LAB/IN VITRO RESEARCH e-ISSN 1643-3750 © Med Sci Monit, 2016; 22: 4644-4650 DOI: 10.12659/MSM.898434
Isoflurane Promotes Non-Small Cell Lung Cancer Malignancy by Activating the Akt-Mammalian Target of Rapamycin (mTOR) Signaling Pathway
Received: 2016.03.10 Accepted: 2016.04.18 Published: 2016.11.29
Authors’ Contribution: Study Design A Data Collection B Statistical Analysis C Data Interpretation D Manuscript Preparation E Literature Search F Funds Collection G
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Wenhua Zhang Xueqian Shao
1 Department of Anesthesiology, The Third Affiliated Hospital of Qiqihar Medical University, Qiqihar, Heilongjiang, P.R. China 2 Department of Surgical Oncology, The Third Affiliated Hospital of Qiqihar Medical University, Qiqihar, Heilongjiang, P.R. China
Xueqian Shao, e-mail
[email protected] Departmental sources
Lung cancer is one of the leading causes of cancer mortalities worldwide, and non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases. Surgery remains one of the front-line treatment options for NSCLC, but events within the perioperative period were found to affect cancer prognosis, such as anesthesia procedures. Isoflurane, a commonly used volatile anesthetic, enhances the malignant potential of renal, prostate, and ovarian cancer cells, but its effects on NSCLC development have not been previously reported. CCK-8 and MTT cell proliferation assays were used to analyze NSCLC cell proliferation. Metastatic ability was examined by wound healing and transwell assays. We used Western blot analysis to study the mechanism of effect of Isoflurane in NSCLC development. We demonstrated that isoflurane promotes proliferation, migration and invasiveness of NSCLC cells, as well as upregulation of the Akt-mTOR signaling pathway in NSCLC cells. Pharmacological inhibition of Akt-mTOR signaling abolished the ability of isoflurane to promote proliferation, migration, and invasion of NSCLC cells, indicating that isoflurane promotes NSCLC cell malignancy by activating the Akt-mTOR signaling pathway. Isoflurane promotes NSCLC proliferation, migration and invasion by activating the Akt-mTOR signaling pathway. Carcinoma, Non-Small-Cell Lung • Isoflurane • Proto-Oncogene Proteins c-akt • TOR Serine-Threonine Kinases http://www.medscimonit.com/abstract/index/idArt/898434
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Zhang W. et al.: Isoflurane promotes NSCLC malignancy © Med Sci Monit, 2016; 22: 4644-4650
LAB/IN VITRO RESEARCH
Background
Akt-mTOR signaling pathway. In addition, isoflurane treatment upregulates the Akt-mTOR signaling pathway in NSCLC cells.
Lung cancer is one of the leading causes of cancer mortalities worldwide in both males and females, with more than 1.5 million deaths annually [1]. Depending on histological characteristics, lung cancer can be categorized into two major types, small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), of which NSCLC accounts for the vast majority of all lung cancer cases. Within the category of NSCLC, there are several subtypes of cancers with distinct cell origins and pathophysiologies, including lung adenocarcinoma, squamous cell carcinoma, and large cell lung cancer. Despite recent research progress in cancer treatment, the clinical prognosis of NSCLC remains still poor, primarily due to NSCLC cases often being diagnosed at advanced stages [2], leaving only limited effective treatment options available, and patients with advanced NSCLC commonly undergoing cancer metastasis and developing chemotherapy resistance [3]. This prompts the urgent need for identification of diagnostic markers, as well as elucidation of the molecular mechanisms responsible for metastasis and drug resistance.
Material and Methods Reagents MK2206 was obtained from Sigma (St. Louis, MO, USA). Antibodies against p-Akt, Akt, p-mTOR, mTOR, Cyclin D1, MMP2, MMP9 and b-actin were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Cell lines NSCLC cell lines A549 and H1299 were cultured in 1640 Medium (Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco-BRL). The cells were maintained in a humidified 37°C incubator with 5% CO2. Isoflurane gas exposure
Currently, surgery remains one of the front-line treatment options for NSCLC [4]. However, events within the perioperative period were found to affect cancer prognosis, including relapse, metastasis, and drug resistance for a variety of cancers [4]. Anesthesia has recently been linked with advancing cancer development through studies comparing regional vs. general anesthesia, with regional anesthesia being less pathogenic for various cancers [5–8]. Moreover, isoflurane, a widely used volatile anesthetic, has been found to enhance the malignant potential of renal [9], prostate [10] and ovarian cancer cells [11]. The mechanisms underlying the effects of isoflurane on cancer malignancy is under debate. Increased expression of insulin-like growth factor (IGF)-1 and vascular endothelial growth factor (VEGF) was suggested to cause enhanced cell cycle progression, cell proliferation and angiogenesis, which could contribute to accelerated cancer progression [11]. Other studies indicated that when the hypoxia-inducible factor (HIF) cellular signaling pathway is activated, it is responsible for isoflurane-induced cancer malignancies [9,10], as HIF signaling transcriptionally regulates various genes that have important roles in cancer activity, including cell growth, angiogenesis, glucose uptake, and metastasis [12].
Isoflurane gas exposure was performed as previously described [9].
To date, the effects of isoflurane on NSCLC development have not been previously evaluated. In our study, we are first to report that isoflurane treatment promotes proliferation, migration and invasion of NSCLC cells, and that pharmacological inhibition of Akt-mTOR signaling abolishes the ability of isoflurane to promote these processes of NSCLC cells, indicating that isoflurane promoted NSCLC cell malignancy via activating the
Wound healing assay
This work is licensed under Creative Common AttributionNonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
CCK-8 assay NSCLC cell proliferation was measured using the CCK-8 cell proliferation kit (Beyotime Biotechnology, Hainan, China) according to manufacturer’s instructions. Briefly, NSCLC cells were seeded into 96-well plates at 2×103 cells per well and cultured for 48 h after treatment. Ten μl CCK-8 (5 mg/ml) was added into the culture medium in each well. After 1 h incubation at 37°C, OD values were read with a microplate reader at the 450-nm wavelength. MTT assay NSCLC cells were plated into 96-well plates at a density of 3×103 cells/well. The cells were incubated with 20 µl MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 4 h at 37°C in the dark. DMSO (150 μl) was added to each well after 4-h incubation. Absorbance at 562 nm was measured with a microplate reader.
NSCLC cells were plated into 6-well plates and reached 80%90% confluence. Artificial wounds were created on the confluent cell monolayer with 200-μL pipette tips, and the detached cells were washed twice with FBS. These cells were grown in complete 1640 medium, and migrating cells were examined
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Zhang W. et al.: Isoflurane promotes NSCLC malignancy © Med Sci Monit, 2016; 22: 4644-4650
LAB/IN VITRO RESEARCH
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Figure 1. Isoflurane promotes NSCLC cell proliferation. (A) CCK-8 proliferation assay of A549 and H1299 cells after isoflurane treatment (0%, 1% or 2%). (B) MTT assay of A549 and H1299 cells at the indicated time points after isoflurane treatment (0%, 1% or 2%). * p
