The non-psychoactive cannabidiol triggers caspase activation and oxidative stress in human glioma cells

Recently, we have shown that the non-psychoactive cannabinoid compound cannabidiol (CBD) induces apoptosis of glioma cells in vitro and tumor regression in vivo. The present study investigated a possible involvement of caspase activation and reactive oxygen species (ROS) induction in the apoptotic effect of CBD. CBD produced a gradual, time-dependent activation of caspase-3, which preceded the appearance of apoptotic death. In addiction, release of cytochrome c and caspase-9 and caspase-8 activation were detected. The exposure to CBD caused in glioma cells an early production of ROS, depletion of intracellular glutathione and increase activity of glutathione reductase and glutathione peroxidase enzymes. Under the same experimental condition, CBD did not impair primary glia. Thus, we found a different sensitivity to the anti-proliferative effect of CBD in human glioma cells and non-transformed cells that appears closely related to a selective ability of CBD in inducing ROS production and caspase activation in tumor cells. Received 6 April 2006; received after revision 31 May 2006; accepted 22 June 2006     

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     

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     

Induction of apoptosis and CD10/neutral endopeptidase expression by jaspamide in HL-60 line cells

Jaspamide (jasplakinolide) is a natural peptide isolated from marine sponges of Jaspis species and has fungicidal and growth-inhibiting activities. We characterized the jasplakinolide-induced loss of viability by programmed cell death in the HL-60 human promyelocytic leukemia cell line and found that this process was accompanied by neutral endopeptidase (NEP)/CD10 expression on the surface of the apoptotic cells. HL-60 cells do not normally express detectable amounts of NEP/CD10 on their surface or intracytoplasmically, but upon jaspamide treatment, CD10 was synthesized de novo, its expression being inhibited by cycloheximide pretreatment. Once synthesized, NEP/CD10 interfered with the jasplakinolide signal delivered to HL-60 cells. Inhibition of NEP/CD10 by the NEP inhibitor phosphoramidon or by an anti-CD10 monoclonal antibody significantly increased apoptosis induction. The appearance of CD10 on the cell surface was blocked by preincubation of the cells with the monocytic/macrophage-differentiating agents vitamin D3 and phorbol 12-myristate 13-acetate, but not by the granulocytic differentiating agents retinoic acid or dimethyl sulfoxide. Moreover, in the promonocytic U937 and mature monocytic THP-1 cell lines, jaspamide induced apoptosis but not CD10 expression. In HL-60 cells, CD10 expression was partially but not totally blocked by the broad-spectrum caspase inhibitor benzyloxacarbonyl-Val-Ala-Asp-fluoromethylketone, indicating a connection between apoptosis induction and CD10 synthesis. Our findings suggest that the CD10 expression is related to the programmed cell death induction by jaspamide, and also with the process of granulocytic differentiation in HL-60 cells. Received 22 April 2002; received after revision 8 June 2002; accepted 10 June 2002     

Selenocystine induces apoptosis of A375 human melanoma cells by activating ROS-mediated mitochondrial pathway and p53 phosphorylation

Selenocystine (SeC), a naturally occurring selenoamino acid, has been shown to be a novel compound with broad-spectrum anticancer activity. In this study, we showed that SeC triggered time- and dose-dependent apoptosis in A375 human melanoma cells by activating the mitochondria-mediated and death receptor-mediated apoptosis pathways. Pretreatment of cells with a general caspase inhibitor z-VAD-fmk significantly prevented SeC-induced apoptosis. A375 cells exposed to SeC showed an increase in levels of total p53 and phosphorylated p53 (serine-15). Silencing of p53 expression with RNA interference significantly suppressed SeC-induced p53 phosphorylation, caspase activation and apoptotic cell death. Moreover, generation of reactive oxygen species and subsequent induction of DNA strand breaks were found to be upstream mediators of p53 activation induced by SeC. In a nude mice xenograft experiment, SeC significantly inhibited the tumor growth of A375 cells via induction of apoptosis. Taken together, these results suggest the potential applications of SeC in cancer chemoprevention.     

