MSAP enhances migration of C6 glioma cells through phosphorylation of the myosin regulatory light chain

A key regulatory mechanism in cell motility is the control of myosin activity, which in non-muscle cells is determined by phosphorylation of the myosin regulatory light chain (MRLC). Here we show that MRLC-interacting protein (MIR)-interacting saposin-like protein (MSAP) enhances cell spreading in fibroblasts and migration of rat C6 glioma cells through increases in MRLC phosphorylation. Overexpression of MSAP enhanced the motility of glioma cells measured in matrigel invasion chambers and using a scratch assay. Downregulation of MSAP by RNA interference significantly decreased glioma cell migration and phosphorylation of MRLC. Inhibition of the corresponding MRLC kinase by ML-7 did not affect migration of MSAP-overexpressing cells. The present results show that MSAP controls glioma cell migration via enhancement of MRLC phosphorylation. This effect is independent of the activity of MRLC kinase. Thus, MSAP is a novel modulator of cell motility that influences migration of glioma cells and possibly other tumors.Received 9 February 2005; received after revision 2 March 2005; accepted 21 March 2005     

Emodin inhibits tumor cell migration through suppression of the phosphatidylinositol 3-kinase-Cdc42/Rac1 pathway

Enhanced cell migration is one of the underlying mechanisms in cancer invasion and metastasis. Therefore, inhibition of cell migration is considered to be an effective strategy for prevention of cancer metastasis. We found that emodin (3-methyl-1,6,8-trihydroxyanthraquinone), an active component from the rhizome of Rheum palmatum, significantly inhibited epidermal growth factor (EGF)- induced migration in various human cancer cell lines. In the search for the underlying molecular mechanisms, we demonstrated that phosphatidylinositol 3-kinase (PI3K) serves as the molecular target for emodin. In addition, emodin markedly suppressed EGF-induced activation of Cdc42 and Rac1 and the corresponding cytoskeleton changes. Moreover, emodin, but not LY294002, was able to block cell migration in cells transfected with constitutively active (CA)-Cdc42 and CA-Rac1 by interference with the formation of Cdc42/Rac1 and the p21-activated kinase complex. Taken together, data from this study suggest that emodin inhibits human cancer cell migration by suppressing the PI3K-Cdc42/Rac1 signaling pathway.Received 7 February 2005; received after revision 11 March 2005; accepted 18 March 2005     

ERK activation by Ca2+ ionophores depends on Ca2+ entry in lymphocytes but not in platelets, and does not conduct membrane scrambling

Rapid Ca²⁺-dependent phospholipid (PL) reorganization (scrambling) at the plasma membrane is a mechanism common to hematopoietic cells exposing procoagulant phosphatidylserine (PS). The aim of this research was to determine whether activation of the extracellular signal-regulated kinase (ERK) pathway was required for PL scrambling, based on a single report analyzing both responses induced by Ca²⁺ ionophores in megakaryoblastic HEL cells. Ca²⁺ ionophore-stimulated ERK phosphorylation was induced in platelets without external Ca²⁺, whereas exogenous Ca²⁺ entry was crucial for ERK activation in Jurkat T cells. In both cells, membrane scrambling only occurred following Ca²⁺ entry and was not blocked by inhibiting ERK phosphorylation. Furthermore, ERK proteins are strongly phosphorylated in transformed B lymphoblastic cell lines, which do not expose PS in their resting state. Overall, the data demonstrated that ERK activation and membrane scrambling are independent mechanisms. A. Arachiche, I. Badirou: These authors contributed equally to this work. Received 18 June 2008; received after revision 24 September 2008; accepted 1 October 2008     

Progesterone-induced blocking factor differentially regulates trophoblast and tumor invasion by altering matrix metalloproteinase activity

Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while—based on our previous data—PIBF might control trophoblast invasion by suppressing proinvasive genes.     

