Constitutive and inducible Akt activity promotes resistance to chemotherapy,trastuzumab, or tamoxifen in breast cancer cells

To evaluate the role of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in breast cancer cell survival and therapeutic resistance, we analyzed a panel of six breast cancer cell lines that varied in erbB2 and estrogen receptor status. Akt activity was constitutive in four cell lines and was associated with either PTEN mutations or erbB2 overexpression. Akt promoted breast cancer cell survival because a PI3K inhibitor, LY294002, or transient transfection of a dominant-negative Akt mutant inhibited Akt activity and increased apoptosis. When combined with therapies commonly used in breast cancer treatment, LY294002 potentiated apoptosis caused by doxorubicin, trastuzumab, paclitaxel, or etoposide. Potentiation of apoptosis by LY294002 correlated with induction of Akt by doxorubicin or trastuzumab alone that occurred before the onset of apoptosis. Similar results were observed with tamoxifen. Combining LY294002 with tamoxifen in estrogen receptor-positive cells greatly potentiated apoptosis, which was correlated with tamoxifen-induced Akt phosphorylation that preceded apoptosis. To confirm that the effects of LY294002 on chemotherapy-induced apoptosis were attributable to inhibition of Akt, we transiently transfected breast cancer cells with dominant-negative Akt and observed increased doxorubicin-induced apoptosis. Conversely, stably transfecting cells with constitutively active Akt increased Akt activity and attenuated doxorubicin-induced apoptosis. These studies show that endogenous Akt activity promotes breast cancer cell survival and therapeutic resistance, and that induction of Akt by chemotherapy, trastuzumab, or tamoxifen might be an early compensatory mechanism that could be exploited to increase the efficacy of these therapies.     

Inhibition of phosphatidylinositol 3-kinase sensitizes ovarian cancer cells to carboplatin and allows adjunct chemotherapy treatment

Signal transduction pathways associated with cancer progression and chemotherapeutic resistance are being investigated as molecular targets for chemotherapy. The phosphatidylinositol 3-kinase (PI3K) pathway has been found to be frequently amplified and have increased activity in ovarian cancer. The current study investigates the efficacy of an antagonist of PI3K, LY294002, in inhibiting ovarian cancer cell growth and survival both in vitro and in vivo. The hypothesis tested is that inhibition of PI3K signaling makes ovarian cancer cells susceptible to the effects of platinum-based chemotherapy. Observations show that LY294002 is an effective inhibitor of ovarian cancer cell growth and survival in vitro. Inhibition of PI3K/Akt signaling increased the sensitivity of ovarian cell cultures to the cytotoxic effects of carboplatin. The combined treatment of LY294002 and carboplatin was needed to optimally promote cellular apoptosis and decrease ovarian cancer cell survival in vitro. To extend these observations, a model involving in vivo i.p. growth of human ovarian tumors in a nude mouse was used. LY294002 in combination with carboplatin was more effective in inhibiting ovarian cancer cell xenograft growth than either agent alone. The results of this study suggest that the combined treatment of carboplatin and LY294002 can effectively decrease ovarian tumor progression and support the use of a PI3K inhibitor (e.g., LY294002) as an adjunct platinum-based drug therapy for treatment of ovarian cancer.     

Inhibition of the phosphatidylinositol 3-kinase/Akt pathway sensitizes MDA-MB468 human breast cancer cells to cerulenin-induced apoptosis

Fatty acid synthase is overexpressed in cancer especially in tumors with a poor prognosis. The specific fatty acid synthase inhibitor cerulenin can induce apoptosis in cancer cells. Likewise, phosphatidylinositol 3-kinase (PI3K)/Akt kinase activities are elevated in primary tumors and cancer cell lines. Here, we tested whether inhibition of PI3K/Akt pathway would sensitize cancer cells to cerulenin-induced apoptosis. We show that LY294002, an inhibitor of PI3K, sensitized MDA-MB468 breast cancer cells to cerulenin-induced apoptosis. In MDA-MB468 cells, cerulenin- and LY294002-mediated apoptosis was associated with caspase-3 activation and the release of cytochrome c from mitochondria to cytosol. In addition, we observed additional species of Bak in mitochondria, suggesting a possible Bak activation. Treatment of cells with cerulenin and LY294002 down-regulated the protein levels of X chromosome-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis 1 (cIAP-1), and Akt, whereas the levels of mitogen-activated protein/extracellular signal-regulated kinase kinase and other antiapoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xl) did not change. Interestingly, the nonspecific caspase inhibitor, z-VAD-FMK, inhibited the down-regulation of Akt, XIAP, and cIAP-1 in cerulenin- and LY294002-treated cells. In conclusion, these studies show that inhibition of PI3K can sensitize cerulenin-induced apoptosis in MBA-MB468 breast cancer cells via activation of caspases, down-regulation of antiapoptotic proteins, such as XIAP, cIAP-1 and Akt, and possibly, activation of Bak in mitochondria.     

