Abstract

Background: Evaluate the anti-tumor activity of ozonide antimalarials using a chemoresistant neuroblastoma cell line, BE (2)-c. Methods: 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 1â ×â 106 BE (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 (Nâ =â 6), and OZ513 treated mice (Nâ =â 5). Results: 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 (IC50â =â 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. Conclusions: 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.

Original languageEnglish (US)
Article number867
JournalBMC cancer
Volume16
Issue number1
DOIs
StatePublished - Nov 8 2016

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Antimalarials
Neuroblastoma
Cell Cycle
Cell Line
Neoplasms
Glycolysis
Oncogenes
Therapeutics
Artemisinins
Western Blotting
Apoptosis
Activity Cycles
Ewing's Sarcoma
Propidium
Growth
Dimethyl Sulfoxide
1,2,4-trioxane
Pharmaceutical Preparations
Inhibitory Concentration 50
Flow Cytometry

Keywords

  • Cell cycle
  • Metabolism
  • Neuroblastoma
  • Ozonide antimalarials

ASJC Scopus subject areas

  • Genetics
  • Oncology
  • Cancer Research

Cite this

Treatment of a chemoresistant neuroblastoma cell line with the antimalarial ozonide OZ513. / Coulter, Donald W; McGuire, Timothy R; Sharp, John G; McIntyre, Erin M.; Dong, Yuxiang; Wang, Xiaofang; Gray, Shawn; Alexander, Gracey R.; Chatuverdi, Nagendra K.; Joshi, Shantaram S; Chen, Xiaoyu; Vennerstrom, Jonathan L.

In: BMC cancer, Vol. 16, No. 1, 867, 08.11.2016.

Research output: Contribution to journalArticle

@article{b83819e4b90548a682c28aecf5f4070d,
title = "Treatment of a chemoresistant neuroblastoma cell line with the antimalarial ozonide OZ513",
abstract = "Background: Evaluate the anti-tumor activity of ozonide antimalarials using a chemoresistant neuroblastoma cell line, BE (2)-c. Methods: 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{\^a} ×{\^a} 106 BE (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{\^a} ={\^a} 6), and OZ513 treated mice (N{\^a} ={\^a} 5). Results: 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{\^a} ={\^a} 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. Conclusions: 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.",
keywords = "Cell cycle, Metabolism, Neuroblastoma, Ozonide antimalarials",
author = "Coulter, {Donald W} and McGuire, {Timothy R} and Sharp, {John G} and McIntyre, {Erin M.} and Yuxiang Dong and Xiaofang Wang and Shawn Gray and Alexander, {Gracey R.} and Chatuverdi, {Nagendra K.} and Joshi, {Shantaram S} and Xiaoyu Chen and Vennerstrom, {Jonathan L}",
year = "2016",
month = "11",
day = "8",
doi = "10.1186/s12885-016-2872-2",
language = "English (US)",
volume = "16",
journal = "BMC Cancer",
issn = "1471-2407",
publisher = "BioMed Central",
number = "1",

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TY - JOUR

T1 - Treatment of a chemoresistant neuroblastoma cell line with the antimalarial ozonide OZ513

AU - Coulter, Donald W

AU - McGuire, Timothy R

AU - Sharp, John G

AU - McIntyre, Erin M.

AU - Dong, Yuxiang

AU - Wang, Xiaofang

AU - Gray, Shawn

AU - Alexander, Gracey R.

AU - Chatuverdi, Nagendra K.

AU - Joshi, Shantaram S

AU - Chen, Xiaoyu

AU - Vennerstrom, Jonathan L

PY - 2016/11/8

Y1 - 2016/11/8

N2 - Background: Evaluate the anti-tumor activity of ozonide antimalarials using a chemoresistant neuroblastoma cell line, BE (2)-c. Methods: 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 1â ×â 106 BE (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 (Nâ =â 6), and OZ513 treated mice (Nâ =â 5). Results: 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 (IC50â =â 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. Conclusions: 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.

AB - Background: Evaluate the anti-tumor activity of ozonide antimalarials using a chemoresistant neuroblastoma cell line, BE (2)-c. Methods: 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 1â ×â 106 BE (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 (Nâ =â 6), and OZ513 treated mice (Nâ =â 5). Results: 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 (IC50â =â 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. Conclusions: 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.

KW - Cell cycle

KW - Metabolism

KW - Neuroblastoma

KW - Ozonide antimalarials

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