| 1. IntroductionLung cancer is one of the most common malignant tumors in the world at present. Small cell lung cancer (SCLC) accounts for around15%of all lung cancers. It is characterized by rapid growth, early dissemination and marked sensitivity to first-line chemotherapy or radiation treatment. However, two thirds of patients present with a high propensity of relapse rate and subsequent poor prognosis because of chemo-and radio-resistance. Although some molecular such as Bmil, MDR1/ABCB1and so on were related to chemo-and radio-resistance, they can not be effectively used in clinical. Accordingly, the treatment of SCLC remains challenging, and the development of more effective treatment targeting molecules associated with resistance is necessary.The aberrant glycosylation of cell-surface glycoproteins is highly associated with a variety of human cancers. Glycosyltransferase, located in the Golgi apparatus, plays a critical role on the protein glycosylation. It includes at least six N-acetylglucosaminyltransferases (GnTs), defined as GnT-I-VI. GnT-V is a key enzyme in the processing of multiantennary N-linked glycoprotein synthesis. Studies have revealed that theβ1,6GlcNAc branching on N-glycans catalyzed by GnT-V is associated with malignant transformation by enhancing cell proliferation, inhibiting cell apoptosis and so on. The relationship between therapy resistance and N-glycans has been investigated in a few studies. For example, inhibition of N-linked glycosylation by tunicamycin enhanced human head-and-neck carcinoma cells sensitivity to cisplatin. The altered surface protein glycosylation of a human glioblastoma cell line could lead to lowered resistance to drugs. Our laboratory has reported that down-regulation of GnT-V led to an enhanced radiosensitivity in human nasopharyngeal carcinoma cell CNE-2cells in vitro and in vivo. However, the correlation of GnT-V with radioresistance in the MDR SCLC cells is not well understood.To further study the role of GnT-V in radioresistance in the MDR SCLC cells, the SCLC subline cells (H69AR GnT-V/1564and H69AR GnT-V/2224) expressing lowered GnT-V was developed. The results indicated that down-regulation of GnT-V markedly sensitised SCLC cells to drugs and radiation. The following studies showed that NF-кB and apoptosis related moleculars was involved in GnT-V regulating radiation resistance. These data revealed that GnT-V may be a potential target for chemo-and radio-resistance in SCLC.2. Objects(1) This experiment chose human small lung cancer cell lines H446, H69cells and MDR subline H69AR cells as object to analyse the discrepancy in the sensitivity to radiation and chemotherapy drugs of SCLC cell lines.(2) To investigate whether GnT-V is involved in the radioresistance of the MDR SCLC, and to analyze the GnT-V expression difference of H69AR, H69and H446cells at expression of protein level.(3) To silent expression of GnT-V in H69AR cells with shRNA, then detect the effect of silencing, and observe the change of radiation and drug resistance of H69AR cell line.(4) To elucidate the role of GnT-V on the radioresistance of MDR SCLC H69AR cells and its underlying mechanism.3. Materials and methods3.1. Cell Lines and Cell CultureThe human small lung cancer cell lines NCI-H446, NCI-H69and the drug-resistant subline H69AR were purchased from the American Type Culture Collection (ATCC, USA). The H69cell line was grown as floating aggregates whereas H69AR or H446cells grown as attached monolayers on plastic. Subculture of H69was achieved by mechanical disaggregation and transfer of small groups of cells to new flasks, whereas for H69AR and H446, the use of0.25%trypsin plus0.02%EDTA is required. All cells were routinely maintained in RPMI1640medium supplemented with (H69AR:20%, H69or H446:10%, v/v) fetal bovine serum (FBS) and L-glutamine in an incubator at37℃with a humidified atmosphere of95%air and5%CO2. H69AR was cultured by alternate feedings with drug-free medium or medium containing0.8μM of Adriamycin (ADM), and overexpresses MRP1/ABCC1. The resistant cell line was checked regularly for maintained resistance to the selected drugs. Growth and morphology of all cell lines were monitored on a weekly basis.3.2. In vitro