- Email: [email protected]
Isoflurane enhances malignancy of head and neck squamous cell carcinoma cell lines: A preliminary study in vitro
Oral Oncology 47 (2011) 329–333
Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Isoflurane enhances malignancy of head and neck squamous cell carcinoma cell lines: A preliminary study in vitro Ren Jun a,1, Zhang Gui-he b,1, Sun Xing-xing a, Zhang Hui a, Xu Li-xian a,⇑ a b
Department of Anesthesiology, School of Stomatology, Fourth Military Medical University, Xi’an 710032, PR China Department of Anesthesiology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, PR China
a r t i c l e
i n f o
Article history: Received 3 January 2011 Received in revised form 22 February 2011 Accepted 1 March 2011 Available online 26 March 2011 Keywords: Cancer of head and neck Squamous cell carcinoma Cell proliferation Apoptosis Isoflurane Inhalation anesthetics Flow cytometry Neoplasm invasion Cell migration Balanced anesthesia Anesthetic drugs
s u m m a r y The objective of this study was to explore the influence of isoflurane on the cell proliferation, apoptosis, and invasion of Tca8113 and HSC2 cell lines in vitro. MTT test was used to detect the cell proliferation. It was performed 72 h after exposure to isoflurane to make sure that a time for normal cell cycle progression was allowed. The cell apoptosis of Tca8113 and HSC2 cell lines were detected by flow cytometry. We used transwell chamber to detect the cell invasion of Tca8113 and HSC2 cell lines. There was a statistically significant increase of cell proliferation in Tca8113 and HSC2 cell lines after exposure to 2% isoflurane for 3 and 6 h. The difference between 3 and 6 h group is statistically significant in Tca8113 and HSC2 cell lines. Flow cytometry showed that there was a decrease of cell apoptosis in Tca8113 and HSC2 cell lines after exposure to 2% isoflurane for 3 and 6 h. Transwell test showed there was a statistically significant increase of cell invasion in Tca8113 and HSC2 cell lines after exposure to 2% isoflurane for 3 and 6 h, and it showed a significant difference between 3 h group and 6 h group of Tca8113 cell line. Our results demonstrated that isoflurane increased malignancy of head and neck squamous cell carcinoma cell lines in vitro. Isoflurane might enhance tumor development and promote metastasis of tumor cells in HNSCC patients. It is suggested that it might be more suitable to choose total intravenous anesthesia for HNSCC patients. Ó 2011 Elsevier Ltd. All rights reserved.
Introduction Head and neck squamous cell carcinoma (HNSCC) is one of the six most common cancers in the world, with approximately 500,000 new cases diagnosed and 250,000 deaths per year.1 Despite much medical improvement over the past two decades, the overall survival rate of HNSCC still has remained poor (5 years survival rate about 50%).2 Among various treatments, surgery is still the mainstream mode of initial definitive treatment for the majority of oral cancers.3 Balanced anesthesia (inhalational combined intravenous anesthesia) is the most widely used anesthesia mode in the operation of head and neck tumor surgery.4 Compared to the total intravenous anesthesia, balanced anesthesia allows to obtain a better haemodynamic stability during the surgery and a
Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; FCM, flow cytometry; FITC, fluorescein isothiocyanate; HNSCC, head and neck squamous cell carcinoma; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; PI, propidium iodide. ⇑ Corresponding author. Address: Department of Anesthesiology, School of Stomatology, Fourth Military Medical University, 145#, Changle Xi Road, Xi’an 710032, PR China. Tel./fax: +86 29 84776115. E-mail address: [email protected] (X. Li-xian). 1 These authors contributed equally to this paper. 1368-8375/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2011.03.002
faster and safer recovery of consciousness after the surgery,5 which is especially benefit for old HNSCC patients. Isoflurane, a commonly used inhalational anesthetic in the balanced anesthesia, induces cytotoxicity in both a concentrationand time-dependent manner in different types of cultured cells.6– 8 At clinical relevant concentrations, isoflurane caused widespread neuronal apoptosis in developing rat brains and apoptosis in human T lymphocytes in vitro.9,10 However, in other studies, isoflurane inhibits cardiac myocyte apoptosis during oxidative and inflammatory stress and attenuates apoptosis in response to ischemia or other forms of tissue injury.11–13 All above indicate that isoflurane might have anti-apoptosis and pro-apoptosis potency according to different circumstances. The pre-operative and post-operative therapies for HNSCC patients have been well considered. However, the risks during the surgery operation period are often neglected. The period of oral cancer surgery is often lasting for 4–6 h, during this time the effect of isoflurane on tumor cells is unknown. It has been reported that clinically relevant concentration of isoflurane induces cell apoptosis in several carcinoma cell lines in vitro,7 but there is no report of influence of isoflurane on HNSCC cells. HNSCC is highly invasive, frequently metastasizing to cervical lymph nodes and corresponds with poor prognosis.14 Therapeutic intervention of tumor invasion is becoming recognized as an
