姜黄素抑制Hedgehog信号诱导胃癌细胞凋亡

目的探讨姜黄素对胃癌细胞MKN45的作用及可能机制。方法不同浓度的姜黄素作用胃癌细胞MKN45后,采用MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,Westernblot技术检测索尼克刺猬信号(Shh)、脑胶质瘤相关癌基因1(GLI1)的表达水平。结果不同浓度的姜黄素与胃癌细胞MKN45共培养后,随着姜黄素浓度的逐渐增加,MKN45细胞的增殖抑制也逐渐增加,经统计学分析发现,姜黄素5μmol/ml组与对照组比较,姜黄素10μmol/ml组与对照组比较,姜黄素20μmol/ml组与10μmol/ml组、5μmol/ml组、对照组比较,差异均有统计学意义(t分别=16.24、14.57、21.16、25.00、38.20,P均<0.05)。胃癌细胞MKN45经姜黄素处理后,姜黄素20μmol/ml组的细胞凋亡率与10μmol/ml组、5μmol/ml组、对照组比较,差异均有统计学意义(t分别=57.05、59.93、61.98,P均<0.05)。经姜黄素处理的MKN45细胞,Shh和GLI1表达均明显降低,与对照组比较,差异均有统计学意义(t分别=3.76、3.55,P均<0.05)。结论姜黄素能通过抑制Hedgehog信号通路,抑制胃癌细胞的增殖,诱导其凋亡。     

Gli1表达抑制对卵巢癌SKOV3细胞增殖的影响

目的 抑制Glioma-associated oncogene homoglog（GLI）基因在卵巢癌SKOV3细胞中的表达,探讨GLI1表达抑制对SKOV3细胞增殖的影响.方法 构建并筛选GLI1基因的RNAi表达质粒,转染SKOV3细胞,CCK-8法比较转染前后卵巢癌细胞增殖变化,流式细胞仪检测紫杉醇诱导转染前后细胞凋亡比例,流式细胞仪检测转染前后SKOV3的细胞周期情况.Westem blot检测cyclinD、Bcl-2及Capase3蛋白表达的影响.结果 转染48 h后,在SKOV3细胞中Control组、N-Control组、GLI1 shRNA-1组、GLI1 shRNA-2组、GLI1shRNA-3组、GLI1 shRNA-4组的GLI1 mRNA水平分别为1.00±0.00、1.03 ±0.02、0.73±0.07、0.98 ±0.08、0.53±0.08、0.68±0.04,与Control组相比,GLI1 shRNA-1、GLI1 shRNA-3、GLI1 shRNA-4组GLI1 mRNA表达量均下降（P＜0.05）,转染GLI1 shRNA-3的SKOV3细胞中GLI1蛋白表达水平低于GLI1 shRNA-1、GLI1shRNA-2、GLI1 shRNA-4组（P＜0.05）,具有显著的干扰效果.转染GLI1 shRNA-3的SKOV3细胞增殖受到明显抑制（P＜0.05）,在紫杉醇的诱导下细胞凋亡比例明显增加,细胞周期主要阻滞在G1/S期.Western blot检测发现Bcl-2蛋白表达下降（P＜0.05）,Capase3蛋白表达升高（P＜0.05）,细胞周期蛋白CyclinD1蛋白表达下降（P＜0.05）.结论 GLI1 shRNh对SKOV3细胞增殖抑制作用明显,促进细胞凋亡,GLI1信号可通过抑制cyclinD1活化抑制细胞恶性增殖.     

Upper urinary tract pacemaker cells join the GLI club

Mutations in GLI3, a component of the Sonic Hedgehog (Shh) signaling pathway, cause a variety of human developmental syndromes. In this issue of the JCI, Cain and colleagues show that tightly regulated GLI3 repressor activity is essential for Shh-dependent differentiation of upper urinary tract pacemaker cells and the efficient flow of urine from the kidney to the bladder. These results link defective pacemaker cell differentiation with hydronephrosis and provide a cellular basis for one of the abnormal renal defects observed in humans with the GLI3-linked disease Pallister-Hall syndrome.     

