G-Quadruplex Binders Induce Immunogenic Cell Death Markers in Aggressive Breast Cancer Cells
"> Figure 1
<p>Chemical Structure of C066-3108 and BRACO-19. The chemical structure of C066-3108 and BRACO-19 is reported.</p> "> Figure 2
<p>Cytotoxic effects of G4 ligands. (<b>A</b>) Cytotoxic effect of G4 ligands determined by SRB assays in MCF-7, MDA-MB231, PC3 and LNCaP cell lines at 72 h of treatment with C066-3108 and BRACO-19 and (<b>B</b>) in MCF-7 and MDA-MB231 cells at six days of treatment. Figures report cell viability (mean of three independent experiments as optical density, O.D.) generated with different concentrations of G4 ligands. The statistical significance was calculated by GraphPad Prism 7 with two-way Analysis of Variance (ANOVA) using Dunnett’s multiple comparisons test (* <span class="html-italic">p</span> < 0.0001, <sup>&</sup> <span class="html-italic">p</span> < 0.005, <sup><span>$</span></sup> <span class="html-italic">p</span> < 0.01). (<b>C</b>) Survival determined by MTT assay of resting and/or PHA-activated PBMC cultured in the presence and in the absence of G4 ligands at the concentrations of 3 and 5 µM for 5 days. Figures report O.D. indicative of cell survival (data are the mean of three independent experiments). No statistical difference was observed with respect to the untreated control as calculated by GraphPad Prism 7.</p> "> Figure 3
<p>G4 ligands induce DNA damage in breast cancer cells. MCF-7 (<b>A</b>) and MDA-MB231 (<b>B</b>) cells were treated with G4 ligands at the indicated concentrations. The dot plot profiles indicate in the upper fluorescein isothiocyanate (FITC) positive panel the amount of DNA damage indicated by yH2AX staining. On our <span class="html-italic">x</span> axis the PI positivity is reported to analyze specific DNA staining. The dot plots reported are representative of a single experiment, whereas the histograms represent the mean ± standard deviation (SD) of at least three independent experiments. The statistical significance was calculated by GraphPad Prism 7 with one-way ANOVA with Dunnett’s multiple comparisons test ((<b>A</b>) * <span class="html-italic">p</span> < 0.005, (<b>B</b>) * <span class="html-italic">p</span> < 0.0001).</p> "> Figure 4
<p>Inhibition of cell cycle progression and apoptosis induction by G4 ligands. MCF-7 (<b>A</b>) and MDA-MB231 (<b>B</b>) cells were treated with G4 ligands at the indicated concentrations. Cells were stained with PI to evaluate cell cycle progression. A representative flow cytometry profile of cell cycle is shown for both cell lines, and the percent of cells in each phase of the cell cycle is indicated for the single experiment, whereas the bars in the histograms represent the mean ± SD of at least three independent experiments. The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Sidak’s multiple comparisons test ((<b>A</b>) * <span class="html-italic">p</span> < 0.0001, (<b>B</b>) * <span class="html-italic">p</span> < 0.05). Apoptosis induction (<b>C</b>) was evaluated by annexin V/PI staining. The percent of early apoptotic cells is reported in the histograms representing the mean ± SD of five independent experiments. The statistical significance was calculated by GraphPad Prism 7 with one-way ANOVA with Dunnett’s multiple comparisons test (* <span class="html-italic">p</span> < 0.005).</p> "> Figure 4 Cont.
