| Lung cancer is the most common cancer worldwide, and non-small cell lung carcinoma accounts for approximately75%～85%of all lung cancers. Surgical resection is a commonly used therapy for patients with non-small cell lung carcinoma. However, tumor recurrence and metastasis after lung cancer surgery often occurs, which is the leading cause of deaths in patients with lung cancer. In the1880s, some scholars found that surgery can promote the growth of tumor and enhance the metastasis potential of tumor, and observed the recurrence and metastasis after cancer surgery. Recently, it is well recognized that anesthetics and anesthesia technique could affect the long-term outcome of patients undergone cancer surgery. Therefore, how to inhibit the progress and metastasis of tumor cells during perioperative period is a new challenge to anesthesiologists, and becoming a new topic of anesthesiology. Choice of anesthetics and anesthesia technique that inhibit invasion and migration ability of cancer cells is important during cancer surgery in order to decrease the risk of recurrence and metastasis.Sevoflurane, a volatile anesthetic agent, has the characteristics of excellent controllability and strong potency. It is used extensively during lung cancer surgery as anesthesia induction and anesthesia maintenance. Bronchi and pulmonary alveoli are exposed to sevoflurane directly during sevoflurane anesthesia. However, the effect of sevofluane on the growth, metastasis potential, and sensitivity to chemotherapy of lung cancer cells remains unclear.Growth of tumor cells is a multi-gene and multi-step process. An imbalance between anti-apoptotic gene expression and pro-apoptotic gene expression happens during tumor progression period. X-linked inhibitor of apoptosis protein (XIAP) and Survivin are two members of inhibitor of apoptosis proteins (IAPs) family, which play an important role in proliferation and apoptosis. XIAP and Survivin can block directly the process of cysteinyl aspartate specific protease-3(Caspase-3, a final effector of apoptosis). The down-regulation of XIAP and Survivin promotes process of Caspase-3, and induce apoptosis of cells. Just like IAP family, Bcl-2family is involved in the regulation of cell apoptosis. Bcl-2family consists of a series of anti-apoptotic and pro-apoptotic members. Bcl-2is an anti-apoptotic member, which prevents apoptosis by inhibiting the release of mitochondrial apoptogenic factors into the cytoplasm. In contrast, Bax is a pro-apoptotic member of this family, which promotes apoptosis by activating caspases. However, the effect of sevoflurane on proliferation and apoptosis of human lung adenocarcinoma A549cells has not been reported. The effect of sevoflurane on the expression of above-mentioned growth related-molecules has not been elucidated.Growth of tumor is also associated with the abnormal regulation of cell cycle. Tumor is a kind of disease that caused by disturbed cell cycle. Development and progress of most tumors are close related to anomalous regulation of cell cycle. Cyclin A, cyclin B1, and Cdc2play critical role in regulating cell cycle progression. The abnormal expression of theses protein is an important mechanism of cell canceration. However, the effect of sevoflurane on cell cycle of human lung adenocarcinoma A549cells has not been reported. The effect of sevoflurane on the expression of Cyclin A, cyclin B1, and Cdc2has not been reported.Metastasis of tumor cells consists of a series of complex, continuous, and multi-step process. It includes mainly three aspects:(1) Tumor cells adhere with basement membrane and extracellular matrix through adhesion molecule;(2) Tumor cells excrete matrix metalloproteinases (MMPs), invade basement membrane and extracellular matrix;(3) Tumor cells migrate through basement membrane and extracellular matrix, and penetrate the blood vessel walls. Finally, new metastasis is formed at another site. MMP-2and MMP-9belong to a family of zinc-dependent proteinases. Their primary function is degradation of proteins in the extracellular matrix, which play an important role in the invasion process of tumor cells. Fascin and Ezrin are of critical importance in tumor cell migration. Tumor cells expressing high levels of Ezrin and Fascin exhibit increased migration ability. Up till now, the effect of sevoflurane on metastasis potential of A549cells remains unclear, and the effect of sevoflurane on the expression of these metastatic-related molecules