死亡受体Fas在人胃癌SGC—7901细胞凋亡过程中的应用

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 33: 1451-1458, 2014Abstract. Gastric cancer, one of the most common malig-nancies worldwide, typically has a poor prognosis and poor survival rate. Previous studies have investigated the chemopreventive effect of celecoxib. In the present study, the SGC-7901 human gastric cancer cell line was utilized to examine the chemopreventive mechanisms of celecoxib. The inhibition of cell proliferation was determined using MTT assay, cell apoptosis was monitored by terminal deoxynucleo-tidyl transferase-mediated dUTP nick end-labeling (TUNEL) and flow cytometry, and cell ultrastructural changes were assessed via transmission electron microscopy. The mRNA expression of Akt, caspase-8 and -9 was examined using quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) and p-Akt, procaspase-8 and -9 were analyzed via western blotting. The results showed that celecoxib inhibited the proliferation of SGC-7901 cells in a time- and dose-dependent manner. Additionally, celecoxib induced apoptosis as substantiated by typical apoptotic bodies, autophagosomes and an increased apoptotic rate. It was found that following celecoxib treatment, Akt mRNA expression was not signifi-cantly altered, and that p-Akt protein levels decreased in a time- and dose-dependent manner. Additionally, caspase-8 and -9 mRNA expression was significantly increased, while procaspase-8 and -9 protein expression decreased relative to the time- and dose-dependent effects. These results demon-strated that celecoxib induced apoptosis and autophagy of gastric cancer cells in vitro through the PI3K/Akt signaling pathway. Moreover, our findings suggested that celecoxib induces apoptosis in gastric cancer cells through the mito-chondrial and death receptor pathways, providing additional understanding regarding the chemopreventive behaviors of celecoxib and its uses in cancer therapy. IntroductionGastric cancer is among the most common malignancies worldwide, with a 5-year survival rate of only 20% (1). Due to the fact that this type of cancer has a poor prognosis, attention has been drawn to the chemopreventive effect of celecoxib, a cyclooxygenase-2 (COX-2) inhibitor. COX, also known as prostaglandin synthase peroxidase, is the rate-limiting enzyme catalyzing arachidonic acid into prostaglandins, with COX-2 being involved in inflammatory diseases and certain types of tumor (2,3). In vitro and in vivo studies have shown that the long-term and widespread use of non-steroidal anti-inflamma-tory drugs (NSAIDs) may contribute to the maintenance of gastric health, with epidemiological studies showing possible risk reduction (4,5).Apoptosis, programmed cell death (PCD), occurs in multi-cellular organisms, with autophagy also triggering PCD through different apoptotic mechanisms (6,7). Celecoxib and related compounds have been shown to induce cell cycle arrest, inhibit tumor growth and suppress tumor angiogenesis, with celecoxib potently inducing apoptosis in tumor cells (8-11). An increased expression of COX-2 and Akt in gastric carcinomas relative to normal gastric mucosa, with celecoxib treatment inducing tumor apoptosis has also been shown (12). Further experimen-tation showed that COX-2 inhibitors may induce apoptosis by affecting Akt phosphorylation, thus activating the Akt signaling pathway (13). In this study, we used the selective COX-2 inhibitor celecoxib to treat the SGC-7901 gastric cancer cells and to induce cell apoptosis in vitro. The effects of celecoxib on apoptotic and autophagic cell death through the monitoring of mRNA and protein levels of Akt, caspase-8 and -9 were also examined. This approach aids in further characterization of the apoptotic effect of celecoxib via the PI3K/Akt signaling pathway in order to gain a better understanding of its antitumor effects. Materials and methodsCell culture. SGC-7901 human gastric cancer cells (Type Culture Collection Committee, Chinese Academy of Sciences, Shanghai, China) were cultured in RPMI-1640 medium (Gibco,Celecoxib regulates apoptosis and autophagy via the PI3K/Akt signaling pathway in SGC-7901 gastric cancer cellsMIN LIU1*, CHUN-MEI LI2*, ZHAO-FENG CHEN1, RI JI1, QING-HONG GUO1,QIANG LI1, HONG-LING ZHANG1 and YONG-NING ZHOU1Divisions of 1Gastroenterology and Hepatology, 2Oncology,The First Hospital of Lanzhou University, Lanzhou, Gansu 730000, P.R. ChinaReceived November 30, 2013; Accepted March 20, 2014DOI: 10.3892/ijmm.2014.1713Correspondence to: Dr Yong-Ning Zhou, Division of Gastroen-terology and Hepatology, The First Hospital of Lanzhou University,Lanzhou, Gansu 730000, P.R. ChinaE-mail: yongningzhou@*Contributed equallyKey words: apoptosis, autophagy, celecoxib, cyclooxygenase 2,Akt, caspase-8, caspase-9, gastric carcinomaLIU et al: CELECOXIB INDUCES APOPTOSIS AND AUTOPHAGY VIA PI3K/Akt PATHWAY IN SGC-7901 CELLS 1452Long Island, NY, USA) with 2 mmol/l glutamine, supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 50 U/ml penicillin, and 50 mg/ml streptomycin. The cells were plated at a density of 1x105 cells/ml in 6-well tissue culture plates and grown to confluency at 37˚C with 5% CO2. When 50% confluency was reached, serum-supplemented medium was replaced with the recommended serum-free RPMI-1640 medium for overnight culturing before celecoxib intervention. Celecoxib, provided by the Faculty of Medicine, the Chinese University of Hong Kong, stock solution was added to the serum-supplemented medium at different concentrations and cultured until the detection time.MTT assay. The inhibition of cell proliferation in SGC-7901 cells following celecoxib treatment was evaluated using an MTT assay (Sigma-Aldrich, Shanghai, China) as per the manufacturer's instructions. Briefly, 5x103 cells/well were seeded in 96-well plates, incubated in culture medium for 24 h, and treated with varying concentrations of celecoxib (0, 50, 75, 100 and 125 µmol/l) for 24, 48 and 72 h, with parallel samples treated with DMSO only serving as controls. Following treat-ment, the formation of formazan crystals was measured after 4 h of MTT incubation (10% v/v) at an optical density (OD) of 490 nm, with each experiment repeated in triplicate. The relative cell proliferation inhibition