C8-Substituted Temozolomide Analogs Overcome O6-Methylguanine-DNA Methyltransferase and Mismatch Repair Precipitating Apoptotic Cancer Cell Death

Zhikuan Yang1, Danping Wei1, Xiaoli Dai1, Malcolm FG Stevens2, Tracey D Bradshaw2, Ying Luo1 and Jihong Zhang1*

1Faculty of Medicine, Kunming University of Science and Technology, Kunming, China

2Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK

*Corresponding Author:
Jihong Zhang
Kunming University of Science and Technology, Kunming, China.
Tel:
8687165194108
E-mail:
[email protected]

Received date: March 31, 2017; Accepted date: April 10, 2017; Published date: April 16, 2017

Citation: Yang Z, Wei D, Dai X, et al. C8-Substituted Temozolomide Analogs Overcome O6-Methylguanine-DNA Methyltransferase and Mismatch Repair Precipitating Apoptotic Cancer Cell Death. Neurooncol Open Access 2017, 1:1. doi: 10.21767/2572-0376.100018

 
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Abstract

Temozolomide (TMZ) is the standard of care chemotherapeutic agent used in the treatment of glioblastoma multiforme. O6-methylguaine lesions mainly formed by TMZ are repaired by O6-methyl-guanine DNA methyltransferase (MGMT), a DNA repair protein that removes alkyl groups located at the O6-position of guanine. Response to TMZ requires low MGMT expression and functional mismatch repair. Resistance to TMZ conferred by MGMT, and tolerance to O6-methylguanine lesions conferred by deficient MMR severely limit TMZ clinical applications. Therefore, development of new TMZ derivatives which can overcome TMZresistance is urgent. In this study, we investigated the anti-tumor mechanism of action of two novel TMZ analogs: C8-imidazolyl (377) and C8-methyl imidazole (465) tetrazines. We found that analogs 377 and 465 display good anticancer activity against the MGMT-overexpressing glioma T98G and MMR deficient colorectal carcinoma HCT116 cell lines with IC50 value of 62.50 μM, 44.23 μM and 33.09 μM, 25.37 μM respectively. Analogs induce cell cycle arrest at G2/M; DNA double strand break damage was detected, preceding apoptosis irrespective of MGMT and MMR status. It was established that analog 377 can ring-open and hydrolyze like TMZ under physiological conditions and its intermediate product is more stable compared to that of TMZ. The following DNA adducts of 377 with calf thymus DNA were identified - N3-methyladenine, N7-methylguanine, O6- methylguanine, N3-methylguanine thymine, N3-methylcytidine deoxynucleotides and N3-methyladenine deoxynucleotides.

Keywords

Glioblastoma; Colorectal carcinoma; O6-Methylguanine-DNA methyltransferase; Mismatch repair; Apoptosis; DNA adducts

Introduction

Temozolomide (TMZ), an oral alkylating agent, has been administered in conjunction with radiation as the standard of care for glioblastoma multiforme (GBM) treatment [1]. TMZ is an imidazotetrazine prodrug that is able to cross the bloodbrain barrier (BBB) [2]. Under normal physiological conditions, TMZ spontaneously hydrolyzes to form the active intermediate 3-methyl-(triazen-1-yl) imidazole-4-carboxamide (MTIC). MTIC rapidly breaks down to form the reactive methyldiazonium ion (diazomethane) which reacts with nucleophilic groups on DNA, resulting in DNA methylation [3]. Approximately 70% of the methyl groups are located on the N7 of guanine (N7-G), 10% on N3-adenine (N3-A) and 5% at O6-guanine (O6-G) sites [2,3]. Interestingly, TMZ exerts cytotoxicity mainly through O6-methylguanosine (O6-MeG) which triggers futile cycles of mismatch repair (MMR), stalled replication forks and lethal DNA double-strand breaks, while N7- and N3-methyl purines are rapidly repaired by base excision repair (BER) [4,5]. However, O6-MeG lesions can be repaired by O6-methyl-guanine DNA methyltransferase (MGMT), a DNA repair protein that removes alkyl groups located at the O6-position of guanine [6]. Thus, response to TMZ requires low MGMT expression and intact MMR [7,8].

Resistance to TMZ conferred by MGMT and tolerance to O6-MeG in the presence of defective MMR limits TMZ clinical applications. Therefore, strategies have been devised to reduce resistance and enhance response to TMZ, including inhibition of DNA repair mechanisms which contribute to TMZ resistance. For example, combining MGMT inhibitors, which consume MGMT and prevent O6-MeG repair, with TMZ sensitizes tumors to TMZ [9]. However, myelosuppression limits the use of MGMT inhibitors and alkylating agent combination chemotherapy [10]. Other DNA repair protein inhibitors have been used in combination with TMZ to treat cancer, such as poly ADP-ribose polymerase (PARP) inhibitors. Although PARP inhibitors (in combination with TMZ) are better tolerated than MGMT inhibitors, myelosuppression and liver toxicity still cause clinical concern [11,12].

