Involvement of PI3K/AKT/GSK3beta pathway in tetrandrine-induced G1 arrest and apoptosis

It has been reported that tetrandrine induces cell cycle arrest and apoptosis in human cancer cells. In the present study, we investigated the role of PI3K/AKT/GSK3beta pathway in tetrandrine- induced G(1) arrest and apoptosis. In HT-29 cells, tetrandrine induced dephosphorylation of AKT, activation and nuclear translocation of GSK3beta as well as upregulation of p27(kip1). Activation of GSK3beta via AKT inhibitoion induced by tetrandrine resulted in enhanced phosphorylation and proteolysis of cyclin D(1), activation of caspase 3 and subsequent cleavage of PARP. Selective GSK3beta inhibitiors and GSK3beta siRNA attenuated tetrandrine-induced G(1) arrest and apoptosis. Similar to tetrandrine, transfection of wild-type GSK3beta led to G(1) arrest and apoptosis via downregulation of cyclin D(1) and cleavage of PARP. These findings suggest that tetrandrine induces G(1) arrest and apoptosis through PI3K/AKT/GSK3beta pathway and identify GSK3beta as an important mediator in the processes.     

RNAi下调AKT1、PI3K P85表达抑制乳腺癌MCF-7细胞的增殖

目的：探讨RNAi（RNA interference）技术抑制乳腺癌MCF7细胞中AKT1和PI3K P85亚基的表达对MCF7细胞增殖和侵袭等的影响。方法：将包含AKT1、PI3K P85两种siRNA开放阅读框的短发夹RNA（shRNA）重组腺病毒质粒表达载体rAd5siAKT1siPI3K转染至乳腺癌MCF7细胞。应用realtime PCR和Western blotting检测转染后目的基因mRNA和蛋白的表达水平,并用Western blotting检测目的基因被沉默后PCNA、cyclin D1和P53的表达情况。应用MTT法、流式细胞术、2D和3D Matrigel实验检测MCF7细胞转染前后的细胞增殖周期和侵袭能力。结果：重组腺病毒质粒表达载体rAd5siAKT1siPI3K介导的靶向〖STBX〗AKT1, PI3K P85 shRNA可以有效抑制目的基因AKT1和PI3 Kp85的mRNA和蛋白表达;下游相关因子PCNA、cyclin D1的表达亦下调,P53表达则上调。MTT法结果显示rAd5siAKT1siPI3K组细胞生长抑制率>50%,与未转染组和rAd5siCtrl转染组比较,出现明显的G1/G0细胞周期阻滞;2D和3D Matrigel实验显示,未转染组和rAd5siCtrl转染组细胞呈正常形态,而rAd5siAKT1siPI3K 转染组细胞贴壁生长能力明显减低,细胞团块明显缩小。结论：靶向AKT1、PI3K P85亚基的shRNA技术可以抑制MCF7细胞中AKT1、PI3K P85亚基的表达,抑制MCF7细胞的体外增殖。     

PI3K/Akt信号通路在RANKL诱导的MCF-7乳腺癌细胞迁移中的作用

目的 核因子-κB受体活化因子配体(RANKL)能促进表达RANK的上皮癌细胞迁移至骨,与乳腺癌骨转移密切相关.本文探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(phosphoinositide3-kinase/serine/threonine protein kinase PI3K/Akt)信号通路在RANKL诱导的乳腺癌MCF-7细胞迁移中的作用.方法 流式细胞仪检测MCF-7细胞表面RANK蛋白的表达;Transwell法测定RANKL刺激后MCF-7细胞迁移能力的改变;Western-blot检测MCF-7细胞RANKL刺激后p-Akt及Akt的表达.SPSS 16.0软件分析实验数据.结果 MCF-7细胞表达RANK 蛋白,RANKL诱导MCF-7细胞迁移能力显著增强,RANKL的圈套受体OPG可阻断RANKL诱导的细胞迁移.RANKL刺激后MCF-7细胞p-Akt表达在1、5分钟时一过性升高,PI3K抑制剂LY294002显著抑制RANKL诱导的细胞迁移.结论 PI3K/Akt信号通路参与RANKL诱导的乳腺癌细胞系MCF-7迁移.     

