Reactive Oxygen Species-Mediated Apoptosis and Cytotoxicity of Newly Synthesized Pyridazin-3-Ones In P815 (Murin Mastocytoma) Cell Line

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Introduction

Currently, classical treatment of cancer offers only limited benefits with systemic cytotoxicity [1–3]. In fact, chemotherapy is the effective method currently available for the treatment of cancer.

However, drug resistance is common and associated several harm- ful side effects which presents major obstacles for the effective treatment of cancer. Consequently, development of new alterna- tive targeted therapies is urgently needed [4].

Bouchmaa Najat et al. Reactive Oxygen Species-Mediated Apoptosis … Drug Res 2018; 00: 00–00

Reactive Oxygen Species-Mediated Apoptosis and Cytotoxicity of Newly Synthesized Pyridazin-3-Ones In P815 (Murin Mastocytoma) Cell Line

Authors

Najat Bouchmaa^{1, 2}, Reda Ben Mrid³, Youness Boukharsa¹, Youssef Bouargalne³, Mohamed Nhiri, Abderrazak Idir², Jamal Taoufik¹, Mʼhammed Ansar¹, Abdelmajid Zyad²

Affiliations

1 Laboratory of Medicinal Chemistry, Faculty of Medicine and Pharmacy, Mohammed V University, Rabat, Morocco 2 Team of Experimental Oncology and Natural Substances, Cellular and Molecular Immuno-pharmacology, Faculty of Science and Technology, Sultan Moulay Slimane

University, Beni-Mellal, Morocco

3 Laboratory of Biochemistry and Molecular Genetics, Faculty of Science and Technology, Abdelmalek Essaâdi University, Tangier, Morocco

Key words

anticancer agent, oxidative stress, thioredoxin system, glutathione system, antioxidant enzymes

received 29.06.2018 accepted 17.10.2018 Bibliography

DOI https://doi.org/10.1055/a-0762-3775 Published online: 2019

Drug Res 2019; 69: 1–9

Correspondence Prof. Abdelmajid Zyad

Faculty of Sciences and Techniques Sultan Moulay Slimane University B.P: 523. Beni Mellal-Morocco

Tel.: + 212/0/523 48 51 12/22/82 ext 289, Fax: + 212/0/523 48 52 01

a.zyad@usms.ma; ab.zyad2@gmail.com

Supporting Information for this article is available online at http://www.thieme-connect.de/products

ABStRAct

Background In cancer cells, the intracellular antioxidant capacity and the redox homeostasis are mainly maintained by the glutathione- and thioredoxin-dependent systems which are considered as promising targets for anticancer drugs. Pyri- dazinones constitute an interesting source of heterocyclic compounds for drug discovery. The present investigation fo- cused on studying the in-vitro antitumor activity of newly synthesized Pyridazin-3(2h)-ones derivatives against P815 (Murin mastocytoma) cell line.

Methods The in-vitro cytotoxic activities were investigated toward the P815 cell line using tetrazolium-based MTT assay.

Lipid peroxidation and the specific activities of antioxidant enzymes were also determined.

Results The newly compounds had a selective dose-dependent cytotoxic effect without affecting normal cells (PBMCs).

Apoptosis was further confirmed through the characteristic apoptotic morphological changes and DNA fragmentation. Two compounds (6f and 7h) were highly cytotoxic and were submit- ted to extend biological testing to determine the likely mechanisms of their cytotoxicity. Results showed that these molecules may induce cytotoxicity via disturbing the redox homeostasis. Importantly, the anticancer activity of 6f and 7h could be due to the intracellular reactive oxygen species hyper- generation through significant loss of glutathione reductase and thioredoxin reductase activities. This eventually leads to oxidative stress-mediated P815 cell apoptosis. Furthermore, the co-administration of 6f or 7h with Methotrexate exhibited a synergistic cytotoxic effect.

Conclusions considering their significant anticancer activity and chemosensitivity, 6f and 7h may improve the therapeutic efficacy of the current treatment for cancer.