An alternative strategy is the development of TMZ analogs which can overcome TMZ-resistance. A series of TMZ derivatives has been synthesized and the anticancer activity investigated in vitro. N3-substituted TMZ analogs were developed and several derivatives of them exhibited a good effect on TMZ-resistant tumor cells in vitro [13,14]. In contrast, C8-substituted TMZ analogs have rarely been reported and compounds` mechanism of action remained unclear [15]. In this study, we investigated the anti-tumor effects of C8-substituted TMZ derivatives on TMZresistant tumor models in vitro. Two new C8-substituted TMZ analogs were synthesized, the C8 carboxamide was replaced by imidazolyl (377) and methyl imidazole (465) respectively (Figure 1). 377 and 465 analogs demonstrated good anticancer activity against MGMT overexpressing glioma T98G and MMR deficient colorectal carcinoma HCT116 cell lines. Moreover, 377 and 465 were able to arrest the cell cycle at G2/M, evoke DNA damage and trigger apoptosis in T98G GBM and HCT116 CRC cells, irrespective of MGMT and MMR status. The mechanism by which analog 377 exacts DNA damage, and specific DNA lesions produced have been identified: like TMZ, analog 377 ring-opens and hydrolyzes under physiological condition leading to DNA adducts formation.

neurooncology-Chemical-structures

Figure 1 Chemical structures of TMZ, 377 and 465.

Materials and Methods

Chemicals

TMZ was provided by Schering-Plough Research Institute (Kenilworth, NJ, USA). New analogs, synthesized at Pharminox Ltd, BioCity, Nottingham, UK, were prepared as 100 mM stock solutions in dimethyl sulfoxide (DMSO) and stored at -20°C. Reagents, unless specified otherwise, originated from Sigma- Aldrich Ltd.

Cell Lines and Culture Conditions

SNB19V (vector) and isogenic MGMT-transfected SNB19M human GBM cell lines were provided by Schering-Plough. Cells were cultured in RPMI 1640 medium supplemented with 2% glutamine, 1% non-essential amino acids, 50 μg/ml gentamicin, 400 g/ml G418 (geneticin) and 10% fetal bovine serum (FBS). HCT116 (hMLH1-) and HT29 CRC cells obtained from the American Type Culture Collection (ATCC), were maintained in RPMI 1640 medium supplemented with 10% FBS. T98G GBM cells (sourced from the ATCC) were grown in DMEM medium supplemented with 10% FBS. Cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C.

MTT Assay

Cells were seeded into wells at a density of 4 × 103 per well in 96-well plates and allowed to attach overnight. TMZ, 377 or 465 dilutions were prepared in culture medium from 100 mM stock solutions and final well concentrations between 0.1 and 1,000 μM were achieved (n=4-8). Following 3-5 days of incubation (37°C, 5% CO2). sterile-filtered 3-(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) (20 μl; 5 mg/ml in phosphate buffered saline) was added to each well (final concentration 0.4 mg/ml). Plates were re-incubated for 4 h allowing metabolism of MTT by viable cells to insoluble formazan crystals. Medium and unconverted MTT were aspirated and DMSO (150 μl) was added to each well to ensure complete formazan solubilization, absorbance was read on a BioTek SynergyH1 microplate reader (490 nm). Compounds concentrations causing 50% inhibition (IC50) values were calculated by interpolation.

Clonogenic Assay

SNB19V and SNB19M cells in the logarithmic growth phase were selected, digested with 0.25% trypsin (AMRESCO, USA) and single-cell suspensions were prepared in RPMI 1640 medium. Cells were then transferred into 6-well plates with 1 ml cell suspension per well, which contained 300 cells per well, and were incubated overnight at 37°C with 5% CO2. Any previous solutions were discarded, and 2.5 ml fresh complete RPMI 1640 medium with TMZ (10, 50, 100, 500 and1000 μM), 377 (10, 50, 100, 200 and 500 μM) and 465 (1, 5, 10, 50 and 100 μM) were added into each well for 16 h. Medium was renewed and cells were incubated at 37°C with 5% CO2 for approximately 11 days. A blank control well was set and each group was cultured and treated in triplicate. When colonies (>50 cells) formed in control wells, the experiment was terminated. Medium was aspirated from wells and 2 ml 100% methanol was added for 20 min to fix cells. Fixative was removed and colonies were stained, 0.5% methylene blue (AMRESCO, USA) was added for 10 min. Colonies were gently washed with water, air-dried and counted; IC50 values were calculated by interpolation.

Detection of γ-H2AX Foci

Cells (2×105) were seeded onto coverslips in 6-well plates and incubated overnight, before being exposed to TMZ (100 μM), 377 (50 and 100 μM) and 465 (20 and 50 μM) for 6, 12, 24 and 48 h. Cells were fixed with ice cold acetone, rinsed with PBS and blocked with 3% BSA and treated with 0.3% Triton-X in PBS for 30 min at room temperature. Primary antibody recognizing phosphorylated H2AX (γH2AX; Cell Signaling) was added overnight at 4°C. The following day, cells were incubated with fluorescence-conjugated secondary antibody Alexa Fluor 488 (Life technologies) at a working concentration of 8 μg/ml diluted in antibody dilution buffer for 60 min at room temperature in the dark. Nuclei were stained with 0.1 μg/ml DAPI. Images were captured using an Immunofluoroscence microscopy (Nikon, Japan; original magnification 1000x).

Pulsed-field gel electrophoresis

Cells (2 x 106) were seeded onto coverslips in plates and incubated overnight, then cells were treated with 100 μM TMZ, 100 μM 377 and 50 μM 465 for 24 and 48 h. Cells (6 x 105) were collected for each sample to make small agarose plugs embedded with cells. The small plugs were then digested with Proteinase K reaction buffer (10 mM Tris, 20 mM NaCl, 50 mM EDTA and 1 mg/ml Proteinase K) at 50°C for 48 h. The plugs were washed four times in 50 ml of wash buffer (20 mM Tris, pH 8.8, 50 mM EDTA) for 30 min each at room temperature with gentle agitation. DNA fragments in plugs were separated on 1% w/v agarose gels in 0.5× Tris/borate/ethylenediaminetetraacetic acid (TBE) buffer at 14°C using a CHEF DRII apparatus (Bio-Rad) with 6 V/cm, pulsed from 60 to 120 s for 24 h. The gels were stained with ethidium bromide (EB). The final results were measured using a gel imager (BIOGEN).