PI3K抑制剂LY294002对弥漫大B细胞性淋巴瘤细胞株化疗增敏作用的研究

目的 研究PI3K抑制剂LY294002对弥漫大B细胞淋巴瘤(DLBCL)细胞株ly1、ly8、ly10的化疗增敏作用.方法 采用LY294002、阿霉素及两者联合、序贯使用分别作用于ly1、ly8、ly10细胞;采用Western blot法观察LY294002处理后ly1、ly8、ly10细胞中磷酸化AKT/PKB(pAKT)的改变;采用流式细胞术结合Brdu法分析LY294002对细胞增殖和细胞周期分布的影响,采用流式细胞术结合AnnexinV-FITC染色检测各组细胞凋亡率.结果 LY294002可显著降低DLBCL细胞中磷酸化AKT的表达水平;并导致S期细胞比例显著降低(P＜0.05);LY294002预处理序贯使用阿霉素组中细胞凋亡的发生率比单独使用LY294002、阿霉素或同时联合使用LY294002、阿霉素组均明显提高,在24、48、72 h(ly1)或48、72 h(ly8)或24 h(ly10)差异均有统计学意义(P值均＜0.05).结论 PI3K抑制剂LY294002对阿霉素诱导DLBCL细胞凋亡的促进作用提示其可能是DLBCL治疗中潜在的具有较好化疗增敏作用的分子靶向药物.     

Inhibition of phosphatidylinositol 3-kinase-Akt signaling blocks growth,promotes apoptosis,and enhances sensitivity of small cell lung cancer cells to chemotherapy

A promising therapeutic alternative to inhibition of growth factor receptors is the inhibition of downstream signal transduction pathways. Such an approach may be especially important in tumors that can use signals from multiple growth factor receptors for growth and survival. Both stem cell factor (SCF) and insulin-like growth factor (IGF)-I, components of prominent small cell lung cancer (SCLC) autocrine loops, as well as FCS, can potently activate phosphatidylinositol 3-kinase (PI3K)-Akt signaling, albeit with different kinetics. SCF-induced PI3K-Akt activation occurs rapidly but fades within 60 min; IGF-I and FCS-induced activation persists for at least 6 h. SCF and IGF-I-mediated growth was potently inhibited by LY294002 in proportion to its ability to inhibit phosphatidylinositol 3-kinase (PI3K)-Akt signaling. A panel of six SCLC cell lines grown in 10% FCS was also very sensitive to LY294002, with average IC50 and LD50 of 5 and 25 microM, respectively. These drug concentrations suppressed the growth of the MRC-5 pulmonary fibroblast cell line and primary bronchial epithelial cells but did not induce significant cell death. Because LY294002 can also inhibit PI3K-related enzymes, we confirmed the role of the PI3K-Akt pathway in SCLC using doxycycline-regulated expression of a dominant-negative (kinase dead) and a constitutively active (CA; myristolated) Akt allele. Expression of dominant-negative Akt, which could only be achieved at relatively low levels, completely inhibited growth in the absence of exogenous growth factors and inhibited SCF-mediated growth but had no effect on IGF-I-mediated growth at the expression levels attained. Expression of CA Akt markedly augmented growth in the absence of exogenous growth factors but had minimal effect on growth in the presence of saturating concentrations SCF or IGF-I. Because PI3K-Akt signaling is known to promote survival under apoptotic stresses, we determined the effect of this pathway on SCLC sensitivity to etoposide. LY294002 potentiated the effect of low concentrations of etoposide in inhibiting growth and inducing apoptosis. The effect of low concentrations of LY294002 could largely be reversed by expression of CA Akt, suggesting that it was mediated by inhibition of Akt signaling. Expression of CA Akt by itself also induced resistance to etoposide-mediated apoptosis. Taken together, these data demonstrate that PI3K-Akt signaling promotes SCLC growth, survival, and chemotherapy resistance. Therefore, selective inhibitors of PI3K or Akt could potentially be useful as novel therapeutic agents in the treatment of SCLC.     