<p>Inhibition of cell cycle progression and apoptosis induction by G4 ligands. MCF-7 (<b>A</b>) and MDA-MB231 (<b>B</b>) cells were treated with G4 ligands at the indicated concentrations. Cells were stained with PI to evaluate cell cycle progression. A representative flow cytometry profile of cell cycle is shown for both cell lines, and the percent of cells in each phase of the cell cycle is indicated for the single experiment, whereas the bars in the histograms represent the mean ± SD of at least three independent experiments. The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Sidak’s multiple comparisons test ((<b>A</b>) * <span class="html-italic">p</span> < 0.0001, (<b>B</b>) * <span class="html-italic">p</span> < 0.05). Apoptosis induction (<b>C</b>) was evaluated by annexin V/PI staining. The percent of early apoptotic cells is reported in the histograms representing the mean ± SD of five independent experiments. The statistical significance was calculated by GraphPad Prism 7 with one-way ANOVA with Dunnett’s multiple comparisons test (* <span class="html-italic">p</span> < 0.005).</p> "> Figure 5
<p>ICD induction by G4 ligands. ICD hallmarks were evaluated in MCF-7 and MDA-MB 231 cells treated with G4 ligands. Calreticulin expression as cell surface marker was evaluated in MCF7 (<b>A</b>) and MDA-MB231 (<b>B</b>) cells. Representative flow cytometry profiles of a single experiment are reported, whereas the histograms represent the mean ± SD of three independent experiments (* <span class="html-italic">p</span> < 0.005). ATP intracellular release by MCF-7 and MDA-MB231 treated with G4 ligands at 5 µM was determined as luminescence by microwell plate reader (<b>C</b>) the histograms represent the mean ± SD of three independent experiments (* <span class="html-italic">p</span> < 0.05). HMGB1 intracellular accumulation is reported in the histograms for MCF-7 and MDA-MB231 cells at the concentration of 3 µM (histograms report the mean ± SD of three independent experiments, <span class="html-italic">p</span> < 0.05) (<b>D</b>). The statistical significance was calculated by GraphPad Prism 7 one-way ANOVA with Dunnett’s multiple comparisons test.</p> "> Figure 5 Cont.
<p>ICD induction by G4 ligands. ICD hallmarks were evaluated in MCF-7 and MDA-MB 231 cells treated with G4 ligands. Calreticulin expression as cell surface marker was evaluated in MCF7 (<b>A</b>) and MDA-MB231 (<b>B</b>) cells. Representative flow cytometry profiles of a single experiment are reported, whereas the histograms represent the mean ± SD of three independent experiments (* <span class="html-italic">p</span> < 0.005). ATP intracellular release by MCF-7 and MDA-MB231 treated with G4 ligands at 5 µM was determined as luminescence by microwell plate reader (<b>C</b>) the histograms represent the mean ± SD of three independent experiments (* <span class="html-italic">p</span> < 0.05). HMGB1 intracellular accumulation is reported in the histograms for MCF-7 and MDA-MB231 cells at the concentration of 3 µM (histograms report the mean ± SD of three independent experiments, <span class="html-italic">p</span> < 0.05) (<b>D</b>). The statistical significance was calculated by GraphPad Prism 7 one-way ANOVA with Dunnett’s multiple comparisons test.</p> "> Figure 6
<p>T cell activation by G4 ligands. CM obtained from the experimental conditions indicated in the figure were used to evaluate T cell activation induced by G4 ligands. The figure reports CD69+CD4+T cells and CD69+CD8+T cells. The histograms represent the mean ± SD of five independent experiments. The statistical significance was calculated by GraphPad Prism 7 with one-way ANOVA with Turkey’s multiple comparisons test (* <span class="html-italic">p</span> < 0.05).</p> "> Figure 7
<p>G4 ligand stabilization of G4 structures. G4 ligand stabilization in MCF-7 (<b>A</b>) and MDA-MB231 (<b>B</b>) cells was evaluated by flow cytometry detecting by gating strategies in each phase of the cell cycle on PI positive cells (PI-W versus PI-H dot plot) the percent of G4 structure positivity. Flow cytometry profiles representative of a single experiment are reported, whereas the histograms represent the mean ± SD of five independent experiments. The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA with Dunnett’s multiple comparisons test (* <span class="html-italic">p</span> < 0.05).</p> "> Figure 7 Cont.
