Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Feb 24:12:837373.
doi: 10.3389/fonc.2022.837373. eCollection 2022.

A Novel Copper(II) Indenoisoquinoline Complex Inhibits Topoisomerase I, Induces G2 Phase Arrest, and Autophagy in Three Adenocarcinomas

Caroline Molinaro  1 , Nathalie Wambang  2 , Till Bousquet  3 , Anne-Sophie Vercoutter-Edouart  1 , Lydie Pélinski  3 , Katia Cailliau  1 , Alain Martoriati  1
Affiliations

A Novel Copper(II) Indenoisoquinoline Complex Inhibits Topoisomerase I, Induces G2 Phase Arrest, and Autophagy in Three Adenocarcinomas

Caroline Molinaro et al. Front Oncol. .

Abstract

Topoisomerases, targets of inhibitors used in chemotherapy, induce DNA breaks accumulation leading to cancer cell death. A newly synthesized copper(II) indenoisoquinoline complex WN197 exhibits a cytotoxic effect below 0.5 µM, on MDA-MB-231, HeLa, and HT-29 cells. At low doses, WN197 inhibits topoisomerase I. At higher doses, it inhibits topoisomerase IIα and IIβ, and displays DNA intercalation properties. DNA damage is detected by the presence of γH2AX. The activation of the DNA Damage Response (DDR) occurs through the phosphorylation of ATM/ATR, Chk1/2 kinases, and the increase of p21, a p53 target. WN197 induces a G2 phase arrest characterized by the unphosphorylated form of histone H3, the accumulation of phosphorylated Cdk1, and an association of Cdc25C with 14.3.3. Cancer cells die by autophagy with Beclin-1 accumulation, LC3-II formation, p62 degradation, and RAPTOR phosphorylation in the mTOR complex. Finally, WN197 by inhibiting topoisomerase I at low concentration with high efficiency is a promising agent for the development of future DNA damaging chemotherapies.

Keywords: adenocarcinoma; autophagy; cell cycle; copper(II) complex; indenoisoquinoline; topoisomerase.

