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].

• Antitumor activity
• drug research
• halogenated boroxine

Introduction

Inorganic cyclic ring systems that are isoelectronic with benzene have been known for many years, of which boroxine are referred to as “inorganic benzene”. Boroxines are the dehydration products of organoboronic acids and their derivatives such as trimethylboroxine, triphenylboroxine and halogenated boroxine dipotassium-trioxohydroxytetrafluorotriborate K₂[B₃O₃F₄OH] also make up a broader class of compounds called boroxines1^,2. Due to their unique electronic structures, these compounds are currently under investigation as possible enzyme inhibitors and therapeutics. In the development of boronic acid-based enzyme inhibitors as potential antitumor drugs, target specificity within a wide family of these compounds is the possibility to avoid different side effects during their application. This intensive area of medicinal chemistry research has recently culminated in the commercialization of the peptidylboronic acid antineoplastic drug Velcade3–5. Some boron-containing anions were observed as inhibitors of enzyme human carbonic anhydrase6–8. Recently, it has been suggested that halogenated boroxines K₂[B₃O₃F₄OH] can be used in the prevention and/or treatment of benign or malignant changes of the epidermis visible in the form of, for example, nevus or skin cancer9^,10. Accidentally, it was found that the dark colored growths after repeated contact with a small amount of K₂[B₃O₃F₄OH], lose color and irreversibly fall from the human skin's surface. This discovered impact on human cells has initiated our research on the effects of this compound on chromosomes, microorganisms, inhibition of enzymes, and proliferation of cancer cells. In previous work11 it was shown its effects on genetic material and inhibition of cell division in human cell cultures. Tested concentrations (0.04, 0.1, 0.2 and 0.4 mg mL⁻¹) 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. Since the presence of potential drugs in the cell can reduce the enzyme activity, in the recent studies12^,13 it was investigated the kinetic parameters and inhibition mechanism of K₂[B₃O₃F₄OH] on enzymes catalase and human carbonic anhydrases. It was shown that its mM concentration could significantly reduce catalase activity and that K₂[B₃O₃F₄OH] is a potent inhibitor of some human carbonic anhydrases with a K_I ranging from of 8.0 to 93 μM. In first study12, it was hypothesized that the local application of K₂[B₃O₃F₄OH]-containing cream or by its intra-tumor injection at level of mM concentration could significantly reduce catalase activity and increase the concentration of H₂O₂ and accordingly produce beneficial effects in tumor tissue alone. In second study13, it was proposed that K₂[B₃O₃F₄OH] binds to the Zn(II) from active site of enzyme carbonic anhydrase, coordinating to the metal ion monodentately through its Boron-OH functionality. It was hypothesized that some of the beneficial antitumor effects reported for K₂[B₃O₃F₄OH] may be due to the inhibition of carbonic anhydrase present in skin tumors.

As there are not enough data in the literature about the antitumor activities of halogenated boroxines (i.e. derivatives of cyclic anhydride of boronic acid), we decided to explore its in vitro and in vivo antitumor activity. For this purpose 4T1 mammary adenocarcinoma, B16F10 melanoma and squamous cell carcinoma SCCVII were used. These cell lines have ability to produce solid tumors in syngeneic mice and are very suitable for testing the antitumor potential of investigated substances. The mouse mammary carcinoma 4T1 was originally isolated as subpopulation derived from a spontaneously arising mammary tumor in BALB/cfC3H mice14^,15. The 6-thioguanine-resistant 4T1 tumor metastasizes via the hematogenous route to liver, lungs, bone and brain, making it a good model of human metastatic breast cancer16. The 4T1 tumor grows progressively and causes a uniformly lethal disease, even after excision of the primary tumor17^,18. Inasmuch as B16F10 murine melanoma cells arrest in lung following intravenous injection19, these cells provided an ideal choice for studying lung-specific metastasis in mice.

Materials and methods

Tested compound and drug preparations

Dipotassium trioxohydroxytetrafluorotriborate (K₂[B₃O₃F₄OH]) is water-soluble white powder. The stock solution was prepared by dissolution 20 mg of K₂[B₃O₃F₄OH] in 1-ml phosphate buffer purchased from Fisher Chemical (Wien, Austria). Tested substance was synthesized in the Laboratory of Department of Chemistry, Faculty of Science, University of Sarajevo, according to the modified method previously described20. The 5-fluorouracil (5-FU) solution for injection (250 mg/5 ml) was purchased from Pliva d.o.o. (Zagreb, Croatia).