has not been reported.The p38mitogen-activated protein kinase (p38MAPK) signaling pathway is a member of the MAPK signaling pathway family. Many studies demonstrated p38MAPK signaling pathway participates in regulating the metastasis of tumor cells. However, it is not clear that the role of p38MAPK signaling pathway in the effect of sevoflurane on invasion and migration of human lung adenocarcinoma A549cell. It also remains unclear whether sevoflurane regulates the expressions of MMP-2, MMP-9, Fascin, and Ezrin of A549cells through p38MAPK signaling pathway.Nowadays, chemotherapy has been used as a routine therapy for patients with lung cancer in order to decrease the incidence of recurrence and metastasis of tumor. It is very commonly to adopt platinum drugs for chemotherapy during perioperative period. Anesthetics used during anesthesia period can exert an impact on the effect of chemotherapy. A study demonstrated that sevoflurane could alleviate the toxicity effect of cisplatin on Ehrlich ascites tumor cells. However, it has not been elucidated whether sevoflurane can affect the anti-cancer potency of cisplatin to A549cells.In this in vitro study, human lung adenocarcinoma A549cells was treated with sevoflurane, and the following three aspects were studied:(1) To investigate the effect of sevoflurane on the growth of A549cells, and to explored the related molecules mechanism;(2) To investigate the effect of sevoflurane on metastasis potential of A549cells, and to explored the related molecules mechanism;(3) To investigate the effect of sevoflurane on sensitivity to chemotherapy of A549cells, and to explored the related molecules mechanism. The results of this study will provide some instructions for clinical anesthesia.Chapter1The effect of sevoflurane on proliferation and apoptosis, and related molecules expression in human lung adenocarcinoma cell line A549Objective To investigate the effect of sevoflurane on proliferation and apoptosis, and related molecules expression in human lung adenocarcinoma cell line A549.Methods A549cells were inoculated in culture plate. After being cultured for24h the cells were randomly divided into4groups:group control (C,0%sevoflurane), group1.7%sevoflurane (S1), group3.4%sevoflurane (S2), and group5.1%sevoflurane (S3). A549cells of group S1-3were exposed to1.7%,3.4%,5.1%sevoflurane for2,4,6h respectively. Cells of group C were exposed to95%O2-5%CO2mixture air, and were then cultured for another48h. The proliferation inhibition rates, colony formation rates, and apoptosis percentages of A549cells were measured by methyl thiazolyl tetrazolium (MTT) assay, colony formation assay, and flow cytometer respectively at48h after exposure to sevoflurane2,4,6h. The protein expressions of XIAP, Survivin, Bcl-2, Bax, and Caspase-3in A549cells were determined by Western blotting at48h after exposure to sevoflurane4h.Data analysis was performed by SPSS13.0software. Data were expressed as mean±standard deviation (SD). Proliferation inhibition rates, colony formation rates, and apoptosis percentages were assessed by using variance analysis of two-way factorial design. The protein expressions of XIAP, Survivin, Bcl-2, Bax, and Caspase-3were assessed by using one-way analysis of variance (ANOVA). LSD test (equal variance) or Dunnett’s T3test (heterogeneity of variance) was used for post hoc comparisons. P<0.05was considered statistically significant.Results (1) The comparison of proliferation inhibition rates:There was statistical significance among groups of treatment concentration (F=128.672, P=0.000). There was statistical significance among groups of treatment duration (F=135.426, P=0.000). There was not interaction between treatment concentration and treatment duration (F=1.854, P=0.135). The proliferation inhibition rates in group S2and group S3of each treatment duration were increased significantly as compared with group S1(P<0.05). The proliferation inhibition rates in group S1-3were increased significantly with treatment duration prolonged(P<0.05); The proliferation inhibition rates in group C of each treatment duration were zero;(2) The comparison of colony formation rates:There was statistical significance among groups of treatment concentration (F=400.176, P=0.000). There was statistical significance among groups of treatment duration (F=65.728, P=0.000). There was interaction between treatment concentration and treatment duration (F=6.081, P=0.000). The simple effect analysis