PubMed Disclaimer

Conflict of interest statement

Author NW was employed by AGAT Laboratories, Intertek. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
WN197 synthesis.
Figure 2
Figure 2
The copper complex WN197 induced DNA damage in cancer cells. MDA-MB-231, HeLa and HT-29 cells were treated with DMSO (0.5%, solvent control), doxorubicin (5 µM, Top2 inhibitor inducing DNA breaks), cisplatin (20 µM, alkylating agent inducing DNA breaks), WN170 (0.5 µM, indenoisoquinoline without metal) or WN197 (0.5 µM). (A) Immunofluorescence of the DNA breaks marker γH2AX was visualized as green foci in nuclei stained with DAPI (blue) on a Leica fluorescent microscope 24 h after treatments. Images were representative of three independent experiments. Scale bar: 20 µm (B) Quantification of γH2AX foci number per cells. (C) Western blot analysis of γH2AX 24 h after treatments. β-actin was used as a loading control and relative γH2AX level was quantified by densitometry using Image J (Fiji Software, v1.52i). (D) Quantification of γH2AX foci number per cells 30 min after treatments, based on immunofluorescence experiments. In B and D, data were expressed as the mean ± SD for 30 nuclei of three independent experiments. Statistical analyses were based on a two-way ANOVA followed by a Dunnett’s test (*p<0.05, **p<0,01, ***p<0,005 and ****p<0,001).
Figure 3
Figure 3
WN197 inhibited human topoisomerase activity in a dose-dependent manner. (A) Top1 activity was determined by in vitro assays after addition of either DMSO (5%, solvent control, lane 4), WN197 at different concentrations (0.2, 0.5, 1 and 2 µM, lanes 5-8), etoposide (VP-16, 50 µM; Top2 poison, lane 9) the negative control of Top1 activity inhibition, or camptothecin (CPT, 10 µM; Top1 poison, lane 10) the positive control of Top1 activity inhibition. Relaxed DNA (RDNA, lane 1) or supercoiled DNA (SCDNA, lane 2) were used as migration controls. SCDNA was used in all other reactions in presence of Top1. The Top1 activity control allowing the relaxation of SCDNA is in lane 3. The addition of proteinase K allowed detection of nicked DNA (NDNA), a witness of the single-strand broken DNA stabilization by a topoisomerase poison. (B) Top2α activity inhibition assay. Migration control of supercoiled DNA (SCDNA) was performed in lane 1. Top2α was present in all other reactions. The Top2α activity control for the relaxation of SCDNA is in lane 2, the first band corresponds to the transitional open circular DNA (OCDNA) and topoisomers correspond to the relaxed DNA. DMSO (5%, solvent control) in lane 3, WN197 (concentrations of 0.2, 0.5, 1 and 2 µM) in lanes 4-7, etoposide (VP-16, 50 µM; Top2 poison) in lane 8, and camptothecin (CPT, 10 µM; Top1 poison) in lane 9. (C) Top2β activity inhibition assay. Migration control of SCDNA was performed in lane 1. Top2β was present in all other reactions. The Top2β activity control for the relaxation of SCDNA is in lane 2, DMSO (5%, solvent control) in lane 3, WN197 (concentrations of 0.2, 0.5, 1 and 2 µM) in lanes 4-7, etoposide (VP-16, 50 µM; Top2 poison) in lane 8, and camptothecin (CPT, 10 µM; Top1 poison) in lane 9. In (A–C) after topoisomerase reactions, DNA was run in a 1% agarose gel, stained with ethidium bromide (0.5 µg/mL), and visualized under UV light.
Figure 4
Figure 4
Activation of the DNA Damage Response (DDR) pathway. Cells were treated for 24 h with doxorubicin (5 µM), cisplatin (20 µM), WN170 (0.5 µM), or WN197 (0.5 µM). Western blots were performed to detect ATM, ATR, Chk1, Chk2, p53 and their phosphorylated forms, and p21. β-actin was used as a loading control and relative protein levels were quantified by densitometry using Image J software (Fiji Software, v1.52i). Results were representative of three independent experiments.
Figure 5
Figure 5
WN197 induced cell cycle accumulation in the G2/M phase. (A) Cytograms (G0/G1 and G2/M first and second peaks respectively), and (B) flow cytometry analysis of MDA-MB-231, HeLa, and HT-29 cells repartition in the cell cycle 24 h after treatments with cisplatin (20 µM, S phase arrest control), nocodazole (84 nM, M phase arrest control), WN170 or WN197 (0.5 µM). (C) Dose-response analysis by flow cytometry of G2/M phase accumulation 24 h after treatments with WN197. (D) Time course analysis by flow cytometry of the cell cycle repartition in cell lines untreated (control) or treated with WN197 (0.5 µM). Statistic were based on two-way ANOVA followed by Dunnett’s test (*p<0,05, **p<0,01, ***p<0,005 and ****p<0,001) on three independent experiments.
Figure 6
Figure 6
WN197 arrested the cell cycle in G2. (A) Western Blot analysis of cells treated for 24 h with doxorubicin (5 µM), cisplatin (20 µM), WN170, WN197 (0.5 µM), or nocodazole (84 nM). β-actin was used as a loading control. For H3 phosphorylation, respective H3 total levels were used as loading controls. (B) 14-3-3 and Cdc25C immunoprecipitations were realized in cell lines treated for 24 h with cisplatin (20 µM) or WN197 (0.5 µM). Relative protein levels were expressed by densitometry using Image J software (Fiji Software, v1.52i). Results were representative of three independent experiments.
Figure 7
Figure 7
WN197-induced autophagy. Cells were treated for 24 h with doxorubicin (5 µM), cisplatin (20 µM), WN170, WN197 (0.5 µM), nocodazole (84 nM) or rapamycin (0.5 µM). (A) Cleaved caspase 3 and PARP analysis by Western blots. Western blot analysis, after 3, 16, (24 or not), 48 and 72 h of treatment with WN197 or doxorubicin for 24 and 48 h, of (B) cleaved PARP or (C) cytosolic cytochrome (C, D) p62, Beclin-1, and LC3 markers analysis by Western blot. LC3 levels were expressed upon the LC3-II/LC3-I ratio. β-actin levels were used as a loading control. Relative protein levels were expressed by densitometry using Image J software (Fiji Software, v1.52i). (E) mTOR immunoprecipitations were realized in cell lines untreated or treated with doxorubicin (5 µM), rapamycin (0.5 µM) or WN197 (0.5 µM) for 24 h and followed by Western blots.
Figure 8
Figure 8
Deciphering of the molecular mechanisms of the novel copper(II) indenoisoquinoline complex WN197. WN197 inhibits topoisomerases I at low doses in a poison mode and forms a ternary complex with the topoisomerase and DNA, leading to strand breaks accumulation. Phosphorylated H2AX (γH2AX) localizes at the sites of DNA damage. The DNA damage response pathway is activated: ATM and ATR kinases are phosphorylated, and subsequently activate Chk1 and Chk2, leading to Cdc25C phosphorylation on serine 216 (S216), and to its binding to 14-3-3. Consequently, Cdk1 remains phosphorylated on tyrosine 15 (Y15), impeding the activation of the MPF (Cdk1/Cyclin B) and the phosphorylation of H3 on serine 10 (S10). Cancer cells arrest in the G2 phase of the cell cycle. The DDR also leads to an increase in p53 and p21 followed by an autophagic cell death characterized by the phosphorylation of RAPTOR on serine 792 (S792) in the mTORC1 complex, the synthesis of Beclin-1, the formation of LC3-II (complex LC3-I/PE (phosphatidylethanolamine)), and the degradation of p62.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin (2018) 68:394–424. doi: 10.3322/caac.21492 - DOI - PubMed
    1. Curtin NJ. DNA Repair Dysregulation From Cancer Driver to Therapeutic Target. Nat Rev Cancer (2012) 12:801–17. doi: 10.1038/nrc3399 - DOI - PubMed
    1. O’Connor MJ. Targeting the DNA Damage Response in Cancer. Mol Cell (2015) 60:547–60. doi: 10.1016/j.molcel.2015.10.040 - DOI - PubMed
    1. Fernández X, Díaz-Ingelmo O, Martínez-García B, Roca J. Chromatin Regulates DNA Torsional Energy via Topoisomerase II-Mediated Relaxation of Positive Supercoils. EMBO J (2014) 33:1492–501. doi: 10.15252/embj.201488091 - DOI - PMC - PubMed
    1. Pommier Y, Sun Y, Huang SYN, Nitiss JL. Roles of Eukaryotic Topoisomerases in Transcription, Replication and Genomic Stability. Nat Rev Mol Cell Biol (2016) 17:703–21. doi: 10.1038/nrm.2016.111 - DOI - PMC - PubMed