Cancer cell growth inhibition by aroylhydrazone derivatives

Abstract Hydrazones have versatile properties that make them promising for a range of possible applications. In this study, we examined a library of 17 aroylhydrazones derived from nicotinic acid hydrazide (1-12) and isonicotinic acid hydrazide (A-E) created by us for their biological activity. The antiproliferative activity of the compounds was investigated on non-tumour MCF-10A cells and cancer cell lines, MCF-7 and MDA-MB-231. Four compounds were selected as most active in cell growth inhibition of the tumour cell lines. These compounds, 5, 11, C and E, were tested on four additional cell lines: non-tumour BJ and cancer cell lines, HeLa, HepG2 and HT-29. Compounds 5 and E exhibited the highest selectivity index on cancer cell lines MDA-MB-231, HeLa and HepG2. High selectivity to MCF-7 cells was demonstrated with compound 5. Compound C was very selective to HepG2 cells as well as to MDA-MB-231 but to a lesser degree. Compound 11 showed selectivity against MDA-MB-231. The obtained results allow assessing the structure–activity relationship of the compounds and provide insight into the further development of this group of aroylhydrazones as more potent and selective anti-neoplastic agents.

In particular, aroylhydrazones, which are easily formed by the condensation of aromatic hydrazides (aroylhydrazides) with carbonyl compounds, are of special interest as they possess especially high and selective antiproliferative activities [22][23][24]. Among the aroylhydrazones, especially effective anti-proliferative agents are the hydrazones derived by condensation reaction of salicylaldehyde and different acid hydrazides. For example, one of the compounds, salicylaldehyde benzoylhydrazone (SBH), has been shown to inhibit DNA synthesis and cell growth in a variety of cultured human and rodent cells [25]. Various derivatives of SBH with a number of functional groups have been created in recent years, in order to discover new bioactive compounds with high antitumour activity and minimal toxicity [26][27][28][29][30][31][32][33].
As a next step, this study aimed to evaluate the biological activity of these compounds. To characterize these analogues, we examined their: (1) antiproliferative activity; (2) selectivity to tumour cells. All compounds were tested on non-tumour MCF-10A cells and on two cancer cell lines, MCF-7 and MDA-MB-231. Four compounds were selected as most active in inhibiting the growth of these tumour cell lines. These four compounds were tested on another four cell lines: nontumour BJ and cancer cell lines HeLa, HepG2 and HT-29. These studies and the results obtained are important to resolve the structure-activity relationships that are essential for the development of more potent biologically active aroylhydrazones.

Chemistry
All the reagents and chemicals used were of analytical and high purity grade. The solvents were purified and dried according to standard procedures. Salicylaldehyde was obtained from Merck (Germany), o-vanillin, nicotinic acid and iso-nicotinic acid hydrazides were purchased from Fluka (Switzerland); benzaldehyde, 2,4-dihydroxybenzaldehyde, 2-hydroxy-4- chlorobenzaldehyde and 3,5-dichloro-2-hydroxy benzaldehyde purchased from Sigma (Switzerland). All hydrazones were synthesized in the University of Zagreb, according to previously described procedures [29,34,[42][43][44][45]. They were obtained by the condensation reaction of equimolar amounts of the corresponding aroylhydrazide (nicotinic hydrazide for 1-12 and isonicotinic hydrazide for A-E) and differently substituted aldehydes or ketones The reactions were carried out in dry alcohol (ethanol or ethanol) under argon atmosphere at 85 C for 20 h. The solvent was evaporated and the solids were suspended in CH 2 Cl 2 , filtered (G-3), rinsed with CH 2 Cl 2 (EtOH was used for 4, 5, 11 and 12), and dried at 50 C over 24 h.

