Calcium-mediated activation of PI3K and p53 leads to apoptosis in thyroid carcinoma cells

The molecular mechanism responsible for cadmium-induced cell death in thyroid cancer cells (FRO) is unknown. We demonstrated that apoptosis of FRO cells induced by cadmium was concentration and time dependent. Cadmium caused the rapid elevation of intracellular calcium and induced phosphorylation of Akt, p53, JNK, ERK and p38. Inhibition of PI3K/Akt attenuated the cadmium-induced apoptosis, but the inhibition of JNK inhibitor, ERK or p38 aggravated it, indicating that activation of PI3K/Akt was a pro-apoptosis signal in response to cadmium treatment, whereas the activation of stress-activated protein kinase JNK, ERK and p38 functioned as survival signals to counteract the cadmium-induced apoptosis. Buffering of the calcium response attenuated mitochondrial impairment, recovered the cadmium-activated Akt, p53, JNK, ERK and p38, and subsequently blocked the apoptosis. These results suggested that apoptosis induced by cadmium in FRO cells was initiated by the rapid elevation of intracellular calcium, followed by calcium-mediated activation of PI3K/Akt and mitochondrial impairment. Received 28 February 2007; received after revision 2 April 2007; accepted 23 April 2007     

N-acetylglucosaminyltransferase V modifies the signaling pathway of epidermal growth factor receptor

Transfection of sense cDNA of N-acetylglucosamyltransferase V (GnTV-S) into human H7721 hepatocarcinoma cells resulted in an increase in the N-acetylglucosamine1,6mannose1,3- branch (GnT-V product) on the N-glycans of epidermal growth factor (EGF) receptor (EGFR), and promotion of its EGF binding and tyrosine autophosphorylation, but showed little effect on the expression of EGFR protein. The phosphorylation at T308, S473 and tyrosine residue(s) and the activity of protein kinase B (Akt/PKB) as well as the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) before and after EGF stimulation were concomitantly increased. Conversely, in the antisense GnT-V (GnTV-AS)-transfected H7721 cells, all the results were the reverse of those with GnTV-S-transfected cells. After the cells were treated with 1-deoxymannojirimycin, an inhibitor of N-glycan processing at high mannose, or antibody against the extracellular glycan domain of EGFR, the differences in PKB activity, p42/44 MAPK and MEK phosphorylation among GnTV-S-, GnTV-AS- and mock-transfected cells were significantly attenuated. These findings indicate that the altered expression of GnT-V will change the glycan structure and function of EGFR, which may modify downstream signal transduction.Received 24 March 2004; received after revision 1 May 2004; accepted 25 May 2004     

Akt-mediated GSK-3β inhibition prevents migration of polyamine-depleted intestinal epithelial cells via Rac1

The rapid migration of intestinal epithelial cells (IEC) is important for the healing of mucosal wounds. We have previously shown that polyamine depletion inhibits migration of IEC-6 cells. Akt activation and its downstream target GSK-3β have been implicated in the regulation of migration. Here we investigated the significance of elevated phosphatidylinositol 3-kinase (PI3K)/Akt signaling on migration of polyamine-depleted cells. Polyamine-depleted cells had high Akt (Ser473) and GSK-3β (Ser9) phosphorylation. Pretreatment with 20 μM LY294002 (PI3K inhibitor) for 30 min inhibited phosphorylation of Akt, increased migration by activating Rac1 in polyamine-depleted IEC-6 cells, and restored the actin structure similar to that in cells grown in control medium. Treatment of cells with a GSK-3β inhibitor (AR-A014418) altered the actin cytoskeleton and inhibited migration, mimicking the effects of polyamine depletion. Thus, our results indicate that sustained activation of Akt in response to polyamine depletion inhibits migration through GSK-3β and Rac1. Received 25 August 2006; received after revision 3 October 2006; accepted 16 October 2006     

Activation of Janus kinase/signal transducer and activator of transcription 3 pathway by growth hormone-releasing hormone

