Discovering simple phenylboronic acid and benzoxaborole derivatives for experimental oncology – phase cycle-specific inducers of apoptosis in A2780 ovarian cancer cells

Summary Objective The aim of the study was to evaluate the antiproliferative potential of simple phenylboronic acid and benzoxaborole derivatives as well as to provide preliminary insight into their mode of action in cancer cells in vitro. Methods The antiproliferative activity was assessed in five diverse cancer cell lines via the SRB method (sulforhodamine B) or MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method after 72 h of treatment. Further studies of the mechanism of action consisted of the influence of the compounds on cell cycle progression and apoptosis induction, which was assessed by flow cytometry, caspase-3 enzymatic activity, fluorescence microscopy and western blot analysis. Results A clear structure-activity relationship was observed for both groups of compounds with several representatives evaluated as highly active antiproliferative agents with low micromolar \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\mathrm{IC}}_{50}^{72\mathrm{h}} $$\end{document}IC5072h values. 2-Fluoro-6-formylphenylboronic acid (18) and 3-morpholino-5-fluorobenzoxaborole (27) exhibited strong cell cycle arrest induction in G2/M associated with caspase-3 activation in an A2780 ovarian cancer cell line. These events were accompanied by a mitotic catastrophe cell morphology and an increased percentage of aneuploid and tetraploid cells. Further experiments indicated that the compounds were phase cycle-specific agents since cells co-treated with hydroxyurea were less sensitive. The observed cell cycle arrest resulted from significant p21 accumulation and was associated neither with cyclin B1 nor β-tubulin degradation. Conclusion Phenylboronic acid and benzoxaborole derivatives were found to be highly promising antiproliferative and proapoptotic compounds with a cell cycle-specific mode of action. The presented data support their candidacy for further studies as a novel class of potential anticancer agents.


Introduction
Boronic acids, which have been known for more than 100 years, have recently gained increasing interest due to their use in organic synthesis, materials' chemistry, supramolecular chemistry, biology and medicine. The ongoing research areas cover both new applications as well as novel classes of compounds. The tetrahedral boron atom geometry in boronic acid closely resembles the enzyme-catalyzed substrate tetrahedral transition state. Thus, the biological activity of boroncontaining compounds is one of the most extensively investigated fields. Since the description of the very first boronic acidderived inhibitor of chymotrypsin [1] in the 1970s, several novel fields of their applications were discovered, including serine protease or histone deacetylase inhibition, and the most successful applicationproteasome inhibition by bortezomib [2]. Recently, benzoxaborolesphenylboronic acids' cyclic internal esters [3] emerged as a particularly interesting class with tavaborole (21), an antifungal drug already approved by the FDA for humans [4]. Additionally, diboronic acids and their derivatives [5] as well as compounds containing boron clusters [6] are receiving increasing attention.
The therapeutic potential of boron-containing compounds has been reviewed by Baker et al. [7]. The issues related to boronic compounds in anticancer, antibacterial and antiviral applications have also been reviewed [8] and mentioned in the recent edition of Hall's book on boronic acids [9]. Recently, several reviews concerning the biological activity of organoboron compounds have been published. Yang et al. described a synthetic strategy for the development of new compounds, revealing antibacterial, antifungal, antiparasitic, antiviral, anti-cancer and anti-inflammatory activities [10]. An extensive study published by Mereddy et al. applies to synthetic methods for benzoxaboroles, along with their recent applications in medicinal chemistry [11].
The substantial interest in boron-containing compounds and their potential in medicinal chemistry has mainly focused on antibacterial, antifungal, antiviral or antiprotozoal activity and generally omitted the field of experimental oncology. In addition to alkylboronic acid-derived compounds, represented mainly by proteolytic enzyme inhibitors [2], the influence of boron-containing moiety incorporation into combretastatin A4 [12], cis-stilbenes [13] or chalcones [14] original structure was recently studied. In some cases, such modifications resulted in compounds with improved biological activity [8]. Concurrently, surprisingly little is known about the antiproliferative activity of simple phenylboronic acid (PBA) and benzoxaborole. In the studies by Plopper et al. [15] and Marasovic et al. [16], phenylboronic acid showed limited activity in prostate and mammary gland cancer models. Some benzoxaborole-derived compounds, such as 6aminobenzoxaboroles, exhibited interesting antiproliferative activity [17], whereas some other derivatives such as βketobenzoxaboroles showed no such potential [18]. Herein, for the first time, we provide comprehensive evidence for the high anticancer potential of such compounds using several diverse cancer cell lines that cover a spectrum of malignancies that are currently commonly diagnosed in humans: leukemia (MV-4-11), breast (MCF7), urinary bladder (5637), ovarian (A2780) and lung cancer (A-549). The antiproliferative studies are supplemented with a preliminary analysis of their plausible mechanisms of action of strong cell cycle arrest at the G 2 /M phase, accompanied by cell death via apoptosis discovered as a major treatment outcome.
