桦木酸对人胃癌SGC-7901细胞增殖的影响*

目的:利用不同浓度的桦木酸对人胃癌SGC-7901细胞增殖的影响。方法:桦木酸设4个不同浓度(0、10、20、30 μg/ml),并采用常规化疗药物5-Fu处理作为阳性对照,以探究其对细胞增殖的影响。采用台盼蓝拒染法和吉姆萨染色法分别检测桦木酸对人胃癌SGC-7901细胞生长抑制率及克隆形成率;EdU法检测SGC-7901的细胞增殖;利用流式细胞术检测细胞周期, 应用qRT-PCR和Western blot分别检测细胞周期蛋白cyclin D1,cyclin B1的mRNA和蛋白表达水平。结果:不同浓度的桦木酸处理人胃癌SGC-7901细胞48 h后,其细胞生长抑制率显著升高(P＜0.05),克隆形成率和细胞增殖率均明显降低(P＜0.01),且呈剂量和时间依赖性;人胃癌SGC-7901细胞被阻滞在G1/G0期,细胞周期蛋白cyclin D1和cyclin B1的mRNA和蛋白表达量也随桦木酸浓度升高而显著降低(P＜0.01)。且与5-Fu对照组相比,桦木酸浓度为20 μg/ml和30 μg/ml时,细胞增殖能力明显降低,细胞周期被抑制,细胞周期蛋白表达量均明显降低(P ＜0.05)。结论:桦木酸通过下调cyclin B1和cyclin D1基因表达,将人胃癌SGC-7901细胞阻滞在G1/G0期,从而抑制细胞增殖。     

Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo

Swainsonine, an extract from Astragalus membranaceus, is known for its anti-cancer effects and could prevent metastases. In order to investigate the effects and mechanisms of swainsonine in C6 glioma cells, we carry out correlated experiments in vitro and in vivo. After treatment with swainsonine, the effective dose and IC₅₀ value of swainsonine in the C6 glioma cell were examined using the MTT assay. Cell cycle distribution and apoptotic rates were analyzed using FCM and [Ca²⁺]_i was measured by LSCM. Expressions of p16 and p53 protein were evaluated by immunocytochemical methods. Simultaneously, glioma-bearing rats were administered swainsonine at doses of 2, 4 and 8 mg/kg body wt. The inhibition rate was calculated and pathological sections were observed. The results indicated that the growth of C6 glioma cells is inhibited by swainsonine in vitro, with an IC₅₀ value within 24 h of 0.05 μg/ml. Increases in swainsonine correlate with S phase percentages of 11.3%, 11.6% and 12.4%, respectively. Moreover, the expression of apoptosis inhibiting p53 and p16 protein decreases gradually. Tumor weight in vivo decreased clearly and HE dyeing of tumor tissue showed gray, its texture was soft, with necrosis and hemorrhagic concentrated inward. Swainsonine could inhibit the proliferation of C6 glioma cells in vitro and the growth of C6 glioma in vivo. The mechanisms of swainsonine-induced apoptosis may relate with the expression of apoptosis-related genes and overloading-[Ca²⁺]_i-induced endoplasmic reticulum stress.     

c—FLIP在DATS诱导人胃癌SGC-7901细胞凋亡中的作用

目的：探讨二烯丙基三硫（diallyl trisulfide,DATS）诱导人胃癌SGC-7901细胞凋亡及凋亡过程中c-FLIP的变化及意义。方法采用MTT、western-blot和细胞免疫组化分别检测DATS对SGC-7901细胞的增殖抑制率及c-FLIP的表达情况。光学显微镜观察凋亡形态,流式细胞术检测凋亡率。结果：MTT结果显示,不同浓度DATS（6、8、10、12、14、16mg.L^-1）DATS处理SGC-7901细胞24、48小时后,生长抑制率分别为20.4%--79%和36%--90%,DATS抑制作用随浓度及时间逐渐增强（P〈0.05）。细胞免疫组化和western-blot显示：9.5mg..L^-1DATS处理SGC-7901细胞24、48小时后,与对照组相比,c-FLIP的表达下调（P〈0.05）。光学显微镜：通过9.5mg..L^-1DATS作用后24、48小时后,胃癌细胞出现了凋亡形态学改变。流式细胞术检测：经过9.5mg..L^-1DATS处理SGC-7901细胞24、48小时后,细胞凋亡率逐渐升高。结论：DATS促进SGC-7901细胞凋亡的机制可能与抑制c-FLIP蛋白的表达有关。     