Growth hormone-releasing hormone (GHRH) can act as a potent growth factor in various cancers. The mitogenic activity of this neuropeptide is exerted through binding to the pituitary type receptors (GHRH-R) or their splice variants (SV). In the present work, we studied whether this hormone can activate the JAK2/STAT3 pathway which plays a crucial role in cancer cell proliferation and is also linked to carcinogenesis. We transfected HeLa human cervical cancer cells, which are not sensitive to GHRH analogs with the pGHRH-R. Transfected cells responded to the GHRH or its antagonist with an increase or a decrease in proliferation, respectively. These results were confirmed by the expression of proliferating cell nuclear antigen. We then showed that these effects are linked to the activation of the JAK2/STAT3 pathway. Our work demonstrates the activation of JAK/STAT3 pathway by GHRH and sheds further light to the mechanisms of the antitumorogenic action of GHRH antagonists.     

胰岛素信号通路PI3K／Akt对海马神经元β-淀粉样前体蛋白裂解酶1mRNA水平的影晌

目的通过胰岛素和磷脂酰肌醇-3激酶（P13K）抑制剂渥曼青霉素（wortmannin）对P13K／丝氨酸苏氨酸蛋白激酶（P13K／Akt）信号通路的激活和抑制作用,观察P13K／Akt信号通路对海马神经元β-淀粉样前体蛋白裂解酶1（BACEl）mRNA水平表达的影响。方法20只sD大鼠随机分为空白对照组、假手术组、胰岛素组和渥曼青霉素组,海马立体定向注射胰岛素和P13K抑制剂渥曼青霉素。逆转录一聚合酶链反应（RT-PCR）检测P13K／Akt信号传导下游蛋白Akt以及BACEImRNA水平。结果注射胰岛素的海马P13K信号通路下游信号分子：AktmRNA表达上调（分别较空白和阴性对照组P=0．047,P=0．002）,而BACElmRNA表达下调（分别较空白和阴性对照组P=0．004,P=0．01）。渥曼青霉素组的P13K下游信号分子AktmRNA表达明显被抑制（分别较空白和阴性对照组P=0．002,P=0．039）,同时BACEImRNA的表达较对照组上调（分别较空白和阴性对照组P=0．039,P=0．018）。结论胰岛素信号通路P13K／AM可以调节BACEl的转录水平参与阿尔茨海默病的发病机制。     

Serine residue 115 of MAPK-activated protein kinase MK5 is crucial for its PKA-regulated nuclear export and biological function

The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38^MAPK, extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38^MAPK, ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phosphomimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38^MAPK, ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.     

Cofilin activity during insulin-like growth factor I-stimulated neuroblastoma cell motility

Insulin-like growth factor I (IGF-I) is a potent stimulator of neuroblastoma cell motility. Cell motility requires lamellipodium extension at the leading edge of the cell through organized actin polymerization, and IGF-I stimulates lamellipodial elaboration in human neuroblastoma cells. Rac is a Rho GTPase that stimulates lamellipodial formation via the regulation of actin polymerization. In this study, we show that IGF-I-stimulated phosphatidylinositol 3-kinase (PI-3K) activity promotes rac activation and subsequent activation of the down- stream effectors LIM kinase and cofilin. Overexpression of wild-type LIM kinase and wild-type Xenopus ADF/cofilin (XAC) suppresses IGF-I-stimulated motility in SH-SY5Y cells, while expression of dominant negative LIM kinase and constitutively active XAC increases SH-SY5Y motility in the absence of IGF-I stimulation. These results suggest that regulation by cofilin of actin depolymerization is important in the process of neuroblastoma cell motility, and IGF-I regulates cofilin activity in part through PI-3K, rac, and LIM kinase.Received 18 October 2004; received after revision 3 December 2004; accepted 16 December 2004     

Simvastatin overcomes the resistance to serum withdrawal-induced apoptosis of lymphocytes from Alzheimer’s disease patients

Statins may exert beneficial effects on Alzheimer’s disease (AD) patients. Based on the antineoplastic and apoptotic effects of statins in a number of cell types, we hypothesized that statins may be able to protect neurons by controlling the regulation of cell cycle and/or apoptosis. A growing body of evidence indicates that neurodegeneration involves the cell-cycle activation in postmitotic neurons. Failure of cell-cycle control is not restricted to neurons in AD patients, but occurs in peripheral cells as well. For these reasons, we studied the role of simvastatin (SIM) on cell survival/death in lymphoblasts from AD patients. We report here that SIM induces apoptosis in AD lymphoblasts deprived of serum. SIM interacts with PI3K/Akt and ERK1/2 signaling pathways thereby decreasing the serum withdrawal-enhanced levels of the CDK inhibitor p21^Cip1 (p21) and restoring the vulnerability of AD cells to trophic factor deprivation.     

