Selected ellipticine derivatives, known to target topoisomerase II, suppress the alternative lengthening of telomere (ALT) pathway in telomerase–negative cells

Abstract

Background

DNA topoisomerase and telomerase enzymes are popular targets of several anti-tumor drugs. Smooth proceeding of telomeric recombination requires Topoisomerase II (Top2), which is involved in telomere-telomere recombination through functioning in relaxation of positive supercoils among the cells adopting telomerase-independent Alternative lengthening of telomere (ALT) pathway. Most of the inhibitors reported so far have been designed to targetsolely telomerase-positive cells, which can potentially lead to therapeutic failure because tumor cells treated with telomerase inhibitors can activate the ALT pathway for telomere maintenance. Knowing that ALT cells are more sensitive against a Top2 inhibitor, ICRF-93 agent, compared to telomerase-positive cells, we analyzed two selected ellipticine derivatives that we recently reported as TopII-targeting compounds, to assess their effects on the formation of DNA breaks and suppression of ALT pathway.

Methods

Cell viability, Comet, C-Circle assays, dot blot, immunofluorescence staining, and telomere fluorescence in situ hybridization (FISH) staining were used for determining the effect of the compounds on ALT status of tumor cells.

Results and conclusions

Treatment of ALT cells with ellipticine derivatives resulted in the formation of DNA breaks and suppression of ALT-associated phenotypes in vitro. Our results will contribute to the development of therapeutic strategies combining telomerase and ALT pathway inhibitors.

Introduction

Telomeres, the DNA–protein complexes located at chromosomal termini are required for protection of ends of linear chromosomes from end-to-end fusions and recombination (Blackburn 1992; Blackburn et al. 2015). In many organisms, telomeric DNAs consist of repetitive G-rich DNA sequences extending in the 5′–3′ direction and are protected by telomere-binding proteins. Telomere shortening due to primer requirement and orientation properties of DNA polymerases is a protective mechanism against the unlimited potential of somatic tissue proliferation. Most immortal cells, including cancer cells, maintain their telomeric length by the expression of a telomere reverse transcriptase, telomerase (Blackburn et al. 2006; Martines and Blasco 2015). Telomerase is composed of an RNA component (hTR) with complementary to the telomeric DNA sequence and catalytic subunit (hTERT) (2). On the other hand, a significant minority of cancer cells ( ~ 15%) can regulate their telomeric structures via a telomerase-independent, recombination-mediated pathway, known as alternative lengthening of telomeres (ALT) (Dunham et al.2000; Reddel et al. 2001; Topcu et al. 2005; Basenko et al. 2011). The latter pathway is effective in many different tumors including sarcomas, pancreatic neuroendocrine tumors, and subtypes of central nervous system related tumors (Bryan et al. 1997; Shay et al. 2012). ALT cells display a heterogeneous telomere length and unique nuclear bodies termed as ALT-associated PML bodies (APBs), which are composed of telomeric DNA, telomere binding proteins (TRF1 and TRF2) and other proteins involved in DNA synthesis, repair, and recombination (Yeager et al. 1999).

Telomeres are protected by a protein complex called Rap1-Rif1-Rif2 complex in yeast (Teng et al. 2000). In telomerase-negative cells bearing long telomeres, the Rap1-Rif1-Rif2 complex blocks the progression of recombinational helicases on telomeres by forming a barrier on telomeric DNA with a large number of supercoils during the process of telomeric recombination (Teng et al. 2000). Therefore, the control of the formation/dissolution steps of DNA supercoils is highly important for the smooth proceeding of telomeric recombination process.

