Borivoj Galic

Dipotassium-trioxohydroxytetrafluorotriborate, K2[B3O3F4OH], is a potent inhibitor of human carbonic anhydrases

Abstract The boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]) was investigated as inhibitor of the zinc enzyme, carbonic anhydrase (CA, EC 4.2.1.1). Eleven human (h) CA isoforms, hCA I–IV, VA, VI, VII, IX and XII–XIV, were included in the investigations. The anion, similar to tetraborate or phenylboronic acid, inhibited most of them. hCA III was not inhibited by K2[B3O3F4OH], whereas hCA VA, hCA VI, hCA IX and hCA XIII were inhibited in the submillimolar range, with KIs of 0.31–0.63 mM. hCA I and II (cytosolic, widespread isoforms), hCA IV (membrane-bound isoform), hCA XII (tumor-associated, transmembrane) and hCA XIV (transmembrane) were much more effectively inhibited by this anion, with inhibition constants ranging from 25 to 93 µM. hCA VII, a cytosolic enzyme present in the brain and associated to oxidative stress, was very effectively inhibited by K2[B3O3F4OH], with a KI of 8.0 µM. We propose that K2[B3O3F4OH] binds to the metal ion from the enzyme active site, coordinating to the Zn(II) ion monodentately through its B-OH functionality. We hypothesize that some of the beneficial antitumor effects reported for K2[B3O3F4OH] may be due to the inhibition of CAs present in skin tumors.

In vitro and in vivo antitumor activity of the halogenated boroxine dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH])

Abstract Dipotassium-trioxohydroxytetrafluorotriborate K2[B3O3F4OH] was listed as a promising new therapeutic for cancer diseases. For in vitro and in vivo investigation of its antitumor effects 4T1 mammary adenocarcinoma, B16F10 melanoma and squamous cell carcinoma SCCVII were used. The detailed in vitro investigation undoubtedly showed that K2[B3O3F4OH] affects the growth of cancer cells. The proliferation of cells depends on the concentration so that aqueous solution of K2[B3O3F4OH], the concentrations of 10−4 M and less, does not affect cell growth, but the concentrations of 10−3 M or more, significantly slows cells growth. B16F10 and SCCVII cells show higher sensitivity to the cytotoxic effects of K2[B3O3F4OH] compared to 4T1 cells. Under in vivo conditions, K2[B3O3F4OH] slows the growth of all three tumors tested compared to the control, and the inhibitory effect was most pronounced during the application of the substance. There is almost no difference if K2[B3O3F4OH] was applied intraperitoneally, intratumor, peroral or as ointment. Addition of 5-FU did not further increase the antitumor efficacy of K2[B3O3F4OH].

A study of the inhibition of catalase by dipotassium trioxohydroxytetrafluorotriborate K2[B3O3F4OH]

Abstract In the development of boronic acid-based enzyme inhibitors as potential pharmaceutical drugs, dipotassium trioxohydroxytetrafluorotriborate K2[B3O3F4OH] was listed as a promising new therapeutic for treatment of these diseases. The catalase-mediated conversion of hydrogen peroxide, in the presence and absence of K2[B3O3F4OH] was studied. The kinetics conformed to the Michaelis–Menten model. Lineweaver–Burk plots were linear and plotted the family of straight lines intersected on the abscissa indicating non-competitive inhibition of the catalase. It appears that in the absence of inhibitor, catalase operates the best at conditions around pH 7.1 and in the presence of K2[B3O3F4OH] the optimum is around pH 6.2. The uncatalyzed reaction of hydrogen peroxide decomposition generally has a value of activation energy of 75 kJ mole−1, whereas catalase, in the absence of inhibitor, lowers the value to 11.2 kJ mole−1, while in the presence 69 mmoles L−1 of K2[B3O3F4OH] it was 37.8 kJ mole−1.

Effects of dipotassium trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]) on genetic material and inhibition of cell division in human cell cultures

We have examined antiproliferative, cytotoxic, and genotoxic potential of a halogenated boroxine dipotassium trioxohydroxytetrafluorotriborate [K2(B3O3F4OH)]. The impact on cell growth was evaluated by alamarBlue assay in basal cell carcinoma culture. Cytostatic, cytotoxic, and genotoxic potential were evaluated in lymphocytes culture, applying cytokinesis-block micronucleus cytome assay and chromosome aberrations analysis. Tested concentrations (0.05, 0.1, 0.2, and 0.4 mg/mL) were correlated with inhibition of cell growth in basal cell carcinoma culture and with the lymphocytes proliferation. Clastogenic activity has been confirmed, without evidences of aneugenic activity, in human lymphocytes.

Inhibitory effects of delphinidin and luteolin on genotoxicity induced by K2(B3O3F4OH) in human lymphocytes in vitro

Abstract The genotoxicity of halogenated boroxine [K2(B3O3F4OH)], a novel compound with the potential for prevention and/or treatment of various skin changes, has been confirmed in human lymphocytes. The potential of luteolin and delphinidin in inhibition of the genotoxic and cytotoxic effects of halogenated boroxine in vitro was analyzed applying the chromosome aberration analysis and the cytokinesis-block micronucleus cytome assay in human lymphocyte cultures. The in vitro treatments included addition of boroxine and flavonoids independently, and combined treatments of boroxine with luteolin or delphinidin. In the concentration of 50 μM, luteolin significantly decreased the frequency of micronuclei and nuclear buds. Delphinidin suppressed the occurrence of aberrant cells in the presence of the halogenated boroxine, but also affected their induction in respective delphinidin treatment. Detection of endoreduplications in combined treatments indicated that these flavonoids are potential inhibitors of cell cycle or topoisomerase II activity. The obtained results have confirmed antigenotoxic activity of selected bioflavonoids in vitro. The side effects of potential therapeutic applications of halogenated boroxine may be inhibited in the presence of bioflavonoids in appropriate dosage.

