Akt2 knock-down reveals its contribution to human lung cancer cell proliferation, growth, motility, invasion and endothelial cell tube formation.

The Akt/PKB serine/threonine protein kinase consists of three isoforms: Akt-1, -2 and -3. Their overexpression has been detected in human cancers, but their roles in cancer progression are unclear. We investigated the impact of specific silencing of Akt1 and Akt2 on human lung cancer cell proliferation, colony growth, motility, and invasion in vitro as well as tumor growth in vivo using human Non-Small Cell Lung Cancer cells LNM35, and on the vascular tube formation using HUVEC cells. Although silencing of Akt1 decreased cellular invasion at least in part via COX-2 inhibition, it had almost no effect on cell motility, proliferation, colony formation, and angiogenesis. Transient as well as stable silencing of Akt2 resulted in a strong inhibition of Rb phosphorylation associated with a decrease in cellular proliferation and colony formation, leading to the inhibition of tumor growth in the xenograft model. Silencing of Akt2 also reduced cellular motility and invasion in vitro, presumably via COX-2 inhibition. Moreover, silencing of Akt2 in the HUVEC cells resulted in the inhibition of their spontaneous angiogenic phenotype. Altogether, these results indicate that Akt2 plays an important role in lung cancer progression and can be a promising target for lung cancer therapy.

Ligand-free estrogen receptor activity complements IGF1R to induce the proliferation of the MCF-7 breast cancer cells.

BACKGROUND: Ligand-dependent activation of the estrogen receptor (ER) as well as of the insulin-like growth factor type 1 (IGF1R) induces the proliferation of luminal breast cancer cells. These two pathways cooperate and are interdependent. We addressed the question of the mechanisms of crosstalk between the ER and IGF1R. METHODS: We evaluated the mitogenic effects of estradiol (E2; agonist ligand of ER) and of insulin (a ligand of IGF1R) in the MCF-7 cells by flow cytometry and by analyzing the cell levels of cell cycle-related proteins (immunoblotting) and mRNA (RT-QPCR). To verify the requirement for the kinase activity of Akt (a downstream target of IGF1R) in the mitogenic action of estradiol, we used shRNA strategy and shRNA-resistant expression vectors. RESULTS: The activation of the ER by E2 is unable to induce the cell cycle progression when the phosphatidyl inositol-3 kinase (PI3K)/Akt signaling is blocked by a chemical inhibitor (LY 294002) or by shRNA targeting Akt1 and Akt2. shRNA-resistant Akt wild-type constructs efficiently complemented the mitogenic signaling activity of E2 whereas constructs with inactivated kinase function did not. In growth factor-starved cells, the residual PI3K/Akt activity is sufficient to complement the mitogenic action of E2. Conversely, when ER function is blocked by the antiestrogen ICI 182780, IGF1R signaling is intact but does not lead to efficient reinitiation of the cell cycle in quiescent, growth factor-starved MCF-7 cells. The basal transcription-promoting activity of ligand-free ER in growth factor-starved cells is sufficient to complement the mitogenic action of the IGF1R-dependent signaling. CONCLUSIONS: The basal ER activity in the absence of ligand is sufficient to allow efficient mitogenic action of IGF1R agonists and needs to be blocked to prevent the cell cycle progression.

Inhibition of cell survival, invasion, tumor growth and histone deacetylase activity by the dietary flavonoid luteolin in human epithelioid cancer cells.

Phytochemical compounds and histone deacetylase (HDAC) inhibitors are emerging as a new generation of anticancer agents with limited toxicity in cancer patients. We investigated the impact of luteolin, a dietary flavonoid, on survival, migration, invasion of cancer cells in vitro, and tumor growth in vivo. Luteolin (25-200µM) decreased the viability of human cancer cell lines originating from the lung (LNM35), colon (HT29), liver (HepG2) and breast (MCF7/6 and MDA-MB231-1833). Luteolin effectively increased the sub-G1 (apoptotic) fraction of cells through caspase-3 and -7 dependent pathways. We provide evidence that luteolin at sub-lethal/non-toxic concentrations inhibited the invasive potential of LNM35, MCF-7/6 and MDA-MB231-1833 cancer cells using Matrigel as well as the chick heart and Oris invasion assays. Moreover, we demonstrate for the first time that luteolin is a potent HDAC inhibitor that potentiates the cytotoxicity of cisplatin in LNM35 cells and decreases the growth of LNM35 tumor xenografts in athymic mice after intraperitoneal injection (20mg/kg/day for 18days) Thus, luteolin, in combination with standard anticancer drugs such as cisplatin, may be a promising HDAC inhibitor for the treatment of lung cancer.

