Protein Expression Levels Of AKT Research Articles

Proanthocyanidins from cranberry (Vaccinium macrocarpon) inhibit matrix metalloproteinase expression in prostate cancer cells by affecting multiple cell signaling pathways

This study examined the effects of a proanthocyanidin‐enriched fraction (PAC) from cranberry (Vaccinium macrocarpon) on matrix metalloproteinase (MMP) activity in DU145 human prostate cancer cells in vitro. MMP‐2/−9 activity was inhibited by PAC in a dose/time dependent manner. PAC (25 ug/mL) decreased cellular viability 30% post 6 h.of treatment and had no significant effect on cellular viability post 3 h. Treatment of DU145 cells with PAC for 3 h. resulted in increased expression of p‐p38, pERK‐1, pERK‐2 protein levels. Increased JNK1 and decreased JNK2 protein expression occurred in response to PAC with corresponding alterations in p‐JNK1 and p‐JNK2. PAC treatment decreased protein expression levels of AKT, p‐AKT, P‐I‐3 kinase p85/p110 proteins. Treatment of DU145 cells with PAC resulted in inhibition of translocation of NF‐kB as suggested by decreased protein expression of p65 in the nuclear fraction. No apparent change in IkB alpha protein levels were noted. Decreased protein levels of p‐IkB alpha were observed. PAC fraction has the ability to inhibit MMP‐2/−9 activity at a concentration which is not cytotoxic to these cells suggesting that PACs inhibit MMP activity levels in DU145 cells in a targeted manner independent of cellular cytotoxicity effects. (N.C.I.C.‐ Canadian Cancer Society, P.E.I. Health Research Program, The Cranberry Institute (Wisconsin Board) funded)

The phosphoinositide 3-kinase/Akt-signal pathway mediates proliferation and secretory function of hepatic sinusoidal endothelial cells in rats after partial hepatectomy

Objective To investigate the role of AKT signaling pathway in hepatic sinusoidal endothelial cells (SECs) early after partial hepatectomy in rats and the regulatory mechanisms involved. Methods The animal model of 70% hepatectomy was made. Hepatic SECs were isolated and cultured according to Braet et al.’s method with some modifications. The cultured hepatic SECs were divided into two groups: 70% partial hepatectomy groups and LY294002 group (LY). We observed the expressions of AKT and NF-κB in cultured hepatic SECs by Western blot, measured the levels of NO, NOs, IL-6, and HGF in the supernatants of hepatic SEC cultures and [ 3H]thymidine incorporation, and analyzed cell cycle of cultured hepatic SECs by flow cytometer. The relationship of the Akt pathway with secretions and proliferation of hepatic SECs after partial hepatectomy was probed. Results The levels of Akt protein expression increased significantly after partial hepatectomy in OG group and with a peak at 24 h post operation. Meanwhile, there was a markedly increase in phosphorylated Akt protein during 2–72 h after operation. But the expression and activity of Akt protein did not change significantly after partial hepatectomy in the LY group. So, partial hepatectomy can marked induce Akt expression and result in rapid and marked phosphorylation of Akt from 2 to 72 h thereafter. The changes of NF-κB expression in cultured hepatic SECs were similar to those of Akt expression after operation. The concentrations of HGF and IL-6 in the supernatants of cultured hepatic SECs were relatively low in the LY group, and were markedly increased after partial hepatectomy, with a peak at 24 h in the OG group. There were significant differences between the OG and LY groups at 6 and 24 h ( P < 0.05). Both NO and NOS secretion was increased in the OG group compared to the LY group within 24 h after partial hepatectomy. But the secretion of NO and NOS was increased more markedly in the LY group than that in the OG beyond 24 h. These findings suggest that the secretion of the cytokines by hepatic SECs is mediated by Akt signaling. Akt signaling pathway in relationship with proliferation of hepatic SECs and suppression of apoptosis. In OG group, the hepatic SECs in S and G2/M obviously increased. The proliferative index of hepatic SECs in OG group had significant differences with that in LY group at 6, 24, and 72 h, P < 0.05. Meanwhile, the cells of apoptosis in OG group were very low, and the cells in LY group gradually increased. Conclusions These results suggest that AKT signaling pathway plays a crucial role in mediating proliferating and secreted signals in hepatic SECs. AKT has been suggested to play a pivotal role in early liver regeneration involved in the induction of secreted cytokines and proliferation of hepatic SECs.