A 50 mM stock solution for each of the tested compounds, as well as for benzyl isothiocyanate (BITC), was prepared in dimethyl sulfoxide (DMSO; Avantor Performance Materials, Gliwice, Poland), and a 500 mM hydroxyurea (HU; Sigma-Aldrich, Poznań, Poland) stock solution was prepared in miliQ water; all solutions were stored at −80°C as singleuse aliquots. Camptothecin (CPT; Sigma-Aldrich, Poznań, Poland) was stored as a 1 mg/mL stock solution in DMSO at −20°C. Cisplatin (CDDP; Ebewe, Unterach am Attersee, Austria) was maintained as a ready-to-use stock solution (1 mg/mL) at room temperature.
For all 96-well plate-based assays, cells were seeded at a pre-optimized density as follows: 10 5 cells/well for MV-4-11 and A2780, 0.75 × 10 5 cells/well for MCF7, 0.5 × 10 5 cells/ well for 5637, and 0.25 × 10 5 cells/well for A-549 in an appropriate culture medium. For all 24-well plate-based assays, A2780 cells were seeded at 5 × 10 5 cells/well density. For western blot sample collection, 10 6 A2780 cells were seeded on 50-mm petri dishes. For experiments utilizing HU, the compound was added 8 h after cell seeding to obtain a 0.5 mM final concentration. Samples described as a co-treatment (+) were further prepared using a culture medium containing 0.5 mM HU; in samples described as a pretreatment (± ), after 16 h of HU treatment, the culture medium was replaced with a fresh one without HU.

Antiproliferative assay
At 24 h after seeding the cells in 96-well plates (Sarstedt, Nümbrecht, Germany), the tested compounds at concentrations ranging from 200 to 5 μM or cisplatin (10-0.01 μg/mL) were added. At the desired points in time, the plates were subjected to the SRB assay (according to a previously described protocol [26] with minor modifications [27], adherent cells) or the MTT assay (according to a previously described protocol [28] with minor modifications [27], non-adherent cells), and the absorbance at 540 nm and 570 nm, respectively, was recorded using a Biotek Hybrid H4 Reader (Biotek Instruments, Bad F r i e d r i c h s h a l l , G e r m a n y ) . C o m p o u n d s a t e a c h concentration/time point were tested in triplicate in a single experiment, and each experiment was repeated at least three times independently. The results are presented as the mean cell proliferation inhibition or IC 50 (half-maximum inhibitory concentration) ± standard deviation (SD), which was calculated using the Prolab-3 system based on the Cheburator 0.4 software [29].

Phase cycle analysis
At 24 h after seeding the cells in 24-well plates (Sarstedt, Nümbrecht, Germany), the tested compounds were applied at various concentrations. At the desired time points, the cells were trypsinized, fixed with ice-cold 70% (v/v) ethanol and analyzed for DNA content according to a previously described procedure [27], using a BD LSRFortessa cytometer (BD Bioscience, San Jose, USA). Compounds at each concentration were tested at least three times. The obtained results were analyzed using Flowing Software 2.5.1 (University of Turku, Turku, Finland) and GraphPad Prism 7.03 (GraphPad Software, Inc., La Jolla, USA).

Apoptosis rate assessment by the caspase-3 activity assay
At 24 h after seeding the cells in 24-well plates (Sarstedt, Nümbrecht, Germany), the tested compounds were applied at various concentrations. At the desired time points, the apoptosis rate was analyzed using a previously described procedure [27] with a Biotek Synergy H4 Reader. Camptothecin (1 μg/mL) applied for 4 h was used as a positive, technical control. Compounds at each concentration were tested at least three times. The obtained results were analyzed using GraphPad Prism 7.03.

