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. 2016 Apr 26;7(17):24027-49.
doi: 10.18632/oncotarget.8210.

Discovery and characterization of Isofistularin-3, a marine brominated alkaloid, as a new DNA demethylating agent inducing cell cycle arrest and sensitization to TRAIL in cancer cells

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Discovery and characterization of Isofistularin-3, a marine brominated alkaloid, as a new DNA demethylating agent inducing cell cycle arrest and sensitization to TRAIL in cancer cells

Cristina Florean et al. Oncotarget. .

Abstract

We characterized the brominated alkaloid Isofistularin-3 (Iso-3), from the marine sponge Aplysina aerophoba, as a new DNA methyltransferase (DNMT)1 inhibitor. Docking analysis confirmed our in vitro DNMT inhibition data and revealed binding of Iso-3 within the DNA binding site of DNMT1. Subsequent increased expression of tumor suppressor gene aryl hydrocarbon receptor (AHR) could be correlated to decreased methylation of CpG sites within the essential Sp1 regulatory region of its promoter. Iso-3 induced growth arrest of cancer cells in G0/G1 concomitant with increased p21 and p27 expression and reduced cyclin E1, PCNA and c-myc levels. Reduced proliferation was accompanied by morphological changes typical of autophagy revealed by fluorescent and transmission electron microscopy and validated by LC3I-II conversion. Furthermore, Iso-3 strongly synergized with tumor-necrosis-factor related apoptosis inducing ligand (TRAIL) in RAJI [combination index (CI) = 0.22] and U-937 cells (CI = 0.21) and increased TRAIL-induced apoptosis via a mechanism involving reduction of survivin expression but not of Bcl-2 family proteins nor X-linked inhibitor of apoptosis protein (XIAP). Iso-3 treatment decreased FLIPL expression and triggered activation of endoplasmatic reticulum (ER) stress with increased GRP78 expression, eventually inducing TRAIL receptor death receptor (DR)5 surface expression. Importantly, as a potential candidate for further anticancer drug development, Iso-3 reduced the viability, colony and in vivo tumor forming potential without affecting the viability of PBMCs from healthy donors or zebrafish development.