Cell lines

Mouse mammary adenocarcinoma 4T1 and B16F10 mouse melanoma cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA), and mouse squamous cell carcinoma SCCVII cell line was obtained from BC Cancer Research Centre (Vancouver, Canada). Cell were grown in a RPMI 1640 medium (Sigma-Aldrich, Buchs, Switzerland) supplemented with 10% FCS (Sigma-Aldrich, Buchs, Switzerland), in a humidified atmosphere of 5% CO₂ in air and temperature 37 °C.

Animals and tumor models

The mice used in the study were obtained from Ruđer Bošković Institute's breeding colony. Three mouse strains, BALB/c syngeneic with 4T1 cells, C57BL/6 syngeneic with B16F10 cells and C3H/H syngeneic with SCCVII cells were used. The animals were about 3 months old and weighed 20–23 g. They were provided standard diet (Mucedola, Italy) and tap water ad libitum. The animals were kept in conventional circumstances: light/dark rhythms 12/12 h, temperature 22 °C, and humidity 55%. Animals were treated according to the Animal Welfare Regulations. Each experimental group consisted of seven animals. Mouse tumor models of mammary adenocarcinoma 4T1, B16F10 melanoma and squamous cell carcinoma SCCVII were established by injecting 5 × 10⁵ of tumor cells subcutaneously into the right thigh of syngeneic mice.

Determination of cytotoxic activity in vitro

Experiments were carried out in microtiter plates with 96 wells and 1 × 10⁴ tumor cells/250 μl of medium was applied in each well. After 24 h, when the cells reached confluence, the old cultured medium was replaced with a fresh one and K₂[B₃O₃F₄OH] was added to the cultures to the final concentrations of 0.125, 0.25, 0.5 and 1.0 mg/ml. The molar concentration is not expressed because K₂[B₃O₃F₄OH] has boroxine ring as “cyclic anhydride”, but it is unknown exactly how is hydrated (n H₂O). Control cells were incubated in RPMI medium without addition of the tested substance. The cells were incubated for the next 24 h and cytotoxicity test with crystal violet was performed to measure cell growth inhibition rate. In short, cells were fixed by the addition of a 3% solution of formalin for 15 min, washed with deionized water and dried in air. After that, cells were stained with 0.1% crystal violet for 20 min, then extensively washed with deionized water and left to dry overnight. The dye was extracted from the cells using a 10% solution of acetic acid and then absorbance was measured at 540 nm using a microplate reader. The absorbance at 590 nm is proportional to the number of surviving cells. Each experiment was done in quadruplicate. Inhibition of cell growth relative to controls was calculated according to the formula: Inhibition of cell growth (% ) = (C − T)/C × 100, where T denotes the mean absorbance of treated cells, and C indicates the mean absorbance of untreated (control) cells. The LC₅₀ concentrations were calculated using probit analysis21^,22.

Tumor treatment and evaluation of antitumor activity in vivo

For adenocarcinoma 4T1, the treatment was started on day 10 after transplantation of tumor cells. Two application route were studied, intraperitoneal (ip), with a dose of 10 mg/kg once a day for five consecutive days starting from day 10 (the total received dose 50 mg/kg), and intratumoral (it), with a dose of 50 mg/kg given once on the 10th day after tumor cell transplantation. The standard cytostatic 5-FU was applied on the same way and dose as K₂[B₃O₃F₄OH] respecting literature data that the lethal dose for a single dose of 5-FU in tumor-free mice was 190 mg/kg of mice23 or a toxic multiple treatment was with 130 mg/kg/week24. Mice with SCCVII squamous cell carcinoma received K₂[B₃O₃F₄OH] either intraperitoneally (10 mg/kg) or orally (50 mg/kg, gavage using a stomach tube) for nine consecutive days starting from day 12. The 5-FU alone or in combination with K₂[B₃O₃F₄OH] was given intraperitoneally applying the same dose and time of applications as the ip application of K₂[B₃O₃F₄OH] alone. Mice with melanoma B16F10 were treated either with intraperitoneal injection of 10 mg/kg of K₂[B₃O₃F₄OH] once a day for nine consecutive days, or with topical application of a cream containing 5% of K₂[B₃O₃F₄OH] on the skin over the tumor once a day for nine consecutive days starting from day 7. For intraperitoneal application, the required dose was injected in 300 µl of saline, while for intratumoral and oral administration the injected volume was 100 µl and 500 µl, respectively. Control mice received on the same way the appropriate volume of saline. Tumor growth was followed by measuring three orthogonal tumor diameters (A,B,C) with a caliper and tumor volume was calculated by the formula V = ABCπ/6. The inhibition of tumor growth was calculated by the formula: TGI (%) = (C − T/C) × 100, where C is the mean tumor volume of control (untreated) group and T is the mean tumor volume of treated groups.