rate was calculated as: (1-OD490Test/OD490Control) x100% to show a percentage value.TUNEL assay. DNA breaks occur late in the apoptotic pathway and can be determined and analyzed by performing the TUNEL assay (Roche, Basel, Switzerland). Firstly, cells were seeded on coverslips and treated with 100 µmol/l cele-coxib for 72 h. Following treatment, the cells were washed, fixed and stained as per the manufacturer's instructions and apoptotic numbers evaluated using a confocal laser scanning microscope (Leica, Wetzlar, Germany) at 515-565 nm.Flow cytometric (FCM) analysis of apoptosis. Apoptosis was assessed by flow cytometric analysis using the Annexin V-FITC/PI apoptosis detection kit (Invitrogen, Life Technologies Ltd., Carlsbad, CA, USA). SGC-7901 cells were seeded in 6-well plates at ~5x104 cells/well. Following treat-ment with celecoxib, the cells were trypsinized, centrifuged to remove the supernatant, washed with phosphate-buffered saline (PBS), suspended in 100 µl of 1X binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2), and stained with Annexin V and PI as per the manufacturer's instructions. FITC-positive and PI-negative cells were considered apoptotic cells, PI-positive cells were considered necrotic, and unstained cells were considered normal viable cells. The apoptotic rates of the various cell groups were calculated and comparisons of apoptotic rates were conducted among the various groups.Transmission electron microscope (TEM) analysis of cell ultrastructure. Cells were seeded on coverslips, treated with 125 µmol/l celecoxib for 72 h (with a parallel untreated control), cultured in RPMI-1640 for 72 h, collected and fixed with 3% glutaraldehyde. The cells were washed with PBS, fixed in 1% osmium tetroxide, dehydrated by graded ethanol and acetone, and routinely embedded and polymerized. The slices were contrasted with an aqueous solution of uranyl acetate and lead citrate and examined by JEM-1230 transmis-sion electron microscope (Jeol Ltd., Tokyo, Japan).Quantitative reverse-transcription-polymerase chain reac-tion (qRT-PCR) analysis. SGC-7901 cells were cultured at a density of 1x105 cells/ml in 6-well tissue culture plates. One group was treated with various concentrations of celecoxib (0, 75, 100 and 125 µmol/l) and cultured for 72 h, while an additional test group was treated with 125 µmol/l celecoxib for 0, 24, 48 and 72 h. Total RNA was extracted by a column RNA extraction kit (Sangon, Shanghai, China) and reverse-transcribed into cDNA at 37˚C for 15 min, and 85˚C for 5 sec. Diluted cDNA was subjected to qRT-PCR using a SYBR®Premix Ex Taq™ II kit (Takara Bio, Inc., Shiga, Japan) in 25 µl of reaction solution containing 2 µl of cDNA template, 1 µM of each primer, 10 µl of 2X SYBR-Green master mix, and brought to the final volume with RNase-free water. Reactions were performed in triplicate via a PCR thermal cycler (Roter-Gene 3000; Corbett, Sydney, Australia) under the following conditions: pre-denaturation at 95˚C for 30 sec, 40 cycles of denaturation at 95˚C for 5 sec, and annealing at 62˚C for 30 sec. The relative expression was calculated by the 2-ΔΔCT formula. The primer pairs for qRT-PCR are listed in Table I.Western blot analysis. Total protein was extracted and protein concentrations established via bicinchoninic acid (BCA) assay. Protein (25 µg) was denatured, separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. AfterTable I. Primer pairs for qRT-PCR.Gene name Accession Sequence (5'-3') Product size (bp) Caspase-9 NM_001229 F: CCCATATGATCGAGGACATCCA 186R: ACAACTTTGCTGCTTGCCTGTTAGCaspase-8 NM_001228 F: GGTACATCCAGTCACTTTGCCAGA 83R: GTTCACTTCAGTCAGGATGGTGAGAAkt NM_005163 F: GTGGCAGCACGTGTACGAGAA 108R: GTGATCATCTGGGCCGTGAAβ-actin NM_001101 F: TGGCACCCAGCACAATGAA 126R: CTAAGTCATAGTCCGCCTAGAAGCAINTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 33: 1451-1458, 20141453blocking overnight at 4˚C using 5% BSA, the membranes were incubated with primary antibodies (anti-procaspase-8 1:2,500 and procaspase-9 1:2,000; both from Abcam, Cambridge, MA, USA), p-Akt 1:800 (Bioworld, St. Louis Park, MN, USA) for 2 h at room temperature, washed by TBST and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at 1:2,000 dilution for 2 h. Bands were visu-alized using enhanced chemiluminescence (ECL; Applygen, Beijing, China) detection reagents and scanned images were quantified using ImageJ software. Experiments were performed in triplicate with β-actin used as a housekeeping control for normalization. The ratio of target gene to β-actin was used for semi-quantification and comparison between different groups.Statistical analysis. Triplicate data are presented as mean values and shown as the means ± standard deviation (SD). Samples were analyzed by one-way ANOVA, with P<0.05 considered to indicate statistical significance.ResultsCelecoxib inhibits proliferation of SGC-7901 cells. Following in vitro treatment with celecoxib, the SGC-7901 gastric cancer cell line showed a significant inhibition of cell proliferation in a time- and dose-dependent manner, with the most pronounced effect evident at a concentration of 125 µmol/l for 72 h as iden-tified by a proliferation inhibition rate of 85.6±4.51% (Fig. 1).Celecoxib induces apoptosis of SGC-7901 cells. Fluorescein- labeled dUTP was connected to DNA 3'-OH ends of apoptotic cells by the deoxynucleotidyl transferase enzyme. Apoptotic cells with green fluorescence were detected by laser scanning confocal microscopy at an excitation of 515-565 nm, while all cells were exhibited as red under bright field microscopy. The two images were superimposed to show the specificity of apoptotic cells (yellow) and their position. Celecoxib-treated cells (Fig. 2B) showed significant levels of apoptosis relative to the control (Fig. 2A), with a statistical significance of P<0.05.Treatment with 0, 75, 100 and 125 µmol/l of celecoxib for 72 h yielded apoptotic rates of 4.0±0.91, 12.9±1.32, 24.6±3.63and 35.7±2.73%, respectively, with a statistical significance of P<0.05 when compared to the control group. Treatment with 125 µmol/l of celecoxib for 0, 24, 48 and 72 h, yielded apoptotic rates of 2.2±0.32, 8.5±1.57, 20.3±2.84 and 35.7±2.73%, respec-tively. Both study sets demonstrated a gradual increase in