Propofol Prevents the Progression of Malignant Pheochromocytoma In Vitro and In Vivo

Abstract

This study aimed to explore the efficacy of propofol to treat malignant pheochromocytoma (PCC) in vitro and in vivo. In vitro, PC12 cells were treated with different concentrations of propofol (0, 1, 5, and 10 μg/mL) for specific times followed by a MTT assay to detect cell proliferation. Transwell assays were performed to assess the function of propofol on the migration and invasion of PC12 cells, and flow cytometry to analyze cell apoptosis and cell cycle progression. Quantitative real-time polymerase chain reaction was carried out to analyze the expression level of mRNA (Bcl-2, Bax, and CyclinE). The levels of Bcl-2, Bax, CyclinE, FOXO1, FOXO3, Bim, procaspase-3, and active caspase-3 were determined by western blotting. In vivo, the effects of propofol on PCC tumor growth were detected by transplanted mouse model. Transferase dUTP nick-end labeling was performed to detect tissue cell apoptosis. The results indicated that propofol inhibited PC12 cell proliferation, prevented cell migration and invasion, and induced the apoptosis of PC12 cells in a dose- and time-dependent manner. Propofol treatment increased the expression of Bax and decreased that of Bcl-2. In addition, propofol significantly induced the G1/S phase arrest in PC12 cells, and the expression of Cyclin E was reduced. Moreover, the levels of FOXO1, FOXO3, Bim, procaspase-3, and active caspase-3 were enhanced by propofol treatment. In vivo, propofol treatment significantly reduced the PCC tumor growth and induced tissue cell apoptosis. In conclusion, propofol has potent anti-PCC activity in vitro and in vivo, and is a potential small-molecule drug for treating malignant PCC.

Introduction

Pheochromocytoma (PCC) is a neuroectodermal tumor stemming from chromaffin cells of the sympathetic nervous system (Ozer et al., 2014; Gimenez-Roqueplo, 2015). PCC is localized in the adrenal medulla in 90% of patients and in the extra-adrenal region in 10% of patients (Ozer et al., 2014). It showed a prevalence of two to eight per one million in a general population (Lentschener et al., 2011). PCC causes clinical symptoms of neuroendocrine tumors through the secretion of catecholamines. It is an important cause of endocrine hypertension, and its most frequent symptoms are paroxysmal hypertension, headache, palpitation, and sweating (Roizn, 2001).

However, it can also follow a course with such atypical symptoms as epigastric distress, nausea, and vomiting (Beattie and Heasman, 1958). An uncontrolled PCC may cause life-threatening hypertensive crisis and cardiac arrhythmia (Voros et al., 1996). Therefore, it is important for PCC to be specified in the preoperative period, and suitable pharmacotherapy to be implemented (Shinn et al., 2012; Björklund and Backman, 2017; Zhang et al., 2017). In recent years, research on PCC has drawn more and more attention. For instance, small activating RNA induces dsP53-285 PCC cell line PC12 cell cycle arrest and apoptosis by enhancing wild-type p53 expression (Lin et al., 2017).

Proteasome inhibitors were considered as a potential medical therapy for malignant PCC (Yu, 2017). PP242, a dual mTORC1/2 kinase inhibitor, showed strong antitumor activity in a PCC PC12 cell tumor model (Zhang et al., 2015). Aplasia Ras homolog member I has been reported to be a novel epigenetic silenced tumor suppressor in sporadic PCC (Wang et al., 2017). Moreover, anthracyclines have been found to suppress PCC cell characteristics, including metastasis, through inhibiting the hypoxia signaling pathway (Pang et al., 2017). Maslinic acid may exert its anticancer activity by promoting autophagy through disrupting the interaction between Bcl2 and Beclin1 in rat PCC PC12 cells (Dong et al., 2017). However, so far, PCC treatment is still a clinically significant challenge.

Propofol is an anesthetic. Recently, many studies have demonstrated that propofol has a variety of other effects, including possible anticancer actions. As the complexity of endoscopic surgery increases, the desire to use propofol and deep sedation are becoming more common in endoscopy kits. Targeted antitumor therapy has been considered a promising new treatment strategy for malignant PCC (Lowery et al., 2013; Mohammed et al., 2014; Fishbein, 2016).