Indirect participation of Hsp90 in the regulation of the cyclin E turnover.

Cyclin E is the Cdk2-regulatory subunit required for the initiation of DNA replication at the G1/S transition. It accumulates in late G1 phase and gets rapidly degraded by the ubiquitin/proteasome pathway during S phase. The degradation of cyclin E is a consequence of its phosphorylation and subsequent isomerization by the peptidyl-prolyl isomerase Pin1. We show that in the colon cancer cells HT-29 the inhibition of the chaperone function of Hsp90 by geldanamycin (GA) enhances the ubiquitinylation of cyclin E and triggers active degradation via the proteasome pathway. As Hsp90 forms multiprotein complexes with and regulates the function and cell contents of numerous signaling proteins, this observation suggests a direct interaction between Hsp90 and cyclin E. However, experiments using cell lysate fractionation did not reveal the presence of complexes containing both Hsp90 and cyclin E. Coupled transcription/translation experiments also failed to detect the formation of complexes between newly synthesized cyclin E and Hsp90. We conclude that Hsp90 can regulate the degradation of cellular proteins without binding to them, by an indirect mechanism. This conclusion postulates a new category of proteins that are affected by the inactivation of Hsp90. Our observations do not support the possible involvement of a PPIase in this indirect mechanism. Besides, we did not observe active geldanamycin-dependent degradation of cyclin E in the prostate cancer-derived cell line DU-145, indicating that the Hsp90-dependent stabilization of cyclin E requires specific regulatory mechanism which may be lost in certain types of cancer cells.

Captopril as a potential inhibitor of lung tumor growth and metastasis.

Lung cancer is the most common form of cancer in the world, and 90% of patients die from their disease. The angiotensin converting enzyme (ACE) inhibitors are used widely as antihypertensive agents, and it has been suggested that they decrease the risk of some cancers, although available data are conflicting. Accordingly, we investigated the anticancer activity of the ACE inhibitor, captopril, in athymic mice injected with highly tumorigenic LNM35 human lung cells as xenografts. Using this model, we demonstrated that daily IP administration of captopril (2.8 mg/mouse) for 3 weeks resulted in a remarkable reduction of tumor growth (58%, P < 0.01) and lymph node metastasis (50%, P= 0.088). There were no undesirable effects of captopril treatment on animal behavior and body weight. In order to determine the mechanism by which captopril inhibited tumor growth, we investigated the impact of this drug on cell proliferation, apoptosis, and angiogenesis. Immunohistochemical analysis demonstrated that captopril treatment significantly reduced the number of proliferating cells (Ki-67) in the tumor samples but was not associated with inhibition of tumor angiogenesis (CD31). Using cell viability and fluorescent activated cell sorting analysis tests, we demonstrated that captopril inhibited the viability of LNM35 cells by inducing apoptosis, providing insight about the mechanisms underlying its antitumorigenic activities. In view of these experimental findings, we conclude that captopril could be a promising option for the treatment of lung cancer.

Mitogenic activity of estrogens in human breast cancer cells does not rely on direct induction of mitogen-activated protein kinase/extracellularly regulated kinase or phosphatidylinositol 3-kinase.

We have addressed the question of rapid, nongenomic mechanisms that may be involved in the mitogenic action of estrogens in hormone-dependent breast cancer cells. In quiescent, estrogen-deprived MCF-7 cells, estradiol did not induce a rapid activation of either the MAPK/ERK or phosphatidylinositol-3 kinase (PI-3K)/Akt pathway, whereas the entry into the cell cycle was documented by the successive inductions of cyclin D1 expression, hyperphosphorylation of the retinoblastoma protein (Rb), activity of the promoter of the cyclin A gene, and DNA synthesis. However, pharmacological inhibitors of the src family kinases, 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP1) or of the PI-3K (LY294002) did prevent the entry of the cells into the cell cycle and inhibited the late G1 phase progression, whereas the inhibitor of MAPK/ERK activation (U0126) had only a partial inhibitory effect in the early G1 phase. In agreement with these results, small interfering RNA targeting Akt strongly inhibited the estradiolinduced cell cycle progression monitored by the activation of the promoter of the cyclin A gene. The expression of small interfering RNA targeting MAPK 1 and 2 also had a clear inhibitory effect on the estradiol-induced activation of the cyclin A promoter and also antagonized the estradiol-induced transcription directed by the estrogen response element. Finally, transfection of the estrogen receptor into NIH3T3 fibroblasts did not confer to the cells sensitivity to a mitogenic action of estradiol. We conclude that the induction of the cell cycle by estradiol does not require a direct activation of MAPK/ERK or PI-3K signaling protein kinase cascades, but that these kinases appear to have a permissive role in the cell cycle progression.