Statistical analysis

The obtained results were expressed as average ± standard deviation (SD). To evaluate differences between the groups, a one-way analysis of variance followed by LSD post hoc test of multiple comparisons was used. Statistical analyses were performed using the Statistica software package (StatSoft, Inc., Tulsa, OK). Significant level was set at p < 0.05.

Results

Cytotoxic activity in vitro

Cytotoxic activity of different concentrations (0.125–1 mg/ml) of K₂[B₃O₃F₄OH] was evaluated on mouse mammary adenocarcinoma 4T1 (Figure 1A and B), squamous cell carcinoma SCCVII (Figure 2A and B) and melanoma B16F10 (Figure 3A and B) tumor cell lines. Generally, in all tested tumor cell lines, K₂[B₃O₃F₄OH] significantly reduced the number of surviving cells compared to the control group and the effect was dose dependent (Figures 1–3). However, some differences in the sensitivity of cells to K₂[B₃O₃F₄OH] were observed. The lowest concentration tested (0.125 mg/ml) did not significantly affect the growth of 4T1 cells (Figure 1), while a 20% inhibition was observed for SCCVII and B16F10 cells (Figures 2 and 3). Likewise, 0.25 mg/ml of K₂[B₃O₃F₄OH] did not inhibit the growth of 4T1 cells (Figure 1), but a significant inhibition of cell growth of ∼ 40 and 80% was obtained in SCCVII and B16F10 cells, respectively (Figures 2 and 3). Concentrations of 0.5 and 1.0 mg/ml were highly cytotoxic to all tumor cell lines (>80%). The LC₅₀ values were calculated and they were 0.415, 0.281 and 0.195 mg/ml for 4T1, SCCVII and B16F10, respectively.

Figure 1. Cytotoxic effect of 0.125, 0.25, 0.5 and 1.0 mg/ml of K₂[B₃O₃F₄OH] on mammary adenocarcinoma 4T1. (A) Cell survival rate measured by crystal violet assay. Absorbance at 590 nm is proportional to the number of surviving cells. Different small letters above bars indicate statistically significant differences among groups (p < 0.05, least significant difference (LSD) post hoc test). (B) Inhibition of cell growth expressed as percentage growth inhibition in reference to control cells.

Figure 2. Cytotoxic effect of 0.125, 0.25, 0.5 and 1.0 mg/ml of K₂[B₃O₃F₄OH] on melanoma B16F10. (A) Cell survival rate measured by crystal violet assay. Absorbance at 590 nm is proportional to the number of surviving cells. Different small letters above bars indicate statistically significant differences among groups (p < 0.05, LSD post hoc test). (B) Inhibition of cell growth expressed as percentage growth inhibition in reference to control cells.

Figure 3. Cytotoxic effect of 0.125, 0.25, 0.5 and 1.0 mg/ml of K₂[B₃O₃F₄OH] on squamous cell carcinoma. (A) Cell survival rate measured by crystal violet assay. Absorbance at 590 nm is proportional to the number of surviving cells. Different small letters above bars indicate statistically significant differences among groups (p < 0.05, LSD post hoc test). (B) Inhibition of cell growth expressed as percentage growth inhibition in reference to control cells.