MicroRNA-128 downregulates Bax and induces apoptosis in human embryonic kidney cells

MicroRNAs (miRNAs) are short ~21-nt non-coding RNA molecules that have been shown to regulate a number of biological processes. Previous reports have shown that overexpression of miR-128 in glioma cells inhibited cell proliferation. Literature also suggests that miR-128 negatively regulates prostate cancer cell invasion. Here, we show that overexpression of hsa-miR-128, a brain-enriched microRNA, induces apoptosis in HEK293T cells as elucidated by apoptosis assay, cell cycle changes, loss of mitochondrial membrane potential and multicaspase assay. By in silico analysis, we identified a putative target site within the 3′ untranslated region (UTR) of Bax, a proapoptotic member of the apoptosis pathway. We found that ectopic expression of hsa-miR-128 suppressed a luciferase reporter containing the Bax-3′ UTR and reduced the levels of Bax in HEK293T cells. Taken together, our study demonstrates that overexpression of hsa-miR-128 not only induces apoptosis in HEK293T cells but also is an endogenous regulator of Bax protein.     

Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis

The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line, metformin blocked the cell cycle progression in G₀/G₁ phase without inducing significant cell death. In confluent C6 cultures, on the other hand, metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation, mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine), while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride, iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C, an inhibitor of AMP-activated protein kinase (AMPK), and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251, while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. Received 14 February 2007; received after revision 26 March 2007; accepted 3 April 2007     

A central role for calcineurin in protein misfolding neurodegenerative diseases

Accumulation of misfolded/unfolded aggregated proteins in the brain is a hallmark of many neurodegenerative diseases affecting humans and animals. Dysregulation of calcium (Ca²⁺) and disruption of fast axonal transport (FAT) are early pathological events that lead to loss of synaptic integrity and axonal degeneration in early stages of neurodegenerative diseases. Dysregulated Ca²⁺ in the brain is triggered by accumulation of misfolded/unfolded aggregated proteins in the endoplasmic reticulum (ER), a major Ca²⁺ storing organelle, ultimately leading to neuronal dysfunction and apoptosis. Calcineurin (CaN), a Ca²⁺/calmodulin-dependent serine/threonine phosphatase, has been implicated in T cells activation through the induction of nuclear factor of activated T cells (NFAT). In addition to the involvement of several other signaling cascades, CaN has been shown to play a role in early synaptic dysfunction and neuronal death. Therefore, inhibiting hyperactivated CaN in early stages of disease might be a promising therapeutic strategy for treating patients with protein misfolding diseases. In this review, we briefly summarize the structure of CaN, inhibition mechanisms by which immunosuppressants inhibit CaN, role of CaN in maintaining neuronal and synaptic integrity and homeostasis and the role played by CaN in protein unfolding/misfolding neurodegenerative diseases.     

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     

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     

Morphine but not fentanyl and methadone affects mitochondrial membrane potential by inducing nitric oxide release in glioma cells

We have observed that treatment of human glioma cells with morphine in the nanomolar range of concentration affects the mitochondrial membrane potential. The effect is specific to morphine and is mediated by naloxone-sensitive receptors, and is thus better observed on glioma cells treated with desipramine; moreover, the mitochondrial impairment is not inducible by fentanyl or methadone treatment and is prevented by the nitric oxide (NO) synthase inhibitor L-NAME. We conclude that in cultured glioma cells, the morphine-induced NO release decreases the mitochondrial membrane potential, as one might expect based on the rapid inhibition of the respiratory chain by NO. The identification of new intra-cellular pathways involved in the mechanism of action of morphine opens additional hypotheses, providing a novel rationale relevant to the therapy and toxicology of opioids.Received 19 August 2004; received after revision 16 September 2004; accepted 7 October 2004     