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increasingly relevant clinical factor.15 However, the impact of isoflurane on the invasion of HNSCC cells has never been reported. Our aim is to observe the influence of clinical relevant concentration of isoflurane on HNSCC cells. Tca8113 and HSC2 are two widely used HNSCC cell lines. So in our study, we observed the influences of isoflurane on the cell viability, cell apoptosis and cell invasion of Tca8113 and HSC2 cell lines in vitro. Materials and methods Cell culture Tca8113 and HSC2 cell lines were grown as monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U penicillin, and 0.1 mg/ml streptomycin. The cultures were equilibrated with humidified 5% CO2 in air at 37 °C in CO2 incubator (Shell Lab, Sheldon Manufacturing, USA). Anesthetic exposure Two percent isoflurane which is the clinically used concentration, 21% O2 and 5% CO2 were delivered from an anesthesia machine to a sealed plastic box in a 37 °C incubator containing sixwell plates seeded with one million cells in 1.5 ml cell culture media. A Datex infrared gas analyzer (Puritan-Bennett, Tewksbury, MA) was used to continuously monitor the delivered carbon dioxide, oxygen, and isoflurane concentrations. A stable gas and volatile anesthetic concentrations were obtained within 5 min. We treated the cells with 2% isoflurane for 3 and 6 h. Control conditions included only 5% CO2 plus 21% O2 in incubator. Cell proliferation study The cell proliferation was determined by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma). The test was performed 72 h after isoflurane exposure. This allowed a time for at least two normal cell cycles. Controls were grown under the same conditions, without anesthetic. Four hours before end of incubation, medium was aspirated and the MTT reduction product, formazan, was dissolved in dimethyl sulfoxide (DMSO) and quantified spectrophotometrically at 570 nm. We presented the changes in the absorbance, as a measure of cell proliferation, in the cells treated with isoflurane as the percentage of those in the cells treated with control conditions. Cell apoptosis analysis To detect phosphatidylserine externalization (on the surface of cell membrane), an indicator of early apoptosis, flow cytometry (FCM, BD Biosciences, USA) was performed with fluorescein isothiocyanate (FITC)-labeled Annexin V (Joincare Biosciences, Zhuhai, China). Propidium iodide (PI), a cell dye, can bind to nucleic acid after penetrating a breached plasma membrane, as occurs in the later stages of cell damage. After treatment, cells were washed with cold PBS at 4 °C. After centrifugation at 1500 rpm for 5 min, 500 lL of 1 binding buffer, 5 lL of FITC-labeled Annexin V and 10 lL of PI were added to the cell suspension and gently mixed. After incubation at 25 °C for 10 min in the dark, the cells were analyzed by FCM. In vitro cell invasion assay Tca8113 and HSC2 cell lines that were exposed to isoflurane were used for the following experiments immediately. A total of
1 105 cells in 0.5 ml serum-free DMEM medium were seeded on a 8 lm-pore polycarbonate membrane Boyden chambers insert in a transwell apparatus (Costar, Cambridge, MA), coated with Matrigel (BD Biosciences, San Jose, CA). 600 lL DMEM containing 10% FBS was added to the lower chamber. After the cells were incubated for 24 h at 37 °C in a 5% CO2 incubator, cells on the top surface of the insert were removed by wiping with a cotton swab. Cells that migrated to the bottom surface of the insert were fixed in 100% methanol for 2 min, stained with HE solution and then subjected to microscopic inspection (200). Values for invasion were obtained by counting five fields per membrane and represent the average of three independent experiments. Statistical analysis The results of cell proliferation and cell invasion were expressed as the mean ± SD. The results of cell apoptosis were expressed as the median and 25–75%. Statistical differences of cell proliferation and cell invasion were estimated by one-way analysis of variance (ANOVA) followed by SNK-q test. Statistical differences of cell apoptosis were estimated by Kruskal–Wallis test. Those P values that were less than 0.05 were considered statistically significant. Analysis of the data and plotting of the figures were performed with the aid of software (Origin Version 7.5 and SPSS Version 13.0).
Results Cell proliferation of Tca8113 and HSC2 cell lines after 2% isoflurane exposure in vitro In order to determine antiproliferative potency of single exposure of isoflurane to Tca8113 and HSC2 cell lines, MTT was performed after 72 h. Thus, a time for normal cell cycle progression was allowed. Both HNSCC cell lines showed growth alterations as a consequence of single exposure to isoflurane. Results of MTT test are shown on Fig. 1. Tca8113 cell line expressed statistically significant increase (P < 0.05) in each particular time of exposure to isoflurane (Fig. 1A). The growth of treated cells in comparison to control was 116% after 3 h, and increased at 127% after 6 h. There is also a significant difference between 3 h group and 6 h group (P < 0.05).HSC2 cell line showed marked growth increase (Fig. 1B), statistically significant in each particular time of exposure to isoflurane (P < 0.05). Cell growth increased to 112% after 3 h and 119% after 6 h. Significant difference existed between 3 h group and 6 h group (P < 0.05). These data demonstrated that isoflurane increased the cell proliferation of Tca8113 and HSC2 cell lines. Cell apoptosis of Tca8113 cell line and HSC2 cell lines after 2% isoflurane exposure in vitro To quantitate cell apoptosis in response to isoflurane, flow cytometric analysis with Annexin V-PI staining is chosen. Early apoptosis is defined by Annexin V+/PI staining (Q4) and late apoptosis is defined by Annexin V+/PI+ staining (Q2) as determined by FCM. As shown in a representative experiment in Fig. 2A, 2% isoflurane exposure decreased the apoptosis rate (Q2 + Q4) of Tca8113 cell line. Fig. 2B showed that the apoptosis rate (Q2 + Q4) of HSC2 cell line decreased after exposed to 2% isoflurane. Compared with control group, the difference of apoptosis rate (Q2 + Q4) of Tca8113 cell line in 6 h group was statistically significant, not in 3 h group (P < 0.05) (Table 1). HSC2 cell line in 3 and 6 h group were statistically significant compared to control group (P < 0.05). As shown in Table 1, there were significant differences between 3 and 6 h group in both Tca8113 and HSC2 cell lines. These data suggested that
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Figure 1 Isoflurane increased cell proliferation of Tca8113 and HSC2 cell lines. Cell proliferation was assessed by MTT in Tca8113 and HSC2 cell lines. The 2% isoflurane treatment increased cell proliferation as compared with control conditions in Tca8113 cell line (A) and HSC2 cell line (B). Significant difference existed between 3 h group and 6 h group (P < 0.05) in both cell lines. Bars represent standard error from mean value. (⁄P < 0.05 as compared with control; #P < 0.05 as compared with 3 h group).
