Delivery of mGluR5 siRNAs by Iron Oxide Nanocages by Alternating Magnetic Fields for Blocking Proliferation of Metastatic Osteosarcoma Cells.

Although osteosarcoma is the most common primary malignant bone tumor, chemotherapeutic drugs and treatment have failed to increase the five-year survival rate over the last three decades. We previously demonstrated that type 5 metabotropic glutamate receptor, mGluR5, is required to proliferate metastatic osteosarcoma cells. In this work, we delivered mGluR5 siRNAs in vitro using superparamagnetic iron oxide nanocages (IO-nanocages) as delivery vehicles and applied alternating magnetic fields (AMFs) to improve mGluR5 siRNAs release. We observed functional outcomes when mGluR5 expression is silenced in human and mouse osteosarcoma cell lines. The results elucidated that the mGluR5 siRNAs were successfully delivered by IO-nanocages and their release was enhanced by AMFs, leading to mGluR5 silencing. Moreover, we observed that the proliferation of both human and mouse osteosarcoma cells decreased significantly when mGluR5 expression was silenced in the cells. This novel magnetic siRNA delivery methodology was capable of silencing mGluR5 expression significantly in osteosarcoma cell lines under the AMFs, and our data suggested that this method can be further used in future clinical applications in cancer therapy.

Riluzole-induced apoptosis in osteosarcoma is mediated through Yes-associated protein upon phosphorylation by c-Abl Kinase.

Our lab has previously demonstrated Riluzole to be an effective drug in inhibiting proliferation and inducing apoptosis in both human and mouse osteosarcoma. Yes-associated protein is a transcription co-activator, known to be involved in cell proliferation or apoptosis depending on its protein partner. In the present study we investigated the role of YAP in apoptosis in osteosarcoma, we hypothesized that YAP may be activated by Riluzole to induce apoptosis in osteosarcoma. By knocking down the expression of YAP, we have demonstrated that Riluzole failed to induce apoptosis in YAP deficient osteosarcoma cells. Riluzole caused translocation of YAP from the cytoplasm to the nucleus, indicating YAP's role in apoptosis. Both Riluzole-induced phosphorylation of YAP at tyrosine 357 and Riluzole-induced apoptosis were blocked by inhibitors of c-Abl kinase. In addition, knockdown of c-Abl kinase prevented Riluzole-induced apoptosis in LM7 cells. We further demonstrated that Riluzole promoted interaction between YAP and p73, while c-Abl kinase inhibitors abolished the interaction. Subsequently, we demonstrated that Riluzole enhanced activity of the Bax promoter in a luciferase reporter assay and enhanced YAP/p73 binding on endogenous Bax promoter in a ChIP assay. Our data supports a novel mechanism in which Riluzole activates c-Abl kinase to regulate pro-apoptotic activity of YAP in osteosarcoma.

Riluzole: A neuroprotective drug with potential as a novel anti­cancer agent (Review).

Riluzole, a glutamate release inhibitor, has been in use for the treatment of amyotrophic lateral sclerosis for over two decades since its approval by the Food and Drug Administration. Recently, riluzole has been evaluated in cancer cells and indicated to block cell proliferation and/or induce cell death. Riluzole has been proven effective as an anti­neoplastic drug in cancers of various tissue origins, including the skin, breast, pancreas, colon, liver, bone, brain, lung and nasopharynx. While cancer cells expressing glutamate receptors frequently respond to riluzole treatment, numerous types of cancer cell lacking glutamate receptors unexpectedly responded to riluzole treatment as well. Riluzole was demonstrated to interfere with glutamate secretion, growth signaling pathways, Ca2+ homeostasis, glutathione synthesis, reactive oxygen species generation and integrity of DNA, as well as autophagic and apoptotic pathways. Of note, riluzole is highly effective in inducing cell death in cisplatin­resistant lung cancer cells. Furthermore, riluzole pretreatment sensitizes glioma and melanoma to radiation therapy. In addition, in triple­negative breast cancer, colorectal cancer, melanoma and glioblastoma, riluzole has synergistic effects in combination with select drugs. In an effort to highlight the therapeutic potential of riluzole, the current study reviewed the effect and outcome of riluzole treatment on numerous cancer types investigated thus far. The mechanism of action and the various molecular pathways affected by riluzole are discussed.

Osteosarcoma cell proliferation and survival requires mGluR5 receptor activity and is blocked by Riluzole.