LY294002对Jeko-1细胞的增殖抑制及作用机制研究

本研究旨在探讨磷脂酰肌醇3-激酶/丝氨酸苏氨酸激酶（PI3K/Akt）特异性抑制剂LY294002对人套细胞淋巴瘤Jeko-1细胞生长、细胞凋亡的影响及其作用机制.四甲基偶氮唑盐（MTT）方法检测细胞增殖率;流式细胞术检测细胞凋亡水平;Western blot检测凋亡相关蛋白Cyclin D1、Bcl-2、procaspase-3及PI3 K/Akt信号通路相关蛋白p-Akt、p-mTOR、p-P70S6K的变化.结果表明,LY294002能抑制Jeko-1细胞的增殖.Jeko-1细胞经LY294002 0、5、10和20 μmol/L作用24 h后,凋亡率分别为（3.25±1.27）％、（11.34±2.35）％、（22.81±2.74）％和（43.61±3.48）％,差异有统计学意义（P＜0.01）;Westem blot检测发现,凋亡相关蛋白Cyclin D1、Bcl-2、procaspase-3表达下降,PI3K/Akt信号通路相关蛋白p-Akt、p-mTOR、p-P70S6K磷酸化水平降低.结论：LY294002可明显抑制Jeko-1细胞增殖,其机制可能通过下调PI3K/Akt信号通路中的p-Akt、p-mTOR、p-P70S6K磷酸化水平,抑制PI3K/Akt信号通路,促进细胞凋亡.     

Blockade of the phosphatidylinositol-3-kinase-Akt signaling pathway enhances the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the pathway is constitutively activated

Constitutive activation of the phosphatidylinositol-3-kinase (PI3K)-Akt signaling pathway is associated with the neoplastic phenotype in many human tumor cell types. Given the antiapoptotic role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although specific blockade of the PI3K-Akt pathway alone with inhibitors such as LY294002 did not induce cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents such as vincristine. This effect was apparent only in tumor cells in which the PI3K-Akt pathway is constitutively activated. Blockade of the PI3K-Akt pathway induced the activation of glycogen synthase kinase-3beta, which phosphorylates microtubule-associated proteins such as tau and thereby reduces their ability to bind and stabilize microtubules. The consequent destabilization of microtubules induced by the inhibition of PI3K-Akt signaling appeared to increase their sensitivity to low concentrations of microtubule-destabilizing agents that alone do not lead to the disruption of cytoplasmic microtubules in tumor cells. Such a synergistic effect on microtubule integrity was not apparent for stable microtubules in the neurites of neuronal cells. These results suggest that the administration of a combination of a PI3K-Akt pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells in which this signaling pathway is constitutively activated.     

Inhibition of PI3K signaling triggered apoptotic potential of curcumin which is hindered by Bcl-2 through activation of autophagy in MCF-7 cells

Curcumin is a natural anti-cancer agent derived from turmeric (Curcuma longa). Curcumin triggers intrinsic apoptotic cell death by activating mitochondrial permeabilization due to the altered expression of pro- and anti-apoptotic Bcl-2 family members. Phosphoinositol-3-kinase (PI3K) and Akt, key molecular players in the survival mechanism, have been shown to be associated with the Bcl-2 signaling cascade; therefore, evaluating the therapeutic efficiency of drugs that target both survival and the apoptosis mechanism has gained importance in cancer therapy. We found that Bcl-2 overexpression is a limiting factor for curcumin-induced apoptosis in highly metastatic MCF-7 breast cancer cells. Forced overexpression of Bcl-2 also blocked curcumin-induced autophagy in MCF-7 cells, through its inhibitory interactions with Beclin-1. Pre-treatment of PI3K inhibitor LY294002 enhanced curcumin-induced cell death, apoptosis, and autophagy via modulating the expression of Bcl-2 family members and autophagosome formation in MCF-7 breast cancer cells. Atg7 silencing further increased apoptotic potential of curcumin in the presence or absence of LY294002 in wt and Bcl-2+ MCF-7 cells. The findings of this study support the hypothesis that blocking the PI3K/Akt pathway may further increased curcumin-induced apoptosis and overcome forced Bcl-2 expression level mediated autophagic responses against curcumin treatment in MCF-7 cells.     

