The Experimental Study Of Influences Of Isoflurane On Human Oral Cancer Cell Lines

The incidence of oral cancer ranks sixth in the body's malignant tumor. Oral cancers are usually located in surface or close to the surface parts, so the physiology functions and social activities of patients are seriously affected. The treatments for oral cancer include surgery, radiotherapy, immunotherapy, gene therapy and so on. Among such therapeutic methods, surgery still holds the most important position, and is the maistream treatment. Balanced anesthesia (inhalational combined intravenous anesthesia) is the most widely used anesthesia mode applied in the operation of head and neck tumor surgery. Compared to the total intravenous anesthesia, balanced anesthesia allows obtaining better haemodynamic stability during the surgery and a faster and safer recovery of consciousness after the surgery, which is especially benefit for old HNSCC patients. The MAC of isoflurane is 1.15, so it is easy to control the depth of anesthesia during the surgery. Isoflurane, a commonly used inhaled anesthetic in the balanced anesthesia, induces cytotoxicity in both a concentration- and time-dependent manner in different types of cultured cells. At clinical relevant concentrations, isoflurane caused widespread neuronal apoptosis in developing rat brains and apoptosis in human T lymphocytes in vitro. 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. All above indicate that isoflurane might have anti-apoptosis and pro-apoptosis potency according to different circumstances. The pre-operative and post-operative therapy for HNSCC patients is well considered. However, the risks during the surgery operation period are often neglected. The period of oral cancer surgery is often lasting for 3 to 6 hours, during this time the effect of isoflurane on tumor cells is unknown. It proposes a question whether isoflurane in the balanced anesthesia is benefit for HNSCC patients? If isoflurane induces the cell apoptosis of HNSCC, it might be benefit to choose balanced anesthesia technique for HNSCC patients. However, if isoflurane inhibits the cell apoptosis of HNSCC, it might be benefit to choose total intravenous anesthesia instead of balanced anesthesia for HNSCC patients. It has been reported that clinically relevant isoflurane induces cell apoptosis in several carcinoma cell lines in vitro, but there is no report of isoflurane on HNSCC cell lines.HNSCC is highly invasive, frequently metastasizing to cervical lymph nodes and corresponds with poor prognosis. Therapeutic intervention of tumor invasion is becoming recognized as an increasingly relevant clinical factor. However, the impact of isoflurane on HNSCC cell lines invasion has not been reported.Objective Our aim is to observe the influence of isoflurane on HNSCC. 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.Methods1. Cell culture was used to observe the cell cycle of Tca8113 and HSC2 cell lines, and also used to observe the cell proliferation of influence of Tca8113 and HSC2 cell lines in vitro.2. Flow cytometry was used to observe the cell apoptosis of Tca8113 and HSC2 cell lines in vitro.3. Transwell chamber was used to observe the cell invasion of Tca8113 and HSC2 cell lines in vitro.4. Western blot was used to observe the pathway of cell apoptosis of Tca8113 and HSC2 cell lines in vitro.Results1. In cell proliferation experiments, Tca8113 and HSC2 cell lines exposed to2.0% isoflurane for 3 to 6 hours, continue to cultivate 72 hours. In orde to observe the changes of cell proliferation capacity more accurate, the test was performed 72 h after isoflurane exposure. This allowed a time for at least two normal cell cycles. Experimental results showed that after exposure to 2.0% isoflurane for 3 to hours, whether Tca8113 lines, or HSC2 lines, cell proliferation capacity were changed. Tca8113 cell line accepted 2.0% isoflurane exposure for3 to 6 hours, and the control group didn't accept any isoflurane exposure. Cell proliferations of experimental groups were significantly higher than controlled group, and the difference was statistically significant. Cell proliferations had a statistic difference between 3 hours group and 6 hours group. Cell proliferation increased obviously in 6 hours exposure group. HSC2 cell line accepted 2.0% isoflurane exposure for 3 to 6 hours, and the control group didn't accept isoflurane exposure. Cell proliferations of experimental groups were significantly higher than controlled group, and the difference was statistically significant. Cell proliferations had a statistic difference between 3 hours group and 6 hours group. Cell proliferation increased obvious in 6 hours exposure group. The results confirmed that 2.0% isoflurane enhanced cell proliferation of oral cancer cell lines in vitro.2. In cell apoptosis study, after exposed to 2.0% isoflurane for 3 to 6 hours, Tca8113 and HSC2 cell lines were digested and rinsed for flow cytometry. To detect phosphatidylserine externalization (on the surface of cell membrane), an indicator of early apoptosis, flow cytometry was performed with fluorescein isothiocyanate (FITC)-labeled Annexin V. 