title: Verticillin A Inhibits Leiomyosarcoma and Malignant Peripheral Nerve Sheath Tumor Growth via Induction of Apoptosis.
abstract: OBJECTIVE
  The heterogeneity of soft tissue sarcoma (STS) represents a major challenge for the development of effective therapeutics. Comprised of over 50 different histology subtypes of various etiologies, STS subsets are further characterized as either karyotypically simple or complex. Due to the number of genetic anomalies associated with genetically complex STS, development of therapies demonstrating potency against this STS cluster is especially challenging and yet greatly needed. Verticillin A is a small molecule natural product with demonstrated anticancer activity; however, the efficacy of this agent has never been evaluated in STS. Therefore, the goal of this study was to explore verticillin A as a potential STS therapeutic.
  
  
  METHODS
  We performed survival (MTS) and clonogenic analyses to measure the impact of this agent on the viability and colony formation capability of karyotypically complex STS cell lines: malignant peripheral nerve sheath tumor (MPNST) and leiomyosarcoma (LMS). The in vitro effects of verticillin A on apoptosis were investigated through annexin V/PI flow cytometry analysis and by measuring fluorescently-labeled cleaved caspase 3/7 activity. The impact on cell cycle progression was assessed via cytometric measurement of propidium iodide intercalation.In vivostudies were performed using MPNST xenograft models. Tumors were processed and analyzed using immunohistochemistry (IHC) for verticillin A effects on growth (Ki67) and apoptosis (cleaved caspase 3).
  
  
  RESULTS
  Treatment with verticillin A resulted in decreased STS growth and an increase in apoptotic levels after 24 h. 100 nM verticillin A induced significant cellular growth abrogation after 24 h (96.7, 88.7, 72.7, 57, and 39.7% reduction in LMS1, S462, ST88, SKLMS1, and MPNST724, respectively). We observed no arrest in cell cycle, elevated annexin, and a nearly two-fold increase in cleaved caspase 3/7 activity in all MPNST and LMS cell lines. Control normal human Schwann (HSC) and aortic smooth muscle (HASMC) cells displayed higher tolerance to verticillin A treatment compared to sarcoma cell lines, although toxicity was seen in HSC at the highest treatment dose.In vivostudies mirrored the in vitro results: by day 11, tumor size was significantly reduced in MPNST724 xenograft models with treatment of 0.25 and 0.5 mg/kg verticillin A. Additionally, IHC assessment of tumors demonstrated increased cleaved caspase 3 and decreased proliferation (Ki67) following treatment with verticillin A.
  
  
  CONCLUSIONS
  Advancement in the treatment of karyotypically complex STS is confounded by the high level of genetic abnormalities found in these diseases. Consequently, the identification and investigation of novel therapies is greatly needed. Our data suggest that verticillin A selectively inhibits MPNST and LMS growth via induction of apoptosis while exhibiting minimal to moderate effects on normal cells, pointing to verticillin A as a potential treatment for MPNST and LMS, after additional preclinical validation.
authors.affiliation: Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA.
authors.forename: A
authors.initials: A
authors.lastname: Zewdu
authors.affiliation: Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA.
authors.forename: G
authors.initials: G
authors.lastname: Lopez
authors.affiliation: Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA.
authors.forename: D
authors.initials: D
authors.lastname: Braggio
authors.affiliation: Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA.
authors.forename: C
authors.initials: C
authors.lastname: Kenny
authors.affiliation: Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA.
authors.forename: D
authors.initials: D
authors.lastname: Constantino
authors.affiliation: Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; Resonant Therapeutics, Inc., Ann Arbor, Michigan, USA.
authors.forename: H K
authors.initials: HK
authors.lastname: Bid
authors.affiliation: Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA.
authors.forename: K
authors.initials: K
authors.lastname: Batte
authors.affiliation: Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; Department of Pathology, Comprehensive Cancer Center, Ohio State University, Columbus, Ohio, USA.
authors.forename: O H
authors.initials: OH
authors.lastname: Iwenofu
authors.affiliation: Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina, USA.
authors.forename: N H
authors.initials: NH
authors.lastname: Oberlies
authors.affiliation: Mycosynthetix, Inc., Hillsborough, North Carolina, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Pearce
authors.affiliation: Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; Department of Cancer Biology and Genetics, Ohio State University, Columbus, Ohio, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Strohecker
authors.affiliation: Surgery B, Sheba Medical Center, Tel Aviv, Israel.
authors.forename: D
authors.initials: D
authors.lastname: Lev
authors.affiliation: Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Pollock
keywords: Apoptosis; Epipolythiodioxopiperazine alkaloid; LMS; Leiomyosarcoma; MPNST; Malignant peripheral nerve sheath tumor; STS; Soft tissue sarcoma; Verticillin A