Sonic hedgehog signaling regulates Bcr-Abl expression in human chronic myeloid leukemia cells

Purpose

Bcr-Abl fusion protein activates tyrosine kinase, resulting in the proliferation of leukemia cells, especially chronic myeloid leukemia (CML) cells. Imatinib (IM) effectively targets Bcr-Abl tyrosine kinase, but development of resistance to IM occurs with varying frequency.

Methods

Elucidation of the common regulatory pathway upstream of Bcr-Abl in IM-sensitive and IM-resistant CML cells is important for developing novel therapeutics against CML.

Results

This study demonstrated that IM preferentially inhibited the viability and Bcr-Abl expression in IM-sensitive K562 (K562) cells, but not in Bcr-Abl overexpressing IM-resistant K562 (K562R) cells. Both K562 and K562R cells expressed Shh preproprotein, cleaved Shh C-terminal and N-terminal peptides, as well as mRNA level of major Shh signaling molecules, including sonic hedgehog (Shh), patched (PTCH), smoothened (Smo) and Gli-1. Moreover, Gli-1 translocation into nucleus was evident in these two cell lines, suggesting that both K562 and K562R cells possess activated and major components of the Shh signaling pathway. Silencing of Gli-1 by interference RNA was accompanied by inhibition of Bcr-Abl protein expression. Pharmacological suppression of Bcr-Abl expression was restored by the Smo agonist purmorpharmine. Treatment of Shh peptide in both K562 and K562R cells not only increased Shh and Gli-1 expression, but also up-regulated Bcr-Abl expression. Resveratrol, a known Bcr-Abl inhibitor, reduced Gli-1 activation and inhibited the viability of CML cells.

Conclusions

Shh signaling may regulate Bcr-Abl expression in human chronic myeloid leukemia cells. Novel compounds inhibiting both Shh signaling and Bcr-Abl expression, such as resveratrol, may have potential to be effective agents against CML independent of IM resistance.     

NCTD通过靶向Hedgehog/SOX2 axis抑制急性髓系白血病HL60细胞的增殖

目的:研究去甲斑蝥素(NCTD)通过抑制Hedgehog信号通路(SHH)关键调节因子及其下游转录因子SOX2对急性髓系白血病细胞增殖的影响.方法:HL60和NB4细胞系经NCTD、SMO和GLI1抑制剂培养24 h后,用CCK8检测细胞数目;HL60细胞系经NCTD处理后,利用免疫印迹法检测GLI1、SMO、SOX2...     

The reduction of IL-6 gene expression,pAKT, pERK1/2, pSTAT3 signaling pathways and invasion activity by gallic acid in prostate cancer PC3 cells

Prostate cancer (PC) is one of the most common cancers among men. Progression of prostate cancer is associated with an increase in cellular level of interleukin-6 (IL-6). Gallic acid (GA) is a polyhydroxy phenolic compound which can inhibit the growth of cancer cells. The aim of this study was to evaluate the effects of GA treatment on cell viability, proliferation, invasion, IL-6 gene expression, IL-6 secretion, cellular levels of pSTAT3, pERK1/2, and pAKT signaling proteins in human prostate cancer PC3 cells. PC3 cells viability after treatment with GA (0–120 μM) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of IL-6 was investigated using real-time polymerase chain reaction. Cellular concentration of pSTAT3, pERK1/2, and pAKT signaling proteins were determined by Western blotting technic. PC3 cells invasion was assessed by invasion assay test. Treatment with GA caused a significant decrease in cell viability, proliferation, invasion, cellular levels of pSTAT3, pERK1/2, and pAKT signaling proteins after 48 h in a dose-dependent manner. The level of IL-6 and its gene expression decreased significantly in PC3 cells treated with GA. Our results show that IL-6 down-regulation and decreased IL-6 protein level in PC3 cells by GA resulted in diminishing of pSTAT3, pERK1/2, and pAKT signaling proteins which lead to the reduction of the cell survival, proliferation, and invasion in PC3 cells. Therefore, it seems that GA can be considered an anticancer agent in the treatment of prostate cancer.     

