Original article

T.P. LEHMANN1, J. KUJAWSKI2, J. KRUK2, K. CZAJA2, M.K. BERNARD2, P.P. JAGODZINSKI1

CELL-SPECIFIC CYTOTOXIC EFFECT OF PYRAZOLE DERIVATIVES ON BREAST CANCER CELL LINES MCF7 AND MDA-MB-231

Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, Poznan, Poland;
Department of Organic Chemistry, Poznan University of Medical Sciences, Poznan, Poland
Pyrazoles and their derivatives belong to a class of compounds that demonstrate a great potential in design of anticancer, antiangiogenic, and antimetastatic drugs. Our earlier studies showed that pyrazole derivatives TOSPYRQUIN and TOSIND diminished viability of colorectal adenocarcinoma cells HT-29. Here we demonstrated for the first time in human mammary gland adenocarcinoma cell lines MCF7 and MDA-MB-231 cells the cytotoxic effects of four pyrazole derivatives: TOSIND, PYRIND, METPYRIND, and DIPYR. Three pyrazoles: PYRIND, METPYRIND, and one novel unpublished derivative DIPYR were tested for the first time in living cells. viability of MCF7 did not significantly change in the presence of TOSIND but it decreased after 72 hours of treatment with PYRIND (IC-50 39.7 ± 5.8 µM). In the presence of METPYRIND the viability was also diminished, while DIPYR increased MCF7 viability after 24 hours of incubation. The viability of MDA-MB-231 cells was strongly decreased by TOSIND (IC-50 17.7 ± 2.7 µM 72 h), and was not influenced by PYRIND and METPYRIND, while DIPYR increased the viability and stimulated the growth of MDA-MB-231 cells. PYRIND, METPYRIND and DIPYR caused a gradual decrease of caspase-3 and caspase-7 activities in MDA-MB-231 cells and there was no influence of TOSIND on the activity of both caspases. Our results open the way to search for other compounds with pendant pyrazole residues in order to increase their cytotoxic activity; especially with regard to its anti-breast cancer activity. It appears that the pyrazoles synthesized by us diminish cell viability in a cell-specific manner. This observation might be useful in designing ‘off-DNA’ anticancer drugs, compounds which are not harmful to the healthy cells.
Key words:
pyrazole, indazole, cytotoxicity, cell viability, apoptosis, caspase, mammary gland adenocarcinoma

INTRODUCTION

Cell-specific pharmacotherapy of cancer constitutes a major challenge for medical chemists due to the heterogeneity of cancer disease (1). Current strategies for anticancer therapy are oriented towards design of a specific molecule which selectively kills cancer cells but is not harmful for normal cells (2). There are two main types of strategies used in the development of anticancer drugs. The first type relies on designing an anticancer drug for treatment of an individual patient, while the second type is based on developing a drug which could be used in many patients.

Pyrazoles and their derivatives, including indazoles, belong to a group of compounds that demonstrate great potential in both types of strategies, enabling treatment of a wide spectrum of cancers. Pyrazole derivatives have attracted considerable attention because of their varied biological activities including, inter alia, anticancer, antiangiogenic, and anti- metastatic action.

In our previous work, we described two condensed pyrazole derivatives TOSPYRQUIN and TOSIND that inhibited proliferation of HT-29 colorectal adenocarcinoma cells. TOSPYRQUIN and TOSIND stimulated also activity of proapoptotic proteins (3). The aim of the present study was to compare for the first time cytotoxic activities of three pyrazoles, with TOSIND in two breast cancer cell lines. We investigated if the indazole ring was essential for the cytotoxic activity of pyrazole derivatives. We have also attempted to show whether the newly synthesised compounds triggered proapoptotic cascade.