UDP-glucose ceramide glucosyltransferase activates AKT,promoted proliferation,and doxorubicin resistance in breast cancer cells

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.     

SNX3-dependent regulation of epidermal growth factor receptor (EGFR) trafficking and degradation by aspirin in epidermoid carcinoma (A-431) cells

Since being introduced globally as aspirin in 1899, acetylsalicylic acid has been widely used as an analgesic, anti-inflammation, anti-pyretic, and anti-thrombotic drug for years. Aspirin had been reported to down-regulate surface expression of CD40, CD80, CD86, and MHCII in myeloid dendritic cells (DC), which played essential roles in regulating the immune system. We hypothesized that the down-regulation of these surface membrane proteins is partly due to the ability of aspirin in regulating trafficking/sorting of endocytosed surface membrane proteins. By using an established epidermoid carcinoma cell line (A-431), which overexpresses the epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR), we show that aspirin (1) reduces cell surface expression of EGFR and (2) accumulates endocytosed-EGFR and -TfnR in the early/sorting endosome (ESE). Further elucidation of the mechanism suggests that aspirin enhances recruitment of SNX3 and SNX5 to membranes and consistently, both SNX3 and SNX5 play essential roles in the aspirin-mediated accumulation of endocytosed-TfnR at the ESE. This study sheds light on how aspirin may down-regulate surface expression of EGFR by inhibiting/delaying the exit of endocytosed-EGFR from the ESE and recycling of endocytosed-EGFR back to the cell surface.     

P16及COX-2蛋白在宫颈癌中表达及意义

目的 探讨P16及COX-2蛋白在宫颈癌中表达及意义.方法 收集宫颈活检或手术标本138例,采用免疫组织化学方法分别检测P16及COX-2蛋白的表达,并分析结果.结果 正常宫颈组织、宫颈低级别上皮内瘤变(LSIL)、宫颈高级别上皮内瘤变(HSIL)、宫颈癌中P16蛋白阳性表达分别为0％(0/30)、42.3％ (11/26)、71.9％(23/32)、92％(46/50).COX-2蛋白阳性表达分别为3.3％(1/30)、50％(13/26)、75％(24/32)、88％ (44/50).随着宫颈病变分级升高,P16及COX-2蛋白阳性率逐渐上升,统计学差异有显著性(p＜0.05);结论 P16及COX-2的高表达在宫颈癌的发生、发展中均起作用.     

TRAIL promotes the survival,migration and proliferation of vascular smooth muscle cells

Human and rat primary sub-cultured vascular smooth muscle cells (VSMCs) showed clear expression of the death receptors TRAIL-R1 and TRAIL-R2; however, recombinant soluble TRAIL did not induce cell death when added to these cells. TRAIL tended to protect rat VSMCs from apoptosis induced either by inflammatory cytokines tumor necrosis factor- + interleukin-1 + interferon- or by prolonged serum withdrawal, and promoted a significant increase in VSMC proliferation and migration. Of note, all the biological effects induced by TRAIL were significantly inhibited by pharmacological inhibitors of the ERK pathway. Western blot analysis consistently showed that TRAIL induced a significant activation of ERK1/2, and a much weaker phosphorylation of Akt, while it did not affect the p38/MAPK pathway. Taken together, these data strengthen the notion that the TRAIL/TRAIL-R system likely plays a role in the biology of the vascular system by affecting the survival, migration and proliferation of VSMCs.Received 6 May 2004; received after revision 7 June 2004; accepted 8 June 2004     

Progesterone-induced blocking factor differentially regulates trophoblast and tumor invasion by altering matrix metalloproteinase activity

Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while—based on our previous data—PIBF might control trophoblast invasion by suppressing proinvasive genes.     

D2A sequence of the urokinase receptor induces cell growth through αvβ3 integrin and EGFR

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvβ3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.