Akt和胞外信号调节激酶通路间的信号交流对内质网应激条件下肝癌细胞周期的调控

目的 研究内质网应激介导的磷脂酰肌醇3激酶(PI3K)/Akt和丝裂原活化蛋白激酶(MEK)/胞外信号调节激酶(ERK)途径间的信号交流及其对内质网应激条件下肝癌细胞周期的调控作用.方法 采用PI3K抑制剂LY294002、Akt激活型突变载体myr-Akt和MEK抑制剂U0126分别阻断或激活内质网应激介导的Akt和ERK活化,并利用Western blot和流式细胞技术分析内质网应激条件下PI3K/Akt和MEK/ERK途径间的信号交流及其对肝癌细胞株SMMC-7721、Hep3B和HepG2细胞周期的调控作用.数据处理采用Sperman等级相关分析,P＜0.05为差异有统计学意义.结果 阻断PI3K/Akt明显促进内质网应激介导的MEK/ERK活化,而过度激活PI3K/Akt则抑制内质网应激介导的MEK/ERK活化.阻断MEK/ERK对内质网应激介导的PI3K/Akt活化无影响.持续活化的Akt突变载体myr-Akt和MEK抑制剂U0126均明显抑制了内质网应激诱导的压力细胞G0/G1期阻滞.结论 PI3K/Akt和MEK/ERK信号途径在内质网应激肝癌细胞中存在信号交流,该信号交流对细胞周期起重要调控作用.     

Stimulation of insulin-like growth factor (IGF) binding protein-3 synthesis by IGF-I and transforming growth factor-alpha is mediated by both phosphatidylinositol-3 kinase and mitogen-activated protein kinase pathways in mammary epithelial cells

IGF binding protein (IGFBP)-3 is an important regulator of mammary epithelial cell (MEC) growth and can enhance the ability of both IGF-I and epidermal growth factor ligands such as TGFalpha to stimulate MEC proliferation. Here we investigate the role of the phosphatidylinositol-3 kinase (PI3K) and MAPK pathways in the regulation of IGFBP-3 expression by IGF-I and TGFalpha in bovine MECs. Both growth factors stimulated DNA synthesis, although IGF-I was the stronger mitogen. IGF-I and TGFalpha also stimulated IGFBP-3 mRNA and protein levels. TGFalpha stimulated rapid, transient activation of Akt that was maximal at 5 min and diminished by 15 min. In contrast, IGF-I-induced Akt activation was maximal between 15 and 90 min and was sustained for 6 h. Although ERK 1/2 was maximally stimulated by TGFalpha between 5 and 15 min, IGF-I did not stimulate discernible activation of ERK 1/2. In addition, TGFalpha but not IGF-I induced rapid phosphorylation of Shc, whereas only IGF-I activated insulin receptor substrate-1. Pretreatment with the PI3K inhibitor LY294002 or knockdown of p85 with small interfering RNA inhibited IGF-I or TGFalpha-stimulated IGFBP-3 expression. Similarly, MAPK kinase-1 inhibitors PD98059 and U0126 each abolished TGFalpha-stimulated increases in IGFBP-3 mRNA levels. In contrast to TGFalpha, IGF-I retained the ability to partially increase IGFBP-3 mRNA levels in the presence of MAPK kinase-1 inhibitors, indicating that IGF-I may activate alternative substrates of the PI3K pathway that are involved in IGFBP-3 regulation. In conclusion, stimulation of IGFBP-3 mRNA levels by mitogens is regulated through both the PI3K and MAPK pathways in bovine MECs.     