Osteosarcomas are malignant tumors of bone, most commonly seen in children and adolescents. Despite advances in modern medicine, the poor survival rate of metastatic osteosarcoma has not improved in two decades. In the present study we have investigated the effect of Riluzole on a human and mouse metastatic osteosarcoma cells. We show that LM7 cells secrete glutamate in the media and that mGluR5 receptors are required for the proliferation of LM7 cells. Riluzole, which is known to inhibit glutamate release, inhibits proliferation, induces apoptosis and prevents migration of LM7 cells. This is also seen with Fenobam, a specific blocker of mGluR5. We also show that Riluzole alters the phosphorylation status of AKT/P70 S6 kinase, ERK1/2 and JNK1/2. Thus Riluzole is an effective drug to inhibit proliferation and survival of osteosarcoma cells and has therapeutic potential for the treatment of osteosarcoma exhibiting autocrine glutamate signaling.

Synergistic inhibition of survival, proliferation, and migration of U87 cells with a combination of LY341495 and Iressa.

Glioblastomas exploit various molecular pathways to promote glutamate- dependent growth by activating the AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid) receptor, the group II metabotropic glutamate receptor, mGluR, and the epidermal growth factor receptor, EGFR. We hypothesized that targeting more than one of these pathways would be more effective in inhibiting glutamate-dependent growth. Using a model of U87 cell line, we show that blocking glutamate release by Riluzole inhibits cell proliferation. Glutamate-dependent growth is effectively inhibited by a combination of Iressa, an inhibitor of EGFR activation and LY341495, a group II mGluR inhibitor. Treatment of U87 cells with a combination of Iressa and LY341495 inhibits proliferation as indicated by Ki-67 staining, induces apoptosis and inhibits migration of U87 cells more effectively than the treatment by Iressa or LY341495 alone. These results demonstrate that a combinatorial therapy with Iressa and LY341495 is more effective due to synergistic effects of these drugs in inhibiting the growth of glioblastoma.

Sucrose ingestion induces rapid AMPA receptor trafficking.

The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.

Molecular cloning of Drosophila HCF reveals proteolytic processing and self-association of the encoded protein.

HCF-1 functions as a coactivator for herpes simplex virus VP16 and a number of mammalian transcription factors. Mature HCF-1 is composed of two subunits generated by proteolytic cleavage of a larger precursor at six centrally-located HCF(PRO) repeats. The resulting N- and C-terminal subunits remain tightly associated via two complementary pairs of self-association domains: termed SAS1N-SAS1C and SAS2N-SAS2C. Additional HCF proteins have been identified in mammals (HCF-2) and Caenorhabditis elegans (CeHCF). Both contain well-conserved SAS1 domains but do not undergo proteolytic processing. Thus, the significance of the cleavage and self-association of HCF-1 remains enigmatic. Here, we describe the isolation of the Drosophila HCF homologue (dHCF) using a genetic screen based on conservation of the SAS1 interaction. The N-terminal beta-propeller domain of dHCF supports VP16-induced complex formation and is more similar to mammalian HCF-1 than other homologues. We show that full-length dHCF expressed in Drosophila cells undergoes proteolytic cleavage giving rise to tightly associated N- and C-terminal subunits. As with HCF-1, the SAS1N and SAS1C elements of dHCF are separated by a large central region, however, this sequence lacks obvious homology to the HCF(PRO) repeats required for HCF-1 cleavage. The conservation of HCF processing in insect cells argues that formation of separate N- and C-terminal subunits is important for HCF function.

Interaction of HCF-1 with a cellular nuclear export factor.

HCF-1 is a cellular protein required by VP16 to activate the herpes simplex virus (HSV) immediate-early genes. VP16 is a component of the viral tegument and, after release into the cell, binds to HCF-1 and translocates to the nucleus to form a complex with the POU domain protein Oct-1 and a VP16-responsive DNA sequence. This VP16-induced complex boosts transcription of the viral immediate-early genes and initiates lytic replication. In uninfected cells, HCF-1 functions as a coactivator for the cellular transcription factors LZIP and GABP and also plays an essential role in cell proliferation. VP16 and LZIP share a tetrapeptide HCF-binding motif recognized by the beta-propeller domain of HCF-1. Here we describe a new cellular HCF-1 beta-propeller domain binding protein, termed HPIP, which contains a functional HCF-binding motif and a leucine-rich nuclear export sequence. We show that HPIP shuttles between the nucleus and cytoplasm in a CRM1-dependent manner and that overexpression of HPIP leads to accumulation of HCF-1 in the cytoplasm. These data suggest that HPIP regulates HCF-1 activity by modulating its subcellular localization. Furthermore, HPIP-mediated export may provide the pool of cytoplasmic HCF-1 required for import of virion-derived VP16 into the nucleus.