表皮生长因子受体在黑色素瘤紫杉醇耐药性中的机制研究

目的探究紫杉醇对黑色素瘤的作用机制及表皮生长因子受体（EGFR）信号通路在调节黑色素瘤侵袭与转移中的作用,揭示该信号通路与紫杉醇耐药的关系及相关机制。 方法以EGFR高表达的恶性黑色素瘤A375细胞为研究对象,采用流式细胞术、细胞迁移实验、MTT法和蛋白质印迹法,研究紫杉醇及表皮生长因子（EGF）通过EGFR信号通路对A375细胞凋亡、增殖和迁移的影响。 结果（1）细胞凋亡：紫杉醇诱导的A375细胞凋亡随浓度升高而增强（P＜0.001）,同时紫杉醇（0.1 μmol/L）抑制Bcl-2并增加了Bax的表达（P＜0.01）;EGFR抑制剂AG1478可明显增加A375细胞凋亡并增强紫杉醇的诱导效果及对Bcl-2和Bax的影响（P＜0.001）;EGF单独处理对A375细胞凋亡无明显影响,但其可抑制紫杉醇诱导的细胞凋亡（P＜0.05）及对Bcl-2和Bax的影响（P＜0.001）。（2）细胞增殖：紫杉醇（0.1 μmol/L）可显著抑制A375细胞增殖（P＜0.001）且该作用可被AG1478进一步增强但被EGF减弱（P＜0.001）。（3）细胞迁移：紫杉醇（0.1 μmol/L）可显著抑制A375细胞的迁移（P＜0.001）,该抑制作用可被AG1478进一步增强但被EGF减弱（P＜0.001）。 结论紫杉醇能够降低Bcl-2并增加Bax表达,从而诱导黑色素瘤A375细胞的凋亡并抑制其增殖和迁移,而这些抑制作用可被EGF激活的EGFR通路减弱。     

miR-152通过调控FOXR2表达抑制人前列腺癌细胞增殖的研究

目的探讨人前列腺癌PC-3细胞中miR-152调控叉头框蛋白R2(FOXR2)对细胞增殖的影响。方法采用实时荧光定量PCR检测人正常前列腺上皮细胞RWPE-1和前列腺癌PC-3细胞中miR-152、FOXR2 mRNA的相对表达水平,Western blot检测FOXR2蛋白水平。miR-152 mimics、miR-152 inhibitor或FOXR2-siRNA转染PC-3细胞后,观察细胞增殖能力的变化。miR-152 mimics、miR-152 inhibitor分别与FOXR23′UTR双荧光素报告基因质粒共转染PC-3细胞,检测荧光素酶活性。miR-152 mimics和miR-152 inhibitor转染PC-3细胞后,检测miR-152、FOXR2 mRNA的相对表达水平及FOXR2蛋白水平。结果与RWPE-1细胞比较,PC-3细胞中miR-152的相对表达水平显著降低(P=0.001),而FOXR2 mRNA的相对表达水平及蛋白水平显著升高(P=0.003、0.003)。miR-152 mimics、miR-152 inhibitor或FOXR2-siRNA转染PC-3细胞后,细胞增殖能力分别显著降低、升高、降低(P=0.022、0.029、0.006)。miR-152 mimics和FOXR23′UTR野生型质粒共转染后荧光素酶活性被显著抑制(P=0.001),而共转染miR-152 inhibitor可显著增加荧光素酶活性(P=0.001)。与对照组比较,miR-152 mimics转染PC-3细胞后,miR-152的相对表达水平显著升高(P<0.001),而FOXR2 mRNA的相对表达水平及蛋白水平显著降低(P<0.001、0.001),细胞活性显著降低(P=0.013);miR-152 inhibitor转染PC-3细胞后,miR-152的相对表达水平显著降低(P<0.001),而FOXR2 mRNA的相对表达水平及蛋白水平显著升高(P<0.001、P=0.007),细胞活性显著升高(P=0.018)。结论miR-152可通过负调控FOXR2表达发挥抑制人前列腺癌PC-3细胞增殖的作用。     