Western Blot Analysis

Cells were lysed in RIPA lysis buffer (25 mM Tris HCl (pH 7.5), 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 100 mM NaCl, 20 mM sodium -glycerophosphate, 10 mM sodium pyrophosphate, 0.5% triton X-100) supplemented with a protease inhibitor cocktail (Roche). Cellular proteins (30 μg) were separated by SDS-PAGE, and electro-transferred onto PVDF membranes. Membranes were blocked in Tris-buffered saline (TBS) containing 5% milk and 0.1% Tween-20 at room temperature. All primary antibodies (γH2AX, actin, tubulin, PARP and caspase 3, from Cell Signaling) were incubated overnight at 4°C; membranes were washed at room temperature before incubation with a secondary antibody (GE) conjugated with horseradish peroxidase for 1 h. Detection was performed with Super Signal Chemiluminescent reagent according to the manufacturer’s protocol (Tanon, China).

Alkaline agarose gel electrophoresis

TMZ, 377 and 465 (1, 10 and 20 μM) were mixed with 1.5 μg pEGFP-N1 plasmid in 40 μl buffer (3 mM NaCl, 1 mM Na3PO4 and 1 mM EDTA, pH 8.0) and reacted at 37°C for 2 h. Then restriction endonuclease BamHI was added to linearize the plasmid, and DNA was precipitated with ethanol. DNA precipitates were dissolved by 1x alkaline agarose electrophoresis buffer (10x alkaline agarose electrophoresis buffer: 500 mM NaOH and 10 mM EDTA, pH 8.0). The desired amount of powdered agarose was thawed in a measured amount of water, cooled to ∼ 55°C, and 0.1x volume of 10x basic agarose electrophoresis buffer was added. Then 20 μl dissolved DNA was mixed with 4 μl 6x basic loading buffer (300 mM NaOH, 6 mM EDTA, 18% Ficoll 400, 0.15% bromocresol green, 0.25% xylene cyanol). Samples were loaded and electrophoresed at a maximum of 5 V/cm at 4°C until the bromocresol green had migrated ∼ two-thirds of the gel length. The gels were placed in a neutralizing solution (1.5 M NaCl and 1 mM Tris-HCl, pH 7.6) and soaked for 45 min at room temperature. The gels were stained with EB and final results measured using a gel imager (BIOGEN).

Cell cycle analysis

Exponentially growing cells were harvested and seeded in 6-well plates (2 x 105 cells/well; 2 ml medium). Cells were incubated overnight, then treated with TMZ (100 μM), 377 (50 and 100 μM) and 465 (20 and 50 μM). Following incubation (24, 48, 72 and 96 h), attached and floating cells were pooled and pelleted by centrifugation (1,200 rpm, at 4°C, 5 min). Pellets were washed (PBS), cells were re-suspended in 0.3 ml hypotonic fluorochrome solution (0.1% sodium citrate, 0.1% Triton X-100, 50 g/ml PI and 0.1 mg/ml ribonuclease A) and stored overnight at 4°C in the dark. Fluorescence of PI-stained DNA was detected on a BD C6 cytometer and data were analyzed using C6 software.

Mitochondrial membrane potential assay

Cells (2 x 105) were seeded in 6-well plates and incubated overnight, then cells were treated with 10, 20 and 50 μM 465 for 9 h. The experimental procedure was carried out according to the JC-10 Mitochondrial Membrane Potential Assay Kit (Sangon, China). Cells (5x105 per tube) were centrifuged and pellets were suspended in 500 μl of JC-10 dye loading solution. Then the dyeloaded cells were incubated for 40 min at room temperature or 37°C in a 5% CO2 incubator. JC-10 monomers and J-aggregates were detected by a flow cytometer on the FL1 and FL2 channels, respectively. Finally, data were analyzed using C6 software.

Sample extraction and purification

Calf thymus DNA (40 μl; 0.2 mg) and 40 μl 377 (0.2 mg) were dissolved in 120 μl NaCl (15 mM) and incubated in 37°C for 24 h. The reaction mixture was then cooled to room temperature, and extracted with 250 μl diethyl ether 4x with centrifugation. The extracted DNA was precipitated with 100 μl 3 M sodium acetate solution (pH 5.2) and 1 ml ice-cold ethanol. The solution was then put on ice for 30 min and centrifuged at 4°C at 10,000 g for 15 min. The resulting DNA pellet was washed twice with 100 μl ethanol (70%) and dried under vacuum. The dried DNA pellet was re-dissolved in 100 μl 2 mM magnesium acetate solution, then 2 μl 50 units benzonase enzyme were added and incubated at 37°C for 4 h. Nuclease S1 enzyme (10 μl; 200 units) was added, and the solution was further incubated at 37°C for 4 h. Finally, solid-phase extraction (SPE) was carried out for these samples as follows: the cartridges were conditioned with 3 ml methanol and 2 ml 15 mM NaCl (pH 7.4). After sample absorption, the cartridges were washed with 3 ml 15 mM NaCl to remove unmodified nucleotides and the modified nucleotides were eluted from the cartridges with 2 ml methanol. The solvent was removed in a vacuum centrifuge and the dried residues were re-dissolved in 100 μl purified water.