chemo-and radio-Sensitivity assayThe chemo-and radiotherapy-induced effects were determined by means of Cell Counting Kit-8(CCK-8) assays. A total of three anticancer drugs [Adriamycin (ADM; Shenzhen, China), Cisplatin (DDP; Shangdong, China) and Etoposide (VP-16; Jiangshu, China)] were obtained from commercial sources and were dissolved according to the manufacturer’s instructions and tested in five concentrations. The ranges of drug concentrations were based on earlier studies and aimed at obtaining an IC50(the concentration that produces50%of growth inhibition) value both for highly sensitive and resistant cases. Growing cultures of cells were exposed to6mV X rays using a linear accelerator (Clinac2300C/D; Varian, United States). Anticancer drugs or radiation-induced cell death was quantified using the CCK-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfoph-enyl)-2H-tetrazolium,monosodiu msalt] assay. Cell survival rate=(value in the treatment group/value in the nontreatment group)×100%.This test was administered as follows. Aliquots of0.1ml of cell suspensions containing104cells/ml were seeded onto96-well plates (104cells/well). After24h of incubation, the cells were submitted either to a new medium containing, or not containing (control), various concentrations of the three antineoplastic drugs tested (i.e. ADM or DDP or VP-16), or to various doses of ionizing radiation(0,2,4,6and8Gy, respectively). This incubation time was chosen for both the chemotherapeutic and radiotherapeutic treatment for the sake of homogeneity in the results. CCK-8reagent was then added and the cells were incubated at37℃for2h. The optical density (OD) value of each sample was measured at a wavelength of450nm on a microplate reader. The assay was conducted in three replicate wells for each sample and three parallel experiments were performed.5.5. Construction and transfection of pGPU6/GFP/Neo GnT-V shRNAThe pGPU6/GFP/Neo vector was obtained from Shanghai GenePharma Co, Ltd. The pGPU6/GFP/Neo GnT-V/1564(plasmid of antisense GnT-V cDNA), pGPU6/GFP/Neo GnT-V/2224(plasmid of antisense GnT-V cDNA) and pGPU6/GFP/Neo GnT-V/NC (control plasmid) were constructed as described previously. The SCLC H69AR cell sublines (H69AR GnT-V/1564and H69AR GnT-V/2224) expressing lower GnT-V and the control for the transfected cell line (H69AR GnT-V/NC) were developed and confirmed. The ways in which the assays were carried out were detailed elsewhere.3.4. Tumorigenicity in athymic mice and fractionated radiationMale BALB/c nu/nu mice aged4-6week-old were obtained from Medical Experimental Animal Center of Guangdong Province. The mice were raised under pathogen-free conditions. Animal experiments were performed under the regulations of the institutional ethical commission (Sun Yat-Sen University).The tumorigenicity of H69AR, H69, H446and H69AR GnT-V/NC, H69AR GnT-V/1564, H69AR GnT-V/2224was determined by s.c. injection of1.0×107viable cells of each type in a volume of0.2ml PBS into the left gluteal region of each mouse. The width and length of tumors were measured with calipers and the tumor size was calculated using the following equation:The tumor volume V=(width2X length)/2. Xenografts were allowed to grow for14days (the tumor volumes reached to about300mm3) before further experimentation. Fractionated radiation was carried out as described previously. The normalized tumor sizes=the tumor volume at day X/the tumor volume at day O in the same group. Twelve days later (two day after the last radiotherapy), the mice from each group were sacrificed. Transplanted tumors were removed, fixed, and stained with routine hemotoxylin and eosin (H&E) and the Bcl-2, Bcl-xl and Bax expression was detected by Western Blot assay.3.4. Tumorigenicity in athymic mice and fractionated radiationThe tumorigenicity of H69AR, H69, H446and H69AR GnT-V/NC, H69AR GnT-V/1564, H69AR GnT-V/2224was determined by s.c. injection of1.0×107viable cells of each type in a volume of0.2ml PBS into the left gluteal region of each mouse. The width and length of tumors were measured with calipers and the tumor size was calculated using the following equation:The tumor volume V= (width2×length)/2. Xenografts were allowed to grow for14days (the