白扁豆多糖对人胃癌细胞凋亡的作用及其机制

目的：探讨白扁豆多糖对人胃癌细胞凋亡的作用及其相关机制。方法：胃癌细胞HGC-27和SGC-7901经终浓度为16、8、4、2、1和0 μg/ml的白扁豆多糖作用24、48和72h,各设3个复孔。MTS法检测其增殖活性;分别取经4、0 μg/ml白扁豆多糖作用24h的HGC-27和SGC-7901细胞（各3个复孔）,JC-1染色观察线粒体膜电位,流式细胞仪分析细胞周期和凋亡率,QPCR法探讨Bcl-2、caspase-3和Bax基因的mRNA转录水平。结果：白扁豆多糖作用后,HGC-27和SGC-7901细胞线粒体膜电位降低;细胞增殖显著受抑制（P<0.01）,且效果与药物作用浓度和时间有关;细胞凋亡率分别为53.15%和38.77%,均较PBS处理组（8.07%和6.03%）明显增加（P<0.01）,而细胞周期无显著变化;同时,细胞内Bcl-2基因的转录水平明显受抑制,Bax和caspase-3基因的转录明显上调（P<0.01）。结论：白扁豆多糖可通过调节Bax-Bcl-2-caspase3通路,诱导胃癌细胞HGC-27和SGC-7901凋亡。     

蛇毒复合酶对人胃癌细胞的抑瘤研究

目的 观察蛇毒复合酶对人胃癌细胞SGC－7901的抑瘤作用及机理。方法 采用体外试验、流式细胞术和细胞形态学检查方法,观察人胃癌细胞的生长抑制率,以及细胞周期和细胞形态的变化。结果 蛇毒复合酶对人胃癌细胞有明显的抑瘤作用。24h抑瘤率达67．5％,接近5－Fu阳性对照组,并且具有明显的时效和量效关系。细胞镜检及涂片染色发现癌细胞胞膜破裂、胞质外溢、细胞坏死。流式细胞术检测蛇毒复合酶对S期细胞有明显杀伤作用,同时阻滞G0／G1期细胞进入S期。结论 蛇毒复合酶对人胃癌细胞具有一定体外抑瘤作用,其作用机理与直接破坏细胞胞膜和干扰细胞增殖周期有关。     

桦木酮酸对肿瘤细胞SGC-7901、HepG-2及S180荷瘤小鼠肿瘤细胞的影响

为研究桦木酮酸体外对SGC-7901、HepG-2及体内对S180荷瘤小鼠的影响,采用MTT与肿瘤细胞集落形成能力实验观察桦木酮酸对HepG-2和SGC-7901作用。通过抑瘤率观察其对S180荷瘤小鼠的影响;HE染色观察其对S180肿瘤细胞形态学的影响。结果显示：桦木酮酸可抑HepG-2和SGC-7901细胞的生长,MTT与肿瘤集落形成能力实验测得其IC50分别为68．14和110．77μmol／L。连续给药8d后,150和75ms／kg／d剂量的桦木酮酸对S180的抑制率分别为68．3％和41．5％（P〈0．05）;HE染色发现肿瘤细胞表现较为明显的胀亡现象：细胞淡染、细胞体积增大,细胞核结构相对完整。提示桦木酮酸体内、外对肿瘤细胞均有一定的抑制作用,其作用可能与其引起肿瘤细胞胀亡有关。     

Effects of antrodia camphorata on viability, apoptosis, and [Ca2+]i in PC3 human prostate cancer cells

Antrodia camphorata (AC) has been used as a health supplement in Asia to control different cancers; however, the cellular mechanisms of its effects are unclear. The effect of AC on cultured human prostate cancer cells (PC3) has not been explored. This study examined the effect of AC on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca2+ handling in PC3 cells. AC at concentrations of 5-50 microg/ml did not affect cell viability, but at 100-200 microg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25-200 microg/ml did not alter basal [Ca2+]i, but at a concentration of 25 microg/ml decreased the [Ca2+]i increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin and histamine increased cell viability whereas thapsigargin decreased it. AC (25 microg/ml) pretreatment inhibited ATP-, bradykinin-, and histamine-induced enhancement on viability, but reversed thapsigargin-induced cytotoxicity. Immunoblotting showed that AC (200 microg/ml) did not induce the phosphorylation of ERK, JNK, and p38 MAPKs. Collectively, in PC3 cells, AC exerted multiple effects on viability and [Ca2+]i, caused apoptosis via pathways unrelated to [Ca2+]i signal and phosphorylation of ERK, JNK and p38 MAPKs.     

