Molecular mechanisms of ZD1839-induced G1-cell cycle arrest and apoptosis in human lung adenocarcinoma A549 cells

Epithelial growth factor receptor (EGFR) has been proposed as a target for anticancer therapy. ZD1839 (Iressa) is a quinazoline derivative that selectively inhibits the EGFR tyrosine kinase activity and is under clinical use in cancer patients. However, the molecular mechanisms involved in ZD1839-mediated anticancer effects remain largely uncharacterized. In this study, exposure of human lung adenocarcinoma A549 cells to ZD1839 caused G1 arrest, and subsequently induced apoptosis. Moreover, ZD1839 increased the protein levels of p27(KIP1) and retinoblastoma-related Rb2/p130 while decreased the expression of cyclin-dependent kinase-2 (CDK2), CDK4, CDK6 and cyclin-D1, cyclin-D3. In vitro kinase assay showed that ZD1839 decreased these CDKs expression in A549 cells, leading to significantly reduce their kinase activities. In addition, ZD1839-induced death of A549 cells with characteristics of apoptosis including apoptotic morphological changes, DNA fragmentation and enhancement of TUNEL-positive cell. These events were accompanied by a marked increase of Fas protein expression, and activation of caspase-2, -3, -8. Co-treatment of cells with Fas antagonist antibody significantly blocked ZD1839-induced apoptosis. Caspase-8 and caspase-3 inhibitors, but not a caspase-9 inhibitor, were also capable of restoring cell viability. Our results indicate that downregulation of the expression and function of CDK2, CDK4, CDK6, cyclin-D1 and cyclin-D3, as well as upregulation of p27(KIP1) and pRb2/p130, are strong candidates for the cell cycle regulator that arrests ZD1839-treated A549 cells at G1 phase. Furthermore, upregulation of Fas appears to play a major role in the initiation of ZD1839-induced apoptosis, activation of caspase-8/caspase-3 cascade is involved in the execution phase of this death program.     

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.     

Induction of apoptosis by R-flurbiprofen in human colon carcinoma cells: involvement of p53

R-flurbiprofen, a non cyclooxygenase inhibiting non-steroidal anti-inflammatory drug (NSAID), has been found to inhibit tumor growth in various animal models. In vitro experiments have shown that this effect is based on the induction of a cell cycle block and apoptosis. Cell cycle inhibition has been explained by activation of the c-Jun-N-terminal kinase (JNK) and downregulation of cyclin D1 expression. However, the molecular mechanism leading to apoptosis is unknown. Here, we show that treatment of the human colon carcinoma cell line HCT116 with different concentrations of R-flurbiprofen leads to an accumulation of p53 protein which is accompanied by an increase in phosphorylated p53 at serine 15. Mutation of serine 15 to alanine by site directed mutagenesis and overexpression of the mutated p53 gene in HCT116 cells, revealed that these cells are significantly less sensitive to apoptosis induced by R-flurbiprofen than pcDNA control cells, as measured by PARP-cleavage and flow cytometry. By contrast, no difference was detected between HCT116p53ser15ala cells and HCT116 pcDNA cells with respect to induction of a cell cycle block after R-flurbiprofen treatment. Moreover, in nude mice HCT116p53ser15ala overexpressing xenografts were significantly less sensitive to R-flurbiprofen than HCT116 pcDNA control xenografts. In conclusion, we were able to show that induction of apoptosis in HCT116 cells after R-flurbiprofen treatment is at least partly dependent on the tumor suppressor gene p53 and that mutation of p53 at serine 15 impairs the apoptotic potency of R-flurbiprofen.     

Induction of apoptosis in two human leukemia cell lines as well as differentiation in human promyelocytic cells by cyanidin-3-O-beta-glucopyranoside

Little is known about the potentially chemopreventive mechanisms of anthocyanins apart from their antioxidant activity. We investigated the in vitro capacity of the anthocyanin cyanidin-3-O-beta-glucopyranoside (Cy-g) to induce apoptosis in T-lymphoblastoid, as well as apoptosis and differentiation in HL-60 promyelocytic cells. Although Cy-g-induced apoptosis (as well as necrosis) in the two systems, HL-60 cells were much less sensitive than T-lymphoblastoid cells. Moreover, treatment of HL-60 cells with Cy-g caused differentiation into macrophage-like cells and granulocytes. Concerning the mechanism of action, the induction of apoptosis in Jurkat T cells can be explained by a modulation of p53 and bax protein expression. At the molecular level, the induction of apoptosis and cytodifferentiation in HL-60 cells involved different proteins, thus suggesting that the effects of Cy-g on apoptosis and cytodifferentiation induction are two distinct events. These interesting biological properties should encourage further investigation into the chemopreventive and/or chemotherapeutic potential of Cy-g.     

Tubulozole-induced G2/M cell cycle arrest in human colon cancer cells through formation of microtubule polymerization mediated by ERK1/2 and Chk1 kinase activation.