IL-2- and IL-15-induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes

We examined functional status, activation mechanisms, and biologic role of the mTORC1 signaling pathway in malignant CD4(+) T cells derived from the cutaneous T-cell lymphoma (CTCL). Whereas the spontaneously growing CTCL-derived cell lines displayed persistent activation of the TORC1 as well as the PI3K/Akt and MEK/ERK pathways, the IL-2-dependent cell lines activated the pathways in response to IL-2 and IL-15 but not IL-21. Activation of mTORC1 and MEK/ERK was nutrient dependent. The mTORC1, PI3K/Akt, and MEK/ERK pathways could also be activated by IL-2 in the primary leukemic, mitogen-preactivated CTCL cells. mTORC1 activation was also detected in the CTCL tissues in the lymphoma stage-dependent manner with the highest percentage of positive cells present in the cases with a large cell transformation. Rapamycin inhibited mTORC1 signaling and suppressed CTCL cell proliferation but showed little effect on their apoptotic rate when used as a single agent. Activation of the mTORC1, PI3K/Akt, and MEK/ERK pathways was strictly dependent on the Jak3 and Jak1 kinases. Finally, mTORC1 activation was transduced preferentially through the PI3K/Akt pathway. These findings document the selective gammac-signaling cytokine-mediated activation of the mTORC1 pathway in the CTCL cells and suggest that the pathway represents a therapeutic target in CTCL and, possibly, other T-cell lymphomas.     

Role of Akt in growth and survival of PANC-1 pancreatic cancer cells.

INTRODUCTION: Akt is involved in different cellular processes such as cell growth, cell differentiation, and anti-apoptosis. AIMS: To investigate the role of Akt in cell growth and survival in PANC-1 pancreatic cancer cells. METHODOLOGY AND RESULTS: Insulin-like growth factor (IGF)-I induced Akt activation in a dose-dependent manner and stimulated anchorage-dependent and anchorage-independent cell growth of PANC-1 cells. In PANC-1 cells infected with adenovirus vectors carrying kinase-deficient Akt, anchorage-dependent and anchorage-independent cell growth was significantly reduced in the presence or absence of IGF-I compared with cells infected with adenovirus vectors carrying wild-type Akt, although IGF-I significantly stimulated cell growth in both transfected cell lines. Conversely, in PANC-1 cells infected with adenovirus vectors carrying kinase-deficient Akt, typical DNA laddering was undetectable in DNA fragmentation assay, and DNA 3;-OH reactivity was not detected in TUNEL assay. We then examined the role of phosphatidylinositol 3-kinase (PI3-K), an upstream mediator of Akt, on cell survival. In PANC-1 cells infected with adenovirus vector carrying a deletion mutant of the 85-kDa regulatory subunit of PI3-K and in cells treated with PI3-K inhibitor wortmannin, typical DNA laddering was evident in DNA fragmentation assay. In TUNEL assay, nuclear condensation and DNA 3;-OH reactivity was observed in approximately 30% of these cells. CONCLUSION: The present results indicate that Akt is implicated in cell growth, but not in survival in PANC-1 cells. These results suggest that there may be an alternative survival signal cascade from PI3-K in PANC-1 cells.     

Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation

Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 approximately 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 microM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.     

PI3K-FRAP/mTOR pathway is critical for hepatocyte proliferation whereas MEK/ERK supports both proliferation and survival

Growth factors are known to favor both proliferation and survival of hepatocytes. In this work, we investigated the role of 2 main signaling pathways, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK), in these processes. First, evidence was provided that the PI3K cascade as well as the MEK/ERK cascade is a key transduction pathway controlling hepatocyte proliferation, as ascertained by arrest of DNA synthesis in the presence of LY294002, a specific PI3K inhibitor. Inhibition of FRAP/mTOR by rapamycin also abrogated DNA replication and protein synthesis induced by growth factor. We showed that expression of cyclin D1 at messenger RNA (mRNA) and protein levels was regulated by this pathway. We highlighted that 4E-BP1 phosphorylation was not activated by epidermal growth factor (EGF) but was under an insulin-regulation mechanism through a PI3K-FRAP/mTOR activation that could account for the permissive role of insulin on hepatocyte proliferation. No interference between the MEK/ERK pathway and 4E-BP1 phosphorylation was detected, whereas p70S6K phosphorylation induced by EGF was under a U0126-sensitive regulation. Last, we established that the antiapoptotic function of EGF was dependent on MEK, whereas LY294002 and rapamycin had no direct effect on cell survival. Taken together, these data highlight the regulation and the role of 2 pathways that mediate growth-related response by acting onto distinct steps. In conclusion, hepatocyte progression in late G1 phase induced by EGF generates survival signals depending on MEK activation, whereas PI3K and MEK/ERK cascades are both necessary for hepatocyte replication.     

