Enhancing Anti-Cancer Therapy with Selective Autophagy Inhibitors by Targeting Protective Autophagy

Autophagy is a process of eliminating damaged or unnecessary proteins and organelles, thereby maintaining intracellular homeostasis. Deregulation of autophagy is associated with several diseases including cancer. Contradictory dual roles of autophagy have been well established in cancer. Cytoprotective mechanism of autophagy has been extensively investigated for overcoming resistance to cancer therapies including radiotherapy, targeted therapy, immunotherapy, and chemotherapy. Selective autophagy inhibitors that directly target autophagic process have been developed for cancer treatment. Efficacies of autophagy inhibitors have been tested in various pre-clinical cancer animal models. Combination therapies of autophagy inhibitors with chemotherapeutics are being evaluated in clinal trials. In this review, we will focus on genetical and pharmacological perturbations of autophagy-related proteins in different steps of autophagic process and their therapeutic benefits. We will also summarize combination therapies of autophagy inhibitors with chemotherapies and their outcomes in pre-clinical and clinical studies. Understanding of current knowledge of development, progress, and application of cytoprotective autophagy inhibitors in combination therapies will open new possibilities for overcoming drug resistance and improving clinical outcomes.

Autophagy Is a Potential Target for Enhancing the Anti-Angiogenic Effect of Mebendazole in Endothelial Cells

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has recently been noted as a repositioning candidate for angiogenesis inhibition and cancer therapy. However, the definite anti-angiogenic mechanism of MBZ remains unclear. In this study, we explored the inhibitory mechanism of MBZ in endothelial cells (ECs) and developed a novel strategy to improve its anti-angiogenic therapy. Treatment of ECs with MBZ led to inhibition of EC proliferation in a dose-dependent manner in several culture conditions in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or FBS, without selectivity of growth factors, although MBZ is known to inhibit VEGF receptor 2 kinase. Furthermore, MBZ inhibited EC migration and tube formation induced by either VEGF or bFGF. However, unexpectedly, treatment of MBZ did not affect FAK and ERK1/2 phosphorylation induced by these factors. Treatment with MBZ induced shrinking of ECs and caused G2-M arrest and apoptosis with an increased Sub-G1 fraction. In addition, increased levels of nuclear fragmentation, p53 expression, and active form of caspase 3 were observed. The marked induction of autophagy by MBZ was also noted. Interestingly, inhibition of autophagy through knocking down of Beclin1 or ATG5/7, or treatment with autophagy inhibitors such as 3-methyladenine and chloroquine resulted in marked enhancement of anti-proliferative and pro-apoptotic effects of MBZ in ECs. Consequently, we suggest that MBZ induces autophagy in ECs and that protective autophagy can be a novel target for enhancing the anti-angiogenic efficacy of MBZ in cancer treatment.

A ROCK Inhibitor Blocks the Inhibitory Effect of Chondroitin Sulfate Proteoglycan on Morphological Changes of Mesenchymal Stromal/Stem Cells into Neuron-Like Cells

Chondroitin sulfate proteoglycan (CSPG) inhibits neurite outgrowth of various neuronal cell types, and CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK pathway. Mesenchymal stromal/stem cells (MSCs) have the potential to differentiate into neuron-like cells under specific conditions and have been shown to differentiate into neuron-like cells by co-treatment with the ROCK inhibitor Y27632 and the hypoxia condition mimicking agent CoCl2. In this study, we addressed the hypothesis that a ROCK inhibitor might be beneficial to regenerate neurons during stem cell therapy by preventing transplanted MSCs from inhibition by CSPG in damaged tissues. Indeed, dose-dependent inhibition by CSPG pretreatment was observed during morphological changes of Wharton's jelly-derived MSCs (WJ-MSCs) induced by Y27632 alone. The formation of neurite-like structures was significantly inhibited when WJ-MSCs were pre-treated with CSPG before induction under Y27632 plus CoCl2 conditions, and pretreatment with a protein kinase C inhibitor reversed such inhibition. However, CSPG treatment resulted in no significant inhibition of the WJ-MSC morphological changes into neuron-like cells after initiating induction by Y27632 plus CoCl2. No marked changes were detected in expression levels of neuronal markers induced by Y27632 plus CoCl2 upon CSPG treatment. CSPG also blocked the morphological changes of human bone marrow-derived MSCs into neuron-like cells under other neuronal induction condition without the ROCK inhibitor, and Y27632 pre-treatment blocked the inhibitory effect of CSPG. These results suggest that a ROCK inhibitor can be efficiently used in stem cell therapy for neuronal induction by avoiding hindrance from CSPG.

Differential inhibition of endothelial cell proliferation and migration by urokinase subdomains: amino-terminal fragment and kringle domain

The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50)= 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.

In vitro Differentiation of Endothelial Precursor Cells Derived from Umbilical Cord Blood

