Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and ⁷Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.     

Biological evaluation of fluorinated p-boronophenylalanine derivatives as a boron carrier

Boron neutron capture therapy (BNCT) and magnetic resonance imaging (MRI) are quite attractive techniques for treatment and diagnosis of cancer, respectively. In order to develop practical materials utilizing both for BNCT and MRI, fluorinated p-boronophenylalanines and their alcohol derivatives had already been designed and synthesized. In the present paper the cytotoxicity, the incorporated amount into cancer cells, and the tumor cell killing effects of these compounds were elucidated to evaluate their usefulness as a boron carrier.     

Therapeutic success of boron neutron capture therapy (BNCT) mediated by a chemically non-selective boron agent in an experimental model of oral cancer: a new paradigm in BNCT radiobiology

The hypothesis of boron neutron capture therapy (BNCT) research has been that the short-range, high-linear energy transfer radiation produced by the capture of thermal neutrons by (10)B will potentially control tumor and spare normal tissue only if the boron compound selectively targets tumor tissue within the treatment volume. In a previous in vivo study of low-dose BNCT mediated by GB-10 (Na(2)(10)B(10)H(10)) alone or combined with boronophenylalanine (BPA) in the hamster cheek pouch oral cancer model that was primarily designed to evaluate safety and feasibility, we showed therapeutic effects but no associated normal tissue radiotoxicity. In the present study, we evaluated the response of tumor, precancerous and normal tissue to high-dose BNCT mediated by GB-10 alone or combined with BPA. Despite the fact that GB-10 does not target hamster cheek pouch tumors selectively, GB-10-BNCT induced a 70% overall tumor response with no damage to normal tissue. (GB-10+BPA)-BNCT induced a 93% overall tumor response with no normal tissue radiotoxicity. Light microscope analysis showed that GB-10-BNCT selectively damages tumor blood vessels, sparing precancerous and normal tissue vessels. In this case, selective tumor lethality would thus result from selective blood vessel damage rather than from selective uptake of the boron compound.     

(Z)-1,4-diamino-2-butene as a vector of boron,fluorine, or iodine for cancer therapy and imaging: synthesis and biological evaluation

Polyamine vectors are attractive for tumor targeting. We envisaged (Z)-1,4-diamino-2-butene (Z-DAB), an unsaturated analogue of putrescine as vector of (10)B, (18)F and (131)I for boron neutron capture therapy (BNCT), and tumor imaging by positron emission tomography or scintigraphy respectively. In the present work, the synthesis and characterization of new derivatives of Z-DAB were reported. Z-DAB was actively transported in cells via the polyamine transport system and converted into the spermidine analogue.(E)-2-iodo-1,4-diamino-2-butene (E-I-DAB) was not taken up by the polyamine transport system and may not be suitable for tumor imaging. In contrast, (Z)-2-[4-(5,5-dimethyl-dioxaborinan-2-yl)phenyl]methyl-1,4-diamino-2-butene (Z-4-Bbz-DAB) was a substrate of the transport system and allowed significant boron accumulation in 3LL cells. Its potential in BNCT will be evaluated.     

Boron uptake in normal melanocytes and melanoma cells and boron biodistribution study in mice bearing B16F10 melanoma for boron neutron capture therapy

Information on (10)B distribution in normal tissues is crucial to any further development of boron neutron capture therapy (BNCT). The goal of this study was to investigate the in vitro and in vivo boron biodistribution in B16F10 murine melanoma and normal tissues as a model for human melanoma treatment by a simple and rapid colorimetric method, which was validated by HR-ICP-MS. The B16F10 melanoma cell line showed higher melanin content than human melanocytes, demonstrating a greater potential for boronophenylalanine uptake. The melanocytes showed a moderate viability decrease in the first few minutes after BNCT application, stabilizing after 75 min, whereas the B16F10 melanoma showed the greatest intracellular boron concentration at 150 min after application, indicating a different boron uptake of melanoma cells compared to normal melanocytes. Moreover, at this time, the increase in boron uptake in melanoma cells was approximately 1.6 times higher than that in normal melanocytes. The (10)B concentration in the blood of mice bearing B16F10 melanoma increased until 90 min after BNCT application and then decreased after 120 min, and remained low until the 240th minute. On the other hand, the (10)B concentration in tumors was increased from 90 min and maximal at 150 min after application, thus confirming the in vitro results. Therefore, the present in vitro and in vivo study of (10)B uptake in normal and tumor cells revealed important data that could enable BNCT to be possibly used as a treatment for melanoma, a chemoresistant cancer associated with high mortality.     

