Polyphenolic Boronates Inhibit Tumor Cell Proliferation: Potential Mitigators of Oxidants in the Tumor Microenvironment

Simple Summary Several plant-based compounds inhibit tumor cell growth. Selective modification of these compounds significantly increases their antitumor potency. The goal of this study was to develop less toxic, more potent natural compounds for use in cancer and immune therapies. To this end, we modified the structures of honokiol and magnolol, the two active components of magnolia extract. The modified compounds—honokiol boronate and mitochondria-targeted honokiol boronate—target the mitochondria of tumor cells and inhibit cell proliferation. These boronate derivatives also react with oxidants that are generated in tumor mitochondria and the tumor microenvironment. During this process, these boronate derivatives are also converted back to the original compounds with antitumor potencies. Thus, boronation of naturally occurring plant-derived compounds could make them more active in tumor cells as well as in the adjoining tumor microenvironment. These novel polyphenolic derivatives may enhance the scope of cancer immunotherapies. Abstract Boronate-based compounds have been used in brain cancer therapy, either as prodrugs or in combination with other modalities. Boronates containing pro-luminescent and fluorescent probes have been used in mouse models of cancer. In this study, we synthesized and developed polyphenolic boronates and mitochondria-targeted polyphenolic phytochemicals (e.g., magnolol [MGN] and honokiol [HNK]) and tested their antiproliferative effects in brain cancer cells. Results show that mitochondria-targeted (Mito) polyphenolic boronates (Mito-MGN-B and Mito-HNK-B) were slightly more potent than Mito-MGN and Mito-HNK in inhibiting proliferation of the U87MG cell line. Similar proliferation results also were observed in other cancer cell lines, such as MiaPaCa-2, A549 and UACC-62. Independent in vitro experiments indicated that reactive nitrogen species (e.g., peroxynitrite) and reactive oxygen species (e.g., hydrogen peroxide) stoichiometrically react with polyphenolic boronates and Mito-polphenolic boronates, forming polyphenols and Mito-polyphenols as major products. Previous reports suggest that both Mito-MGN and Mito-HNK activate cytotoxic T cells and inhibit immunosuppressive immune cells. We propose that Mito-polyphenolic boronate-based prodrugs may be used to inhibit tumor proliferation and mitigate oxidant formation in the tumor microenvironment, thereby generating Mito-polyphenols in situ, as well as showing activity in the tumor microenvironment.


Introduction
Boronates are a novel class of molecules containing an electron-deficient boron atom. Borax and boronophenylalanine have been used in boron neutron capture therapy (BNCT) to treat glioma [1][2][3][4][5]. There is extensive literature indicating the antiproliferative and

Materials and Methods
All chemicals and organic solvents were commercially available and were used as supplied. The reactions were monitored by thin layer chromatography (TLC) using silica gel Merck 60 F254. Crude materials were purified by flash chromatography on Merck silica gel 60 (0.040-0.063 mm). 31 P nuclear magnetic resonance (NMR), 1 H NMR, and 13 C NMR spectra were recorded with spectrometers at 400.13 MHz and 75.54 MHz, respectively. 1 H NMR spectra were recorded at 400.13 MHz using a Bruker DPX AVANCE 400 spectrometer equipped with a quattro nucleus probe. 1 H NMR and 31 P NMR were taken in deuterated chloroform (CDCl3) using CDCl3 and tetramethylsilane as internal references, respectively. Chemical shifts (δ) are reported in ppm and J values in hertz. Synthetic procedures for preparing polyphenolic and Mito-polyphenolic boronates are shown in Schemes 1-5. NMR spectra are presented in Appendix A.

Materials and Methods
All chemicals and organic solvents were commercially available and were used as supplied. The reactions were monitored by thin layer chromatography (TLC) using silica gel Merck 60 F254. Crude materials were purified by flash chromatography on Merck silica gel 60 (0.040-0.063 mm). 31 P nuclear magnetic resonance (NMR), 1 H NMR, and 13 C NMR spectra were recorded with spectrometers at 400.13 MHz and 75.54 MHz, respectively. 1 H NMR spectra were recorded at 400.13 MHz using a Bruker DPX AVANCE 400 spectrometer equipped with a quattro nucleus probe. 1 H NMR and 31 P NMR were taken in deuterated chloroform (CDCl 3 ) using CDCl 3 and tetramethylsilane as internal references, respectively. Chemical shifts (δ) are reported in ppm and J values in hertz. Synthetic procedures for preparing polyphenolic and Mito-polyphenolic boronates are shown in Schemes 1-5. NMR spectra are presented in Appendix A.