PI3K/Akt信号通路在RANKL诱导的MCF-7乳腺癌细胞迁移中的作用

目的：核因子-κB受体活化因子配体（RANKL）能促进表达RANK的上皮癌细胞迁移至骨,与乳腺癌骨转移密切相关。本文探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶（phosphoinositide3-kinase/serine/threonine protein kinase PI3K/Akt）信号通路在RANKL诱导的乳腺癌MCF-7细胞迁移中的作用。方法：流式细胞仪检测MCF-7细胞表面RANK蛋白的表达;Transwell法测定RANKL刺激后MCF-7细胞迁移能力的改变;Western-blot检测MCF-7细胞RANKL刺激后p-Akt及Akt的表达。SPSS 16.0软件分析实验数据。结果：MCF-7细胞表达RANK蛋白,RANKL诱导MCF-7细胞迁移能力显著增强,RANKL的圈套受体OPG可阻断RANKL诱导的细胞迁移。RANKL刺激后MCF-7细胞p-Akt表达在1、5分钟时一过性升高,PI3K抑制剂LY294002显著抑制RANKL诱导的细胞迁移。结论：PI3K/Akt信号通路参与RANKL诱导的乳腺癌细胞系MCF-7迁移。     

EBV-BZLF1通过激活PI3K/AKT信号通路促进胃癌细胞生长

  目的   明确EB病毒（Epstein-Barr virus,EBV）BZLF1基因（EBV-BZLF1）对EB病毒阴性胃癌细胞生物学行为的影响及可能的分子机制。   方法   运用过表达BZLF1的慢病毒感染胃癌细胞系AGS和HGC27,分别采用CCK8、细胞凋亡检测、细胞迁移和侵袭实验及Western blot法检测细胞的增殖能力、细胞凋亡与迁移侵袭能力及细胞信号通路变化情况。将过表达BZLF1的胃癌细胞HGC27注射于非肥胖糖尿病/重症联合免疫缺陷（NOD/SCID）小鼠背部皮下构建移植瘤模型,观察BZLF1对肿瘤生长的影响。   结果   过表达BZLF1的慢病毒感染组AGS-BZLF1与HGC27-BZLF1相比亲本细胞,细胞中BZLF1蛋白表达明显上调;体外细胞增殖与小鼠体内成瘤能力均显著增强（P < 0.05）;细胞凋亡受到BZLF1蛋白的抑制,其中AGS-BZLF1和HGC27-BZLF1凋亡率为（2.40± 0.14）%和（3.90±0.14）%,显著低于AGS和HGC细胞的（5.75±0.35）%和（9.70±0.42）%（P < 0.05）;但细胞迁移和侵袭能力无明显改变。深入分子机制研究发现,BZLF1表达上调后,PI3K/AKT信号通路明显激活,表现为pAKT和pS6蛋白表达上调。使用BEZ235抑制剂阻断PI3K/AKT信号通路后,HGC27-BZLF1和AGS-BZLF1细胞的生长均受到抑制。   结论   EBV-BZLF1可通过激活PI3K/ AKT信号通路促进胃癌细胞生长,靶向PI3K/AKT通路抑制剂或可成为EBV阳性胃癌的治疗选择。      

RICTOR involvement in the PI3K/AKT pathway regulation in melanocytes and melanoma

Several studies have highlighted the importance of the PI3K pathway in melanocytes and its frequent over-activation in melanoma. However, little is known about regulation of the PI3K pathway in melanocytic cells. We showed that normal human melanocytes are less sensitive to selective PI3K or mTOR inhibitors than to dual PI3K/mTOR inhibitors. The resistance to PI3K inhibitor was due to a rapid AKT reactivation limiting the inhibitor effect on proliferation. Reactivation of AKT was linked to a feedback mechanism involving the mTORC2 complex and in particular its scaffold protein RICTOR. RICTOR overexpression in melanocytes disrupted the negative feedback, activated the AKT pathway and stimulated clonogenicity highlighting the importance of this feedback to restrict melanocyte proliferation. We found that the RICTOR locus is frequently amplified and overexpressed in melanoma and that RICTOR over-expression in NRAS-transformed melanocytes stimulates their clonogenicity, demonstrating that RICTOR amplification can cooperate with NRAS mutation to stimulate melanoma proliferation. These results show that RICTOR plays a central role in PI3K pathway negative feedback in melanocytes and that its deregulation could be involved in melanoma development.     

Inhibition of activated phosphatidylinositol 3-kinase/AKT pathway in malignant pleural mesothelioma leads to G1 cell cycle arrest

Phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway plays pivotal roles in fundamental cellular functions including cell proliferation and cell survival. Its deregulation has been implicated in many types of human malignancies. We investigated the role of PI3K/AKT signaling pathway in human malignant pleural mesothelioma (MM). Here, we report that aberrant activation of the PI3K/AKT signaling pathway is associated with cell cycle progression in MM cells. Inhibition of the PI3K activity by its small molecule inhibitor LY294002 led to significant G1 cell cycle arrest and suppression of cell proliferation in all MM cell lines that we examined. In addition, we found that the protein level of p27Kip1 was up-regulated and the protein level of cyclin D1 was down-regulated following LY294002 treatment in those MM cell lines. However, no noticeable apoptosis induction was observed following 24 h of LY294002 treatment in those MM cell lines. These results confirm that the PI3K/AKT signaling pathway is aberrantly active and plays a critical role for the cell cycle progression in human MM cells.     