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Original Article Thieme

Reactive oxygen species (ROS) are a group of molecules (hy- droxyl radicals, peroxides, H2O2, nitric oxide) that are maintained, at an optimal level, through enzymatic and non-enzymatic systems [5]. The increase of ROS production or the decrease of the antioxidant defence system efficiency is responsible for the cell’s oxidative stress [6]. In cancer cells, the intracellular antioxidant capacity is mainly conferred by the glutathione- and thioredoxin-dependent systems. In fact, the important enzymes implicated in the regulation of redox homeostasis such as peroxidases and thiol reductases, are limited to the pool of GSH and thioredoxin as source of reducing equivalents. Glutathione reductase (GR) which catalyses the reduction of GSSG to GSH and thioredoxin reductase (TrxR) which is responsible for the reduction of thioredoxin were reported to be up-regulated in a number of cancers [7, 8]. Therefore, reducing GSH and thioredoxin pool in cancer cells, through the inhibition of GR and TrxR enzymes, may be considered as an effective strat- egy to combat these tumor cells by altering their ability to elimi- nate ROS and to cope with oxidative damage. Both glutathione- and thioredoxin-dependent systems require NADPH, as cofactor, to combat ROS generated from oxidative stress [9]. It has been sys- tematically showed that NADPH is a limiting factor for oxidative stress management in cancer cells. Thus, it was reported that cells exposed to oxidative stress exhibited reduced NADPH levels, which may be a result of elevated activity of NADPH-dependent antioxidant systems [10]. NADP + − ICDH is one of the main enzymes implicated in the provision of NADPH [11]. In fact, it was reported that the over-expression of NADP + − ICDH is responsible for high resistance to oxidative stress, however, decreased NADP + − ICDH expression lead to a low resistance [12, 13]. Furthermore, IDH-1 knockdown was reported to depress cancer cell proliferation [14].

Pyridazin-3(2h)-one derivatives constitute an interesting class of heterocyclic lead compounds for drug discovery and research into anticancer molecules [15–17]. Recently, it was reported that some pyridazin-3(2h)-one derivatives specifically interact with and inhibit PIM (potential tumor targets) kinases with low nanomolar potency [18–21]. In addition, pyridazin-3(2h)-ones are endowed by many biological activities [22–24]. In this context, the present study was aimed at synthesizing new pyridazin-3(2h)-one derivatives with cytotoxic activity. The obtained molecules were tested for their antitumor activity against P815 murin mastocytoma cells (P815). Thereafter, 2 highly cytotoxic molecules were chosen and compared for their ability to induce oxidative stress, in the same cell line, in an attempt to reveal the likely mechanisms involved in this cytotoxic effect.

Materials and Methods

Chemicals and cell lines

All chemicals and biochemicals reagents were purchased from Sigma-Aldrich except DMEM medium and L-Glutamin were from Lonza, Fetal Bovine Serum were from Gibco BRL, (Cergy Pontoise, France), Bradford reagent was obtained from Bio-Rad (Hercules, CA, USA) and β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) was purchased from Roche Diagnostics (Mannheim, Germany). Tumor cell line P815 (Murine mastocytoma) was from the stock cultures of the Labora-

tory of Biological Engineering, Faculty of Science & Technology, Sultan Moulay Slimane University, Morocco.

Cell culture

P815 (Murin matosytoma) cell line was cultured in Dulbecco’s modi fied Eagle’s medium (DMEM) supplemented with 5 % heat- inactivated fetal bovine serum, penicillin G-streptomycin (1 %), and sodium bicarbonate (0.2 %). Incubation was performed at 37 °C in a humidified atmosphere containing 5 % CO2.

Synthesis of drug molecules

Melting points were determined on a Büchi SMP 20 apparatus and are not corrected. Infrared (IR) spectra were recorded with an IR VERTEX 70 FT-IR (Bruker Optics) spectrometer. 1h Nuclear mag- netic resonance (1h NMR) spectra were recorded on a Bruker Avance (300 MHz) spectrometer, using tetramethylsilane (TMS) as internal standard and, CDCl3 and DMSOd6 as solvent. Mass spectra was recorded on a API 3200 LC/MS/MS mass spectrometer using electrospray ionization (ESI) in positive polarity.