Overexpression of insulin-like growth factor binding protein-5 helps accelerate progression to androgen-independence in the human prostate LNCaP tumor model through activation of phosphatidylinositol 3'-kinase pathway

Although insulin-like growth factor (IGF) binding protein-5 (IGFBP-5) is highly up-regulated in normal and malignant prostate tissues after androgen withdrawal, its functional role in castration-induced apoptosis and androgen-independent progression remains undefined. To analyze the functional significance of IGFBP-5 overexpression in IGF-I-mediated mitogenesis and progression to androgen-independence, IGFBP-5-overexpressing human androgen-dependent LNCaP prostate cancer cells were generated by stable transfection. The growth rates of IGFBP-5-transfected LNCaP cells were significantly faster, compared with either the parental or vector-only transfected LNCaP cells in both the presence and absence ofdihydrotestosterone. IGFBP-5-induced increases in LNCaP cell proliferation occurs through both IGF-I-dependent and -independent pathways, with corresponding increases in the cyclin D1 messenger RNA expression and the fraction of cells in S + G2/M phases of the cell cycle. Changes in Akt/protein kinase B, a downstream component of phosphatidylinositol 3'-kinase (PI3K) pathway, in the LNCaP sublines also paralleled changes in their growth rates. Although treatment with a PI3K inhibitor induced apoptosis in both control and IGFBP-5-overexpressing LNCaP cells, this PI3K inhibitor-induced apoptosis was prevented by exogenous IGF-I treatment only in IGFBP-5 transfectants, suggesting that IGFBP-5 overexpression can potentiate the antiapoptotic effects of IGF-I. Furthermore, tumor growth and serum prostate-specific antigen levels increased several fold faster in mice bearing IGFBP-5-transfected LNCaP tumors after castration, despite having similar tumor incidence and tumor growth rates with controls when grown in intact mice before castration. Collectively, these data suggest that IGFBP-5 overexpression in prostate cancer cells after castration is an adaptive cell survival mechanism that helps potentiate the antiapoptotic and mitogenic effects of IGF-I, thereby accelerating progression to androgen independence through activation of the PI3K-Akt/ protein kinase B signaling pathway.     

HB-EGF enhances restitution after intestinal ischemia/reperfusion via PI3K/Akt and MEK/ERK1/2 activation

BACKGROUND & AIMS: Early recovery of intestinal function after injury occurs by restitution, a complex process with a poorly understood molecular basis. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent chemotactic factor that is induced during ischemia/reperfusion in vivo and intestinal wounding in vitro. The role of HB-EGF in intestinal restitution and the underlying intracellular signaling pathways involved were investigated. METHODS: Adult rats were subjected to intestinal ischemia, with histologic and biochemical damage assessed during the first 3 hours of reperfusion. The effect of recombinant HB-EGF (rHB-EGF) on structural and functional recovery of the intestine by restitution was evaluated in vivo. Scrape wounding of intestinal epithelial cell monolayers was used to elucidate the mechanisms of intrinsic and rHB-EGF-induced restitution. RESULTS: Early structural recovery occurred within 3 hours of reperfusion and was attributed to restitution rather than proliferation. HB-EGF treatment significantly improved structural recovery and accelerated functional recovery of the gut barrier. In vivo restitution was preceded by activation of Akt and extracellular signal-regulated kinase (ERK) 1/2, which were accelerated and enhanced by HB-EGF treatment. Blocking of ErbB-1, phosphatidylinositol 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase/ERK kinase (MEK)/ERK activity resulted in significant reduction in intrinsic and HB-EGF-induced restitution in vitro. Endogenous HB-EGF was shown to play an essential role in wound-induced ErbB-1 and ERK1/2 activation and in intrinsic restitution. CONCLUSIONS: Endogenous HB-EGF, ErbB-1, PI3K/Akt, and MEK/ERK are involved in intrinsic restitution. rHB-EGF enhances restitution in vivo and in vitro in a PI3K/Akt- and MEK/ERK1/2-dependent fashion.     