of treatment concentration revealed that the colony formation rates in group S1-3of each treatment duration were decreased significantly as compared with group C (P<0.05); The simple effect analysis of treatment duration revealed that the colony formation rates in group S1-3were decreased significantly with treatment duration prolonged (P<0.05);(3) The comparison of apoptosis percentages:There was statistical significance among groups of treatment concentration (F=278.06, P=0.000). There was statistical significance among groups of treatment duration (F=94.230, P=0.000). There was interaction between treatment concentration and treatment duration (F=11.440, P=0.000). The simple effect analysis of treatment concentration revealed that the apoptosis percentages in group S1-3of each treatment duration were increased significantly as compared with group C (P<0.05); The simple effect analysis of treatment duration revealed that the apoptosis percentages in group S1-3were increased significantly with treatment duration prolonged (P<0.05);(4) The comparison of XIAP, Survivin, and caspase-3expression:Compared with group C, the expression of XIAP and Survivin was significantly down-regulated with sevoflurane concentration increased(P<0.05). Compared with group C, the expression of cleaved caspase-3was significantly up-regulated with sevoflurane concentration increased (P<0.05);(5) The comparison of Bcl-2and Bax expression:Compared with group C, the expression of Bcl-2and Bax was not changed significantly in group S1-3(P>0.05).Conclusion Sevoflurane can inhibit the proliferation and induce apoptosis of A549cells through down-regulating the expression of XIAP and Survivin, and up-regulating the expression of caspase-3, but not depend on Bcl-2and Bax pathway.Chapter2The effect of sevoflurane on cell cycle and related molecules expression in human lung adenocarcinoma cell line A549Objective To investigate the effects of sevoflurane on cell cycle and related molecules expression in human adenocarcinoma cell line A549.Methods A549cells were inoculated in culture plate. After being cultured for24h the cells were randomly divided into4groups:group control (C,0%sevoflurane), group1.7%sevoflurane (SI), group3.4%sevoflurane (S2), and group5.1%sevoflurane (S3). A549cells of group Sl-3were exposed to1.7%,3.4%,5.1%sevoflurane for4h respectively. Cells of group C were exposed to95%O2-5%CO2mixture air, and were then cultured for another48h. Cell cycle in A549cells were detected with flow cytometer at48h after exposure to sevoflurane4h. The protein expressions of Cyclin A, Cyclin B1, and Cdc2in A549cells were determined by Western blotting at48h after exposure to sevoflurane4h. Data analysis was performed by SPSS13.0software. Data were expressed as mean±standard deviation (SD). Data was assessed by using one-way analysis of variance (ANOVA). LSD test was used for post hoc comparisons. P<0.05was considered statistically significant.Results (1) The comparison of cell cycle:Compared with group C, the percentage of cells in G2/M phase of A549cells in group S1-3were increased significantly with sevoflurane concentration increased (P<0.05);(2) The comparison of Cyclin A, Cyclin B1, and Cdc2expression:Compared with group C, the expression of Cyclin A, Cyclin B1, and Cdc2was significantly down-regulated with sevoflurane concentration increased (P<0.05). Conclusion Sevoflurane can block cell cycle of A549cells arrest at G2/M phase through down-regulating the protein expressions of Cyclin A, Cyclin B1, and Cdc2.Chapter3The effect of sevoflurane on metastasis potential and related molecules expression in human lung adenocarcinoma cell line A549Objective To investigate the effect of sevoflurane on metastasis potential and related molecules expression in human lung adenocarcinoma cell line A549.Methods A549cells were inoculated in culture plate. After being cultured for24h the cells were randomly divided into4groups:group control (C,0%sevoflurane), group1.7%sevoflurane (S1), group3.4%sevoflurane (S2), and group5.1%sevoflurane (S3). A549cells of group S1-3were exposed to1.7%,3.4%,5.1%sevoflurane for2,4,6h respectively. Cells of group C were exposed to95%O2-5%CO2mixture air, and were then cultured for another24h. Cell invasion was assessed by Transwell invasion assay at24h after exposure to sevoflurane2,4,6h. Cell migration was evaluated by wound healing assay at24h after exposure to sevoflurane4h. The expression MMP-2, MMP-9, Fascin and Ezrin in