HPLC

The LC system consisted of an Agilent (USA) Accela HPLC pumping system, coupled with an Accela Autosampler and Degasser. Chromatographic separation of the DNA-adducts was achieved by reverse phase chromatography and gradient elution. Separation of the DNA-adducts was carried out on a Perfluorophenyl column (Phenomenex Luna, USA), kept at 25°C. The mobile phase was a gradient prepared from 1% ethanoic acid, pH 5.5 (Eluent A) and methanol (Eluent B). Gradient program: 0 min 95% A, 0-6 min up to 95% B, 6-8 min 95% B, 8-8.1 min back to 95% A, 8.1-11 min equilibration. The flow rate was 1 ml/min and the injected volume was 5 μl.

Compound Stability Test

TMZ and 377 (20 μl; 5 mg/ml) were taken into the vials, then 80 μl pure water was added and mixed thoroughly in a 37°C incubator. At different time points (0-48 h), samples were taken for detection, and filtered with a 13 mm 0.2 μm polytetrafluoroethylene (PTFE) needle prior to HPLC detection. The data processing was carried out with Agilent 1260 Infinity.

UPLC-Q-TOF/MS Analysis

The Agilent 6540 UPLC-Q-TOF/MS (USA) was used with the electrospray ionization for DNA adducts qualitative detection. Chromatographic separation of the DNA adducts was achieved by reverse phase chromatography and gradient elution. The detection conditions of UPLC were the same as those for HPLC. Analysis was performed on a triple quadrupole time of flight mass spectrometer, fitted with a heated electrospray ionization source Jet Stream operating in the positive ion mode with the following working conditions: dry gas N2 temperature of 350°C and flow rate of 9 l/min; nebulizer pressure of 40 psig; capillary voltage of 3500 V; nozzle voltage of 1000 V; fragmentor voltage of 135 V; secondary mass spectral collision pool voltage of 20 V. Finally, data processing was carried out with MassHunter software.

Statistical analysis

Statistical significance was measured using the analysis of t-test. P ≤ 0.05 was considered significant and all statistical tests were two-sided.

Results

Growth inhibition

The activity of TMZ and analogs 377 and 465 was tested against a pair of isogenic human GBM cell lines: SNB19V (vector control) and SNB19M (stable MGMT transfection). TMZ was active in MGMT negative/low cell lines, with an IC50 value of 38.01 μM in SNB19V cells, but inactive in MGMT-overexpressing cells. SNB19M cell line transfected with MGMT demonstrated 13.39-fold inherent resistance to TMZ with a mean IC50 value of 508.84 μM. As can be seen in Table 1, C8-imidazolyl 377 showed anticancer activity in SNB19V and SNB19M cells with mean IC50 values of 32.96 μM, and 65.92 μM respecively. Methyl imidazole analog 465 gave IC50 values of 14.34 μM and 31.83 μM in SNB19V and SNB19M cells respectively. In addition, 377 and 465 showed good anticancer activity against the T98G GBM cell line which maintains inherently high MGMT expression, with IC50 values of 62.50 μM and 33.09 μM respectively. Furthermore, the MMR-deficient HCT116 CRC cell line, resistant to TMZ (IC50 values >500 μM), responded to 377 (IC50 value 44.23 μM) and 465 (IC50 value 25.37 μM).

in vitro, the antitumor activity of TMZ derivatives was further corroborated by clonogenic assays. SNB19V and SNB19M cells were briefly (16 h) exposed to TMZ (50, 100, 200, 500 and1000 μM), 377 (10, 50, 100, 200 and 500 μM) and 465 (1, 5, 10, 50 and 100 μM). As shown in Table 2, compared to SNB19V cells (IC50 value 32.2 μM), SNB19M cells demonstrated ∼ 27-fold inherent resistance to TMZ (IC50 value 854.65 μM). Compared to SNB19V cells (IC50 value 17.86 μM), SNB19M cells showed >6-fold resistance to C8-imidazole analog 377 (IC50 value >100 μM). However, C8-methylimidazole analog 465 revealed better anticancer activity against SNB19V cells (IC50 value 14.34 μM) and moreover, potent anti-clonogenic activity against MGMTexpressing SNB19M cells (IC50 31.83 μM).

These results indicate that novel imidazolyl/methyl imidazole imidazotetrazinones 377 and 465 inhibit the growth of tumor cells, of note, 465 demonstrated superior activity against TMZresistant cells.

Generation of DNA double-strand breaks by imidazotetrazine analogs

It is well known that TMZ generates O6-MeG adducts which trigger futile cycles of mismatch repair (MMR) in MMR-proficient cells and ultimately lead to lethal DNA double-strand breaks (DSBs). DSBs induce rapid phosphorylation of Ser139 at the carboxy terminus of histone H2AX, namely γH2AX; γH2AX has become the ‘gold standard’ marker of DNA DSBs. In order to determine whether 377 and 465 evoke DNA DSBs, the formation of γH2AX foci was examined in HCT116 cells treated with 377 (50 and 100 μM) and 465(20 and 50 μM) and visualized by immunofluorescence microscopy following 3, 9, 12 and 24 h exposure. Representative foci in HCT116 cells are shown in Figure 2A and foci quantification of treated cells is shown in Figure 2B. As demonstrated, new analogs 377 (50 and 100 μM) and 465 (20 and 50 μM) induce time- and concentration-dependent DNA DSBs in HCT116 cells. In addition, T98G cells were treated with 100 μM 377 and 465 for 3, 6 and 12 h, cellular γH2AX foci increased timedependently (Figures 2C-2D).