Cell Culture
The U87MG (ATCC Cat# HTB-14, human glioblastoma cancer cells), A549 (ATCC Cat# CCL-185, human lung cancer cells), and MiaPaCa-2 (ATCC Cat# CRL-1420, human pancreatic cancer cells) cell lines were purchased from the American Tissue Culture Collection (Manassas, VA, USA). The UACC-62 melanoma cell line was purchased from AddexBio (San Diego, CA; Cat# C0020003). All cell lines were regularly authenticated. All cell lines were grown at 37°C in 5% carbon dioxide. The U87MG, A549, and UACC-62 cells were maintained in Roswell Park Memorial Institute Medium 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA; Cat# 11875) supplemented with 10% fetal bovine serum. MiaPaCa-2 cells were maintained in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific, Waltham, MA, USA; Cat# 11965) and supplemented with 10% fetal bovine serum. All cells were stored in liquid nitrogen and used within 20 passages after thawing.

Cell Proliferation
The IncuCyte Live-Cell Analysis System was used to continuously monitor cell proliferation, as described in earlier publications [43,45,46].

Cell Culture
The U87MG (ATCC Cat# HTB-14, human glioblastoma cancer cells), A549 (ATCC Cat# CCL-185, human lung cancer cells), and MiaPaCa-2 (ATCC Cat# CRL-1420, human pancreatic cancer cells) cell lines were purchased from the American Tissue Culture Collection (Manassas, VA, USA). The UACC-62 melanoma cell line was purchased from AddexBio (San Diego, CA; Cat# C0020003). All cell lines were regularly authenticated. All cell lines were grown at 37 • C in 5% carbon dioxide. The U87MG, A549, and UACC-62 cells were maintained in Roswell Park Memorial Institute Medium 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA; Cat# 11875) supplemented with 10% fetal bovine serum. MiaPaCa-2 cells were maintained in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific, Waltham, MA, USA; Cat# 11965) and supplemented with 10% fetal bovine serum. All cells were stored in liquid nitrogen and used within 20 passages after thawing.

Cell Proliferation
The IncuCyte Live-Cell Analysis System was used to continuously monitor cell proliferation, as described in earlier publications [43,45,46].

Calculated Values of the Octanol/Water Partition Coefficients
The calculated octanol/H 2 O partition coefficients (logP) of boronate analogs were assessed using a quantitative structure-activity relationship analysis and rational drug design as a measure of molecular hydrophobicity ( Figure 1). This method also uses a consensus model built using the ChemAxon software (San Diego, CA, USA) [47,48].

LC/MS Analysis
High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1200 apparatus equipped with ultraviolet-visible spectroscopy absorption and a mass spectrometry detector (single quadrupole).
In the studies on the reaction profile of ONOO − oxidation of Mito-HNK-B and derivatives, 2 µL of sample was injected into the HPLC system equipped with a C18 column (Phenomenex, Kinetex Evo, 100 × 2.1 mm, 1.7 µm) equilibrated with 5% MeCN containing 0.1% (v/v) formic acid. The compounds were separated by a linear increase in MeCN phase concentration from 5% to 100% over 8 min and until 15 min at 100% MeCN using a flow rate of 0.21 mL/min. The peak areas detected by monitoring the absorption at 260 nm were used for the quantitation.