Anticancer activities of a chemically sulfated polysaccharide obtained from Grifola frondosa and its combination with 5-Fluorouracil against human gastric carcinoma cells

Chemically sulfated polysaccharide (S-GAP-P) was derived from water-insoluble polysaccharide of Grifola frondosa mycelia. In this research, we investigated the anticancer effects of S-GAP-P and its combination with 5-fluorouracil (5-FU) on human gastric carcinoma SGC-7901 cells. Results showed that S-GAP-P distinctly inhibited SGC-7901 cells growth in a dose-dependent manner and induced cell apoptosis evidenced by characteristic DNA ladder and sub-G₀/G₁ peak. Furthermore, the combination of S-GAP-P (10–50 μg/ml) with 1 μg/ml 5-FU resulted in a significant inhibition on SGC-7901 cells growth, meaning the beneficial interaction between the two drugs. All these results suggested that S-GAP-P has evident anticancer activity through apoptotic induction and could significantly accelerate the anticancer activity of 5-FU.     

原花青素诱导胃癌SGC-7901细胞凋亡及其机制的研究

为探讨原花青素对人胃癌SGC-7901细胞增殖及凋亡的影响及其可能的作用机制,以体外培养的SGC-7901细胞为研究对象,经一定浓度的原花青素作用后,用MTT法及流式细胞仪检测细胞增殖抑制及凋亡情况,Real-time PCR技术及免疫组化法检测Bcl-2、Bax mRNA和相关蛋白表达的含量。结果表明,不同浓度的原花青素不仅能有效抑制SGC-7901细胞增殖,还可诱导细胞凋亡,且抑制增殖及促进凋亡作用呈浓度和时间依耐性;Real-time PCR及免疫组化试验中显示,随着原花青素浓度的增加,Bcl-2mRNA及相应蛋白表达逐渐减少,Bax mRNA和相关蛋白表达逐渐增加。因此,原花青素对人胃癌SGC-7901细胞具有明显的抑制作用,其作用机制可能与上调Bcl-2蛋白和下调Bax蛋白水平有关。     

rmhTNF-α联合抗IL-6抗体对胃癌SGC-7901细胞杀伤作用的研究

目的：研究rmhTNF-α联合抗IL-6抗体对胃癌SGC-7901细胞的杀伤作用。方法：（1）将已培养的SGC-7901细胞株分为a、b、c、d、e组,a、b、c、d组分别加入浓度为10 ng/ml、20 ng/ml、50 ng/ml、100 ng/ml的rmhTNF-α,e组加入等量PBS。ELISA检测不同时间点SGC7901细胞上清液中IL-6的分泌量,MTT法检测细胞培养24 h、32 h、40 h、48 h的凋亡情况。（2）将SGC-7901细胞分为f、g、h、i组,f组加入rmhTNF-α,浓度分别为10 ng/ml、20 ng/ml、50 ng/ml、100 ng/ml;g组加入不同浓度的rmhTNF-α与浓度为100 ng/ml的IL-6,h、i组加入不同浓度的rmhTNF-α与浓度分别为1：62.5和1：125的抗IL-6抗体。MTT法测细胞培养48 h的凋亡情况。荧光显微镜观察细胞凋亡情况。结果：（1）IL-6分泌五组差别具有统计学意义（P〈0.05）,a、b、c、d组IL-6浓度均高于e组（P〈0.05）,a、b、c组相比各时间点IL-6浓度逐渐升高（P〈0.05）,c、d组差别无统计学意义。（2）细胞存活率a、b、c、d组与e组差别具有统计学意义（P〈0.05）,a、b、c、d组内比较差别有统计学意义（P〈0.05）,显示杀伤作用40 h后减弱,c、d组差别无统计学意义。（3）细胞存活率f、g组比较差别具有统计学意义（P〈0.05）,h、i组与f组差别有意义（P〈0.05）,h、i组比较,i组细胞存活率较低。结论：rmhTNF-α可以促进SGC-7901细胞分泌IL-6;抗IL-6抗体可以增强rmhTNF-α对SGC-7901细胞的杀伤作用。     

Effect of the organotin compound triethyltin on Ca2+ handling in human prostate cancer cells

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.     