miR-203a-3p 靶向GLS1 调控HCC 细胞增殖、迁移、侵袭的机制研究

目的　探究微小核糖核酸（microRNAs, miRNA, miR）-203a-3p 对肝细胞癌（hepatocellular carcinoma,HCC）细胞增殖、迁移、侵袭的影响及其潜在分子机制。方法　采用实时荧光定量PCR（quantitative real-time PCR,qRTPCR）检测HCC 细胞、人正常肝细胞以及临床HCC 组织中miR-203a-3p 相对表达;采用细胞增殖实验、细胞划痕实验和Transwell 实验分别检测miR-203a-3p 对HCC 细胞增殖、迁移、侵袭的影响;生物信息学网站预测miR-203a-3p 的潜在靶基因（GLS1）,双荧光素酶实验进行验证;探究HCC 细胞对谷氨酰胺的依赖性及抑制谷氨酰胺酶1(glutaminase 1,GLS1) 对HCC 细胞增殖、迁移、侵袭的影响;蛋白免疫印迹（Western blot）实验检测Wnt/β-catenin 信号通路关键蛋白β-catenin,p-GSK-3β 和c-Myc 表达。结果　HCC 癌组织中miR-203a-3p（0.32±0.07）表达明显低于癌旁正常组织（1.02±0.03）,差异有统计学意义（t ＝ 41.105,P ＜ 0.001）;HCC 细胞HepG2（0.34±0.05）,HCCLM3（0.58±0.06）,Huh7（0.43±0.05）,Hep3B（0.29±0.04）中miR-203a-3p 相对表达水平明显低于人正常肝细胞LO2（1.01±0.02）中miR-203a-3p 表达,差异有统计学意义（F ＝ 119.080,P ＜ 0.001）。与Blank 组相比,miR-203a-3p 过表达组HCC细胞增殖（0.61±0.05 vs 1.24±0.06）, 迁移率（21.43%±2.01% vs 60.22%±3.14%） 及侵袭能力（76.54±13.56 vs221.06±16.54）均明显降低,差异有统计学意义（t ＝ 14.849,13.900,10.562,均P ＜ 0.001）。GLS1 是miR-203a-3p的靶基因,miR-203a-3p 靶向负调控GLS1 表达。HCC 细胞中GLS1 高表达呈现高酶活性,HCC 细胞对谷氨酰胺存在明显依赖性。GLS1 抑制组α-KG,谷氨酸水平均较Blank 组和siRNA-NC 组明显降低,差异有统计学意义（F ＝ 64.754,35.627,均P ＜ 0.001）。GLS1 抑制组细胞增殖（0.59±0.04）、迁移率（30.15%±1.02%）和侵袭能力（69.59±15.74）较Blank 组明显降低（1.29±0.07,59.67%±1.45%,202.14±13.52）,差异均有统计学意义（t ＝ 16.499,16.278,11.215,均P ＜ 0.001）。miR-203a-3p 过表达和GLS1 表达抑制明显抑制了Wnt/β-catenin 信号通路关键蛋白β-catenin,p-GSK-3β 和c-Myc 的表达,差异均有统计学意义（t ＝ 11.129 ～ 28.213,均P ＜ 0.001）。转染GLS1 可逆转miR-203a-3p 对HCC 细胞生物学行为及Wnt/β-catenin 信号通路关键蛋白表达的抑制作用。结论　HCC 中miR-203a-3p 显著低表达,其过表达能够抑制HCC 细胞的增殖、迁移和侵袭,可能与GLS1 调控谷氨酰胺代谢及miR-203a-3p 靶向GLS1调控Wnt/β-catenin 信号通路活性有关。     