neurooncology-imidazotetrazine-analogs

Figure 2 C8-imidazotetrazine analogs 377 and 465 induce cancer cell DNA damage. (A) Induction of γH2AX foci in HCT116 cells following exposure to 377 and 465. HCT116 cell was treated with vehicle control, 50 μM 377 and 465 for 3, 9, 12 and 24 h; (B) Foci of treated HCT116 cells were quantified by densitometry (Image J software). (C) Induction of γH2AX foci in T98G cells. T98G cells were treated with vehicle control, 100 μM 377 and 465 for 6, 12 and 24 h, then immunocytochemically labeled with γH2AX antibody and the secondary antibody Alexa Fluor 488; DNA was counterstained with DAPI. (D) γH2AX quantification was performed by the ratio of DNA damaged cells to total T98G cells (cellular foci number >10 was considered to be damaged cell). (E) Pulsed-field gel electrophoresis (PFGE) directly detected DSBs in HCT116 cells. HCT116 cells were treated with TMZ (100 μM), 377 (100 μM), 465 (50 μM) and DDP (10 μM, as a positive control) for 24 and 48 h. (F) Detection of total γH2AX expression by Western blot. T98G cells were treated with TMZ, 377 or 465 (50 and 100 μM) for 6, 12, 24 and 48 h. (G) Detection of DNA cross-linking by alkaline gel electrophoresis. The double strand DNA was incubated with 1, 10 and 20 μM TMZ, 377 and 465, DDP (10 μM) was used as a positive control. The arrow marked the cross-linked DNA.

A more direct method, pulsed-field gel electrophoresis (PFGE) was adopted to detect DNA DSBs. HCT116 cells were treated with TMZ (100 μM), 377 (100 μM), 465 (50 μM) and DDP (10 μM; positive control) for 24 and 48 h, then disruption of genomic DNA was detected by PFGE (Figure 2E). TMZ and 377 both induced DSBs in HCT116 cells and the extent of genomic DNA breakage increased with time. Moreover, 50 μM 465 was able to achieve effects equivalent to or greater than those of 100 μM TMZ and 50 μM 377. Furthermore, global γH2AX expression was examined by Western blot following exposure of T98G cells to TMZ (100 μM), 377 (50 and 100 μM) and 465 (50 and 100 μM) for 6, 12, 24 and 48 h. As shown in Figure 2F, TMZ only slightly increased the expression of γH2AX in T98G cells, whereas, the same concentration of 377 and 465 caused more γH2AX expression with treatment time. Together these data indicate that new analogs 377 and 465 cause DNA damage in TMZ-resistant cells.

It is known that the TMZ analogue mitozolomide results in DNA cross-linking [16], which causes severe and unpredictable bone marrow suppression [17]. Therefore, we examined by alkaline gel electrophoresis whether the TMZ derivatives caused DNA damage by DNA cross-linking. Double-stranded DNA was treated with 1, 10 and 20 μM TMZ, 377 and 465 (1 and 10 μM DDP was used as a positive control); as Figure 2G demonstrates, imidazotetrazine analogs TMZ, 377 and 465 did not lead to DNA cross-linking.

Cell cycle perturbation by imidazotetrazine analogs

Futile DNA repair cycles triggered by TMZ result in cell cycle arrest in the absence of MGMT and the presence of MMR. To compare the effects of TMZ and analogs on cell cycle progression, DNA flow-cytometric analyses of SNB19V and SNB19M cells following exposure to agents (24-96 h) were performed. After 48 h treatment with 100 μM TMZ, 28.7% of the SNB19V cells arrested at G2/M (compared with 17.7% of control cells in G2/M; Figure 3A). Further significant (P<0.01) and prolonged accumulation of DNA in G2/M phases was observed after 72 and 96 h of exposure (31.8 and 48.5%, respectively). In contrast, 100 μM TMZ failed to perturb the SNB19M cell cycle, a result attributed to competent MGMT activity (Figure 3A).

neurooncology-caused-cell-cycle

Figure 3 377 and 465 caused cell cycle arrest in SNB19V and SNB19M cells. Representative DNA histograms of SNB19V and SNB19M cells demonstrating the effects of TMZ (A), 377 (B) and 465 (C) on cell cycle progression. Following desired exposure periods, cellular DNA was stained with propidium iodide and analyzed by flow cytometry. Control samples were treated with equal volume DMSO. For each sample, 10,000 events were recorded and experiments were performed in triplicate; n ≥ 3 independent trials.

Exposure of SNB19V cells to 377 (50 μM) significantly (P<0.001) increased S-G2/M events by >15% during the first 24 h period, persistent G2/M accumulation was maintained from 48 to 96 h, and a small pre-G1 peak indicative of apoptosis induction emerged; 6.6% pre-G1 phase at 96h (0.9% pre-G1 in control) (Figure 3B). Compared to SNB19V cells, SNB19M cells also exhibited significant (P<0.001) S-G2/M arrest at 24 h exposure, but the S-G2/M block was transient - reversing ≥ 48 h (Figure 3B). The cell cycle perturbations of 377 (100 μM) in both SNB19V and SNB19M were similar to those elicited by 50 μM 377.

SNB19V and SNB19M cells exposed to 465 (50 μM) revealed obvious (P<0.01) S-G2/M arrest at 24 h. Further significant (P<0.001) and prolonged accumulation of DNA in G2/M phase was observed from 48-72 h (38.1% and 37.3% in SNB19V; 53.0% and 48.2% in SNB19M, respectively) (Figure 3C). The percentage of the cell population in G2/M was higher in SNB19M cells than SNB19V cells, 53.0% compared to 38.1% at 48 h and 48.2% compared to 37.3% at 72 h. Analog 465 (20 μM) minimally perturbed SNB19V and SNB19M cell cycles (Figure 3C). The effect of TMZ derivative 465 on the cycle of MMR-deficient HCT116 cells was also examined. It was evident that events in G2/M accrued at both 48 and 72 h (50 μM 465) while 50 μM TMZ did not interfere with the cell cycle (Figure S1). These data indicate that TMZ analogs 377 and 465 cause cell cycle perturbation in TMZ-resistant cells.