General procedures for the formylation of phenols 2a-f, the synthesis of 2-formylphenoxyacetadehyde diethyl acetals 3a-f, the synthesis of substituted benzo[b]furan-2-carbaldehydes 4a-f, the synthesis of substituted 3-benzo[b]furan-2-ylmethylene-levulinic acids 5a-f, the synthesis of substituted 5-(benzo[b]furan- 2-ylmethyl)-6-methylpyridazin-3(2h)-ones 6a-f have been described in our paper [25].

General procedures for the synthesis of substituted 5-(benzo[b]

furan-2-ylmethyl)-6-methylpyridazin-3(2h)-thiones 7g-h: The mixture of substituted 6-methylpyridazin-3(2h)-ones 6 and diphosphore pentasulfure solution in pyridin was refluxed for 4h; the precipitate formed is filtered and recrystallized from an appropriate solvent.

The analytical data of the products 7g-7h is depicted below:

5-[(5-Chlorobenzo[b]furan-2-yl)methyl]-6-methylpyridazin-3(2h)- thione 7g. This compound was obtained as yellow solid, yield 65 %, mp 165–167 °C (ethanol); IR (KBr νmax cm^{− 1}), 1605 (C = N), 1080 (C = S); ¹H NMR (DMSOd6, 300 MHz, J Hz), δ = 2.30 (s, 3h, -N = C-CH3), 4.15 (s, 2h, -CH2-), 6.76 (s, 1h, H3’), 7.28 (dd, 1h, J6’-7’ = 8.70 and J6’-4’ = 2.40, H6’), 7.32 (s, 1h, H4), 7.58 (d, 1h, J7’-6’ = 8.70, H7’), 7.65 (d, 1h, J4’-6’ = 2.40, H4’), 14.51 (ls, 1h, NH).

5-[(7-Chlorobenzo[b]furan-2-yl)methyl]-6-methylpyridazin- 3(2h)-thione 7h. This compound was obtained as yellow solid, yield 62 %, mp 188–190 °C (ethanol); IR (KBr νmax cm^{− 1}), 1606 (C = N), 1082 (C = S) ; ¹H NMR (DMSOd6, 300 MHz, J Hz), δ = 2.33 (s, 3h, -N = C-CH3), 4.19 (s, 2h, -CH2-), 6.86 (s, 1h, H3’), 7.20–7.57 (m, 4h, H4, H4’, H5’ and H6’), 14.51 ( ls, 1h, NH).

MS : m/z; 290.7 [M-H]⁺, 313.10 [M-Na]⁺.

Cytotoxic activity evaluation

P815 cell line was harvested from starting cultures at the exponen- tial growth phase. After PBS wash, the harvested cells were poured in flat-bottomed 96-well microliter plates containing 100 µL of complete medium per well (4.3 × 10⁴ cells/mL). 3h later, several concentrations of pyridazin-3(2h)-one derivatives in DMSO completed to 100 µL with complete DMEM were added. Control cells were treated with DMSO alone. In all cases, the final concentration of DMSO never exceeded 0.1 %. After 48h incubation in humidified atmosphere at 37 °C and 5 % CO2, 100 µL of medium was carefully

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(5 mg/mL PBS). After 4h incubation under the same conditions, the cleavage of MTT to formazan by metabolically active cells was quantified by scanning the plates at 540 nm using a Multiskan EX (Finland) apparatus. 3 independent sets of experiments performed in dupli- cate were evaluated. The relative percentage of cell viability was calculated by the following formula:

% Viability = 100 × (A/A0),

Where A0 and A are the absorbance of negative control and test culture, respectively.

The cytotoxic effects of pyridazin-3(2h)-one derivatives against the cell line were compared using their IC50 values (concentration of tested molecules leading to 50 % inhibition of cell viability).