贝母素乙通过调控PI3K/Akt信号通路诱导乳腺癌MCF-7细胞凋亡

目的：探究贝母素乙（Peiminine）对乳腺癌细胞MCF-7细胞凋亡的影响及其可能作用机制。方法：采用不同浓度贝母素乙或联合PI3K抑制剂LY294002干预MCF-7细胞,MTT法检测细胞增殖能力;Hoechst33258染色和Annexin V-FITC/PI流式细胞术检测细胞凋亡情况;JC-1染色法检测细胞线粒体膜电位变化;Western blotting检测细胞中PI3K(p110α)、Akt、p-Akt(ser473)、Bad、Bax、Bcl-2、cleaved-Caspase-3以及线粒体和胞浆中细胞色素C(Cyt C)等蛋白表达水平。结果：贝母素乙可呈时间-浓度依赖性抑制MCF-7细胞增殖,诱导细胞出现凋亡形态改变,促进细胞凋亡,并降低线粒体膜电位,上调细胞中Bad、Bax、cleaved-Caspase-3及胞浆中Cyt C蛋白表达水平,下调PI3K(p110α)、p-Akt、Bcl-2和线粒体中Cyt C蛋白表达水平,而Akt蛋白表达水平无显著变化。然而,联合LY294002干预可增强贝母素乙对MCF-7细胞凋亡的促进作用。结论：贝母素乙可诱导乳腺癌MCF-7细胞凋...     

Transforming growth factor beta induces apoptosis through repressing the phosphoinositide 3-kinase/AKT/survivin pathway in colon cancer cells

FET cells, derived from an early-stage colon carcinoma, are nontumorigenic in athymic mice. Stable transfection of a dominant-negative transforming growth factor beta (TGFbeta) type II receptor (DNRII) into FET cells that express autocrine TGFbeta shows loss of TGFbeta signaling and increased tumorigenicity in vivo indicating tumor suppressor activity of TGFbeta signaling in this model. The ability of tumorigenic cells to withstand growth factor and nutrient deprivation stress (GFDS) is widely regarded as a key attribute for tumor formation and progression. We hypothesized that increased tumorigenicity of FET/DNRII cells was due to loss of participation of autocrine TGFbeta in a "fail-safe" mechanism to generate cell death in response to this stress. Here, we document that loss of autocrine TGFbeta in FET/DNRII cells resulted in greater endogenous cell survival in response to GFDS due to activation of the phosphoinositide 3-kinase (PI3K)/Akt/survivin pathway. Treatment of FET DNRII cells with a PI3K inhibitor (LY294002) inhibited Akt phosphorylation and reduced survivin expression resulting in increased apoptosis in FET/DNRII cells. We also show that exogenous TGFbeta increased apoptosis in FET cells through repression of the PI3K/Akt/survivin pathway during GFDS. These results indicate that the PI3K/Akt/survivin pathway is blocked by TGFbeta signaling and that loss of autocrine TGFbeta leads to increased cell survival during GFDS through the novel linkage of TGFbeta-mediated repression of survivin expression. Inhibition of survivin function by dominant-negative approaches showed that this inhibitor of apoptosis family member is critical to cell survival in the FET/DNRII cells, thus indicating the importance of this target for TGFbeta-mediated apoptosis.     

PFKFB3通过调控PI3K/AKT信号通路促进骨肉瘤细胞的增殖及转移

目的:探讨6-磷酸果糖2-激酶/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3,PFKFB3)对骨肉瘤细胞增殖、凋亡、迁移及侵袭的影响,及可能的作用机制.方法:采用实时荧光定量PCR法、蛋白质印迹法及免疫组织化学法检测骨肉瘤组织及其相...     