TRAIL induces proliferation of human glioma cells by c-FLIPL-mediated activation of ERK1/2

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in TRAIL-sensitive human malignant glioma cells. We show for the first time that TRAIL stimulates cell growth in TRAIL-resistant glioma cells. TRAIL-induced cell growth in resistant cells occurred through increased cell cycle progression as determined by flow cytometry and Western blot analysis of retinoblastoma protein phosphorylation. Western blot analysis of TRAIL-treated resistant cells revealed phosphorylation of ERK1/2 proteins and in vitro kinase analysis confirmed the activation of the ERK1/2 kinases. Inhibition of MEK1 eliminated both TRAIL-induced ERK1/2 activation and cell proliferation. In addition, siRNA inhibition of c-FLIP expression eliminates TRAIL-induced ERK1/2 activation and proliferation. Furthermore, overexpression of c-FLIP_L potentiates TRAIL-induced ERK1/2 activation and proliferation of resistant glioma cells. Our results have shown for the first time that TRAIL-induced ERK1/2 activation and proliferation of TRAIL-resistant human glioma cells is dependent upon the expression of the long form of the caspase-8 inhibitor c-FLIP_L. Received 2 November 2007; received after revision 14 December 2007; accepted 21 December 2007     

Targeting mitochondria by α-tocopheryl succinate overcomes hypoxia-mediated tumor cell resistance to treatment

Rapidly proliferating tumor cells easily become hypoxic. This results in acquired stability towards treatment with anticancer drugs. Here, we show that cells grown at 0.1 % oxygen are more resistant towards treatment with the conventionally used anticancer drugs doxorubicin and cisplatin. The stimulation of apoptosis, as assessed by the number of cells in the SubG1 fraction of the cell cycle, release of cytochrome c into the cytosol, activation of caspase-3, and cleavage of PARP, was markedly suppressed under low oxygen content or when hypoxia was mimicked by deferoxamine. Hypoxia or deferoxamine treatment was accompanied by stabilization of the hypoxia-inducible factor (HIF-1). The downregulation of HIF-1 using siRNA technique restored cell sensitivity to treatment under hypoxic conditions to the levels detected under normoxic conditions. In contrast to cisplatin or doxorubicin, α-tocopheryl succinate (α-TOS), a compound that targets mitochondria, stimulated cell death irrespective of the oxygen concentration. Moreover, under hypoxic condition cell death induced by α-TOS was even enhanced. Thus, α-TOS can successfully overcome resistance to treatment caused by hypoxia, which makes α-TOS an attractive candidate for antitumor therapy via mitochondrial targeting.     

Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment

The fatality of cancer predominantly results from the dissemination of primary tumor cells to distant sites and the subsequent formation of metastases. During tumor progression, some of the primary tumor cells as well as the tumor microenvironment undergo characteristic molecular changes, which are essential for the metastatic dissemination of tumor cells. In this review, we will discuss recent insights into pro-metastatic events occurring in tumor cells themselves and in the tumor stroma. Tumor cell-intrinsic alterations include the loss of cell polarity and alterations in cell-cell and cell-matrix adhesion as well as deregulated receptor kinase signaling, which together support detachment, migration and invasion of tumor cells. On the other hand, the tumor stroma, including endothelial cells, fibroblasts and cells of the immune system, is engaged in an active molecular crosstalk within the tumor microenvironment. Subsequent activation of blood vessel and lymph vessel angiogenesis together with inflammatory and immune-suppressive responses further promotes cancer cell migration and invasion, as well as initiation of the metastatic process. Received 4 July 2005; received after revision 3 November 2005; accepted 14 November 2005     

MAPK/ERK1/2 signaling mediates endothelial-like differentiation of immature DCs in the microenvironment of esophageal squamous cell carcinoma

Endothelial-like differentiation of dendritic cells (DCs) is a new phenomenon, and the mechanism is still elusive. Here, we show that the tumor microenvironment derived from the human esophageal squamous cell carcinoma (ESCC) cell line EC9706 can induce immature DCs (iDCs) differentiate toward endothelial cells, and become endothelial-like cells, but it has no obvious influence on mature DCs. During the course of endothelial-like differentiation of iDCs, a sustained activation of mitogen-activated protein kinase/extracelluar signal-regulated kinase1/2 (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) was detected. Incubation of iDCs with MEK phosphorylation inhibitor PD98059 blocked the MAPK/ERK1/2 and CREB phosphorylation as well as the endothelial-like differentiation of iDCs. Inhibition of vascular endothelial growth factor-A (VEGF-A) in the microenvironment with its antibody blocked the endothelial-like differentiation and the phosphorylation of MAPK/ERK1/2 and CREB. These data suggest that MAPK/ERK1/2 signaling pathway activated by VEGF-A could mediate endothelial-like differentiation of iDCs in the ESCC microenvironment.     