tumor volumes reached to about300mm3) before further experimentation. Fractionated radiation was carried out as described previously.The normalized tumor sizes=the tumor volume at day X/the tumor volume at day0in the same group. Twelve days later (two day after the last radiotherapy), the mice from each group were sacrificed. Transplanted tumors were removed, fixed, and stained with routine hemotoxylin and eosin (H&E) and the Bcl-2, Bcl-xl and Bax expression were detected by Western Blot assay.3.5. Cell cycle assayFor cell cycle distribution analysis, SCLC cells were harvested after radiation (0Gy,6Gy) for24hours, washed with phosphate buffer saline (PBS) twice and then fixed with70%cold ethanol for12h. The fixed cells were spun down and re-suspended in PBS at1×106cells/ml. After being washed with PBS, the cells were treated with PBS containing RNase. Next, DNA was stained with propidium iodide (PI) for30min on ice followed by flow cytometry analysis of cell cycle (BD Biosciences, Oxford, United Kingdom). Each test was repeated in triplicate.3.6. Cell apoptosis assayFor Hoeschst33258staining, cells were seeded on24-well plates and grown to monolayer. Cells were treated with radiation (0Gy,6Gy) for24hours. After removing medium, cells were rinsed twice in4℃ice-cold phosphate buffered saline (PBS) and fixed in4%formaldehyde at4℃for10min. After washing, the cells were incubated using0.5ml Hoechst33258solution staining at room temperature for10min in the dark, and washed three times with PBS. Cells were observed and imaged under a fluorescence microscope to determine cell apoptosis.Cells were harvested after radiation (0Gy,6Gy) for24hours. Detection was performed according to instructions of the cell apoptosis detection kit, and analysis was carried out using a flow cytometer (BD Biosciences, Oxford, United Kingdom).Cells were harvested after radiation (OGy,6Gy) for24hours. Further characterization of apoptosis in cells was performed using a commercially available in situ cell death detection kit (Boehringer Mannheim) to find DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reagent according to the manufacturer’s protocol. The number of TUNEL-positive cells was counted in five different fields by an observer blinded to cell treatment or transfection status.Caspase-3activity was assessed via a colorimetric assay utilizing specific substrates. After radiation (OGy,6Gy) for24hours, the cells were washed once with ice-cold PBS and collected by trypsinization followed by centrifugation. The cellular pellet was resuspended in a cell lysis buffer and incubated on ice for10min. Lysates were centrifuged for10min at16,000rpm, and the supernatants were assayed for Caspase-3activity in assay buffer. The cell lysate was incubated with caspase substrate Ac-DEVE-pNA (Caspase-3substrate1) for60min at37℃. An absorbance at405nm was measured in spectrophotometer. The relative increase of Caspase-3activity was determined by comparing the absorbance of pNA from radiation group to the corresponding control group.3.7. Dual-Luciferase Reporter Assay for NF-кB activityTo determine the effect of radiation exposure and GnT-V knockdown on radiation-induced NF-kB transcriptional activity, cells in70-80%confluent were seeded on24-well dishes and allowed to grow for24hours. Cells were then co-transfected with pNF-icB Luc reporter plasmid and pRL-CMV Vector (kind gift from Metastasis Research Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.) using Lipofectamine2000(Invitrogen, California, USA), according to the manufacturer’s procedures. At24hours after transfection, transfected cells were treated with OGy,6Gy radiations for24hours. Subsequently, cells were harvested and NF-кB-mediated transcription was evaluated by measuring luminescence using the Dual-Luciferase(?) Reporter Assay System (Promega, Madison, WJ). A luminometer (Berthold Lumat LB9507) was used to monitor luciferase activity.3.8. Immunofluorescence.Cultured cells were treated with OGy,6Gy radiations for24hours, and stained for NF-кB p65followed by Cy3-labeled secondary antibody. DAPI was used to visualize the nuclei. Stained cells were visualized and fluorescence images were captured using a Leica DMIRE2fluorescence microscope (Leica, Wetzlar, Germany). Briefly, after splitting the two channels (Cy3-stained p65and DAPI-stained nuclei), segmentation of individual nuclei and definition of the overall cellular area is then performed.3.9. Western Blot Analysis.Cells (107) were harvested after radiation (OGy,6Gy) and incubation for24h. After incubation, the cell proteins were extracted with RIPA lysis buffer and then measured using the standard BCA method (BCATM Protein Assay Kit, Pierce, USA).Each protein sample (20ug/well) was homogenized in the loading buffer and boiled for5min, then separated on10%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes using a semi-dry transfer apparatus. The membranes were blocked with5%nonfat dried milk for2h, then treated with primary antibodies (1:200-diluted antibody of Bcl-2,1:300-diluted antibody of Bcl-xl and1:200-diluted antibody of Bax, all from Santa Cruz Biotechnology, Inc., California, USA and1:1000diluted antibody of GAPDH, from Abmart, Inc., China) for12h at4℃. The membranes were washed again with TBST (10mM Tris, pH8.0,150mM NaCl, and0.1%Tween20), followed by incubation with horseradish peroxidase-labeled secondary antibody (1:4000-diluted antibody, from Beijing Biosynthesis Biotechnology Co., Ltd.) for45min at room temperature. Finally, after being developed with super echochemiluminescence (ECL) plus detection reagents, the protein bands of membranes were visualized by exposure to X-ray film. Ratios of protein band intensity were obtained using Quantity One software.3.10. Statistical analysisThe results of at least three independent experiments are displayed as means±SDs. Comparison of means between two samples was determined by Student’s t test. Statistical comparisons of more than two groups were performed using one-way analysis of variance (ANOVA), and then least-significant difference (LSD) for multiple comparisons. In all analysis, statistical significance was defined as P<0.05. Differences between values were analyzed using SPSS ver.13.0statistical software.4. Results4.1. Drug and radiation sensitivity in small cell lung cancer cell linesTo determine the drug sensitivity of SCLC cells to chemotherapeutic agents, the IC50values of the chemotherapeutic drugs including adriamycin, cisplatin and etoposide were evaluated by CCK-8assay. The growth inhibitory IC50values to chemotherapeutic drugs in the H69AR was larger than that in H69or H446cells (P<0.05).The radiosensitivity of SCLC cells was investigated by CCK-8assay. Cells were irradiated with4single doses (2,4,6and8Gy, respectively). Compared with H69AR cells, the survival rate of H69or H446cells decreased significantly at2,4,6and8Gy (P<0.05). The result revealed that H69AR cells is resistant to radiation.4.2. Comparison of the levels of GnT-V in small cell lung cancer cell lines To evaluate GnT-V expression at the mRNA and protein level in H69AR, H69and H446cells, qRT-PCR and Western Blot were performed. The results showed that the mRNA and protein level of GnT-V in H69AR cells was higher than H69or H446cells (P<0.05).H69AR cells showing higher GnT-V and more resistance to chemotherapy and radiation suggested that GnT-V may be involved in radio-and chemo-resistance of SCLC.4.3. Development of down-expressing GnT-V H69AR cells by GnT-V shRNATo further study the role of GnT-V in radioresistance in H69AR cells, the H69AR cells expressing lowered GnT-V was developed. The pGPU6/GFP/Neo GnT-V/NC, pGPU6/GFP/Neo GnT-V/1564and pGPU6/GFP/Neo GnT-V/2224were transfected into H69AR cells. The transfected cells (H69AR GnT-V/NC, H69AR GnT-V/1564and H69AR GnT-V/2224) were obtained under G418, screened for1month. Fluorescent microscopy showed that the transfected cells all emitted green fluorescence.The expression of GnT-V mRNA and protein in H69AR and H69AR GnT-V/NC cells has no significant difference. The expression of GnT-V mRNA in H69AR GnT-V/1564and H69AR GnT-V/2224cells were decreased by (62.00±4.35)%and (73.12±3.52)%respectively compared to H69AR GnT-V/NC by qRT-PCR. The GnT-V protein in H69AR GnT-V/1564and H69AR GnT-V/2224cells was decreased by (68.24±3.53)%and (76.52±4.67)%respectively compared with H69AR GnT-V/NC by Western Blot assay. It confirmed the expression of GnT-V at both the