The involvement of PI 3-K/Akt-dependent up-regulation of Mcl-1 in the prevention of apoptosis of Hep3B cells by interleukin-6

Interleukin-6 (IL-6) is a pleitrophic cytokine that not only regulates growth and differentiation of many cell types, but also induces production of acute phase proteins (AAP) in hepatocytes. Our previous works have demonstrated that both PI 3-K/Akt and STAT3 pathways were concomitantly activated and cooperatively mediated the anti-apoptotic effect of IL-6. This investigation reports that IL-6 protected cells against apoptosis induced by a variety of agents including, TGF-beta, UV and retinoic acid (RA) in Hep3B cells, suggesting that IL-6 is a fundamental determinant of hepatic cell survival. Mcl-1, but not other Bcl-2 family members, was rapidly up-regulated by IL-6, with a peak (approximately 3-4-fold) appearing at 4 h. Transient transfection of cells with a mcl-1 antisense vector, resulting in a 50-60% reduction of the anti-apoptotic effect of IL-6, indicating that Mcl-1 is a downstream effector of IL-6. Which signaling pathway transduced by IL-6 responsible for the Mcl-1 up-regulation was further investigated. In Hep3B cells, the JAK/STAT3, ERK, and PI 3-K/Akt pathways were activated by IL-6 stimulation. Blocking JAK/STAT3 activation with a dominant-negative mutant STAT3F or a JAK inhibitor AG490 could not influence IL-6-mediated Mcl-1 up-regulation. Similarly, PD98059 treatment, a MEK specific inhibitor, also failed to inhibit Mcl-1 expression. However, the IL-6-induced Mcl-1 up-regulation was effectively attenuated in the presence of PI 3-K inhibitors, LY294002 and wortmannin. Expression of dominant-negative Akt, but not Etk, could abrogate the IL-6-induced increase of Mcl-1. In conclusion, our results suggest that the anti-apoptotic effect of IL-6 is mediated, at least in part, by Mcl-1 expression and that is mainly through the PI 3-K/ Akt-dependent pathway.     

Inositol pentakisphosphate promotes apoptosis through the PI 3-K/Akt pathway

Phosphoinositide 3-kinase (PI 3-K) is implicated in a wide array of biological and pathophysiological responses, including tumorigenesis, invasion and metastasis, therefore specific inhibitors of the kinase may prove useful in cancer therapy. We propose that specific inositol polyphosphates have the potential to antagonize the activation of PI 3-K pathways by competing with the binding of PtdIns(3,4,5)P3 to pleckstrin homology (PH) domains. Here we show that Ins(1,3,4,5,6)P5 inhibits the serine phosphorylation and the kinase activity of Akt/PKB. As a consequence of this inhibition, Ins(1,3,4,5,6)P5 induces apoptosis in ovarian, lung and breast cancer cells. Overexpression of constitutively active Akt protects SKBR-3 cells from Ins(1,3,4,5,6)P5-induced apoptosis. Furthermore, Ins(1,3,4,5,6)P5 enhances the proapoptotic effect of cisplatin and etoposide in ovarian and lung cancer cells, respectively. These results support a role for Ins(1,3,4,5,6)P5 as a specific inhibitor of the PI 3-K/Akt signalling pathway, that may sensitize cancer cells to the action of commonly used anticancer drugs.     

姜黄素对人结肠癌细胞PI3-K/Akt和MEK/ERK信号通路的影响

目的:探讨姜黄素对人结肠癌RKO细胞PI3-K/Akt和MEK/ERK通路的影响。方法:MTT法检测细胞活力,Western blot检测p-Akt、Akt、p-ERK,ERK及凋亡相关蛋白Bcl-2、Bax的表达。结果:姜黄素作用人结肠癌RKO细胞,24h和48h的IC50值分别为51.69μg/ml和36.12μg/ml。选用50μg/ml的姜黄素分别作用24h和48h,RKO细胞凋亡百分比为26.79%和42.16%,与对照组相比有显著差异(P＜0.05)。进一步检测发现姜黄素（50μg/ml）显著下调了p-Akt 和p-ERK的表达,同时Bcl-2与Bax的比值显著下调。结论:姜黄素可能通过抑制PI3-K/Akt和MEK/ERK信号通路的活化、下调Bcl-2与Bax的比值,从而抑制RKO细胞增殖和诱导细胞凋亡。     