Figure 2 Isoflurane decreased cell apoptosis of Tca8113 and HSC2 cell lines by flow cytometry analysis using Annexin V-FITC and propidium iodide. The experiments were performed after treatment with 2% isoflurane for 3 and 6 h. Cell populations in bottom right and top right quadrants represented the proportion of late apoptotic and early apoptotic, respectively. The figure demonstrated the decrease of apoptosis rate (Q2 + Q4) in Tca8113 (A) and HSC2 (B) cell lines.
isoflurane could significantly decrease cell apoptosis in both Tca8113 and HSC2 cell lines. In vitro cell invasion of Tca8113 and HSC2 cell lines after isoflurane exposure To measure the effect of isoflurane on cell invasion of Tca8113 and HSC2 cell lines, cells growing in the log phase exposed to isoflurane were collected and cultured on Transwell apparatus. Data
Table 1 Quantification of apoptosis by Annexin V-FITC/PI staining.
Tca8113 HSC2
Control
3h
6h
8.8 (6.5–11.1) 11.9 (7–15.3)
7.8 (4.4–8.7) 5.95 (4.7–9.7)*
6.3 (4.2–7.3)*# 4.7 (3.6–6.6)*#
Data are presented as median (interquartile range). * P < 0.05 as compared with control, # P < 0.05 as compared with 3 h group.
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Figure 3 Isoflurane increased cell invasion of Tca8113 and HSC2 cell lines. Representative photos and statistic plots of invasion assay in Tca8113 cell line and HSC2 cell line (200). The invasion cell number of Tca8113 cell line increased in the isoflurane treated groups compared with that of control (P < 0.05) (A). The invasion cell number of HSC2 cell line increased in the isoflurane exposure groups compared with that of control (P < 0.05) (B). There was a significant difference in cell invasion number between 3 h group and 6 h group in Tca8113 cell line, but not in HSC2 cell line. Bars represent standard error from mean value. (⁄P < 0.05 as compared with control; #P < 0.05 as compared with 3 h group).
are shown on Fig. 3. After 12 h incubation, the cell number of Tca8113 cell line was significantly increased in 3 and 6 h group than that in control group (P < 0.05) (Fig. 3A). It also showed a significant difference between 3 h group and 6 h group (P < 0.05) of Tca8113 cell line. There was a significant increase of cell number of HSC2 cell line in 3 and 6 h group, compared with the control group (P < 0.05) (Fig. 3B). However, there was not a significant difference in cell invasion number between 3 h group and 6 h group in HSC2 cell line. These results demonstrated that isoflurane increased the cell invasion in both Tca8113 and HSC2 cell lines.
Discussion General anesthesia is a drug-induced, reversible condition which provides amnesia, analgesia, immobility, and control of autonomic responses during surgical procedures.16 Though total intravenous anesthesia developed rapidly in recent years, balanced anesthesia combined with isoflurane is still adopted in the majority of general anesthesia.17 Clinical relevant concentration of isoflurane showed little or no influence on normal cell lines in growth inhibition and cell apoptosis.7,18 Concerning for tumor cell lines after exposure to inhalational anesthetics, the results were not consistent. The study of O’Leary has found that inhalational anesthetic suppressed the growth of tumor cells.19 Moudgil and Singal reported the opposite results that inhalational anesthetic enhanced tumor metastasis in mice.20 In our study, we found that 2% isoflurane increased cell proliferation in both HNSCC cell lines after single exposure for 3 and 6 h. And this clinical relevant concentration of isoflurane
inhibited cell apoptosis in our experiment. These results are different from that mentioned in the study of O’Leary. Furthermore, we firstly found that 2% isoflurane increased cell invasion in HNSCC cell lines in vitro. There are a number of factors in the perioperative period that may result in immunosuppression.21 Manipulation of the primary tumor during surgery is associated with release of tumor cells into the circulation.22 In addition, surgery induces a profound stress response, including neuroendocrine, cytokine, and metabolic response.23 Anesthesia itself contributes to perioperative immunosuppression.21,24,25 Inhalational anesthetics used in the general anesthesia can modulate immune defense, especially when applied in higher concentrations or doses or for longer period of time.26 Isoflurane has been shown to induce caspase-dependent, mitochondria-mediated apoptosis in human T cells.10 This, in turn, causes suppression of natural killer cell activity. This compromises host immune function, which has been shown to enhance tumor development and promote metastasis of tumor cells in patients.27 Beside the above indirect influences of inhalational anesthetics on cancer cells, there are also direct influences of isoflurane on cancer cells. As shown in our study, 2% isoflurane has a direct enhancement on the cell proliferation of head and neck squamous cell carcinoma cell lines in vitro. A tumor cell proliferation after exposure to clinically relevant concentrations of isoflurane appears important in the view of altered metastatic capability of tumor cells that enter into circulation during tumor and organ resections.7 As the time for at least two normal cell cycles was allowed, our results provided an indication as to the consequence on ongoing and subsequent proliferative effects. Spontaneous apoptosis in tumor cells reflect cellular metabolism and growth condition.7 The inhibition
