Cancerous phenotypes associated with hypoxia-inducible factors are not influenced by the volatile anesthetic isoflurane in renal cell carcinoma

The possibility that anesthesia during cancer surgery may affect cancer recurrence, metastasis, and patient prognosis has become one of the most important topics of interest in cancer treatment. For example, the volatile anesthetic isoflurane was reported in several studies to induce hypoxia-inducible factors, and thereby enhance malignant phenotypes in vitro. Indeed, these transcription factors are considered critical regulators of cancer-related hallmarks, including “sustained proliferative signaling, evasion of growth suppressors, resistance to cell death, replicative immortality, angiogenesis, invasion, and metastasis.” This study aimed to investigate the impact of isoflurane on the growth and migration of derivatives of the renal cell line RCC4. We indicated that isoflurane treatment did not positively influence cancer cell phenotypes, and that hypoxia-inducible factors (HIFs) maintain hallmark cancer cell phenotypes including gene expressions signature, metabolism, cell proliferation and cell motility. The present results indicate that HIF activity is not influenced by the volatile anesthetic isoflurane.


Introduction
The hypothesis that anesthesia during cancer surgery may affect tumor recurrence, metastasis, and patient prognosis [1,2] is gaining currently increasing importance [3]. Accordingly, numerous recent in vitro, in vivo, retrospective, and translational studies have investigated the effect of anesthetics on perioperative immunity and cancer metastatic potential. For example, isoflurane was reported in several studies to induce hypoxia-inducible transcription factors (HIFs), and thereby enhance malignant phenotypes in vitro [4][5][6]. HIF-1 was originally cloned as a driver of erythropoietin expression [7][8][9][10]; however, shortly thereafter, it was reportedly associated with the tumor grade in various cancers [11]. Indeed, HIFs are now well-known as critical regulators of cancer hallmarks, including "sustained proliferative signaling, evasion of Immunoblotting Whole-cell lysates were prepared by incubating cells for 30 min in cold radioimmune precipitation assay buffer with cOmplete Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Tokyo, Japan). Samples were then centrifuged at 10,000 ×g to sediment the cell debris, and 35 μg total protein from the resulting supernatant was resolved via 7.5% sodium dodecyl sulfate-polyacrylamide, gel electrophoresis and electro-transferred to membranes, probed with 1:2,000 of the indicated primary antibodies, probed with 1:8,000 of donkey anti-rabbit IgG (GE Healthcare, Piscataway, NJ) or sheep anti-mouse IgG (GE Healthcare) conjugated with horseradish peroxidase, and visualized with enhanced Chemi-Lumi One Super (Nacalai Tesque, Kyoto, Japan). Bands were quantified via densitometric analysis using Image Studio Lite (LI-COR Biosciences, Lincoln, NE, USA) [15,20,21]. Experiments were performed in triplicate, and representative blots are shown. Detailed protocols are available at protocols.io (dx. doi.org/10.17504/protocols.io.x9mfr46).

Assessment of cell growth
Cell growth was assessed using Proliferation Assay Kit in accordance with the manufacturer's instructions (Promega, Madison, WI, USA) [15,21,22]. Briefly, cells were seeded in 96-well

Cellular oxygen consumption and extracellular acidification rate
Cellular oxygen consumption rate and extracellular acidification rate were determined with XF Cell Mito Stress Test and XF Glycolysis Stress Test, respectively, using an XFp Extracellular Flux Analyzer (Seahorse Bioscience, USA) [15,21,22]. RCC4-EV and RCC4-VHL cells (1 × 10 4 cells/ well) were seeded in an XFp cell culture microplate, cultured overnight, treated with or without 2% isoflurane for 2 h, and incubated in 5% CO 2 and 95% air for 6 h. Oxygen consumption was then assessed in glucose-containing XF base medium in accordance with the manufacturer's instructions, using a sensor cartridge hydrated at 37˚C in a non-CO 2 incubator the day before use. Injection port A was loaded with 1.0 μM oligomycin (complex V inhibitor), port B was loaded with 2.0 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and port C was loaded with 0.5 μM rotenone/antimycin A (inhibitors of complex I and complex III). The sensor was calibrated using cells incubated at 37˚C in a non-CO 2 incubator and in 180 μL of assay medium (XF base medium with 25 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine, pH 7.4

Cell migration assay
Cell migration was analyzed using Oris Cell Migration Assay (Platypus Thechnologies, Madison, WI). Cells (2 × 10 4 cells/well)) were seeded in wells plugged with stoppers to restrict seeding to outer areas only. Cells were then exposed for 8 h to 21% oxygen and 5% carbon dioxide balanced with nitrogen with or without 2% isoflurane. Stoppers were then removed to expose unseeded sites, into which cells could migrate during subsequent incubation at 37˚C in 5% CO 2 and 95% air for indicated times. Cell migration was imaged on a BZ-9000 Fluorescence Microscope (KEYENCE, Itasca, IL), and non-colonized areas were quantified in pixels in ImageJ 1.51 (National Institutes of Health), corrected for total unseeded area, and expressed as percentage of colonized areas in reference wells.