Keywords: DNMT inhibitor; TSG hypermethylation; autophagy; cell cycle arrest; leukemia.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Iso-3 is a new DNMT1 inhibitor interacting with the DNA binding site of DNMT1
(A) Chemical structures of Iso-3, Psammaplin A and bisoxasolidinone. (B) In vitro activity of purified DNMT1 was tested in the presence of increasing concentrations of Iso-3. Data are reported as percentage of DNMT1 activity respect to the control. (C) Iso-3 inhibitory activity against DNMT1 was measured in the presence of increasing concentrations of SAM. (D) In vitro activity of purified DNMT1 in the presence of increasing concentrations of bisoxasolidinone. Histograms represent the mean ± SD of three independent experiments. (E) Docking poses of Iso-3, aerothionin, and bisoxasolidinone on the crystal structure of DNMT1 (PDB-Code: 3SWR). DNMT1 protein is represented as cartoon and stick models with carbon, nitrogen, oxygen, and sulphur in white, blue, red, and yellow, respectively. Sinefungin, Iso-3, aerothionin, and bisoxasolidinone are shown as stick models with carbon colored in green, magenta, cyan, and yellow; nitrogen, oxygen, and bromide atoms colored in blue, red, and brown, respectively. Double-stranded DNA model was adopted from the crystal structure of DNMT1-DNA complex (PDB-Code: 3PTA) and colored in orange. Electrostatic potential surface of DNMT1 was calculated and represented as negatively and positively charged surfaces in red and blue shade, respectively.
Figure 2
Figure 2. Iso-3 increases AHR expression and induces AHR promoter demethylation
(A) mRNA levels of AHR in RAJI cells measured after 72 h of treatment with 1 μM DAC or 25 μM Iso-3. (B) DNA sequence of the AHR promoter region selected for methylation analysis. The underlined sequence corresponds to SNP rs71010234. (C) The methylation pattern of AHR promoter in RAJI cells was revealed upon 72 h of treatment with Iso-3 or DAC. Results show the methylation status for each of the 12 CpGs upstream of the transcription start site (horizontal numbering), in the different clones sequenced (vertical numbering). Open and closed circles indicate unmethylated and methylated CGs, respectively. (D) mRNA levels of AHR in SH-SY5Y cells measured after 72 h of treatment with 1 μM DAC or indicated Iso-3 concentrations. (E) Protein expression levels of DNMT isoforms in RAJI cells treated 72 h with Iso-3 at indicated doses or 1 μM DAC. (F) Top panel: in vitro total HDAC activity in presence of indicated Iso-3 doses or 2 μM SAHA. Data are reported as percentage of HDAC activity respect to the control. Bottom panel: acetylated histone 4 (H4ac) levels in Iso-3- or 2 μM SAHA-treated RAJI cells (24 h). Histone H1 (H1) was used as a loading control. All histograms represent the mean ± SD of three independent experiments. All blots are representative of three independent experiments.
Figure 3
Figure 3. Iso-3 arrests cancer cells in the G0/G1 phase of cell cycle
(A) RAJI and U-937 cells were treated with Iso-3 and cell number was evaluated after 24 and 72 h. (B) RAJI and U-937 cells treated with Iso-3 for 24 h were analyzed for DNA content by flow cytometry. Bars represent the percentage of cells in each cell cycle phase. (C) mRNA and (D) protein expression levels of cell cycle-related genes in RAJI cells treated with Iso-3 for 24 h. Histograms represent the mean ± SD of three independent experiments. Blots are representative of three independent experiments.
Figure 4
Figure 4. Iso-3 induces autophagy in lymphoma cells
(A) Morphological analysis was performed by Diff-Quik staining and microscopic observation of RAJI and U-937 cells after 24 h of treatment with Iso-3. (B) Cellular size (forward scatter, FSC) and granularity (side scatter, SSC) were measured by flow cytometry in RAJI cells treated for 24 h with Iso-3. (C) Western blot analysis of LC3 conversion in RAJI cells treated with different concentrations of Iso-3 for 24 h. Where indicated, bafilomycin A1 (40 nM) was added 2 h before harvesting. Blots are representative of three independent experiments. (D) RAJI cells where treated or not with Iso-3 for 24 h, then stained with Cyto-ID® Green dye as described in the materials and methods section, and appearance of autophagosome-related vesicles was observed by fluorescence microscopy. Representative images and quantification of autophagosome-positive cells are provided. (E) Representative images of electron microscopy analysis of RAJI and U-937 cells treated or not for 24 h with Iso-3. (1) Phagophores, (2) autolysosomes of different maturity, (3) residual bodies, (4) lysosomes, (5) multivesicular body. All histograms represent the mean ± SD of three independent experiments. All blots are representative of three independent experiments.
Figure 5
Figure 5. Effect of Iso-3 on cell viability
