflurane alter mRNA expression levels of tumour metastasis genes shown by array analysis

SK-OV3 cells were treated with air (N₂) or 2% Isoflurane (Iso) or 3.6% sevoflurane or 10.3% Desflurane for 2 hours, and then recovered in the normal cell incubator for up to 24 hours. Six hours after exposure analysis of the tumour metastasis PCR array was carried out. Unsupervised hierarchical cluster analysis using Euclidean distance from TaqMan low-density arrays. Gas treatment induced changes in the expression of 70 out of 81 mRNAs relative to the controls and, in comparison to sevoflurane and isoflurane, desflurane led to greater increases in mRNA (N=4). All data is relative to endogenous control, β-Actin. Red and green colours indicate relatively high and low expression, respectively.

Figure 2. Isoflurane, Sevoflurane and Desflurane alter mRNA expression levels of tumour metastasis genes shown by RT-PCR

SK-OV3 cells were treated with air (N₂) or 2% isoflurane (Iso) or 3.6% sevoflurane (Sevo) or 10.3% desflurane (Des) for 2 hours, and then recovered in the normal cell incubator for up to 24 hours. Six hours after exposure analysis of the tumour metastasis PCR array was carried out. Results obtained from the tumour metastasis PCR array and RT-PCR analysis are well correlated. All data is displayed as relative to the endogenous control, β-Actin (n = 4). Data are expressed as mean ± SD. NC: naïve control. Iso: isoflurane, Sevo: sevoflurane, Des: desflurane.

Figure 3. Expression of CXCR2 is increased in ovarian cancer upon exposure to volatile anaesthetics

SK-OV3 cells were treated with air (N₂) or 2% isoflurane (Iso) or 3.6% sevoflurane or 10.3% desflurane for 2 hours, and then recovered in the normal cell incubator for up to 24 hours. CXCR2 siRNA or scrambled siRNA was administered 6 hours before the gas exposure. Expression of A. CXCR2 (red) was assessed with immunofluorescent staining (nuclei counter-stained with DAPI) at 24 hour after gas exposure. Statistical analysis of fluorescent intensity of B. CXCR (n = 8). C. Negative control (secondary antibody with no primary antibody is added) demonstrated the specificity of the staining. D. Western blotting analysis of CXCR2 and GAPDH (n=4). Data are expressed as mean ± SD. *p<0.05 and ***p<0.001. Scale bar: 10μm. NC: naïve control. Iso: isoflurane. Sevo: sevoflurane, Des: desflurane. Scr Si: Scrambled siRNA, C Si: CXCR2 SiRNA. #: comparison between air and anaesthetic treated cells, *comparison between scrambled siRNA and CXCR2 siRNA treated cells.

Figure 4. Upregulation of VEGF-A, MMP11 and TGF-β in ovarian cancer cells upon exposure to volatile anaesthetics

SK-OV3 cells were treated with air (N₂), 2% isoflurane (Iso) or 3.6% sevoflurane or 10.3% desflurane for 2 hours, and then recovered in the normal cell incubator for up to 24 hours. CXCR2 siRNA or scrambled siRNA was administered 6 hours before the gas exposure. Expression of A. MMP-11 (red), C. VEGF-A (red) and E. TGF-β (red) was assessed with immunofluorescent staining (nuclei counter-stained with DAPI) at 24 hour after gas exposure. Statistical analysis of fluorescent intensity of B. MMP-11, D. VEGF-A, and F. TGF-β (n = 8). Data are expressed as mean ± SD. *p<0.05 and ***p<0.001. Scale bar: 50μm. NC: naïve control. Iso: isoflurane. Sevo: sevoflurane, Des: desflurane. Scr Si: Scrambled siRNA, C Si: CXCR2 SiRNA. #: comparison between air and anaesthetic treated cells, *comparison between scrambled siRNA and CXCR2 siRNA treated cells.

Figure 5. Migration of SKOV3 cells is increased after exposure to volatile anaesthetics

SK-OV3 cells were treated with air (N₂) or 2% isoflurane (Iso) or 3.6% sevoflurane or 10.3% desflurane for 2 hours, and then recovered in the normal cell incubator for up to 72 hours. CXCR2 siRNA or scrambled siRNA was administered 6 hours before the gas exposure. A. Cell migration at 0, 24, 48 and 72 hours after gas exposure, assessed by scratch assay (wound healing assay). B. % healing (gap closure) after gas exposure (n=8). Data are expressed as mean ± SD, *p<0.05, **p<0.01 and ***p<0.001. NC: naïve control. Iso: isoflurane, Sevo: sevoflurane, Des: desflurane.

Figure 6. CXCR-2 siRNA abolished effects of inhalational anaesthetics on SK-OV3 cell migration

SK-OV3 cells were treated with air (N₂) or 2% isoflurane (Iso) or 3% sevoflurane or 10.3% desflurane for 2 hours, and then recovered in the normal cell incubator for up to 72 hours. A. Cell migration at 0, 24, 48 and 72 hours after gas exposure, assessed by scratch assay (wound healing assay). B, C, D, E. % healing (gap closure) after gas exposure (n=8). Data are expressed as mean ± SD, *p<0.05, **p<0.01 and ***p<0.001. NC: naïve control. Iso: isoflurane, Sevo: sevoflurane, Des: desflurane. Scr Si: Scrambled siRNA, C Si: CXCR2 SiRNA.