This study aimed to investigate the effects of propofol on PCC in vivo and in vitro.

Materials and Methods

Materials

Rat PCC PC12 cells were obtained from American Type Culture Collection ATCC (Manassas, VA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were from Gibco. All primary antibodies (anti-β-actin, anti-cyclinE, anti-Bax, anti-CyclinE; anti-FOXO1, anti-FOXO3, anti-Bim, anti-procaspase-3, and anti-active caspase-3) were acquired from Cell Signaling Technology (Boston, MA).

Cell culture and treatment

Rat PCC PC12 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (Sigma-Aldrich). Cells were incubated in a humidified incubator at 37°C with 5% CO₂. Propofol was dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C.

Rat PCC PC12 cells were treated in the following manner: (1) Cells were treated with different concentrations (0, 1, 5, and 10 μg/mL; 0 μg/mL as control) of propofol for 24, 48, and 72 h at 37°C with 5% CO₂, and subsequently analyzed by the MTT assay. (2) Cells were treated with different concentrations (0, 1, 5, and 10 μg/mL; 0 μg/mL as control) of propofol for 48 h, and subsequently detected by flow cytometry and Transwell assay.

Western blotting

Following treatment, cells were collected and total proteins were extracted with 40 mM Tris–HCl (pH 7.4) containing 150 mM NaCl and 1% (v/v) Triton X-100, supplemented with protease inhibitors. The protein concentration was determined using the bicinchoninic acid protein assay (Pierce, IL). Equal amounts of protein were resolved on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, and then transferred to a polyvinylidene difluoride membrane (Millipore, MA). The blots were blocked with 5% skimmed milk in Tris Buffered Saline with Tween-20 (TBST) and then probed with antibodies. The blots were washed three times and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies.

Immunoreactive bands were visualized using the enhanced chemiluminescence detection system, and images were captured with a Fuji LAS3000-mini imaging system (Fujiflm, Tokyo, Japan). Furthermore, the immunoreactive bands were quantified by ImageJ software (v1.41; NIH, Bethesda, MD). The protein levels of the stripes were normalized based on the gray value of β-actin.

RNA isolation and quantitative real-time polymerase chain reaction

Total RNA was extracted from the PC12 cells (blank and propofol groups) using TRIzol reagent. The concentration of RNA was detected by NanoDrop 2000. The RNA samples were stored at −80°C for future use. Then, cDNAs were synthesized with the miScript Reverse Transcription Kit (GmbH, Qiagen) according to the manufacturer's protocol. The QuantiFast SYBR Green PCR Kit (Qiagen) was used to perform quantitative real-time polymerase chain reaction (qRT-PCR) under a CFX Connect Real-Time System (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase was used as the internal control. The 2^−ΔΔCq method was applied for the quantification of relative gene expression. Primer sequences were obtained from GenScript (Nanjing) as required.

MTT assay

A total of 5 × 10³ cells were seeded into the wells of 96-well plates in triplicate and incubated for 24 h before treatment to determine cell growth. MTT (20 μL; 5 mg/mL; Sigma-Aldrich) was added to each well 24, 48, and 72 h after propofol treatment. Subsequent to incubation for 4 h, the MTT medium was removed, and 150 μL DMSO was added. After shaking for 15 min at room temperature, the optical density (OD) of each sample was determined with an Enzyme Immunoassay Instrument. The cell inhibition rate = (1 − OD value of treatment/OD value of control) × 100%.

Cell migration and invasion assays

The effects of propofol on cell migration and its invasive capability were detected using a 24-well Transwell plate (8-mm pore size; NY). Briefly, chamber inserts were coated with or without 200 mg/mL of Matrigel and dried overnight under sterile conditions. The cells (1 × 10⁴ cells) were suspended in different concentrations of propofol–1640 medium (0, 1, 5, and 10 μg/mL), and then seeded into the top chamber; 1640 medium supplemented with 20% FBS was added to the lower chamber. Following incubation for 48 h at 37°C, the top chambers were wiped with cotton wool to remove the nonmigratory or noninvasive cells and subsequently fixed with 100% methanol for 10 min. Following Hematoxylin–Eosin staining, the migratory and invasive cells on the underside of the membrane were counted at five random fields under a microscope (Olympus).

