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Molecular Cancer Therapeutics
Molecular Cancer Therapeutics
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Small Molecule Therapeutics

Verticillin A Causes Apoptosis and Reduces Tumor Burden in High-Grade Serous Ovarian Cancer by Inducing DNA Damage

Amrita Salvi, Chiraz Soumia M. Amrine, Julia R. Austin, KiAundra Kilpatrick, Angela Russo, Daniel Lantvit, Esther Calderon-Gierszal, Zachary Mattes, Cedric J. Pearce, Mark W. Grinstaff, Aaron H. Colby, Nicholas H. Oberlies and Joanna E. Burdette
Amrita Salvi
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois.
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  • ORCID record for Amrita Salvi
Chiraz Soumia M. Amrine
2Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina.
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Julia R. Austin
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois.
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KiAundra Kilpatrick
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois.
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Angela Russo
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois.
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Daniel Lantvit
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois.
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Esther Calderon-Gierszal
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois.
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Zachary Mattes
3Departments of Chemistry, Biomedical Engineering, and Medicine, Boston University, Boston, Massachusetts.
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Cedric J. Pearce
4Mycosynthetix Inc., Hillsborough, North Carolina.
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Mark W. Grinstaff
3Departments of Chemistry, Biomedical Engineering, and Medicine, Boston University, Boston, Massachusetts.
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Aaron H. Colby
3Departments of Chemistry, Biomedical Engineering, and Medicine, Boston University, Boston, Massachusetts.
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Nicholas H. Oberlies
2Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina.
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  • ORCID record for Nicholas H. Oberlies
Joanna E. Burdette
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois.
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  • For correspondence: joannab@uic.edu
DOI: 10.1158/1535-7163.MCT-19-0205 Published January 2020
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  • Figure 1.
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    Figure 1.

    Verticillin A induces apoptosis in HGSOC cell lines in vitro. A, OVSAHO, OVCAR4, and OVCAR8 cells were treated with vehicle (DMSO), verticillin A (50 nmol/L), and chemotherapeutic control taxol (10 nmol/L) for 72 hours. Dose–response curves were generated and normalized to vehicle control. IC50 values are denoted in the table. B, OVCAR4 and OVCAR8 cells were treated with vehicle and verticillin A (50 nmol/L) for 8 hours. Following drug incubation, media was changed and cells were incubated for 2 weeks to form colonies. Representative images of 2D foci assay performed in OVCAR4 and OVCAR8 cells are shown. Each experiment was performed in three biological replicates, and data represent mean ± SEM. Significance tested by Student t test in comparison to vehicle control. C, OVCAR8 spheroids were treated with vehicle, verticillin A, taxol, and cisplatin for 72 hours. Spheroid viability was determined using CellTiter-Glo Cell Viability assay. IC50 values are denoted in the table. Data represent mean ± SEM from two biological replicates.

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    Figure 2.

    RNA-sequencing analysis shows upregulation of apoptosis and oxidative stress in verticillin A–treated OVCAR8 cells. A, OVCAR4 and OVCAR8 cells were treated with vehicle (DMSO), verticillin A (50 nmol/L), and taxol (10 nmol/L) for 24 hours, stained with Annexin V-FITC and propidium iodide, and analyzed by Nexcelom Cellometer. Each experiment was performed in three biological replicates, and data represent mean ± SEM. Statistics were generated with one-way ANOVA with Dunnett multiple comparisons with vehicle control within each group. B, Immunoblot analysis of whole-cell lysates of OVCAR4 and OVCAR8 cells probed for apoptotic marker (cPARP), and actin was used as a loading control. C, OVCAR8 cells were treated with vehicle and verticillin A for 24 hours. Chart denotes pathway analysis of the upregulated genes by PANTHER analysis. D, Table represents pathways identified by GSEA for OVCAR8 cells treated with vehicle control and verticillin A. E, Gene set enrichment plots based upon GSEA of transcripts altered by verticillin A treatment in OVCAR8 cells.