Akt is a major downstream target of PI3-kinase involved in angiotensin II-induced proliferation

Different signal transduction cascades have been implicated in angiotensin II (Ang II)-mediated cell growth, such as the extracellular signal-regulated kinase 1/2 (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K) pathways. To identify the downstream targets of PI3K involved in Ang II-induced proliferation, we used both rat aortic smooth muscle (RASM) cells and a CHO cell line stably expressing the rat AT1A receptor. The ERK1/2 and PI3K pathways are independently activated and implicated in Ang II-mediated DNA synthesis and cell number increase in these 2 cell lines. In addition, a specific inhibitor of Akt inhibited Ang II-induced Akt phosphorylation, DNA synthesis and proliferation in CHO-AT1A or RASM cells. A dominant-negative mutant of Akt was also found to selectively block Ang II-induced proliferation of CHO-AT1A cells. To further elucidate the signaling events leading to Akt activation, we used an AT1 receptor mutant (AT1AD74E), deficient for Gq protein coupling, and the intracellular calcium chelator BAPTA-AM. Although altered Akt and ERK1/2 activation was observed in the CHO-AT1AD74E cell line, blockade of intracellular calcium elevation did not affect phosphorylation of these kinases. These results provide the first evidence of a specific and necessary role of Akt in Ang II-induced proliferation through a Gq protein-dependent calcium-independent pathway.     

The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells

Sustained engagement of the B-cell receptor (BCR) increases apoptosis resistance in chronic lymphocytic leukemia (CLL) B cells, whereas transient stimulation usually has an opposite effect. The antiapoptotic BCR signal has been associated with prolonged activation of the PI3K/Akt and MEK/ERK pathways, which are key regulators of survival and proliferation in various cell types. To further define the relative contribution of the Akt and ERK kinases in regulating CLL B-cell survival, we introduced constitutively active mutants of Akt and MEK in primary CLL B cells and evaluated changes in the expression of relevant pro- and antiapoptotic proteins. Sustained activation of Akt resulted in increased leukemic cell viability and increased expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and X-linked inhibitor of apoptosis protein (XIAP), thus largely recapitulating the effects of sustained BCR stimulation. Constitutively active MEK2 also up-regulated XIAP, but did not show a significant impact on leukemic cell survival. Down-regulation of Mcl-1 by siRNA treatment induced rapid and potent apoptosis in CLL B cells and blocked the antiapoptotic effect of sustained BCR stimulation, whereas down-regulation of Bcl-xL and XIAP did not affect leukemic cell viability. These data demonstrate that Akt and Mcl-1 are major components of a survival pathway that can be activated in CLL B cells by antigen stimulation.     

Aging is associated with decreased pancreatic acinar cell regeneration and phosphatidylinositol 3-kinase/Akt activation

BACKGROUND & AIMS: The effects of aging on pancreatic acinar cell proliferation have not been clearly defined. Phosphatidylinositol 3-kinase (PI3K)-mediated phosphorylation of Akt is a critical step for proliferation of various cell types and insulin secretion from pancreatic endocrine cells; however, its role in acinar cell proliferation is not known. The purpose of this study was to (1) delineate the effects of aging on pancreatic regeneration after partial pancreatectomy (Px) and (2) define the involvement of the PI3K/Akt pathway in pancreatic regeneration. METHODS: Following partial Px, pancreatic regeneration and activation of the PI3K pathway were compared in young and aged mice. Activation of the PI3K/Akt pathway was evaluated by Akt phosphorylation (pAkt). The role of the PI3K pathway in pancreatic regeneration after partial Px was assessed by effects of a pharmacologic PI3K inhibitor wortmannin or small interfering RNA (siRNA) to the p85alpha regulatory subunit. To confirm further the critical role of the PI3K/Akt pathway in pancreatic acinar cell proliferation, IGF-1-mediated cell proliferation was determined in cultured acinar cells pretreated with wortmannin or p85alpha siRNA. RESULTS: Pancreatic regeneration and pAkt expression after partial Px were significantly decreased with aging. Treatment with wortmannin or p85alpha siRNA reduced pancreatic regeneration after partial Px. The IGF-1-mediated cell proliferation in vitro was completely blocked by wortmannin or p85alpha siRNA but not by the MEK/ERK inhibitor PD98059. CONCLUSIONS: PI3K/Akt activation plays a critical role in the regeneration of pancreatic acini after resection. Furthermore, pancreatic regeneration is markedly attenuated in the aged pancreas most likely because of decreased PI3K/Akt activation.     