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. Experimental results found that after exposure to 2.0% isoflurane for 3 and 6 hours, whether Tca8113 lines, or HSC2 lines, cell apoptosis were changed. Tca8113 cell line was exposed to 2.0% isoflurane for 3 to 6 hours, and the controlled group didn't receive any isoflurane exposure. The cell apoptosis of 6 hours group reduced compared to controlled group, and the difference was significant. But the cell apoptosis between 3 hours group and controlled group, there was not a significant difference. There was also a significant difference of cell apoptosis between 3 hours group and 6 hours group. HSC2 cell line was exposed to 2.0% isoflurane for 3 to 6 hours, and the controlled group didn't receive any isoflurane exposure. The cell apoptosis of 3 hours and 6 hours groups reduced compared to controlled group, and the differences were significant. There was also a significant difference of cell apoptosis between 3 hours group and 6 hours group. These results confirmed that 2.0% isoflurane inhibited cell apoptosis of Tca8113 and HSC2 cell lines in votro. 3. In cell invasion study, Tca8113 and HSC2 cell lines accepted 2.0% isoflurane exposure for 3 to 6 hours. After exposure, cell lines were used for cell invasion study immediately. A total of 1 x 105 cells in 0.5 ml serum-free DMEM medium were seeded on a 8μm-pore polycarbonate membrane Boyden chambers insert in a transwell apparatus, coated with Matrigel. 600μL DMEM containing 10% FBS was added to the lower chamber. After the cells were incubated for 24 hours at 37℃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 minutes, stained with HE solution and then subjected to microscopic inspection. Experimental results found that after exposure to 2.0% isoflurane for 3 and 6 hours, whether Tca8113 line, or HSC2 line, cell invasion ability was changed. Tca8113 cell line accepted 2.0% isoflurane exposure for 3 to 6 hours, and the control group didn't accept isoflurane exposure. Cell invasions of experimental groups are significantly higher than controlled group, and the difference is statistically significant. Cell invasion had a statistic difference between 3 hours group and 6 hours group. Cell invasion increased obviously in 6 hours exposure group. HSC2 cell line accepted 2.0% isoflurane exposure for 3 to 6 hours, and the control group didn't accept any isoflurane exposure. Cell invasions of experimental groups were significantly higher than controlled group, and the difference is statistically significant. There was no statistical difference of cell invasion between 3 hours group and 6 hours group. The results confirmed that 2.0% isoflurane enhanced cell invasion of oral cancer cell lines in vitro.4. In molecular experiment associated to cell apoptosis pathway, after Tca8113 and HSC2 cell lines accepted 2.0% isoflurane exposure for 3 to 6 hours. Tca8113 and HSC2 cell lines were immediately collected and prepared for western blot. Tca8113 cell line accepted 2.0% isoflurane exposure for 3 to 6 hours, and the control group didn't accept any isoflurane exposure. Bcl-2 level increased in both 3 hours group and 6 hours group, and the increase had statistical differences compared to the control group. In 6 hours group, the Bcl-2 level had a statistical increase compared to 3 hours group. HSC2 cell line accepted 2.0% isoflurane exposure for 3 to 6 hours, and the control group didn't accept any isoflurane exposure. Bcl-2 level increased in both 3 hours group and 6 hours group, and the increase had statistical differences compared to the control group of HSC2 cell line. In 6 hours group, the Bcl-2 level had a statistical increase compared to 3 hours group.The results showed that 2.0% isoflurane inhibited cell apoptosis in vitro might through Bcl-2/Bax pathway.ConclusionTo sum up, 2.0% isoflurane increased cell proliferation, cell invasion of HNSCC cell lines, and inhibited cell apoptosis of HNSCC cell linese. These results show that 2% isoflurane enhanced malignancy of HNSCC cell lines. 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 in the future.