mRNA and protein level in H69AR GnT-V/1564and H69AR GnT-V/2224cells were lower than those in the control groups.4.4. Suppression of GnT-V sensitizes H69AR cells to chemotherapeutic drugs and radiation The viability of the transfected H69AR cells exposed to chemotherapeutic drugs was analyzed by CCK-8assay. The growth inhibitory IC50values to chemotherapeutic drugs in the H69AR GnT-V/1564or H69AR GnT-V/2224cells was smaller than that in H69AR GnT-V/NC cells (P<0.05). These data indicated that down regulation of GnT-V sensitised SCLC cells to chemotherapeutic drugs.Cell radiosensitivity was also studied by CCK-8assay in H69AR GnT-V/NC, H69AR GnT-V/1564and H69AR GnT-V/2224. The cells were irradiated with4single doses (2,4,6and8Gy, respectively). Compared with the control cells, the survival rates of H69AR GnT-V/1564or H69AR GnT-V/2224cells decreased significantly at any radiation dose (P<0.05). These data indicated that the radiation sensitivity of SCLC cells was elevated after down-regulation of GnT-V.^4.5. Down-regulation of GnT-V enhances cell radiosensitivity in vivoThe effect of GnT-V on tumor growth and radiosensitivity was further studied in vivo. The tumor size of H69AR, H69and H446was measured after subcutaneous (s.c.) inoculation of1.0×107cells. The H69, H446and H69AR radiation groups presented smaller tumor sizes than their nonradiation groups (P<0.05), however, the decreased extent in H69or H446cells was more significant than that in H69AR cells (P<0.05). H69AR cells were less sensitivity to radiation than H69and H446cells.After radiation, the tumor sizes were all smaller, but H69AR GnT-V/1564or H69AR GnT-V/2224decreased more significantly than H69AR GnT-V/NC groups(P<0.05). Thus, the results suggested that down-regulation of GnT-V can enhance H69AR tumor radiosensitivity in vivo.4.6. Inhibition of GnT-V enhanced radiation-induced G2-M phase arrestThe cell cycle analysis was performed using flow cytometry to evaluate the differences in SCLC cells. The G2-M phase proportion of H69, H446and H69AR cells in the radiation group were increased compared with the corresponding nonradiation group (P<0.05), but the G2-M phase proportion of H69or H446cells was increased more significantly than H69AR cells (P<0.05).The G2-M phase proportion of the stable transfected H69AR cells were all enhanced in radiation group compared with corresponding nonradiation group, but the increased extent in H69AR GnT-V/1564or H69AR GnT-V/2224cells was more than that in H69AR GnT-V/NC cells (P<0.05). This result showed that suppression of GnT-V enhanced radiation-induced G2-M phase arrest in the cell cycle progression.4.7. Effect of GnT-V on radiation-induced apoptosis and apoptotic proteins expressionThe effect of GnT-V on radiation-induced cell apoptosis was evaluated by the following experiments:Hoechst33258staining, flow cytometry, Tunel assay in situ detection and Caspase-3activity measurement.Hoechst33258staining revealed that chromatic agglutination, enhanced fluorescent staining and apoptotic body formation could be observed in the all SCLC cells after radiation. However, radiation induced more cell apoptosis in H69or H446cells than in H69AR cells. Compared with H69AR GnT-V/NC, more radiation induced apoptosis could be observed in H69AR GnT-V/1564or H69AR GnT-V/2224cells.The cell apoptosis was studied by flow cytometric analysis in SCLC cells treated with OGy and6Gy. The cell apoptosis rates were all increased after radiation (P<0.05), however, the increased extent was different. H69AR cells presented less cell apoptosis after radiation than H69or H446cells, and also less than H69AR GnT-V/1564and H69AR GnT-V/2224cells. Consistent with the result of flow cytometry detection, TUNEL-staining showed the cell apoptosis rates were all increased in the radiation groups compared with the corresponding nonradiation(P<0.05). More radiation induced cell apoptosis were observed in H69or H446than in H69AR cells, and more in H69AR GnT-V/1564or H69AR GnT-V/2224cells than in H69AR GnT-V/NC.Caspase-3plays a pivotal role in the terminal execution phase of apoptosis. The Caspases-3activity of H69AR, H69and H446was increased by (210.39±32.16)%,(499.93±42.74)%and (515.63±32.72)%in radiation groups