Flow cytometry analysis

Following treatment with propofol for 48 h, PC12 cells were harvested and washed with phosphate-buffered saline (PBS). The cells were subsequently fixed with 70% methanol at −20°C overnight and then washed with PBS twice. Then, the cells were stained with RNase A Propidium Iodide (PI), and flow cytometry was used to detect cell cycle distribution.

Cells were stained with Annexin V and PI to detect cell apoptosis (Apoptosis Detection Kit, BD Biosciences). After incubation for 15 min in the dark, apoptotic cells were detected by flow cytometry according to the manufacturer's protocol.

Establishment of a PCC mouse xenograft model

The present study was performed according to the principles and procedures of the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. This study was approved by the Animal Care and Use Committee of the 3rd Affiliated Hospital of Harbin Medical University. A PCC mouse xenograft model was established as previously described (Lian et al., 2017). In brief, PC12 cells (1 × 10⁶ cells/100 μL) were subcutaneously implanted into the left posterior abdomen of 4- to 5-week-old female athymic nude mice. Vernier calipers were used to measure the volume of the tumor.

When the tumor volume reached 100–200 mm³ (∼8 days after injection), the mice were randomly divided into two groups (n = 5 per group). (1) Sham group: the mice were intraperitoneally injected with saline; (2) propofol group: the mice were intraperitoneally injected with propofol (35 mg/kg). Mice were treated every 2 days for 14 days. All mice were sacrificed at the end of the experiment, and the tumor was stripped and measured.

Terminal deoxynucleotidyl transferase dUTP nick end labeling assay

The tumors were taken out, fixed with 4% neutral-buffered paraformaldehyde, and then paraffin embedded. Paraffin blocks were cut into 4-μm sections and histologically examined after Hematoxylin and Eosin staining. Paraffin slices were deparaffinized and then rehydrated (Shim et al., 2017). The cell death detection kit was used for staining with terminal deoxynucleotidyl transferase dUTP nick end labeling. ProLong Gold Antifade Mountant was performed and DAPI was used to mount the samples.

Statistical analysis

All experiments were repeated at least for three times. Results were expressed as mean values ± SD. Statistical analysis was performed by Student's t-test. A level of p < 0.05 was considered to be significant.

Results

Propofol inhibited PC12 cell proliferation, migration, and invasion

The present study showed that propofol inhibited PC12 cell proliferation and prevented PC12 cell migration and invasion in a dose-dependent and time-dependent manner (Fig. 1). The results obtained from the Transwell assays showed that the number of migratory and invasive PC12 cells significantly decreased in propofol-treated groups (Fig. 2).

FIG. 1.

Effect of propofol on PC12 cell proliferation. PC12 cells were treated with different concentrations of propofol (0, 1, 5, and 10 μg/mL) for 24, 48, 72 h, respectively. Then the proliferation inhibition rate of cells from different groups was assessed by MTT assay. Data are reported as mean ± SD. *p < 0.05, **p < 0.01 versus control group.

FIG. 2.

Effect of propofol on PC12 cell migration and invasion. PC12 cells were treated with different concentrations of propofol (0, 1, 5, and 10 μg/mL) for 48 h, respectively. Then the migration and invasion of cells in different groups were determined by transwell assay. Mean number of migrated and invasive cells was determined in triplicate. (A) Effect of propofol on PC12 cell migration; (B) Effect of propofol on PC12 cell invasion. Data are reported as mean ± SD. *p < 0.05, **p < 0.01 versus control group.

Propofol induced cell cycle arrest in PC12 cells

The cell cycle analysis was used to explore the possible mechanism by which propofol inhibited PC12 cell proliferation. The result demonstrated that treatment with propofol for 48 h significantly increased the percentage of PC12 cells in the G1 phase and decreased that in the G2 phase in a dose-dependent manner (Fig. 3A, B). Meanwhile, the western blotting results showed that the expression of cyclin E, a cell cycle-associated protein, was blocked by propofol treatment (Fig. 3C, D).

FIG. 3.

