MT1-MMP downregulation via the PI3K/Akt signaling pathway is required for the mechanical stretching-inhibited invasion of bone-marrow-derived mesenchymal stem cells

Mobilization from the bone marrow and the migration of bone-marrow-derived mesenchymal stem cells (BMSCs) through the peripheral circulation to injured tissue sites are regulated by multiple mechanical and chemical factors. We previously demonstrated that mechanical stretching promotes the migration but inhibits the invasion of BMSCs. However, the involved mechanisms, especially the mechanism of stretching-inhibited BMSC invasion, have not been thoroughly elucidated to date. In this study, we found that mechanical stretching with a 10% amplitude at a 1-Hz frequency for 8 hr significantly reduces BMSC invasion and downregulates the expression of membrane type-1 matrix metalloproteinases (MT1-MMP) at both the messenger RNA and protein levels. The overexpression of MT1-MMP restores mechanical stretching-reduced BMSC invasion. Moreover, phosphatidylinositol 3-kinase (PI3K)-dependent Akt phosphorylation in BMSCs was found to be inactivated by mechanical stretching. Pharmacological inhibitors of PI3K/Akt signaling (LY294002 or A443654) reduced the expression of MT1-MMP and impaired BMSC invasion. In addition, the upregulation of Akt phosphorylation by a pharmacological activator (SC79) increased MT1-MMP expression and suppressed mechanical stretching-reduced BMSC invasion. Taken together, our results suggest that mechanical stretching inhibits BMSC invasion by downregulating MT1-MMP expression by suppressing the PI3K/Akt signaling pathway.     

Arachidonic acid promotes migration and invasion through a PI3K/Akt-dependent pathway in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n−6 cis polyunsaturated fatty acid that under physiological conditions is present in an esterified form in cell membrane phospholipids, however it might be present in the extracellular microenvironment. AA and its metabolites mediate FAK activation, adhesion and migration in MDA-MB-231 breast cancer cells. However, it remains to be investigated whether AA promotes invasion and the signal transduction pathways involved in migration and invasion. Here, we demonstrate that AA induces Akt2 activation and invasion in MDA-MB-231 cells. Akt2 activation requires the activity of Src, EGFR, and PIK3, whereas migration and invasion require Akt, PI3K, EGFR and metalloproteinases activity. Moreover, AA also induces NFκB-DNA binding activity through a PI3K and Akt-dependent pathway. Our findings demonstrate, for the first time, that Akt/PI3K and EGFR pathways mediate migration and invasion induced by AA in MDA-MB-231 breast cancer cells.     

Downregulation of the PI3K/Akt survival pathway in cells with deregulated expression of c-Myc

Oncogenic transformation leads to an increased sensitivity to apoptosis, a characteristic that is selectively lost during tumor progression. The sensitization process affects the mitochondrial pathway of apoptosis through signaling events that are poorly defined. We previously showed that a deregulated expression of c-Myc in cells treated with toxic agents caused an enhanced activation of p38 that acts in a death-promoting pathway. Here, we show that deregulated expression of c-Myc causes a severe reduction in the basal activity of Akt, which was further accelerated by serum deprivation. Furthermore, c-Myc expression repressed the activation of Akt induced by the toxic agents doxorubicin, cisplatin and H₂O₂, and also by the physiological agonists PDGF and insulin. We determined that the activation of Akt was inhibited as a result of the action of c-Myc upstream of phosphatidylinositol 3-kinase (PI3K) activation. c-Myc overexpression impaired the induced association of the p85 subunit of PI3K with phosphotyrosine containing proteins, causing a reduction in the activation of PI3K and recruitment of Akt to the membrane. Inhibiting Akt in addition to enhancing p38 further exacerbate the imbalance between the death and survival signals and results in an enhanced sensitivity to apoptosis. This study was supported by the Canadian Institutes of Health Research Grant MOP-37860 to J.L. and K.B. and the Canada Research Chair in Stress Signal Transduction (to J.L.).     