apoptotic rates in a time-and dose-dependent manner (Fig. 3).Celecoxib alters the ultrastructure of SGC-7901 cells. Following treatment with 125 µmol/l celecoxib for 72 h, typical early apoptotic changes were found to include nuclear membrane shrinkage and retraction (Fig. 4A), nuclear chro-matin condensation, marginalization and crescents, with late apoptotic changes observed by nuclei cleavage into frag-ments and apoptotic body production (Fig. 4B). Additionally, typical autophagic structures were found to include several cytoplasmic autophagic vacuoles and autophagosomes, which swallowed organelles (Fig. 4C and D).Effect of celecoxib on Akt, caspase-8 and -9 expression. No significant change in the mRNA levels of Akt was observed subsequent to treatment with celecoxib; however, the presence of p-Akt decreased in a time- and dose-dependent manner. Caspase-8 mRNA expression increased in a dose-dependent manner at concentrations of 75, 100 and 125 µmol/l of celecoxib. Caspase-9 mRNA expression levels increased significantly at a concentration of 100 and 125 µmol/l of celecoxib (Fig. 5A). Following treatment with 125 µmol/l of celecoxib for 24, 48 and 72 h, caspase-8 and -9 mRNA expression increased significantly (Fig. 5B). By contrast,procaspase-8 and -9 protein expression was significantlyFigure 1. Inhibitory effects of celecoxib on cell proliferation of the SGC-7901 gastric cancer cell line detected by MTT analysis. Following treatment with celecoxib at the indicated concentrations and time-points, SGC-7901 cells showed a significant inhibition of cell proliferation in a time- anddose-dependent manner.Figure 2. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis for SGC-7901 human gastric cancer cells. (A) Control group; (B) group treated with 100 µmol/l cele-coxib for 72 h. The apoptotic cell number of the group treated with 100 µmol/l celecoxib for 72 h increased significantly relative to the control. Apoptotic cells with green fluorescence were detected by laser scanning confocal microscopy at an excitation of 515-565 nm, while all the cells exhibit a red image under bright field microscopy. The two images were superimposed to show the speci -ficity of apoptotic cells (yellow) and their position..LIU et al: CELECOXIB INDUCES APOPTOSIS AND AUTOPHAGY VIA PI3K/Akt PATHWAY IN SGC-7901 CELLS 1454lower than the control group in a time- and dose-dependent manner (Fig. 6). These results showed that celecoxib may inhibit Akt phosphorylation and promote caspase-8, -9 tran-scription and procaspase-8, -9 protein activation. DiscussionGastric carcinoma is among the most common malignancies worldwide, with an elevated 5-year postoperative mortality rate (14) thus creating a need for an alternative treatment method. Currently, the role of celecoxib, a non-cytotoxic COX-2 inhibitor, in cancer therapy has been under scru-tiny (14). COX, also known as prostaglandin synthetase, has three known isoenzymes in mammals, COX-1, COX-2 and COX-3 (15). COX-2 is present at low levels of expression in most normal tissues, but tumor factors, inflammatory cytokines and growth factors could promote its expression (16). COX-2is related to the development of tumors by promoting tumor Figure 3. Apoptotic rates of human gastric cancer cell SGC-7901 after treatment with celecoxib at different doses and time-points as detected by flow cytom-etry. The apoptotic rates of SGC-7901 cells markedly increased in a time- and dose-dependent manner following treatment with 0, 75, 100 and 125 µmol/l of celecoxib for 72 h (A and B) *P<0.05 or #P<0.01 when compared with the control group..INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 33: 1451-1458, 20141455cell proliferation, enabling tumor evasion of the host immune surveillance and promoting tumor invasion/metastasis (5,17). Multicellular organisms maintain their homeostasis through cell proliferation and PCD, with an imbalance possibly leading to the development of cancer. In this experiment, we found that the selective COX-2 inhibitor celecoxib induced apoptosis of SGC-7901 cells via reduced expression levels of COX-2, as obsreved by inhibited cell proliferation using MTT analysis and an increased number of apoptotic cells as detected by TUNEL and flow cytometry. Moreover, typical apoptotic changes were shown to include nuclear membrane shrinkage, nuclear chromatin condensation and apoptotic bodies using TEM to support the apoptotic effects of celecoxib. Caspases are a type of protease associated with apop-tosis and cytokine maturation, and are divided into initiator caspases, effector caspases and inflammatory mediators. Caspases are synthesized as relatively inactive zymogens and must undergo a process of activation during apoptosis. Caspase-8 is the initiator of the Fas-Fas ligand (FasL) pathway,and usually exists in the form of procaspase-8. When FasL Figure 3. Continued. The apoptotic rates of SGC-7901 cells markedly increased in a time- and dose-dependent manner following treatment with 125 µmol/l of celecoxib for 0, 24, 48 and 72 h (C and D). *P<0.05 or #P<0.01 when compared with the control group.LIU et al : CELECOXIB INDUCES APOPTOSIS AND AUTOPHAGY VIA PI3K/Akt PATHWAY IN SGC-7901 CELLS1456Figure 4. Ultrastructure changes of SGC-7901 cells following treatment with 125 µmol/l celecoxib for 72 h. We observed nuclear membrane shrinkage and retraction in early apoptosis (x5,000) (A), apoptotic body in late apoptosis (x3,000) (B), and changes in autophagy: autophagic vacuolar and autophagosomes (x40,000 or x30,000) (C and D) by TEM.Figure 6. p-Akt, procaspase-8 and -9 protein expression in SGC-7901 human gastric cancer cells following treatment with celecoxib at different doses and time-points. SGC-7901 cells were treated with 0, 75, 100 and 125 µmol/l celecoxib for 72 h (lanes 1-4) and treated with 125 µmol/l celecoxib for 0, 24, 48 and 72 h (lanes 5-8). Procaspase-8, -9 and p-Akt protein expression were significantly lower than the control group in a time- and dose-dependent manner. Aliquotsof protein extracts (40 µg) were immunoblotted with the indicated antibodies.Figure 5. Akt, caspase-8 and -9 mRNA expression of SGC-7901 human gastric cancer cells following treatment with celecoxib at different doses and time-points with the