Oxidation of Boronates Derivatives by ONOO − , H 2 O 2 , and HOCl
The stock solutions of oxidants (HOCl and H 2 O 2 ) were prepared freshly before each experiment; their concentrations were determined by spectrophotometry by reacting nitrite with H 2 O 2 , using the procedure described previously [15]. ONOO − was prepared according to the published procedure. Typically, ONOO − was synthesized in a reaction of 0.6 M nitrite with 0.7 M H 2 O 2 at pH 13. Excess H 2 O 2 was removed by passage through a column of MnO 2 and the solution was frozen at −20 • C. The liquid over the frozen solid was collected and stored at −80 • C. Immediately prior to each experiment, the concentration of ONOO − was determined spectrally at 302 nm (ε = 1.7 × 103 M −1 cm −1 ) after dilution in 0.1 M sodium hydroxide to~10 mM concentration [49].
Stock solutions of HNK-B, HNK-B 2 , and Mito-HNK-B were prepared in dimethyl sulfoxide at a 10 mM concentration and this solution was added directly to the buffer to obtain the desired concentration. HPLC analysis indicated that HNK-B, HNK-B 2 , and Mito-HNK-B (pinacolate ester) undergo fast hydrolysis to the corresponding boronic acid formed upon dilution in the aqueous phosphate buffer. Thus, the boronic acid species were tested with the oxidants. When studying HNK-B and Mito-HNK-B oxidations by HOCl, stock solutions (10 mM) in MeCN were added to the phosphate buffer (100 mM, pH 7.4) to obtain a final concentration of the probe of 500 µM. Dimethyl sulfoxide solvent was avoided due to known rapid quenching of HOCl by dimethyl sulfoxide [50].
A study of the reactivity of HNK-B 2 in the presence of sodium hypochlorite (NaOCl) was not possible because its solubility is too low in MeCN and water.  Figure 2 shows the dose-dependent effects of HNK and HNK-B in glioblastoma (U87MG), pancreatic cancer (MiaPaCa-2), and lung cancer (A549) cells. In these cell lines, the antiproliferative effects of HNK and HNK-B were nearly the same. Figure 3 shows the dose-dependent effects of Mito-HNK and Mito-HNK-B in these cell lines, and the antiproliferative effects of Mito-HNK and Mito-HNK-B were nearly the same. As shown in Figure Figures 6 and 7 show the HPLC traces and mass spectral parameters for HNK/HNK-B, Mito-HNK/Mito-HNK-B, and corresponding intermediates and products formed from the reaction with oxidants. In the presence of ONOO -, both HNK-B and Mito-HNK-B were rapidly oxidized to HNK and Mito-HNK. In contrast, in the presence of H2O2, smaller conversion of HNK-B to HNK (60%) and Mito-HNK-B to Mito-HNK (37%) was observed. However, at higher concentrations of H2O2, this conversion was increased. These results are consistent with our original finding that ONOOreacts with boronates considerably faster than H2O2 [51]. Nitrated HNK and Mito-HNK were likely formed in minor quantities. HPLC results also show the formation of the hydroxy intermediate that triggers the self-immolative pathway (see Discussion, Figure 8).

Oxidative Cleavage of Polyphenolic Boronates and TPP + -Conjugated Polyphenolic Boronates
The polyphenolic boronate compounds consist of an arylboronic ester head group and a quinone methide group linked via a carbamate group to the parent polyphenolic compound (e.g., HNK or Mito-HNK). In the presence of oxidants such as H2O2, HOCl, and ONOO -, boronates are oxidized through insertion of an oxygen atom into the carbonboron bond, forming the corresponding phenol intermediate. The phenoxide ion eliminates the quinone methide, releasing the polyphenolic compound via a decomposition process known as self-immolation ( Figure 8). As shown in our previous publications [13,25,26], ONOOreacts with boronates about one million times faster than H2O2 and one thousand times faster than HOCl. Phenols are the major product. Whereas the H2O2/boronate reaction is catalase-sensitive, the ONOO -/boronate reaction is catalase-insensitive. Peroxy-caged luciferin (PCL-1) is oxidized by H2O2, HOCl, and ONOOto luciferin as a major product through a self-immolation mechanism [15,20,27]. In an in vivo setting, PCL-1 has been used in luciferase-transfected mice xenografts to monitor oxidant formation through measurement of bioluminescence and in PET using a boronate-based positron emitting probe [16,[20][21][22].