Induction of apoptosis by imidazotetrazine analogs

In order to further investigate the cellular effects of 377 and 465, we characterized their impact on cell death at the molecular level. Lysates from compound-treated cells were subjected to Western blot analyses for expression of two apoptosis markers, caspase 3 activation and cleaved PARP. Initially, HCT116 cells were treated with the same concentration (50 μM) of TMZ and its derivatives for xx h. As shown in Figure S2, we found that only 465 induced PARP cleavage. HCT116 cells were then treated with 20 and 50 μM 465 for 24, 48 and 72 h. 465 induced HCT116 cells apoptosis in a time- and concentration-dependent manner, 20 μM 465 analog induced PARP cleavage from 48 h, however, 50 μM 465 caused apoptosis ≥ 24 h (Figure 4A). In addition, we also treated MGMT-overexpressing T98G cells with 100 μM TMZ, 50 and 100 μM 377 and 465 for 24 and 48 h. As shown in Figure 4B, neither significant caspase 3 nor PARP cleavage were induced by either TMZ, 377, or 465 cells after 24 h. However, ≤ 48 hours later, TMZ, 377 and 465 induced apoptosis; compared to TMZ, 377 and 465 induced enhanced apoptotic effects. These data indicate that new analogs 377 and 465 can cause apoptosis in TMZ resistant cells.

neurooncology-Induction-apoptosis

Figure 4 Induction of apoptosis by C8-imidazotetrazine analogs 377 and 465. Detection of apoptosis proteins (PARP and caspase3) by Western blot in (A) HCT116 cells and (B) T98G cells following treatment of cells with different concentrations TMZ, 377 or 465 for 24, 48 or/and 72 h. (C) HCT116 cells were dye-loaded with JC-10 dye-loading solution along with 10, 20 and 50 μM 465 for 9 h. The fluorescent intensities for both J-aggregates and monomeric forms of JC-10 were measured by flow cytometry using FL1 (green) and FL2 (red) channels. Data were analyzed using C6 software. (D) Quantitative assessment of JC-10 monomer signals (*P<0.05, **P<0.01).

In addition, the change in HCT116 cells` mitochondrial membrane potential was determined after exposure of cells to 465 concentrations associated with apoptosis [18]. As shown in Figure 4C, when HCT116 cells were treated with 10, 20 and 50 μM 465 for 9 h; the JC-10 monomer signals increased obviously (P<0.05) with increasing 465 concentrations (Figures 4C and 4D).

Hydrolysis of imidazotetrazine analog 377

Under physiological conditions, TMZ is able to hydrolyze to form MTIC, then MTIC rapidly breaks down to form the diazomethane and 5-aminoimidazole-4-carboxamide (AIC); diazomethane causes methylation of DNA, leading to cell death. We hypothesize that 377 could ring-open and alkylate DNA like TMZ, resulting in DNA damage to tumor cells and subsequent apoptosis. Analog 377 was dissolved in water, incubated at 37°C and analyzed by HPLC. As shown in Figure 5A, three picks were observed at retention times tR=2.12 min, tR=2.43 min and tR=4.43 min respectively. The hydrolyzate of 377 was then identified using a highly sensitive UPLC-Q-TOF/MS. As shown in Figure 5B, the absorption peaks can be detected in the total ion flow chromatogram at tR=1.45 min, tR=1.99 min and tR=3.57 min. The structures were assigned on the basis of MS/MS data of m/z 218, m/z 192 and m/z 150. The extracted ion chromatogram at retention time tR=3.736 (m/z 218, tR=1.431 (m/z 192 and tR=1.898 (m/z 150) was found to be 377 (C8H7N7O), intermediate (C7H9N7) and metabolite (C6H7N5) by high resolution mass spectrometry analysis (Figures 5C, 5D and 5E). Therefore, we speculate that 377 can undergo a hydrolysis reaction like TMZ. These data indicate that 377 is rapidly hydrolyzed to form intermediate 5-(3-methyltriazen- 1-yl) imidazole-4-imidazole under physiological conditions, which further hydrolyzes to 5-aminoimidazole-4-imidazole and diazonium (Figure 5F).

neurooncology-Reaction-scheme

Figure 5 Decomposition of C8-imidazotetrazine analog 377. (A) Chromatogram of 377 by HPLC. (B) Total ion chromatogram of 377 by UPLCQ- TOF / MS. (C) Mass spectra of m/z 218. (D) Mass spectra of m/z 192. (E) Mass spectra of m/z 150. (F) Reaction scheme for the decomposition of 377 in H2O.

Stability of imidazotetrazine analog

The stability of 377 in H2O was monitored by HPLC. TMZ or 377 solutions (1 mg/ml) were incubated at 37°C for 0-48 h. As shown in Figure 6A, TMZ was easily hydrolyzed at 37°C and almost completely hydrolyzed to form AIC at 48 h. The intermediate product MTIC was very unstable and we did not find the absorption of MTIC by UV detector. The peak area of TMZ was decreased from 2758 mAU*s to 475 mAU*s at 37°C from 0 to 24 h, and reached 50% after 10 h. The absorption peak of TMZ could not be detected at 48 h, but the absorption peak of AIC was increased with time (Figure 6B). However, the peak area of 377 was decreased from 279 mAU*s to 0 mAU*s at 37°C from 0 to 20 h, and reduced to 50% at 6 h. The peak of 377 could not be observed at 20 h (Figures 6C-6D) and the intermediate accumulated over time. The peak area of the intermediate increased from 87 mAU*s to 518 mAU*s and that of the hydrolyzate increased from 21 mAU*s to 2491 mAU*s from 0 to 20 h. These data suggest that 377 hydrolates faster and the intermediate is more stable than that of TMZ.

neurooncology-absorption-spectra

Figure 6 The stability of TMZ and 377. (A) The UV absorption spectra of TMZ, TMZ was incubated at 37°C for different times. (B) Quantitative peak area of TMZ. (C) The UV absorption spectra of 377, 377 was incubated at 37°C for different times. (D) Quantitative peak area of 377.