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of isoflurane in HNSCC cells might attribute to the increasing of cell proliferation. Though there were differences between early (Q4) and late apoptosis (Q2) in both cell lines, the total apoptosis (Q2 + Q4) in different time points is more meaningful in consideration. The exact mechanism of isoflurane in inhibiting apoptosis of head and neck cell lines is unknown, but isoflurane might influence caspase-dependent apoptotic cell death pathway to inhibit HNSCC cell apoptosis as in T lymphocyte.10 Increased cell migration is one of the critical steps in the development of metastasis.28 In cell invasion study, we found significant differences compared to control after exposed to isoflurane in both cell lines. The above results in our study provided some evidences that isoflurane enhanced malignancy of HNSCC cell lines in vitro. It is a controversial which anesthesia mode is more suitable for HNSCC patients.17,29 Most anesthesiologists focused their studies on the influences of different anesthesia techniques on haemodynamic stability during the surgery and recovery of consciousness after the surgery.5 Balanced anesthesia has better haemodynamic stability and fewer drugs required during the surgery, so it becomes the mainstream anesthesia mode in the head and neck tumor surgery.4 However, the influences of different anesthesia techniques and anesthetic drugs on tumor cell of HNSCC are often ignored. The anesthesia technique may not impact the recovery of patients underwent ordinary head and neck surgery such as benign tumor, mandible fracture and plastic surgery. However, for cancer patients, the least impaction of anesthesia technique and anesthesia drugs on tumor cells should be chosen. As demonstrated in our study, isoflurane has a direct influence on the biological features of HNSCC cell lines in vitro, so it might not be suitable to choose isoflurane in the surgery for HNSCC patients. Though it might increase the possible implications on hemodynamic stability and safer recovery of consciousness after surgery, total intravenous anesthesia might be a suggested choice for HNSCC patients now. If there are some other inhalational anesthetics emerged in future which do not show adverse influences on HNSCC cell lines, balanced anesthesia with inhalational anesthetic is still a better choice for its less implications during the surgery. Our studies also have limitations. We used in vitro HNSCC cell lines cultures. These cells are obviously different from in situ HNSCC. It is also impossible to provide cell cultures with an environment identical to what the in vivo carcinoma cells have, such as local pH, oxygen, and electrolyte concentrations, and the support and interaction from other cells. Furthermore, the mechanisms, which isoflurane enhances malignancy of Tca8113 and HSC2 cell lines after exposure, need to be investigated. In conclusion, our data provided some evidences in vitro that balanced anesthesia combined with isoflurane might not be suitable for HNSCC patients. It might be more suitable to choose total intravenous anesthesia for HNSCC patients during the surgery. Conflicts of interest statement None declared. Acknowledgment The study was supported by the National Natural Science Foundation of China (No. 30772073). References 1. Molinolo AA, Amornphimoltham P, Squarize CH, Castilho RM, Patel V, Gutkind JS. Dysregulated molecular networks in head and neck carcinogenesis. Oral Oncol 2009;45(4–5):324–34.
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2. Fuller CD, Wang SJ, Thomas Jr CR, Hoffman HT, Weber RS, Rosenthal DI. Conditional survival in head and neck squamous cell carcinoma: results from the SEER dataset 1973–1998. Cancer 2007;109(7):1331–43. 3. Kalavrezos N, Bhandari R. Current trends and future perspectives in the surgical management of oral cancer. Oral Oncol 2010;46(6):429–32. 4. Lopez Correa T, Sanchez Hernandez MV, Briz Sanchez E, Estevez Amores FI. Anesthesia in head and neck paragangliomas. Acta Otorrinolaringol Esp 2009; 60(Suppl 1):76–9. 5. Juckenhofel S, Feisel C, Schmitt HJ, Biedler A. TIVA with propofol-remifentanil or balanced anesthesia with sevoflurane-fentanyl in laparoscopic operations. Hemodynamics, awakening and adverse effects. Anaesthesist 1999;48(11): 807–12. 6. Wei H, Kang B, Wei W, Liang G, Meng QC, Li Y, et al. Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently. Brain Res 2005;1037(1–2): 139–47. 7. Kvolik S, Glavas-Obrovac L, Bares V, Karner I. Effects of inhalation anesthetics halothane, sevoflurane, and isoflurane on human cell lines. Life Sci 2005;77(19): 2369–83. 8. Xie Z, Dong Y, Maeda U, Alfille P, Culley DJ, Crosby G, et al. The common inhalation anesthetic isoflurane induces apoptosis and increases amyloid beta protein levels. Anesthesiology 2006;104(5):988–94. 9. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, et al. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003;23(3):876–82. 10. Loop T, Dovi-Akue D, Frick M, Roesslein M, Egger L, Humar M, et al. Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro. Anesthesiology 2005;102(6):1147–57. 11. Jamnicki-Abegg M, Weihrauch D, Pagel PS, Kersten JR, Bosnjak ZJ, Warltier DC, et al. Isoflurane inhibits cardiac myocyte apoptosis during oxidative and inflammatory stress by activating Akt and enhancing Bcl-2 expression. Anesthesiology 2005;103(5):1006–14. 