Antitumor Activity of MTDs: Activation of Immune Cells
We showed that mitochondria-targeted atovaquone (Mito-ATO) targets both granulocytic-myeloid-derived suppressor cells (G-MDSCs) and regulatory T cells (Tregs) in the TME, resulting in significant decreases of both G-MDSCs and Tregs as determined by flow cytometry analysis [45]. Intratumoral injection of Mito-ATO into primary tumors in a spontaneous tumor model triggered potent T cell immune responses locally and in distant tumor sites. Single-cell RNA sequencing revealed that Mito-ATO inhibits the expression of genes for OXPHOS and glycolysis in G-MDSCs and Tregs and facilitates the infiltration of CD4 + T cells in the tumor microenvironment [52]. Other studies also revealed the immuno-modulatory and tumor-preventing effects of MTDs [53,54].

Oxidants in the Tumor Microenvironment: Potential Inhibition by Polyphenolic Boronates
The TME changes dynamically. The TME consists of tumor cells, cancer-associated macrophages, lymphocytes, neutrophils, cancer-associated fibroblasts, endothelial cells, and vascular pericytes. The extracellular matrix in the TME consist of protein and polysaccharides. The dynamic interaction or the lack thereof between the tumor cells and the many cells and components in the TME regulates metabolism. MDSCs generate reactive

Oxidative Cleavage of Polyphenolic Boronates and TPP + -Conjugated Polyphenolic Boronates
The polyphenolic boronate compounds consist of an arylboronic ester head group and a quinone methide group linked via a carbamate group to the parent polyphenolic compound (e.g., HNK or Mito-HNK). In the presence of oxidants such as H 2 O 2 , HOCl, and ONOO − , boronates are oxidized through insertion of an oxygen atom into the carbonboron bond, forming the corresponding phenol intermediate. The phenoxide ion eliminates the quinone methide, releasing the polyphenolic compound via a decomposition process known as self-immolation ( Figure 8). As shown in our previous publications [13,25,26], ONOO − reacts with boronates about one million times faster than H 2 O 2 and one thousand times faster than HOCl. Phenols are the major product. Whereas the H 2 O 2 /boronate reaction is catalase-sensitive, the ONOO − /boronate reaction is catalase-insensitive. Peroxycaged luciferin (PCL-1) is oxidized by H 2 O 2 , HOCl, and ONOO − to luciferin as a major product through a self-immolation mechanism [15,20,27]. In an in vivo setting, PCL-1 has been used in luciferase-transfected mice xenografts to monitor oxidant formation through measurement of bioluminescence and in PET using a boronate-based positron emitting probe [16,[20][21][22].

Antitumor Activity of MTDs: Activation of Immune Cells
We showed that mitochondria-targeted atovaquone (Mito-ATO) targets both granulocyticmyeloid-derived suppressor cells (G-MDSCs) and regulatory T cells (T regs ) in the TME, resulting in significant decreases of both G-MDSCs and T regs as determined by flow cytometry analysis [45]. Intratumoral injection of Mito-ATO into primary tumors in a spontaneous tumor model triggered potent T cell immune responses locally and in distant tumor sites. Single-cell RNA sequencing revealed that Mito-ATO inhibits the expression of genes for OX-PHOS and glycolysis in G-MDSCs and T regs and facilitates the infiltration of CD4 + T cells in the tumor microenvironment [52]. Other studies also revealed the immuno-modulatory and tumor-preventing effects of MTDs [53,54].