MATERIALS AND METHODS

General Procedures

Melting points were determined on a Boetius apparatus and are uncorrected. 1H NMR and 13C NMR spectra were recorded in CDCl3 at 400 or 100 MHz on a Varian VNMR-S spectrometer with TMS as an internal standard. Low resolution mass spectra were recorded on a Bruker 320MS/420GC instrument operating at 75 eV. IR spectrum was run in KBr on a Mattson Genesis II FTIR apparatus. Elemental analyses were performed on a Vario EL III instrument. Compound 1 (4) as well as TOSIND, METPYRIND, and DIPYR were obtained by previously described methods (3, 5, 6).

1-[3-(4-Methylphenylsulfonylmethyl)-4-nitrophenyl]-1H-pyrazole (compound 2)

Finely powdered KOH (2.00 g) was added in one portion to a stirred solution of sulfone (compound 4) (1.02 g, 5 mmol) and nitroarene (compound 1) (0.88 g, 5 mmol) in 5 mL of dry DMSO. The resulting dark coloured mixture was vigorously stirred for 45 min at 22°C, quenched with 3% HCl (100 mL) and extracted with dichloromethane (3 × 30 mL). The extract was washed with water, dried over MgSO4, and the solvent was removed under vacuum. The residue was crystallized from ethanol to give compound 2 (0.34 g, 19%) as yellow needles (m.p. 197 – 199°C).

1H NMR δ: 2.43 (s, 3H, CH3), 5.01 (s, 2H, CH2), 6.56 (dd, J = 2 and 2 Hz, 1H, 4-H, pyrazole), 7.27 – 7.30 (m, 2H, 3“-H and 5“-H, Ts), 7.59 – 7.62 (m, 2H, 2“-H and 6“-H, Ts), 7.79 (d, J = 2 Hz, 2H, 5-H, pyrazole), 7.81 (d, J = 2 Hz, 1H, 3-H, pyrazole), 7.88 (dd, J = 8 and 2 Hz, 1H, 6’-H), 8.01 (d, J = 2 Hz, 1H, 2’-H), 8.11 (d, J = 8 Hz, 1H, 5’-H).

13C NMR δ: 21.65 (CH3), 58.70 (CH2), 109.50, 118.95, 123.19, 125.50, 127.12, 127.46, 128.43, 129.93, 134.75, 142.92, 142.96, 145.49, 146.04.

Anal. Calcd for C17H15N3O4S (357.38): C 57.13; H 4.23 N; 11.76. Found: C 57.37; H 4.19; N 11.89.

1-[4-Amino-3-(4-methylphenylsulfonylmethyl)phenyl]-1H-pyrazole (compound 3)

Hydrazine hydrate (4.49 mL, 8.2 mmol) was added dropwise to a warm solution of pyrazole (compound 2) in isopropanol (90 mL), then 10% palladium on charcoal (0.21 g) was added, the mixture was refluxed for 4 hours and filtered through Celite. The solvent was distilled off and the residue was crystallized from ethanol to give compound 3 (1.79 g, 78%) as light brown plates (m.p. 152 – 154°C).

1H NMR δ: 2.40 (s, 3H, CH3), 4.33 (bs, 2H, NH2, exchangeable with D2O), 4.36 (s, 2H, CH2), 6.35 (dd, J = 2.4 and 1.8 Hz, 1H, 4-H, pyrazole), 6.80 (d, J = 9 Hz, 1H, 5’-H), 6.88 (d, J = 2 Hz, 1H, 2’-H), 7.28 – 7.30 (m, 2H, 3“-H and 5“-H, Ts), 7.42 (dd, J = 9 and 2 Hz, 1H, 6’-H), 7.51 (dd, J = 2.4 and 0.6 Hz, 1H, 5-H, pyrazole), 7.60 (d, J = 1.8 Hz, 1H, 3-H, pyrazole), 7.66 (m, 2H, 2’’-H and 6’’-H, Ts).

13C NMR δ: 21.58 (CH3), 59.95 (CH2), 106.95, 114.92, 118.34, 121.60, 123.97, 126.38, 128.46, 129.78, 132.74, 134.75, 140.38, 145.17, 145.45.