Inhibition of Horseradish Peroxidase Activity by Boroxine Derivative, Dipotassium-trioxohydroxytetrafluorotriborate K2[B3O3F4OH]

Recently research shows that horseradish peroxidase, HRP, when combined with other compounds, is highly reactive toward different human tumour cells and that better understanding of catalytic mechanism and inhibition HPR could lead to a new targeted cancer therapy. Thus, the inhibition of HRP activity by dipotassium-trioxohydroxytetrafluorotriborate K2[B3O3F4OH] was investigated for possible explanation of previously observed antitumour activities of this promising drug. HRP activity was studied under steady-state kinetic conditions by a spectrophotometric method. In the absence of the inhibitor values of = 0.47 mM and = 0.34 mM min−1, respectively, were determined. The hydrogen peroxide H2O2 kinetic measurements show a competitive inhibition with the inhibition constant = 2.56 mM. The activation energy values were found to be very similar for both reactions; in the absence of inhibitor activation energy was 17.7 kJ mol−1 and in the presence of inhibitor activation energy was 16.3 kJ mol−1. The values of Arrhenius constants were found to be different; = 4.635 s−1 was measured in the absence of inhibitor while in the presence of inhibitor Arrhenius constant was 1.745 s−1 showing that K2[B3O3F4OH] initiates conformational change in the structure of the HRP and subsequently reduces its activity.

In vitro and in vivo antitumour effects of phenylboronic acid against mouse mammary adenocarcinoma 4T1 and squamous carcinoma SCCVII cells.

Abstract The cytotoxic activity of phenylboroxine acid was evaluated in vitro on mouse mammary adenocarcinoma 4T1, mouse squamous cell carcinoma SCCVII, hamster lung fibroblast V79 and mouse dermal fibroblasts L929 cell lines. The cytotoxic effects were dose dependent for all tested tumour and non-tumour cell lines. Under in vivo conditions, three application routes of phenylboronic acid were studied: intra-peritoneal (i.p.), intra-tumour (i.t.) and per-oral. After tumour transplantation in syngeneic mice, phenylboronic acid was shown to slow the growth of both tumour cell lines (4T1 and SCCVII) compared with the control. The inhibitory effects were pronounced during the application of phenylboronic acid. For both tested tumour cell lines, the most prominent antitumour effect was obtained by intraperitoneal administration, followed significantly by oral administration.

Effects of dipotassium-trioxohydroxytetrafluorotriborate, K2[B3O3F4OH], on cell viability and gene expression of common human cancer drug targets in a melanoma cell line.

Abstract Recently it was found that dipotassium-trioxohydroxytetrafluorotriborate, K2(B3O3F4OH), is a potent and highly specific inhibitor of precancerous cell processes. We conducted gene expression profiling of human melanoma cells before and after treatment with two concentrations (0.1 and 1 mM) of this boron inorganic derivative in order to assess its effects on deregulation of genes associated with tumor pathways. Parallel trypan blue exclusion assay was performed to assess the cytotoxicity effects of this chemical. Treatment with K2(B3O3F4OH) induced a significant decrease of cell viability in melanoma cellline at both tested concentrations. Furthermore, these treatments caused deregulation of more than 30 genes known as common anti-tumor drug targets. IGF-1 and hTERT were found to be significantly downregulated and this result may imply potential use of K2(B3O3F4OH) as an inhibitor or human telomerase and insulin-like growth factor 1, both of which are associated with various tumor pathways.

Electrochemical Investigation of the Influence of K2[B3O3F4OH] on the Activity of Immobilized Superoxide Dismutase

It is known that oxidoreductase is responsible for the regulation of oxidative stress in organisms, and pathological changes occur within the cell in the form of accumulation or lack of superoxide and peroxide radicals if the oxidoreductase activity is disturbed. Currently, the development of drugs that target the affected cells while leaving healthy cells unaffected is of great interest. The action of potential drugs is based on the inhibition / activation of oxidoreductase. In this work, we studied the electrochemical parameters of superoxide dismutase as well as the action of the potential drug of boroxine - dipotassium trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]) as a target therapeutic for enzyme activity. Electrochemical tests were carried out in a classical three-electrode system using cyclic voltammetry and chronoamperometry techniques, and the obtained results were presented in the form of the kinetic parameters with the maximum value of the current obtained when the solution was saturated with the substrate (Imax) and the Michaelis-Menten constant (Km). K2[B3O3F4OH] was proven to be a reversible inhibitor, and the obtained Imax without inhibitor value of 0.014 mM and Km = 12.09 mM. The results from the Lineweaver - Burk diagram show that the inhibition is a partial noncompetitive inhibition type.

Advantages of an Electrochemical Method Compared to the Spectrophotometric Kinetic Study of Peroxidase Inhibition by Boroxine Derivative

In this study, boroxine derivative (K2[B3O3F4OH]) was tested as an inhibitor of horseradish peroxidase (HRP) by spectrophotometric and electrochemical methods. The activity of horseradish peroxidase was first studied under steady-state kinetic conditions by a spectrophotometric method which required the use of guaiacol as a second substrate to measure guaiacol peroxidation. The results of this method have shown that, by changing the concentration of guaiacol as the literature suggests, a different type of inhibition is observed than when changing the concentration of hydrogen peroxide as the substrate. This suggests that guaiacol interferes with the reaction in some way. The electrochemical method involves direct electron transfer of HRP immobilized in Nafion nanocomposite films on a glassy carbon (GC) electrode, creating a sensor with an electro-catalytic response to the reduction of hydrogen peroxide. The electrochemical method simplifies kinetic assays by removing the requirement of reducing substrates.