Isoginkgetin inhibits tumor cell invasion by regulating phosphatidylinositol 3-kinase/Akt-dependent matrix metalloproteinase-9 expression

Matrix metalloproteinase (MMP)-9 plays a key role in tumor invasion. Inhibitors of MMP-9 were screened from Metasequoia glyptostroboides (Dawn redwood) and one potent inhibitor, isoginkgetin, a biflavonoid, was identified. Noncytotoxic levels of isoginkgetin decreased MMP-9 production profoundly, but up-regulated the level of tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of MMP-9, in HT1080 human fibrosarcoma cells. The major mechanism of Ras-dependent MMP-9 production in HT1080 cells was phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor-kappaB (NF-kappaB) activation. Expression of dominant-active H-Ras and p85 (a subunit of PI3K) increased MMP-9 activity, whereas dominant-negative forms of these molecules decreased the level of MMP-9. H-Ras did not increase MMP-9 in the presence of a PI3K inhibitor, LY294002, and a NF-kappaB inhibitor, SN50. Further studies showed that isoginkgetin regulated MMP-9 production via PI3K/Akt/NF-kappaB pathway, as evidenced by the findings that isoginkgetin inhibited activities of both Akt and NF-kappaB. PI3K/Akt is a well-known key pathway for cell invasion, and isoginkgetin inhibited HT1080 tumor cell invasion substantially. Isoginkgetin was also quite effective in inhibiting the activities of Akt and MMP-9 in MDA-MB-231 breast carcinomas and B16F10 melanoma. Moreover, isoginkgetin treatment resulted in marked decrease in invasion of these cells. In summary, PI3K/Akt is a major pathway for MMP-9 expression and isoginkgetin markedly decreased MMP-9 expression and invasion through inhibition of this pathway. This suggests that isoginkgetin could be a potential candidate as a therapeutic agent against tumor invasion.     

Spectrum of activity and molecular correlates of response to phosphatidylinositol ether lipid analogues, novel lipid-based inhibitors of Akt

The serine/threonine kinase Akt is a promising target in cancer. We previously identified five phosphatidylinositol ether lipid analogues (PIA) that inhibited Akt activation and selectively killed lung and breast cancer cells with high levels of Akt activity. To assess the spectrum of activity in other cell types and to compare PIAs with other inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, we compared growth inhibition by PIAs against the PI3K inhibitors LY294002 and wortmannin and the mTOR inhibitor rapamycin in the NCI60 cell line panel. Although each of these compounds inhibited the growth of all the cell lines, distinct patterns were observed. The PIAs were the least potent but the most cytotoxic. The broad spectrum of activity of PIAs was confirmed in vivo in hollow fiber assays. The response to PIAs was significantly correlated with levels of active but not total Akt in the NCI60, as assessed using COMPARE analysis. However, a number of molecular targets were identified whose expression was more highly correlated with sensitivity to PIAs than active Akt. Expression of these molecular targets did not overlap with those that correlated with sensitivity to LY294002, wortmannin, or rapamycin. A COMPARE analysis of the National Cancer Institute chemical screening database revealed that the patterns of activity of PIAs correlated best with patterns of activity of other lipid-based compounds. These studies show that although PIAs are widely active in cancer cells, which correlates with the presence of its intended target, active Akt, PIAs are biologically distinct from other known inhibitors of the PI3K/Akt/mTOR pathway.     

Effects induced by inhibitors of the phosphatidylinositol 3‐kinase/Akt and nitric oxide synthase/guanylyl cyclase pathways on the isometric contraction in rat aorta: a comparative study

