New pterocarpanquinones: Synthesis,antineoplasic activity on cultured human malignant cell lines and TNF-α modulation in human PBMC cells

A new pterocarpanquinone (5a) was synthesized through a palladium catalyzed oxyarylation reaction and was transformed, through electrophilic substitution reaction, into derivatives 5b–d. These compounds showed to be active against human leukemic cell lines and human lung cancer cell lines. Even multidrug resistant cells were sensitive to 5a, which presented low toxicity toward peripheral blood mononuclear cells (PBMC) cells and decreased the production of TNF-α by these cells. In the laboratory these pterocarpanquinones were reduced by sodium dithionite in the presence of thiophenol at physiological pH, as NAD(P)H quinone oxidoredutase-1 (NQO1) catalyzed two-electron reduction, and the resulting hydroquinone undergo structural rearrangements, leading to the formation of Michael acceptors, which were intercepted as adducts of thiophenol. These results suggest that these compounds could be activated by bioreduction.     

Furanodienone inhibits cell proliferation and survival by suppressing ERα signaling in human breast cancer MCF-7 cells

Estrogen receptor alpha (ERα) plays an important role in the development and progression of breast cancer and thus the attenuation of ERα activities is a promising treatment strategy. Furanodienone is one of the main bioactive chemical components of Rhizoma Curcumae which is commonly used in Chinese medicine for the treatment of cancer. In this study, we investigated the effects of furanodienone on human breast cancer MCF‐7, T47D, and MDA‐MB‐231 cells. Our results showed that furanodienone could inhibit MCF‐7, T47D, and MDA‐MB‐231 cells proliferation in a dose (10–160 µM) dependent manner. ERα‐negative MDA‐MB‐231 cells were less sensitive to furanodienone than ERα‐positive MCF‐7 and T47D cells. Furanodienone could effectively block 17β‐estradiol (E2)‐stimulated MCF‐7 cell proliferation and cell cycle progression and induce apoptosis evidenced by the flow cytometric detection of sub‐G1 DNA content and the appearance of apoptotic nuclei after DAPI staining. Furanodienone specifically down‐regulated ERα protein and mRNA expression levels without altering ERβ expression. Furanodienone treatment inhibited E2‐stimulation of estrogen response element (ERE)‐driven reporter plasmid activity and ablated E2‐targeted gene (e.g., c‐Myc, Bcl‐2, and cyclin D1) expression which resulted in the inhibition of cell cycle progression and cell proliferation, and in the induction of apoptosis. Knockdown of ERα in MCF‐7 cells by ERα‐specific siRNA decreased the cell growth inhibitory effect of furanodienone. These findings suggest that effects of furanodienone on MCF‐7 cells are mediated, at least in part, by inhibiting ERα signaling. J. Cell. Biochem. 112: 217–224, 2011. © 2010 Wiley‐Liss, Inc.     

Interleukin-1β regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1β is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1β on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling.Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1β. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays.Pig heart cells express receptors for IL-1 and application of IL-1β resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1β (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1β (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively).Our in vitro data suggest that IL-1β plays a major role in the events of tissue remodelling in the heart. Combined with our recently published in vivo data (Meybohm et al., PLoS One, 2009), the results presented here strongly suggest IL-1β as a key molecule guiding tissue remodelling events after myocardial infarction.     

Exposure of vital cells to necrotic cell lysates induce the IRE1α branch of the unfolded protein response and cell proliferation

Necrosis is a form of cell death that is detrimental to the affected tissue because the cell ruptures and releases its content (reactive oxygen species among others) into the extracellular space. Clusterin (CLU), a cytoprotective extracellular chaperone has been shown to be upregulated in the face of necrosis. We here show that in addition to CLU upregulation, necrotic cell lysates induce JNK/SAPK signaling, the IRE1α branch of the unfolded protein response (UPR), the MAPK/ERK1/2, and the mTOR signaling pathways and results in an enhanced proliferation of the vital surrounding cells. We name this novel response mechanism: Necrosis-induced Proliferation (NiP).     

Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.     

The effects of estrogen receptors α- and β-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.     