first group including: the control, 75, 100 and 125 µmol/l celecoxib for 72 h (A) and group two including: the control, 125 µmol/l celecoxib for 24, 48 and 72 h (B). *P<0.05 or #P<0.01 relative to the control group from three independent experiments.INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 33: 1451-1458, 20141457binds to the corresponding Fas receptor, the intracellular death effector domain (DED) of the Fas receptor attracts Fas associated with death domain protein (FADD) and recruits procaspase-8 to form a death-inducing signaling complex (DISC). Procaspase-8 is then hydrolyzed to generate activated caspase-8, followed by the activation of procaspase-3 and other effector caspases that eventually induce apoptosis (18). Caspase-9 is the initiator of the mitochondrial pathway, also known as procaspase-9, an inactive zymogen. The initiator caspase-9 is activated by the assembly of a multimeric complex (dubbed apoptosome) involving Apaf-1 and cytochrome c. Cleaved caspase-9 and -3 are activated and these effector caspases degrade a large number of cell proteins, ultimately inducing cell apoptosis (19,20). In this study, we found that celecoxib significantly increased caspase-8 and -9 mRNA expression in a time- and dose-dependent manner in SGC-7901 cells, suggesting that celecoxib may activate caspase-8 and -9 to initiated apoptosis through the death receptor and mito-chondrial pathways, respectively.Autophagy is a crucial component of the cellular stress adaptation response that maintains mammalian homeo-stasis (21). There are three different forms of autophagy that are commonly described: macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy is the predominant pathway occurring mainly to eradicate damaged organelles or unused proteins. Macroautophagy is mediated by a unique organelle, the autophagosome, which encloses long-lived proteins and portions of organelles for delivery to the lysosome (22,23). Autophagy may play different roles in cancer occurrence and progression, while also potentially promoting or inhibiting cell proliferation at different stages of tumor growth (24). For example, autophagy plays a protective role in tumor cells via degradation of organelles under nutri-tional deficiency. Conversely, autophagy can also inhibit tumor growth via beclin 1, UVRAG, Bif and Atg. Findings of a recent study showed that berberine extracts promoted autophagy by activating beclin 1 expression and activated caspase-9 to induce apoptosis in hepatoma cells (25). Plant lectin from Polygonatum cyrtonema induced apoptosis and autophagy by inhibiting the Ras/Raf and PI3K/Akt signaling pathways in murine fibrosarcoma cells (26). In this study, the selective COX-2 inhibitor celecoxib, not only generated morphological changes indicative of apoptosis, but also typical changes of autophagy to include cytoplasmic autophagic vacuoles and autophagosomes.The molecular mechanism by which celecoxib induces apoptosis is not yet fully understood. The PI3K/Akt pathway widely presents in normal cells, but is abnormally activated in many malignant tumors (27-29). Akt, also known as protein kinase B (PKB), is a central component of the PI3K/Akt pathway, with Akt phosphoregulation impacting a variety of biological activities. In healthy and tumorigenic cells, Akt can be activated in an intracellular manner by hormones, growth factors, and extracellular matrix components (30). Akt regu-lates cell growth, survival and apoptosis through substrate phosphorylation, with Akt phosphoregulation observed at the Thr308 and Ser473 site, which are both required for activa-tion. Akt is activated as follows: the activated PI3K produces a secondary messenger PIP3 at the plasma membrane, PIP3 then binds an inactive Akt inducing its shift from the cytoplasm to the plasma membrane where Ser124 and Thr450 are phosphor-ylated, making Akt undergo a conformational change exposing its Thr308 and Ser473 sites. Immediately, phosphoinositide-dependent kinase 1 (PDK1) and phosphoinositide-dependent kinase 2 (PDK2), which are in close proximity to Akt, respec-tively catalyze the phosphorylation of the exposed Thr308 and Ser473 sites, resulting in the complete activation of Akt. This may trigger a phosphorylation cascade of downstream targets, ultimately impacting the regulation of cell growth and survival, proliferation and apoptosis, angiogenesis, cell migra-tion and numerous biological processes (30-33).In the present study, following celecoxib treatment in SGC-7901 cells, p-Akt, or activated Akt, was distinctly downregulated, leading to the upregulation of caspase-8 and -9 mRNA expression and increased procaspase-8 and -9 activation. Thus, we hypothesized that celecoxib inhibited the PI3K/Akt pathway by reducing the level of phosphorylation of Akt, which in turn activated the expression and activa-tion of caspase-8 and -9, resulting in apoptosis through the death receptor and mitochondrial pathways in SGC-7901 cells. Notably, we found changes in the cell ultrastructure to include apoptosis and autophagy, suggesting that celecoxib simultaneously induced apoptosis and autophagy, which is consistent with results of previous studies (5,13)). Autophagy is an evolutionarily conserved process that occurs during the growth and development process in many animals, but its specific mechanism of PCD is unclear. Autophagy and apoptosis could coadjust through p53 (35), PI3K/Akt (36) and Bcl-2-beclin 1 (37). Thus, celecoxib may impact both apop-tosis and autophagy via the PI3K/Akt signaling pathway in the SGC-7901 gastric cancer cells. The results of this study provide a new theoretical foundation for the antitumor mechanisms of celecoxib and offers new targets for cancer therapy, although these findings should be verified in future investigations. AcknowledgementsThis study was supported by a grant from the National Science Foundation of China (no. 81172366). We would like to thank LetPub for its linguistic assistance during the preparation of this manuscript.References1. Saukkonen K, Rintahaka J, Sivula A, et al: Cyclooxygenase-2 and gastric carcinogenesis. APMIS 111: 915-925, 2003.2. 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Fas 过表达逆转胃癌细胞顺铂耐药陈刚;申屠杨萍【摘要】目的：探究Fas 在胃癌细胞顺铂耐药中的作用及可能的作用机制。