Geldanamycin, an inhibitor of the chaperone activity of HSP90, induces MAPK-independent cell cycle arrest.

The effects of GA, an ansamycin antibiotic in development as a lead anticancer drug, were studied in mouse BP-A31 fibroblasts and in human cancer-derived cell lines. GA and related molecules act by inhibiting the chaperone function of the Hsp90 protein through competition for ATP binding. The antiproliferative effects of GA have been attributed to destabilization of the Raf-1 protein, one of the targets of Hsp90, and to the resulting inhibition of MAPK. Addition of GA to BP-A31 cells, synchronously progressing through the G(1) phase, inhibited Rb hyperphosphorylation and G(1)/S transition irrespective of the time of addition. The G(1) arrest was accompanied by a progressive decrease in Raf-1 content, especially of the phosphorylated form; however, GA caused only partial inhibition of MAPK phosphorylation. We show that GA triggers a rapid and marked decrease in the kinase activity of the cyclin E/cdk2 complex coupled with a decline in both total and cdk2-associated cyclin E. In transient transfection experiments, inhibition of cyclin E expression by GA was correlated with inhibition of the transcriptional activity of the cyclin E gene promoter. Inhibition of cdk4 activity by GA was observed 3 hr after addition of the drug to late G(1) cells but not after a short (1 hr) exposure, as revealed by the phosphorylation of Rb on the Ser(780) residue. In human cancer-derived cell lines expressing or not a functional Rb protein, GA blocked proliferation and inhibited the transcriptional activity of the cyclin E gene promoter. In these cell lines, the antiproliferative effect of GA was not limited to the G(1) phase, suggesting the existence of multiple cellular targets of the drug.

Rapamycin inhibits cdk4 activation, p 21(WAF1/CIP1) expression and G1-phase progression in transformed mouse fibroblasts.

Rapamycin, a bacterial macrolide antibiotic, is a potent immunosuppressant agent that blocks cell proliferation by inhibiting the G1/S transition in several cell types. In sensitive cells, rapamycin inhibits the phosphorylation of p70 S6K and of Rb; however, the precise mechanisms involved have not been elucidated. In the mouse BP-A31 fibroblasts, synchronised in G0/G1 phase by serum starvation and induced to reinitiate the G1-phase progression, rapamycin inhibited the entry into S phase. The effect of rapamycin was situated in early G1 phase. The assembly of the cyclin D1/cdk4 complexes that phosphorylate Rb early in the G1 phase was not modified by the drug. Nevertheless, an inhibition of the activation of cyclin D1/cdk4 and cyclin E/cdk2 as well as of Rb phosphorylation accompanied the cell cycle arrest. Remarkably, rapamycin reduced the level of total p21(WAF1/CIP1) as well as that of p21(WAF1/CIP1) associated with the cyclin D1/cdk4 complexes. Besides its inhibitory activity toward cdk, p21(WAF1/CIP1) has been recently found to participate in the formation/stabilisation/nuclear translocation of cyclin D1/cdk4 complexes. We propose that the inhibition of the expression of p21(WAF1/CIP1) is a mechanism by which rapamycin inhibits the triggering of the cdk cascade in the BP-A31 cells.

Cellular effects of purvalanol A: a specific inhibitor of cyclin-dependent kinase activities.

We have studied the effects of purvalanol A on the cell cycle progression, proliferation and viability. In synchronized cells, purvalanol A induced a reversible arrest the progression in G1 and G2 phase of the cell cycle, but did not prevent the completion of DNA synthesis in S-phase cells. The specificity of action of the drug was supported by the selective inhibition of the phosphorylation of cyclin-dependent kinase (cdk) substrates such as Rb and cyclin E. The cell contents of cyclins D1 and E were lower in cells incubated with purvalanol A compared to controls, but the level of the cdk inhibitory protein p21(WAF1/CIP1) was increased, indicating that the drug did not cause a general inhibition of gene expression. Purvalanol A did not inhibit transcription under cell-free conditions. This compound, however, caused an inhibition of the estradiol-induced expression of an integrated luciferase gene, suggesting that cdk or related enzymes may participate in the regulation of the activity of certain promoters. When exponentially growing cells, both mouse fibroblasts and human cancer cell lines, were incubated with purvalanol A for prolonged periods of time (24 hr), a lasting inhibition of cell proliferation as well as cell death were observed. In contrast, a 24 hr incubation of quiescent (non-transformed) cells with purvalanol A did not prevent their resumption of cell cycle after removal of the drug.