IR v (cm–1): 3460, 3376 (NH2), 1629, 1601, 1519, 1501, 1316, 1302, 1148, 1083, 1037, 954, 901, 812, 772, 756, 720, 549, 505.

MS m/z (%): 328 (6), 327 (32, M+), 173 (12), 172 (100, M – PhSO2), 145 (3), 143 (2), 118 (2), 117 (2), 105 (2), 104 (5), 91 (10), 77 (4), 69 (3), 65 (7), 59 (3), 57 (4), 55 (3), 51 (2).

Anal. Calcd for C17H17N3O2S (327.4): C 62.36; H 5.23; N 12.83. Found: C 62.18; H 5.30; N 12.99.

3-(4-Methylphenylsulfonyl)-5-(pyrazol-1-ylo)indazole (PYRIND)

Amine 3 (0.27 g, 0.9 mmol) was dissolved in small volume of acetone, adsorbed on silica gel (0.70 g), and transferred into a pressure tube. The mixture was moistened with acetic acid (ca 1 mL) and isopentyl nitrite (0.24 mL, 1.8 mmol), and heated in a microwave reactor (PLAZMATRONIKA RM800, Poland) for 10 min. (reaction conditions: KT = 240, Tmin = 100°C, Tmax = 110°C, P = 400 W). The reaction mixture was extracted with dichloromethane, silica gel was filtered off, and the solvent was distilled off. The residue was crystallized from ethanol to give compound PYRIND (0.42 g, 54%) as pale yellow plates, (m.p. 98 – 101°C).

1H NMR δ: 2.38 (s, 3H, CH3), 6.54 (dd, J = 2 and 2 Hz, 1H, 4’-H, pyrazole), 7.28 – 7.30 (m, 2H, 3“-H and 5“-H, Ts), 7.75 (d, J = 9 Hz, 1H, 7-H, indazole), 7.78 (d, J = 2 Hz, 1H, 3’-H, pyrazole), 7.97 – 7.99 (m, 2H, 2’’-H and 6’’-H, Ts), 8.00 (dd, 1H, J = 9 and 2 Hz, 6-H, indazole), 8.05 (d, J = 2 Hz, 1H, 4-H, indazole) 8.33 (d, 1H, J = 2 Hz, 5’-H, pyrazole), 11,76 (bs, 1H, NH, exchangeable with D2O).

13C NMR δ: 21.63 (CH3), 108.02, 109.74, 112.10, 120.68, 122.08, 127.56, 127.75, 129.99, 136.86, 137.82, 139.70, 141.43, 144.86, 145.51.

MS m/z (%): 339 (17), 338 (100, M+), 337 (4) 273 (8), 245 (4), 183 (36, M – PhSO2), 172 (3), 156 (6), 155 (4), 129 (5), 91 (24), 88 (4), 78 (3), 77 (3), 65 (5).

Anal. Calcd for C17H14N4O2S (338.4): C 60.34; H 4.17; N 16.56. Found: C 60.62; H 4.08; N 16.43

Cell lines and culture

MCF7 breast cancer cells belong to a luminal molecular type of breast cancer and MDA-MB-231 cell line is a triple negative, metastatic form of breast cancer (7).

The cells were centrifuged at 300 g for 5 min, seeded into a tissue culture flask and cultured at 37°C in 5% CO2/95% air, in a (1:1 v/v) Dulbecco’s Modified Eagle’s Medium/Nutrient F-12 Ham (DMEM/F-12) supplemented with 10% foetal bovine serum, 100 units penicillin, 100 µg streptomycin and 25 ng amphotericin B per mL (ABAM). All reagents were from (Sigma-Aldrich, St. Louis, MO, USA). The MCF7 and MDA-MB-231 cells were incubated with 2.5 to 100 µM of PYRIND, METPYRIND, and DIPYR, and their viability was determined (8).