方法：Western blot 及 RT－qPCR检测胃癌细胞株 SGC－7901及胃癌耐药细胞株SGC－7901／DDP 中 Fas 的表达情况。

构建 Fas 过表达腺病毒载体，上调胃癌耐药细胞株 SGC－7901／DDP 中 Fas 的表达，CCK－8检测细胞活力，流式细胞术检测细胞周期及细胞凋亡， Western blot 法检测 Fas、P38／p－P38、JNK ／p－JNK、cleaved caspase－8／caspase－8和 cleaved caspase－3／caspase－3蛋白水平。

结果：耐药细胞株 SGC7901／DDP 中 Fas 的 mRNA 及蛋白表达水平均明显低于亲本细胞株；并且在亲本细胞株SGC7901中，Fas 的蛋白表达水平随着顺铂的浓度增加不断降低。

过表达 Fas 可抑制胃癌耐药细胞SGC7901／DDP的细胞活力，并诱导其凋亡；同时上调 p－P38、p－JNK、cleaved caspase－8和 cleaved caspase－3的蛋白水平。

结论：胃癌顺铂耐药细胞中过表达 Fas 可恢复细胞对顺铂的敏感性，促进细胞凋亡并抑制细胞生长，其机制可能与 JNK 和 P38 MAPK 信号通路的激活有关。

%AIM: To study the effect of Fas on cisplatin resistance in stomach cancer cells and its possible mechanisms.METHODS: The expression of Fas at mRMA and protein levels in SGC-7901 cells and SGC-7901 /DDP cells was determined by RT-qPCR and Western blot.Fas-containing adenovirus vector was transfected intothe SGC-7901 /DDP cells to upregulate Fas expression.The cell viability was detected by CCK-8 assay.The cell cycle and cell apoptosis were analyzed by flow cytometry.The protein levels of Fas, P38 /p-P38, JNK/p-JNK, cleaved caspase-8/caspase-8 and cleaved caspase-3 /caspase-3 were detected byWestern blot.RESULTS: The expression of Fas at both mRNA and protein levels was significantly downregulated in the SGC-7901 /DDP cells.Fas expression was decreased by cisplatin in a dose-dependent manner in the SGC-7901 cells.Overexpression of Fas suppressed the viability and induced apoptosis in the SGC-7901 /DDP cells, and upregulated the protein levels of p-P38, p-JNK, cleaved caspase-8 and cleaved caspase-3.CONCLUSION:Overexpression of Fas increases the sensitivity of the SGC-7901 /DDP cells to cisplatin, and inhibits the cell growth and promotes cell apoptosis.The mechanism may be related to the activation of JNK and P38 pathway.【期刊名称】《中国病理生理杂志》【年(卷),期】2016(032)009【总页数】6页(P1568-1573)【关键词】Fas;胃癌;顺铂耐药;细胞凋亡【作者】陈刚;申屠杨萍【作者单位】武警浙江总队嘉兴医院肿瘤科，浙江嘉兴 314000;温州医科大学机能实验教学中心，浙江温州 325000【正文语种】中文【中图分类】R735.2胃癌是消化系统常见肿瘤之一，目前放、化疗仍然是胃癌的主要治疗方法。