Topoisomerases, on the other hand, solve the problems associated with the topological constraints of genetic materials by reversibly nicking one (Type I topoisomerases) or both strands (Type II topoisomerases) through transient enzyme/DNA covalent intermediates (Wang 1996; McClendon and Osheroff 2007; Sarikaya et al. 2012). Topoisomerase II (TopII) is also involved in telomere-telomere recombination, and in ALT cells in which TopII isoforms TopIIα and TopIIβ enzymes were silenced. The number of APB nuclear bodies was reported to decrease and associated with an increase of telomeric dysfunction-induced foci (TIFs) (Hsieh et al. 2015). Moreover, ALT cells are reported to be more sensitive against TopII inhibitor ICRF-93 agent compared to telomerase-positive cells as evidenced by telomere shortening among ALT cells treated with the ICRF-93 agent (Hsieh et al. 2015). The data on the suppressed proliferation of ALT-specific tumor cells upon TopII inhibition led the idea of TopII as prospectively important protein in the ALT pathway through its functions in relaxing recombination-dependent positive supercoils in ALT cells. Although DNA damage induced by drugs in conventional chemotherapy applications is thought to occur in the genome, various genotoxic molecules such as cisplatin, anti-TopI and anti-TopII and replication inhibitors are also effective on telomere stability (Lu et al. 2013). For this reason, genotoxic agents of the broad spectrum may trigger telomere-specific DNA damage by binding to telomeric DNA directly or blocking the binding sites of telomeric nucleoproteins on telomeres. In this regard, TopII, can be considered as one of the important targets in the development of therapeutics for the targeting ALT-specific cancer cells.

Ellipticine (5,11-dimethyl-6H-pyridol[4,3b]carbazole), a naturally occurring plant alkaloid from Apocyanaceae plants, is a widely known anti-cancer agent (Miller and McCarthy 2012; Itoh et al. 2018). Several key mechanisms of action including DNA intercalation and topoisomerase II inhibition contribute to the activities of ellipticine (Vann et al. 2016). However, it is not a widely-accepted drug due to its poor water-solubility, low specificity, potential to lead secondary malignancies, and drug resistance. Therefore, many natural and synthetic variants of this compound product were developed (Miller and McCarthy 2012; Stiborova and Frei 2014; Vann et al. 2016). In this study, we showed that two selected ellipticine derivatives that we recently reported as TopII-targeting compounds (Kuskucu et al. 2020), have a considerable effect on the formation of DNA breaks and the suppression of ALT pathway.

Materials and methods

Chemistry

N²-Ethyl N-methyl 5-demethyl ellipticinium bromide (Z1) and N²-Hexyl N-methyl 5-demethyl ellipticinium bromide (Z2) were synthesized and characterized as described (Kuskucu et al. 2020). The compounds were dissolved in 100% DMSO and diluted up to 0.01% using deionized water. A concentration below 5% DMSO in test compounds is not influential in the assays (Vann et al. 2016; Kuskucu et al. 2020).

Cell viability assay

The cytotoxicity of the compounds was tested with MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay using two different cell lines; HeLa (Telo⁺) and Saos-2 (ALT⁺) (Lin et al. 2016). The compounds were tested in different concentrations and cell viability was determined after incubating the mixture of 100 μL of culture and 20 μL of MTS at 37 °C for 1 h. The cells (5–100 × 10³/well) were cultured in a 96-well microtiter plate in a final volume of 200 µL/well. The absorbance was measured at 490 nm using a microplate reader (Biorad, California, USA). Stock solutions of the tested compounds (10 mM) were prepared in DMSO. Cell viability was normalized as a percentage of control and the experiments were performed in duplicates.

Comet assay

Comet assay was performed to determine the effect of the compounds on chromosomal DNA breaks as described (Huang et al. 2010; Lee et al. 2012). Nuclear DNA on slides was stained with SybrGold and visualized under a fluorescent microscope (Nikon Eclipse 80i, Japan). Images were captured by a CCD camera (Nikon DS-Ri1, Japan) to determine the percentage (%) of cells revealing a comet tail.

C-Circle assay, dot blot, and hybridization

C-circle amplification assay was performed essentially as described (Henson et al. 2009, 2017). Genomic DNA was digested with HinfI and RsaI restriction enzymes. Following the extraction, Klenow (TAKARA, Mountain View, USA) reaction was performed and reaction products were diluted in 2xSSC and dot-blotted onto a 2XSSC soaked Hybond-N⁺ nylon membrane (GE Healthcare Bio-Sciences, Pittsburgh, USA). DNA was UV-cross-linked onto the membrane, hybridized at 37 °C with end-labeled ³² P-(CCCTAA)₃ oligo probe in Church buffer (0.5 M NaHPO4 pH: 7.2, 1 mM EDTA, 1% bovine serum albumin, 7% SDS) and visualized by using Alphalnnotech photodocumentation system.