Cell viability test

Cell viability was measured with the use of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based Cell Growth Determination Kit, (Sigma-Aldrich). After incubation with pyrazoles, the cells (in 24-well plates) were incubated in serum free DMEM/F-12 containing 10% MTT (0.5 mg/ml). After 4 hours of incubation, the media were removed and the cells were treated with 0.1 N HCl in isopropanol. After gentle mixing, the absorbance was recorded at 630 nm, and the background absorbance was measured at 405 nm using a Stat-Fax 2100 plate reader (Awareness Technology Inc., Palm City, FL, USA).

Determination of caspase activity

The activities of caspase-3 and caspase-7 were measured with the Caspase-Glo 3/7 Assay Kit (Promega, Madison, WI, USA). The cells were incubated with tested pyrazoles for 24 h in 96-well plates and the incubation was completed by addition of the Caspase-Glo 3/7 Reagent to the culture medium in a ratio 1:4 (v/v) respectively. After 1 hour of incubation, luminescence was measured in a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA).

Statistical analyses

All results presented in the diagrams are means of at least three assays ± S.D. The significance of differences was calculated using the one-way analysis of variance (ANOVA) and post-hoc test Student-Newman-Keuls multiple comparisons test.

RESULTS

Our earlier studies showed that TOSPYRQUIN and TOSIND diminished viability of colorectal adenocarcinoma cells HT-29 (3). In the current study, we investigated the effects of TOSIND and three pyrazole derivatives. PYRIND, METPYRIND, and DIPYR which were not tested earlier in on the viability of human mammary gland adenocarcinoma cell lines MCF7 and MDA- MB-231 (Fig. 1) (3).

Figure 1
Fig. 1. Chemical formulas of the tested compounds.

Fig. 2 shows the time- and dose-dependent effects of TOSIND, PYRIND, METPYRIND and DIPYR on the viability of breast cancer cells. viability of MCF7 did not significantly change in the presence of TOSIND but it decreased after 72 hours of treatment with PYRIND (IC-50 39.7 ± 5.8 µM). In the presence of METPYRIND the viability was also diminished, while DIPYR increased MCF7 viability after 24 h incubation.

Figure 2
Fig. 2. Comparison of cytotoxicity of the newly synthesized pyrazoles to the known cytotoxic pyrazole, TOSIND in breast cancer cells. The MCF7 and MDA-MB-231 cells were treated for 24, 48 and 72 hours with equimolar concentrations of PYRIND, METPYRIND and DIPYRIND and their effects were compared with that of TOSIND. Viability of the cells, determined by the MTT assay, was expressed as the mean ± S.D. from four experiments and was related to the untreated control. It was assumed that the viability of the control cells equals 100%. Significant at: aP < 0.05, bP < 0.01, and cP < 0.001.

The viability of MDA-MB-231 cells was strongly decreased by TOSIND (IC-50 17.7 ± 2.7 µM 72 h), and was not influenced by PYRIND and METPYRIND, while DIPYRIND increased the viability and stimulated the growth of MDA-MB-231 cells.

Comparison of our previous and current studies showed that TOSIND was relatively a stronger cytotoxic agent for colorectal adenocarcinoma cells than for the breast cancer cells (3). PYRIND and METPYRIND that contain a distal pyrazole ring were more effective in decreasing the viability of MCF7 cells than DIPYR and TOSIND, which were devoid of this heterocycle residue.