  • Figure 3.
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    Figure 3.

    Verticillin A causes oxidative stress and DNA damage in HGSOC cells. A, Verticillin A–induced oxidative stress in OVCAR4 and OVCAR8 cells was measured with CellROX Green reagent in cells treated with vehicle (DMSO), verticillin A (50 nmol/L for 6 hours), and H2O2 (50 μmol/L for 1 hour). Images were acquired using 40× objective of a fluorescent microscope. Scale bar, 20 μm. B, OVCAR4 and OVCAR8 cells were treated with vehicle, verticillin A (50 nmol/L), and H2O2 (50 μmol/L), and alkaline comet assay was performed to analyze DNA damage. Comet tail moment was used to quantify DNA damage by TriTek CometScore software. Each experiment was performed in three biological replicates, and data represent mean ± SEM. Statistics were generated with one-way ANOVA with Dunnett multiple comparisons with vehicle control within each cell line. Scale bar, 20 μm. C, Representative images of immunofluorescence staining of γH2A.X foci for OVCAR4 and OVCAR8 cells treated with vehicle and verticillin A (50 nmol/L) for 24 hours. Nuclei were stained by DAPI (0.1 μg/mL). Scale bar, 20 μm. D, Immunoblot analysis of whole-cell lysates of OVCAR4 and OVCAR8 cells treated with vehicle and verticillin A (50 nmol/L) for different timepoints. Lysates were probed for DNA damage marker γH2A.X, and actin was used as a loading control.

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    Figure 4.

    Verticillin A–mediated apoptosis and DNA damage are reversed by free radical quencher NAC. A, Immunoblot analysis of whole-cell lysates of OVCAR4 and OVCAR8 cells treated with vehicle (DMSO), verticillin A (50 nmol/L), and combination of verticillin A (50 nmol/L) and NAC (1 mmol/L) for 24 hours. Lysates were probed for apoptosis and DNA damage markers. Actin was used as a loading control. B, OVCAR4 and OVCAR8 cells were treated with vehicle, verticillin A (50 nmol/L), and combination of verticillin A (50 nmol/L) and NAC (1 mmol/L) for 24 hours, stained with Annexin V-FITC (AV) and propidium iodide (PI), and analyzed by Nexcelom Cellometer. Each experiment was performed in three biological replicates, and data represent mean ± SEM. Statistics were generated with one-way ANOVA with Tukey multiple comparisons within each group. C, OVCAR4 and OVCAR8 cells were treated with vehicle, verticillin A (50 nmol/L), and combination of verticillin A (50 nmol/L) and NAC (1 mmol/L) for 24 hours. Alkaline comet assay was performed to analyze DNA damage. Comet tail moment was used to quantify DNA damage by TriTek CometScore software. Each experiment was performed in three biological replicates, and data represent mean ± SEM. Statistics were generated with one-way ANOVA with Tukey multiple comparisons within each cell line. Scale bar, 20 μm; ns, not significant.

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    Figure 5.

    eNP-VA demonstrate reduced liver toxicity relative to free drug. A, OVCAR8-RFP cells were xenografted intraperitoneally to form tumors. Mice were dosed with verticillin A and vehicle (Cremophor EL/EtOH) once in 7 days (dosage: 0.5 mg/kg). Representative images show liver damage in verticillin A–treated animals. B, Four ovarian cell lines (IOSE80, FT33, OVCAR4, and OVCAR8) were treated with vehicle (DMSO) and verticillin A (50 nmol/L) for 72 hours. Dose–response curves were generated and normalized to vehicle control. Each experiment was performed in three biological replicates, and data represent mean ± SEM. Statistics were generated with Student t test for day 0 and day 3 within each cell line. C, OVSAHO, OVCAR4, and OVCAR8 cells were treated with eNP-VA and empty nanoparticles (eNP) for 72 hours. Dose–response curves were generated and normalized to eNP. Data represent mean ± SEM from three biological replicates. IC50 values are denoted in the table.

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    Figure 6.