Protein level assessment by western blot analysis
At 24 h after seeding the cells on 50-mm petri dishes (Sarstedt, Nümbrecht, Germany), the tested compounds were applied at various concentrations. After 48 h of treatment, the cells were washed with PBS (HIIET, PAS, Wrocław, Poland) and lysed with RIPA Buffer supplemented with protease and phosphatase cocktails (all Sigma-Aldrich, Poznań, Poland) for 30 min at 4°C. After centrifuging the samples (15 min, 4°C, 15,000×g; whole cell lysate), the collected supernatant was assessed for protein content using Pierce™ Coomassie Plus (Thermo Fisher Scientific, Warsaw, Poland) and stored at −80°C.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 7.03. All results are reported as the mean ± SD. One-way ANOVA analyses with the appropriate post hoc tests described in the figure captions were performed. p-values less than 0.05 were considered statistically significant.

Phenylboronic acids and benzoxaborole derivatives show high antiproliferative activity in selected cancer cell lines
A series of eighteen phenylboronic acid and nine benzoxaborole structurally diverse derivatives was preliminarily evaluated for antiproliferative activity in five cancer cell lines ( Table 1). The activity of the compounds was assessed using the SRB/MTT method after 72 h of treatment and expressed as the half-maximal inhibitory concentration (IC 72h 50 ). In the case of compounds for which the IC 72h 50 exceeded 200 μM, the mean proliferation inhibition is provided.
Phenylboronic acid (1) was almost completely inactive in all cell lines used, even at concentration as high as 200 μM. Among all PBA modifications introduced at the 2-position, the formyl moiety (4) was the only group that significantly increased the biological activity. Further studies indicated that the substituent position affected the antiproliferative potential of the compound. For example, the introduction of the formyl group at position "4" in 11 resulted in an IC 72h 50 rise compared with 4 with the greatest increase observed in the A2780 cell line. Concomitantly, modifications utilizing trifluoromethyl group at various positions (6-8) revealed the 4-position as the most promising. Further studies applying various moieties indicated that the mercapto group (9) was the most potent enhancer of 1 activity among the para-modified derivatives in all cell lines. It should be noted that 9 was the only PBAderived compound that showed at least moderate antiproliferative activity in the MCF7 cell line and was the most active derivative in the A-549 cell line.
Di-substituted derivatives of 1 provided further insights into the compound structure-activity relationship and belonged to the most active derivatives tested. In comparison to 4, a second formyl group introduced at the 2-position (14)   substituent at the 5-position (21) resulted in a pronounced rise in activity towards all cell lines, with the highest IC 72h 50 drop in 5637 and A-549. Additionally, 5-fluorobenzoxaborole (21) was the most active benzoxaborole-based compound tested on the MCF7, 5637 and A-549 cell lines. The benzoxaborole substitution at the 3-position (compounds 24-26) had little (on A2780 and A-549 cell lines) or no effect (on MCF7 and 5637 cell lines). MV-4-11 was the only cell line that responded positively to such modifications. Finally, di-substituted 3-morpholino-5-fluorobenzoxaborole (27) proved to be the most active benzoxaborole-based compound in A2780 and MV-4-11 cell lines. None of the tested compounds showed activity comparable to a widely used cytostaticcisplatin (CDDP) or an additional reference compoundbenzyl isothiocyanate (BITC). However, it should be noted that these reference agents plausibly do not share a common mechanism of action with the tested compounds.