Our studies demonstrated that human colon cancer cells (COLO 205), with higher expression level of check point kinase 1 (Chk1), were more sensitive to microtubule damage agent Tubulozole (TUBU) induced G2/M phase arrest than normal human colon epithelial (CRL) cells. TUBU (10 microM, for 3h) treatment resulted in rapid and sustained phosphorylation of Cdc25C (Ser-216) leading to increased 14-3-3beta binding. This resulted in increased nuclear translocation. In addition, TUBU induced phosphorylation of the Cdc25C (Ser-216) and Bad (Ser-155) proteins were blocked by Chk1 SiRNA-transfection. Surprisingly, cellular apotosis was observed in cells treated with TUBU after Chk1 SiRNA inhibition. We further demonstrated that extracellular signal-regulated kinase (ERK) activation by TUBU was needed for Chk1 kinase activation and microtubule formation as shown by the attenuation of these responses by the ERK1/2 specific inhibitor PD98059. However, TUBU induced ERK1/2 phosphorylation was not blocked in the Chk1 SiRNA-transfected COLO 205 cells. These results imply that ERK1/2 mediated Chk1 activation may be play an important role in determining TUBU induced G2/M arrest or apoptosis in COLO 205 cells.     

Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondrial pathway

Genipin, the aglycone of geniposide, exhibits anti-inflammatory and anti-angiogenic activities. Here we demonstrate that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells, detected by morphological cellular changes, caspase activation and release of cytochrome c. During genipin-induced apoptosis, reactive oxygen species (ROS) level was elevated, and N-acetyl-l-cysteine (NAC) and glutathione (GSH) suppressed activation of caspase-3, -7 and -9. Stress-activated protein kinase/c-Jun NH2-terminal kinase 1/2(SAPK/JNK1/2) but neither MEK1/2 nor p38 MAPK was activated in genipin-treated hepatoma cells. SP600125, an SAPK/JNK1/2 inhibitor, markedly suppressed apoptotic cell death in the genipin-treated cells. The FaO cells stably transfected with a dominant-negative c-Jun, TAM67, was less susceptible to apoptotic cell death triggered by genipin. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, inhibited ROS generation, apoptotic cell death, caspase-3 activation and JNK activation. Consistently, the stable expression of Nox1-C, a C-terminal region of Nox1 unable to generate ROS, blocked the formation of TUNEL-positive apoptotic cells, and activation of caspase-3 and JNK in FaO cells treated with genipin. Our observations imply that genipin signaling to apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of ROS, which leads to downstream of JNK.     

Anti-proliferation effect of 3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one (BJ-601) on human vascular endothelial cells: G0/G1 p21-associated cell cycle arrest

The aim of this study was to examine the anti-proliferation effect of 3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one (BJ-601) on human vascular endothelial cells and its possible molecular mechanism underlying. Our data showed that BJ-601 at a range of concentrations (0-40 microM) dose- and time-dependently decreased cell number in cultured human dermal microvascular endothelial cells (HDMVECs), but not human fibroblasts. The BJ-601-induced growth inhibition in HDMVECs was reversible. [3H]thymidine incorporation demonstrated that BJ-601 arrested the HDMVECs at the G0/G1 phase of the cell cycle. Western blot analysis revealed that BJ-601 (0-40 microM) dose-dependently increased the levels of the protein p21, but not of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase 2 (CDK2), and CDK4 in HDMVECs. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not CDK2-p27, CDK4-p21 and CDK4-p27 complexes, was increased in the BJ-601-treated HDMVECs. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in a dose-dependent manner in the BJ-601-treated HDMVECs. Pretreatment of HDMVECs with a p21 antisense oligonucleotide, which blocked the expression of p21 protein, reversed the BJ-601-induced inhibition of [3H]thymidine incorporation into HDMVECs. Moreover, cotreatment of the endothelial cells with protein kinase C (PKC) inhibitor, staurosporine, prevented the BJ-601-induced decrease of [3H]thymidine incorporation into HDMVECs. Administration of BJ-601 dose-dependently inhibited capillary-like tube formation of HDMVECs in Matrigel. In conclusion, these data suggest that BJ-601 inhibits HDMVECs proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally causes retardation of the cell cycle at the G0/G1 phase.     

The on-off action of Forkhead protein O3a in endotoxin tolerance of Kupffer cells depends on the PI3K/AKT pathway

BackgroundThe endotoxin tolerance (ET) of Kupffer cells (KCs) is an important protective mechanism for limiting endotoxin shock. As a key anti-inflammatory molecule, the roles and mechanism of Forkhead protein O3a (Foxo3a) in ET of KCs are not yet well understood.MethodsET and nonendotoxin tolerance (NET) KCs models were established in vitro and in vivo. The levels of cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The protein expression and phosphorylation levels were detected by western blotting (WB). Changes in the localization of nuclear factor kappa B (NF-κB) and Foxo3a in KCs were detected by immunofluorescence assays. KCs apoptosis and survival rates were detected by flow cytometry and an automatic cell counter, respectively.ResultsThe activity of NF-κB and the levels of p-Foxo3a and tumor necrosis factor (TNF-α) in the ET group were significantly lower than those in the NET group, while the levels of Foxo3a and interleukin 10 (IL-10) in the ET group were significantly higher than those in the NET group. Overexpression of Foxo3a or the use of a phosphatidylinositol-3-hydroxykinase (PI3K) inhibitor suppressed the activation of NF-κB by decreasing the levels of p-Foxo3a by inhibiting the activity of PI3K/AKT, which improved the tolerance of KCs and mice to endotoxin. In contrast, silencing Foxo3a or the use of a PI3K agonist reduced the tolerance of KCs and mice to endotoxin. The PI3K agonist counteracted the inhibitory effects of Foxo3a overexpression on NF-κB, impairing the tolerance of KCs to endotoxin.ConclusionsThe on-off action of Foxo3a in the ET of KCs depends on the PI3K/AKT pathway.