β1整合素表达下调对人结肠癌细胞增殖和化疗敏感性影响

目的 观察β1整合素表达下调对人结肠癌HT-29细胞增殖和化疗敏感性的影响.方法 采用反义寡核苷酸（ASODN）技术转染HT-29细胞,逆转录-聚合酶链反应（RT-PCR）检测β1整合素表达下调情况.MTT法检测HT-29细胞相对存活率.给予梯度浓度氟尿嘧啶（5-FU）,计算IC50.结果 实验组β1整合素条带吸光度积分（IAD）值/β-actin条带IAD值的比值及HT-29细胞的存活率与对照组相比显著降低（F=1 630.08,q=72.40,P〈0.01;t=4.37,P〈0.05）.5-FU＋ASODN组HT-29细胞对5-FU的IC50与5-FU组比较显著下降（t′=37.71,P〈0.05）.结论 β1整合素表达下调可抑制结肠癌细胞增殖,增强结肠癌细胞对化疗药物敏感性.     

Glucocorticoids and neonatal brain injury: the hedgehog connection

Glucocorticoids (GCs) play a critical role in neural development; however, their prenatal or neonatal therapeutic use can have detrimental effects on the developing brain. In this issue of the JCI, Heine and Rowitch report that the molecular mechanisms underlying these detrimental effects involve the sonic hedgehog (Shh) signaling pathway, a crucial regulator of brain development and neural stem/progenitor cells (see the related study beginning on page 267). They show that GCs suppress Shh-induced proliferation of cerebellar progenitor cells in postnatal mice and that, conversely, Shh signaling is protective against GC-induced neonatal cerebellar injury by inducing the enzyme 11βHSD2, which inactivates the GCs corticosterone and prednisolone, but not dexamethasone. The data provide a rationale for the therapeutic use of 11βHSD2-sensitive GCs, but not dexamethasone, or for the exploitation of the neuroprotective effect of Shh agonists to prevent GC-induced pre- or neonatal brain injury.     

Inhibition of IRAK1/4 sensitizes T cell acute lymphoblastic leukemia to chemotherapies

Signaling via the MyD88/IRAK pathway in T cells is indispensable for cell survival; however, it is not known whether this pathway functions in the progression of T acute lymphoblastic leukemia (T-ALL). Here, we determined that compared with thymic and peripheral T cells, T-ALL cells from patients have elevated levels of IRAK1 and IRAK4 mRNA as well as increased total and phosphorylated protein. Targeted inhibition of IRAK1 and IRAK4, either with shRNA or with a pharmacological IRAK1/4 inhibitor, dramatically impeded proliferation of T-ALL cells isolated from patients and T-ALL cells in a murine leukemia model; however, IRAK1/4 inhibition had little effect on cell death. We screened several hundred FDA-approved compounds and identified a set of drugs that had enhanced cytotoxic activity when combined with IRAK inhibition. Administration of an IRAK1/4 inhibitor or IRAK knockdown in combination with either ABT-737 or vincristine markedly reduced leukemia burden in mice and prolonged survival. IRAK1/4 signaling activated the E3 ubiquitin ligase TRAF6, increasing K63-linked ubiquitination and enhancing stability of the antiapoptotic protein MCL1; therefore, IRAK inhibition reduced MCL1 stability and sensitized T-ALL to combination therapy. These studies demonstrate that IRAK1/4 signaling promotes T-ALL progression through stabilization of MCL1 and suggest that impeding this pathway has potential as a therapeutic strategy to enhance chemotherapeutic efficacy.     

Cilostazol enhances casein kinase 2 phosphorylation and suppresses tumor necrosis factor-alpha-induced increased phosphatase and tensin homolog deleted from chromosome 10 phosphorylation and apoptotic cell death in SK-N-SH cells