Dependence receptors: between life and death

The recently described family of dependence receptors is a new family of functionally related receptors. These proteins have little sequence similarity but display the common feature of inducing two completely opposite intracellular signals depending on ligand availability: in the presence of ligand, these receptors transduce a positive signal leading to survival, differentiation or migration, while in the absence of ligand, the receptors initiate or amplify a negative signal for apoptosis. Thus, cells that express these proteins manifest a state of dependence on their respective ligands. The mechanisms that trigger cell death induction in the absence of ligand are in large part unknown, but typically require cleavage by specific caspases. In this review we will present the proposed mechanisms for cell death induction by these receptors and their potential function in nervous system development and regulation of tumorigenesis.Received 19 December 2003; received after revision 19 February 2004; accepted 26 February 2004     

Proteasome inhibition overcomes the resistance of renal cell carcinoma cells against the PPARγ ligand troglitazone

In order to analyze the effects of peroxisome proliferator-activated receptor-γ (PPARγ) activation on renal cell carcinomas we utilized several cell lines that were treated with the high affinity PPARγ agonist, troglitazone. Incubation of RCC cells with troglitazone resulted in reduced secretion of growth factors that was due to the inhibition of MAP kinase signaling and reduced nuclear localized expression of relB and HIF1alpha. Interestingly, the cell lines used showed a different sensitivity towards apoptosis induction that did not correlate with the inhibition of growth factors or expression of pro- and antiapoptotic molecules. To overcome this resistance the cells were treated with a combination of troglitazone and the proteasome inhibitor, bortezomib. The combination of both compounds induced apoptosis even in cells resistant to both agents alone, due to increased induction of ER-stress and caspase-3 mediated cell death. Received 03 September 2009; received after revision 02 February 2009; accepted 10 February 2009     

Inhibition of proliferation by 1-8U in interferon-α-responsive and non-responsive cell lines

Interferon (IFN)-inducible proteins of the 1-8 gene family mediate homotypic adhesion and transduction of antiproliferative signals. Their induction correlates with inhibition of cell growth while they are often repressed in the course of malignant transformation and tumor development. Ras-mediated transformation of mouse mast cells is associated with downregulation of 1-8U expression and interferon-α (IFN-α) treatment reverts the proliferation rate to normal levels together with induction of 1-8U. Conversely, the antiproliferative responses of IFN-α in sensitive human melanoma cells are accompanied by 1-8U induction. Here we provide direct evidence that recombinant expression of 1-8U in human cell lines is sufficient to block cell proliferation. Based on the abundant expression and subcellular localization to the plasma membrane and exosome-like structures, we propose a model capable of explaining the pleiotropic functions of 1-8 family proteins in tumor cells and during normal development. Received 15 January 2003; received after revision 21 March 2003; accepted 25 March 2003 RID="*" ID="*"Corresponding author.     

Mitochondrial dysfunction-induced amphiregulin upregulation mediates chemo-resistance and cell migration in HepG2 cells

The aim of this study was to investigate the contribution of mitochondrial dysfunction to chemoresistance and migration of hepatoma cells. We found that inhibition of mitochondrial respiration and mitochondrial DNA (mtDNA) depletion resulted in induction of amphiregulin (AR) expression in HepG2 cells. Upon oligomycin treatment of HepG2 cells, the cytosolic Ca²⁺ was significantly raised after 30 min, and the intracellular level of reactive oxygen species (ROS) was elevated 2.2-fold after 4 h. Moreover, the condition medium of oligomycin-treated HepG2 cells was found to stimulate the migration of SK-Hep-1 cells. On the other hand, oligomycin-induced cisplatin-resistance and cell migration of HepG2 cells were attenuated by AR-specific RNA interference (#L-017435, Dharmacon) and a neutralizing antibody (MAB262, R&D Systems), respectively. Together, these findings suggest that mitochondrial dysfunction induced Ca²⁺ mobilization, and ROS overproduction, which modulated the chemo-resistance and migration of hepatoma cells through the induction and activation of AR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Y.-H. Wei, H.-C. Lee: These authors contribute equally to this work. Received 02 December 2008; received after revision 16 March 2009; accepted 17 March 2009     