Selected phenylboronic acid and benzoxaborole derivatives induce cell cycle arrest and apoptosis in A2780 ovarian cancer cells
Further studies focused on the basic mechanism of action of 18 and 27 as the most active representatives of phenylboronic acids and benzoxaboroles, respectively. The A2780 ovarian cancer cell line was used in all subsequent studies because of its high sensitivity to the tested compounds. First, the influence of the compound on the cell cycle was assessed using a standard, RNAse/propidium iodide-based protocol (Fig. 2a, d, e). Treatment of the cells with various compound concentrations for 48 h significantly modulated cell cycle progression with G 2 /M phase arrest. The 2-fluoro-6formylphenylbronic acid (18) significantly increased the percentage of cells in G 2 /M phase even at the lowest 5 μM concentration used (40.4 ± 8.8% in comparison to 21.0 ± 4.6% in the Ctrl), with highest increase observed for 10 μM (59.12 ± 6.56%). In both cases, the G 2 /M phase arrest was accompanied by a pronounced decrease in G 0 /G 1 and S phase cells percentage and significant increase in the percentage of tetraploid cells (>4 N)from 0.9 ± 0.1% in the control to 15.6 ± 2.8% in the sample treated with 18 at a 10 μM concentration. Surprisingly, at a high 25 μM concentration, the cell cycle progression arrest caused by 18 shifted towards significant S phase arrest (53.9 ± 6.3% cells in comparison to 18.9 ± 2.8% in the Ctrl) accompanied by almost complete decay of G 0 /G 1 phase (6.8 ± 1.2% in comparison to 59.2 ± 6.9% in the Ctrl). The tetraploid cell population decreased in comparison to the sample treated with 18 at the 10 μM concentration, but they remained significantly higher than in control samples (5.1 ± 2.8% in comparison to 0.9 ± 0.1% in Ctrl). The 3-morpholin-5-fluorobenzoxaborole (27)   Representative histograms of the cell cycle analysis performed using 18 (d) and 27 (e). 2 N stands for cells in G 0 /G 1 phase, 4 N stands for cells in G 2 /M phase, 8 N stands for tetraploid cells. f Representative western blot images. * -p < 0.05, ** -p < 0.01 -one-way ANOVA with Dunnett's multiple comparisons test compared to the Ctrl G 2 /M phase and 8.3 ± 0.6% tetraploid cells), but not with 5 μM. In both cases, the G 2 /M phase arrest was associated with a significant decrease in G 0 /G 1 cell percentage, with the highest of more than 3.8-times decay in the sample treated with 27 at a concentration of 25 μM (13.2 ± 2.6% in comparison to 50.5 ± 6.9% in the Ctrl).
Subsequently, the pro-apoptotic activity of the compounds was assessed after 48 h of A2780 cell treatment by caspase-3 enzymatic activity analysis (Fig. 2b). Caspase-3 is an effector enzyme that is activated regardless of the upstream events that caused apoptosis [30]. Such experiments provide insights concerning whether a tested agent is able to induce apoptosis without distinguishing the intra-and extracellular apoptotic pathways. Both compounds exhibited dose-dependent potential as pro-apoptotic agents, with the highest activation observed for the 18 at 25 μM concentration (9.7 ± 1.9fold caspase-3 activity increase in comparison to the Ctrl). Similarly to the cell cycle analysis, 27 caused apoptosis of a slightly lower scope, with a 5.6 ± 1.8fold caspase-3 activity increase with 25 μM.
To provide further insight into the compound mode of action, β-tubulin, p21 and cyclin B1 protein levels were assessed by western blot analysis after 48 h of A2780 cell treatment with various compound concentrations (Fig. 2c, f). These proteins were selected because of their crucial role in microtubule assembly and mitosis (β-tubulin) [31], S-G 2 -M cell cycle progression checkpoints (cyclin B1) [32] or as a master inhibitor of cyclin kinases responsible for cell cycle progression control (p21) [32]. A remarkable, dose-dependent increase in p21 level was observed when cells were treated with 18, even at the highest concentration used (25 μM), which caused S, not G 2 / M phase cell cycle arrest. Compound 27 induced a slightly less pronounced increase in the p21 protein level, but similarly to 18, a clear dose-dependence was demonstrated. Cyclin B1 level was significantly increased when cells were treated with 18 at 10 μM (1.8 ± 0.3fold level increase) and 27 at 25 μM (1.8 ± 0.3fold level increase), but its level remained equal to the control sample when 18 was applied at 25 μM, which correlates with its ability to suppress cell cycle progression in S phase. β-tubulin level did not change significantly as a result of the compound treatment regardless of the concentration used.

A2780 cells treated with phenylboronic acid and benzoxaborole derivatives exhibit a mitotic catastrophe-like morphology
A clear correlation between cell cycle arrest in G 2 /M phase, the ability to induce formation of the tetraploid cell population, and the ability of compounds to induce apoptosis was observed. This finding indicated that the compound mode of action was associated with mitotic catastrophe processes, characterized by aberrant mitosis, mitotic cell death, and as a result of aberrant mitosis slippage, the presence of aneuploid and tetraploid cells [33]. To address this hypothesis, A2780 cells treated for 48 h were stained with phalloidin, which binds to and stabilizes F-actin as well as DAPI to label DNA/nucleus, and examined by fluorescence microscopy (Fig. 3). In contrast to healthy, untreated cells, those treated with various compound concentrations demonstrated features characteristic of apoptosis, e.g., cell membrane blebbing, apoptotic bodies, mitotic catastrophelike aberrant mitotic figures and enlarged, multinucleated cellsthe result of failed karyo-and/or cytokinesis. Disruption of cell microfilaments was also often observed, especially actin filament decomposition and the presence of regions that were intensely stained by phalloidin.