A stable meta‐carborane enables the generation of boron‐rich peptide agonists targeting the ghrelin receptor

Boron neutron capture therapy (BNCT) is a binary cancer therapy, which combines the biochemical targeting of a boron‐containing drug with the regional localization of radiation treatment. Although the concept of BNCT has been known for decades, the selective delivery of boron into tumor cells remains challenging. G protein‐coupled receptors that are overexpressed on cancer cells in combination with peptidic ligands can be potentially used as shuttle system for a tumor‐directed boron uptake. In this study, we present the generation of short, boron‐rich peptide conjugates that target the ghrelin receptor. Expression of the ghrelin receptor on various cancer cells makes it a viable target for BNCT. We designed a novel hexapeptide super‐agonist that was modified with different specifically synthesized carborane monoclusters and tested for ghrelin receptor activation. A meta‐carborane building block with a mercaptoacetic acid linker was found to be optimal for peptide modification, owing to its chemical stability and a suitable activation efficacy of the conjugate. The versatility of this carborane for the development of peptidic boron delivery agents was further demonstrated by the generation of highly potent, boron‐loaded conjugates using the backbone of the known ghrelin receptor ligands growth hormone releasing peptide 6 and Ipamorelin.     

Boron neutron capture therapy (BNCT) for the treatment of liver metastases: biodistribution studies of boron compounds in an experimental model

We previously demonstrated the therapeutic efficacy of different boron neutron capture therapy (BNCT) protocols in an experimental model of oral cancer. BNCT is based on the selective accumulation of (10)B carriers in a tumor followed by neutron irradiation. Within the context of exploring the potential therapeutic efficacy of BNCT for the treatment of liver metastases, the aim of the present study was to perform boron biodistribution studies in an experimental model of liver metastases in rats. Different boron compounds and administration conditions were assayed to determine which administration protocols would potentially be therapeutically useful in in vivo BNCT studies at the RA-3 nuclear reactor. A total of 70 BDIX rats were inoculated in the liver with syngeneic colon cancer cells DHD/K12/TRb to induce the development of subcapsular tumor nodules. Fourteen days post-inoculation, the animals were used for biodistribution studies. We evaluated a total of 11 administration protocols for the boron compounds boronophenylalanine (BPA) and GB-10 (Na(2)(10)B(10)H(10)), alone or combined at different dose levels and employing different administration routes. Tumor, normal tissue, and blood samples were processed for boron measurement by atomic emission spectroscopy. Six protocols proved potentially useful for BNCT studies in terms of absolute boron concentration in tumor and preferential uptake of boron by tumor tissue. Boron concentration values in tumor and normal tissues in the liver metastases model show it would be feasible to reach therapeutic BNCT doses in tumor without exceeding radiotolerance in normal tissue at the thermal neutron facility at RA-3.     

Catalytic asymmetric synthesis of α-methyl-p-boronophenylalanine

p-Boronophenylalanine (l-BPA) is applied in clinical settings as a boron carrier for boron neutron capture therapy (BNCT) to cure malignant melanomas. Structural modification or derivatization of l-BPA, however, to improve its uptake efficiency into tumor cells has scarcely been investigated. We successfully synthesized (S)-2-amino-3-(4-boronophenyl)-2-methylpropanoic acid in enantioenriched form as a novel candidate molecule for BNCT. Key steps to enhance the efficiency of this synthesis were enantioselective alkylation of N-protected alanine tert-butyl ester with a Maruoka catalyst and Miyaura borylation reaction to install the boron functionality.     