Cell cultures
MCF-10A (human mammary epithelial cell line), MCF-7 (human breast adenocarcinoma cell line) and MDA-MB-231 (human breast adenocarcinoma cell line) were cultured in Dulbecco Modified Eagle's medium (DMEM; Gibco, Austria) supplemented with 10% foetal bovine serum (Gibco, Austria), 100 U/mL penicillin (Lonza, Belgium) and 0.1 mg/mL streptomycin (Lonza, Belgium) under a humidified 5% CO 2 atmosphere at 37 C. The complete growth medium for MCF-10A was additionally supplemented with: human insulin, cholera toxin, epidermal growth factor, hydrocortisone at the final concentrations recommended by Merck. Plastic flasks supplied by Greiner, Germany, were used to grow the cells. Cells were trypsinized using Trypsin-EDTA (Gibco, Austria) when they reached approximately 80% confluence.
In the experiments, cells in the exponential phase of growth were treated with Trypsin-EDTA prior to seeding into 96-well plates (Greiner, Germany) in a concentration of 2 Â 10 3 cells/well. The cells were incubated for 24 h post seeding (under a humidified 5% CO 2 atmosphere at 37 C) to allow them to attach to the wells. Four cell lines were used in additional experiments: HeLa (human epithelioid cervix carcinoma), HepG2 (human hepatocellular carcinoma), HT-29 (human colorectal adenocarcinoma) and BJ (human fibroblast cell line). They were processed in the same manner as described above.

Antitumour activity
The cells were treated with the compounds in a wide concentration range (4-2000 mmol/L). Untreated cells were used as controls. Empty wells were blank controls. Cell viability was measured by a colourimetric assay based on tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma Chemical Co.). The MTT assay is based on the protocol first described by Mosmann [46]. In this assay, living cells reduce the yellow MTT to insoluble purple formazan crystals. The compounds were dissolved in dimethyl sulphoxide (DMSO). The final concentration of DMSO in the samples did not affect the viability of the cells. The assay was performed 72 h after treatment with the compounds. For this purpose, MTT solution was prepared at 5 mg/mL in PBS and was filtered through a 0.22 mm filter. Then 1 mL of MTT solution was added to 10 mL DMEM and 100 mL of this solution were added into each well, including the cell-free blank wells. Then the plates were further incubated for 3 h to allow MTT to be metabolized. The supernatant was removed and 100 mL/well DMSO/ethanol (1/1) was added. The plates were placed in a microtiter plate shaker for 10 min at room temperature to thoroughly mix the purple formazan into the solvent. An ELISA plate reader (TECAN, Sunrise TM, Grodig/Salzburg, Austria) was used for reading the results. Optical density (OD) was determined at a wavelength of 540 nm and a reference wavelength of 620 nm. Cell viability determined by MTT assay was expressed as: The effects of all tested hydrazones were compared to the activity of the referent cytostatic oxaliplatin (Ox), which is a drug widely used in the clinic [47,48].

Selectivity index (SI)
In the present study, the degree of selectivity of the synthetic compounds is expressed as selectivity index (SI). SI ¼ IC 50 of pure compound in a normal non-tumour cell line/IC 50 of the same pure compound in a cancer cell line, where IC 50 is the half maximal inhibitory concentration of the compounds over cell viability [49].  Cell antiproliferative activity determined by MTT assay is expressed as per cent of viable cells versus the control group (C, control) and is presented as mean ± SD (n ¼ 6), ÃÃÃ p < 0.001, ÃÃ p < 0.01, Ã p < 0.05, ANOVA-test.