Effect of propofol on PC12 cell cycle. Forty-eight hours after treatment with propofol (0, 1, 5, and 10 μg/mL), PC12 cell cycle was determined. (A) PC12 cell cycle progression was evaluated by Propidium Iodide staining and flow cytometry; (B) Percentage of PC12 cells in G1, S, or G2 phase after treatment with propofol; (C) Protein level of Cyclin E after exposure to propofol; (D) relative protein expression level of Cyclin E was analyzed. *p < 0.05, **p < 0.01 versus control group.

Propofol induced apoptosis in PC12 cells

The effect of propofol on cell apoptosis was measured using the Annexin V-FITC/PI staining method. The results demonstrated that the apoptosis rate of PC12 cells significantly increased after 48 h of propofol treatment (Fig. 4A, B). Consistent with the results of apoptosis, the protein and mRNA expression of Bax increased and the Bcl-2 level decreased dose dependently in response to propofol treatment for 48 h (Fig. 4C, D). Thus, the findings of the present study clearly revealed that propofol induced apoptosis in PC12 cells.

FIG. 4.

Effect of propofol on PC12 cell apoptosis. Forty-eight hours after treatment with propofol (0, 1, 5, and 10 μg/mL), PC12 cell apoptosis was measured. (A) PC12 cell apoptosis was detected using flow cytometry; (B) Percentage of apoptotic PC12 cells after exposure to propofol; (C–E) Protein/mRNA expression levels of Bax and Bcl-2 after exposure to propofol for 48 h. *p < 0.05, **p < 0.01 versus control group.

Propofol enhanced the expression of FOXO1, FOXO3, Bim, procaspase-3, and active caspase-3 in PC12 cells

Studies have reported that FOXO transcription factors play critical roles in regulating various cell functions such as cell survival, cell proliferation, apoptosis, DNA repair, and metabolic processes (Furukawa-Hibi et al., 2005; Paik et al., 2007; Liu et al., 2009). FOXO is involved in cell growth and apoptosis by directly inducing FOXO3a-dependent apoptotic protein Bim expression (Gilley et al., 2003) and by activating caspase family (Brunet et al., 1999). A previous study showed that propofol significantly increased the protein levels of FOXO1, FOXO3, Bim, procaspase-3and activated caspase-3 in lung cancer cells (Yang et al., 2017). Therefore, in the present study, we explored whether propofol could affect the protein levels of FOXO1, FOXO3, Bim, procaspase-3, and activated caspase-3 in PC12 cells. Our data indicated that the expression levels of FOXO1, FOXO3, Bim, procaspase-3, and active caspase-3 were found to increase in a dose-dependent manner in response to propofol treatment for 48 h (Fig. 5).

FIG. 5.

Effect of propofol on FOXO1, FOXO3, Bim, procaspase-3, and active caspase-3 expression in PC12 cells. Forty-eight hours after treatment with different concentrations of propofol (0, 1, 5, and 10 μg/mL), mRNA (B–D) and protein (A) expression level of FOXO1, FOXO3, and Bim expression in PC12 cells were determined by quantitative real-time polymerase chain reaction and western blotting, respectively. The protein expression level of procaspase-3 and active caspase-3 was detected by western blotting (A). Data are reported as mean ± SD. *p < 0.05, **p < 0.01 versus control group.

Propofol reduced the growth of PCC tumors in a PC12 xenograft model

A previous study indicated that propofol has potential anticancer activity in vitro, and the present study hypothesized that propofol could inhibit PCC tumor growth in vivo. A PC12 cell xenograft model was established in this study. The PCC tumor volume and tumor weight were detected 14 days after treatment, and the results showed that the PCC tumor volume and weight of the propofol treatment group were significantly lower than that of the sham group (Fig. 6A–C). Furthermore, propofol was found to significantly induce cell apoptosis in PCC tumors compared with the sham group (Fig. 6D).

FIG. 6.

Propofol inhibits the growth of pheochromocytoma tumors. (A) Representative tumor images after vehicle or propofol treatment; (B) Tumor volume is expressed in cubic millimeters, and calculated as 0.5236 × (length × width × height) in either vehicle or propofol (35 mg/kg, 14 days)-treated mice; (C) The average tumor weights of mice treated with propofol or vehicle for 14 days; (D) Percentage of apoptotic cells in PC12 xenografts of mice treated with propofol or vehicle for 14 days. **p < 0.01.