Cytotoxic effect against peripheral blood mononuclear cells (PBMCs)

This test was realized in order to evaluate the effect of synthesized pyridazin-3(2h)-one derivatives against normal cells using the MTT colorimetric assay described above. To isolate the human PBMCs, blood samples were collected from healthy donors in heparinized tubes and the PBMCs were isolated using standard Ficoll-hypaque density centrifugation. The interface lymphocytes were washed twice with phosphate buffer solution (PBS). Cells were incubated in 96-well microtiter plates in the presence of the same concentrations of synthesized pyridazin-3(2h)-one derivatives in the same conditions in the tumor cells.

Combination study

The combination effect between 6f or 7h and Methotrexate (MTX) against P815 cell line was measured as described by Chou and Talalay [26]. Using combination index (CI) analysis at a non-constant ratio, i. e., drug combinations were made by varying the concentrations of one drug (MTX) while keeping the second drug (6f or 7h) concentration fixed to IC30. The IC30 values of 6f and /or 7h derivatives were determined on the basis of the dose-response curve. Finally, a series of dilutions of MTX were mixed to the IC30 to generate the dose-response curve in the P815 cell proliferation. The combination effects were an- alyzed by the combination index (CI) as described below:

CI = (D1)/(Dx1) + (D2)/(Dx2),

Where (D)1 and (D)2 are the doses of 6f or 7h and MTX, respectively, in the combination system; (Dx)1 and (Dx)2 are the doses of 6f or 7h and MTX alone, respectively. For data analysis of combinations an average CI < 1 indicates synergism, CI > 1 indicates antagonism and an average CI = 1 indicates additivity.

Apoptosis test via DNA fragmentation

P815 were treated with 6f and 7h (IC50). After 24h incubation in the same conditions described for cell culture, the cells were washed in PBS and treated with the lysis buffer (100 mM TRIS, 0.5 M EDTA, 10 % SDS, 5 M NaCl and 20 mg/mL Proteinase K). Samples were then incubated at 37 °C for 3h with agitation. Then, DNA was precipitated by ispropanol and was recovered and dispersed in

solved DNA were subjected to electrophoresis at 60 V for 2h in 1.5 % (w/v) agarose gels. Electrophoresis bands showing DNA fragmentation (180–200 bp) provide confirmation of programmed cell death. This characteristic of DNA laddering was visualized with Ethidium bromide staining using a UV trans-illuminator Biorad XR.

Malondialdehyde (MDA) content

Lipid peroxidation measured as MDA content in P815 cells was determined using thiobarbituric acid (TBA) according to the method described previously [27] with slight modifications. In brief, cell homogenate was mixed with trichloroacetic acid (TCA) (20 %) and TBA (0.67 %). The mixture was heated at 95 °C for 1h. After cooling, 1 mL of n-butanol was added to the mixture followed by centrifugation at 12,000g for 10 min. Organic supernatant was collected to measure the absorbance at 532 nm.

Preparation of cell extracts for antioxidant enzyme assays

P815 were treated with 6f or 7h for 24 and 48h. After washing once with phosphate buffer solution (10 mM, pH 7.4), the cells were harvested and centrifuged at 1,200g for 10 min. The pellet was sus- pended in 500 μL of lysis buffer composed of 50 mM Tris-HCl, 1 mM phenylmethanesulfonyl (PMSF), 0,1 % (v/v) Triton X-100 and maintained in constant agitation at 4 °C for 30 min. The homogenate was then centrifuged (12,000g, 20 min) at 4 °C. The supernatant (enzyme extract solution) was kept at − 80 °C or used for the determination of glutathione peroxidase (GPx), glutathione reductase (GR), isocitre dehydrogenase and thioredoxin reductases (TrxR) activities.

Antioxidant enzyme assays

GPx activity was measured by the method of Lawrence and Burk [28] with some modifications. The reaction mixture contained 0.1 M potassium phosphate, pH 7.0, 1 mM EDTA, 1 mM sodium azide, 1 mM GSH, GR (10 µg/ml), 0.25 mM NADPH and enzyme extract. The mixture was incubated at 25˚C for 3 min and completed by adding 0.25 mM of H₂O₂. The rate of NADPH oxidation was monitored at 340 nm for 5 min. GPx activity was calculated and expressed as µmol of NADPH oxidized/min/mg protein by using the extinction coefficient of 6.2 mM^–1cm^–1.