Insights into erlotinib action in pancreatic cancer cells using a combined experimental and mathematical approach

AIM:To gain insights into the molecular action of erlotinib in pancreatic cancer (PC) cells. METHODS:Two PC cell lines, BxPC-3 and Capan-1, were treated with various concentrations of erlotinib, the specific mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and protein kinase B (AKT) inhibitor XIV. DNA synthesis was measured by 5-bromo-2'-deoxyuridine (BrdU) assays. Expression and phosphorylation of the epidermal growth factor receptor (EGFR) and downstream signaling molecules were quantified by Western blot analysis. The data were processed to calibrate a mathematical model, based on ordinary differential equations, describing the EGFRmediated signal transduction. RESULTS:Erlotinib significantly inhibited BrdU incorporation in BxPC-3 cells at a concentration of 1 mol/L, whereas Capan-1 cells were much more resistant. In both cell lines, MEK inhibitor U0126 and erlotinib attenuated DNA synthesis in a cumulative manner, whereas the AKT pathway-specific inhibitor did not enhance the effects of erlotinib. While basal phosphorylation of EGFR and extracellular signal-regulated kinase (ERK) did not differ much between the two cell lines, BxPC-3 cells displayed a more than five-times higher basal phospho-AKT level than Capan-1 cells. Epidermal growth factor (EGF) at 10 ng/mL induced the phosphorylation of EGFR, AKT and ERK in both cell lines with similar kinetics. In BxPC-3 cells, higher levels of phospho-AKT and phospho-ERK (normalized to the total protein levels) were observed. Independent of the cell line, erlotinib efficiently inhibited phosphorylation of EGFR, AKT and ERK. The mathematical model successfully simulated the experimental findings and provided predictions regarding phosphoprotein levels that could be verified experimentally. CONCLUSION:Our data suggest basal AKT phosphorylation and the degree of EGF-induced activation of AKT and ERK as molecular determinants of erlotinib efficiency in PC cells.     

Growth factor-induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle cells: induction of cell proliferation and inhibition of cell death

OBJECTIVE: The signaling pathways mediating proliferation and apoptosis in vascular smooth muscle cells (VSMC) are not well established. It has previously been shown that activation of the phosphoinositide 3-OH kinase (PI3K)/Akt pathway or the ERK 1/2 pathway can mediate anti-apoptotic function in different cell types. This study determined the specific contribution of the PI3K/Akt and ERK pathway in the regulation of apoptosis and proliferation of VSMC. METHODS AND RESULTS: Incubation of rat VSMC with FCS, insulin or IGF-1 time-dependently stimulated the phosphorylation of Akt, however FCS but not insulin or IGF-1 activated the MAP-kinase ERK 1/2. Moreover, insulin inhibited H(2)O(2)-induced apoptosis via the Akt pathway as demonstrated by pharmacological inhibition of the PI3K or overexpression of a dominant negative Akt mutant. In contrast, FCS inhibited H(2)O(2)-induced apoptosis via the Akt and also the ERK pathway. FCS, but not insulin or IGF-1 induced VSMC proliferation, suggesting that Akt activation is necessary but not sufficient for VSMC proliferation. FCS-induced proliferation of VSMC was only mediated via the Akt pathway and not the ERK pathway. CONCLUSIONS: These results define a link between cell proliferation and programmed cell death in VSMC via the same signal transduction pathway, namely activation of the serine/threonine kinase Akt, which may have significant implication for the development of vascular diseases or remodeling.     

Phosphatidylinositol 3-kinase mediates proliferative signals in intestinal epithelial cells

