actinonin and Glioblastoma

Glioblastoma multiforme (GBM) is a common intracranial primary tumor of the central nervous system with high malignancy, poor prognosis, and short survival. Studies have shown that mitochondrial energy metabolism plays an important role in GBM chemotherapy resistance, suggesting that interrupting mitochondrial oxidative phosphorylation (OXPHOS) may improve GBM treatment. Human peptide deformylase (HsPDF) is a mitochondrial deformylase that removes the formylated methionine from the N-terminus of proteins encoded by mitochondrial DNA (mtDNA), thereby contributing to correct protein folding and participating in the assembly of the electron respiratory chain complex. In this study, we found that the expression of mtDNA-encoded proteins was significantly downregulated after treatment of GBM cells U87MG and LN229 with the HsPDF inhibitor, actinonin. In combination with temozolomide, a preferred chemotherapeutic medicine for GBM, the OXPHOS level decreased, mitochondrial protein homeostasis was unbalanced, mitochondrial fission increased, and the integrated stress response was activated to promote mitochondrial apoptosis. These findings suggest that HsPDF inhibition is an important strategy for overcoming chemoresistance of GBM cells.

Topics: Amidohydrolases; Antineoplastic Agents, Alkylating; Brain Neoplasms; Cell Line, Tumor; DNA, Mitochondrial; Drug Resistance, Neoplasm; Glioblastoma; Humans; Hydroxamic Acids; Methionine; Mitochondrial Proteins; Temozolomide

Pre-clinical trials for cancer therapeutics support the anti-neoplastic properties of the lectin from Canavalia ensiformis (Concanavalin-A, ConA) in targeting apoptosis and autophagy in a variety of cancer cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP), a plasma membrane-anchored matrix metalloproteinase, is a glycoprotein strongly expressed in radioresistant and chemoresistant glioblastoma that mediates pro-apoptotic signalling in brain cancer cells, we investigated whether MT1-MMP could also signal autophagy. Among the four lectins tested, we found that the mannopyranoside/glucopyranoside-binding ConA, which is also well documented to trigger MT1-MMP expression, increases autophagic acidic vacuoles formation as demonstrated by Acridine Orange cell staining. Although siRNA-mediated MT1-MMP gene silencing effectively reversed ConA-induced autophagy, inhibition of the MT1-MMP extracellular catalytic function with Actinonin or Ilomastat did not. Conversely, direct overexpression of the recombinant Wt-MT1-MMP protein triggered proMMP-2 activation and green fluorescent protein-microtubule-associated protein light chain 3 puncta indicative of autophagosomes formation, while deletion of MT1-MMP's cytoplasmic domain disabled such autophagy induction. ConA-treated U87 cells also showed an upregulation of BNIP3 and of autophagy-related gene members autophagy-related protein 3, autophagy-related protein 12 and autophagy-related protein 16-like 1, where respective inductions were reversed when MT1-MMP gene expression was silenced. Altogether, we provide molecular evidence supporting the pro-autophagic mechanism of action of ConA in glioblastoma cells. We also highlight new signal transduction functions of MT1-MMP within apoptotic and autophagic pathways that often characterize cancer cell responses to chemotherapeutic drugs.

Topics: Acridine Orange; Autophagy; Brain Neoplasms; Cell Line, Tumor; Concanavalin A; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioblastoma; Humans; Hydroxamic Acids; Indoles; Mannose; Matrix Metalloproteinase 14; Neoplasm Proteins; Protein Structure, Tertiary; RNA, Small Interfering; Signal Transduction; Vacuoles

Recent profiling has identified the aminopeptidase N/CD13 inhibitor actinonin as a selective soluble secreted matrix metalloproteinase (MMP) inhibitor. Given that actinonin's effects against membrane-bound MMPs remain unknown and that MT1-MMP has been linked to chemo- and radio-therapy resistance in brain tumor development, we therefore assessed MT1-MMP functional inhibition by actinonin in U87 glioblastoma cells. We show that actinonin inhibits concanavalin-A (ConA)-induced proMMP-2 activation, while it does not inhibit ConA-induced MT1-MMP gene expression suggesting post-transcriptional effects of the drug possibly mediated through the membrane-anchored protease regulator RECK. Specific gene silencing of MT1-MMP with siRNA abrogated the ability of ConA to activate proMMP-2. Functional recombinant MT1-MMP whose constitutive expression led to proMMP-2 activation was also efficiently antagonized by actinonin. We provide evidence for actinonin's new therapeutic application in the direct targeting of MT1-MMP-mediated proMMP-2 activation, an essential step in both brain tumor infiltration and in brain tumor-associated angiogenesis.

Topics: Antineoplastic Agents; CD13 Antigens; Cell Line, Tumor; Concanavalin A; Enzyme Activation; Enzyme Precursors; Glioblastoma; Humans; Hydroxamic Acids; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; PHEX Phosphate Regulating Neutral Endopeptidase; Protein Binding