(A) The viability of RAJI and U-937 cells was measured by trypan blue exclusion assay after 24 and 72 h of exposure to Iso-3. (B) Western blot analyses of caspase activation and PARP-1 cleavage in RAJI and U-937 cells treated for 72 h with Iso-3. U-937 cells, untreated or treated with 100 μM VP-16 for 3 h, were used as negative and positive controls for caspase cleavage, respectively. (C) Hoechst-PI staining of RAJI and U-937 cells treated with 50 μM Iso-3 for 72 hours. Apoptotic and non-apoptotic PI-positive nuclei are reported as a percentage of the total number of cells. ZVAD-FKM (50 μM) and Necrostatin-1 (Nec-1; 30 μM) were added 1 h before Iso-3 treatment, where indicated. White arrows: apoptotic cells. Red arrow: PI-positive cells. Pictures are representative of three independent experiments (D) RAJI, U-937 and PC-3 cells were grown in the presence of Iso-3 for 10 days and colony formation was then scored. (E) Trypan blue scored viability of a panel of cancer cell lines (left) and of PBMCs from healthy donors after Iso-3 treatment at the indicated time points and doses. (F) Representative images of Zebrafish embryos after 24 h treatment with the indicated Iso-3 doses (right panel) and corresponding quantification of viable embryos percentage (left panel). Ethanol 3% (EtOH) was used as a positive control for toxicity. (G) Fluorescent SH-SY5Y or PC-3 cells were treated or not in vitro at different concentrations of Iso-3 for 24 h and then injected in the zebrafish yolk sac. Fluorescence was quantified. Representative images from a total of six to nine fish per condition. Fluorescence intensity quantification graphs are shown. PBS injection was used as a control for injection toxicity. All histograms represent the mean ± SD of three independent experiments. All blots are representative of three independent experiments.
Figure 6
Figure 6. Iso-3 sensitizes cancer cells to TRAIL-induced apoptosis
(A) RAJI and U-937 cells were treated with the indicated concentrations of Iso-3 during 24 h, then increasing concentrations of TRAIL were added for additional 24 h and cell viability was assessed. Significant differences between combination treatments, untreated controls and single agents are indicated with a $. (B) Viability of RAJI cells co-treated with the indicated doses of Iso-3 and TRAIL for 48 h. (C) Healthy donors' PBMCs were treated with Iso-3 during 24 h, then TRAIL was added for additional 24 h and viability was measured. (D) RAJI and U-937 cells were treated with Iso-3 (15 μM) and Z-VAD-FKM (50 μM) for 24 h, before TRAIL addition for 24 h (50 ng/ml for RAJI, 5 ng/ml for U-937). Cell death was measured by trypan blue exclusion assay (left panel) and Hoechst-PI staining (right panel). Significant differences between combination treatments, untreated controls and single agents are indicated with a $; significant differences between combination treatments with and without ZVAD-FKM are indicated with a £. (E) Cells were treated as in panel D and caspase activation was measured by luminescent caspase 3/7 assay (right panel). Significant differences are indicated as in panel D. Western blot analysis of caspases and PARP-1 cleavage was performed in RAJI cells (left panel). CF = cleaved fragments. (F) Expression levels of a panel of anti-apoptotic proteins implicated in TRAIL resistance in RAJI cells after 24 h treatment with Iso-3. (G) Survivin, FLIP and GRP78 expression levels after 24 h of Iso-3 treatment (upper panel); FLIP and GRP78 levels after 48 h of 15 μM Iso-3 treatment (lower panel) in RAJI cells. Blots are representative of three independent experiments. (H) mRNA expression levels of CHOP and DR5 in RAJI cells after treatment with 15 μM Iso-3 for 48 h. (I) FACS analysis of DR4 and DR5 surface levels in RAJI cells after 48 h treatment with 15 μM Iso-3. Representative histograms (left panel) and quantification of mean fluorescence intensity (MFI) levels (right panel) relative to matched control PE-conjugated IgG antibodies. All histograms represent the mean ± SD of three independent experiments. All blots are representative of three independent experiments.

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References

    1. Schnekenburger M, Florean C, Dicato M, Diederich M. Epigenetic alterations as a universal feature of cancer hallmarks and a promising target for personalized treatments. Curr Top Med Chem. 2016;16:745–776. - PubMed
    1. Florean C, Schnekenburger M, Grandjenette C, Dicato M, Diederich M. Epigenomics of leukemia: from mechanisms to therapeutic applications. Epigenomics. 2011;3:581–609. - PubMed
    1. Schnekenburger M, Diederich M. Epigenetics Offer New Horizons for Colorectal Cancer Prevention. Curr Colorectal Cancer Rep. 2012;8:66–81. - PMC - PubMed
    1. Seidel C, Florean C, Schnekenburger M, Dicato M, Diederich M. Chromatin-modifying agents in anti-cancer therapy. Biochimie. 2012;94:2264–2279. - PubMed
    1. Yoo CB, Jones PA. Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov. 2006;5:37–50. - PubMed

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