Statistical analysis
Experiments were repeated at least twice with triplicates of each sample. Data are mean ± SD. Groups were compared in Prism 7 (GraphPad Software, Inc. La Jolla, CA) by one-way analysis of variance or Dunnett's test for post hoc analysis. P-values less than 0.05 indicated statistical significance.

C D
to isoflurane regardless of subsequent exposure to 20% or 1% O 2 , but was suppressed in RCC4-VHL cells subsequently exposed to 1% O 2 . Similar trends were observed in cells exposed to isoflurane for 8 h at 20% and 1% O 2 (Fig 1E and 1F). Nonetheless, HIF-1β expression was stable in both cells regardless of isoflurane treatment. Furthermore, we investigated expression of the HIFs-α subunits including HIF-1α and HIF-2α and HIF-downstream genes such as glucose transporter 1(SLC2A1) and vascular endothelial growth factor A (VEGFA) by semi-quantitative RT-PCR. SLC2A1 and VEGFA were more abundant in RCC4-EV cells than in RCC4-VHL cells, but were induced in the latter at 1% O 2 (Fig 2A and 2B). However, expression in RCC4-VHL cells at 1% O 2 was suppressed by isoflurane. Interestingly, HIF1A and EPAS1 (HIF-2α) mRNAs were less abundant in RCC4-EV cells, but were insensitive to isoflurane (Fig 2C and 2D). These results show that two different protocols for isoflurane treatment did not activate HIF-1 or HIF-2 under 20% O 2 conditions.

Effect of isoflurane on cell growth
Next, the critical phenotype of cancer cell growth was investigated. MTS assay is based on the reduction of MTS tetrazolium compound by viable cells to generate a colored formazan product that is soluble in cell culture media. This conversion is considered to be carried out by NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells. Thus, the assay is correlate with cell growth. Cell growth was higher in RCC4-EV cells than in RCC4-VHL cells, but was insensitive to isoflurane (Fig 3A). Cell proliferation of RCC4-EV was suppressed by treatment with the HIF inhibitor YC-1 at 24 h (Fig 3B). Cells were subjected to another method for determining the number of viable cells in culture based on quantitation of the ATP present. Similarly, cellular ATP was more abundant in the former than in the latter, and was also insensitive to isoflurane treatment (Fig 3C). In contrast, ATP levels were also decreased by treatment with YC-1 at 24 h (Fig 3D). Our result strongly suggest that isoflurane treatment did not affect cell growth. However, HIFs are involved in cell growth and cellular energy metabolism. Isoflurane treatment did not affect assay systems for MTS or ATP assay (S2 Fig).

Effect of isoflurane on cell migration
High cell motility is also one of the most significant feature of cancer cells. Therefore we examined the effect of isoflurane and HIFs on cell migration ability. RCC4-EV cells migrated significantly faster than RCC4-VHL cells over 12 h (Fig 4A), although exposure to 2% isoflurane for 2 h significantly suppressed migration in both cells (Fig 4B). The effect of isoflurane was concentration-dependent (S3 Fig). Furthermore, the involvement of HIF was examined. The HIF inhibitor YC-1 canceled HIF-dependent facilitation of cell migration but not the isofluranedependent suppression (Fig 4C). Similar to cell growth, cell motility depended on HIF activity but was not affected by isoflurane treatment.

Effect of isoflurane on glucose metabolism
In comparison to normal cells, cancer cells exhibit the Warburg effect, and thus preferentially metabolize glucose by glycolysis, producing lactate as an end product, despite availability of isoflurane, (C) and after 8 h at 1% O 2 with or without 2% isoflurane. Cells were harvested, and 35 μg whole-cell lysates were immunoblotted using primary antibodies for the indicated proteins. (D) A schematic representation of two protocols of isoflurane treatment. (E and F) Cells were exposed to 2% isoflurane by two different protocols as indicated. HIF-1α expression was analyzed by densitometry and normalized to that in RCC4-VHL cells at 20% O 2 , which was considered 100%. Data represent the mean ± SD value (n = 5).; #, p < 0.05; NS, not significant.
https://doi.org/10.1371/journal.pone.0215072.g001 oxygen. Using an Extracellular Flux Analyzer, the mitochondrial oxygen consumption rate was found to be lower in RCC4-EV cells in comparison to RCC4-VHL cells (Fig 5A), but was insensitive to isoflurane regardless of protocol ( Fig 5B). However, extracellular acidification rate was higher in RCC4-EV cells relative to RCC4-VHL cells (Fig 5C), but was also insensitive to isoflurane (Fig 5D). Key parameters determining the mitochondrial oxygen consumption rate, including basal oxygen consumption rate, maximum respiration, proton leak, and nonmitochondrial respiration, were also calculated from Cell Mito Stress Test data (Fig 6). These parameters were significantly different between RCC4-EV and RCC4-VHL cells, but were insensitive to isoflurane.

