Differential effects of sevoflurane on the metastatic potential and chemosensitivity of non-small-cell lung adenocarcinoma and renal cell carcinoma in vitro

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Differential effects of sevoflurane on the metastatic potential and chemosensitivity of non-small-cell lung adenocarcinoma and renal cell carcinoma in vitro S. Ciechanowicz, H. Zhao, Q. Chen, J. Cui, E. Mi, E. Mi, Q. Lian, D. Ma British Journal of Anaesthesia Volume 120, Issue 2, Pages 368-375 (February 2018) DOI: 10.1016/j.bja.2017.11.066 Copyright © 2017 Terms and Conditions

Fig 1 Sevoflurane inhibited cell viability of A549 cells. (A) Alamar Blue reduction of naïve, control and sevoflurane-treated A549 cells at 24 h post gas exposure (n=3). (B) Alamar Blue absorbance after inhibition of TGF-βRI/II with LY2109761 (LY21) at 24h post gas exposure (n=3). (C) Wound healing at 24 h post sevoflurane exposure in cultures with and without 24 h of 40 μM DDP treatment. Images are X4 magnification. Dashed-line indicates initial wound at t = 0h. (D) Wound/gap closure expressed as percentage remaining wound area at 24h post gas exposure. 24h data naive (n=4), control (n=4), sevo (n=4), naive + DDP (n=3), control + DDP (n=3), sevo + DDP (n=3). N=number of independent experiments. (E) Wound healing after siRNA knockdown of TGF-β1 and OPN at 24h post gas exposure (n=6). SSi=scramble RNA negative control, TSi=TGF-β1 SiRNA-treated, OSi=OPN SiRNA-treated. Data are expressed as mean (SEM) and analysed using one-way ANOVA with Tukey's post hoc test. *P<0.05, **P<0.01, ***P<0.001. British Journal of Anaesthesia 2018 120, 368-375DOI: (10.1016/j.bja.2017.11.066) Copyright © 2017 Terms and Conditions

Fig 2 Effect of sevoflurane on TGF-β1 expression in A549 cells. TGF-β1 expression in A549 cells detected by immunofluorescence staining. Mean fluorescent intensity is displayed as a box and whisker plot with mean plus or minus upper and lower limits (n=6). Data are expressed as mean (SEM) and analysed using Kruskal-Wallis with Dunn's post hoc test. British Journal of Anaesthesia 2018 120, 368-375DOI: (10.1016/j.bja.2017.11.066) Copyright © 2017 Terms and Conditions

Fig 3 Sevoflurane exposure enhanced nuclear Smad3 expression in A549 cells. (A) Nuclear Smad3 expression in DDP-treated A549 cells as detected by immunofluorescence staining at 24 h post sevoflurane exposure. Fluorescent intensity of Smad3 displayed as a box-and-whisker plot with mean upper and lower limits. Naive + DDP (n=3), Control + DDP (n=3) Sevo + DDP (n=4). Data were analysed with Kruskal-Wallis with Dunn's post hoc test. ***P<0.001. British Journal of Anaesthesia 2018 120, 368-375DOI: (10.1016/j.bja.2017.11.066) Copyright © 2017 Terms and Conditions

Fig 4 Sevoflurane promoted cell viability, chemoresistance and migration of RCC4 cells. (A) Alamar Blue reduction of control and sevoflurane-treated RCC4 cells with and without treatment with DDP at 24 h post gas exposure. (B) Alamar Blue absorbance after LY21 treatment at 24 h post gas exposure, (C) Wound healing at 24h post sevoflurane exposure in cultures with and without 24h of 40 μM DDP treatment. Images are X4 magnification. Dashed-line indicates initial wound at t = 0h. (D) Wound/gap closure expressed as percentage remaining wound area at 24 h post gas exposure. (E) Wound healing after siRNA knockdown of TGF-β1 and OPN at 24h post gas exposure (n=3). SSi=scramble RNA, TSi=TGF-β1 siRNA-treated, OSi=OPN siRNA-treated. Data expressed as mean (SEM) and analysed using ANOVA with Tukey's post hoc test. *P<0.05, **P<0.01, ***P<0.001. British Journal of Anaesthesia 2018 120, 368-375DOI: (10.1016/j.bja.2017.11.066) Copyright © 2017 Terms and Conditions

Fig 5 Sevoflurane increased TGF-β1 and OPN expression in RCC4 cells. (A) TGF-β1 expression in RCC4 cells was detected by immunofluorescence staining at 24h post sevoflurane exposure. Fluorescent intensity of TGF-β1 displayed as a box-and-whisker plot with mean plus or minus upper and lower limits. Naive (n=3), Control (n=4), Sevo (n=4). (B) OPN in RCC4 cells was detected by immunofluorescence staining at 24 h post sevoflurane exposure. Fluorescent intensity of OPN displayed as a box-and-whisker plot with mean plus or minus upper and lower limits (n=4). Data were analysed with Kruskal-Wallis with Dunn's post hoc test. ***P<0.001. British Journal of Anaesthesia 2018 120, 368-375DOI: (10.1016/j.bja.2017.11.066) Copyright © 2017 Terms and Conditions

Fig 6 Sevoflurane enhanced TGF-βRII and reduced nuclear Smad3 expression in RCC4 cells. (A) TGF-βRII expression in DPP-treated RCC4 cells was detected by immunofluorescence staining at 24 h post sevoflurane exposure. Fluorescent intensity of TGF-βRII displayed as box-and-whisker plot with mean plus or minus upper and lower limits. Naive + DDP (n=3), Control + DDP (n=4), Sevo + DDP (n=4). (B) Nuclear Smad3 expression in DDP-treated RCC4 cells was detected by immunofluorescence staining at 24 h post sevoflurane exposure. Fluorescent intensity of Smad3 displayed as a box-and-whisker plot with mean plus or minus upper and lower limits. Naive + DDP (n=4), Control + DDP (n=4), Sevo + DDP (n=3). Data were analysed using Kruskal-Wallis with post hoc Dunn's test. **P<0.01, ***P<0.001. British Journal of Anaesthesia 2018 120, 368-375DOI: (10.1016/j.bja.2017.11.066) Copyright © 2017 Terms and Conditions