Evaluation of the anti-angiogenic effect of aloe-emodin

The present study identified aloe-emodin (AE, a hydroxyanthraquinone from Aloe vera and other plants) as a new anti-angiogenic compound with inhibitory effects in an in vivo angiogenesis assay and evaluates its effects on specific key steps of the angiogenic process. AE inhibits endothelial cell proliferation, but this effect is not cell specific, since AE also inhibits tumor cell proliferation. Cell migration and invasion are not remarkably affected by AE. On the other hand, AE has different effects on endothelial and tumor cell gelatinases. Two main targets of the pharmacological action of AE as an anti-angiogenic compound seem to be urokinase secretion and tubule formation of endothelial cells. Finally, AE produces a remarkable photocytotoxic effect on tumor cells. Taken together, our data indicate that AE can behave both as an anti-tumor and an anti-angiogenic compound and suggest that AE could be a candidate drug for photodynamic therapy. Received 7 September 2006; received after revision 17 October 2006; accepted 31 October 2006     

Calcium-mediated activation of PI3K and p53 leads to apoptosis in thyroid carcinoma cells

The molecular mechanism responsible for cadmium-induced cell death in thyroid cancer cells (FRO) is unknown. We demonstrated that apoptosis of FRO cells induced by cadmium was concentration and time dependent. Cadmium caused the rapid elevation of intracellular calcium and induced phosphorylation of Akt, p53, JNK, ERK and p38. Inhibition of PI3K/Akt attenuated the cadmium-induced apoptosis, but the inhibition of JNK inhibitor, ERK or p38 aggravated it, indicating that activation of PI3K/Akt was a pro-apoptosis signal in response to cadmium treatment, whereas the activation of stress-activated protein kinase JNK, ERK and p38 functioned as survival signals to counteract the cadmium-induced apoptosis. Buffering of the calcium response attenuated mitochondrial impairment, recovered the cadmium-activated Akt, p53, JNK, ERK and p38, and subsequently blocked the apoptosis. These results suggested that apoptosis induced by cadmium in FRO cells was initiated by the rapid elevation of intracellular calcium, followed by calcium-mediated activation of PI3K/Akt and mitochondrial impairment. Received 28 February 2007; received after revision 2 April 2007; accepted 23 April 2007     

Interleukin-17 stimulates inducible nitric oxide synthase-dependent toxicity in mouse beta cells

The influence of the proinflammatory cytokine interleukin (IL)-17 on inducible nitric oxide (NO) synthase (iNOS)-mediated NO release was investigated in the mouse insulinoma cell line MIN6 and mouse pancreatic islets. IL-17 markedly augmented iNOS mRNA/protein expression and subsequent NO production induced in MIN6 cells or pancreatic islets by different combinations of interferon-γ, tumor necrosis factor-α, and IL-1β. The induction of iNOS by IL-17 was preceded by phosphorylation of p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK activation completely abolished IL-17-stimulated NO release. IL-17 enhanced the NO-dependent toxicity of proinflammatory cytokines toward MIN6 cells, while IL-17-specific neutralizing antibody partially reduced the NO production and rescued insulinoma cells and pancreatic islets from NO-dependent damage induced by activated T cells. Finally, a significant increase in blood IL-17 levels was observed in a multiple low-dose streptozotocin model of diabetes, suggesting that T cell-derived IL-17 might be involved in NO-dependent damage of beta cells in this disease. Received 14 June 2005; received after revision 17 September 2005; accepted 21 September 2005     

Protein kinase C-dependent NF-κB activation is altered in T cells by chronic stress

Chronic stress has been associated with impaired immune function. In this work we studied the effect of chronic mild stress (CMS) exposure on the early intracellular pathways involved in T cells after stimulation with mitogen. We found that mitogen stimulation of T lymphocytes from CMS-exposed mice resulted in a reduction of the intracellular [Ca²⁺] rise, an impairment of growth-promoting protein kinase C (PKC) activation, a lower NF-κB activation and an increase in the inhibitory cAMP-protein kinase A (PKA) pathway activity with respect to those found in control lymphocytes. However, T cell activation with the direct PKC activator phorbol 12-myristate 13-acetate plus calcium ionophore led to a similar proliferative response in both CMS and control lymphocytes, indicating that signals downstream of PKC would not be affected by stress. In summary, our results show that chronic stress induced an alteration in T cell early transduction signals that result in an impairment of the proliferative response.Received 11 February 2005; received after revision 20 May 2005; accepted 6 June 2005     