Discussion

Due to the low incidence of PCC tumors and its complex pathogenesis, the treatment of malignant PCC is quite limited (Nilsson et al., 2009; Lian et al., 2017). Therefore, a few effective chemotherapeutic drugs are used to treat malignant PCC (Casaccia et al., 2017; Caza et al., 2017). Finding a new effective diagnosis and treatment method has been a hot issue in the study of malignant PCC. Chromogranin A and plasma-free metanephrine or normetanephrine determined by radioimmunoassay have been considered as the effective biomarkers of PCC or paraganglioma (Bílek et al., 2017). The report of Yu (2017) suggested that the use of proteasome inhibitors may be a potential medical therapy for malignant PCC. Bortezomib alone or in combination with salinosporamide A can promote PCC cell death (Bullova et al., 2017).

Studies have shown that propofol has not only an anesthetic effect but also an anticancer function. Propofol is one of the most common intravenous anesthetics used in the tumor resection operation (Siddiqui et al., 2005; Spangenberg et al., 2008; Venook et al., 2010). The inhibitory effect of propofol on the growth and metastasis of tumor cells and its effect on tumor immunity have been gradually noticed (Wang et al., 2015; Liu et al., 2016). The purpose of this study was to investigate the effect of propofol on malignant PCC in vivo and in vitro.

This study demonstrated that propofol could effectively suppress tumor growth and induced cell apoptosis in malignant PCC in vitro and in vivo. In view of the effect of propofol drug, an alternative therapy for malignant PCC may be considered using propofol alone or combined with other methods. First, the proliferation ability of PC12 cells was found to be significantly inhibited by propofol in a dose- and time-dependent manner. In addition, the data suggested that propofol dose dependently repressed PC12 cell migration and invasive capability. Furthermore, the findings of this study indicated that propofol prevented PC12 cell proliferation and metastasis through inducing cell cycle arrest and cell apoptosis through regulating cell growth and cell apoptosis-related genes, which were consistent with previous studies (Hsu et al., 2017; Meng et al., 2017; Yang et al., 2017).

Finally, a PC12 xenograph model, which has been used in a variety of studies on PCC, was established to perform our in vivo study, and the results from animal experiment were consistent with results from cell experiments.

PC12 cells are widely used in in vitro studies of neurological diseases. However, using PC12 cell xenograft model to study the PCC in vivo has some limitations. (1) PCC is a tumor derived from chromogranules, most PCCs originate from the adrenal medulla while PCCs outside the adrenal gland originate from the sympathetic ganglia and parasympathetic ganglia. PC12 cells are derived from a transplantable rat adrenal medullary PCC, so PC12 xenografts cannot represent all kinds of PCCs. (2) PC12 cells are derived from rattus norvegicus adrenal PCC; thus, this is in fact a rat PCC model, and because of the differences in species, the actual effect of propofol on human PCC may be different. Therefore, the effect of propofol on human PCC, therefore, needs a large number of experimental studies.

Taken together, this study showed that propofol effectively inhibited PCC cell proliferation, migration, and invasion; induced cell cycle arrest and apoptosis; and suppressed PCC tumor growth in the xenograft mouse model. In addition, these effects were mediated by the downregulation and inactivation of proteins involved in cell survival and cell apoptosis. Propofol is a promising new chemotherapeutic small molecule for treating malignant PCC.

Conclusion

Propofol prevents the progression of malignant PCC in vitro and in vivo, and it is a promising new chemotherapeutic small molecule for the treatment of malignant PCC.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Beattie

M.K.

, and Heasman

M.A.

(1958). The pituitary and adrenal glands of elderly mental hospital patients with and withouthypertension. J Pathol Bacteriol, 75, 83–94.

Björklund

, and Backman

(2017). Epigenetics of pheochromocytoma and paraganglioma. Mol Cell Endocrinol. PMID: 28630023; DOI: 10.1016/j.mce.2017.06.016.

Brunet

, Bonni

, Zigmond

M.J.

, Lin

M.Z.

, Juo

, Hu

L.S.

, et al. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell, 96, 857–868.

Bílek

, Zelinka

, Vlček

, et al. (2017). Radioimmunoassay of chromogranin A and free metanephrines in diagnosis of pheochromocytoma. Physiol Res, 66, S397–S408.