PYRIND, METPYRIND and DIPYR caused a gradual decrease of caspase-3 and caspase-7 activities in MDA-MB-231 cells and there was no influence of TOSIND on the activity of both caspases (Fig. 3). However, in our previous studies, TOSIND promoted a cleavage of caspases-8 and -9, what precedes an increase of caspase-3 and -7 activation in the cascade of caspases (3). In the present work, the activity of caspases-3 and -7 were measured directly by chemiluminescence assay and were normalized to a relative cell viability determined by the MTT assays. This enabled us to show caspases activity in relation to the number of the living cells and to exclude the cells that die as a result of treatment with pyrazoles. Our results, showing a decrease in the activity of caspases-3 and -7 after treatment with DIPYR, suggest that a positive effect of this pyrazole on cell viability might have been accompanied by decreased apoptosis.

Figure 3
Fig. 3. The effect of the newly synthetized pyrazoles and a known cytotoxic agent, TOSIND on caspase-3 and caspase-7 activities in MDA-MB-231 cells. After 72 h treatment with PYRIND, METPYRIND, DIPYRIND, and TOSIND, caspase-3 and caspase-7 activities were determined in triplicate, as described in the Materials and Methods. Results were the mean ± S.D., of four separate experiments, expressed as percentage of untreated control assuming that the viability of the control cells equals 100%. Significant at: bP < 0.01, and cP < 0.001.

Our results open the way to search for other compounds with pendant pyrazole residues in order to increase their cytotoxic activity; especially with regard to its anti-breast cancer activity, with negligible side effects. However doubtful the hypothesis that the three pyrazoles presently tested in breast cancer cells intercalate with DNA, it should be elucidated finally using other methods. It appears that TOSIND, PYRIND and METPYRIND diminish cell viability in a cell-specific manner. This observation might be useful in designing ‘off-DNA’ anticancer drugs which are not harmful to the healthy cells.

DISCUSSION

Our report compares cytotoxic effects of three condensed pyrazole derivatives: PYRIND, METPYRIND, and DIPYR with TOSIND. We evaluated the impact of compounds on cell viability and the activity of executioner caspases -3 and -7 in the mammary gland adenocarcinoma lines MCF7 and MDA-MB-231.

Our studies have shown that PYRIND and METPYRIND, with a pyrazole ring linked to indazole, are stronger inhibitors of the viability of MCF7 than MDA-MB-231 cells. Indazole derivative, TOSIND, with a chlorine substituent instead of pyrazole moiety, also decreased the viability of MDA-MB-231 cells. The latter observation confirms previously described results concerning the growth-inhibitory potency of this compound on the HT-29 colon carcinoma cells.

Surprisingly, DIPYR, that contains two fused pyrazole rings instead of indazole, increased the viability of both breast cancer cell lines MCF7 and MDA-MB-231. This finding indicates that the pendant indazole ring seems to be an important element in biological activity of pyrazoles. It also means that the interactions between these compunds and amino acids in the protein structure include rather a hydrophobic contact (the benzene ring of indazole) than the two additional hydrogen bonds (the fused pyrazole ring in DIPYR).

The compounds tested, namely PYRIND, METPYRIND, and DIPYR, did not stimulate but rather decrease the activity of caspase-3 and -7. There are known caspase-3 and -7 inhibitors such as isatin sulphonamides (9). Since caspases were not stimulated we thought that cell viability might have been decreased either due to authophagy or to necroptosis rather than apoptosis as the latter process involves executioner caspases-3 and -7 (10, 11).

Authophagy is a catabolic process engaged under the metabolic stress. This process is referred to as caspase independent cell safe mode (12). It has been shown that pyrazole derivatives could inhibit growth of A549 lung cancer cells inducing authophagy (13). A wide range of pyrazole derivatives, differing in residues linked to the aromatic ring, such as methyl groups or chlorine atoms, could exhibit distinct biological effects either apoptotic or non-apoptotic (14). The same pyrazole derivative may have also a multi-target activity by interaction with several proteins (15). Particular type of necrosis, called necroptosis could be also a form of programmed cell death, and differs from apoptosis by the disruption of a plasma membrane and a lack of nuclear fragmentation (10). In the lung cancer cell line A549, one of pyrazole derivatives described by Zhang et al. increased lactate dehydrogenase activity indicating the possibility of necrosis induction (14). A derivative of 1-tert-butyl-5-methylpyrazole, with a pendant pyrazole ring inhibited caspase-3 activity, measured by the Caspase-Glo 3/7 Assay Kit in rat pancreatic beta-cells (16). Our results, show decrease of executioner caspases-3 and -7 activity and non-antiproliferative effect of DIPYR. This small molecule of pyrazolopyrazole will possibly open the way to design related compounds protecting non-cancerous cells against premature cell death.