Antidepressant-like effect of saponins extracted from Chaihu-jia-longgu-muli-tang and its possible mechanism

In this study, we investigated the antidepressant-like effect of saponins (SCLM) extracted from a traditional Chinese medicine, Chaihu-jia-longgu-muli-tang (CLM), in mice and rats using the tail suspension test (TST) and forced swimming test (FST). Subchronic administration of 100 and 200 mg/kg (p.o.) SCLM for 7 days reduced immobility time in the TST and FST in mice and also decreased immobility time at 70 and 140 mg/kg (p.o.) in the FST in rats. The results also showed that the anti-immobility activity of SCLM in these two tests is dose-dependent, without accompanying significant effects on locomotor activity. In addition, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase (LDH) assays showed that 25, 50 and 100 microg/ml SCLM or 10 microM fluoxetine (FLU), protected PC12 cells from the lesion induced by 10 microM corticosterone (Cort) treatment for 48 h. In the fura-2/AM (acetoxymethyl ester) labeling assay, 50 and 100 microg/ml SCLM, 10 microM FLU attenuated the intracellular Ca2+ overloading induced by 200 microM Cort treatment for 48 h in PC12 cells. Using RT-PCR, the mRNA level of nerve growth factor (NGF) was also detected. Treatment with SCLM (50, 100 microg/ml) for 48 h elevated the NGF mRNA expression in PC12 cells. In summary, these results suggest that SCLM possesses an antidepressant-like activity in behavioral models that might be mediated via the cytoprotective action shown in PC12 cells.     

Validation of sensitivity to praziquantel using Schistosoma mansoni worm muscle tension and Ca2+-uptake as possible in vitro correlates to in vivo ED50 determination

Schistosome worm muscle tension and [45Ca2+]-uptake were tested as possible correlates of susceptibility to praziquantel (PZQ) assessed by estimating the drug ED50. Schistosoma mansoni cercariae of PZQ sensitive (S-CD, S-MOC and S-GP) and insensitive S. mansoni isolates (I-EE2, I-BANL and I-Senegal 47) were used to infect batches of CD-1 Swiss albino mice. Seven weeks after infection, animals of each batch were divided into six groups. Five of them received PZQ in doses of 12.5, 25, 50, 100 or 200 mg/kg PZQ, respectively, for five consecutive days, while the sixth was left as untreated controls. Two weeks after treatment mice were sacrificed, perfused and PZQ ED50's were estimated. Male worms recovered from infected untreated controls were examined for their muscle tension increase in response to PZQ using a physiological recorder coupled to a photooptic transducer. [45Ca2+]-uptake of male worms in the presence and absence of PZQ was determined using a liquid scintillation beta counter. Data revealed that PZQ insensitive isolates had significantly higher drug ED50 (>130 mg/kg) than PZQ sensitive isolates with ED50's <100 mg/kg. Moreover, in response to PZQ they were found to possess significant reductions in their worm muscle tension and their [45Ca2+]-uptake were <100%. Both parameters showed a significant negative correlation to PZQ ED50 in vivo and a significant positive correlation to each other.     