TNF-α and TGF-β synergistically stimulate elongation of human endothelial cells without transdifferentiation to smooth muscle cell phenotype

We earlier reported synergy between tumor necrosis factor-α (TNF-α) and transforming growth factor-β₁ (TGF-β₁) for apoptosis in human umbilical vein endothelium (HUVEC). Here, we study morphological change by circularity measurement of HUVEC surviving this cytokine induced synergistic apoptosis. Contrasting with reports by others studying bovine endothelium, HUVEC did not change morphology in response to TGF-β₁. TNF-α markedly elongated cells (p < 0.001) and this further increased with combination of the two cytokines (p < 0.001), while elongation was accompanied by increased actin stress fibres. Transdifferentiation of HUVEC to a smooth muscle cell phenotype as reported elsewhere was excluded in the current study.     

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca²⁺ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell–endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell–ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell–cell contact region, and thus inhibits cell–cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca²⁺-CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell–cell contact regions might be essential for this process under hypoxic stress. W.-G. Shen and W.-X. Peng Contributed to this paper equally     

Activation of protein kinase C-α is essential for stimulation of cell proliferation by ceramide 1-phosphate

We previously demonstrated that ceramide-1-phosphate (C1P) stimulates fibroblast and macrophage proliferation, but the mechanisms involved in this action have only been partially described. Here we demonstrate that C1P induces translocation of protein kinase C-alpha (PKC-α) from the soluble to the membrane fraction of bone marrow-derived macrophages. Translocation of this enzyme was accompanied by its phosphorylation on Ser 657 residue. Activation of PKC-α was independent of prior stimulation of phosphatidylinositol-dependent or phosphatidylcholine-dependent phospholipase C activities, but required activation of sphingomyelin synthesis. Inhibition of PKC-α activation also blocked C1P-stimulated macrophage proliferation indicating that this enzyme is essential for the mitogenic effect of C1P.     

Study on the effect of IRE1α on cell growth and apoptosis via modulation PLK1 in ER stress response

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. Inositol-requiring enzyme-1a (IRE1a), as one of three unfolded protein sensors in UPR signaling pathways, senses ER unfolded proteins through an ER lumenal domain that becomes oligomerized during ER stress. It is known to be important for ER stress-mediated apoptosis and cell growth, but the exact molecular mechanism underlying these processes remains unexplored. In this study, we report that knockdown of IRE1a by an siRNA silencing approach enhanced, whereas its overexpression inhibited, cell proliferation in Hepatoma cells. Besides, overexpression of IRE1a induced, while its repression inhibited, ER stress-mediated apoptosis in Hepatomas cells. Furthermore, we found that overexpressed IRE1a can down-regulate Polo-like kinase 1(PLK1) from mRNA and protein two levels. IRE1a-mediated induction of apoptosis and inhibition of proliferation in response to ER stress is through downregulation PLK1, an early trigger for G2/M transition known to be participated in regulating cell proliferation and cell apoptosis. Collectively, these findings reveal a novel critical role of IRE1a in ER stress-mediated apoptosis and the molecular mechanisms involved. IRE1a may be a useful molecular target for the development of novel predictive and therapeutic strategies in cancer.     

Cell proliferation and drug sensitivity of human glioblastoma cells are altered by the stable modulation of cytosolic 5′-nucleotidase II

Cytosolic 5′-nucleotidase II (cN-II) has been reported to be involved in cell survival, nucleotide metabolism and in the cellular response to anticancer drugs. With the aim to further evaluate the role of this enzyme in cell biology, we stably modulated its expression the human glioblastoma cell ADF in which the transient inhibition of cN-II has been shown to induce cell death. Stable cell lines were obtained both with inhibition, obtained with plasmids coding cN-II-targeting short hairpin RNA, and stimulation, obtained with plasmids coding Green Fluorescence Protein (GFP)-fused wild type cN-II or a GFP-fused hyperactive mutant (GFP-cN-II-R367Q), of cN-II expression. Silenced cells displayed a decreased proliferation rate while the over expressing cell lines displayed an increased proliferation rate as evidenced by impedance measurement using the xCELLigence device. The expression of nucleotide metabolism relevant genes was only slightly different between cell lines, suggesting a compensatory mechanism in transfected cells. Cells with decreased cN-II expression were resistant to the nucleoside analog fludarabine confirming the involvement of cN-II in the metabolism of this drug. Finally, we observed sensitivity to cisplatin in cN-II silenced cells and resistance to this same drug in cN-II over-expressing cells indicating an involvement of cN-II in the mechanism of action of platinum derivatives, and most probably in DNA repair. In summary, our findings confirm some previous data on the role of cN-II in the sensitivity of cancer cells to cancer drugs, and suggest its involvement in other cellular phenomenon such as cell proliferation.