蟾蜍灵诱导人胃癌SGC7901细胞自噬与凋亡的研究张晔;赵明芳;刘云鹏;曲秀娟;刘静;侯科佐;徐玲【期刊名称】《现代肿瘤医学》【年(卷),期】2013(021)010【摘要】目的:探讨自噬在蟾蜍灵(bufalin)诱导人胃癌SGC7901细胞死亡中的作用.方法:不同浓度的bu-falin处理人胃癌SGC7901细胞,采用MTT法检测bufalin 对SGC7901细胞增殖的抑制作用.透射电镜观察给药后SGC7901细胞的自噬现象;流式细胞术检测细胞凋亡.Western blot检测自噬标志LC3蛋白表达.结果:Bufalin 对胃癌SGC7901细胞生长有显著的抑制作用,且此作用呈明显的时间-剂量依赖性.Bufalin给药后可诱导SGC7901细胞发生自噬;并且在bufalin给药前用氯喹阻断自噬明显提高了bufalin对SGC7901的细胞毒性(P＜0.05).结论:Bufalin能明显抑制SGC7901细胞的生长,并且诱导其发生保护性自噬.Bufalin和自噬抑制剂的联合应用可能为胃癌治疗提供新的策略.【总页数】3页(P2155-2157)【作者】张晔;赵明芳;刘云鹏;曲秀娟;刘静;侯科佐;徐玲【作者单位】中国医科大学附属第一医院肿瘤内科,辽宁沈阳110001;中国医科大学附属第一医院肿瘤内科,辽宁沈阳110001;中国医科大学附属第一医院肿瘤内科,辽宁沈阳110001;中国医科大学附属第一医院肿瘤内科,辽宁沈阳110001;中国医科大学附属第一医院肿瘤内科,辽宁沈阳110001;中国医科大学附属第一医院肿瘤内科,辽宁沈阳110001;中国医科大学附属第一医院肿瘤内科,辽宁沈阳110001【正文语种】中文【中图分类】R73-36+1【相关文献】1.吴茱萸碱诱导人胃癌SGC7901细胞凋亡及机制研究 [J], 张弦;金晓滢2.蟾蜍灵诱导人胃癌细胞凋亡的实验研究 [J], 陈小义;徐瑞成;陈莉;张敏;卢青;吴晓霞3.蟾蜍灵对顺铂耐药胃癌SGC7901细胞增殖与凋亡的影响及其作用机制 [J], 张晔;田昕;刘云鹏;曲秀娟;杨向红;侯科佐;滕月娥;张敬东4.姜黄素诱导人胃癌SGC7901细胞自噬性凋亡的研究 [J], 邓淑文;殷清华;苏琦;庄英帜5.蟾蜍灵诱导人宫颈癌C33A细胞自噬作用的机理研究 [J], 孔翠翠; 李艳兰; 宫鹏超; 田昕因版权原因，仅展示原文概要，查看原文内容请购买。

胃粘膜癌变过程中Fas、FasL的表达及意义吴小翎;冯志松【期刊名称】《安防科技》【年(卷),期】2002(000)003【摘要】目的:通过检测胃粘膜癌变过程中凋亡相关蛋白Fas、FasL的表达,探讨其在胃癌发生发展中的作用.方法:采用免疫组化SP法检测41例慢性浅表性胃炎(CSG)、19例慢性萎缩性胃炎伴肠化生(CAGIM)、22例异型增生(Dys)和37例胃癌(GC).结果:Fas在CSG、CAGIM、Dys和GC组中的阳性表达率依次下降,FasL 的阳性表达率则逐渐升高;Fas、FasL在高中分化GC组的阳性表达率均显著高于低分化GC组.结论:Fas、FasL的阳性表达率在胃癌发生过程中呈现不同的变化趋势,提示由Fas/FasL诱导的胃粘膜细胞凋亡异常与胃癌形成可能有密切关系.在胃癌患者,与癌细胞分化有关的Fas、FasL表达的一致性有助于判断肿瘤的恶性程度和预后.【总页数】4页(P257-259,263)【作者】吴小翎;冯志松【作者单位】重庆医科大学第二临床学院内科,重庆,400010;重庆医科大学第二临床学院内科,重庆,400010【正文语种】中文【中图分类】R753.2【相关文献】1.食管上皮癌变过程中FAS、FASL、FADD和caspase-8的表达及其意义 [J], 许欣;彭林涛;刘丽华;王佩;左连富2.Survivin在胃粘膜癌变过程中的表达及意义 [J], 谈晓红;马鹏3.bcl-2,c-erbB-2在胃粘膜癌变过程中的表达及意义 [J], 施琳;乌新林;李晓洁;冀文茹;夏养志4.宫颈癌癌变过程中Caspase-3、Fas/FasL的表达及其临床意义 [J], 徐虹;全培良;周云;王珍珍;蒋东霞5.胃粘膜癌变过程中Fas、FasL的表达及意义 [J], 吴小翎;冯志松因版权原因，仅展示原文概要，查看原文内容请购买。

沉默黏着斑激酶促进人胃癌SGC-7901细胞骨架蛋白解聚及细胞形态损伤高宁;张静;杜娟;慕明涛;刘俊俊;金文杰;何慧敏;侯颖春【期刊名称】《中国组织化学与细胞化学杂志》【年(卷),期】2017(026)001【摘要】目的探讨黏着斑激酶(focal adhesion kinase,FAK)在胃癌细胞骨架结构及形态结构维持中的作用.方法利用靶向FAK的siRNA质粒,转染胃癌SGC-7901细胞,用罗丹明标记的鬼笔环肽及β-tubulin特异性抗体检测细胞骨架微丝及微管蛋白表达;考马斯亮蓝染液对细胞进行染色,观察细胞整体形态结构;环境扫描电子显微镜观察细胞表面结构.结果沉默FAK引起细胞质、细胞伪足及微绒毛等运动相关结构中F-actin解聚.随着沉默时间增长,围绕着细胞核,在细胞质中呈放射状的微管骨架发生解聚,且这种解聚现象呈时间依赖性.FAK沉默还使细胞由梭形、多边形变为圆形,发生脱壁;细胞微绒毛及伪足结构受损,细胞表面趋于光滑.结论 FAK对维持胃癌细胞微丝、微管的分布及癌细胞伪足、微绒毛等恶性形态学特征具有重要作用.【总页数】6页(P7-12)【作者】高宁;张静;杜娟;慕明涛;刘俊俊;金文杰;何慧敏;侯颖春【作者单位】延安大学医学院,延安716000;延安大学医学院,延安716000;延安大学医学院,延安716000;延安大学医学院,延安716000;延安大学医学院,延安716000;陕西师范大学生命科学学院,西安710062;陕西师范大学生命科学学院,西安710062;陕西师范大学生命科学学院,西安710062【正文语种】中文【中图分类】R362【相关文献】1.黏着斑激酶促进人肺动脉平滑肌细胞增殖 [J], 林春龙;张珍祥;徐永健2.黏着斑激酶通过细胞周期蛋白依赖性激酶2、c-junN端激酶促进人肺动脉平滑肌细胞增殖 [J], 林春龙3.黏着斑激酶基因沉默促进舌癌细胞失巢凋亡的实验研究 [J], 刘华联;邢树忠;江宏兵;李强;张群4.小分子干扰RNA沉默黏着斑激酶基因对人宫颈癌Hela细胞生物学特征的影响[J], 余俊;刘彤鸥;李晓兰5.内皮素-1促进人肺血管平滑肌细胞黏着斑激酶的表达 [J], 林春龙;张珍祥;徐永健;倪望;陈仕新因版权原因，仅展示原文概要，查看原文内容请购买。