蛋白激酶B/Akt抑制剂

磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB/Akt)信号通路在细胞生长与存活中起着关键作用,PI3K/Akt通路的过度激活在多种肿瘤中常见。Akt激酶本身以及Akt激酶上游调节分子,例如PTEN和PI3K,在超过50%的人类肿瘤中均有异常变化。因此Akt成为肿瘤预防和肿瘤靶向治疗的热点之一。许多小分子化合物通过不同机制抑制Akt活性,根据小分子抑制剂与激酶的结合部位和化学结构不同,主要分为ATP竞争性抑制剂、Akt变构抑制剂和磷脂酰肌醇类似物抑制剂。本文综述了PI3K/Akt通路与肿瘤的关系和Akt抑制剂的研究现状,为新型抗癌药物的设计研究提供参考。     

PI3K/Akt信号通路相关的生物学调控机制研究进展

PI3K/Akt信号通路是由酶联受体介导的信号转导通路,该通路不仅参与多种生长因子、细胞因子和细胞外基质等的信号转导,同时还参与细胞增殖、分化、凋亡和葡萄糖转运等多种细胞功能的调节,特别是在细胞凋亡、细胞存活以及调控细胞糖代谢等方面具有重要作用。本研究综述了PI3K-Akt信号通路的结构组成、通路活化、通信过程、调控机制及其生物学功能等方面的研究进展,为进一步研究PI3K/Akt信号通路的生物学调控作用机制提供启示。     

LncRNA MYU对胶质瘤细胞周期分布、增殖、转移和凋亡以及PI3K/Akt信号通路的影响

摘要 目的：探讨长链非编码RNA（LncRNA）MYU对胶质瘤细胞周期分布、细胞增殖、迁移、侵袭和凋亡的影响,并初步探讨其作用机制。方法：实时荧光定量PCR（RT-qPCR）检测人脑正常胶质细胞HEB和胶质瘤细胞（U-251MG、A172、SHG139）中LncRNA MYU的表达情况。选取SHG139细胞,分为正常对照（NC）组、si-con组、si-LncRNA MYU组进行转染实验,行RT-qPCR检测转染效果。分别采用流式细胞术、细胞计数试剂盒（CCK-8）、Transwell实验检测沉默LncRNA MYU对SHG139细胞周期分布和凋亡、细胞增殖、细胞迁移和侵袭的影响。蛋白免疫印迹（Western blot）法检测基质金属蛋白酶2（MMP-2）、MMP-9、裂解的半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase-3）、Cleaved caspase-9以及磷脂酰肌醇-3-羟激酶/蛋白激酶B（PI3K/Akt）信号通路相关蛋白表达情况。结果：LncRNA MYU在胶质瘤细胞株中比人脑正常胶质细胞中的表达水平显著升高（P＜0.05）,因此选择表达量最高的SHG139细胞进行转染实验。沉默LncRNA MYU能够显著诱导SHG139细胞G0-G1期阻滞、抑制细胞增殖、迁移和侵袭并诱导细胞凋亡（P＜0.05）。沉默LncRNA MYU可显著抑制MMP-2、MMP-9、p-PI3K和p-AKT表达并促进Cleaved caspase-3、Cleaved caspase-9表达（P＜0.05）。结论：沉默LncRNA MYU可诱导胶质瘤细胞G0-G1期阻滞,抑制细胞增殖、迁移和侵袭,促进细胞凋亡,其机制可能与抑制PI3K/AKT信号通路有关。     

RhoGDI2与PI3K/Akt/mTOR信号通路在肺癌细胞中的相关性研究

目的探讨肿瘤转移相关因子RhoGDI2与PI3K/Akt/mTOR信号通路在肺癌侵袭转移过程中的作用及相关机制。方法利用PI3K/Akt/mTOR信号通路上特异性的抑制剂,采用MTT法,伤口愈合实验及侵袭实验观察不同浓度药物对肺癌95D细胞生长侵袭转移能力的影响,通过Western Blot方法观察RhoGDI2蛋白水平的变化。结果PI3K抑制剂LY294002及mTOR抑制剂Rapamycin都能抑制肺癌细胞95D的侵袭转移能力,联合应用抑制作用更强。PI3K抑制剂LY294002处理组RhoGDI2蛋白的表达量增加,且随浓度增加RhoGDI2蛋白表达也增加。mTOR抑制剂Rapamycin组,在低浓度时增加RhoGDI2蛋白的表达,但增大Rapamycin的浓度,RhoGDI2蛋白的表达反而降低。低浓度LY294002组和Rapa-mycin组联合应用可以明显增加RhoGDI2蛋白的表达。结论PI3K/Akt/mTOR信号通路中Akt的活化与RhoGDI2密切相关,RhoGDI2可能直接或间接通过与Akt的相互作用参与调节肺癌的侵袭转移的过程。     

hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway

hnRNP A1 acts as a critical splicing factor in regulating many alternative splicing events in various physiological and pathophysiological progressions. hnRNP A1 is capable of regulating UVB-induced hdm2 gene alternative splicing according to our previous study. However, the biological function and underlying molecular mechanism of hnRNP A1 in cell survival and cell cycle in response to UVB irradiation are still unclear. In this study, silencing hnRNP A1 expression by siRNA transfection led to decreased cell survival after UVB treatment, while promoting hnRNP A1 by lentiviruse vector resulted in increased cell survival. hnRNP A1 remarkably enhanced PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of Akt, mTOR and P70S6 protein. Inhibition of PI3K/Akt signaling by LY294002 suppressed the expression of hnRNP A1. While mTOR signaling inhibitors, rapamycin and AZD8055, did not influence hnRNP A1 expression in HaCaT cells, suggesting that hnRNP A1 may be an upstream mediator of mTOR signaling. Furthermore, hnRNP A1 could alleviate UVB-provoked cell cycle arrest at G0/G1 phase and promoted cell cycle progression at G2/M phase. Our results indicate that hnRNP A1 promotes cell survival and cell cycle progression following UVB radiation.     

Knockdown of insulin receptor substrate 1 reduces proliferation and downregulates Akt/mTOR and MAPK pathways in K562 cells

BCR-ABL kinase activates downstream signaling pathways, including the PI3K-Akt/mTOR and the MAPK pathway. IRS1 has been previously described as constitutively phosphorylated and associated with BCR-ABL in K562 cells, suggesting that IRS1 has role in the BCR-ABL signaling pathways. In this study, we analyzed the effect of IRS1 silencing, by shRNA-lentiviral delivery, in K562 cells, a CML cell line that presents the BCR-ABL. IRS1 silencing decreased cell proliferation and colony formation in K562 cells, which correlates with the delay of these cells at the G0/G1 phase and a decrease in the S phase of the cell cycle. Furthermore, IRS1 silencing in K562 cells resulted in a decrease of Akt, P70S6K and ERK1/2 phosphorylation. Nevertheless, apoptosis was unaffected by IRS1 knockdown and no alterations were found in the phosphorylation of BAD and in the expression of BCL2 and BAX. BCR-ABL and CRKL phosphorylation levels remained unaffected upon IRS1 silencing, and no synergistic effect was observed with imatinib treatment and IRS1 knockdown, indicating that IRS1 is downstream from BCR-ABL. In conclusion, we demonstrated that inhibition of IRS1 is capable of inducing the downregulation of Akt/mTOR and MAPK pathways and further decreasing proliferation, and clonogenicity and induces to cell cycle delay at G0/G1 phase in BCR-ABL cells.     

FOXO-independent suppression of programmed cell death by the PI3K/Akt signaling pathway in Drosophila

Signaling through the PI3K/Akt/FOXO pathway plays an important role in vertebrates in protecting cells from programmed cell death. PI3K and Akt have been similarly shown to be involved in survival signaling in the invertebrate model organism Drosophila. However, it is not known whether PI3K and Akt execute this function by controlling a pro-apoptotic activity of Drosophila FOXO. In this study, we show that elevated signaling through PI3K and Akt can prevent developmentally controlled death in the salivary glands of the fruit fly. We further show that Drosophila FOXO is not required for normal salivary gland death and that the rescue of salivary gland death by PI3K occurs independent of FOXO. These results give support to the notion that FOXOs have acquired pro-apoptotic functions after separation of the vertebrate and invertebrate lineages.     

KAI1/CD82 decreases Rac1 expression and cell proliferation through PI3K/Akt/mTOR pathway in H1299 lung carcinoma cells

Although the KAI1/CD82 protein has been reported to inhibit cell metastasis in many studies, its mechanism of action has not yet been fully elucidated. In the present study, we investigated the possible effects of KAI1/CD82 on the metastatic phenotype in H1299 lung carcinoma cells. These studies were based on the pivotal role that the acquisition of motile phenotype plays on the initial steps of metastasis. KAI1/CD82‐mediated morphological changes were observed using phase contrast microscopy. We report here, that a KAI1/CD82‐induced phenotypic change was involved in the decrease of Rac1 expression and GTPase activity. However, we found that KAI1/CD82 did not regulate Rac1 mRNA levels. This suggests the existence of another regulatory mechanism of Rac1 protein maturation or activation. To identify the signaling pathway of Rac1 regulation, we investigated the PI3K/Akt/mTOR pathway, since the PI3K/Akt pathway regulates Rac1 activation and mTOR is known to play a regulatory role in protein translation. H1299/CD82‐transfectants showed lower mTOR expression and cell growth than the control group. The data obtained from this study suggested that KAI1/CD82 decreased the metastatic phenotype of H1299 lung carcinoma cells by down‐regulating Rac1 expression through the PI3K/Akt/mTOR pathway. Copyright © 2008 John Wiley & Sons, Ltd.     

