Treatment of a chemoresistant neuroblastoma cell line with the antimalarial ozonide OZ513.
Evaluate the anti-tumor activity of ozonide antimalarials using a chemoresistant neuroblastoma cell line, BE (2)-c.



The activity of 12 ozonides, artemisinin, and two semisynthetic artemisinins were tested for activity against two neuroblastoma cell-lines (BE (2)-c and IMR-32) and the Ewing's Sarcoma cell line A673 in an MTT viability assay. Time course data indicated that peak effect was seen 18 h after the start of treatment thus 18 h pre-treatment was used for all subsequent experiments. The most active ozonide (OZ513) was assessed in a propidium iodide cell cycle flow cytometry analysis which measured cell cycle transit and apoptosis. Metabolic effects of OZ513 in BE (2)-c cells was evaluated. Western blots for the apoptotic proteins cleaved capase-3 and cleaved PARP, the highly amplified oncogene MYCN, and the cell cycle regulator CyclinD1, were performed. These in-vitro experiments were followed by an in-vivo experiment in which NOD-scid gamma immunodeficient mice were injected subcutaneously with 1 × 106BE (2)-c cells followed by immediate treatment with 50-100 mg/kg/day doses of OZ513 administered IP three times per week out to 23 days after injection of tumor. Incidence of tumor development, time to tumor development, and rate of tumor growth were assessed in DMSO treated controls (N = 6), and OZ513 treated mice (N = 5).



It was confirmed that five commonly used chemotherapy drugs had no cytotoxic activity in BE (2)-c cells. Six of 12 ozonides tested were active in-vitro at concentrations achievable in vivo with OZ513 being most active (IC50 = 0.5 mcg/ml). OZ513 activity was confirmed in IMR-32 and A673 cells. The Ao peak on cell-cycle analysis was increased after treatment with OZ513 in a concentration dependent fashion which when coupled with results from western blot analysis which showed an increase in cleaved capase-3 and cleaved PARP supported an increase in apoptosis. There was a concentration dependent decline in the MYCN and a cyclinD1 protein indicative of anti-proliferative activity and cell cycle disruption. OXPHOS metabolism was unaffected by OZ513 treatment while glycolysis was increased. There was a significant delay in time to tumor development in mice treated with OZ513 and a decline in the rate of tumor growth.



The antimalarial ozonide OZ513 has effective in-vitro and in-vivo activity against a pleiotropic drug resistant neuroblastoma cell-line. Treatment with OZ513 increased apoptotic markers and glycolysis with a decline in the MYCN oncogene and the cell cycle regulator cyclinD1. These effects suggest adaptation to cellular stress by mechanism which remain unclear.
College of Medicine, Division of Pediatrics, University of Nebraska Medical Center, Omaha, NE, USA.
Don W
DW
Coulter
Department of Pharmacy Practice and Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA. trmcguir@unmc.edu.
Timothy R
TR
McGuire
Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.
John G
JG
Sharp
Department of Pharmacy Practice and Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA.
Erin M
EM
McIntyre
Department of Pharmacy Practice and Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA.
Yuxiang
Y
Dong
Department of Pharmacy Practice and Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA.
Xiaofang
X
Wang
Department of Pharmacy Practice and Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA.
Shawn
S
Gray
Department of Pharmacy Practice and Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA.
Gracey R
GR
Alexander
College of Medicine, Division of Pediatrics, University of Nebraska Medical Center, Omaha, NE, USA.
Nagendra K
NK
Chatuverdi
Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.
Shantaram S
SS
Joshi
Department of Pharmacy Practice and Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA.
Xiaoyu
X
Chen
Department of Pharmacy Practice and Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA.
Jonathan L
JL
Vennerstrom
D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D037621:Artemisinins / Q000494:pharmacology; D015415:Biomarkers; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D006571:Heterocyclic Compounds / Q000494:pharmacology*; D006801:Humans; D055442:Metabolome; D055432:Metabolomics / Q000379:methods; D051379:Mice; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism / Q000401:mortality / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
C503429:1,2,4-trioxane; D000962:Antimalarials; D000970:Antineoplastic Agents; D037621:Artemisinins; D015415:Biomarkers; D006571:Heterocyclic Compounds; C031327:artemisinin; D053148:Caspase 3
Cell cycle; Metabolism; Neuroblastoma; Ozonide antimalarials