GR activity was estimated by a modified method of Carlberg and Mannervik [29]. Briefly, the reaction mixture contained 0.1 M phosphate buffer, pH 7.6, 1 mM GSSG and 0.2 mM NADPH. The contents were incubated at 25 °C for 3 min and the reaction was initiated by adding enzyme extract. The rate of NADPH oxidation was monitored at 340 nm. GR activity was expressed as nmol of NADPH oxidized per min per mg of protein by using the extinction coefficient of 6.2 mM^–1cm^–1

TrxR activity was measured as reported by Lim et al. [30] and NADP + -ICDH activity was estimated according to the procedure of Leterrier et al. [31].

Protein content determination

Total protein content of the samples was determined following the method of Bradford [32] using BSA as a protein standard.

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Statistical analysis

The used data are mean values ± S.D. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the

Tukey test using PASW statistics (version 18). Different letters indicate significant differences at the level of 5 %.

▶Fig 1 Cytotoxicity towards P815 after treatment with 6a, 6b, 6c, 6d, 6e, 6f, 7g and 7h Pyridazin-3(2h)-one derivatives for 48h. Each value represents the mean of 3 replicates of 3 independents measurement. Bars represent the standard error. Different letters indicate significant differences among treatments. p-value < 0.05 indicate significant difference.

0 20 40 60 80 100

1.5625 3.125 6.25 12.5 25 50 100 200

Compunnd 6a 6a

MTX

Compund 6c 6c

MTX

Compund 6b 6b

MTX

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6d MTX Compund 6d

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Compund 6e 6e

MTX

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Compund 6f 6f

MTX

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Concentration (µM) Concentration (µM)

Compund 7g

% of Cell Viability

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% of Cell Viability

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% of Cell Viability

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7g MTX

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Compund 7h 7h

MTX

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Chemistry

As described in our paper Bouchmaa et al. [25], the pyridazinone derivatives incorporating Benzofuran moiety studied in this work (compounds 6a-f and 7g-h) were synthesized according to the general procedures outlined in ▶Supplementary Fig.1 (supple- mented materials). In the first step, salicylaldehydes 2a-f were synthesized via Reimer Thieman [33] formylation of the appropriate substituted phenol 1a-f with CHCl3 and NaOH. Aldehydes 2a-f has been previously synthesized with very low yield. Briefly, reaction of intermediates 2a-f with bromacetaldehyde diethyl acetal in the presence of potassium carbonate in DMF (dimethylformamide) yielded compounds 3a-f. Next, these compounds 3a-f were cyclized to benzo[b]furan-2- ylcarboxaldehydes 4a-f by heating in concen- trated acetic acid. Benzo[b]furanaldehydes 4a-f were prepared according to the methods described in the literature [34]. Then, treatment of substituted aldehydes 4a-f with levulinic acid in the presence of HCl (chlorhydric acid) in acetic acid gave adducts 5a-f, which were heated by hydrazine hydrate to afford the pyridazin- 3(2h)-ones 6a-f. Finally, the mixture of substituted 6-methylpyridazin-3(2h)-ones 6 and diphosphore pentasulfure solution in pyridin gave 7g-h[35].

Cytotoxcity

As shown in ▶Fig. 1, the treatment of P815 with different concentrations of Pyridazin-3(2h)-one derivatives 6a-f and 7g-h showed that the survival of cells gradually reduced when the concentration increases.Indeed, the cell proliferation was inhibited by more than 70 %. The most cytotoxic molecules were 6f and 7h, with an IC50 of 14.50 µM and 20 µM respectively. Whereas the compounds 6a, 6d and 7g showed high cytotoxicity between 20 and 37.5 µM. Con- cerning 6b and 6c, they showed low cytotoxicity (48.5 and 70.3 µM). Comparison of data suggested that the activity is dose dependent manner (▶table 1).

The majority of clinically approved anticancer drugs are character- ized by a narrow therapeutic window that results mainly from a high systemic toxicity of drugs. Thus, we tested the most important compounds (which are the most cytotoxic against tumor cells) against the human peripheral blood mononuclear cells (PBMCs) in order to determine their effects against normal cells. Interestingly, our results represented in Figure depicted no cytotoxic effect on normal PBMCs indicating a selective cytotoxicity of our molecules.