Bullova

, Cougnoux

, Marzouca

, Kopacek

, and Pacak

(2017). Bortezomib alone and in combination with salinosporamid a induces apoptosis and promotes pheochromocytoma cell death in vitro and in female nude mice. Endocrinology, 158, 3097–3108.

Casaccia

, Macina

, and Fornaro

(2017). Single-stage laparoscopic adrenalectomy for pheochromocytoma and enucleation of a pancreatic neuroendocrine tumor in Von Hippel-Lindau disease: a case report. Mol Clin Oncol, 6, 799–801.

Caza

, Manwaring

, and Riddell

(2017). Recurrent, bilateral, and metastatic pheochromocytoma in a young patient with Beckwith-Wiedemann syndrome: a genetic link?. Can Urol Assoc J, 11, E240–E243.

Dong

, Zhang

, Zhou

, Ye

, Chen

, Yuan

, et al. (2017). Maslinic acid promotes autophagy by disrupting the interaction between Bcl2 and Beclin1 in rat pheochromocytoma PC12 cells. Oncotarget, 8, 74527–74538.

Fishbein

(2016). Pheochromocytoma and paraganglioma: genetics, diagnosis, and treatment. Hematol Oncol Clin North Am, 30, 135–150.

10.

Furukawa-Hibi

, Kobayashi

, Chen

, and Motoyama

(2005). FOXO transcription factors in cell-cycle regulation and the response to oxidative stress. Antioxid Redox Signal, 7, 752–760.

11.

Gilley

, Coffer

P.J.

, and Ham

(2003). FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. J Cell Biol, 162, 613–622.

12.

Gimenez-Roqueplo

A.P.

(2015). Pheochromocytoma and paraganglioma. Rev Prat, 65, 826–830.

13.

Hsu

S.S.

, Jan

C.R.

, and Liang

W.Z.

(2017). Evaluation of cytotoxicity of propofol and its related mechanism in glioblastoma cells and astrocytes. Environ Toxicol, 32, 2440–2454.

14.

Lentschener

, Gaujoux

, Tesniere

, and Dousset

(2011). Point of controversy: perioperative care of patients undergoing pheochromocytoma removal-time for a reappraisal?. Eur J Endocrinol, 165, 365–373.

15.

Lian

, Lin

, Xie

, Xu

, Meng

, et al. (2017). nVP-aUY922, a novel hsP90 inhibitor, inhibits the progression of malignant pheochromocytoma in vitro and in vivo. Onco Targets Ther, 10, 2219–2226.

16.

Lin

, Meng

, Xu

, Lian

, Xu

, Xie

, et al. (2017). Enhanced wild-type p53 expression by small activating RNA dsP53-285 induces cell cycle arrest and apoptosis in pheochromocytoma cell line PC12. Oncol Rep, 38, 3160–3166.

17.

Liu

, Rudd

M.D.

, Hernandez-Gonzalez

, Gonzalez-Robayna

, Fan

H.Y.

, Zeleznik

, et al. (2009). FSH and FOXO1 regulate genes in the sterol/steroid and lipid biosynthetic pathways in granulosa cells. Mol Endocrinol, 23, 649–661.

18.

Liu

, Zhang

, Hong

, Quan

, Zhang

, Yu

, et al. (2016). Propofol inhibits growth and invasion of pancreatic cancer cells through regulation of the miR-21/Slug signaling pathway. Am J Transl Res, 8, 4120–4133.

19.

Lowery

A.J.

, Walsh

, McDermott

E.W.

, and Prichard

R.S.

(2013). Molecular and therapeutic advances in the diagnosis and management of malignant pheochromocytomas and paragangliomas. Oncologist, 18, 391–407.

20.

Meng

, Song

, Wang

, et al. (2017). Propofol induces proliferation partially via downregulation of p53 protein and promotes migration via activation of the Nrf2 pathway in human breast cancer cell line MDA-MB-231. Oncol Rep, 37, 841–848.

21.

Mohammed

A.A.

, El-Shentenawy

A.M.

, Sherisher

M.A.

, and El-Khatib

H.M.

(2014). Target therapy in metastatic pheochromocytoma: current perspectives and controversies. Oncol Rev, 8, 249.

22.