compared with the corresponding nonradiation groups (P<0.05). The Caspases-3activity of H69AR GnT-V/NC, H69AR GnT-V/1564and H69AR GnT-V/2224was increased by (225.47±27.84)%,(595.09±38.63)%and (609.12±20.28)%after radiation(P<0.05). These data showed that the Caspases-3activity in cells was all increased in radiation group compared with the corresponding nonradiation. The increased extent of apoptosis rate in H69or H446was more than in H69AR groups, and in H69AR GnT-V/1564or H69AR GnT-V/2224cells it was greater than in H69AR GnT-V/NC cells.Because over-expression of Bcl-2family members has been strongly associated with radioresistance and apoptosis, we examined the expression level of Bcl-2, Bcl-xl and Bax in the SCLC cell lines with or without radiation in vitro and in vivo. The level of Bcl-2and Bcl-xl was higher, and the level of Bax was lower in H69AR cells compared with H69and H446cells. The differences were more obvious after radiation (P<0.05).To further investigate whether Bcl-2family was closely associated with radiosensitivity regulated by GnT-V, we detected these proteins in the transfected H69AR cell lines at24h after treatment with or without radiation (6Gy) in vitro and in vivo. The protein level of Bcl-2and Bcl-xl was greatly reduced, while Bax was increased in H69AR GnT-V/1564or H69AR GnT-V/2224, and the expression level was further decreased or increased after radiation respectively.(P<0.05). But there was no significant difference in H69AR GnT-V/NC with or without radiation. The results showed that down-regulation of GnT-V promoted H69AR radiation sensitivity possibly through suppression of Bcl-2and Bcl-xl, and enhancement of Bax.4.8. Effect of GnT-V on radiation-induced NF-кB signalingNF-кB transcriptional activity in SCLC cells was investigated using a NF-кB luciferase reporter construct. SCLC cells were co-transfected with pNF-кB Luc reporter plasmid and pRL-CMV Vector. Cells were harvested after radiation (OGy,6Gy) for24hours, NF-кB luciferase activity in SCLC cells was assessed by luminometer. According to the results, radiation induced an increase (4.60,2.35,2.26-fold and4.50,1.87,1.65-fold respectively) in NF-кB activity of H69AR, H69, H446and H69AR GnT-V/NC, H69AR GnT-V/1564, H69AR GnT-V/2224. The data indicated that radiation-induced NF-кB activity in H69AR cells was higher than that in H69and H446cells, but it was lower in H69AR GnT-V/1564or H69AR GnT-V/2224cells than that in H69AR GnT-V/NC cells, indicating that down-regulation of GnT-V in H69AR cells reduced radiation-induced NF-кB signaling.The results were confirmed by immunofluorescence staining of p65. SCLC cells were harvested after radiation (OGy,6Gy) for24hours, and the effect of radiation exposure on p65translocation was investigated by immunofluorescence staining. More nuclear translocation of p65would be observed if the NF-кB activity was increased after radiation. The results revealed that H69AR cells showed more radiation induced nuclear translocation of p65than H69and H446cells, and also more than H69AR GnT-V/1564or H69AR GnT-V/2224cells, consistent with the result of Dual-Luciferase Reporter Assay for NF-кB. 5. Conclusions(1) Human SCLC MDR cell line H69AR is resistant to chemotherapeutic drugs and radiation.(2) Compared with SCLC cell lines H69and H446, GnT-V level of H69AR cells increases obviously. The shRNA expression vectors aimed at GnT-V gene can down-regulate the expression of GnT-V both in the level of mRNA and protein obviously.(3) After down-regulation of GnT-V expression of H69AR, the sensitivity to chemotherapeutic drugs and radiation of H69AR increases greatly, to further confirm that GnT-V may stand up to chemo-and radio-therapeutics induced cell apoptosis, and may play an important role in SCLC drugs and radiation resistance%enerating course.(4) The drugs and radiation resistance of H69AR generates from G2-M phase arrest of the cell cycle; increasing of GnT-V level, NF-кB activity and Caspase-3activity; increasing expression of protein Bcl-2and Bcl-xl of Bcl-2family and decreasing expression of protein Bax.(5) GnT-V may be a potential biomarker for predicting SCLC response to chemo-and radio-therapy. |
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