Combination study

It is noteworthy that when combined, 6f or 7h and MTX induced a strong cytotoxic activity as it is shown in ▶table 2. A synergistic activity was observed at concentrations of MTX from 0.58 μM to 75 μM combined with the IC30 of 6f (5 μM) and 7h (6 μM). Howev- er, at very low concentrations ( < 0.58 μM) of MTX, an antagonistic effect was observed in 6f effect. However, the synergistic effect induced by 7h molecule did not decrease even up to 0.14 μM MTX.

DNA fragmentation assay

DNA fragmentation generally occurs during programmed cell death and is a landmark for apoptosis [36]. The conventional agarose gel electrophoresis was performed on the isolated DNA sam- ple from cells treated with 6f and 7h for 24h. The ▶Fig. 3 showed an appearance of intranucleosomal DNA cleaved products with ladder like patterns of 180- 200 bp, with no significant fragmentation detected in the untreated cells (▶Fig. 3).

Lipid peroxidation

The lipid peroxidation, which is an indicator of oxidative stress, was quantified by measuring the MDA content. The 2 Pyridazin-3(2h)- one derivatives tested here, were able to induce an increase in the MDA content compared to the negative control. However, increase

▶table 1 Inhibitory Concentration 50 (IC50) in µM of the Pyridazin-3(2h)-one derivatives tested against P815 tumor cell line. The differences between means are statistically significant at p < 0.05.

Molecules series

6a 6b 6c 6d 6e 6f 7g 7h MtX

Ic50 (µM) of cytotoxicity 24 ± 2.4 48.5 ± 5.3 70.2 ± 29.6 30.5 ± 4.5 37.5 ± 0.2 14.5 ± 3 36 ± 3.1 20 ± 1.7 3.15 ± 2.8

▶table 2 Combination index (IC) of 6f or 7h Pyridazin-3(2h)-one combined with MTX against P815 tumor cell line.

[MtX] µM [Ic30 of 6f] µM cI Description [MtX] µM [Ic30 of 7h] µM cI Description

75 5 ≅ 0 Synergism 75 6 ≅ 0 Synergism

37.5 5 ≅ 0 Synergism 37.5 6 ≅ 0 Synergism

18.75 5 ≅ 0 Synergism 18.75 6 0.37 Synergism

9.37 5 ≅ 0 Synergism 9.37 6 0.75 Synergism

4.68 5 0.017 Synergism 4.68 6 0.48 Synergism

0.29 5 1.14 Antagonism 0.29 6 0,51 Synergism

0.14 5 1.18 Antagonism 0.14 6 0.81 Synergism

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in the MDA content caused by 6f was similar to that induced by the positive control, MTX (▶Fig. 4).

Antioxidant enzyme activity

P815 tumor cells exposed to 7h and 6f negatively affected the GR and TrxR activities. In fact, the 2 Pyridazin-3(2h)-one derivatives decreased the GR activity compared to the untreated control cells (▶Fig. 6, A).

However, the decrease was more pronounced after 48h.

Concerning the result obtained for TrxR enzyme, results showed that the 7h molecule caused a slight decrease in the activity of this enzyme. However, 6f seems to be an important inhibitor of TrxR.

In fact, 6f decreased the TrxR activity by 66 % after 48h (▶Fig. 6, B).

The effect of the 2 compounds, 6f and 7h, on the GPx and ICDH activities is represented in ▶Fig. 5. In one hand, the tested molecules affected positively the GPx activity, however, the increase

▶Fig 2 Effect of Pyridazin-3(2h)-one derivatives on the viability of PBMCs, were treated with 6a, 6d, 6e, 6f, 7g and 7h for 48h. Cell viability was calculated as described in materials and methods. Each value represents the mean of 3 replicates. Bars represent the standard error. Different letters indicate significant differences among treatments. p-value < 0.05 indicate significant difference.