12. Zaugg M, Jamali NZ, Lucchinetti E, Shafiq SA, Siddiqui MA. Norepinephrineinduced apoptosis is inhibited in adult rat ventricular myocytes exposed to volatile anesthetics. Anesthesiology 2000;93(1):209–18. 13. Wise-Faberowski L, Raizada MK, Sumners C. Oxygen and glucose deprivationinduced neuronal apoptosis is attenuated by halothane and isoflurane. Anesth Analg 2001;93(5):1281–7. 14. Kramer RH, Shen X, Zhou H. Tumor cell invasion and survival in head and neck cancer. Cancer Metastasis Rev 2005;24(1):35–45. 15. Dolgin E. Cancer metastasis scrutinized. Nature 2009;461(7266):854–5. 16. Brown EN, Lydic R, Schiff ND. General anesthesia, sleep, and coma. N Engl J Med 2010;363(27):2638–50. 17. Lerman J, Johr M. Inhalational anesthesia vs total intravenous anesthesia (TIVA) for pediatric anesthesia. Paediatr Anaesth 2009;19(5):521–34. 18. Sakahira H, Enari M, Ohsawa Y, Uchiyama Y, Nagata S. Apoptotic nuclear morphological change without DNA fragmentation. Curr Biol 1999;9(10): 543–6. 19. O’Leary G, Bacon CL, Odumeru O, Fagan C, Fitzpatrick T, Gallagher HC, et al. Antiproliferative actions of inhalational anesthetics: comparisons to the valproate teratogen. Int J Dev Neurosci 2000;18(1):39–45. 20. Moudgil GC, Singal DP. Halothane and isoflurane enhance melanoma tumour metastasis in mice. Can J Anaesth 1997;44(1):90–4. 21. Deegan CA, Murray D, Doran P, Ecimovic P, Moriarty DC, Buggy DJ. Effect of anaesthetic technique on oestrogen receptor-negative breast cancer cell function in vitro. Br J Anaesth 2009;103(5):685–90. 22. Ben-Eliyahu S. The price of anticancer intervention. Does surgery promote metastasis? Lancet Oncol 2002;3(9):578–9. 23. Tsuchiya Y, Sawada S, Yoshioka I, Ohashi Y, Matsuo M, Harimaya Y, et al. Increased surgical stress promotes tumor metastasis. Surgery 2003;133(5): 547–55. 24. Marsh L, Dawson TM. Treatment of early Parkinson’s disease. BMJ 2000; 321(7252):1–2. 25. Rodgers A, Walker N, Schug S, McKee A, Kehlet H, van Zundert A, et al. Reduction of postoperative mortality and morbidity with epidural or spinal anaesthesia: results from overview of randomised trials. BMJ 2000;321(7275): 1493. 26. Loop T, Scheiermann P, Doviakue D, Musshoff F, Humar M, Roesslein M, et al. Sevoflurane inhibits phorbol-myristate-acetate-induced activator protein-1 activation in human T lymphocytes in vitro: potential role of the p38-stress kinase pathway. Anesthesiology 2004;101(3):710–21. 27. Ben-Eliyahu S, Page GG, Yirmiya R, Shakhar G. Evidence that stress and surgical interventions promote tumor development by suppressing natural killer cell activity. Int J Cancer 1999;80(6):880–8. 28. Minard ME, Kim LS, Price JE, Gallick GE. The role of the guanine nucleotide exchange factor Tiam1 in cellular migration, invasion, adhesion and tumor progression. Breast Cancer Res Treat 2004;84(1):21–32. 29. Theodossy T, Chapman M, Mitchell V, Hopper C. Anaesthetic considerations for patients receiving photodynamic therapy in head and neck surgery. Eur J Anaesthesiol 2007;24(3):225–9.
Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Isoflurane enhances malignancy of head and neck squamous cell carcinoma cell lines: A preliminary study in vitro Ren Jun a,1, Zhang Gui-he b,1, Sun Xing-xing a, Zhang Hui a, Xu Li-xian a,⇑ a b
Department of Anesthesiology, School of Stomatology, Fourth Military Medical University, Xi’an 710032, PR China Department of Anesthesiology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, PR China
a r t i c l e
i n f o
Article history: Received 3 January 2011 Received in revised form 22 February 2011 Accepted 1 March 2011 Available online 26 March 2011 Keywords: Cancer of head and neck Squamous cell carcinoma Cell proliferation Apoptosis Isoflurane Inhalation anesthetics Flow cytometry Neoplasm invasion Cell migration Balanced anesthesia Anesthetic drugs
s u m m a r y The objective of this study was to explore the influence of isoflurane on the cell proliferation, apoptosis, and invasion of Tca8113 and HSC2 cell lines in vitro. MTT test was used to detect the cell proliferation. It was performed 72 h after exposure to isoflurane to make sure that a time for normal cell cycle progression was allowed. The cell apoptosis of Tca8113 and HSC2 cell lines were detected by flow cytometry. We used transwell chamber to detect the cell invasion of Tca8113 and HSC2 cell lines. There was a statistically significant increase of cell proliferation in Tca8113 and HSC2 cell lines after exposure to 2% isoflurane for 3 and 6 h. The difference between 3 and 6 h group is statistically significant in Tca8113 and HSC2 cell lines. Flow cytometry showed that there was a decrease of cell apoptosis in Tca8113 and HSC2 cell lines after exposure to 2% isoflurane for 3 and 6 h. Transwell test showed there was a statistically significant increase of cell invasion in Tca8113 and HSC2 cell lines after exposure to 2% isoflurane for 3 and 6 h, and it showed a significant difference between 3 h group and 6 h group of Tca8113 cell line. Our results demonstrated that isoflurane increased malignancy of head and neck squamous cell carcinoma cell lines in vitro. Isoflurane might enhance tumor development and promote metastasis of tumor cells in HNSCC patients. It is suggested that it might be more suitable to choose total intravenous anesthesia for HNSCC patients. Ó 2011 Elsevier Ltd. All rights reserved.