Results and discussion
A common route was used for the synthesis of the investigated aroylhydrazones. The aroylhydrazones 1-12 ( Figure 1) and A-E ( Figure 2) contain different substituents (e.g. 2-Cl, 3-Cl, -OH, NO 2 ) at the salicilyc aroyl part. We also varied the position of the pyridine N atom (e.g. at position 2 or 3), while keeping hydrazone spacer between the pyridine and salicylaldehyde moieties.
The antiproliferative activity of the compounds was tested over different cancer cell lines and non-tumour cells. The effects were compared to the activity of Ox, a referent cytostatic, which is widely used in the clinic [47,48].
The results of the antiproliferative study of the compounds 5, 11, C and E on non-tumour MCF-10A cells and cancer cell lines MCF-7 and MDA-MB-231 are shown in Figure 3. The cells were exposed to different concentrations (ranging from 4 to 2000 mmol/L) of the compounds for 72 h. The treatment of the cell lines with the compounds resulted in a concentrationdependent reduction in the number of the viable cells ( Figure 3).
The antiproliferative activity of the compounds based on the half maximal inhibitory concentrations (IC 50 values) is shown in Table 1. Compound 11 had a stronger inhibitory effect on the growth of the three cell lines compared to its parent compound. Compounds 5, C and E also showed promising results.
The main problem of the drugs in clinical use for cancer treatment is selectivity. The selectivity index (SI) is an important measure to identify substances with promising biological activity. As the SI demonstrates the relation between IC 50 of the respective pure compound on a non-tumour cell line and its IC 50 on a cancer cell line, the greater the SI value is, the more selective the compound is to tumour cells. An SI value of less than 2 indicates general toxicity of the pure compound [50]. Based on this, the SI data shown in Table 2 (Table 2).
In order to investigate more closely the antiproliferative activity of the compounds with the highest selectivity index, additional cell lines were used: HeLa, HepG2 and HT-29 (cancer cell lines) and BJ (nontumour cell line). The IC 50 values of compounds 5, 11, C and E are shown in Table 3. The highest cell growth inhibitory effect was observed with the substances 11 and C in the cell line HepG2. In this case, the IC 50 value of 11 was 14.46 mmol/L and the IC 50 value of C was 19.11 mmol/L (whereas the respective value of Ox was 2.39 mmol/L). These results are comparable with previously reported activities of aroylhydrazones [32]. Hristova-Avakumova et al. [32] investigated the cytotoxic activity of 3-methoxy aroylhydrazone on HepG2 (hepatocellular carcinoma cell line), HEK-293 (human Table 1. Antiproliferative activity of the hydrazones on MCF-7, MDA-MB-231 and MCF-10A cells after 72 h of treatment (MTT-dye reduction assay).  embryonic kidney cell line) and SH-SY-5Y (human neuroblastoma cells). Their compounds also showed selectivity for tumour cell lines. The IC 50 values that they report for HepG2 cells are in micromolar concentrations in the range of 3.7-13.2 lmol/L [32].
In our experiments, the selectivity index of compounds 5, 11, C, E and Ox to the additional cell lines (Table 4) showed that none of these substances showed considerable SI on HT-29 cells. It may be concluded that compounds 5, 11, C and E do not exhibit selective antiproliferative activity against the cells of this cancer cell line. We observed a considerably higher SI value for compounds 5 and E on HepG2 cells, as well as for compound E on HeLa cells. The SI value in the latter case was 5.319, even higher than the SI of oxaliplatin (4.594). The SI of oxaliplatin with HepG2 and HT-29 cells was not reached by any of the compounds tested.
Taken together, the results from the antiproliferative activity screening of a library of aroylhydrazones (1-12 and A-E) indicate that the compounds 5 and E exhibit highest SI for the cancer cell lines MDA-MB-231, HeLa and HepG2. High selectivity to MCF-7 cells was demonstrated with compound 5. Compound C was selective to MDA-MB-231 and HepG2 cells. Compound 11 showed selectivity against MDA-MB-231. These compounds would be of interest for further study for the needs of pharmacology.
In terms of structure-activity relationship, we demonstrated the importance of the substituents at the salicylidene part of the molecule. For example, the introduction of substituents into the aromatic nucleus led to an increase in the cell growth inhibition activity of the compounds to varying degrees. The presence of two hydroxyl groups in positions 2 and 5 (substances 5 and E), as well as the introduction of the nitro group in position 5 (substances 11 and C) increased the selectivity of the compounds for the cancer lines. However, the introduction of a chlorine atom at position 5 (substance 9) reduced the selectivity versus selectivity of substances 5 and 11. It should also be noted that the position of the nitrogen atom in the aromatic nucleus in nicotinic or isonicotinic residue was not relevant for the activity of the compounds tested.

Conclusions
The results obtained with all tested substances from a library of aroylhydrazones showed that their biological activity depended particularly on the substituents at the salicylidene part of the molecule. The compounds of interest that showed selectivity for cancer cell lines have two hydroxyl groups in positions 2 and 5 in the aromatic nucleus (substances 5 and E) and a nitro group in position 5 (substances 11 and C). A modification that reduced the selectivity versus selectivity of substances 5 and 11 is a chlorine atom at position 5 (substance 9). The obtained results give insight into the structure-activity relationships of the tested group of compounds and lay the ground for further development of more potent and selective antitumour agents.

Funding
This study was financially supported by the National Science

Disclosure statement
No potential conflict of interest was reported by the authors.