0 20 40 60 80

100 ^6f

MTX

0 20 40 60 80 100

Viability (%)Viability (%)Viability (%)

Compound 7h 7h

MTX

a a a

b b

c b

cc

cd de

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100 Compound 6e 6e

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7g MTX 0

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1.95 3.9 7.81 15.62 31.25 62.5 125 250 Concentration in µM

6a MTX

Compound 6f

a a a

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c cd cd cd cd

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caused by the 6h molecule was more pronounced compared to that of the 7h compound. After 48h exposure, we observed a decrease in the GPx activity for both positive control and 6f molecule.

In the other hand, the ICDH activity was negatively affected by 6f and 7h, however, the effect of the 6f compound was more pronounced after 48h.

Discussion

During the last decade, many interesting functional structure of pharmacophore moieties in pyridazinones makes them an attrac- tive source of new active derivatives [24, 37]. Combination of benzofuran and pyridazinones has led to good results for the first time.

From the results of cytotoxicity, it has been shown that newly synthesized pyridazin-3(2h)-ones derivatives exhibit a higher inhibitory

▶Fig 3 DNA extracted from P815 cells treated with 6e (line 1), DNA extracted from P815 cells treated with 7h (lane 2), DNA extracted from P815 cells treated with 6f (lane 3), DNA extracted from P815 cells untreated (lane 4), DNA extracted from treated P815 cells treated with MTX as reference drug (lane 5) and DNA ladder/marker of 100 base pair (lane 6).

▶Fig 4 Concentration of malondialdehyde (MDA) in the P815 cell line after treatment with 14.5 uM of 6f and 20 uM of 7h Pyridazin- 3(2h)-one derivatives. Each value represents the mean of 6 replicates. Bars represent the standard error. Different letters indicate significant differences among treatments. p-value value < 0.05 indicate significant difference.

a

b

a

b a

c

a

c

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07

NT MTX 7 h 6 f

MDA content (nmol.mg–1protein)

24 h 48 h

▶Fig 5 Activities of antioxidative enzymes; Gluthatione peroxy- dase (GPx, A) and (ICDH, B) in the P815 cell line after treatment with 14.5 uM of 6f and 20 uM of 7h Pyridazin-3(2h)-one derivatives. Each value represents the mean of 6 replicates. Bars represent the standard error. Different letters indicate significant differences among treatments. p-value < 0.05 indicate significant difference.

a

c c

a

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e

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 0.0 1.0 2.0 3.0 4.0 5.0

a

d

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a

c

d

e

NT MTX 7 h 6 f

GPx activity (µmol.min–1 mg–1protein)ICDH activity (µmol.min–1 mg–1protein)

48 h

24 h 48 h

▶Fig 6 Activities of antioxidative enzymes; glutathione reductase (GR, A) and thioredoxin reductase (TrxR, B) in the P815 cell line after treatment with 14.5 uM of 6f and 20 uM 7h Pyridazin-3(2h)-one derivatives. Each value represents the mean of 6 replicates. Bars represent the standard error. Different letters indicate significant differences among treatments. p-value < 0.05 indicate significant difference.

a

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0.0 10.0 20.0 30.0 40.0 50.0 60.0

NT MTX 7 h 6 f

GR activity (nmol.min–1 mg–1 protein)

24 h 48 h

24 h 48 h 14.000

12.000 10.000 8.000 6.000 4.000 2.000 0.000 TrxR activity (nmol.min–1 mg–1protein)

NT MTX 7 h 6 f

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activity toward murin mastocytoma P815 tumor cells (▶Fig. 1).

These results corroborate recent findings showing a cytotoxic activity of other pyridazin-3(2h)-one derivatives against different cell lines [23, 38, 39]. The lowest IC₅₀ value was around 14.5 µM for 6f and 20 µM for 7h derivatives (▶table 2). One of the important re- sults of this study was that pyridazinones molecules especially 6f and 7h did not show cytotoxicity towards human PBMCs (▶Fig. 2).

Furthermore, combination between 6f or 7h with MTX exhibited synergistic effect (▶table 2). This finding suggests a selectively and an effectively killing ability of these molecules against tumor cells (P815) without posing impact on normal cells.