Introduction Head and neck squamous cell carcinoma (HNSCC) is one of the six most common cancers in the world, with approximately 500,000 new cases diagnosed and 250,000 deaths per year.1 Despite much medical improvement over the past two decades, the overall survival rate of HNSCC still has remained poor (5 years survival rate about 50%).2 Among various treatments, surgery is still the mainstream mode of initial definitive treatment for the majority of oral cancers.3 Balanced anesthesia (inhalational combined intravenous anesthesia) is the most widely used anesthesia mode in the operation of head and neck tumor surgery.4 Compared to the total intravenous anesthesia, balanced anesthesia allows to obtain a better haemodynamic stability during the surgery and a
Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; FCM, flow cytometry; FITC, fluorescein isothiocyanate; HNSCC, head and neck squamous cell carcinoma; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; PI, propidium iodide. ⇑ Corresponding author. Address: Department of Anesthesiology, School of Stomatology, Fourth Military Medical University, 145#, Changle Xi Road, Xi’an 710032, PR China. Tel./fax: +86 29 84776115. E-mail address: [email protected] (X. Li-xian). 1 These authors contributed equally to this paper. 1368-8375/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2011.03.002
faster and safer recovery of consciousness after the surgery,5 which is especially benefit for old HNSCC patients. Isoflurane, a commonly used inhalational anesthetic in the balanced anesthesia, induces cytotoxicity in both a concentrationand time-dependent manner in different types of cultured cells.6– 8 At clinical relevant concentrations, isoflurane caused widespread neuronal apoptosis in developing rat brains and apoptosis in human T lymphocytes in vitro.9,10 However, in other studies, isoflurane inhibits cardiac myocyte apoptosis during oxidative and inflammatory stress and attenuates apoptosis in response to ischemia or other forms of tissue injury.11–13 All above indicate that isoflurane might have anti-apoptosis and pro-apoptosis potency according to different circumstances. The pre-operative and post-operative therapies for HNSCC patients have been well considered. However, the risks during the surgery operation period are often neglected. The period of oral cancer surgery is often lasting for 4–6 h, during this time the effect of isoflurane on tumor cells is unknown. It has been reported that clinically relevant concentration of isoflurane induces cell apoptosis in several carcinoma cell lines in vitro,7 but there is no report of influence of isoflurane on HNSCC cells. HNSCC is highly invasive, frequently metastasizing to cervical lymph nodes and corresponds with poor prognosis.14 Therapeutic intervention of tumor invasion is becoming recognized as an
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increasingly relevant clinical factor.15 However, the impact of isoflurane on the invasion of HNSCC cells has never been reported. Our aim is to observe the influence of clinical relevant concentration of isoflurane on HNSCC cells. Tca8113 and HSC2 are two widely used HNSCC cell lines. So in our study, we observed the influences of isoflurane on the cell viability, cell apoptosis and cell invasion of Tca8113 and HSC2 cell lines in vitro. Materials and methods Cell culture Tca8113 and HSC2 cell lines were grown as monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U penicillin, and 0.1 mg/ml streptomycin. The cultures were equilibrated with humidified 5% CO2 in air at 37 °C in CO2 incubator (Shell Lab, Sheldon Manufacturing, USA). Anesthetic exposure Two percent isoflurane which is the clinically used concentration, 21% O2 and 5% CO2 were delivered from an anesthesia machine to a sealed plastic box in a 37 °C incubator containing sixwell plates seeded with one million cells in 1.5 ml cell culture media. A Datex infrared gas analyzer (Puritan-Bennett, Tewksbury, MA) was used to continuously monitor the delivered carbon dioxide, oxygen, and isoflurane concentrations. A stable gas and volatile anesthetic concentrations were obtained within 5 min. We treated the cells with 2% isoflurane for 3 and 6 h. Control conditions included only 5% CO2 plus 21% O2 in incubator. Cell proliferation study The cell proliferation was determined by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma). The test was performed 72 h after isoflurane exposure. This allowed a time for at least two normal cell cycles. Controls were grown under the same conditions, without anesthetic. Four hours before end of incubation, medium was aspirated and the MTT reduction product, formazan, was dissolved in dimethyl sulfoxide (DMSO) and quantified spectrophotometrically at 570 nm. We presented the changes in the absorbance, as a measure of cell proliferation, in the cells treated with isoflurane as the percentage of those in the cells treated with control conditions. Cell apoptosis analysis To detect phosphatidylserine externalization (on the surface of cell membrane), an indicator of early apoptosis, flow cytometry (FCM, BD Biosciences, USA) was performed with fluorescein isothiocyanate (FITC)-labeled Annexin V (Joincare Biosciences, Zhuhai, China). Propidium iodide (PI), a cell dye, can bind to nucleic acid after penetrating a breached plasma membrane, as occurs in the later stages of cell damage. After treatment, cells were washed with cold PBS at 4 °C. After centrifugation at 1500 rpm for 5 min, 500 lL of 1 binding buffer, 5 lL of FITC-labeled Annexin V and 10 lL of PI were added to the cell suspension and gently mixed. After incubation at 25 °C for 10 min in the dark, the cells were analyzed by FCM. In vitro cell invasion assay Tca8113 and HSC2 cell lines that were exposed to isoflurane were used for the following experiments immediately. A total of
1 105 cells in 0.5 ml serum-free DMEM medium were seeded on a 8 lm-pore polycarbonate membrane Boyden chambers insert in a transwell apparatus (Costar, Cambridge, MA), coated with Matrigel (BD Biosciences, San Jose, CA). 600 lL DMEM containing 10% FBS was added to the lower chamber. After the cells were incubated for 24 h at 37 °C in a 5% CO2 incubator, cells on the top surface of the insert were removed by wiping with a cotton swab. Cells that migrated to the bottom surface of the insert were fixed in 100% methanol for 2 min, stained with HE solution and then subjected to microscopic inspection (200). Values for invasion were obtained by counting five fields per membrane and represent the average of three independent experiments. Statistical analysis The results of cell proliferation and cell invasion were expressed as the mean ± SD. The results of cell apoptosis were expressed as the median and 25–75%. Statistical differences of cell proliferation and cell invasion were estimated by one-way analysis of variance (ANOVA) followed by SNK-q test. Statistical differences of cell apoptosis were estimated by Kruskal–Wallis test. Those P values that were less than 0.05 were considered statistically significant. Analysis of the data and plotting of the figures were performed with the aid of software (Origin Version 7.5 and SPSS Version 13.0).