This work was aimed to determine whether isometric contraction in Wistar rat aorta is related to the phosphatidylinositol 3‐kinase (PI3K)/Akt‐dependent activation of endothelial nitric oxide synthase (eNOS). Basically, we hypothesized that additional increases in active tone occur after the pharmacological inhibition of a transduction pathway involved in NO synthesis or action. In intact aortic rings contracted with phenylephrine or high K⁺, the cumulative administration of the PI3K inhibitor, LY294002, elicited significant decreases – but not supplementary increases – in tone. In endothelium‐intact tissues, on the other hand, the Akt1/2 kinase inhibitor did not alter phenylephrine‐ and K⁺‐induced isometric contractions. The PI3K inhibitor wortmannin (1 × 10^?7 m ) produced a significant supplementary contraction only in endothelium‐intact aortic rings precontracted with phenylephrine. Higher concentrations of this inhibitor produced relaxations of phenylephrine and high K⁺‐constricted endothelium‐intact and endothelium‐denuded aortic rings. LY294002 and wortmannin did not cause any potentiating effect on phenylephrine‐ and angiotensin II‐induced concentration‐dependent contractile responses in endothelium‐intact tissues. In intact aortic rings contracted with phenylephrine or high K⁺, the addition of the NOS inhibitor, L‐NAME, or the guanylyl cyclase inhibitor, ODQ, further augmented tone in a concentration‐dependent manner, and these supplementary contractions were significantly reduced by endothelium removal. Taken together, our data suggest that the PI3K/Akt pathway is not counteracting aortic isometric contractions by activation of the eNOS. It appears, on the other hand, that the smooth muscle PI3K can stimulate contraction without activation of the protein kinase Akt in response to GPCR agonists and high K⁺.     

Blocking of PI3K/AKT induces apoptosis by its effect on NF-κB activity in gastric carcinoma cell line SGC7901

NF-κB plays an important role in many aspects of tumorigenesis and tumor progression by its antiapoptosis effect. Hence, NF-κB has been regarded as a therapeutic target in cancer, because inhibition of NF-κB not only induces enhancing apoptosis but also causes increasing sensitivity to radiation or chemotherapy in several tumor cells. The activation of NF-κB is presumed to be associated with PI3K/Akt signal pathway in gastric carcinoma, but the underlying molecular mechanism remains unclear. Our work demonstrates that blocking PI3K/Akt by LY294002 inhibits the NF-κB activity with significantly increased apoptosis in gastric cancer cell. Furthermore, when the cells were pretreated with IKK siRNA and/or IκB siRNA then exposed to LY294002, the results suggest that the regulatory significantly increased apoptosis in gastric cancer cell. Furthermore, when the cells were pretreated, effect of PI3K/AKT on NF-κB activity is associated with the influence of PI3K/AKT on IKK/IκB. The apoptosis induced by blocking PI3K/AKT might be ascribed to inhibition of NF-κB activity through IKK/IκB at least in part.     

A role for Akt in epidermal growth factor-stimulated cell cycle progression in cultured hepatocytes: generation of a hyperproliferative window after adenoviral expression of constitutively active Akt

Epidermal growth factor (EGF) stimulation of cell cycle progression in cultured primary hepatocytes has previously been reported to be dependent on the mammalian target of rapamycin (mTOR) elements of the phosphoinositide 3-kinase (PI3K) signaling cascade and not the Akt pathway. Here we have established conditions of combined treatment of rat hepatocytes with insulin and EGF that favor cell cycle progression. The resulting cell population expresses albumin and retains receptor regulation of the signaling pathways leading to glycogen phosphorylase activation. We then investigated the hypothesis that the Akt limb of the PI3K pathway plays a central role in this insulin/EGF enhancement of cell cycle progression. The phosphorylation of Akt, central to the PI3K pathway, was increased by both insulin (sustained) and EGF (transient). The stimulation of Akt phosphorylation was inhibited in a concentration-dependent manner by the PI3K inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Cell cycle progression in these cultures was reduced, but not abolished, by this inhibitor. The mTOR inhibitor, rapamycin, also inhibited entry into S phase. The novel Akt inhibitor A-443654 [(S)-1-(1H-indol-3-ylmethyl)-2-[5-(3-methyl-1H-indazol-5-yl)-pyridin-3-yloxy]-ethylamine] blocked both EGF-stimulated cell cycle progression and phosphorylation of the Akt substrate glycogen synthase kinase-3. Infection of cells with an adenoviral vector expressing a constitutively active form of Akt but not a kinase-dead form increased hepatocyte proliferation probably through enhanced cell cycle progression and reduced apoptosis. These results show that the Akt element of the PI3K cascade is necessary for EGF-stimulated cell cycle progression and provide evidence that the sustained elevation of Akt alone generates a hyperproliferative window in hepatocyte cultures.     