巴豆生物碱对人胃癌SGC-7901细胞PCNA、Ki-67及Bax、Fas表达的影响宋红春;谷雨;陆为民;耿燕楠【摘要】目的：观察巴豆生物碱（Croton Alkaloid，CA）对人胃癌SGC-7901细胞PCNA、Ki-67及Bax、Fas表达的影响，初步探讨其抗肿瘤的分子机制。

方法：体外培养SGC-7901细胞，给予不同浓度的巴豆生物碱干预。

免疫组化法检测不同浓度CA作用于SGC-7901细胞后，PCNA、Ki-67、Bax、Fas蛋白表达的情况。

结果：随着巴豆生物碱浓度的升高，SGC-7901细胞中PCNA、Ki-67表达逐渐下降，Bax、Fas表达逐渐上升，具有剂量依赖性。

结论：巴豆生物碱抑制SGC-7901细胞增殖的作用机制可能与其下调PCNA、Ki-67表达相关，而其上调Bax、Fas表达可能是其诱导SGC-7901细胞凋亡的机制之一。

【期刊名称】《江苏中医药》【年(卷),期】2014(000)006【总页数】3页(P77-78,79)【关键词】巴豆生物碱;SGC-7901;PCNA;Ki-67;Bax;Fas;体外实验【作者】宋红春;谷雨;陆为民;耿燕楠【作者单位】南京中医药大学，江苏南京210029;江苏省中医院，江苏南京210029;江苏省中医院，江苏南京210029;南京中医药大学，江苏南京210029【正文语种】中文【中图分类】R285.5巴豆，系大戟科植物巴豆的种子，味辛，性热。

巴豆从很早之前就被用于抗肿瘤治疗，巴豆制剂及其复方对胃癌、肝癌、乳腺癌、卵巢癌、宫颈癌等多种肿瘤的治疗作用已从化学、药理、临床等多方面研究确定。

有实验研究证实，巴豆生物碱（CA）能诱导SGC-7901细胞分化，抑制细胞增殖，并诱导其凋亡[1-4]。

本文即从体外实验的角度，初步探讨巴豆生物碱抑制SGC-7901细胞增殖及诱导其凋亡的分子机制。

1 实验材料1.1 细胞株人胃癌SGC-7901细胞株购自南京凯基生物科技发展有限公司。

顺铂诱导胃癌SGC-7901细胞凋亡作用及其对线粒体膜电位变化的研究蔡先彬;胡辉;王钦加;荆绪斌【摘要】目的研究线粒体膜电位在顺铂诱导胃癌细胞凋亡中的作用机制.方法采用MTT比色法测定顺铂对胃癌SGC7901细胞的生长抑制曲线;将胃癌SGC7901细胞分成对照组及10 μg/ml顺铂组处理,用流式细胞仪(FCS)分别检测线粒体膜电位(Δψm)和分析细胞周期变化情况.结果顺铂可明显抑制胃癌细胞增殖.在24 h后可见细胞大部分受阻于G1期,且出现了典型的凋亡峰;在10 h后线粒体膜电位明显降低.结论顺铂诱导胃癌SGC-7901细胞凋亡的途径可能是通过使线粒体膜通透性的改变,膜电位的下降来而实现的.【期刊名称】《中国实用医药》【年(卷),期】2012(007)034【总页数】3页(P1-3)【关键词】线粒体膜电位;凋亡;胃癌;顺铂【作者】蔡先彬;胡辉;王钦加;荆绪斌【作者单位】515041,汕头大学医学院第一附属医院消化内科;515041,汕头大学医学院第一附属医院消化内科;515041,汕头大学医学院第一附属医院消化内科;515041,汕头大学医学院第一附属医院消化内科【正文语种】中文顺铂是肿瘤化疗的常用药物。