<p>G4 ligand stabilization of G4 structures. G4 ligand stabilization in MCF-7 (<b>A</b>) and MDA-MB231 (<b>B</b>) cells was evaluated by flow cytometry detecting by gating strategies in each phase of the cell cycle on PI positive cells (PI-W versus PI-H dot plot) the percent of G4 structure positivity. Flow cytometry profiles representative of a single experiment are reported, whereas the histograms represent the mean ± SD of five independent experiments. The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA with Dunnett’s multiple comparisons test (* <span class="html-italic">p</span> < 0.05).</p> ">
Abstract
:1. Introduction
2. Results
2.1. Anti-Proliferative Effects of G4 Ligands in Cancer Cells
2.2. C066-3108 and BRACO-19 Induce DNA Damage in Breast Cancer Cells
2.3. C066-3108 and BRACO-19 Arrest Cell Cycle Progression
2.4. Apoptosis Induction in MCF-7 Cells by Flow Cytometry
2.5. Increase of Calreticulin Surface Exposure in MDA-MB-231 Cells
2.6. C066-3108 and BRACO-19 Decrease Intracellular ATP Release
2.7. C066-3108 and BRACO-19 Effects on Intracellular HMGB1
2.8. BRACO-19 Induces T-Cell Activation
2.9. G4 Structure Induction/Stabilization in Breast Cancer Cells
3. Discussion
4. Materials and Methods
4.1. Cells and Drugs
4.2. Sulforhodamine B (SRB) and MTT Proliferation Assays
4.3. γH2AX and/or G4 and PI Intracellular Staining
4.4. Cell cycle, Annexin V/PI and Calreticulin Staining
4.5. ATP Intracellular Concentration
4.6. HMGB1 Intracellular Staining
4.7. T-cell Activation Assays
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
G-quadruplex | G4 |
High mobility group box 1 | HMGB1 |
Immunogenic cell death | ICD |
Estrogen receptor | ER |
Progesterone receptor | PR |
Human epidermal growth factor receptor 2 | HER2 |
Damage-associated molecular patterns | DAMPS |
Peripheral blood mononuclear cells | PBMC |
Sulforhodamine B | SRB |
Phytohemagglutinin | PHA |
Conditioned media | CM |
Propidium iodide | PI |
Telomere repeat binding factor 1 | TRF1 |
References
- Burge, S.; Parkinson, G.N.; Hazel, P.; Todd, A.K.; Neidle, S. Quadruplex DNA: Sequence, topology and structure. Nucleic Acids Res. 2006, 34, 5402–5415. [Google Scholar] [CrossRef] [PubMed]
- Siddiqui-Jain, A.; Grand, C.L.; Bearss, D.J.; Hurley, L.H. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc. Natl. Acad. Sci. USA 2002, 99, 11593–11598. [Google Scholar] [CrossRef] [PubMed]
- Dexheimer, T.S.; Sun, D.; Hurley, L.H. Deconvoluting the structural and drug-recognition complexity of the G-quadruplex-forming region upstream of the Bcl-2 P1 promoter. J. Am. Chem. Soc. 2006, 128, 5404–5415. [Google Scholar] [CrossRef] [PubMed]
- Cogoi, S.; Xodo, L.E. G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription. Nucleic Acids Res. 2006, 34, 2536–2549. [Google Scholar] [CrossRef] [PubMed]
- Castor, K.J.; Liu, Z.; Fakhoury, J.; Hancock, M.A.; Mittermaier, A.; Moitessier, N.; Sleiman, H.F. A platinum(II) phenylphenanthroimidazole with an extended side-chain exhibits slow dissociation from a c-Kit G-quadruplex motif. Chemistry 2013, 19, 17836–17845. [Google Scholar] [CrossRef] [PubMed]