Ultraviolet irradiation activates PI 3-kinase/AKT survival pathway via EGF receptors in human skin in vivo

Growth factors interact with their cell surface receptors and activate the enzyme PI 3-kinase (PI 3-K) resulting in the formation of 3-phosphorylated phosphatidylinositols, which in turn activate the serine/threonine kinase AKT/PKB. AKT functions, in part, to promote cell survival by phosphorylating the BCL-2 family member BAD and the cell death pathway enzyme, caspase-9. Although induction of apoptosis by ultraviolet (UV) irradiation is well documented, little is known about UV activation of cell survival pathways in human skin cells. We have investigated whether UV activates the PI 3-K/AKT pathway in human skin in vivo. UV irradiation (2MED from UVB source) stimulated PI 3-kinase activity within 15 min. PI 3-K activity was maximal (2.5-fold, n=6) 30 min post UV and remained elevated for 4 h. UV stimulated AKT activity within 30 min. Maximal activity (4-fold, n=11) was observed 1 h post UV. UV also stimulated phosphorylation of the downstream AKT effectors, S6 kinase and BAD. S6 kinase was maximally stimulated 4 h post UV (15-fold, n=6). Increased BAD phosphorylation was observed 1 h post UV and remained elevated for 4 h. Western blot analysis revealed that UV-induced phosphorylation of BAD at Ser112, a site known to be phosphorylated by AKT. Inhibitors of EGFR and PI 3-kinase blocked UV-induced phosphorylation of BAD, suggesting that EGFR mediates UV-activated cell survival pathway. Collectively, both positive and negative roles for UV activation of the PI 3-K/AKT pathway in human skin can be envisioned. The PI 3-K/AKT pathway likely plays a critical role in balancing UV-induced apoptotic signals, thereby preventing widespread skin cell death. Conversely UV activation of the PI 3-K/AKT pathway may enhance survival of mutated cells, thereby promoting skin cancer, as has been found in several other types of cancer.     

TRPM7通过PI3K/AKT/ERK信号途径影响脑胶质瘤细胞增殖、上皮-间质转化

目的 探讨沉默M型顺时受体电位通道7(melastatin transient receptor potential channel 7,TRPM7)对脑胶质瘤细胞增殖、上皮-间质转化的影响及其作用机制.方法 采用qRT-PCR和Western blot检测TRPM7在人正常星形胶质HA1800细胞株以及脑胶质瘤细胞株...     

Downregulation of HER2 by RIG1 involves the PI3K/Akt pathway in ovarian cancer cells

Interferon-gamma (IFN-gamma) is known to downregulate HER2 oncoprotein (p185(HER2) or briefly p185) in prostate cancer cells. We demonstrate that the IFN-gamma-induced retinoid-inducible gene 1 (RIG1) acts as a transrepressor of p185. Furthermore, we exhibit that RIG1 downregulates the activated (phosphorylated) form of p185 and phosphoinositide-3 kinase (PI3K)/serine/threonine-specific protein kinase (Akt) and the mammalian target of rapamycin (mTOR), downstream substrates of HER2. We also elucidate that heregulin (HRG) specifically restores the activation of p185 and Akt after their activities are reduced by RIG1. Additionally, expression of vascular endothelial growth factor (VEGF) increases through the HER2- and Akt/mTOR-signaling pathways, indicating that VEGF is downregulated by RIG1 within the cell. These findings suggest that RIG1 plays a role in IFN-gamma-mediated therapy by downregulating p185 and its downstream PI3K/Akt/mTOR/VEGF-signaling pathway. These results may provide a new therapeutic mechanism for the clinical use of IFN-gamma and RIG1.     

Suppression of esophageal tumor growth and chemoresistance by directly targeting the PI3K/AKT pathway

Esophageal cancer is the sixth most common cause of cancer-related deaths worldwide. Novel therapeutic intervention is urgently needed for this deadly disease. The functional role of PI3K/AKT pathway in esophageal cancer is little known. In this study, our results from 49 pairs of human esophageal tumor and normal specimens demonstrated that AKT was constitutively active in the majority (75.5%) of esophageal tumors compared with corresponding normal tissues. Inhibition of the PI3K/AKT pathway with specific inhibitors, wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, significantly reduced Bcl-xL expression, induced caspase-3-dependent apoptosis, and repressed cell proliferation and tumor growth in vitro and in vivo without obvious toxic effects. Moreover, significantly higher expression level of p-AKT was observed in fluorouracil (5-FU)-resistant esophageal cancer cells. Inactivation of PI3K/AKT pathway markedly increased the sensitivity and even reversed acquired resistance of esophageal cancer cells to chemotherapeutic drugs in vitro. More importantly, the resistance of tumor xenografts derived from esophageal cancer cells with acquired 5-FU resistance to chemotherapeutic drugs was significantly abrogated by wortmannin treatment in animals. In summary, our data support PI3K/AKT as a valid therapeutic target and strongly suggest that PI3K/AKT inhibitors used in conjunction with conventional chemotherapy may be a potentially useful therapeutic strategy in treating esophageal cancer patients.     