DNA adducts of imidazotetrazine analog 377

To investigate DNA adducts of 377, calf thymus DNA (ctDNA) was incubated with 377 in vitro and the resulting modifications were enzymatically cleaved with benzonase and nuclease S1. Compared with the chromatograms of 377 and ctDNA alone, four new absorption peaks were observed at the retention time tR=5.241, tR=5.418, tR=5.702 and tR=5.885 by HPLC analysis (Figures 7A-C). We speculated that DNA adducts may be formed, as described above in Figure 5F, 377 can ring-open and form an intermediate which then further breaks down to a reactive diazonium, we assume that the diazonium will alkylate DNA. Then UPLC-Q-TOF/MS was used to analyze the structures of DNA adducts. As shown in Figure 8A, there are new absorption peaks in the total ion current profile and the UV absorption pattern. From analysis, the peak corresponding to m/z 152 was assigned to N3-methyladenine, m/z 168 was identified as N7- methylguanine, m/z 168 was O6-methylguanine, m/z 141 was established as N3-methylguanine thymine, m/z 324 was assigned as N3-methylcytidine deoxynucleotides and m/z 348 was N3- methyladenine deoxynucleotides (Figures 8B-8F).

neurooncology-Chromatogram

Figure 7 Chromatogram of 377 with ctDNA by HPLC. (A) Chromatogram of 377. (B) Chromatogram of ctDNA. (C) Chromatogram of 377 with ctDNA.

neurooncology-Mass-spectra

Figure 8 Mass spectra of 377. The incubation of 377 with ctDNA for 24h followed by enzymatically digestion with benzonase and nuclease S1. (A) Total ion chromatogram of 377 with ctDNA. (B) Mass spectra of m/z 152. (C) Mass spectra of m/z 168. (D) Mass spectra of m/z 141. (E) Mass spectra of m/z 324. (F) Mass spectra of m/z 348.

Discussion

TMZ plays a key role in GBM therapy; indeed, radiotherapy, and concomitant and adjuvant chemotherapy with temozolomide is the standard of care for this devastating malignancy. TMZ has also been used in the treatment of other cancers (e.g. malignant melanoma) [19], but the ultimate benefit is limited by inherent, or emergence of resistance. Expression of the DNA repair protein MGMT accounts for TMZ resistance; inactivation or down-regulation of MMR leads to acquired tolerance to TMZinduced lesions [2,7] and moreover, can promote a hypermutator phenotype [20]. Therefore, developing new TMZ derivatives which can overcome TMZ-resistance may provide significant therapeutic advantage.

A variety of TMZ derivatives has been previously reported [13-15,21-26]. The most common modification sites for these analogs are the N3 or C8 positions of TMZ. Among them, N3- modified TMZ analogs can be divided into two categories: one class of derivatives is incorporation of other groups to link two TMZ molecules through the N3 site, for example, derivatives DP68 are two TMZ molecules linked together by methylaniline. DP68 binds two DNA strands together by hydrolysis to cross-link the DNA and kill the cells [21]. Another class of analogs results from replacement of the methyl group at the N3 position with other substituents, such as chloromethyl and propargyl. These derivatives allow the new group to alkylate DNA, thereby exerting a cytotoxic effect [13,14]. In addition, C8-modified TMZ analogs can also be divided into two categories: one class may be derived through conjugation of two TMZ molecules through the C8 position. For example, the analog 2T-P400 links two TMZs by polyethylene glycol, thereby enhancing the solubility and stability of TMZ [22]. Another type of derivative may be formed following replacement of the C8 carboxamide group of TMZ by another group. For instance, NEO212 is a covalent bond of perilla alcohol to the C8 site of TMZ, thereby enhancing the efficacy of TMZ [23-25], while TMZ hexyl ester which incorporates a hexyl ester bound to the C8 site of TMZ can improve TMZ skin delivery potency and antitumor activities [26]. In addition, other C8- substituted derivatives such as 8-cyano-imidazotetrazine and 8-thiotemozolomide have been developed, but their antitumor mechanism(s) remain to be defined [15].

In this study, the anti-cancer activity of two novel C8-substituted TMZ analogs C8 imidazolyl (377) and C8 methyl imidazole (465) has been evaluated. Isogenic GBM cell lines, SNB19V (vector control) and SNB19M (stable MGMT transfection), were used to detect the activity of TMZ and its derivatives. TMZ is active in the MGMT-low cell line, with a mean IC50 value of 38.01 μM, but inactive in MGMT-overexpressing SNB19M cells, with a mean IC50 value of 508.84 μM. SNB19M cells overexpressing MGMT revealed 13-fold resistance to TMZ (Table 1). However, TMZ analogs 377 and 465 are active in SNB19 GBM cell lines, irrespective of MGMT activity. Moreover, relative to TMZ, 377 and 465 show greater potency (~5-24-fold) in MGMT overexpressing glioma T98G and MMR deficient CRC HCT116 cells. These results appear to indicate that the C8 carboxamide moiety of TMZ is more stable after being replaced by imidazolyl and methyl imidazole. Moreover, in clonogenic assays, after brief exposure of cells to inidazotetrazine analogs, we found that the IC50 value of 377 against SNB19M cells was at least 6-fold greater than that in SNB19V cells by clonal formation assay while the activity of 465 against SNB19M (compared with SNB19V) cells was only 2-fold greater (Table 2). These data suggest that the lesions imparted by 377 can be repaired by MGMT over time, whereas inclusion of a methyl group within the C8 imidazolyl (465), imparts structural changes that lead to more stable DNA adducts, less susceptible to MGMT repair.