Towards improved boron neutron capture therapy agents: evaluation of in vitro cellular uptake of a glutamine-functionalized carborane

Abstract  Sodium borocaptate (BSH) is widely used for boron neutron capture therapy (BNCT) of brain tumors. One drawback is the large uptake by the liver causing a decrease of its availability at the tumor region as well as bringing about toxicity problems. A novel carborane-based compound containing a boron payload very similar to that of BSH has been synthesized and tested on rat glioma (C6) cells, hepatoma tissue culture (HTC) cells, and hepatocytes. The newly synthesized system consists of an o-carborane unit (C₂B₁₀H₁₁, o-CB) conjugated to a glutamine residue through a proper spacer, namely, o-CB-Gln. As compared with BSH, it showed the same uptake by C6 cells, but a 50% decrease in uptake by HTC cells and an 80% decrease in uptake by healthy hepatocytes. On this basis o-CB-Gln appears an interesting candidate for BNCT of brain tumors as it is expected to have a therapeutic index analogous to that of BSH accompanied by a much lower liver toxicity. Graphical Abstract  A novel carborane based compound, consisting in an o-carborane unit (C2B10H11, o-CB) conjugated to a glutamine residue through a proper spacer (namely o-CB-Gln) has been synthesized, characterized and tested on rat glioma (C6), hepatoma (HTC) and hepatocytes. As compared to sodium borocaptate (BSH), widely used for boron neutron capture therapy (BNCT) of brain tumors, the newly synthesized system showed the same uptake by C6 cells, but a 50% decrease by HTC and 80% decrease by healthy hepatocytes. On this basis o-CB-Gln appears an interesting candidate for BNCT of brain tumors as it is expected to have a therapeutic index analogous to BSH accompanied by a much lower liver toxicity.      

Towards new boron carriers for boron neutron capture therapy: metallacarboranes and their nucleoside conjugates

Thymidine conjugates containing metallacarborane, {8-[5-(N(3)-thymidine)-3-oxa-pentoxy]-3-cobalt bis(1,2-dicarbollide)}- (5) and {8-[5-(O(4)-thymidine)-3-oxa-pentoxy]-3-cobalt bis(1,2-dicarbollide)}- (6) ions and several simple [3-cobalt bis(1,2-dicarbollide)]- ion (1) derivatives have been studied as potential boron carriers for BNCT. Compound 6 and some nonnucleoside derivatives of 1 were not toxic above 100 microM. The partition coefficient for both metallacarborane bearing thymidine conjugates 5 and 6 was more than 500 times higher than that of unmodified nucleoside. The cellular uptake studies showed accumulation of compounds 6 in V79 Chinese hamster cells but not of compound 5. The low toxicity of conjugate type of 6 together with its high partition coefficient suggest that judicially designed derivatives of metallacarboranes can be considered as potential boron carriers for BNCT.     

Synthesis and in vitro evaluation of thiododecaborated α, α- cycloalkylamino acids for the treatment of malignant brain tumors by boron neutron capture therapy

Boron-neutron capture therapy (BNCT) is an attractive technique for cancer treatment. As such, α, α-cycloalkyl amino acids containing thiododecaborate ([B₁₂H₁₁]^2?-S-) units were designed and synthesized as novel boron delivery agents for BNCT. In the present study, new thiododecaborate α, α-cycloalkyl amino acids were synthesized, and biological evaluation of the boron compounds as boron carrier for BNCT was carried out.     

The Anti-Proliferative Effect of Boron Neutron Capture Therapy in a Prostate Cancer Xenograft Model

Purpose

Boron neutron capture therapy (BNCT) is a selective radiation treatment for tumors that preferentially accumulate drugs carrying the stable boron isotope, ¹⁰B. BNCT has been evaluated clinically as an alternative to conventional radiation therapy for the treatment of brain tumors, and more recently, recurrent advanced head and neck cancer. Here we investigated the effect of BNCT on prostate cancer (PCa) using an in vivo mouse xenograft model that we have developed.