BACKGROUND AND OBJECTIVES: Ex vivo expansion of endothelial cells is important when applying cell therapy to therapeutic angiogenesis in ischemic tissues. Endothelial precursor cells (EPCs) from the umbilical cord blood are one of adult stem cell. In order to establish the culture system for EPCs, we examined the effects of the media and matrix on the differentiation of a subset of mononuclear cells to endothelial cells, and analyzed their endothelial-lineage phenotype. MATERIALS AND METHODS: Mononuclear cells isolated from human umbilical cord blood were cultured in a chamber slide coated with fibronectin or gelatin in a M199 medium supplemented with 10% fetal bovine serum (FBS) (the normal medium) or with 20% FBS and ECGS (the rich medium). Changes in the morphology and the attainment of DiI-ac-LDL uptake ability were examined during a 7 day period. The attached cells were immunostained for CD31, KDR, and vWF. RESULTS: The fibronectin matrix gave rise to more attached cells than the gelatin matrix (about 1.5 fold). The numbers of attached cells were no different between the normal medium and the rich medium at day 3 and 7, and were about 12% of the seeded mononuclear cells. However, the cell size and the numbers of longer spindle-shaped cells increased with the rich medium. Moreover, there was no increase in cellular population, but a 2-3 fold increase in the cellular size between day 3 and 7. About 20-40% of the attached cells acquired the DiI-ac-LDL uptake ability at day 3, whereas more than 85% of the attached cells could be stained with fluorescent DiI-ac-LDL at day 7 (p<0.001). The attached cells after being cultured for 7 days were stained moderately with the antibodies of CD31, or KDR. However, the cells at day 7 were only weakly immunostained with the vWF antibody, whereas more than 90% of cells were strongly stained at day 14. CONCLUSION: These results suggest that a subset of mononuclear cells derived from cord blood cells can give rise to cells with an endothelial cell-like phenotype, in vitro, at high percentages, which could be applied to in vivo vasculogenesis.

Potentials and limitations of adenovirus-p53 gene therapy for brain tumors

We investigated the antineoplastic potentials of recombinant adenovirus containing wild-type p53 cDNA (Ad5CMV-p53) for malignant gliomas. In four human glioma cell lines (U-251 and LG expressing endogenous mutant p53, and U-87 and EFC-2 expressing wild-type p53) and two rat glioma cell lines (9L and C6, each expressing mutant and wild-type p53), gene transfer efficiency determined by X-gal staining and Western blotting was varied (10-99% at 10-500 multiplicity of infection, MOI). Growth inhibitory effect was drastic (>90% at 100 MOI) in U-251 cells and only moderate or minimal in other cell lines harboring wild-type p53 or low gene transfer efficiency. Ex vivo transduction of U-251 cells with Ad5CMV-p53 suppressed the in vivo tumorigenicity of the cells. Histopathologic examination for Ad5CMV-p53 toxicity to rat brains showed inflammatory reactions in half of the tested brains at 10(8) MOI. U-251 cells were inoculated intracerebrally in nude mice and injected Ad5CMV-p53 into the tumor, in which neither the tumor suppression nor the survival benefit was observed. In conclusion, heterogeneity of the cellular subpopulations of malignant glioma in p53 status, variable and insufficient gene delivery to tumor, and adenoviral toxicity to brain at higher doses may be limiting factors to be solved in developing adenovirus-p53 gene therapy for malignant gliomas.

Potentials and limitations of adenovirus-p53 gene therapy for brain tumors

We investigated the antineoplastic potentials of recombinant adenovirus containing wild-type p53 cDNA (Ad5CMV-p53) for malignant gliomas. In four human glioma cell lines (U-251 and LG expressing endogenous mutant p53, and U-87 and EFC-2 expressing wild-type p53) and two rat glioma cell lines (9L and C6, each expressing mutant and wild-type p53), gene transfer efficiency determined by X-gal staining and Western blotting was varied (10-99% at 10-500 multiplicity of infection, MOI). Growth inhibitory effect was drastic (>90% at 100 MOI) in U-251 cells and only moderate or minimal in other cell lines harboring wild-type p53 or low gene transfer efficiency. Ex vivo transduction of U-251 cells with Ad5CMV-p53 suppressed the in vivo tumorigenicity of the cells. Histopathologic examination for Ad5CMV-p53 toxicity to rat brains showed inflammatory reactions in half of the tested brains at 10(8) MOI. U-251 cells were inoculated intracerebrally in nude mice and injected Ad5CMV-p53 into the tumor, in which neither the tumor suppression nor the survival benefit was observed. In conclusion, heterogeneity of the cellular subpopulations of malignant glioma in p53 status, variable and insufficient gene delivery to tumor, and adenoviral toxicity to brain at higher doses may be limiting factors to be solved in developing adenovirus-p53 gene therapy for malignant gliomas.

Production of Soluble VEGF Receptor Mutants for Inhibition of Angiogenesis / 대한암학회지

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent angiogenic factor of many solid tumors, promoting vascularization and formation of metastases. In an attempt to generate effective VEGF inhibitors, the authors constructed the VEGF receptor mutants, expressed in E. coli and Sf9 insect cells, and examined their binding to VEGF. MATERIALS AND METHODS: The cDNA fragment encoding FLT-1 extracellular domain was cloned from human umbilical vein endothelial (HUVE) cell total RNA using RT-PCR. PCR- subcloning was performed using this template, in order to generate the deletion mutants by introducing FLT-1 partial sequences into E.coli expression vector pET-21d and baculovirus transfer vactors, pBAC-1 and pBAC-3. Two mutant proteins from baculovirus-infected insect cells were purified by heparin sepharose chromatography and immobilized into nitrdegrees Cellulose membrane followed by 125I-VEGF binding assay. RESULTS: Two mutant receptors, sFLT (1~7) and sFLT (2~4) expressed in E.coli appeared in inclusion body as insoluble proteins. The soluble mutant receptors were produced in low yield by baculovirus/insect cell expression system. Both immobilized mutant receptors, sFLT (1~7) and sFLT (2~4) were able to bind VEGF. CONCLUSION: These results suggest that a small soluble mutant receptor, sFLT (2~4), as well as sFLT (1~7) may be used effectively for bldegrees Cking angiogenic function of VEGF.

Purification and characterization of recombinant murine endostatin in E. coli

Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent. Copyright 2000 Academic Press.