Immunofluorescence staining and telomere fluorescence in situ hybridization (FISH) staining

U2OS and Saos-2 cells were grown on glass coverslips and then growth-arrested by withdrawal of methionine for 4 days, fixed in 4% paraformaldehyde at room temperature for 10 min, followed by permeabilization in 0.05% Triton X buffer at room temperature for 10 min (Hsieh et al. 2015). For dual immunostaining, slides were blocked with 1% bovine serum albumin and incubated at 4 °C for overnight with mouse anti-TRF2 (Millipore, Massachusetts, USA), rabbit anti-PML (Santa Cruz Biotechnology, Dallas, USA) antibodies. The primary antibodies were detected using Rhodamine Red-conjugated goat anti-rabbit and fluorescein isothiocyanate-conjugated goat anti-mouse antibodies (1:500). DNA was stained with DAPI at room temperature. Immunofluorescence was analyzed with a Zeiss Axioplan fluorescence microscope. Cells were scored as APB positive if they contained two or more large PML bodies co-localized with TRF2 or PML.

Statistical analyses

The chi-square test was used to compare the effect of two Ellipticine derivatives, Z1 and Z2, with negative control groups on occurrence of DNA damage and formation of C-circle and APBs. All data were presented as frequency and percent, and results were considered statistically significant when p < 0.05. The statistical analysis was performed using the SPSS software, version 11.0.

Results and discussion

Following the cloning of human and yeast telomerase, the discovery of telomerase inhibitors has become an important strategy in anti-cancer drug development studies. However most of the inhibitors reported so far have been designed to target solely telomerase-positive cells. Studies have shown that tumor cells treated with telomerase inhibitors can activate the ALT pathway for telomeric maintenance through recombination (Cesare and Reddel 2010). This approach potentially leads to therapeutic failures and/or a resistance against telomerase inhibition-based anti-cancer therapy. Therefore developing new therapeutics targeting proteins known to be involved in the latter pathway will be crucial for the targeting of ALT-specific cells. TopII, having an active function in the ALT pathway in this regard, is one of the important therapeutic targets in treatment approaches for ALT-specific cancer cells.

We recently reported a number of Ellipticine derivatives as potent inhibitors of topoisomerase IIα (Kuskucu et al. 2020). Here, we examined the effectivity of two of these Ellipticine derivatives in both telomerase-positive and telomerase-deficient human cells. We first assessed the cytotoxicity of the compounds using HeLa (Telo⁺) and Saos-2 (ALT⁺) cells using an MTS assay. Although both Z1 and Z2 were profoundly toxic in either cell lines, we observed a more dramatic toxic effect on Saos-2 cells compared to HeLa cells (Fig. 1).

Fig. 1.

Dose-dependent cytotoxic effect of the Z compounds in vitro. Replicate plates of equally seeded cells were treated as indicated. The cell viability was measured by an MTS assay in HeLa (a), and Saos-2 (b) cells treated with Z1 and Z2 compounds in a range of concentrations from 1 to 10 μM following the incubation for 24 h. Results are expressed as the mean from a representative experiment performed in triplicate

We carried out comet assay, a sensitive technique to detect early DNA damage for measuring chromosomal DNA strand breaks among the cells exposed to Z compounds. Chromosomal DNA strand breaks were significantly increased in HL-60 cells exposed to the compounds (5 μM for 60 min) compared to untreated HL-60 cells. The mitoxantrone (MX), one of the known TOP2-targeting anthracenediones (19), and parental compound Ellipticine were used as control, and the cells treated with Z1 and Z2 caused 100% of HL-60 cells bearing DNA damage, which was significantly greater than the induction by MX (% of cells showing comet image, p < 0.001, Fig. 2). The two ellipticine derivatives, Z1 and Z2, were around twice more effective on the formation of chromosomal DNA strand breaks than the parental ellipticine (100% and 46%, respectively) (Fig. 2). These results indicate that Z1 and Z2 compounds, like MX, induce chromosomal DNA strand breaks. However, these two compounds did not yield detectable DNA cleavage patterns in our previous report, which was carried out using covalent complex analysis through trapping the enzyme–DNA complex with SDS, followed by Proteinase K treatment (Kuskucu et al. 2020). Both Comet assay and cleavage assays using SDS and Proteinase K treatment are useful techniques in detection of DNA damages however the former one is known to be more sensitive assay as reported by other studies covering comparisons of comet assay to other approaches (Yasuhara et al. 2003; Yu et al. 2006). Comet assay has been shown to reveal consistent results in several studies investigating the cytotoxicity of potentially important pharmaceutical compounds (Isloor et al. 2013; Dar et al. 2017). Given that covalent complex analysis based on SDS-trapping among Proteinase K-treated and untreated enzyme–DNA intermediates is profoundly influenced by the amount of enzyme, one might expect circumstantial results depending on the enzyme as this approach requires several times more topoisomerase to be included in reaction mixture for exerting a detectable amount of cleavage upon ethidium bromide staining. Moreover, detection of DNA breaks requires binding of Topoisomerase prior its relegation of DNA strands in the method we employed in our earlier report.