Materials and Methods

Mice bearing the xenotransplanted androgen-independent human PCa cell line, PC3, were divided into four groups: Group 1: untreated controls; Group 2: Boronophenylalanine (BPA); Group 3: neutron; Group 4: BPA-mediated BNCT. We compared xenograft growth among these groups, and the body weight and any motility disturbance were recorded. Immunohistochemical (IHC) studies of the proliferation marker, Ki-67, and TUNEL staining were performed 9 weeks after treatment.

Results

The in vivo studies demonstrated that BPA-mediated BNCT significantly delayed tumor growth in comparison with the other groups, without any severe adverse events. There was a significant difference in the rate of freedom from gait abnormalities between the BPA-mediated BNCT group and the other groups. The IHC studies revealed that BNCT treatment significantly reduced the number of Ki-67-positive cells in comparison with the controls (mean±SD 6.9±1.5 vs 12.7±4.0, p<0.05), while there was no difference in the number of apoptotic cells, suggesting that BPA-mediated BNCT reduced PCa progression without affecting apoptosis at 9 weeks post-treatment.

Conclusions

This study has provided the first preclinical proof-of-principle data to indicate that BPA-mediated BNCT reduces the in vivo growth of PCa. Although further studies will be necessary, BNCT might be a novel potential treatment for PCa.     

Microdosimetry for boron neutron capture therapy.

Preclinical studies for boron neutron capture therapy (BNCT) using epithermal neutrons are ongoing at several laboratories. The absorbed dose in tumor cells is a function of the thermal neutron flux at depth, the microscopic boron concentration, and the size of the cell. Dosimetry is therefore complicated by the admixture of thermal, epithermal, and fast neutrons, plus gamma rays, and the array of secondary high-linear-energy-transfer particles produced within the patient from neutron interactions. Microdosimetry can be a viable technique for determining absorbed dose and radiation quality. A 2.5-cm-diameter tissue-equivalent gas proportional counter has been built with 50 parts per million (ppm) 10B incorporated into the walls and counting gas to simulate the boron uptake anticipated in tumors. Measurements of lineal energy (y) spectra for BNCT in simulated volumes of 1-10 microns diameter show a dose enhancement factor of 4.3 for 30 ppm boron, and a "y" of 250 keV/microns for the boron capture process. Chamber design plus details of experimental and calculated linear energy spectra will be presented.     

Study on the compounds containing 19F and 10B atoms in a single molecule for the application to MRI and BNCT

Magnetic resonance imaging (MRI) and boron-neutron capture therapy (BNCT) are quite attractive techniques for diagnosis and treatment of cancer, respectively. In order to progress the study on both MRI and BNCT, the novel compounds containing 19F and 10B atoms in a single molecule were designed and synthesized. In the present paper, the syntheses and the internalization rates into tumor cells of these compounds are elucidated.     

Detection of γH2AX foci in mouse normal brain and brain tumor after boron neutron capture therapy

Aim

In this study, we investigated γH2AX foci as markers of DSBs in normal brain and brain tumor tissue in mouse after BNCT.

Background

Boron neutron capture therapy (BNCT) is a particle radiation therapy in combination of thermal neutron irradiation and boron compound that specifically accumulates in the tumor. ¹⁰B captures neutrons and produces an alpha (⁴He) particle and a recoiled lithium nucleus (⁷Li). These particles have the characteristics of extremely high linear energy transfer (LET) radiation and therefore have marked biological effects. High LET radiation causes severe DNA damage, DNA DSBs. As the high LET radiation induces complex DNA double strand breaks (DSBs), large proportions of DSBs are considered to remain unrepaired in comparison with exposure to sparsely ionizing radiation.

Materials and methods

We analyzed the number of γH2AX foci by immunohistochemistry 30 min or 24 h after neutron irradiation.