The antiproliferative and pro-apoptotic activity of the compound is largely a phase cycle-specific feature A strong correlation between the compound-induced cell cycle inhibition and apoptosis rate raised the question of whether both events were not only correlated but also dependent. Additionally, the compound IC 50 assessment at various time points indicated that prolonged treatment was necessary to reveal the high antiproliferative potential (Fig. 4a) Fig. 4a). BITC is characterized by multimodal activity that is not necessarily associated with a specific cell cycle phase [34]. To address the hypothesis that the tested derivatives act as cell cycle specific agents in a subsequent experiments, we used A2780 cells treated with 0.5 mM HUa ribonucleotide reductase inhibitor that is commonly used to arrest cell cycle progression in G 0 /G 1 or early S phase [35]. Pre-treatment with HU significantly decreased cell growth, whereas the co-treatment almost completely abrogated it (Fig. 4b). Caspase-3 activation assessed under three different conditions (Fig. 4c) showed that continuous treatment with HU alone caused a significant increase in caspase-3 activity (9 ± 1.7 fold change compared with the untreated control), whereas the ability of 18 and 27 to induce apoptosis was completely abolished under these conditions since no further increase in caspase-3 activity was observed (8.3 ± 1.0 and 9 ± 0.6fold change compared with the untreated control for 18 and 27, respectively), irrespectively of the concentration used (representative results shown). Such a negative effect was not observed when HU co-treated cells were additionally treated with camptothecin (40 ± 5.8fold change compared with the   control). In the samples that were pre-treated with HU, the ability of 18 and 27 to induce apoptosis was also significantly reduced. Particularly in the case of 27 at 25 μM, which caused a 3.8 ± 0.8fold increase in caspase-3 activity in HU pre-treated cells (2.1 ± 0.4fold change for HU pre-treated control cells) in contrast to 6.2 ± 2.0fold change in untreated cells.
The cell cycle analysis confirmed the above-mentioned observation (Fig. 4d, e, f). Compounds 18 and 27 caused a significant increase in G 2 /M and > 4 N cell percentage, whereas these effects almost completely disappeared when samples were co-treated with HU and were significantly reduced when cells were pre-treated with HU. The described correlation was observed regardless of the used concentrations of compounds (representative results are presented). This phenomenon was most striking for 27, which applied alone at 25 μM caused a massive accumulation of cells in G 2 /M phase (64.4 ± 1.3% cell percentage) in comparison to the untreated control (22.6 ± 3.5% cells); however, after concurrent usage with HU, its impact was abolished (16.5 ± 2.0% cell percentage in G 2 /M). Additionally, HU treatment significantly reduced the capability of the tested compounds to induce tetraploidy, e.g., compared with the 16.8 ± 2.5% >4 N cell percentage originally observed for 18-treated cells (at 10 μM), only 3.2 ± 0.3% cells were observed following co-treatment with HU (1.0 ± 0.1% cell percentage in untreated control). Taken together, the abovedescribed observations clearly indicate that phenylboronic acid and benzoxaborole derivatives act as G 2 /M phase cycle-specific pro-apoptotic agents.

Discussion
The first boron-containing anticancer drug was bortezomiba dipeptide boronic acid that was featured as a strong proteasome inhibitor and approved by FDA for multiple myeloma treatment in 2003. Since then, much effort was undertaken to search for novel boron-containing compounds that are useful in medicinal chemistry, resulting in a large set of bioactive molecules at various stages of development (including clinical trials) [6]. In the area of experimental oncology, these efforts were focused mainly on boronic acid derivatives [8] with ixazomib, a second generation proteasome inhibitor that was approved for multiple myeloma in 2015 [36]. Concurrently, the (1) anticancer properties of phenylboronic acid were studied only by the Plopper group [15,37]. The authors identified 1 as a cell growth and migration inhibitor, but the required compound concentrations to observe a biological effect exceeded 500 μM in most cases. Recently, moderate anticancer activity of PBA was described in 4T1 murine mammary gland adenocarcinoma and SCCVII squamous carcinoma but also at high concentrations [16].