Green tea impedes dyslipidemia, lipid peroxidation, protein glycation and ameliorates Ca2+ -ATPase and Na+/K+ -ATPase activity in the heart of streptozotocin-diabetic rats

Diabetes-induced hyperlipidemia, oxidative stress and protein glycation impair cellular calcium and sodium homeostasis associated with abnormal membrane-bound enzyme activities resulting in cardiac dysfunction in diabetes. To explore the cardioprotective mechanism of green tea in diabetes, we measured the changes in the levels of calcium, sodium, potassium and the activities of Na+/K+ -ATPase and Ca2+ -ATPase in green tea treated diabetic rat hearts. The effect of green tea on triglycerides, lipid peroxidation and protein glycation in diabetic heart were also measured to elucidate the underlying mechanisms. Diabetes was induced by streptozotocin (STZ, 60 mg/kg i.p.). Six weeks after the induction of diabetes, some of the diabetic rats were treated orally with green tea extract (GTE) (300 mg/kg/day) for 4 weeks. GTE produced reduction in blood glucose and lowered the levels of lipid peroxides, triglycerides and extent of protein glycation in the heart of diabetic rats. GTE blunted the rise in cardiac [Ca2+] and [Na+] whereas increased the activities of Ca2+ -ATPase and Na+/K+ -ATPase in diabetic rats. In conclusion, the data provide support to the therapeutic effect of GTE and suggest that a possible mechanism of action may be associated with the attenuation of the rise in [Ca2+] and [Na+] by ameliorating Ca2+ -ATPase and Na+/K+ -ATPase activities.     

gamma-Tocotrienol induces mitochondria-mediated apoptosis in human gastric adenocarcinoma SGC-7901 cells

Tocotrienols are naturally occurring isoprenoid compounds highly enriched in palm oil, rice bran, oat, wheat germ, barley and rye. Tocotrienols have antioxidant properties as well as potent anticancer properties. In this study, the mechanisms underlying the apoptosis of gamma-tocotrienol on human gastric adenocarcinoma SGC-7901 cells were further studied, especially in correlation with the involvement of the apoptotic pathway. gamma-Tocotrienol inhibited SGC-7901 cell growth in a concentration- and time-dependent manner. The inhibitory effects of SGC-7901 cells were correlated with the DNA damage and arresting cell cycle at G(0)/G(1) phase in a time-dependent manner at 60 mumol/L concentration of gamma-tocotrienol. gamma-Tocotrienol induced activation of caspase-3 and increased the cleavage of the downstream substrate poly(ADP-ribose) polymerase. Furthermore, gamma-tocotrienol-induced apoptosis on SGC-7901 cells was mediated by activation of caspase-9. The data in this study suggested that gamma-tocotrienol could induce the apoptosis on human gastric cancer SGC-7901 cells via mitochondria-dependent apoptosis pathway. Thus, our findings revealed gamma-tocotrienol as a potential, new chemopreventive agent for human gastric cancer.     

桦木酸对胃癌SGC-7901细胞凋亡的影响*

目的: 以人胃癌SGC-7901细胞为研究对象,探究桦木酸对其凋亡的影响。方法: 将人胃癌SGC-7901细胞分为4组,每组设置3个复孔,对照组未加入桦木酸,而三组实验组分别加入浓度为10 mg/L、20 mg/L及30 mg/L的桦木酸,将各组细胞放入5%的CO₂培养箱中培养48 h,激光共聚焦显微镜观察细胞形态变化;流式细胞术检测细胞凋亡率和线粒体膜电位变化;qRT-PCR和Western blot分别检测SGC-7901细胞凋亡相关基因Bcl-2、Bax和Caspase-3在mRNA和蛋白水平的表达。结果: 与对照组相比,终浓度为10 mg/L、20 mg/L、30 mg/L的桦木酸处理组,细胞发生皱缩、细胞核裂解并出现凋亡小体;细胞早期凋亡与晚期凋亡率显著增加(P＜0.05 or P＜0.01),线粒体膜电位明显降低(P＜0.05 or P＜0.01);细胞凋亡相关基因Bax和Caspase-3的mRNA与蛋白表达水平均显著上升(P＜0.01),而Bcl-2的mRNA与蛋白表达水平显著降低(P＜0.01)。结论: 在一定浓度范围内,桦木酸通过调节凋亡相关基因Bcl-2、Bax和Caspase-3的表达诱导人胃癌SGC-7901细胞凋亡。     