Oxidants in the Tumor Microenvironment: Potential Inhibition by Polyphenolic Boronates
The TME changes dynamically. The TME consists of tumor cells, cancer-associated macrophages, lymphocytes, neutrophils, cancer-associated fibroblasts, endothelial cells, and vascular pericytes. The extracellular matrix in the TME consist of protein and polysaccharides. The dynamic interaction or the lack thereof between the tumor cells and the many cells and components in the TME regulates metabolism. MDSCs generate reactive oxy-gen and nitrogen species in the TME via activation of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and inducible nitric oxide synthase (iNOS) [55].
We have previously reported the anti-inflammatory effects of MTDs in Mito-Park mice [56,57]. MTDs such as Mito-apocynin decreased the expression of iNOS and NOX2, resulting in decreased oxidative and nitrative damage in neuronal cells. In another study, administration of an MTD (i.e., mitochondria-targeted carboxy-proxyl [Mito-CP]) inhibited cisplatin-induced renal toxicity by inhibiting pro-inflammatory mediators (iNOS in macrophages) in the kidney [58]. Mitochondria-targeted compounds inhibit oxidants generated from NOX2 [56,59]. Inhibition of NOX4 (which presumably generates only H 2 O 2 ) potentiates cancer immunotherapy [60,61]. Recent clinical trials using iNOS inhibitors in breast cancer patients who were resistant to other forms of cancer therapies showed tumor shrinkage and enhanced patient survival [62]. Inhibition of nitric oxide generated in the TME from tumor-associated macrophages decreased inflammatory mediators in the TME and enhanced killing of cancer cells by T cells.
In the present study, the reaction between polyphenolic boronates and reactive oxygen/nitrogen species released the parent polyphenolic compounds in situ. When MTD is released, it may inhibit iNOS and nitric oxide generation in the TME, thus decreasing inflammation. Figure 9 shows the antioxidant potentials of Mito-HNK-B/Mito-MGN-B and HNK-B/MGN-B in the TME, especially the direct and nearly stoichiometric scavenging of ONOO − . The compound 3-(aminocarbonyl) furoxan-4-yl) methyl salicylate (AT38) was used to enhance the efficacy of immunotherapy in pancreatic cancer [40]. AT38 inhibited the expression of arginase-1 and NOS-2 (or iNOS) in myeloid cells in the TME [41]. AT38 mitigated chemokine nitration, promoting intra-tumoral infiltration of T cells. It is not clear if AT38 had any effect on NOX2 expression or on formation of superoxides and H 2 O 2 . Boronate-based drugs are likely to be more potent scavengers of ONOO − (and HOCl), even in the presence of cellular reductants such as glutathione. Detection of the minor nitrated or chlorinated product from polyphenolic boronates can serve as a very reliable and diagnostic marker product of ONOO − or HOCl generated in the TME. Preliminary results obtained from our laboratory indicate that Mito-HNK inhibits oxidant formation from NOX2 (Zielonka J and Kalyanaraman B, unpublished data). At present, the inhibitory effects of Mito-HNK or Mito-MGN or their boronate analogs on iNOS and NOX2 expression in cells are not known.

Mitochondria-Targeted Boronates as Radiosensitizers
Boronated agents (e.g., boronophenylalanine) are used to enhance the therapeutic ratio in neutron treatment of cancers. This BNCT modality involves selective delivery of boron-10 to tumors followed by irradiation with neutrons. During this process, lithium-7 and an alpha particle that are generated destroy the tumor tissues [1][2][3][4][5]. BNCT has been used clinically to treat cancers, including glioblastoma multiforme, head and neck cancers, lung cancer, and breast cancer. A critical requirement for enhanced therapeutic success is selective localization of boron-containing compounds in tumor tissues. Mitochondriatargeted boronates (Mito-HNK-B and Mito-MGN-B) that are selectively sequestered in tumor mitochondria could be more effective in capturing neutrons in mitochondria of tumors and enhance tumor killing.
Inhibition of mitochondrial respiration could alleviate tumor and TME hypoxia and act as radiation sensitizers. Mitochondria-targeted metformin and X-radiation sensitized the killing of pancreatic cancer cells [46,63]. Clinical trials are ongoing with atovaquone and radiation therapy [64]. Tumor hypoxia inhibits the efficacy of radiation and immunotherapies in cancer. Targeting mitochondrial metabolism in non-small cell lung cancer patients was reported to modify both the tumor and TME through a decrease in hypoxia and hypoxic gene expression [65,66]. Mitochondria-targeted drugs decrease tumor hypoxia (or increase tumor oxygenation) by inhibiting mitochondrial respiration in tumors. Bloodbrain-barrier-permeable aromatic boronates were effective in treating brain tumors using neutron capture therapy [1,6]. Although it is not known whether mitochondria-targeted polyphenolic boronates (Mito-MGN-B and Mito-HNK-B) are blood-brain-barrier permeable, Mito-HNK inhibited metastasis of lung tumors to the brain in mice xenografts and showed blood-brain-barrier permeability [43].