In our studies, we demonstrated for the first time that the introduction of simple substituents into 1 led to highly active phenylboronic acid-derived compounds such as 9, 16 and 18 with low micromolar IC 72h 50 values in several different types of cancer cells. Clear structure-activity relationships were observed, highlighting a significant influence of the substituent type and position. Markedly higher activity of 9 was observed in all cancer cell lines among all para-substituted derivatives, which indicated that resonance or inductive effects might play an important role in this matter since the mercapto group was the only electron donating group tested. Moreover, compound pK a modulation appeared to be crucial, at least in the case of the di-substituted compounds 15-18, of which the least active was 17 with a pK a = 6.72; the other compounds exhibiting pK a = 5.74, 6.42 and 6.05 (for 15, 16, and 18, respectively [23]; pK a = 8.72 for 1 [38]) were significantly more active.
Notable differences were observed in the sensitivity of the cell lines to the tested compounds, with MCF7 breast cancer cells recognized as generally resistant, and ovarian cancer (A2780) as well as leukemia cells (MV-4-11) identified as the most sensitive. The molecular origin of the observed discrepancies remains unknown but identifies biological activity of the compounds as a result of interactions with specific molecular targets rather than their overall toxicity. Moreover, our studies of HU-treated A2780 cells clearly indicated that the compound activity was focused on proliferating cells, with cells in S and/or G 2 /M phases identified as those targeted by 18 and 27.
Since the discovery of the sugar-binding properties of benzoxaboroles in 2006 [39], this class of compounds has received much attention as a potential antifungal, antibacterial, antiviral, antiprotozoal and even anti-inflammatory agent, with tavaborole (21) already approved for the treatment of onychomycosis as the most spectacular example [3]. This interest largely omitted experimental oncology, with few recently published papers focused on this field. Zhang et al. described a set of interesting, highly active chalcone-benzoxaborole hybrids [14], but further studies are needed to reveal to what extent the observed activity resulted from the introduction of the boron-containing moiety. Suman et al. synthesized a series of 6-aminobenzoxaborole derivatives, of which some showed high antiproliferative activity in pancreatic cancer cells (MIA-PaCa-2) [17], whereas some other examples of 6-aminobenzoxaborole-derived compounds showed no activity in MCF7 at a concentration of 50 μM [40], similarly to a set of β-ketobenzoxaboroles synthesized by Sravan Kumar et al. [18]. In both cases, the described compounds had a much more complex chemical structure than the derivatives reported herein. These results clearly indicate that further development of benzoxaborole-based anticancer agents requires a careful selection and evaluation of simple substituents rather than the introduction of additional, complex moieties. A perfect example of such an approach can be the antifungal drug tavaborole (21), a derivative comprising a single fluorine atom as a substituent.
In our studies, we not only observed relatively poor activity of the tested benzoxaboroles in MCF7 but also in some other cancer cell lines (data not shown). Concomitantly, several tested compounds exhibited high antiproliferative activity in A2780 and MV-4-11 cell lines, and, to a lesser extent, the 5637 urinary bladder cell line. Similar to PBA derivatives, the acidity of the compounds appeared to play an important role since pK a lowering from 7.39 and 7.42 for 19 and 23, respectively, to 6.97 observed for 21 was accompanied by a significant increase in biological activity. However, 20 and 22 showed poor antiproliferative potential despite their even lower pK a (6.36 and 6.57, respectively) [21,24], indicating the presence of at least several factors affecting the structure-activity relationships. Interestingly, we observed similar dependencies on several fungal strains [24], which might suggest the compounds' common molecular target (potentially leucyl-tRNA synthetase) in both cases. Clearly, a much larger set of compounds must be examined in future studies spanning a wider set of cancer cell lines to establish definitive rules responsible for the compound activities. Additionally, future studies focused on the disclosure of molecular features of cell lines determining their sensitivity to phenylboronic acid and benzoxaborole derivatives will be of high importance. Nevertheless, our preliminary evaluation clearly demonstrated extensive capabilities for further improvements of the compound biological activity that should provide a set of agents with sub-micromolar activities suitable for in vivo studies of anticancer activity.