A549cells were determined by RT-PCR or Western blotting at24h after exposure to sevoflurane4h. Data analysis was performed by SPSS13.0software. Data were expressed as mean±standard deviation (SD). The number of invasive cells was assessed by using variance analysis of two-way factorial design. Cells migration rates, expressions of mRNA and protein of MMP-2, MMP-9, Fascin, and Ezrin were assessed by using one-way analysis of variance (ANOVA). LSD test (equal variance) or Dunnett’s T3test (heterogeneity of variance) were used for post hoc comparisons.P<0.05was considered statistically significant.Results (1) The comparison of cell invasion:There was statistical significance among groups of treatment concentration (F=241.554, P=0.000). There was statistical significance among groups of treatment duration (F=31.188, P=0.000). There was interaction between treatment concentration and treatment duration (F= 3.801, P=0.003). The simple effect analysis of treatment concentration revealed that the invasive cells number in group S1-3of each treatment duration were decreased significantly as compared with group C (P<0.05). The simple effect analysis of treatment duration revealed that the number of invasive cells in group S1-3were decreased significantly with treatment duration prolonged (P<0.05);(2) The comparison of invasion-related molecules expression:Compared with group C, the mRNA and protein expressions of MMP-2and MMP-9in group S1-3were significantly down-regulated with sevoflurane concentration increased (P<0.05);(3) The comparison of cell migration:Compared with group C, the migration rates in group S1were not changed significantly(P>0.05). Compared with group C, the migration rates in group S1and group S2were decreased significantly (P<0.05);(4) The comparison of migration-related molecules expression:Compared with group C, the mRNA and protein expressions of Fascin and Ezrin in group S1-3were significantly down-regulated with sevoflurane concentration increased (P<0.05).Conclusion Sevoflurane can inhibit invasion of human lung adenocarcinoma A549cells through down-regulating the mRNA and protein expression of MMP-2and MMP-9. Sevoflurane can inhibit migration of A549cells through down-regulating the mRNA and protein expression of Fascin and Ezrin.Chapter4The role of p38MAPK signaling pathway in sevoflurane inhibiting metastasis potential of human lung adenocarcinoma cell line A549Objective To investigate the role of p38MAPK signaling pathway in sevoflurane inhibiting metastasis potential of human lung adenocarcinoma cell line A549.Methods A549cells were inoculated in culture plate. After being cultured for24h the cells were randomly divided into4groups:group control (C), group3.4%sevoflurane (Sev), group3.4%sevoflurane combined with SB203580(Sev+SB), and group SB203580(SB). A549cells of group Sev were exposed to3.4%sevoflurane for4h, cells of group Sev+SB were pretreat with20μmol/L SB203580for8h before exposed to3.4%sevoflurane for4h, cells of group SB were treat with20μmol/L SB203580for8h, cells of group C were exposed to95%O2-5%CO2mixture air, and were then cultured for another24h. Cell invasion was assessed by Transwell invasion assay. Cell migration was evaluated by wound healing assay. The expression MMP-2, MMP-9, Ezrin, Fascin, p38MAPK, phospho-p38MAPK (p-p38MAPK) in A549cells were determined by Western blotting at24h. Data analysis was performed by SPSS13.0software. Data were expressed as mean±standard deviation (SD). Differences among groups were assessed by using variance analysis of two-way factorial design, followed by LSD test (equal variance) or Dunnett’s test (heterogeneity of variance) for post hoc comparisons. P<0.05was considered statistically significant.Results (1) The comparison of cell invasion:The invasive cells number were decreased significantly after sevoflurane treatment (F=87.188, P=0.000). The invasive cells number were decreased significantly after SB203580treatment (F=124.866, P=0.000). There was interaction between sevoflurane treatment and SB203580treatment (P=15.840, P=0.001). One way analysis of variance revealed that the invasive cells number in group Sev and SB were decreased significantly as compared with group C (P<0.05). The invasive cells number in group Sev+SB were decreased more significantly than that of the other3groups (P<0.05);(2) The comparison of cell migration:The migration rates were decreased significantly after sevoflurane treatment (F=79.419,P=0.000). The