PI3K/AKT/mTOR pathway inhibitors inhibit the growth of melanoma cells with mTOR H2189Y mutations in vitro

mTOR is an important therapeutic target in many types of cancers. In melanoma, the mTOR nonsynonymous mutation rate is up to 10.4%. However, mTOR inhibitors have shown limited effects in clinical trials of melanoma. Because mTOR mutations are distributed, not selecting patients with specific mTOR mutations may be the main reason for therapeutic failures. Our previous research found that mutations in the mTOR P2213S and S2215Y kinase domains resulted in gain-of-function and were sensitive to specific inhibitors. The purpose of this study was to test the effect of heterozygous/homozygous H2189Y mutations on downstream pathways and sensitivity to inhibitors. mTOR kinase activity was analyzed by western blot and a K-LISA? mTOR activity kit. The sensitivity of melanoma cells to inhibitors was tested by a proliferation assay. The expression of downstream pathway proteins was also analyzed by western blot. The results showed that heterozygous/homozygous H2189Y mutations were gain-of-function. The heterozygous H2189Y mutation was sensitive to the AKT inhibitor, AZD5363, and the phosphoinositide 3-kinase inhibitor, LY294002. The homozygous H2189Y mutation was sensitive to the mTOR inhibitor, everolimus, and the AKT inhibitors AZD5363 and MK-2206 2HCL,and the phosphoinositide 3-kinase inhibitor, LY294002. These results indicated that homozygous mutations in the kinase domain have a greater effect on protein function than heterozygous mutations. The mTOR kinase domain may play an important role in mTOR kinase activity and may be the target of selective inhibitors. Our study can facilitate the selection of appropriate inhibitors for patients in clinical trials.     

The phosphoinositide 3-kinase/Akt pathway is essential for the retinoic acid-induced differentiation of F9 cells

Retinoic acid (RA) induces cell growth arrest and differentiation through two families of nuclear receptors, the RARs and the RXRs. The phosphoinositide 3-kinase (PI3K)/Akt pathway also plays key roles in these processes, that is, cell cycle progression, cell differentiation and cell survival. We report that, in mouse embryocarcinoma cells (F9 cells), RA induces an early activation of PI3K and Akt via an increase in the expression of the p85alpha regulatory subunit. This effect is followed by an inhibition of Akt. Both effects require the integrity of the RA pathway as they are not observed in RA-resistant RARgamma null cells. We propose a model through which RA induces a biphasic regulation of Akt with an activation participating to the differentiation process, followed by an inhibition, which has been correlated to the RA-induced growth arrest.     

KIT oncogenic signaling mechanisms in imatinib-resistant gastrointestinal stromal tumor: PI3-kinase/AKT is a crucial survival pathway

Most gastrointestinal stromal tumor (GIST) patients respond to KIT inhibition with imatinib, yet will eventually exhibit resistance. Imatinib-resistance mechanisms are heterogeneous, and little is known about KIT functional roles in imatinib-resistant GIST. Biological consequences of biochemical inhibition of KIT, phosphatidyl-inositol-3-kinase (PI3-K), PLCgamma, MAPK/ERK kinase/mitogen-activated protein kinase (MEK/MAPK), mammalian target of rapamycin (mTOR) and JAK were determined by immunoblotting for protein activation, and by cell proliferation and apoptosis assays in GIST cell lines from imatinib-sensitive GIST (GIST882), imatinib-resistant GISTs (GIST430 and GIST48) and KIT-negative GIST (GIST62). KIT activation was 3- to 6-fold higher in GIST430 and GIST48 than in GIST882, whereas total KIT expression was comparable in these three GIST lines. In addition to the higher set point for KIT activation, GIST430 and GIST48 had intrinsic imatinib resistance. After treatment with 1 muM imatinib, residual KIT activation was 6- and 2.8-fold higher in GIST430 and GIST48, respectively, compared to GIST882. In all GIST lines, cell growth arrest resulted from PI3-K inhibition, and - to a lesser extent - from MEK/MAPK and mTOR inhibition. Inhibition of JAK/STAT or PLCgamma did not affect cell proliferation. Similarly, only PI3-K inhibition resulted in substantial apoptosis in the imatinib-resistant GISTs. We conclude that GIST secondary KIT mutations can be associated with KIT hyperactivation and imatinib resistance. Targeting critical downstream signaling proteins, such as PI3-K, is a promising therapeutic strategy in imatinib-resistant GISTs.