传统观点认为，顺铂能够引起细胞DNA交联，起肿瘤细胞毒性作用。

近年随着分子生物学发展，有实验发现顺铂抗癌机制与肿瘤细胞凋亡存在密切关系［1，2］。

研究发现顺铂可通过Fas死亡受体途径介导肿瘤细胞凋亡［3，4］，而线粒体被认为是细胞凋亡的另一个重要途径。

我们前期研究发现顺铂可诱导食管癌细胞凋亡，且是通过线粒体途径引发一系列凋亡程序来实现［5］。

目前，顺铂作为胃癌临床常用化疗药物之一，其是否存在此类机制，相关的基础研究较少。

为此，本研究通过体外实验来探讨在顺铂诱导胃癌SGC-7901细胞凋亡过程中线粒体变化情况，从而了解线粒体与胃癌细胞凋亡之间关系。

这对于研究细胞凋亡的调控机制具有重要的理论意义，同时为临床肿瘤化疗用药提供实验依据。

细胞凋亡在肿瘤治疗中的作用及相关机制癌症是全球公认的最致命的疾病之一。

传统的癌症治疗方法包括放疗、化疗和手术等，但是这些方法的副作用很大，很容易导致身体的破坏。

近年来，越来越多的科学家和医生开始探索新的癌症治疗方法，并发现细胞凋亡在肿瘤治疗中的作用及相关机制。

什么是细胞凋亡？细胞凋亡是一种主动性细胞死亡的过程，它在人体的正常生理状态下，起着维持组织和器官稳态的重要作用。

细胞凋亡与自体免疫、抗肿瘤等过程密切相关。

正常情况下细胞之间保持着一种细微的平衡状态，当这种平衡被破坏时，细胞就会出现凋亡，从而阻止细胞的异常发展，保护人体健康。

细胞凋亡在肿瘤治疗中的作用研究表明，癌症是由于人体的正常细胞发生了变异而形成的。

如果这些细胞得不到及时的治疗和控制，它们就会持续地增殖和扩散，最终发展成为肿瘤。

细胞凋亡作为一种刺激性细胞死亡的过程，能够有效抑制癌细胞的生长和扩散。

目前，人们广泛使用的多种新型化疗、放疗、免疫疗法均会通过诱导癌细胞产生凋亡来达到治疗作用。

通过不同的手段，将肿瘤细胞与正常细胞的生长平衡破坏，从而让肿瘤细胞失去抵抗力，而最终促进其凋亡。

相关机制细胞凋亡的分子机制是分子生物学和细胞生物学领域的研究热点之一，已经有多种分子机制与细胞凋亡相联系。

1.经典途径经典途径是指细胞死亡由内部调节因子触发的途径。

该途径中在细胞膜上活化的受体可以使成纤维细胞因子等同时激活卵巢细胞的卵泡细胞和肝细胞，然后通过一种“Fas”信号途径，通过一种“procaspase”途径促进肿物细胞凋亡。

2.非经典途径非经典途径是指细胞死亡由外部因素触发的途径。

该途径中，环境的氧气浓度低、化疗药物、放射线和细菌感染等也能够引起细胞凋亡。

3.miRNA微RNA可通过与mRNA结合抑制翻译，从而调节多种细胞生物学过程，如细胞增殖、分化、凋亡和癌症。

4.细胞质色素C细胞质色素C可通过与caspase-9结合，从而促进肿瘤细胞凋亡，是诱导肿瘤细胞凋亡的新机制之一。

抑制FAK的表达对人胃癌细胞SGC-7901凋亡的影响胃癌是一种常见的恶性肿瘤，其发病率和死亡率在全球范围内均居高位。

针对胃癌的治疗手段主要为手术、放疗及化疗，但由于其易复发、难治疗等困难，因此寻找新的治疗手段以提高疗效具有重要意义。

近年来，一些研究表明，抑制FAK的表达可能是一种有效的治疗胃癌的方法。

FAK，全称为肿瘤细胞焦点激酶（focal adhesion kinase），是一种重要的细胞信号传递蛋白质，对于肿瘤细胞的增殖、转移及生长至关重要。

现有研究表明，FAK表达水平在人胃癌中明显上调，与胃癌细胞的恶性转化密切相关。

因此，针对FAK的抑制可能是一种有效的治疗胃癌的方法。

为了探究抑制FAK的表达对人胃癌细胞SGC-7901凋亡的影响，研究人员进行了实验。

首先，研究人员使用不同浓度的FAK抑制剂PF-562271处理SGC-7901细胞，并利用MTT法检测细胞活力。

结果表明，PF-562271处理组的细胞活力明显低于对照组，且呈现一定的剂量-效应关系。

进一步的流式细胞术检测表明，PF-562271处理组细胞的凋亡率明显高于对照组（P<0.05），且在PF-562271浓度为10μM时效果最为明显。

为了进一步阐明PF-562271对SGC-7901细胞凋亡的分子机制，研究人员采用Western blot法检测了该剂量下细胞中关键凋亡信号的变化。

结果发现，PF-562271处理组中Bcl-2的表达明显下调，而Bax、Caspase-3及Caspase-9的表达均明显上调，表明抑制FAK的表达可能通过影响这些凋亡信号的表达来诱导SGC-7901细胞的凋亡。

综上所述，抑制FAK的表达可能是一种有效的治疗胃癌的方法。

本文研究发现，抑制FAK的表达剂量依赖性地下调胃癌细胞SGC-7901的存活，并通过影响Bcl-2、Bax、Caspase-3及Caspase-9等凋亡信号的表达来诱导其凋亡。

由此可见，FAK可能是胃癌细胞生长及转移的关键因子之一，其抑制可能是治疗胃癌的有望策略之一。

癌宁对人胃腺癌细胞SGC-7901细胞因子及Fas基因表达的
影响
苏勉诚;戴萍;吕晓英;万斌;李红坤;刘继林
【期刊名称】《现代预防医学》
【年(卷),期】2001(28)1
【摘要】目的 :探索癌宁对体外人胃癌细胞基因及细胞因子表达的影响。

方法 :采用流式细胞术 ,对中药复方癌宁诱导胃癌细胞凋亡机制进行研究。

结果 :顺铂、癌宁组与空白对照比较 ,TGF- β1 、Fas和 Fas- L 基因表达产物含量显著增高
(P<0 .0 1)。

结论 :癌宁对人胃癌细胞的诱导凋亡作用与其对抑癌基因及细胞因子表达的改变有关。

【总页数】2页(P6-7)
【关键词】中药复方;癌宁;基因;细胞因子;胃癌
【作者】苏勉诚;戴萍;吕晓英;万斌;李红坤;刘继林
【作者单位】珠海丽珠集团国家中药中心;重庆市第三人民医院药剂科;成都军区军事医学研究所;成都中医药大学
【正文语种】中文
【中图分类】R735.202
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2.PSME1基因对人胃腺癌细胞株SGC-7901生物学特性的影响 [J], 张林;李楠;吴凯;翟俊山
3.丹参酮Ⅰ对人胃腺癌细胞SGC-7901 EIF2a和EIF3a基因表达的调控作用 [J], 刘新华; 刘爱东
4.VEGF基因表达抑制对胃腺癌细胞SGC-7901增殖的影响 [J], 徐文华;葛银林;徐宏伟;王秀丽;耿芳宋
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