This study shows the signaling pathway by which cilostazol suppresses tumor necrosis factor-alpha (TNF-alpha)-induced the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) phosphorylation and apoptosis via casein kinase 2 (CK2) phosphorylation in the SK-N-SH cells (neuroblastoma cells). Cilostazol (10 microM) fully restored cell proliferation with suppression of DNA fragmentation induced by TNF-alpha and emodin, a CK2 inhibitor, which were antagonized by iberiotoxin, a maxi-K channel blocker. Under application of TNF-alpha or emodin, increased PTEN phosphorylation and decreased phosphorylation of CK2/Akt/cyclic AMP response element-binding protein (CREB), and CK2 activity were significantly reversed by cilostazol (approximately 1-100 microM), all of which were antagonized by iberiotoxin. 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619) and (3S)-(+)-(5-chloro-2-methoxyphenyl-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one (BMS 204352) maxi-K channel openers significantly elevated CK2 activities that were reversible by iberiotoxin. SK-N-SH cells treated with antisense CK2 oligodeoxynucleotide showed a prominent DNA fragmentation with little responsiveness to TNF-alpha in the phosphorylation of PTEN, indicative of the essential role of p-CK2/CK2 in cell proliferation, and the decreased cell viability of these cells was not restored by cilostazol. It is suggested that the action of cilostazol promoting cell survival is ascribed to the maxi-K channel opening-coupled up-regulation of CK2 phosphorylation and down-regulation of PTEN phosphorylation with resultant increased phosphorylation of Akt and CREB.     

Hedgehog signaling has a protective effect in glucocorticoid-induced mouse neonatal brain injury through an 11βHSD2-dependent mechanism

Glucocorticoids (GCs) are administered to human fetuses at risk of premature delivery and to infants with life-threatening respiratory and cardiac conditions. However, there are ongoing concerns about adverse effects of GC treatment on the developing human brain, although the precise molecular mechanisms underlying GC-induced brain injury are unclear. Here, we identified what we believe to be novel cross-antagonistic interactions of Sonic hedgehog (Shh) and GC signaling in proliferating mouse cerebellar granule neuron precursors (CGNPs). Chronic GC treatment (from P0 through P7) in mouse pups inhibited Shh-induced proliferation and upregulation of expression of N-myc, Gli1, and D-type cyclin protein in CGNPs. Conversely, acute GC treatment (on P7 only) caused transient apoptosis. Shh signaling antagonized these effects of GCs, in part by induction of 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2). Importantly, 11βHSD2 antagonized the effects of the GCs corticosterone, hydrocortisone, and prednisolone, but not the synthetic GC dexamethasone. Our findings indicate that Shh signaling is protective in the setting of GC-induced mouse neonatal brain injury. Furthermore, they led us to propose that 11βHSD2-sensitive GCs (e.g., hydrocortisone) should be used in preference to dexamethasone in neonatal human infants because of the potential for reduced neurotoxicity.     

rhG-CSF对健康供者外周血和骨髓免疫特性影响的比较

本研究探讨rhG—CSF对健康供者外周血采集物（G—PB）和骨髓采集物（G—BM）免疫学特性影响的异同。用MTT法和夹心酶联免疫复合物（ELISA）法检测G—PB和G—BM中T淋巴细胞增殖能力和IL-4、IFN-7的分泌，并用流式细胞术测定两种移植物的T细胞亚群、树突状细胞（DC）亚群、单核细胞及共刺激分子CD28的表达。结果表明：G—PB中淋巴细胞，CD3^＋、CD4^＋、CD8^＋T淋巴细胞，DC1、DC2以及单核细胞的含量，CD4／CD8的比值高于G—BM（P〈0，001）。G—PB的T淋巴细胞增殖能力高于G—BM（P〈0．05）；每微升G—PB移植物中T淋巴细胞分泌细胞因子IFN-γ,IL-4的量均明显高于G—BM，且G—PB中IL-4／IFN-γ比值小于G-BM（P〈0．001），DC2与T淋巴细胞的比值也低于G—BM（P〈0．01）；而G-PB中CD4^＋、CD8^＋细胞上CD28表达的百分比和总体表达均高于G—BM（P〈0．001）。结论：rhG—CSF体内应用诱导G—PB和G—BM产生的T细胞免疫低反应性是有差异的，而两者之间的差异是G—PB和G—BM移植后移植物抗宿主病（GVHD）发生率和程度不同的免疫学基础。