Snai1 miRNA干扰质粒构建及胃癌细胞系稳定株筛选

目的：设计并构建针对Snai1的微小干扰核糖核酸（miRNA）,最终鉴定出有效干扰质粒并筛选稳定转染的胃癌细胞株SGC-7901。方法：设计并构建4对Snai1的pcDNATM6.2-GW/EmGFPmiR microRNA及1对无效对照microRNA干扰质粒。将干扰质粒用罗氏BD转染试剂转染胃癌细胞株SGC-7901,通过倒置荧光显微镜观察绿色荧光确定转染效率。分别用不同浓度l的杀稻瘟菌素作用于SGC-7901细胞,得到杀稻瘟菌素对SGC-7901细胞的筛选浓度。Westernblot检测4对干扰质粒、阴性对照质粒对snai1蛋白水平表达的影响。结果：测序表明,Snai1干扰序列及读码框完全正确,干扰质粒瞬时转染的SGC-7901细胞系在倒置荧光显微镜下观察绿色荧光达85%以上。杀稻瘟菌素对于SGC-7901细胞的筛选浓度为5μg/ml。Westernblot结果显示,干扰序列Mi-1对Snai1有较强的干扰效果。结论：成功构建了Snai1干扰真核表达载体,同时筛选出有效干扰质粒及稳定转染株,为进一步研究Snai1在胃癌中的作用奠定了基础。     

Upregulation of periostin prevents P53-mediated apoptosis in SGC-7901 gastric cancer cells

Periostin is frequently upregulated in human cancers including gastric cancer and implicated in cancer cell proliferation, invasion, and epithelial–mesenchymal transition. This study was undertaken to investigate the effects of periostin overexpression on the chemosensitivity of gastric cancer cells. We constructed a stable cell line overexpressing periostin in SGC-7901 human gastric cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that periostin had no influence on the proliferation of SGC-7901 cells. Compared to empty vector-transfected cells, overexpression of periostin rendered SGC-7901 cells more resistant to cisplatin or 5-fluorouracil (5-FU)-induced apoptosis, accompanying with less release of cytochrome c from mitochondria and diminished cleavage of caspase-3 and poly (ADP-ribose) polymerase. Periostin-overexpressing cells treated with cisplatin or 5-FU showed significantly (p < 0.05) decreased expression of Bax and p53 proteins and increased expression of Bcl-2 protein, when compared to drug-treated mock counterparts. Restoration of p53 expression by delivering wild-type p53 gene resulted in a marked increase in drug-induced apoptosis in periostin-overexpressing SGC-7901 cells. Periostin overexpression elevated the phosphorylation of Akt. Pretreatment of periostin-overexpressing cells with an Akt inhibitor, MK-2206, partially rescued periostin-mediated inhibition of p53 expression and drug resistance. Taken together, our data indicate that periostin confers protection against cisplatin or 5-FU-induced apoptosis in SGC-7901 cells, likely through modulating the Akt/p53 pathway, and thus represents a potential therapeutic target in gastric cancer.     

Protease-activated receptor-2-mediated Ca2+ signaling in guinea pig tracheal epithelial cells

The protease-activated receptor-2 (PAR-2), a G protein-coupled receptor activated by trypsin, contributes to the pathogenesis of inflammatory disease including asthma. Here, we examined the mechanisms by which stimulation of PAR-2 induces an increase in intracellular Ca2+ concentration ([Ca2+]i) in guinea pig tracheal epithelial cells. Trypsin (0.01-3 units/ml) dose-dependently induced a transient increase in [Ca2+]i, the increase being blocked by soybean trypsin inhibitor (SBTI 1 microM). An increase in [Ca2+]i was also induced by an agonist peptide for PAR-2 (SLIGRL-NH2, 0.001-10 microM) but not by thrombin (3 units/ml, an activator for PAR-1, PAR-3 or PAR-4). Repeated or cross stimulation of trypsin or SLIGRL-NH2 caused marked desensitization of the [Ca2+]i response. These responses of [Ca2+]i to trypsin and SLIGRL-NH2 were attenuated by a phospholipase C inhibitor, U-73122, and a Ca2+-ATPase inhibitor, thapsigargin (100 nM), while removal of Ca2+ and a L-type Ca2+-channel blocker, verapamil, were without significant effects. Further, trypsin was without effect on the rate of fura 2 quenching by Mn2+ entry as an indicator of Ca2+ influx. Thus, stimulation of PAR-2 appears to increase [Ca2+]i through the mobilization of Ca2+ from intracellular stores probably via phospholipase Cbeta-linked generation of a second messenger.