Antitumor activity in vivo

Antitumor activity in vivo was assessed on the same tumor models as in vitro. The effects of intraperitoneal and intratumoral application of K₂[B₃O₃F₄OH] on the growth of mouse mammary adenocarcinoma are presented in Figures 4 and 5, respectively. Preliminary experiments showed that the application of 50 mg/kg of K₂[B₃O₃F₄OH], when administered intraperitoneally in a single dose was lethal for more than 50% of mice. However, there were no deaths if the dose was divided in five doses of 10 mg/kg. Also, the dose of 50 mg/kg was not lethal if it was applied as a single dose intratumorally. As shown in Figure 4, intraperitoneal application significantly reduced the growth of adenocarcinoma 4T1 compared to the control and inhibition rate was between 20 and 30%. Reduction was also significant compared to the 5-FU treatment, and 5-FU alone had no significant effect on tumor growth compared to the control (Figure 4). Intratumoral application of K₂[B₃O₃F₄OH] was even more effective than intraperitoneal one and tumor growth inhibition of about 40% compared to control was achieved (Figure 5). The same dose in mg/kg of 5-FU caused better inhibitory effect than intratumoral application of K₂[B₃O₃F₄OH] (probably because molecular weight of 5-FU is 130 and molecular weight of K₂[B₃O₃F₄OH is 251,6 and therefore the molar concentration is twice times greater). However, it should be noted that after the 23^rd day of the experiment a high mortality of 57% in the 5-FU group was observed, whereas in the K₂[B₃O₃F₄OH] group, there were no deaths. The effect of intraperitoneal application of K₂[B₃O₃F₄OH], applied separately or in combination with 5-FU, on the growth of SCCVII squamous cell carcinoma is shown in Figure 6. The K₂[B₃O₃F₄OH] alone significantly inhibited the growth of carcinoma SCCVII, with a maximum inhibition of 42% compared to the control achieved on the 20^th day after tumor transplantation. The 5-FU alone reduced tumor volume in a similar way as the K₂[B₃O₃F₄OH] treatment. The combination of K₂[B₃O₃F₄OH] and 5-FU, except during the first days of treatment, did not result with significantly better antitumor efficacy than each treatment separately. Oral administration of K₂[B₃O₃F₄OH] also caused significant inhibition of tumor growth compared to control (Figure 7). However, the inhibition rate was slightly lower than in the intraperitoneal administration, although the differences were not statistically significant. The effect of intraperitoneal and topical application of K₂[B₃O₃F₄OH] on the growth of mouse melanoma B16F10 is shown in Figure 8. In the period from day 12 until the end of the experiment, both administration routes significantly inhibited tumor growth compared to control. Achieved inhibition rates were in the range 25–30% and 35–40% for the intraperitoneal and topical application, respectively. However, there were no statistically significant differences in antitumor activity between the treatments.

Figure 4. The effect of intraperitoneal application of K₂[B₃O₃F₄OH] and 5-fluorouracil (5-FU) on the growth of mammary adenocarcinoma 4T1 transplanted into mouse thigh. K₂[B₃O₃F₄OH] and 5-fluorouracil (5-FU) were injected in a dose of 10 mg/kg once a day for five consecutive days starting from day 10 after tumor transplantation. Significant differences (p < 0.05, LSD post hoc test) between each treatment and control on a particular day are indicated with an asterisk (*), and significant differences between K₂[B₃O₃F₄OH] and 5-FU treatments are shown with the sign (#). Each experimental group consisted of seven animals.

Figure 5. The effect of intratumoral application of K₂[B₃O₃F₄OH] and 5-fluorouracil (5-FU) on the growth of mammary adenocarcinoma 4T1 transplanted into mouse thigh. K₂[B₃O₃F₄OH] and 5-fluorouracil (5-FU) were injected as a single dose of 50 mg/kg at day 10 after tumor transplantation. Significant differences (p < 0.05, LSD post hoc test) between each treatment and control on a particular day are indicated with an asterisk (*), and significant differences between K₂[B₃O₃F₄OH] and 5-FU treatments are shown with the sign (#). Each experimental group consisted of seven animals.

Figure 6. The effect of intraperitoneal application of K₂[B₃O₃F₄OH] and 5-fluorouracil (5-FU), applied alone or in a combination (K₂[B₃O₃F₄OH] + 5-FU), on the growth of squamous cell carcinoma SCCVII transplanted into mouse thigh. K₂[B₃O₃F₄OH] and 5-fluorouracil (5-FU) were injected in a dose of 10 mg/kg once a day for nine consecutive days starting from day 12 after tumor transplantation. Significant differences (p < 0.05, LSD post hoc test) between each treatment and control on a particular day are indicated with an asterisk (*). The sign (#) indicates a significant difference between combined treatment and each individual treatment. Each experimental group consisted of seven animals.