Understanding microtubule dynamics for improved cancer therapy

Microtubules (MTs), key components of the cytoskeleton, are dynamic polymers of tubulin that form a well-organized network of polarized tube filaments. MT dynamics are highly regulated both spacially and temporally by several MT-related proteins, themselves regulated by several kinases and phosphatases via signaling cascades, and also by coordinated interactions with actin cytoskeleton and adhesion sites. Regulation of MT dynamics is crucial for mitosis, cell migration, cell signaling and trafficking. MT-targeted drugs (MTDs), which constitute a major anticancer drug family with antimitotic and antiangiogenic properties, inhibit tumor progression mainly by altering MT dynamics in both cancer and endothelial cells. Identification of proteins regulating the MT network will lead to a better understanding of tumor progression regulators and will be helpful in improving cancer therapy. Received 22 July 2005; received after revision 8 September 2005; accepted 12 September 2005     

Targeting mitochondria by α-tocopheryl succinate kills neuroblastoma cells irrespective of MycN oncogene expression

Amplification of the MycN oncogene characterizes a subset of highly aggressive neuroblastomas, the most common extracranial solid tumor of childhood. However, the significance of MycN amplification for tumor cell survival is controversial, since down-regulation of MycN was found to decrease markedly neuroblastoma sensitivity towards conventional anticancer drugs, cisplatin, and doxorubicin. Here, we show that a redox-silent analogue of vitamin E, α-tocopheryl succinate (α-TOS), which triggers apoptotic cell death via targeting mitochondria, can kill tumor cells irrespective of their MycN expression level. In cells overexpressing MycN, as well as cells in which MycN was switched off, α-TOS stimulated rapid entry of Ca(2+) into the cytosol, compromised Ca(2+) buffering capacity of the mitochondria and sensitized them towards mitochondrial permeability transition and subsequent apoptotic cell death. Prevention of mitochondrial Ca(2+) accumulation or chelation of cytosolic Ca(2+) rescued the cells. Thus, targeting mitochondria might be advantageous for the elimination of tumor cells with otherwise dormant apoptotic pathways.     

Survival of docetaxel-resistant prostate cancer cells in vitro depends on phenotype alterations and continuity of drug exposure

We evaluated in vitro the effect of paclitaxel and docetaxel on PC-3 and DU-145 prostate cancer cell lines to understand better the downstream events in drug-induced tumor cell death. Taxane treatments of DU-145 cells induced rapid cell death by apoptosis, but in PC-3 cells, treatments achieved growth arrest, followed by extensive karyokinesis resulting in multinucleation, giant-cell formation and delayed cell death. To determine if the giant multinucleated cells were able to produce proliferating and drug-resistant survivors, we first delineated the kinetics of drug activity and cytotoxic dose range. Analysis of both lines by colorimetric and cell viability assays demonstrated improved cytotoxicity of taxanes applied continuously. Selected doses and schedules of docetaxel were used to induce giant multinucleated cells that gave rise to docetaxel-resistant survivors, which remained sensitive to paclitaxel and other chemotherapeutics. Growth and morphology of the recovered clones was similar to parental cells. The resistant phenotype of these clones determined by immunofluorescence and immunoblot was associated with transient expression of the β-tubulin IV isoform and was independent of P-glycoprotein, bcl-2 and bcl-xL. Resistant clones will be useful to model progression of resistance to taxanes and to identify unknown and clinically important molecular mechanisms of cell death and resistance. Received 15 March 2002; received after revision 25 April 2002; accepted 27 May 2002     

Mitochondria and calpains mediate caspase-dependent apoptosis induced by doxycycline in HeLa cells

Doxycycline (Dc) has been demonstrated to inhibit cell growth and induce apoptosis in tumor cells, although its mechanism of action is not fully understood. The present study demonstrates that apoptosis can be induced in HeLa cells. Western blot data demonstrated that cytochrome c (Cyt c), Smac (the second mitochondria-derived activator of caspase), calpain I, caspase-9, −3 and −8 were involved in the apoptotic process, while the pan caspase inhibitor zVAD-fmk almost completely inhibited Dc-induced apoptosis. We further demonstrated that the release of mitochondrial proteins and the activation of calpains occurred upstream of the caspase cascade, in which caspase-9 was activated in response to the release of Cyt c, that caspase-8 activation was caspase and calpain dependent, and that caspase-3 was activated mainly by caspase-8 and −9. Caspase-8 played important roles in the activation of caspase-3 and induction of apoptosis, whereas the role of the caspase-9 was limited. Received 26 November 2005; received after revision 14 February 2006; accepted 1 March 2006