Alternatively, when the use of a new cancer drug is expanded to malignant brain tumors, especially GBM, their distribution, pharmacokinetics, and BBB permeability become an issue

Alternatively, when the use of a new cancer drug is expanded to malignant brain tumors, especially GBM, their distribution, pharmacokinetics, and BBB permeability become an issue. The concept of drug repositioning has recently drawn attention and involves old clinical drugs being put to practical use for another disease and another target. and glioma stem cells by suppressing both FAK and Akt/mammalian target of rapamycin signaling. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same dose of anti-depressant effect. In conclusion, fluvoxamine is usually a promising anti-invasive treatment against GBM with reliable approach. Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, with a median survival of approximately 1 year. Despite advances in diagnostics and treatment, the prognosis for GBM has not significantly improved in recent decades1. This poor prognosis is mainly due to the highly invasive nature of GBM cells. Diffused GBM cell invasion into surrounding normal brain tissue prevents complete surgical resection of GBM tumors and results in recurrence. Furthermore, in the central nervous system, most anti-cancer drugs, including molecular-targeted drugs, forming a first line of treatment against various cancers are ineffective because the BBB prevents their delivery into the brain2. Therefore, the development of novel anti-invasive drugs that can permeate the BBB is essential for treatment of GBM. Recent studies have identified CD133+ glioma-initiating cells (GICs) that exhibit stem cell-like properties3,4.These GICs possess capacities for tumorigenesis, self-renewal, and differentiation into multiple cell types, such as neurons, astrocytes, and oligodendrocytes4,5. GICs have been shown to be highly invasive and resistant to chemotherapy and radiotherapy6,7,8. Therefore, GICs are thought to be responsible for the poor prognosis of GBM and constitute a potential target for GBM therapy. Tumor cell migration and invasion require dynamic reorganization of the actin cytoskeleton9,10. Migrating cells produce membrane protrusions, such as filopodia, lamellipodia, invadopodia, focal adhesions, and stress fibers11. Because these structures of migrating cells require precise regulation of actin polymerization and depolymerization, control of actin polymerization in tumor cells around the leading edge of the tumor may inhibit invasion and migration of GBM cells into normal brain. In terms of drug development and clinical applications, the cost of development and unexpected side effects just before clinical use obstruct the process from basic research to clinical use. As a result, obtaining new uses for existing clinically used drugs, termed drug repositioning or repurposing, is an alternative strategy for drug discovery and development12. This approach has been widely attempted and has been successful in some cases (e.g., aspirin as an anti-platelet medication, sildenafil for erectile dysfunction, etc.)12,13. Because the pharmacokinetics of most existing clinically used drugs have already been studied, the effective dose, possible side effects, cost are already known and the time required to bring these drugs to market can be reduced14. Results Fluvoxamine found to inhibit actin polymerization using a new screening method for quantitative determination of actin polymerization Reorganization of the actin cytoskeleton is essential for cancer cell migration and invasion. Therefore, we established a new drug screening method for quantitative determination of actin polymerization and screened clinically used drugs that can penetrate the BBB. To test the new screening method, we first screened inhibitors of actin polymerization from among 18 clinically used drugs that can permeate the BBB (Table 1) using a pyrene-actin-based actin polymerization assay. This assay is based on enhancement of the fluorescence of pyrene-labeled G-actin (monomer) that occurs during polymerization (Fig. 1aCc). Each drug was added to the reaction mixture at a concentration of 40?M, and the fluorescence of pyrene-actin was measured. We found that drug No. 16, the antidepressant fluvoxamine, exhibited the most potent inhibition against actin polymerization (Fig. 1d). Open in a separate window Physique 1 Pyrene-actin-based screening identified fluvoxamine as a potent inhibitor of actin polymerization.(aCc) Schematic diagram of Rolitetracycline screening performed. Pyrene-labeled G-actin was polymerized by stimulation with liposomes (50% phosphatidylcholine, 50% phosphatidylserine) in the reaction buffer made up of mouse brain cytosol, ATP, and GTP. Ex/Em: 365/407?nm. (d) Drug No. 16 (fluvoxamine, arrow) effectively inhibited actin polymerization. Each drug (40?M) was added to the reaction mixture to assess its effect on actin polymerization. The fluorescence intensity of each sample was normalized to the DMSO control at 2000?s. (e) Actin polymerization was monitored by a visual assay. Mouse brain cytosol pretreated with 80?M fluvoxamine (middle panel) or 80?M dynasore (bottom panel) for 30?min before incubation. Control cytosol was pretreated with 0.1% DMSO (upper panel). Scale bar,.4b). Open in a separate window Figure 4 Fluvoxamine inhibited invasion of hGICs and prolonged the survival of GBM mice.