migration rates were decreased significantly after SB203580treatment (F=84.304,P=0.000). There was interaction between sevoflurane treatment and SB203580treatment (F=33.305, P=0.000). One way analysis of variance revealed that the migration rates in group Sev and SB were decreased significantly as compared with group C (P<0.05). The migration rates in group Sev+SB were decreased more significantly than that of the other3groups (P<0.05);(3) The comparison of p-p38MAPK level:The p-p38MAPK level were decreased significantly after sevoflurane treatment (F=51.124, P=0.000). The p-p38MAPK level were decreased significantly after SB203580treatment (F= 82.749, P=0.000). There was interaction between sevoflurane treatment and SB203580treatment (F=5.463, P=0.03). One way analysis of variance revealed that the p-p38MAPK levels in group Sev and SB were decreased significantly as compared with group C (P<0.05). The p-p38MAPK level in group Sev+SB were decreased more significantly than that of the other3groups (P<0.05).(4) The comparison of MMP-2and MMP-9protein expressions:The MMP-2protein expressions were down-regulated significantly after sevoflurane treatment (F=103.042, P=0.000). The MMP-2protein expressions were down-regulated significantly after SB203580treatment (F=243.337, P=0.000). There was interaction between sevoflurane treatment and SB203580treatment (F=5.656, P=0.03). One way analysis of variance revealed that the protein expressions of MMP-2in group Sev and SB were decreased significantly as compared with group C (P<0.05). The protein expressions of MMP-2in group Sev+SB were decreased more significantly than that of the other3groups (P<0.05); The MMP-9protein expressions were down-regulated significantly after sevoflurane treatment (F=88.369, P=0.000). The MMP-9protein expressions were down-regulated significantly after SB203580treatment (F=271.388, P=0.000). There was interaction between sevoflurane treatment and SB203580treatment (F=5.968, P=0.03). One way analysis of variance revealed that the protein expressions of MMP-9in group Sev and SB were decreased significantly as compared with group C (P<0.05). The protein expressions of MMP-9in group Sev+SB were decreased more significantly than that of the other3groups (P<0.05);(5) The comparison of Fascin and Ezrin protein expressions:The Fascin protein expressions were down-regulated significantly after sevoflurane treatment (F=104.422, P=0.000). The Fascin protein expressions were down-regulated significantly after SB203580treatment (F=364.239, P=0.000). There was interaction between sevoflurane treatment and SB203580treatment (F=10.168, P=0.005). One way analysis of variance revealed that the protein expressions of Fascin in group Sev and SB were decreased significantly as compared with group C (P<0.05). The protein expressions of Fascin in group Sev+SB were decreased more significantly than that of the other3groups (P <0.05); The Ezrin protein expressions were down-regulated significantly after sevoflurane treatment (F=78.411, P=0.000). The Ezrin protein expressions were down-regulated significantly after SB203580treatment (F=169.365, P=0.000). There was interaction between sevoflurane treatment and SB203580treatment (F=21.122,P=0.000). One way analysis of variance revealed that the protein expressions of Ezrin in group Sev and SB were decreased significantly as compared with group C (P<0.05). The protein expressions of Ezrin in group Sev+SB were decreased more significantly than that of the other3groups (P<0.05);Conclusion The effects of sevoflurane inhibiting invasion and migration of A549cells may be in part through inactivating p38MAPK signaling pathway.Chapter5The effect of sevoflurane on sensitivity to chemotherapy of human lung adenocarcinoma cell line A549Objective To investigate the effect of sevoflurane on sensitivity to chemotherapy of human lung adenocarcinoma cell line A549.Methods A549cells were inoculated in culture plate. After being cultured for24h the cells were randomly divided into4groups:group control (C), group2.5%sevoflurane (Sev), group cisplatin (DDP), and group sevoflurane combined with DDP (Sev+DDP). A549cells of group Sev were exposed to2.5%sevoflurane for4h, cells of group DDP were treat with10μmol/L DDP and exposed to95%O2-5%CO2mixture air4h, cells of group Sev+DDP were treat with10μmol/L DDP and exposed to exposed to2.5%sevoflurane for4h, cells of group C were exposed to95%O2-5%CO2mixture air, and were then cultured for