Results

In both normal brain and brain tumor, γH2AX foci induced by ¹⁰B(n,α)⁷Li reaction remained 24 h after neutron beam irradiation. In contrast, γH2AX foci produced by γ-ray irradiation at contaminated dose in BNCT disappeared 24 h after irradiation in these tissues.

Conclusion

DSBs produced by ¹⁰B(n,α)⁷Li reaction are supposed to be too complex to repair for cells in normal brain and brain tumor tissue within 24 h. These DSBs would be more difficult to repair than those by γ-ray. Excellent anti-tumor effect of BNCT may result from these unrepaired DSBs induced by ¹⁰B(n,α)⁷Li reaction.     

Synthesis and evaluation of cyclic RGD-boron cluster conjugates to develop tumor-selective boron carriers for boron neutron capture therapy

Boron-containing agents play a key role in successful boron neutron capture therapy (BNCT). Icosahedral boron cluster-Arg-Gly-Asp (RGD) peptide conjugates were designed, synthesized, and evaluated for the biodistribution to develop tumor-selective boron carriers. Integrin αvβ3 is an attractive target for anti-tumor drug delivery because of its specific expression in proliferating endothelial and tumor cells of various origins. We, therefore, selected a c(RGDfK) moiety recognizing αvβ3 as an active tumor-targeting device to conjugate with icosahedral boron-10 clusters, disodium mercaptododecaborate (BSH) or o-carborane as a thermal neutron-sensitizing unit. Preparation of o-carborane derivatives involved microwave irradiation, and resulted in high yields in a short time. An in vitro cell adhesion assay on αvβ3-positive U87MG and SCCVII cells demonstrated the high binding affinity of conjugates to integrin αvβ3 (IC(50)=0.19-2.66 μM). Biodistribution experiments using SCCVII-bearing mice indicated that GPU-201 showed comparable tumor uptake and a significantly longer retention in tumors compared with BSH. These results suggest that GPU-201 is a promising candidate for use in BNCT.     

Hyaluronan as carrier of carboranes for tumor targeting in boron neutron capture therapy

Boron neutron capture therapy (BNCT) represents a promising approach for tumor therapy. A critical requirement for BNCT is tumor targeting, a goal that is currently addressed with the development of low and high molecular weight agents capable of interacting with receptors expressed by cancer cells. Here, we describe a new bioconjugate (HApCB) composed by n-propyl carborane linked to hyaluronan (HA) via an ester linkage for a degree of substitution of approximately 30%, leading to a water-soluble derivative. The structure and main physicochemical characteristics of the new HA derivative were determined by means of Fourier transform infrared, fluorescence, and 1H, 13C, and 10B NMR analysis and are herein reported in detail. As HA is recognized by the CD44 antigen, densely populating the surface of many tumor cells, HApCB is expected to deliver boron atoms from the locally released carborane cages directly to target cells for antitumor application in BNCT. In vitro biological experiments showed that HApCB was not toxic for a variety of human tumor cells of different histotypes, specifically interacted with CD44 as the native unconjugated HA, and underwent uptake by tumor cells, leading to accumulation of amounts of boron atoms largely exceeding those required for a successful BNCT approach. Thus, HApCB may be regarded as a promising new BNCT agent for specific targeting of cancer cells overexpressing the CD44 receptor.     

closo-borane conjugated regulatory peptides retain high biological affinity: synthesis of closo-borane conjugated Tyr(3)-octreotate derivatives for BNCT

Despite the improvements in cancer therapy during the past years, high-grade gliomas and many other types of cancer are still extremely resistant to current forms of therapy. Boron neutron capture therapy (BNCT) provides a promising way to destroy cancer cells without damaging healthy tissue. However, BNCT in practice is still limited due to the lack of boron-containing compounds that selectively deliver boron to cancer cells. Since many neuroendocrine tumors show an overexpression of the somatostatin receptor, it was our aim to synthesize compounds that contain a large number of boron atoms and still show high affinity toward this transmembrane receptor. The synthetic peptide Tyr (3)-octreotate (TATE) was chosen as a high-affinity and internalizing tumor targeting vector (TTV). Novel boron cluster compounds, containing 10 or 20 boron atoms, were coupled to the N-terminus of TATE. The obtained affinity data demonstrate that the use of a spacer between TATE and the closo-borane moiety is the option to avoid a loss of biological affinity of closo-borane conjugated TATE. For the first time, it was shown that closo-borane conjugated regulatory peptides retain high biological affinity and selectivity toward their transmembrane tumor receptors. The results obtained and the improvement of spacer and boron building block chemistry may stimulate new directions for BNCT.     