Results Cell proliferation of Tca8113 and HSC2 cell lines after 2% isoflurane exposure in vitro In order to determine antiproliferative potency of single exposure of isoflurane to Tca8113 and HSC2 cell lines, MTT was performed after 72 h. Thus, a time for normal cell cycle progression was allowed. Both HNSCC cell lines showed growth alterations as a consequence of single exposure to isoflurane. Results of MTT test are shown on Fig. 1. Tca8113 cell line expressed statistically significant increase (P < 0.05) in each particular time of exposure to isoflurane (Fig. 1A). The growth of treated cells in comparison to control was 116% after 3 h, and increased at 127% after 6 h. There is also a significant difference between 3 h group and 6 h group (P < 0.05).HSC2 cell line showed marked growth increase (Fig. 1B), statistically significant in each particular time of exposure to isoflurane (P < 0.05). Cell growth increased to 112% after 3 h and 119% after 6 h. Significant difference existed between 3 h group and 6 h group (P < 0.05). These data demonstrated that isoflurane increased the cell proliferation of Tca8113 and HSC2 cell lines. Cell apoptosis of Tca8113 cell line and HSC2 cell lines after 2% isoflurane exposure in vitro To quantitate cell apoptosis in response to isoflurane, flow cytometric analysis with Annexin V-PI staining is chosen. Early apoptosis is defined by Annexin V+/PI staining (Q4) and late apoptosis is defined by Annexin V+/PI+ staining (Q2) as determined by FCM. As shown in a representative experiment in Fig. 2A, 2% isoflurane exposure decreased the apoptosis rate (Q2 + Q4) of Tca8113 cell line. Fig. 2B showed that the apoptosis rate (Q2 + Q4) of HSC2 cell line decreased after exposed to 2% isoflurane. Compared with control group, the difference of apoptosis rate (Q2 + Q4) of Tca8113 cell line in 6 h group was statistically significant, not in 3 h group (P < 0.05) (Table 1). HSC2 cell line in 3 and 6 h group were statistically significant compared to control group (P < 0.05). As shown in Table 1, there were significant differences between 3 and 6 h group in both Tca8113 and HSC2 cell lines. These data suggested that
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Figure 1 Isoflurane increased cell proliferation of Tca8113 and HSC2 cell lines. Cell proliferation was assessed by MTT in Tca8113 and HSC2 cell lines. The 2% isoflurane treatment increased cell proliferation as compared with control conditions in Tca8113 cell line (A) and HSC2 cell line (B). Significant difference existed between 3 h group and 6 h group (P < 0.05) in both cell lines. Bars represent standard error from mean value. (⁄P < 0.05 as compared with control; #P < 0.05 as compared with 3 h group).
Figure 2 Isoflurane decreased cell apoptosis of Tca8113 and HSC2 cell lines by flow cytometry analysis using Annexin V-FITC and propidium iodide. The experiments were performed after treatment with 2% isoflurane for 3 and 6 h. Cell populations in bottom right and top right quadrants represented the proportion of late apoptotic and early apoptotic, respectively. The figure demonstrated the decrease of apoptosis rate (Q2 + Q4) in Tca8113 (A) and HSC2 (B) cell lines.
isoflurane could significantly decrease cell apoptosis in both Tca8113 and HSC2 cell lines. In vitro cell invasion of Tca8113 and HSC2 cell lines after isoflurane exposure To measure the effect of isoflurane on cell invasion of Tca8113 and HSC2 cell lines, cells growing in the log phase exposed to isoflurane were collected and cultured on Transwell apparatus. Data
Table 1 Quantification of apoptosis by Annexin V-FITC/PI staining.
Tca8113 HSC2
Control
3h
6h
8.8 (6.5–11.1) 11.9 (7–15.3)
7.8 (4.4–8.7) 5.95 (4.7–9.7)*
6.3 (4.2–7.3)*# 4.7 (3.6–6.6)*#
Data are presented as median (interquartile range). * P < 0.05 as compared with control, # P < 0.05 as compared with 3 h group.
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Figure 3 Isoflurane increased cell invasion of Tca8113 and HSC2 cell lines. Representative photos and statistic plots of invasion assay in Tca8113 cell line and HSC2 cell line (200). The invasion cell number of Tca8113 cell line increased in the isoflurane treated groups compared with that of control (P < 0.05) (A). The invasion cell number of HSC2 cell line increased in the isoflurane exposure groups compared with that of control (P < 0.05) (B). There was a significant difference in cell invasion number between 3 h group and 6 h group in Tca8113 cell line, but not in HSC2 cell line. Bars represent standard error from mean value. (⁄P < 0.05 as compared with control; #P < 0.05 as compared with 3 h group).
are shown on Fig. 3. After 12 h incubation, the cell number of Tca8113 cell line was significantly increased in 3 and 6 h group than that in control group (P < 0.05) (Fig. 3A). It also showed a significant difference between 3 h group and 6 h group (P < 0.05) of Tca8113 cell line. There was a significant increase of cell number of HSC2 cell line in 3 and 6 h group, compared with the control group (P < 0.05) (Fig. 3B). However, there was not a significant difference in cell invasion number between 3 h group and 6 h group in HSC2 cell line. These results demonstrated that isoflurane increased the cell invasion in both Tca8113 and HSC2 cell lines.