Fig. 2.

DNA breakage analysis by comet assay for HL-60 cells treated with Z1 and Z2 compounds. HL-60 cells were treated with topoisomerase II-targeting compounds Z1 and Z2 for 60 min and monitored using comet assay. Representative images of the comet assay are shown on the left panel. The percentage (%) of cells with a comet tail was determined for randomly selected 100 cells (right panel)

To investigate whether Z compounds have any effect on the suppression of ALT pathway, we examined the occurrence of APBs and C-circles as the hallmarks of the ALT cells (Henson and Reddel 2010). These include the existence of extra-chromosomal telomeric DNA in the form of double-stranded linear/circular DNA or single-stranded circular DNA such as C-circles, an increase in the number of telomeric sister chromatid exchanges as well as the presence of APBs (Yeager et al. 1999; Henson and Reddel 2010). C-circle structures in non-ALT cells were found to be below the basal level and in normal human fibroblast cells 1000-fold less than ALT⁺ cells (Henson et al. 2017). We observed that Z1 and Z2 treatment resulted in a significant loss in the level of C-circles in Saos-2 cells (p < 0.05, Fig. 3a). Our immunofluorescence analyses also revealed that Z1 and Z2 treatment resulted in a significant decrease in the frequency of APBs foci among ALT-positive U2OS and Saos-2 cells (p < 0.05, Fig. 3b). Similarly, it was reported that treatment of ALT cells with a hTOP2 inhibitor ICRF-193 leading to suppression of ALT-associated phenotypes in vitro, causes telomere shortening, and inhibits ALT cell proliferation in mice (Hsieh et al. 2015). There are a number of studies suggesting that ellipticine and some of its analogs inhibit telomerase via association with the DNA secondary structure, the G-quadruplex, in telomeric DNA sequences (Sassano et al. 2012; Ghosh et al. 2013).

Fig. 3.

Suppression of the ALT telomere phenotypes by Z1 and Z2 compounds. a Detection of ALT activity by C-circle assay; Representative dot blot of C-circle assay products for the ALT cells (U2OS) treated with Z1 and Z2 (Left panel). Genomic DNA (100 ng) were isolated from U2OS and Saos-2 cells and subjected to C-circle assay by using the Klenow reaction. The presence of C-circles indicates that the cell line utilizes the ALT mechanism. b Fluorescence imaging of APBs in ALT cells treated with Z1 and Z2; ALT cells (U2OS and Saos-2) and telomerase-positive cells (HeLa) were treated with Z1 and Z2. The APBs were examined according to colocalization of TRF2 (red) and PML (green). Representative photos from U2OS cells are shown on the left panel. A total of 200 cells were observed. At least four colocalized PML/TRF2 signals in an individual cell nucleus were scored. Cell nuclei are stained with DAPI (blue). Quantification of the results from the cells shown on the right panel

Taken together, our results demonstrated that treatment of ALT cells with N²-Ethyl N-methyl 5-demethyl ellipticinium bromide and N²-Hexyl N-methyl 5-demethyl ellipticinium bromide suppressed ALT-associated phenotypes in vitro. TopII might play a role in opening recombination-dependent positive supercoils intensively observed in ALT cells and might be a novel therapeutic target for inhibition of the ALT pathway. Knowing that Z1 and Z2 are effective on topoisomerase II inhibition can be significant against ALT cancer cells. Since telomeres and DNA topoisomerases are ideal targets of anti-tumor drugs, our results will contribute to the development of therapeutic strategies combining telomerase and ALT pathway inhibitors.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial and non-financial interests.