Combined Analysis of Molecular and Clinical Predictors of Gefitinib Activity in Advanced Non–Small Cell Lung Cancer: Epidermal Growth Factor Receptor Mutations Do Not Tell the Whole Story

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been introduced in the standard therapy of non-small-cell lung cancer (NSCLC), but they benefit a minority of patients. The study of molecular markers may identify the subset of patients who are the most appropriate to treat with these agents. We analyzed 43 patients with advanced NSCLC who were treated with gefitinib, an oral EGFR tyrosine kinase inhibitor, were included in analysis. We evaluated EGFR in tumor tissue by using immunohistochemistry and fluorescence in situ hybridization. We also studied downstream molecules (AKT, ERK, p38 MAPK) and their activation status and the presence of EGFR mutations in tumor tissue in exons 18-21. Three patients had tumors with EGFR mutations, all of which had EGFR gene amplification with a ratio of 2 or greater (p= 0.001). There was no correlation between EGFR protein expression and gene amplification. Six patients (14%) achieved an objective response and nine (21%) had stable disease; the median survival was 162 days. EGFR mutations, high levels of AKT protein expression, rash of any grade, and no history of smoking were predictive of disease control (objective response plus stable disease). Only 3 of 15 patients (20%) with disease control had an EGFR mutation. On multivariate analysis, rash and AKT were independent predictors of disease control. Patients with rash survived longer than patients without rash. EGFR mutation-positive tumors are present in a small fraction of patients who achieve disease control with gefitinib. Other molecular markers, such as AKT, need to be further evaluated. Clinical parameters remain major determinants of gefitinib activity in NSCLC.

Development of Herceptin resistance in breast cancer cells.

Herceptin, a humanized antibody to HER-2, is now utilized in the clinic for metastatic breast cancer treatment. The response rate for HER-2+ patients is only 30% and little is known as to mechanisms of resistance. The mechanism of Herceptin action is also unknown but has been related to cell cycle inhibition. The effects of Herceptin and other antibody treatments were determined by cell counting and cell cycle analysis. HER-2 and p27 expression levels were analyzed by flow cytometry and levels of activated AKT were compared by Western blot analysis. Cellular HER-2 and p27 expression was measured by immunofluorescence. Herceptin treatment of BT-474 cells results in inhibition of cell growth and arrest in the G1 phase. The efficacy of growth arrest was not directly correlated to the binding affinity of antibodies to Her-2. Our laboratory has developed cell lines that are resistant to Herceptin treatment. In resistant cell lines, binding of antibodies is not hindered. However, Herceptin has completely lost the ability to inhibit cell proliferation. Yet, the mouse isotype 4D5 maintains significant inhibitory activity upon Herceptin-resistant clones. Herceptin binds effectively to Her-2 on the cell surface of Herceptin-resistant cell lines and the level of Her-2 expression on the cell surface is not downregulated. Herceptin resistance is not due to downregulation of levels of AKT protein expression, although, phosphorylation of AKT is enhanced in resistant lines and could have a role in resistance. Resistance appears to correlate with the loss of nuclear expression of the cyclin-dependent kinase inhibitor, p27, as defined by immunofluorescence and flow cytometry studies and cdk-2 binding studies.

Pleotropic effects of genistein on MCF-7 breast cancer cells

Soy isoflavone, genistein has been shown to induce growth inhibition, cell cycle arrest and apoptosis in cultured cancer cell lines derived from head and neck, breast, lung, and prostate cancers and showed antitumor activity against tumors in multiple animal models. In the present study we show that genistein inhibits the growth of MCF-7 breast cancer cell line in a dose dependent manner. The genistein induced growth inhibition is accompanied by the reduction in the number of mitotic cells and overexpression of cyclin dependent kinase inhibitor p21WAF1 leading to cell cycle arrest. In addition, the telomeric area was significantly reduced in genistein treated MCF-7 cells. Analysis of multiple genes involving the apoptotic pathway reveals inhibition of Akt activity without affecting the steady state levels of Akt protein expression and the down regulation of proapoptotic gene BAD expression. From these results, we conclude that genistein-induced inhibition of cell division is partly mediated by decreased telomere length, reduced mitosis and inhibition of Akt activation, leading to induction of apoptosis.