The mode of antiproliferative activity of phenylboronic acids is largely unknown. Inhibition of GTPases by the Rho family in the DU-145 prostate cancer cell line was suggested by McAuley et al. as a plausible mechanism [37]. Additionally, a limited G 2 /M cell cycle arrest was observed for various cell lines, including DU-145 and PC-3 prostate cancer cells after treatment with 1 [15]. The antibacterial, antifungal and antiprotozoal activity of benzoxaboroles mainly derives from β-lactamase, PDE4 nucleotide phosphodiesterase, D , D -carboxypeptidase and leucyl-tRNA synthetase (LeuRS) inhibition [3]. Little is known about the mechanisms of action underlying their antiproliferative potential in cancer cells. Studies by Gao et al. indicated LeuRS as a potential target for 21 and some of its derivatives in human osteosarcoma (U2OS) and ovarian cancer (SKOV-3) cells. The apoptotic morphology associated with the ambiguous transcriptional activity of the p21 promotor and no significant impact on the cell cycle was reported [41].
In the present work, we performed a preliminary evaluation of the mechanism of action underlying phenylboronic acid and benzoxaborole derivative antiproliferative activity. The two most potent representatives -2-fluoro-6-formylphenylboronic acid (18) and 3-morpholine-5-fluorobenzoxaborole (27)and the A2780 ovarian cancer cell line were used. A very evident cell cycle arrest at G 2 /M phase associated with a significantly increased level of p21 protein probably resulted from the binding of p21 to cyclin B1-CDK1 and its inhibition. p21 acts as a cyclin-dependent kinase inhibitor that binds to various cyclin-CDK (cyclin dependent kinase) complexes and regulates cell cycle progression [32]. This process also includes binding to cyclin A-CDK1/CDK2 and the cyclin E-CDK2 complexes responsible for S phase progression, which explains the ability of 18 to induce S phase arrest at a concentration of 25 μM. Such activity could also result from p21 binding to the proliferating cell nuclear antigen (PCNA) that is also responsible for S phase arrest [42]. This hypothesis requires further studies, but it is supported by our observations that the p27 protein level was not markedly increased (data not shown) in any of the compound-treated samples. p27 is another member of the Cip/ Kip family of cyclin-dependent kinase inhibitors that lacks a PCNA binding domain [42]. The upstream mechanisms responsible for the increase in p21 as a result of compound treatment remain unknown but will be a subject of our future studies. Since the p21 level is also tightly controlled by proteasomedependent degradation [43], the activity of the tested compounds as its inhibitors as well as a multi-targeted mode of action cannot be excluded.
Another possible mechanism underlying such strong G 2 /M arrest might be tubulin polymerization disruption (a feature of antimitotic agents such as vinblastine or colchicine [44]) or αand β-tubulin degradation (a feature of isothiocyanates such as benzyl or allyl isothiocyanate [45]); however, our preliminary studies excluded this possibility since the β-tubulin level was not markedly reduced in cells treated with the tested compounds even at high concentrations (Fig. 2c, f), and we did not observe any influence on the tubulin polymerization process (data not shown). Cyclin B1 level increases during cell progression through G 2 phase, reaching a maximal level at prophase of mitotic division, and is associated with cyclin B1 shift to the nucleus and cyclin B1-CDK1 activity. In the samples treated with 18 and 27 (at 10 μM and 25 μM, respectively), a significant increase in cyclin B1 was accompanied by a high level of aberrant tetraploid cellsplausibly a result of failed mitotic death that led to mitotic catastrophe slippage and an increased number of multinucleated cells. Such cells are increasingly susceptible to cell death with every cell cycle progression and eventually undergo apoptosis [33].
In conclusion, we identified phenylboronic acid and benzoxaborole derivatives as potent antiproliferative agents acting as cell cycle arrest and apoptosis inducers. Great possibilities for further modifications makes them a promising new class of anticancer agents. Additionally, the tested compounds were evaluated as phase cycle-specific agents with activity focused on proliferating cells in late S/early G 2 phases. The upstream events caused by 18 and 27 that led to the above discussed image of A2780 cells remain unknown, but based on these preliminary studies, establishment of the exact mechanisms of action of phenylboronic acid and benzoxaborole derivatives should be possible in the future. Cell line features responsible for the specificity of the observed compounds will also be an important part of further studies.