BACKGROUND AND AIMS: Determination of intracellular signalling pathways that mediate intestinal epithelial proliferation is fundamental to the understanding of the integrity and function of the intestinal tract under normal and diseased conditions. The phosphoinositide 3-kinase (PI3K)/Akt pathway transduces signals initiated by growth factors and is involved in cell proliferation and differentiation. In this study, we assessed the role of PI3K/Akt in transduction of proliferative signals in intestinal epithelial cells. METHODS: A rat intestinal epithelial (RIE) cell line and human colorectal cancer HCA-7 and LS-174 cell lines served as in vitro models. The Balb/cJ mouse was the in vivo model. RESULTS: PI3K activation was critical for G1 cell cycle progression of intestinal epithelial cells. Ectopic expression of either active p110alpha or Akt-1 increased RIE cell proliferation. In vivo experiments demonstrated that PI3K activation was closely associated with the proliferative activity of intestinal mucosa. Treatment of mice with PI3K inhibitors blocked induction of PI3K activity and attenuated intestinal mucosal proliferation associated with oral intake. Epidermal growth factor and transforming growth factor alpha stimulated PI3K activation which was required for growth factor induced expression of cyclin D1. CONCLUSIONS: The PI3K/Akt pathway transduces mitogenic signals from growth factor receptors to the cell cycle machinery and plays a critical role in regulation of intestinal epithelial proliferation.     

Grb2-associated binder-1 plays a central role in the hepatocyte growth factor enhancement of hepatoma growth inhibition by K vitamin analog compound 5

Compound 5 (Cpd 5), a K vitamin analog, has been shown to inhibit Hep3B human hepatoma cell growth in cultures and rat hepatoma growth in vivo through prolonged epidermal growth factor receptor (EGFR)-extracellular response kinase (ERK) phosphorylation, and hepatocyte growth factor (HGF) synergizes with Cpd 5 to enhance the inhibition of Hep3B cell and rat hepatoma growth. To explore the mechanisms mediating the HGF/Cpd 5 synergy, we examined the possible involvement of the Grb2-associated binder-1 (Gab1) docking protein because it interacts with both EGFR and HGF receptor c-Met pathways. We found that HGF enhanced Cpd 5-induced c-Met phosphorylation at Tyr-1349, a binding site for Gab1, resulting in increased c-Met binding to Gab1, and induced strong and prolonged Gab1 tyrosine phosphorylation. Prolonged Gab1 phosphorylation by HGF/Cpd 5 in turn enhanced the ability of Gab1 to bind to protein tyrosine phosphatase SHP2 and enhanced the activation of its downstream mitogen-activated protein kinase pathway. In contrast, this same HGF/Cpd 5 treatment inhibited Gab1 binding to phosphatidylinositol 3-kinase (PI3K), leading to the inactivation of the PI3K-Akt pathway. The inhibition of Akt phosphorylation by HGF/Cpd 5 further activated the Raf-MEK-ERK signaling cascade via an Akt-Raf1 interaction, leading to strong and prolonged ERK phosphorylation. The transfection of Hep3B cells with mutated Gab1 (Gab1 Y627F), which had lost its ability to bind SHP2, antagonized HGF/Cpd 5-induced ERK phosphorylation, whereas the transfection of Hep3B cells with mutated Gab1 3YF, which lost its ability to bind PI3K, further enhanced HGF/Cpd 5-induced ERK phosphorylation and cell growth inhibition. CONCLUSION: Gab1 plays a central role in regulating HGF/Cpd 5 synergy in their actions on Hep3B cell growth inhibition.     

Role of the phosphatidylinositol 3'-kinase-Akt signal pathway in the proliferation of human pancreatic ductal carcinoma cell lines

: Phosphatidylinositol 3'-kinase (PI3K) and Akt mediate survival signals and allow the cells to escape apoptosis in various human cancers. We postulated that LY294002, a PI3K inhibitor, might inactivate Akt, consequently inhibiting cell proliferation in 3 human pancreatic ductal carcinoma cell lines, PSN-1, PANC-1, and KP-4. LY294002 (50 micromol/L) caused a decrease in phosphorylated Akt and inhibition of cell proliferation in a time-dependent manner, but there was no obvious induction of apoptosis. Flow cytometric analysis revealed that pancreatic cancer cells treated with 50 micromol/L LY294002 underwent G1 arrest, which was associated with dephosphorylation of the ppRB protein, a decrease in the protein expression of cyclin D and E, and their activating partners Cdk2, 4, and 6 with simultaneous accumulation of P27/Kip1. Our data indicate that P27/Kip1 accumulation by Akt inactivation could induce cell cycle arrest in the G1 phase and suggest that the PI3K-Akt pathway plays an important role in cell proliferation in human pancreatic ductal carcinoma cells.