IL 6经PI3K/Akt通路诱导卵巢癌细胞对他莫西芬耐药

目的:探讨IL-6诱导卵巢癌细胞对他莫西芬(tamoxifen,TAM)耐药的分子机制.方法:构建内源性过表达IL-6的人卵巢癌A2780细胞系和内源性抑制IL-6表达的人卵巢癌CAOV-3细胞,50 ng/ml外源性IL-6预处理A2780细胞(A2780/preIL-6细胞),Western blotting检测内/外源IL-6对卵巢癌细胞ERα Ser167位磷酸化水平的影响;IL-6与PI3K抑制剂Wort-mannin单独或联合作用于A2780细胞,Western blotting检测其对A2780细胞Akt磷酸化和ERα磷酸化的影响;MTT法检测Wortmannin和内/外源IL-6对A2780细胞TAM敏感性的影响;荧光素酶报告基因检测卵巢癌细胞ERα的转录活性,并分析其可能涉及的信号通路.结果:外源性及内源性过表达IL-6可明显促进A2780细胞ERα Ser167位点磷酸化水平(均P＜0.01),而内源性抑制IL-6表达则可降低CAOV-3细胞ERα Ser167位点的磷酸化水平(P＜0.01);Wortmannin可阻断IL-6诱导的A2780细胞对TAM的耐药及ERα的磷酸化(P ＜0.05);IL-6可促进细胞ERα的转录活性(P＜0.01),而Wortmannin并不能阻断IL-6对ERα的转录活性的影响(P＞0.05).结论:IL-6可经PI3 K/Akt通路引起ERα磷酸化从而活化ER信号通路,进而诱导卵巢癌细胞对TAM耐药.     

Constitutive NF-kappaB DNA-binding activity in AML is frequently mediated by a Ras/PI3-K/PKB-dependent pathway.

In the present study, we aimed to elucidate the mechanism responsible for constitutive NF-kappaB DNA-binding activity in AML cells. Intervening in aberrant signaling pathway provides a rational approach for in vivo targeting of AML cells. Constitutive NF-kappaB DNA-binding activity was observed in 16 of 22 (73%) investigated AML cases and was, in general, associated with resistance to spontaneous apoptosis. Indeed, inhibition of NF-kappaB activity by the NF-kappaB inhibitor SN-50 peptide resulted in enhanced chemotherapy-induced apoptosis. In the majority of cases, constitutive NF-kappaB activity was mediated by a Ras/PI3 kinase (PI3-K)/protein kinase B (PKB)-mediated pathway. The PI3-K inhibitor Ly294002 and the Ras inhibitor L-744832 both inhibited PKB phosphorylation and NF-kappaB DNA-binding activity. The constitutive activation of Ras GTP-ase was caused by mutations in the gene encoding for N-Ras in 29% of the cases. The constitutive NF-kappaB activity could so far not be ascribed to the autocrine production of growth factors or to mutations in the Flt3 receptor, since anti-GM-CSF, -IL-1, -IL6, -TNFalpha or the tyrosine kinase inhibitor AG1296 did not affect the NF-kappaB DNA-binding activity. The present study demonstrates that Ras activation is an important pathway for triggering the NF-kappaB pathway in AML cells.     

ICAM-3-induced cancer cell proliferation through the PI3K/Akt pathway

ICAM-3 interacts with LFA1, and is involved in the intercellular adhesion of leukocytes as well as in the mainenance of cell survival. It has also been suggested to induce cancer cell proliferation but the precise signaling pathway is unclear. The aim of this study was to determine the ICAM-3-activated downstream pathway in H1299 lung cancer cells. The level of ICAM-3-induced cell growth was examined using BrdU incorporation, which is a colony-forming assay, FACS analysis, and cell counting. The results showed that ICAM-3 expression induces cancer cell proliferation. In addition, FAK, Akt, PDK1, GSK-3beta, BAD, and PTEN were phosphorylated by ICAM-3-overexpression, resulting in enhanced cell proliferation. In conclusion, ICAM-3 expression induces cancer cell proliferation, and an increase in ICAM-3 expression can contribute to cancer progression.     

FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in acute myeloid leukemia

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4–11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway.     

SIRT3 inhibits prostate cancer by destabilizing oncoprotein c-MYC through regulation of the PI3K/Akt pathway

SIRT3 is involved in aging-related diseases including cancer, but its role in prostate cancer and detailed regulatory function are not known. We found that SIRT3 was moderately down-regulated in prostate carcinomas. Overexpression of SIRT3 by lentiviral transfection inhibited prostate cancer growth both in vitro and in vivo, whereas knockdown of SIRT3 increased prostate tumor growth. Mechanistically, the tumor suppression effect of SIRT3 was achieved via its inhibition of the PI3K/Akt pathway. Notably, upregulation of SIRT3 suppressed the phosphorylation of Akt, leading to the ubiquitination and degradation of oncoprotein c-MYC; this could be attenuated by constitutive activation of PI3K/Akt signaling. Collectively, our results unveiled SIRT3''s tumor suppressive function and the underlying mechanism in prostate cancer, which might provide therapeutic implications for the disease.     

JAK/STAT信号转导途径活化和Mcl-1表达上调介导IL-6在人骨髓瘤细胞系XG-7上的凋亡抑制效应

背景和目的：IL－6能够抑制多种因素诱发的骨髓瘤细胞凋亡反应,在此过程中涉及到胞浆内一系列信号蛋白分子的参与。本研究旨在确定能够介导IL-6尖人骨髓瘤细胞系XG－7上凋亡抑制效应的Bcl-2家族抗凋亡蛋白（Bcl-2,Bcl-xL,Mcl-1)和IL－6信号转导途径（JAK／STAT,Ras,MAPK,PI-3K/Akt途径）。方法：采用碘化丙腚（PI）染色和流式细胞术分析XG－7细胞的凋亡情况;采用免疫印变方法检测Bcl-2家族分子在XG－7细胞中的表达情况;分别采用针对JAK／STAT,Ras/MAPK和PI－3K／Akt途径的特异性抑制剂AG490,PD98059和LY294002处理XG－7细胞,从而确定哪条信号转导途径能够介导IL－6在XG－7细胞上的凋亡抑制效应。结果：IL－6能够抑制XG－7细胞凋亡,同时上调Bcl-2家族抗凋亡蛋白Mcl-1表达。AG490处理的XG－7细胞中Mcl-1表达上调被明显抑制;但PD98059和LY294002处理后对Mcl-1表达没有显著影响。结论：IL－6在XG－7细胞上的凋抑制效应是通过上调Mcl-1表达和激活JAK／STAT途径而介导的,与Ras/MAPK和PI-3K/Akt途径的活化状态无关。     

CUEDC2通过活化 PI3K／Akt 信号通路促进乳腺癌细胞的生长

目的：探讨 CUE 结构域2（CUE domain －containing 2,CUEDC2）蛋白在人乳腺癌细胞中的生物学作用。方法：人乳腺肿瘤细胞 MCF －7转染的 Myc －CUEDC2真核表达载体,提取转染细胞的总蛋白检测磷酸化 Akt 和总 Akt 的表达变化。利用 CCK －8检测过表达 CUEDC2后 MCF －7细胞的生长。结果：CUEDC2能够活化 Akt,使磷酸化 Akt 表达增加,能够促进人乳腺癌细胞生长。结论：当乳腺癌中 CUEDC2表达增高时,能够引起 PI3K／Akt 信号通路活化,促进肿瘤细胞生长。     

Deciphering the role of PI3K/Akt/mTOR pathway in breast cancer biology and pathogenesis

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway mediates multiple cellular functions critical to tumor initiation, progression, and outcomes, including growth and proliferation, metabolism, motility, migration, invasion, angiogenesis, survival, and autophagy. Tight regulation of this pathway is paramount to ensure that multiple cellular inputs are integrated for appropriate cellular outcomes. Frequent deregulation and aberrations of this pathway have been implicated in breast cancer development and progression. This review focuses on the biology of this pathway and its role in breast cancer pathogenesis. The role of therapies directed at targeting mTOR in the PI3K/Akt/mTOR pathway, which are currently being evaluated in clinical trials, will also be reviewed.     

