| Introduction:Lung cancer is one of the most common malignant tumors. For those with advanced or recurring lung cancer patients, chemotherapy forms the foundation of their treatment. However, lung cancer cells usually acquire resistance to current standard chemotherapy based on platinum agents, leading to a poor curative effect. Therefore, it is meaningful to explore the potential causes that are involved in resistance to chemotherapeutics. It has been reported that fibroblast growth factor2(FGF-2) plays part in tumors resistance to chemotherapeutics through inhibiting cell apoptosis, however the underlying mechanism is unclear. Recent studies have shown that FGFR1(fibroblast growth factor receptor1) specific amplification is common in squamous cell lung cancer, and that FGFR1may represent a promising therapeutic target for squamous cell lung cancer. FGFR1exerts functions in binding with its high affinitive ligand FGF-2, therefore it is significant to investigate the role of FGF-2/FGFR1signaling in the development of squamous cell lung cancer. Previous researches have indicated that thioredoxin (TRX) can promote cancer development. However, there is no research to investigate whether TRX is involved in apoptotic process regulated by FGF-2in squamous cell lung cancer.Objective:(1) To investigate the effects of FGF-2on expression levels of TRX in lung squamous cancer cell line SK-MES-1;(2) To explore the role of FGF-2in cisplatin-induced apoptosis in SK-MES-1cell line;(3) To investigate the function of TRX in the process that FGF-2regulates SK-MES-1apoptosis.Methods:(1) Western blot assay was performed to examine the expression levels of TRX protein in SK-MES-1cells, which were treated by FGF-2at concentration gradients (0,5,10,25,50,75ng/ml) and different time points (0,4,8,12,24h) to determine the optional concentration and time of FGF-2.(2) RT-PCR assay was used to detected the levels of TRX transcript in SK-MES-1.(3) MTT assay was used to detect IC50(inhibition concentration50%) of cisplatin in SK-MES-1.(4) To analyse the effects of FGF-2on apoptosis, five methods were carried out:Western blot for the expression levels of TRX and PARP proteins, Immunofluorescence cytochemistry for the subcellular position and relative quantity of TRX, Ac-DEVD-pNA method for the activity of caspase-3enzyme, flow cytometry (FCM) for apoptotic rates and RT-PCR assay for the expression levels of TRX mRNA.(5) To make thioredoxin gene silence, TRX-siRNA was trasfected into SK-MES-1cells. Fluorescence microscope was used to examine the transfection efficiency of siRNA and Western blot was carried out to determine the inhibition efficiency of TRX-siRNA. Annexin V-FITC/PI and FCM were performed to detect the rates of early and advanced stage apoptosis in SK-MES-1cells.Results:(1) SK-MES-1cells incubated at different concentrations of FGF-2, and the expression levels of TRX protein was obviously up-regulated (1.89±0.08) fold (P<0.05) in the10ng/ml FGF-2group.(2) SK-MES-1cells incubated with10ng/ml FGF-2for different time ranges, and the expression levels of TRX protein obviously increased (2.31±0.04) times (P<0.05) in the group, which was treated with10ng/ml FGF-2for8hours.(3) There were no changes in TRX mRNA transcript in SK-MES-1cells between treated with and without10ng/ml FGF-2, and between with FGF-2+cisplatin (DDP) and with only DDP (P>0.05). This suggested that FGF-2may increase the expression levels of TRX protein through post-transcriptional regulation.(4) SK-MES-1cells were incubated at different concentrations of DDP for24h, including0,3,10,30,75,150,300mg/L DDP. The survival rate of SK-MES-1decreased when the dose of DDP increased, then DDP concentrations and survival rates of SK-MES-1were used to calibrate IC50(50%of inhibition concentration) of DDP, which was approximately30mg/L(5) FGF-2could obviously inhibit the activites of caspase-3enzyme and its substrate PARP, and the inhibition rates were (54.13±1.57)%and (34.46±0.53)%, respectively (P<0.05). FGF-2could clearly up-regulate the expression of TRX protein to (1.87±0.09) fold (P<0.05)(6) In subcellular structures, TRX protein was located in cytoplasm and cellular nucleus in SK-MES-1, and the tendency of relative quantities of TRX was consistent with that detected by western blot assay.(7) FGF-2could obviously suppress apoptosis induced by cisplatin, and the inhibition rate was (41.77±2.42)%.(8) TRX-siRNA could effectively inhibit the expression levels of TRX, and the inhibition rates of TRX-siRNA-1and TRX-siRNA-3were (50.49±0.06)%and (54.25±0.03)%, respectively.(9) FGF-2could obviously inhibit apoptosis induced by low serum culture medium in SK-MES-1, and the inhibition rate was (41.77±2.42)%(P<0.05); TRX-siRNA could cause apoptosis and partly block the anti-apoptotic effect of FGF-2. Compared with normal control, the rate of apoptosis in TRX-siRNA group increased (1.55±0.02) fold (P<0.05), in addition, the apoptotic rate in TRX-siRNA+low serum group was up-regulated (1.42±0.03) fold (P<0.05), relative to low serum group. However, there was no differentce in apoptotic rate between TRX-siRNA+FGF2+low serum group and TRX-siRNA+low serum group (P>0.05).Conclusions:(1)FGF-2can up-regulate the expression level of TRX protein in lung squamous cell cancer line SK-MES-1.(2)FGF-2can suppress apoptosis of lung squamous cell cancer line SK-MES-1induced by cisplatin.(3) TRX protein may partly play part in the anti-apoptotic effect of FGF-2on lung squamous cell cancer line SK-MES-1. |
PDF Full Text Request