- Tauchi, T.; Shin-Ya, K.; Sashida, G.; Sumi, M.; Nakajima, A.; Shimamoto, T.; Ohyashiki, J.H.; Ohyashiki, K. Activity of a novel G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), against human leukemia cells: Involvement of ATM-dependent DNA damage response pathways. Oncogene 2003, 22, 5338–5347. [Google Scholar] [CrossRef] [PubMed]
- Li, Q.; Xiang, J.F.; Yang, Q.F.; Sun, H.X.; Guan, A.J.; Tang, Y.L. G4ldb: A database for discovering and studying G-quadruplex ligands. Nucleic Acids Res. 2013, 41, D1115–D1123. [Google Scholar] [CrossRef]
- Monchaud, D.; Teulade-Fichou, M.P. A hitchhiker’s guide to G-quadruplex ligands. Org. Biomol. Chem. 2008, 6, 627–636. [Google Scholar] [CrossRef]
- Maji, B.; Bhattacharya, S. Advances in the molecular design of potential anticancer agents via targeting of human telomeric DNA. Chem. Commun. 2014, 50, 6422–6438. [Google Scholar] [CrossRef]
- Pennarun, G.; Granotier, C.; Gauthier, L.R.; Gomez, D.; Hoffschir, F.; Mandine, E.; Riou, J.F.; Mergny, J.L.; Mailliet, P.; Boussin, F.D. Apoptosis related to telomere instability and cell cycle alterations in human glioma cells treated by new highly selective G-quadruplex ligands. Oncogene 2005, 24, 2917–2928. [Google Scholar] [CrossRef]
- Rodriguez, R.; Miller, K.M.; Forment, J.V.; Bradshaw, C.R.; Nikan, M.; Britton, S.; Oelschlaegel, T.; Xhemalce, B.; Balasubramanian, S.; Jackson, S.P. Small-molecule-induced DNA damage identifies alternative DNA structures in human genes. Nat. Chem. Biol. 2012, 8, 301–310. [Google Scholar] [CrossRef] [PubMed]
- Xiong, Y.X.; Su, H.F.; Lv, P.; Ma, Y.; Wang, S.K.; Miao, H.; Liu, H.Y.; Tan, J.H.; Ou, T.M.; Gu, L.Q.; et al. A newly identified berberine derivative induces cancer cell senescence by stabilizing endogenous G-quadruplexes and sparking a DNA damage response at the telomere region. Oncotarget 2015, 6, 35625–35635. [Google Scholar] [CrossRef] [PubMed]
- Gluszynska, A.; Juskowiak, B.; Rubis, B. Binding study of the fluorescent carbazole derivative with human telomeric G-quadruplexes. Molecules 2018, 23, 3154. [Google Scholar] [CrossRef] [PubMed]
- Kaulage, M.H.; Maji, B.; Pasadi, S.; Ali, A.; Bhattacharya, S.; Muniyappa, K. Targeting G-quadruplex DNA structures in the telomere and oncogene promoter regions by benzimidazolecarbazole ligands. Eur. J. Med. Chem. 2018, 148, 178–194. [Google Scholar] [CrossRef]
- Xu, H.; di Antonio, M.; McKinney, S.; Mathew, V.; Ho, B.; O’Neil, N.J.; Santos, N.D.; Silvester, J.; Wei, V.; Garcia, J.; et al. Cx-5461 is a DNA G-quadruplex stabilizer with selective lethality in Brca1/2 deficient tumours. Nat. Commun. 2017, 8, 14432. [Google Scholar] [CrossRef]
- Brooks, T.A.; Hurley, L.H. Targeting MYC expression through G-quadruplexes. Genes Cancer 2010, 1, 641–649. [Google Scholar] [CrossRef]
- Read, M.; Harrison, R.J.; Romagnoli, B.; Tanious, F.A.; Gowan, S.H.; Reszka, A.P.; Wilson, W.D.; Kelland, L.R.; Neidle, S. Structure-based design of selective and potent G quadruplex-mediated telomerase inhibitors. Proc. Natl. Acad. Sci. USA 2001, 98, 4844–4849. [Google Scholar] [CrossRef]
- Harrison, R.J.; Cuesta, J.; Chessari, G.; Read, M.A.; Basra, S.K.; Reszka, A.P.; Morrell, J.; Gowan, S.M.; Incles, C.M.; Tanious, F.A.; et al. Trisubstituted acridine derivatives as potent and selective telomerase inhibitors. J. Med. Chem. 2003, 46, 4463–4476. [Google Scholar] [CrossRef]
