Prostate cancer cell malignancy via modulation of HIF-1α pathway with isoflurane and propofol alone and in combination

Abstract

Background: Surgery is considered to be the first line treatment for solid tumours. Recently, retrospective studies reported that general anaesthesia was associated with worse long-term cancer-free survival when compared with regional anaesthesia. This has important clinical implications; however, the mechanisms underlying those observations remain unclear. We aim to investigate the effect of anaesthetics isoflurane and propofol on prostate cancer malignancy.

Methods: Prostate cancer (PC3) cell line was exposed to commonly used anaesthetic isoflurane and propofol. Malignant potential was assessed through evaluation of expression level of hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, cell proliferation and migration as well as development of chemoresistance.

Results: We demonstrated that isoflurane, at a clinically relevant concentration induced upregulation of HIF-1α and its downstream effectors in PC3 cell line. Consequently, cancer cell characteristics associated with malignancy were enhanced, with an increase of proliferation and migration, as well as development of chemoresistance. Inhibition of HIF-1α neosynthesis through upper pathway blocking by a PI-3K-Akt inhibitor or HIF-1α siRNA abolished isoflurane-induced effects. In contrast, the intravenous anaesthetic propofol inhibited HIF-1α activation induced by hypoxia or CoCl2. Propofol also prevented isoflurane-induced HIF-1α activation, and partially reduced cancer cell malignant activities.

Conclusions: Our findings suggest that modulation of HIF-1α activity by anaesthetics may affect cancer recurrence following surgery. If our data were to be extrapolated to the clinical setting, isoflurane but not propofol should be avoided for use in cancer surgery. Further work involving in vivo models and clinical trials is urgently needed to determine the optimal anaesthetic regimen for cancer patients.

Figures

Figure 1

Effect of isoflurane exposure on expression and translocation of HIF-1α in cultured human prostate cancer cells (PC3 cells). Cultured cells were exposed to 0.5–2% for 2 h and were harvested immediately or 2–24 h after exposure for western blotting. (A, B) A time- and concentration-dependent increase in HIF-1α expression after isoflurane exposure; (C) immunostaining further revealed isoflurane exposure promoted the translocation of HIF-1α from cytoplasm to nucleus where its downstream effects are initiated; (D, E) HIF-1β expression was unchanged after exposure to isoflurane. Results are expressed as mean±s.d.; *P<0.05; **P<0.001 vs naïve control. Bar=25 μm.

Figure 2

Propofol abolished increased HIF-1α expression induced by various treatments in human prostate cancer cells. (A) Propofol abolished increased HIF-1α production triggered by hypoxia (less than 1% oxygen for 18 h); (B) propofol antagonised CoCl2-induced upregulation of HIF-1α (CoCl2 at 100 μM for 6 h); propofol in intralipid (C) or DMSO (D) at clinically relevant concentrations (2–4 μg ml−1) effectively prevented isoflurane-induced activated expression of HIF-1α. Results are mean±s.d. (n=4); *P<0.05; **P<0.01 vs naïve control. #P<0.05; ##P<0.01 vs isoflurane or CoCl2 alone.

Figure 3

Isoflurane exposure increases human prostate cancer cell growth-promoting signalling and proliferation markers. Exposure to isoflurane induced activation of markers for angiogenesis (VEGF, (A)) and proliferation (Ki67, (B)), while propofol at 4 μg ml−1 or HIF-1α-specific siRNA (20 nM) abolished these changes. Fluorescent intensity of VEGF (C) and percentage of Ki-67+(D) cells were shown. *P<0.01 vs naïve control; **P=0.001 vs naïve control; ###P<0.001 naive control vs isoflurane alone. Abbreviations: HSi=HIF-1α siRNA; Iso=isoflurane; Pro=propofol; SSi=scrambled siRNA. Bar=50 μm.