Cell line MGMT MMR IC50 (M) 3 days
TMZ 377 465
SNB19V - proficent 38.01 ± 16.35 32.96 10.8 14.34 2.92
SNB19M + proficent 508.84 ± 142.32 65.92 7.68 31.83 8.66
HTC116 + MLH1­ 592.88 ± 16.10 44.23 12.72 25.37 2.21
T98G + proficent 286.96 ± 19.61 62.50 6.93 33.09 3.42

Table 1 MTT assay IC50 values of TMZ and C8-imidazotetrazine analogs 377 and 465 against human cancer cell lines.

TMZ exerts cytotoxicity primarily through O6-MeG, which triggers ineffective cycles of MMR, stalled replication forks and lethal DNA double-strand breaks, leading to cell death by autophagy or apoptosis [2,4]. We found through immunofluorescence, that new analogs 377 and 465 can induce DSB in T98G and HCT116 cells after 6 h treatments (Figures 2A and 2C). Further study found that TMZ and analogs could induce DSBs in HCT116 and T98G cells. The lower concentration of 465 (50 μM) in HCTl16 cells can result in DNA damage equal to, or greater than that inflicted by the higher concentration (100 μM) of TMZ and 377 (Figure 2D). γH2AX expression in T98G cells was also examined; compared with TMZ, the same concentration of 377 and 465 can upregulate the expression of γH2AX (Figure 2E). Moreover, we found that TMZ derivatives 377 and 465 did not cause DNA cross-linking like the TMZ analog mitozolomide which can lead to serious side effects such as myelosuppression (Figure 2F). These results indicate that analogs 377 and 465 can cause DNA damage in TMZ-resistant cells without leading to DNA cross-linking. In addition, we found that TMZ analogs 377 and 465 can lead to G2/M arrest irrespective of MGMT status, and 465 had a greater effect on the cell cycle of MGMT highly-expressing SNB19M cells (Figure 3). Further study found that 465 can cause G2/M block in HCT116 cells (Figure S1). In addition, 50 μM 465 can induce apoptosis in HCT116 cells, while the same concentration of TMZ and 377 did not induce PARP cleavage (Figures 4A and S2). We also found that 377 and 465 led to significant T98G cell apoptosis; indeed, compared to TMZ, 377 and 465 induced greater apoptosis in T98G cells (Figure 4B). Moreover, further study showed that 465 can cause mitochondrial membrane potential changes, which indicates that TMZ derivatives may lead to apoptosis dependent on mitochondrial pathways (Figure 4C). Together, these data demonstrate that novel C8-substitured TMZ analogs can circumvent TMZ resistance in CRC and GBM cells. We further observe that TMZ analog 377 can ring-open and be hydrolyzed like TMZ, it is rapidly hydrolyzed to form a stable intermediate 5-(3-methyltriazen-1-yl) imidazole-4- imidazole under physiological conditions, which further forms 5-aminoimidazole-4-imidazole and diazonium (Figure 5F). Active diazonium then causes DNA lesions to occur at multiple sites of methylation, includng N3-methyladenine, N7-methylguanine and O6-methylguanine (Figures 8B and 8C). In addition, as shown in Figures 6A-6C, 377 hydrolysis is faster than TMZ, and the intermediate is more stable which may explain why 377 exerts activity in TMZ-tolerant or -resistant HCT116 and T98G cells; however, in the long term, damage accrued from exposure of cells to 377 can be repaired by MGMT and 377 activity is therefore attenuated (Table 2 and Figure 3B). The replacement of C8 caroxamide by imidazolyl (377) and methyl imidazole (465) may contribute to the stability of intermediates which might induce DNA damage and apoptosis in MGMT overexpressing and MMR deficient CRC cells. However, analyses of stability, and DNA adduct formation by analog 465 was thwarted by precipitation which occurred during incubation of 465 with ctDNA.

Compound SNB19V SNB19M IF*
TMZ 32.2 ± 10.35 854.65 ± 142.32 27
377 17.86 ± 7.3 >100 >6
465 14.34 ± 2.92 31.83 ± 8.66 2

Table 2 Clonogenic assay IC50 values of TMZ and C8-imidazotetrazine analogs 377 and 465 against human cancer cell lines.

In summary, the novel C8-substituted TMZ analogs 377 and 465 elicit in vitro antitumor activity irrespective of MGMT and MMR status. DNA DSBs were generated in MGMT-overexpressing glioma SNB19M and T98G cells and MMR deficient CRC HCT116 cells. TMZ derivatives perturbed and arrested SNB19V and SNB19M cell cycle progression. Moreover, compared to TMZ, analogs 377 and 465 were able to induce more severe apoptosis in HCT116 and T98G cells. And similar to TMZ, analog 377 is subjected to ring opening and hydrolysis leading to DNA adducts formation which likely underlies the anticancer effects. These analogs may offer potential for TMZ-resistant GBM and broader spectrum malignancies.

Acknowledgements

Financial support for all work described herein was provided by the National Natural Science Foundation of China (No. 81260501; 81560601).

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