Inhibition of the Akt/survivin pathway synergizes the antileukemia effect of nutlin-3 in acute lymphoblastic leukemia cells

The phosphatidylinositol 3-kinase (PI3K)/Akt and p53 pathways play antiapoptotic and proapoptotic roles in cell death, respectively. Cancer cell growth and progression are associated with high levels of PI3K/Akt activation by loss of PTEN expression and the inactivation of p53 by MDM2 overexpression. We report that inhibition of PI3K/Akt, either by the PI3K inhibitor Ly294002 or by expression of PTEN, synergized the ability of the MDM2 antagonist nutlin-3 to induce apoptosis in acute lymphoblastic leukemia (ALL). We used a set of ALL cell lines with wild-type p53 and MDM2 overexpression, but different status of PTEN expression/PI3K/Akt activation, to test the ability of nutlin-3 to induce p53 and apoptosis. Nutlin-3 activated p53 in all the ALL cell lines; however, induction of apoptosis was dependent on PTEN status. Nutlin-3 induced potent apoptosis in cells with PTEN expression but not in those without PTEN, suggesting that PTEN/PI3K/Akt pathway may play a role in this process. Furthermore, nutlin-3 significantly down-regulated survivin expression in PTEN-positive cells but not in PTEN-negative cells. When these nutlin-3-resistant cells were either pretransfected with the PTEN gene or simultaneously treated with the PI3K inhibitor Ly294002, survivin was down-regulated and sensitivity to nutlin-3 was increased. Furthermore, direct silencing of survivin by small interfering RNA also increased the proapoptotic effect of nutlin-3 on the PTEN-negative, nutlin-3-resistant ALL cells. Our results suggest that Akt-mediated survivin up-regulation in PTEN-negative ALL cells may counteract the proapoptotic effect of nutlin-3, and indicate that a combination of MDM2 antagonist and PI3K/Akt inhibitor may be a promising approach for treating refractory ALL.     

Tissue inhibitor of metalloproteinase-1 decreased chemosensitivity of MDA-435 breast cancer cells to chemotherapeutic drugs through the PI3K/AKT/NF-кB pathway

TIMP-1 is well known to be capable of inhibiting apoptosis. Elevated levels of TIMP-1 in tumor tissue have been shown to be strongly associated with a poor response to chemotherapy. In this study, using conventional cytotoxic drugs commonly used in the treatment of breast cancer, we investigated how TIMP-1 influenced the efficacy using breast cell lines. Our data demonstrated that overexpression of TIMP-1 could significantly decrease the sensitivity of MDA-435 breast cancer cells to epirubicin and paclitaxel. TIMP-1 can potently activate phosphatidylinositol 3-kinase (PI3 K)/Akt and nuclear factor-kappaB (NF-кB) signaling. Furthermore, the TIMP-1-induced attenuation of the effect of epirubicin and paclitaxel was reversed by the PI3K/Akt chemical inhibitor LY294002 and the NF-кB inhibitor pyrrolidine dithiocarbamate (PDTC), showing that the PI3 K/Akt and NF-кB signaling pathway was involved in the TIMP-1-induced effect on chemoresistance. Taken together, our results indicate that TIMP-1 decreased chemosensitivity through the PI3 K/Akt/NF-кB signal transduction pathway in MDA-435 breast cancer cells.     

新生大鼠支气管上皮衍生舒张因子信号传导

目的研究支气管上皮衍生舒张因子（BrEpDRF）是否经一氧化氮（NO）-3＇，5＇-鸟嘌呤核苷环一磷酸（cGMP）及磷脂酰三肌醇（PI3）／蛋白激酶B（Akt）通路传导。方法支气管结合的肺动脉经血栓烷A2（TXA2）类似物U46619预刺激肺动脉收缩，在PI-3激酶／Akt抑制剂LY294002和NO-GMP抑制剂ODQ存在／不存在下，测定乙酰甲胆碱（Mch）引起的血管舒张比率。结果结合支气管的肺动脉在经U46619预刺激后，Mch引起血管舒张率为39．37％；Mch中加入PI-3激酶／Akt抑制剂引起的血管舒张率为11．72％，两者比较差异有显著性，（P〈0．01）。Mch中加入NO-GMP抑制剂引起的血管舒张率为2．15％，与单一Mch比较统计学上有差异，（P〈0．01）。结论新生大鼠在生理条件下BrEpDRF经NO-cGMP及PI3／Akt通路传导。     