Streptococcus pyogenes CAMP factor promotes bacterial adhesion and invasion in pharyngeal epithelial cells without serum via PI3K/Akt signaling pathway

Streptococcus pyogenes is a bacterium that causes systemic diseases, such as pharyngitis and toxic shock syndrome, via oral- or nasal-cavity infection. S. pyogenes produces various molecules known to function with serum components that lead to bacterial adhesion and invasion in human tissues. In this study, we identified a novel S. pyogenes adhesin/invasin. Our results revealed that CAMP factor promoted streptococcal adhesion and invasion in pharyngeal epithelial Detroit562 cells without serum. Recombinant CAMP factor initially localized on the membranes of cells and then became internalized in the cytosol following S. pyogenes infection. Additionally, CAMP factor phosphorylated phosphoinositide 3-kinase and serine–threonine kinase in the cells. ELISA results demonstrate that CAMP factor affected the amount of phosphorylated phosphoinositide 3-kinase and serine–threonine kinase in Detroit562 cells. Furthermore, CAMP factor did not reverse the effect of phosphoinositide 3-kinase knockdown by small interfering RNA in reducing the level of adhesion and invasion of S. pyogenes isogenic cfa-deficient mutant. These results suggested that S. pyogenes CAMP factor activated the phosphoinositide 3-kinase/serine–threonine kinase signaling pathway, promoting S. pyogenes invasion of Detroit562 cells without serum. Our findings suggested that CAMP factor played an important role on adhesion and invasion in pharyngeal epithelial cells.     

Apoptosis in medfly hemocytes is regulated during pupariation through FAK, Src, ERK, PI-3K p85a, and Akt survival signaling

Focal adhesion kinase (FAK) and its downstream signaling targets are implicated in the process of apoptosis induced by external stimuli, in several mammalian systems. In this report, we demonstrate, that medfly (Ceratitis capitata) hemocytes do undergo apoptosis during larval development. In particular, we show using Western blot, ELISA and flow cytometry analysis, that FAK expression silencing in transfected by FAK double-stranded RNA (dsRNA) hemocytes, enhances twofold hemocyte apoptosis, by signaling through Src, MEK/ERK, and PI-3K/Akt signaling pathways. FAK expression silencing, in response to FAK dsRNA treatment, blocks partially the phosphorylation of its downstream targets. Pre-incubation of hemocytes, with specific inhibitors of FAK downstream signaling molecules, demonstrated that all these inhibitors reduced hemocyte viability and enhanced the magnitude of apoptosis about threefold. This data suggest that these pathways contribute to hemocyte survival and/or death during development. The expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK signaling molecules appear to be dependent upon developmental stages. The expression and phosphorylation of the above signaling molecules, in annexin-positive and annexin-negative hemocytes is also distinct. The maximum expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK appeared in annexin-positive hemocytes, in both early and late apoptotic hemocytes. The novel aspect of this report is based on the fact that hemocytes attempt to suppress apoptosis, by increasing the expression/phosphorylation of FAK and, hence its downstream targets signaling molecules Src, ERK, PI-3K p85a, and Akt. Evidently, the basic survival pathways among insects and mammals appear to remain unchanged, during evolution.     

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.     

Rhabdastrellic acid-A inhibited PI3K/Akt pathway and induced apoptosis in human leukemia HL-60 cells

Increasing evidence suggests that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer treatment. Our investigation indicates that Rhabdastrellic acid-A, an isomalabaricane triterpenoid isolated from the sponge, Rhabdastrella globostellata, inhibits proliferation of HL-60 cells with an IC(50) value of 0.68mug/ml, and induces apoptosis. Rhabdastrellic acid-A also induces cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and caspase-3. Pretreatment of HL-60 cells with the caspase-3 specific inhibitor, DEVD-CHO, prevents Rhabdastrellic acid-A-induced DNA fragmentation and PARP cleavage. Activated PI3K and Akt significantly decreases after treatment with Rhabdastrellic acid-A in HL-60 cells. Expression levels of protein bcl-2, bax remain unchanged in response to Rhabdastrellic acid-A treatment in HL-60 cells. These results suggest that Rhabdastrellic acid-A inhibits PI3K/Akt pathway and induces caspase-3 dependent-apoptosis in HL-60 human leukemia cells.