Discussion General anesthesia is a drug-induced, reversible condition which provides amnesia, analgesia, immobility, and control of autonomic responses during surgical procedures.16 Though total intravenous anesthesia developed rapidly in recent years, balanced anesthesia combined with isoflurane is still adopted in the majority of general anesthesia.17 Clinical relevant concentration of isoflurane showed little or no influence on normal cell lines in growth inhibition and cell apoptosis.7,18 Concerning for tumor cell lines after exposure to inhalational anesthetics, the results were not consistent. The study of O’Leary has found that inhalational anesthetic suppressed the growth of tumor cells.19 Moudgil and Singal reported the opposite results that inhalational anesthetic enhanced tumor metastasis in mice.20 In our study, we found that 2% isoflurane increased cell proliferation in both HNSCC cell lines after single exposure for 3 and 6 h. And this clinical relevant concentration of isoflurane
inhibited cell apoptosis in our experiment. These results are different from that mentioned in the study of O’Leary. Furthermore, we firstly found that 2% isoflurane increased cell invasion in HNSCC cell lines in vitro. There are a number of factors in the perioperative period that may result in immunosuppression.21 Manipulation of the primary tumor during surgery is associated with release of tumor cells into the circulation.22 In addition, surgery induces a profound stress response, including neuroendocrine, cytokine, and metabolic response.23 Anesthesia itself contributes to perioperative immunosuppression.21,24,25 Inhalational anesthetics used in the general anesthesia can modulate immune defense, especially when applied in higher concentrations or doses or for longer period of time.26 Isoflurane has been shown to induce caspase-dependent, mitochondria-mediated apoptosis in human T cells.10 This, in turn, causes suppression of natural killer cell activity. This compromises host immune function, which has been shown to enhance tumor development and promote metastasis of tumor cells in patients.27 Beside the above indirect influences of inhalational anesthetics on cancer cells, there are also direct influences of isoflurane on cancer cells. As shown in our study, 2% isoflurane has a direct enhancement on the cell proliferation of head and neck squamous cell carcinoma cell lines in vitro. A tumor cell proliferation after exposure to clinically relevant concentrations of isoflurane appears important in the view of altered metastatic capability of tumor cells that enter into circulation during tumor and organ resections.7 As the time for at least two normal cell cycles was allowed, our results provided an indication as to the consequence on ongoing and subsequent proliferative effects. Spontaneous apoptosis in tumor cells reflect cellular metabolism and growth condition.7 The inhibition
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of isoflurane in HNSCC cells might attribute to the increasing of cell proliferation. Though there were differences between early (Q4) and late apoptosis (Q2) in both cell lines, the total apoptosis (Q2 + Q4) in different time points is more meaningful in consideration. The exact mechanism of isoflurane in inhibiting apoptosis of head and neck cell lines is unknown, but isoflurane might influence caspase-dependent apoptotic cell death pathway to inhibit HNSCC cell apoptosis as in T lymphocyte.10 Increased cell migration is one of the critical steps in the development of metastasis.28 In cell invasion study, we found significant differences compared to control after exposed to isoflurane in both cell lines. The above results in our study provided some evidences that isoflurane enhanced malignancy of HNSCC cell lines in vitro. It is a controversial which anesthesia mode is more suitable for HNSCC patients.17,29 Most anesthesiologists focused their studies on the influences of different anesthesia techniques on haemodynamic stability during the surgery and recovery of consciousness after the surgery.5 Balanced anesthesia has better haemodynamic stability and fewer drugs required during the surgery, so it becomes the mainstream anesthesia mode in the head and neck tumor surgery.4 However, the influences of different anesthesia techniques and anesthetic drugs on tumor cell of HNSCC are often ignored. The anesthesia technique may not impact the recovery of patients underwent ordinary head and neck surgery such as benign tumor, mandible fracture and plastic surgery. However, for cancer patients, the least impaction of anesthesia technique and anesthesia drugs on tumor cells should be chosen. As demonstrated in our study, isoflurane has a direct influence on the biological features of HNSCC cell lines in vitro, so it might not be suitable to choose isoflurane in the surgery for HNSCC patients. Though it might increase the possible implications on hemodynamic stability and safer recovery of consciousness after surgery, total intravenous anesthesia might be a suggested choice for HNSCC patients now. If there are some other inhalational anesthetics emerged in future which do not show adverse influences on HNSCC cell lines, balanced anesthesia with inhalational anesthetic is still a better choice for its less implications during the surgery. Our studies also have limitations. We used in vitro HNSCC cell lines cultures. These cells are obviously different from in situ HNSCC. It is also impossible to provide cell cultures with an environment identical to what the in vivo carcinoma cells have, such as local pH, oxygen, and electrolyte concentrations, and the support and interaction from other cells. Furthermore, the mechanisms, which isoflurane enhances malignancy of Tca8113 and HSC2 cell lines after exposure, need to be investigated. In conclusion, our data provided some evidences in vitro that balanced anesthesia combined with isoflurane might not be suitable for HNSCC patients. It might be more suitable to choose total intravenous anesthesia for HNSCC patients during the surgery. Conflicts of interest statement None declared. Acknowledgment The study was supported by the National Natural Science Foundation of China (No. 30772073). References 1. Molinolo AA, Amornphimoltham P, Squarize CH, Castilho RM, Patel V, Gutkind JS. Dysregulated molecular networks in head and neck carcinogenesis. Oral Oncol 2009;45(4–5):324–34.
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