Fenretinide induces mitochondrial ROS and inhibits the mitochondrial respiratory chain in neuroblastoma

Fenretinide induces apoptosis in neuroblastoma by induction of reactive oxygen species (ROS). In this study, we investigated the role of mitochondria in fenretinide-induced cytotoxicity and ROS production in six neuroblastoma cell lines. ROS induction by fenretinide was of mitochondrial origin, demonstrated by detection of superoxide with MitoSOX, the scavenging effect of the mitochondrial antioxidant MitoQ and reduced ROS production in cells without a functional mitochondrial respiratory chain (Rho zero cells). In digitonin-permeabilized cells, a fenretinide concentration-dependent decrease in ATP synthesis and substrate oxidation was observed, reflecting inhibition of the mitochondrial respiratory chain. However, inhibition of the mitochondrial respiratory chain was not required for ROS production. Co-incubation of fenretinide with inhibitors of different complexes of the respiratory chain suggested that fenretinide-induced ROS production occurred via complex II. The cytotoxicity of fenretinide was exerted through the generation of mitochondrial ROS and, at higher concentrations, also through inhibition of the mitochondrial respiratory chain.     

Matrix metalloproteinase 19 processes the laminin 5 gamma 2 chain and induces epithelial cell migration

In this study we analyzed the proteolytic activity of MMP-19 and its impact on keratinocyte migration. In the HaCaT keratinocyte cell line overexpressing wild-type MMP-19 (HaCaT-WT), transmigration through fibrin and type IV collagen matrices was significantly increased compared to cells harboring a catalytically inactive mutant (HaCaT-EA). Studying the expression of MMP-19 in early stages of squamous cell cancer (SCC), we found co-localization of MMP-19 and laminin 5 at the invading tumor front but not in suprabasal epidermis of the tumor. Examination of laminin 5 processing revealed increased processing of the 2 chain in the medium and matrix of HaCaT-WT cells and degradation by recombinant human MMP-19 to 105-kDa and 80-kDa fragments. Parental HaCaT grown on the matrix of HaCaT-WT and HaCaT-EA cells displayed differential tyrosine phosphorylation. Using integrin blocking and stimulating antibodies we could attribute these differences to a shift from 4-integrin-dependent signaling on the HaCaT-EA matrix toward 3-integrin-dependent signaling on the HaCaT-WT matrix. As a consequence, parental HaCaT showed increased migration on the matrix of HaCaT-WT cells. These data suggest that the MMP-19-dependent processing of the 2 chains leads to the integrin switch favoring epithelial migration and that MMP-19 actively participates in the early stages of SCC invasion.Received 29 October 2004; received after revision 7 December 2004; accepted 17 January 2005     

Inhibitor of growth tumor suppressors in cancer progression

The inhibitor of growth (ING) family of tumor suppressors has five members and is implicated in the control of apoptosis, senescence, DNA repair, and cancer progression. However, little is known about ING activity in the regulation of cancer progression. ING members and splice variants seem to behave differently with respect to cancer invasion and metastasis. Interaction with histone trimethylated at lysine 4 (H3K4me3), hypoxia inducible factor-1 (HIF-1), p53, and nuclear factor kappa-B (NF-κB) are potential mechanisms by which ING members exert effects on invasion and metastasis. Subcellular mislocalization, rapid protein degradation, and to a lesser extent ING gene mutation are among the mechanisms responsible for inappropriate ING levels in cancer cells. The aim of this review is to summarize the different roles of ING family tumor suppressors in cancer progression and the molecular mechanisms involved.