The increase in the ROS scavenging enzymes is known to be a protective mechanism to cope with oxidative stress. In cancer cells, the intracellular antioxidant capacity and the redox homeostasis are mainly maintained by 2 antioxidant systems, the glutathione- and thioredoxin-dependent systems [8, 40]. In fact, the important enzymes implicated in the regulation of redox homeostasis such as peroxidases and thiol reductases, are limited to the pool of GSH and thioredoxin as source of reducing equivalents [41–43]. Indeed, the increase of ROS or the decrease of free radical scavengers such as GSH and Trx was reported to be toxic to tumor cells [44, 45].

In the present work, the determination of lipid peroxidation levels, measured as MDA content, showed a profound increase after cells exposure to 6f compared to 7h. The increase in the MDA content caused by 6f was similar to that induced by the positive control, MTX. Lipid peroxidation is a major indicator of oxidative stress and its increase after 6f treatment indicate that this compound induced apoptosis in the P815 cells through an oxidative stress. When exposed to an oxidative stress, cells use GPx to catalyze the reduction of H₂O₂ to H₂O in the presence of GSH. The GSH consumed is re- generated from GSSG by the action of GR. In this work, the 2 molecules tested affected positively the GPx activity, however, the increase caused by the 6h molecule was more pronounced compared to that of the 7h compound. After 48h exposure, we observed a decrease in the GPx activity for both, positive control and 6f molecule. This result may be due to a decrease in the GSH content. In fact, the GR activity decreased after exposure to 6f, 7h and MTX after 24h and the decrease was more pronounced after 48h. Con- cerning the TrxR enzyme, results showed that the 7h molecule caused a slight decrease in TrxR activity; however, 6f seems to be an important inhibitor of TrxR. In fact, 6f decreased the TrxR activity by 66 % after 48h. It was reported that the overexpression of TrxR was common in a number of human cancers and that the down-regulation of its expression decreased the tumor growth [7].

The results presented here, suggest that 6f and 7h have an important anticancer capacity. These results indicated also that the anticancer activity of the 6f compound could be due to the increase of intracellular ROS through the inhibition of GR and TrxR activities.

Both glutathione- and thioredoxin-dependent systems require NADPH, as cofactor, to combat ROS generated from oxidative stress [9]. Thus, deficit in the NADPH content may affect the cell response to ROS. In fact, it was reported that cells exposed to oxidative stress exhibited reduced NADPH levels, which may be a result of elevated activity of NADPH-dependent antioxidant systems [10]. NADP⁺- ICDH is one of the main enzymes implicated in the provision of NADPH [11]. In fact, it was reported that the over-expression of NADP⁺-ICDH is responsible for high resistance to oxidative stress, however, de-

creased NADP⁺-ICDH expression lead to a low resistance [12, 13]. Fur- thermore, IDH-1 knockdown was reported to depress cancer cell proliferation [14]. In the present study, 6f and 7h affected negatively the ICDH activity, however, the effect of the 6f compound was more pronounced after 48h. The decrease in the ICDH activity may be responsible for the depletion of NADPH used to cope with oxidative stress through the glutathione- and thioredoxin-dependent systems.

Considering the structure–activity relationship, the substitu- tion at C5 in the benzofuran ring was of interest. Taken together, results of our experiments confirmed that molecules possessing Br or Cl in the radical 1 or 2 in the C5 of benzofuran are stronger cytotoxic agents of pyridazin-3(2h)-one derivatives against P815 cell line which is connected with induction of oxidative stress in these tumor cells. Furthermore, as shown in ▶Fig. 1, molecules 6f was the most active among all tested pyridazin-3(2h)-ones. The interesting cytotoxic effect of 6f can probably be attributed to the Br in position 1 (R1) and/or Cl in position 2 (R2).

Acknowledgments

This work was supported by Lalla Salma Foundation: Prevention and Treatment of Cancer. Research project no. 09/AP 2013 (Rabat- Morocco). The Authors gratefully acknowledge Dr. Amorette Barber Professor of Biology from Longwood University of Virginia, USA, for reading the paper.

Conflict of Interest

No conflict of interest has been declared by the authors.

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