二氢青蒿素通过PTEN/PI3K/Akt通路抑制胃癌细胞的增殖

  目的  探讨二氢青蒿素(dihydroartemisinin, DHA)通过PTEN/PI3K/Akt通路对人胃癌细胞株SGC7901细胞周期的影响及其分子机制。  方法  不同浓度(6.25、12.5、25、50、100μmol/L)DHA作用SGC7901细胞24、48、72h后, 细胞计数法检测SGC7901细胞增殖的情况。不同浓度DHA作用SGC7901细胞24 h后, 流式细胞术测定细胞周期的分布; RT-PCR和Western blotting分别测定cyclin D1、P27的mRNA和蛋白的表达水平; Western blotting测定PTEN、PI3K、p-Akt的表达水平。分别以PTEN特异性小干扰RNA(PTEN-siRNA)及无关序列对照siRNA(non-specific siRNA, NS-siRNA)转染细胞, 加入100μmol/L DHA, 作用SGC7901细胞24 h后, Western blotting测定cyclin D1、P27、PTEN、PI3K、p-Akt的表达水平。  结果  DHA剂量和时间依赖性抑制SGC7901细胞的增殖, 使细胞周期阻滞于G1期(P < 0.05)。RT-PCR和Western blotting分析结果显示, 100μmol/L DHA作用SGC7901细胞24 h后, cyclin D1 mRNA和蛋白表达显著下降, P27 mRNA和蛋白表达显著上升(P < 0.05)。PTEN的蛋白表达显著增加, PI3K和p-Akt的表达水平逐渐下降(P < 0.05)。敲低PTEN表达后, DHA对PI3K和p-Akt的表达水平的影响明显减弱, 与此同时, cyclin D1表达水平升高, P27表达有所下降(P < 0.05)。  结论  DHA通过抑制PTEN/PI3K/Akt信号通路的活化, 影响细胞增殖相关基因cyclin D1和P27的表达, 进而使细胞阻滞于G₀/G₁期, 抑制人胃癌SGC7901细胞的增殖。      

Modulation of PI3K/Akt pathway by E1a mediates sensitivity to cisplatin

In order to investigate the molecular mechanisms implicated in the induction of chemo sensitivity by adenovirus E1a gene expression, we decided to investigate which signal transduction pathways could be affected by the E1a gene in Human Normal Fibroblast (IMR90). No effect was observed in SAPK pathways (p38MAPK and JNK), but E1a was able to affect the Akt activation mediated by insulin. This result was confirmed by transient transfection experiments performed in Cos-7 cells and also observed in other transformed cell lines such as A431. Furthermore, E1a expression induces a decrease in the basal status of Akt activity. Finally we demonstrated that E1a is able to block the Akt activation mediated by cisplatin and correlates with a sensitive phenotype. In summary, our data demonstrate that specific inhibition of the PI3K/Akt pathway mediates some of the biological properties of E1a such as induction of chemosensitivity.     

The antiapoptotic effect of IL-6 autocrine loop in a cellular model of advanced prostate cancer is mediated by Mcl-1

Levels of the proinflammatory cytokine interleukin-6 (IL-6) are increased in therapy-resistant prostate cancer. IL-6 has been considered a positive growth factor in late-stage prostate cancer cells and a potential target for therapeutic interference. Effects of inhibition of IL-6 on cell survival were studied in LNCaP-IL6+ cells, a model system for advanced prostate cancer, which produce IL-6. We show that the autocrine IL-6 loop is responsible for resistance to apoptosis and increased cellular levels of myeloid cell leukemia-1 (Mcl-1) protein, an antiapoptotic member of the Bcl-2 family. Treatment of cells with a chimeric anti-IL-6 antibody (CNTO 328) led to the induction of apoptosis and downregulation of Mcl-1 protein levels. Specific knockdown of Mcl-1 gene expression by small interfering RNA also yielded an increase in apoptosis of LNCaP-IL-6+ cells. Vice versa, inactivation of IL-6 autocrine loop had no influence on apoptosis levels in the absence of Mcl-1, thus suggesting this molecule as a mediator of the survival action of IL-6. Mcl-1 protein regulation by the endogenous cytokine directly involved the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway. Our data support the concept of anti-IL-6 targeted therapy in therapy-resistant prostate cancer.