"Sequential" boron neutron capture therapy (BNCT): a novel approach to BNCT for the treatment of oral cancer in the hamster cheek pouch model

In the present study the therapeutic effect and potential toxicity of the novel "Sequential" boron neutron capture therapy (Seq-BNCT) for the treatment of oral cancer was evaluated in the hamster cheek pouch model at the RA-3 Nuclear Reactor. Two groups of animals were treated with "Sequential" BNCT, i.e., BNCT mediated by boronophenylalanine (BPA) followed by BNCT mediated by sodium decahydrodecaborate (GB-10) either 24 h (Seq-24h-BNCT) or 48 h (Seq-48h-BNCT) later. In an additional group of animals, BPA and GB-10 were administered concomitantly [(BPA + GB-10)-BNCT]. The single-application BNCT was to the same total physical tumor dose as the "Sequential" BNCT treatments. At 28 days post-treatment, Seq-24h-BNCT and Seq-48h-BNCT induced, respectively, overall tumor responses of 95 ± 2% and 91 ± 3%, with no statistically significant differences between protocols. Overall response for the single treatment with (BPA + GB-10)-BNCT was 75 ± 5%, significantly lower than for Seq-BNCT. Both Seq-BNCT protocols and (BPA + GB-10)-BNCT induced reversible mucositis in the dose-limiting precancerous tissue around treated tumors, reaching Grade 3/4 mucositis in 47 ± 12% and 60 ± 22% of the animals, respectively. No normal tissue toxicity was associated with tumor response for any of the protocols. "Sequential" BNCT enhanced tumor response without an increase in mucositis in dose-limiting precancerous tissue.     

Boronated dipeptide borotrimethylglycylphenylalanine as a potential boron carrier in boron neutron capture therapy for malignant brain tumors.

Takagaki, M., Ono, K., Masunaga, S-I., Kinashi, Y., Oda, Y., Miyatake, S-I., Hashimoto, N., Powell, W., Sood, A. and Spielvogel, B. F. Boronated Dipeptide Borotrimethylglycylphenylalanine as a Potential Boron Carrier in Boron Neutron Capture Therapy for Malignant Brain Tumors. Radiat. Res. 156, 118-122 (2001).A boronated dipeptide, borotrimethylglycylphenylalanine (BGPA), was synthesized as a possible boron carrier for boron neutron capture therapy (BNCT) for malignant brain tumors. In vitro, at equal concentrations of (10)B in the extracellular medium, BGPA had the same effect in BNCT as p-boronophenylalanine (BPA). Boron analysis was carried out using prompt gamma-ray spectrometry and track-etch autoradiography. The tumor:blood and tumor:normal brain (10)B concentration ratios were 8.9 +/- 2.1 and 3.0 +/- 1.2, respectively, in rats bearing intracranial C6 gliosarcomas using alpha-particle track autoradiography. The IC(50), i.e. the dose capable of inhibiting the growth of C6 gliosarcoma cells by 50% after 3 days of incubation, was 5.9 x 10(-3) M BGPA, which is similar to that of 6.4 x 10(-3) M for BPA. The amide bond of BGPA is free from enzymatic attack, since it is protected from hydrolysis by the presence of a boron atom at the alpha-carbon position of glycine. These results suggest promise for the use of this agent for BNCT of malignant brain tumors. Further preclinical studies of BGPA are warranted, since BGPA has advantages over both BPA and BSH.