Figure 7. The effect of intraperitoneal (ip, 10 mg/kg once a day for nine consecutive days) and peroral (50 mg/kg once a day for nine consecutive days) application of K₂[B₃O₃F₄OH] on the growth of squamous cell carcinoma SCCVII transplanted into mouse thigh. Treatments were started at day 12 after tumor transplantation. Significant differences (p < 0.05, LSD post hoc test) between each treatment and control on a particular day are indicated with an asterisk (*). Each experimental group consisted of seven animals.

Figure 8. The effect of intraperitoneal (ip, 10 mg/kg once a day for nine consecutive days) and topical (35 mg of cream with 5% of K₂[B₃O₃F₄OH] once a day for nine consecutive days) application of K₂[B₃O₃F₄OH] on the growth of melanoma B16F10 transplanted into mouse thigh. Treatments were started at day 7 after tumor transplantation. Significant differences (p < 0.05, LSD post hoc test) between each treatment and control on a particular day are indicated with an asterisk (*). Each experimental group consisted of seven animals.

Discussion

In this article, as presented in the “Results” section, the detailed in vitro investigation on 4T1 mammary adenocarcinoma, B16F10 melanoma and squamous cell carcinoma SCCVII undoubtedly showed that K₂[B₃O₃F₄OH] affects the growth of cancer cells. The proliferation of cells depends on the concentration in an unusual manner 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 cell growth (Figures 1–3). This effect is a general phenomenon which was observed in a large number of preliminary studies of different types of cancer cells and non-tumor cells. The preliminary effects of K₂[B₃O₃F₄OH] have been tested in vitro on 81 different types of cells (performed at the National Cancer Institute, Division of Cancer Treatment and Diagnosis, Betheseda, MD and Rudjer Boskovic Institute, Zagreb, Croatia) and similar results were obtained. The only exceptions were the cancer cells SK-MEL-28, CAKI-1, and HS 578T where the opposite effect was observed, i.e. the cell growth was significantly increased (at concentration 10⁻⁵ M).

Under in vivo conditions, K₂[B₃O₃F₄OH] slows the growth of all three tumor tested (B16F10 melanoma, squamous cell carcinoma SCCVII, and 4T1 mammary adenocarcinoma) compared to the control (Figures 4–8). Slower growth of tumor is associated with the duration of therapy. During the course of therapy, tumor growth was slowed, and after the cessation of treatment, it suddenly accelerated compared to the control. Different methods of application lead to a similar effect on tumor growth. There is almost no difference if K₂[B₃O₃F₄OH] was applied intraperitoneally, intratumoral, peroral or as ointment. This result suggests that K₂[B₃O₃F₄OH] is stable in various body fluids (blood, plasma, etc.), and regardless of the way of applications, inside or over the surface of the skin, it almost identically suppresses the growth of skin cancer cells. The comparison with well known anti-cancer drug (5-fluorouracil, 5-FU) shows that, in identical conditions, both compounds have nearly identical effects on the growth of cancer cells, although twice as many molecules of 5-FU were used in the experiments.

Conclusions

Based on the results of this study, it has undoubtedly found that K₂[B₃O₃F₄OH] shows antitumor activity. In some unknown way, it recognizes cancer cells or cancer cells have a general feature that is attractive to K₂[B₃O₃F₄OH] molecules. In support of this assumption is the fact that a small number of these molecules finds and operates on a small number of cancer cells, but in the environment of a huge number of normal cells. At the same time, it recognizes and affects cell growth of skin cancer regardless of whether it was administered intraperitoneally or peroral. Since it has been shown as an efficient inhibitor of the enzymes catalase and carbonic anhydrases, as a compound promising as a new therapeutic for treatment of cancer, K₂[B₃O₃F₄OH] need further investigation of its inhibitory properties on activity of other enzymes such as tyrosinases and especially tyrosine kinases. As known several oncogenic tyrosine kinases have been detected in human malignancies. Therefore, targeting the active sites of these enzymes with small anion [B₃O₃F₄OH]²⁻, with the potential ability to inhibit their intracellular activities, could explain the hypothesis of anticancer properties of K₂[B₃O₃F₄OH].

Acknowledgements

The authors are grateful to the Ministry of Science, Education and Sports of the Republic of Croatia.

Declaration of interest

This research was supported by Ministry of Science, Education and Sports of the Republic of Croatia (project No. 011-2160547-1330). The authors report no declarations of interest. The authors confirm that they are alone responsible for the content and writing of the article.