Our studies showed unexpectedly that DIPYR stimulated the viability of both breast cancer cell lines: MCF7 and MDA-MB-231. The literature search has revealed that 1-(2,4-dichlorobenzyl)-3-nitro-1H-pyrazole was reported to inhibit the receptor interacting protein 1 (RIP1) kinase (17). This enzyme plays an important role in necroptosis regulated cell death with morphological features resembling passive non-regulated necrosis (10). Inhibitors of the RIP1 kinase are of potential therapeutic value in the treatment of diseases related to necrosis (17, 18). An increase of breast cancer cell viability could be explained by inhibition of apoptosis by pyrazole derivative DIPYR. The literature data search has shown that another pyrazole derivative, i.e. ethyl 3-(2-chlorophenyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxylate, is an inhibitor of apoptosis in non-cancerous cell, and it acts by down-regulating the levels of integrin b4, p53, and the reactive oxygen species (19).

We have not determined caspase activities in MCF7 cells because of their resistance to apoptotic stimuli. These cells are deficient in the expression of caspase-3, as a result of a 47 bp deletion in exon 3 of CASP3 gene (20, 21). The apoptosis in MCF7 cells proceeds by activation of caspases-9, -7 and -6 (22, 23). On the other hand, the MDA-MB-231 cells express functional caspase-3 and -7 (24).

The main conclusion from this work is that the novel class of relatively structurally simple pyrazole derivatives could provide compounds of increased bioactivity and this would encourage further studies on designing novel anticancer drugs.

In this study, we used breast cancer cell lines MCF7 and MDA-MB-231 corresponding to two molecular types of breast cancer (luminal and basal). The viability of these lines was differently regulated by PYRIND, METPYRIND, and DIPYR. To obtain selective anticancer drug, the candidate substances should be tested on noncancerous cells interacting with cancer such as macrophages and other healthy cells (25). The proapoptotic or pro-autophagic activity should be also tested using another method since caspase-3 and -7 were downregulated in our tests. Measurement of the oxidative status after treatment with pyrazoles may reveal if reactive oxygen species are involved in observed decrease in cell viability (26). Although an interaction with DNA was observed for some classes of pyrazoles, our results showed a different effect of three new pyrazoles on the viability of MCF7 and MDA-MB-231 cells. Such observation indicates that DNA is not necessarily the main target for the studied compounds; however, such assumption needs further confirmation.

Improvement of the efficiency of selective anticancer activity and resistance of normal cells to the treatment should be the matter of future studies. It seems that proteins specifically over-expressed in cancer, that are absent from normal cells, are usually better targets for anticancer drugs than DNA. A potential anticancer drug should rather be then an off-DNA, selective inhibitor of cancer-specific proteins or RNA. Novel pyrazole class described here could be candidates for such molecules.

Acknowledgements: The present study was supported by the Polish National Science Centre (grant no. NN403598538). The authors would like to thank Professor Wieslaw H. Trzeciak for his indispensable assistance during the manuscript correction and Ms. Bogumila Ratajczak (Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, Poznan, Poland) for her essential assistance during the experiments preparation.

Conflict of interests: None declared.

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R e c e i v e d : October 5, 2016
A c c e p t e d : March 28, 2017
Author’s address: Dr. Tomasz Lehmann, Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, 6 Swiecickiego Street, Poznan, Poland. E-mail: tlehmann@ump.edu.pl