- Gowan, S.M.; Harrison, J.R.; Patterson, L.; Valenti, M.; Read, M.A.; Neidle, S.; Kelland, L.R. A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumor activity. Mol. Pharmacol. 2002, 61, 1154–1162. [Google Scholar] [CrossRef]
- Zhou, G.; Liu, X.; Li, Y.; Xu, S.; Ma, C.; Wu, X.; Cheng, Y.; Yu, Z.; Zhao, G.; Chen, Y. Telomere targeting with a novel G-quadruplex-interactive ligand braco-19 induces T-loop disassembly and telomerase displacement in human glioblastoma cells. Oncotarget 2016, 7, 14925–14939. [Google Scholar] [CrossRef]
- di Leva, F.S.; Zizza, P.; Cingolani, C.; D’Angelo, C.; Pagano, B.; Amato, J.; Salvati, E.; Sissi, C.; Pinato, O.; Marinelli, L.; et al. Exploring the chemical space of G-quadruplex binders: Discovery of a novel chemotype targeting the human telomeric sequence. J. Med. Chem. 2013, 56, 9646–9654. [Google Scholar] [CrossRef] [PubMed]
- Galluzzi, L.; Buque, A.; Kepp, O.; Zitvogel, L.; Kroemer, G. Immunogenic cell death in cancer and infectious disease. Nat. Rev. Immunol. 2017, 17, 97–111. [Google Scholar] [CrossRef] [PubMed]
- Mouw, K.W.; Goldberg, M.S.; Konstantinopoulos, P.A.; D’Andrea, A.D. DNA damage and repair biomarkers of immunotherapy response. Cancer Discov. 2017, 7, 675–693. [Google Scholar] [CrossRef] [PubMed]
- Rufo, N.; Garg, A.D.; Agostinis, P. The unfolded protein response in immunogenic cell death and cancer immunotherapy. Trends Cancer 2017, 3, 643–658. [Google Scholar] [CrossRef] [PubMed]
- Yatim, N.; Cullen, S.; Albert, M.L. Dying cells actively regulate adaptive immune responses. Nat. Rev. Immunol. 2017, 17, 262–275. [Google Scholar] [CrossRef] [PubMed]
- Nakhjavani, M.; Hardingham, J.E.; Palethorpe, H.M.; Price, T.J.; Townsend, A.R. Druggable molecular targets for the treatment of triple negative breast cancer. J. Breast Cancer 2019, 22, 341–361. [Google Scholar] [CrossRef] [PubMed]
- Laezza, C.; Malfitano, A.M.; di Matola, T.; Ricchi, P.; Bifulco, M. Involvement of Akt/Nf-κB pathway in N6-isopentenyladenosine-induced apoptosis in human breast cancer cells. Mol. Carcinog. 2010, 49, 892–901. [Google Scholar] [CrossRef]
- Amato, J.; Madanayake, T.W.; Iaccarino, N.; Novellino, E.; Randazzo, A.; Hurley, L.H.; Pagano, B. Hmgb1 binds to the KRAS promoter G-quadruplex: A new player in oncogene transcriptional regulation? Chem. Commun. 2018, 54, 9442–9445. [Google Scholar] [CrossRef]
- Amato, J.; Cerofolini, L.; Brancaccio, D.; Giuntini, S.; Iaccarino, N.; Zizza, P.; Iachettini, S.; Biroccio, A.; Novellino, E.; Rosato, A.; et al. Insights into Telomeric G-Quadruplex DNA Recognition by Hmgb1 Protein. Nucleic Acids Res. 2019, 47, 9950–9966. [Google Scholar] [CrossRef]
- Debray, J.; Zeghida, W.; Jourdan, M.; Monchaud, D.; Dheu-Andries, M.L.; Dumy, P.; Teulade-Fichou, M.P.; Demeunynck, M. Synthesis and evaluation of fused bispyrimidinoacridines as novel pentacyclic analogues of quadruplex-binder braco-19. Org. Biomol. Chem. 2009, 7, 5219–5228. [Google Scholar] [CrossRef]