(a) Daily treatment with fluvoxamine inhibited tumor cell invasion analysis, and high-throughput screening for the identification of new drugs against new cancer targets; however, these techniques may require a large developmental budget and long-term development. both FAK and Akt/mammalian target of rapamycin signaling. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same dose of anti-depressant effect. In conclusion, fluvoxamine is a promising anti-invasive treatment against GBM with reliable approach. Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, with a median survival of approximately 1 year. Despite advances in diagnostics and treatment, the prognosis for GBM has not significantly improved in recent decades1. This poor prognosis is mainly due to the highly invasive nature of GBM cells. Diffused GBM cell invasion into surrounding normal brain tissue prevents complete surgical resection of GBM tumors and results in recurrence. Rolitetracycline Furthermore, in Rolitetracycline the central nervous system, most anti-cancer drugs, including molecular-targeted drugs, forming a first line of treatment against various cancers are ineffective because the BBB prevents their delivery into the brain2. Therefore, the development of novel anti-invasive drugs that can permeate Rolitetracycline the BBB is essential for treatment of GBM. Recent studies have identified CD133+ glioma-initiating cells (GICs) that exhibit stem cell-like properties3,4.These GICs possess capacities for tumorigenesis, self-renewal, and differentiation into multiple cell types, such as neurons, astrocytes, and oligodendrocytes4,5. GICs have been shown to be highly invasive and resistant to chemotherapy and radiotherapy6,7,8. Therefore, GICs are thought to be responsible for the poor prognosis of GBM and constitute a potential target for GBM therapy. Tumor cell migration and invasion require dynamic reorganization of the actin cytoskeleton9,10. Migrating cells produce membrane protrusions, such as filopodia, lamellipodia, invadopodia, focal adhesions, and stress fibers11. Because these structures of migrating cells require precise regulation of actin polymerization and depolymerization, control of actin polymerization in tumor cells on the leading edge of the tumor may inhibit invasion and migration of GBM cells into normal brain. In terms of drug development and clinical applications, the cost of development and unexpected side effects just before clinical use obstruct the process from basic research to clinical use. As a result, finding new uses for existing clinically used drugs, termed drug repositioning or repurposing, is an alternative strategy for drug discovery and development12. This approach has been widely attempted and has been successful in some cases (e.g., aspirin as an anti-platelet medication, sildenafil for erectile dysfunction, etc.)12,13. Because the pharmacokinetics of most existing clinically used drugs have already been studied, the effective dose, possible side effects, cost are already known and the time required to bring these drugs to market can be reduced14. Results Fluvoxamine found to inhibit actin polymerization using a new screening method for quantitative determination of actin polymerization Reorganization of the actin cytoskeleton is essential for cancer cell migration and invasion. Therefore, we established a new drug screening method for quantitative determination of actin polymerization and Rabbit polyclonal to PNLIPRP1 screened clinically used drugs that can penetrate the BBB. To test the new screening method, we first screened inhibitors of actin polymerization from among 18 clinically used drugs that can permeate the BBB (Table 1) using a pyrene-actin-based actin polymerization assay. This assay is based on enhancement of the fluorescence of pyrene-labeled G-actin (monomer) that occurs during polymerization (Fig. 1aCc). Each drug was added to the reaction mixture at a concentration of 40?M, and the fluorescence of pyrene-actin was measured. We found that Rolitetracycline drug No. 16, the antidepressant fluvoxamine, exhibited the most potent inhibition against actin polymerization (Fig. 1d). Open in a separate window Figure 1 Pyrene-actin-based screening identified fluvoxamine as a potent inhibitor of actin polymerization.(aCc) Schematic diagram of screening performed. Pyrene-labeled G-actin was polymerized by stimulation with liposomes (50% phosphatidylcholine, 50% phosphatidylserine) in the reaction buffer containing mouse brain cytosol, ATP, and GTP. Ex/Em: 365/407?nm. (d) Drug No. 16 (fluvoxamine, arrow) effectively inhibited actin polymerization. Each drug (40?M) was added to the reaction mixture to assess its effect on actin polymerization. The fluorescence intensity of each sample was normalized to the DMSO control at 2000?s. (e) Actin polymerization was monitored by a visual assay. Mouse brain cytosol pretreated with 80?M fluvoxamine (middle panel) or 80?M dynasore (bottom panel) for 30?min before incubation. Control cytosol was pretreated with 0.1% DMSO (upper panel). Scale bar, 10?mm. (f) Representative time course of actin polymerization in the presence of DMSO (black line) or 40?M fluvoxamine (red line). (g) Fluvoxamine inhibited actin polymerization in a concentration-dependent manner, and its IC50 was ~30?M. Values are mean??SEM from three experiments. Table 1 List of drugs used in this study. actin assembly assay, we.