another24h. Cell invasion was assessed by Transwell invasion assay. Cell migration was evaluated by wound healing assay. The expressions of MMP-2, MMP-9, Ezrin, and Fascin in A549cells were detected by Western blotting. Data analysis was performed by SPSS13.0software. Data were expressed as mean±standard deviation (SD). Differences among groups were assessed by using variance analysis of two-way factorial design, followed by LSD test for post hoc comparisons. P<0.05was considered statistically significant.Results:(1) The comparison of cell invasion:The invasive cells number were decreased significantly after sevoflurane treatment (F=80.695, P=0.000). The invasive cells number were decreased significantly after DDP treatment (F=127.681, P=0.000). There was interaction between sevoflurane treatment and DDP treatment (F=7.871, P=0.011). One way analysis of variance revealed that the invasive cells number in group Sev and DDP were decreased significantly as compared with group C (P<0.05). The invasive cells number in group Sev+DDP were decreased more significantly than that of the other3groups (P<0.05);(2) The comparison of MMP-2and MMP-9protein expressions:The MMP-2protein expressions were down-regulated significantly after sevoflurane treatment (F=68.510, P=0.000). The MMP-2protein expressions were down-regulated significantly after DDP treatment (F=205.571, P=0.000). There was interaction between sevoflurane treatment and DDP treatment (F=8.351,P=0.009). One way analysis of variance revealed that the protein expressions of MMP-2in group Sev and DDP were decreased significantly as compared with group C (P<0.05). The protein expressions of MMP-2in group Sev+DDP were decreased more significantly than that of the other3groups (P<0.05); The MMP-9protein expressions were down-regulated significantly after sevoflurane treatment (F=86.277, P=0.000). The MMP-9protein expressions were down-regulated significantly after DDP treatment (F=365.709, P=0.000). There was interaction between sevoflurane treatment and DDP treatment (F=18.754, P=0.000). One way analysis of variance revealed that the protein expressions of MMP-9in group Sev and DDP were decreased significantly as compared with group C (P<0.05). The protein expressions of MMP-9in group Sev+DDP were decreased more significantly than that of the other3groups (P<0.05);(3) The comparison of cell migration:The migration rates were decreased significantly after sevoflurane treatment (F=133.671, P=0.000). The migration rates were decreased significantly after DDP treatment (F=209.764, P=0.000). There was interaction between sevoflurane treatment and DDP treatment (F=52.888, P=0.000). One way analysis of variance revealed that the migration rates in group Sev and DDP were decreased significantly as compared with group C (P<0.05). The migration rates in group Sev+DDP were decreased more significantly than that of the other3groups (P<0.05);(4) The comparison of Ezrin and Fascin protein expressions:The Ezrin protein expressions were down-regulated significantly after sevoflurane treatment (F=74.205, P=0.000). The Ezrin protein expressions were down-regulated significantly after DDP treatment (F=282.359, P=0.000). There was interaction between sevoflurane treatment and DDP treatment (F=9.516, P=0.006). One way analysis of variance revealed that the protein expressions of Ezrin in group Sev and DDP were decreased significantly as compared with group C (P<0.05). The protein expressions of Ezrin in group Sev+DDP were decreased more significantly than that of the other3groups (P<0.05); The Fascin protein expressions were down-regulated significantly after sevoflurane treatment (F=128.451,P=0.000). The Fascin protein expressions were down-regulated significantly after DDP treatment (F=603.874, P=0.000). There was interaction between sevoflurane treatment and DDP treatment (F=52.328, P=0.000). One way analysis of variance revealed that the protein expressions of Fascin in group Sev and DDP were decreased significantly as compared with group C (P<0.05). The protein expressions of Fascin in group Sev+DDP were decreased more significantly than that of the other3groups (P<0.05)Conclusion Sevoflurane can enhance sensitivity to DDP of human lung adenocarcinoma cell line A549, and strengthen the effect of inhibiting invasion and migration of DDP, which is associated with down-regulating the expressions of MMP-2, MMP-9, Ezrin, and Fascin. |
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