Mitochondria Metabolism and Racial Disparity in Cancer
Recent publications suggest that race and ethnicity are factors that affect mitochondrial metabolism in cancer cells, and that mitochondrial metabolism in white cancer patients is considerably different than in patients from other racial and ethnic groups [67][68][69][70]. Studies have revealed distinct differences in the TME in Black cancer patients. In Black patients with bladder cancer, mitochondrial metabolism is considerably higher [71]. Although more studies are needed to fully substantiate this trend, the differential metabolism provides a compelling rationale for testing mitochondria-targeted drugs in Black patients with bladder cancer.

Mitochondria-Targeted Boronates as Radiosensitizers
Boronated agents (e.g., boronophenylalanine) are used to enhance the therapeutic ratio in neutron treatment of cancers. This BNCT modality involves selective delivery of boron-10 to tumors followed by irradiation with neutrons. During this process, lithium-7 and an alpha particle that are generated destroy the tumor tissues [1][2][3][4][5]. BNCT has been used clinically to treat cancers, including glioblastoma multiforme, head and neck cancers, lung cancer, and breast cancer. A critical requirement for enhanced therapeutic success is selective localization of boron-containing compounds in tumor tissues. Mitochondria-tar- The existence of racial and ethnic disparities has been identified in the breast cancer microenvironment [72][73][74]. The TME of Black cancer patients with breast cancer exhibits in-creased level pro-tumorigenic factors (e.g., macrophages, T regs , exhausted T cells) compared with white counterparts [67,68,72,75]. Upregulation of OXPHOS genes was detected in tumor samples isolated from Black cancer patients [76]. Tumors obtained from Black cancer patients have more mitochondria and PGC-1α (proliferation-activated receptor gamma coactivator 1-alpha) [69]. Identification of biomarkers (e.g., hypoxia-induced genes) in these patients may provide additional insights [65]. Black cancer patients were reported to exhibit a higher level of pro-inflammatory cytokines [76]. Clinical trials targeting mitochondrial metabolism in cancer should include Black patients and patients from diverse racial and ethnic backgrounds. Presently, metformin is used as the sole drug of choice to test the mechanistic role of mitochondria in cancer disparity studies [67,77]. Metformin is one of the most prescribed drugs for treating diabetes. Despite increased safety, its bioavailability is poor. Clinical trials revealed that Black cancer patients respond better to mitochondrial OXPHOS inhibitors (e.g., metformin) than white cancer patients [67,77]. The present study opens the possibility of testing a new class of mitochondria-targeted drugs that target both the tumor mitochondria and TME to treat Black cancer patients.

Conclusions
The development of drugs that target both the tumor mitochondria and the TME would be a value-adding therapeutic advancement [78][79][80][81][82]. Higher expression of OXPHOS in triple-negative breast cancer patients who received neoadjuvant chemotherapy was associated with worse treatment outcome [83]. In this study, we developed a novel class of mitochondria-targeted polyphenolic boronates that inhibit tumor cell proliferation and scavenge oxidants such as H 2 O 2 , HOCl, and ONOO − that are generated in the immunosuppressive TME. During this reaction, immunoactive parent polyphenols are regenerated. Specific polyphenols include HNK and MGN, which are active components of magnolia plant extract. Borono-L-phenylalanine was used to treat brain cancer in humans, so it is likely that polyphenolic boronates and the mitochondria-targeted boronate analogs are nontoxic and have high clinical and translational potential. We hope that the redoxbased mitochondria-targeted polyphenolic boronates will be tested in the appropriate mice models (e.g., KPC mice) to assess their ability to inhibit reactive oxygen species/reactive nitrogen species-mediated nitration in the TME.           Figure A4. NMR spectra of Mito-HNK-B. Figure A4. NMR spectra of Mito-HNK-B.  Figure A5. NMR Spectra of MGN-B. Figure A5. NMR Spectra of MGN-B.