    Verticillin A reduces tumor burden in vivo. A, OVCAR8-RFP cells were xenografted intraperitoneally to form tumors. Mice were dosed with eNP-VA and eNP once every 2 days for total 12 days (dosage: 0.5 mg/kg). Representative images show IVIS images of tumors in mice on days 0, 4, 8, and 12 of drug treatment. B, Quantification of tumor burden (average radiant efficiency as measured with IVIS imaging) normalized to day 0. Statistics were performed using two-way ANOVA relative to vehicle control. C, Quantification of average body weight (BW) of the mice during the course of treatment with eNP and eNP-VA. D, H & E staining showing the histomorphology of paraffin-embedded tumor tissue sections of eNP and eNP-VA–treated animals. E, IHC of γH2A.X and cPARP staining as markers of DNA damage and apoptosis is shown from tumors of animals treated with eNP and eNP-VA. Scale bar, 20 μm.

Tables

  • Figures
  • Table 1.

    qRT-PCR primers.

    Target geneForward primer sequence (5′-3′)Reverse primer sequence (5′-3′)
    TXNTGAAGCAGATCGAGAGCAAGACTTCATTAATGGRGGCRRCAAGC
    DUSP6CCTGAGGCCATTTCTTTCATAGAGTCACAGTGACTGAGCGGCTAAT
    NFKB2GAACAGCCTTGCATCTAGCCTCCCAGTCGCTATCAGAGG
    RELBTCCCAACCAGGATGTCTAGCAGCCATGTCCCTTTTCCTCT
    TXNRD1GAAGATCTTCCCAAGTCCTATGACATTTGTTGCCTTAATCCTGTGAGG
  • Table 2.

    Primary antibodies.

    AntibodySourceDilution for WBDilution for immunofluorescence/IHC
    Anti-rabbit PARPCST #95421:1,000—
    Anti-rabbit cPARPCST #9541—1:100 (IHC)
    Anti-rabbit γH2A.XCST #97181:5001:100 (IHC, IF)
    Anti-rabbit BaxCST #50231:500—
    Anti-rabbit actinSigma #A20661:5,000—
    • Abbreviations: CST, Cell Signaling Technology; IF, immunofluorescence; WB, Western blotting.

  • Table 3.

    Secondary antibodies.

    AntibodySourceDilution for WBDilution for immunofluorescence
    Anti-rabbit IgG-HRPCST #70741:10,000—
    Anti-rabbit Alexa Fluor 488Invitrogen #A-11034—1:1,000
    • Abbreviations: CST, Cell Signaling Technology; WB, Western blotting.

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Molecular Cancer Therapeutics: 19 (1)
January 2020
Volume 19, Issue 1
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Verticillin A Causes Apoptosis and Reduces Tumor Burden in High-Grade Serous Ovarian Cancer by Inducing DNA Damage
Amrita Salvi, Chiraz Soumia M. Amrine, Julia R. Austin, KiAundra Kilpatrick, Angela Russo, Daniel Lantvit, Esther Calderon-Gierszal, Zachary Mattes, Cedric J. Pearce, Mark W. Grinstaff, Aaron H. Colby, Nicholas H. Oberlies and Joanna E. Burdette
Mol Cancer Ther January 1 2020 (19) (1) 89-100; DOI: 10.1158/1535-7163.MCT-19-0205

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Verticillin A Causes Apoptosis and Reduces Tumor Burden in High-Grade Serous Ovarian Cancer by Inducing DNA Damage
Amrita Salvi, Chiraz Soumia M. Amrine, Julia R. Austin, KiAundra Kilpatrick, Angela Russo, Daniel Lantvit, Esther Calderon-Gierszal, Zachary Mattes, Cedric J. Pearce, Mark W. Grinstaff, Aaron H. Colby, Nicholas H. Oberlies and Joanna E. Burdette
Mol Cancer Ther January 1 2020 (19) (1) 89-100; DOI: 10.1158/1535-7163.MCT-19-0205
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