Figure 4

Isoflurane exposure enhanced human prostate cancer cell cycle progression and cell proliferation. (A and B) Dual labelling of cyclin D (green) or cyclin E (green) with HIF-1 (red). Exposure to isoflurane induced activation of markers for cell cycle (cyclin D, (A) and cyclin E, (B)), while propofol at 4 μg ml−1 or HIF-1α-specific siRNA (20 nM) abolished these changes. Percentage of cyclin D+ (C) and cyclin E+ cells (D) after treatment. (E) MTT assay (n=6) further indicated that propofol at 4 μg ml−1 effectively blocked isoflurane-induced cell proliferation. Results are mean±s.d. (n=6); *P<0.01 vs naïve control; **P=0.001 vs naïve control; ###P<0.001 navie control vs isoflurane alone. Abbreviations: HSi=HIF-1α siRNA; Iso=isoflurane; Pro=propofol; SSi=scrambled siRNA. Bar=50 μm. The full colour version of this figure is available at British Journal of Cancer online.

Figure 5

Propofol attenuated chemotherapy-resistance development induced by isoflurane exposure via inhibition of HIF-1α in human prostate cancer cells. (A) Exposure to 2% isoflurane induced resistance against docetaxel (an anti-mitotic chemotherapy medication), shown as positive staining for Ki67, a marker for active mitosis (mean±s.d., n=8; **P<0.001 vs docetaxel; ##P<0.001 vs isoflurane+docetaxel); (B) MTT assay also demonstrated that with isoflurane pre-treatment, cells had a higher proliferation rate, which was prevented with propofol (mean±s.d., n=6; **P<0.01 vs docetaxel; #P<0.05 vs isoflurane+docetaxel); (C) cytochrome-c release, as a marker of cellular injury caused by decetaxel treatment, was reduced in isoflurane-exposed cells, while treatment with propofol at 4 μg ml−1 or HIF-1α-specific siRNA (20 nM) restored the sensitivity to Dectaxel treatment; (D) anti-apopotic protein, Bcl-2 was also overexpressed following isoflurane exposure before chemotherapy challenge. All these changes in favour of cell survival were blocked by co-administration of propofol at 4 μg ml−1 or HIF-1α-specific siRNA (20 nM) with isoflurane (n=4; **P<0.001 vs naïve control; ##P<0.001 vs isoflurane alone). Abbreviations: Doc=docetaxel; Iso=isoflurane; Pro=propofol. Bar=50 μm.

Figure 6

Molecular mechanisms of isoflurane (Iso)-induced hypoxia-inducible factor (HIF)-1α expression in human prostate cancer cells. Cultured cells were treated with a PI-3K inhibitor, LY294002 (LY) (50 μM), or an ERK inhibitor, U0126 (U) (50 μM), dissolved in DMSO (D), for 16 h prior to 2% isoflurane, or 2% isoflurane combined with propofol (Pro) (4 μg ml−1) for 2 h and then cell extracts were used for western blotting 24 h after treatment. Other cohort cultured cells were exposed to 2% isoflurane or propofol (Pro) (4 μg ml−1) for 2 h and were then harvested immediately or 2–24 h after exposure. (A) HIF-1α; (B) p-Akt; (C) p-ERK; (D, E) p-Akt vs time course. Results are mean±s.d. (n=5); *P<0.05; **P<0.01 vs naïve control. #P<0.05; ##P<0.01 vs Iso.

Figure 7

Isoflurane exposure enhanced human prostate cancer cell migration and invasion. (A and B) ELISA assessment of MMP-2 and MMP-9 in the cell culture medium. (C and D) Scratch assay of PC3 cells before and 24 h after exposure to isoflurane demonstrated accelerated gap closure, while propofol at 4 μg ml−1 or HIF-1α-specific siRNA (20 nM) abolished these changes. Results are mean±s.d. (n=4); *P<0.01 and ***P=0.001; #P<0.001, naive control vs isoflurane alone. Abbreviations: HSi=HIF-1α siRNA; Iso=isoflurane; NC=naive control; Pro=propofol; SSi=scrambled siRNA.