血小板源性生长因子-DD通过Akt信号通路促进膀胱癌T24细胞增殖

目的研究外源性血小板源性生长因子-DD(PDGF-DD)对膀胱癌T24细胞增殖及Akt信号转导通路的影响,阐述factor其诱导细胞增殖的机制。方法外源性PDGF-DD蛋白作用T24细胞,采用MTT法分析细胞的增殖;流失细胞仪检测细胞周期的变化;Western blot法观测膀胱癌T24细胞Akt、p-Akt、mTOR、p-mTOR以及核因子NF-κB蛋白表达的变化。结果 PDGF-DD促进T24细胞的增殖,并且具有浓度依赖性;DNA合成前期(G0/G1期)细胞比例下降,合成期(S期)细胞比例上升;Akt、mTOR表达变化不明显,而p-Akt、p-mTOR及NF-κB p65的表达均上调。LY294002抑制PI3K/Akt及其下游靶位蛋白磷酸化;雷帕霉素和PDTC分别抑制p-mTOR和NF-κBp65的表达。结论外源性PDGF-DD刺激T24细胞的增殖,其机制可能是通过Akt/mTOR和Akt/NF-κB两条独立的信号通路实现的。     

骨髓间充质干细胞分泌基质细胞衍生因子1保护心肌细胞

背景：有研究显示表达CXCR4的干细胞能够沿着基质细胞衍生因子1的浓度梯度迁移到心肌梗死部位再生心肌和血管而改善心脏的功能。目的：探索间充质干细胞通过其分泌的基质细胞衍生因子1对心肌细胞的保护作用。方法：收集培养2d的间充质干细胞条件培养基。在缺氧条件,利用基质细胞衍生因子1受体CXCR4阻断剂AMD3100或P13．K／Akt途径阻断剂LY294002预处理H9C2细胞后,利用AnnexinV／PI双标法流式细胞术分析间充质干细胞条件培养基作用下H9C2细胞凋亡的变化;Western blotting分析H9C2细胞磷酸化Akt蛋白的表达;RT-PCR分析间充质干细胞基质细胞衍生因子1的表达。结果与结论：RT-PCR结果显示间充质干细胞表达基质细胞衍生因子1,Westernblotting结果显示间充质干细胞条件培养基增加了H9C2细胞磷酸化Akl蛋白的水平。AnnexinV／P1分析发现间充质干细胞条件培养基明显降低了H9C2细胞缺氧复氧后的凋亡,且这种抗凋亡作用能被CXCR4阻断剂AMD3100或P13-K／Akt途径阻断剂LY294002所阻断。说明间充质干细胞通过其分泌的基质细胞衍生因子1通过激活P13-K／Akt途径保护H9C2细胞,增加H9C2细胞的幸存能力。     

血管生成素-1对HCT-8细胞的作用及其可能机制的研究

目的观察血管生成素-1（Angiopoietin-1,Ang-1）蛋白对无血清DMEM培养基培养的结肠癌细胞（HCT-8）存活率的影响,探讨其与PI3-’kinase/Akt通路的关系。方法将不同浓度（0、0.05、0.2、0.4、0.82、.0 mg/L）的Ang-1蛋白作用于HCT-8细胞,用MTT法检测细胞增殖,根据实验结果选定Ang-1蛋白的后续实验浓度,加入Ang-1及LY294002,应用Western blotting蛋白免疫印记法分析相关蛋白（Tie-2、PI3K、Akt）的变化。结果 Ang-1（0.05、0.2、0.4、0.82、.0 mg/L）＋DMEM组与无血清DMEM培养基组比较,Tie-2、PI3K、Akt三种蛋白在HCT-8细胞中的表达均增强（P〈0.01,P〈0.01,P〈0.01）,DMEM＋Ang-1＋LY294002组三种蛋白的表达均减弱（P〉0.05,P〈0.01,P〈0.01）。结论较低浓度（0.05 mg/L）的血管生成素-1蛋白在结肠癌细胞中即有抗凋亡作用,并且随着剂量的增加抗凋亡作用平稳,其诱导凋亡的机理可能与Tie-2/PI3-’kinase/Akt调节的通路有关,应用该途径的抑制剂LY294002可抑制结肠癌细胞的生长,实现抗肿瘤作用。