Effect of isoflurane on global gene expression
Persistent effects of isoflurane on the cancer cell phenotypes would result in significant changes in the gene expression landscape. Genome-wide gene expression patterns were assessed via RNA-Seq, using next-generation sequencing.
Clustering of RNA sequencing data (S1 File) indicated that transcriptomic bias due to isoflurane was smaller than transcriptomic variations due to VHL expression ( Fig 7A). Indeed, more than 200 genes were differentially expressed between RCC4-EV and RCC4-VHL cells, as inferred from Wilcoxon signed rank test of FPKM values at significance level 0.05 (Fig 7B). However, only one gene was differentially expressed in RCC4-VHL cells exposed to isoflurane, while no such gene was identified in RCC4-EV cells. Pairwise scatter plots comparing log 10 [FPKM+1] values from four experiments further confirmed this result (Fig 7C). Furthermore, enrichment analysis also revealed that GO:0001666 (response to hypoxia), GO:0010035 (response to inorganic substance), hsa05230 (central carbon metabolism in cancer), GO:00 3198 (extracellular matrix organization), and GO:0097190 (apoptotic signaling pathway) were significantly enriched in RCC4-EV cells (Fig 7D) regardless of isoflurane exposure (Fig 7E). Finally, only 42 genes annotated to cancer hallmark gene ontologies were sensitive to isoflurane, although the effects were negligible (Fig 8). Indeed, only CITED1 was strongly responsive to isoflurane.

Discussion
This study shows that transcription factor HIFs maintain hallmarks of cancer cell phenotypes including gene expression signatures, metabolism, cell proliferation, and cell motility, and HIF activity is not influenced by the volatile anesthetic isoflurane.
The hallmarks of cancer were originally proposed by Hanahan and Weinberg in 2000 [12], and included sustained proliferative signaling, evasion of growth suppressors, resistance to cell death, replicative immortality, angiogenesis, invasion, and metastasis. Subsequent conceptual progress has revealed two additional emerging hallmarks, namely reprogramming of energy metabolism and immune evasion [13]. Accordingly, we investigated the impact of isoflurane and HIF on these hallmarks through global gene expression. Since HIF has been extensively investigated in the context of cancer biology [4,11], we used RCC4-EV cells, which are derived from human renal cell carcinoma. As these cells are VHL-deficient, both HIF-1 and HIF-2 are activated even under normoxic conditions, but are suppressed upon forced expression of VHL, as in RCC4-VHL cells [14]. Accordingly, RCC4-EV cells proliferated and migrated faster than RCC4-VHL cells, and exhibited metabolic reprogramming from oxidative phosphorylation to glycolysis. These results clearly indicate that HIFs are critically involved in cancerous phenotypes, as previously reported.
However, isoflurane treatment did not upregulate HIF-1α and HIF-2α expression in RCC4-EV cells. In contrast, isoflurane suppressed HIF-1α expression in RCC4-VHL cells at 1% O 2 . Accordingly, isoflurane also suppressed SLC2A1 and VEGFA, which are downstream of HIF-1, under hypoxic conditions, although this effect is minor to that of VHL expression. A precedent study described that 2h isoflurane exposure upregulated both HIF-1α and HIF-2α protein expression in a PTEN-Akt-dependent manner in RCC4 cells lacking VHL, which is equivalent to RCC4-EV cells in this study, expression within 6 h [24]. In the study, HIF-1α and HIF-2α proteins are barely expressed without isoflurane treatment in RCC4-EV cells. However, the results are concurrent with previous reports including the landmark study although the reason is unknown [14,[25][26][27]. Hence, we investigated potential protocol-dependent effects; however, we found that neither exposure to isoflurane for 8 h, nor for 2 h followed by culture for another 6 h, affected HIF-1 and HIF-2 in RCC4-EV cells. In 2006, Exadaktylos et al. proposed that anesthesia and analgesia during cancer surgery may affect tumor recurrence or metastasis [2], a hypothesis that was subsequently supported by several clinical studies [1]. Potential underlying mechanisms may include direct cellular effects, as well as indirect effects on patient immunity and on cancer metastasis. In this study, we focused on hallmarks of cancer phenotypes in relation to HIF activity. The effect of isoflurane on proliferation, cell growth and cell migration were investigated. The present results clearly indicate that isoflurane treatment did not exert positive effects on cancer cells positively. In addition, metabolism and global gene expression appeared to be sensitive to HIFs but not to isoflurane. This study has several limitations. The present data are derived entirely from in vitro experiments in established cell lines; however, xenografts may be required to elucidate the impact of anesthetics on cancer progression in vivo. In addition, since surgical procedures are aimed at eliminating most of the cancer, there is the complication that the remaining cells may be in an environment containing damaged tissue and inflammation; anesthetics may alter ongoing cell-cell interactions in such an environment.
In summary, this study shows that demonstrated that isoflurane does not affect HIF activity in renal carcinoma cells, nor the expression of genes associated with cancer hallmarks. However, we confirmed that HIFs help maintain cancerous phenotypes.