Nilsson

, Nilsson

, and Ahlman

(2009).Treatment of gastrointestinal stromal tumor after imatinib and sunitinib—and then?. Expert Opin Investig Drugs, 18, 457–468.

23.

Ozer

A.B.

, Demirel

, Duzgol

, Ayten

, and Erhan

O.L.

(2014). Management of anesthesia in unspecified extra-adrenal pheochromocytoma patient who used beta-blocker. Saudi J Anaesth, 8, S105–S108.

24.

Paik

J.H.

, Kollipara

, Chu

, Ji

, Xiao

, Ding

, et al. (2007). FoxOs are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell, 128, 309–323.

25.

Pang

, Yang

, Schovanek

, Wang

, Bullova

, Caisova

, et al. (2017). Anthracyclines suppress pheochromocytoma cell characteristics, including metastasis, through inhibition of the hypoxia signaling pathway. Oncotarget, 8, 22313–22324.

26.

Roizn

M.F.

(2001). Anesthetic Implications of Concurrent Diseases/ Miller

, eds. Anesthesia, 5th ed. (San Francisco, Harcourt Asia Churchill Livingstone) pp. 906–910.

27.

Shim

I.K.

, Yi

H.J.

, Yi

H.G.

, Lee

C.M.

, Lee

Y.N.

, Choi

Y.J.

, et al. (2017). Locally-applied 5-fluorouracil-loaded slow-release patch prevents pancreatic cancer growth in an orthotopic mouse model. Oncotarget, 8, 40140–40151.

28.

Shinn

H.K.

, Jung

J.K.

, Park

J.K.

, Kim

J.H.

, Jung

I.Y.

, and Lee

H.S.

(2012). Hypertensive crisis during wide excision of gastrointestinal stromal cell tumor (GIST): undiagnosed paraganglioma-A case report. Korean J Anesthesiol, 62, 289–292.

29.

Siddiqui

R.A.

, Zerouga

, Wu

, et al. (2005). Anticancer properties of propofol-docosahexaenoate and propofol-eicosapentaenoate on breast cancer cells. Breast Cancer Res, 7, R645–R654.

30.

Spangenberg

H.C.

, Thimme

, and Blum

H.E.

(2008). Evolving therapies in the treatment of hepatocellular carcinoma. Biologics, 2, 453–462.

31.

Venook

A.P.

, Papandreou

, Furuse

, et al. (2010). The incidence and epidemiology of hepatocellular carcinoma: a global and regional perspective. Oncologist, 15, 5–13.

32.

Voros

D.C.

, Smyrniotis

, Argyra

, et al. (1996). Undiagnosed phaeochromocytoma in the perioperative period. Eur J Surg, 162, 985–987.

33.

Wang

, Song

, Wang

, Zhao

, Xiang

, Li

, et al. (2017). ARHI is a novel epigenetic silenced tumor suppressor in sporadic pheochromocytoma. Oncotarget, 8, 86325–86338.

34.

Wang

, Lin

, Zhang

, et al. (2015). Effects of propofol on pulmonary metastasis of intravenously injected MADB106 tumor cells and expression of E-cadherin and β-catenin in rats. Nan Fang Yi Ke Da Xue Xue Bao, 35, 852–856.

35.

Yang

, Gao

, Yan

, et al. (2017). Propofol inhibits the growth and survival of gastric cancer cells in vitro through the upregulation of ING3. Oncol Rep, 37, 587–593.

36.

Yang

, Liang

, Yang

, and Jiang

(2017). Propofol inhibits lung cancer cell viability and induces cell apoptosis by upregulatingmicroRNA-486 expression. Braz J Med Biol Res, 50, e5794.

37.

(2017). Proteasome inhibitors: a potential medical therapy for malignant pheochromocytoma. Endocrinology, 158, 3083–3085.

38.

Zhang

, Gupta

, and Albert

S.G.

(2017). Pheochromocytoma as a reversible cause of cardiomyopathy: analysis and review of the literature. Int J Cardiol, 249, 319–323.

39.

Zhang

, Wang

, Qin

, Xu

, Zhu

, Zhong

, et al. (2015). The dual mTORC1 and mTORC2 inhibitor PP242 shows strong antitumor activity in a pheochromocytoma PC12 cell tumor model. Urolog, 85, 273.e1–e7.