- Golden, E.B.; Frances, D.; Pellicciotta, I.; Demaria, S.; Barcellos-Hoff, M.H.; Formenti, S.C. Radiation fosters dose-dependent and chemotherapy-induced immunogenic cell death. Oncoimmunology 2014, 3, e28518. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kang, R.; Chen, R.; Zhang, Q.; Hou, W.; Wu, S.; Cao, L.; Huang, J.; Yu, Y.; Fan, X.G.; Yan, Z.; et al. Hmgb1 in health and disease. Mol. Asp. Med. 2014, 40, 1–116. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pagano, B.; Amato, J.; Iaccarino, N.; Cingolani, C.; Zizza, P.; Biroccio, A.; Novellino, E.; Randazzo, A. Looking for efficient G-quadruplex ligands: Evidence for selective stabilizing properties and telomere damage by drug-like molecules. ChemMedChem 2015, 10, 640–649. [Google Scholar] [CrossRef] [PubMed]
- Hato, S.V.; Khong, A.; de Vries, I.J.; Lesterhuis, W.J. Molecular pathways: The immunogenic effects of platinum-based chemotherapeutics. Clin. Cancer Res. 2014, 20, 2831–2837. [Google Scholar] [CrossRef]
- Ren, J.; Chaires, J.B. Sequence and structural selectivity of nucleic acid binding ligands. Biochemistry 1999, 38, 16067–16075. [Google Scholar] [CrossRef]
- Wang, A.H.; Ughetto, G.; Quigley, G.J.; Rich, A. Interactions between an anthracycline antibiotic and DNA: Molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2-a resolution. Biochemistry 1987, 26, 1152–1163. [Google Scholar] [CrossRef]
- Malfitano, A.M.; Sosa, S.; Laezza, C.; de Bortoli, M.; Tubaro, A.; Bifulco, M. Rimonabant reduces keratinocyte viability by induction of apoptosis and exerts topical anti-inflammatory activity in mice. Br. J. Pharmacol. 2011, 162, 84–93. [Google Scholar] [CrossRef] [Green Version]
- Di Natale, C.; la Manna, S.; Malfitano, A.M.; di Somma, S.; Florio, D.; Scognamiglio, P.L.; Novellino, E.; Netti, P.A.; Marasco, D. Structural insights into amyloid structures of the C-terminal region of nucleophosmin 1 in type A mutation of acute myeloid leukemia. Biochim. Biophys. Acta Proteins Proteom. 2019, 1867, 637–644. [Google Scholar] [CrossRef]
G4 Ligand Effects | MCF-7 | MDA-MB231 |
---|---|---|
Anti-proliferative effects | √ | √ |
yH2AX phosphorylation | √ | √ |
Apoptosis | √ | x |
Immunogenic cell death markers: Reduction of ATP Calreticulin exposure Enhanced intracellular HMGB1 | √ x x | √ √ √ |
T-cell activation | not investigated | √ |
G4 structure induction | x | √ |
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Di Somma, S.; Amato, J.; Iaccarino, N.; Pagano, B.; Randazzo, A.; Portella, G.; Malfitano, A.M. G-Quadruplex Binders Induce Immunogenic Cell Death Markers in Aggressive Breast Cancer Cells. Cancers 2019, 11, 1797. https://doi.org/10.3390/cancers11111797
Di Somma S, Amato J, Iaccarino N, Pagano B, Randazzo A, Portella G, Malfitano AM. G-Quadruplex Binders Induce Immunogenic Cell Death Markers in Aggressive Breast Cancer Cells. Cancers. 2019; 11(11):1797. https://doi.org/10.3390/cancers11111797
Chicago/Turabian StyleDi Somma, Sarah, Jussara Amato, Nunzia Iaccarino, Bruno Pagano, Antonio Randazzo, Giuseppe Portella, and Anna Maria Malfitano. 2019. "G-Quadruplex Binders Induce Immunogenic Cell Death Markers in Aggressive Breast Cancer Cells" Cancers 11, no. 11: 1797. https://doi.org/10.3390/cancers11111797