A photo-inducible DNA cross-linking agent with potent cytotoxicity and selectivity toward triple negative breast cancer cell line

Abstract

DNA interstrand cross-links (ICLs) are the sources of the cytotoxicity of many anticancer agents. Selenium compounds showed great potential as anticancer drugs. In this work, we synthesized a binaphthalene analog 1 containing phenyl selenide (-SePh) as the leaving group and investigated its photochemical reactivity towards DNA as well as cytotoxicity and selectivity. DNA ICLs were not observed with binaphthalene phenyl selenide 1 without UV irradiation, while ~15% DNA ICL products were detected with UV irradiation, indicating a photo-responsive property of 1. The trapping reactions with TEMPO and MeONH₂, respectively, suggested that free radicals and carbocations are involved in the DNA cross-linking process induced by photoirradiation of 1. The photochemical reactivity of 1 toward DNA was sequence-dependent. DNA interstrand cross-linking occurred mainly at dG/dC base pairs, while mono-alkylations occurred at dGs and dAs. Additionally, we have demonstrated that 1 alone without UV irradiation did not inhibit cancer cell growth even with a concentration of 100 μM, while the cytotoxicity of 1 toward cancer cells was significantly enhanced upon 350 nm irradiation with an IC₅₀ of 1.7 μM. No cytotoxicity was observed towards normal epithelial MCF 10A cells, regardless of UV exposure, in the presence or absence of 1. The alkaline comet assay suggested that the photo-induced cytotoxicity of 1 is correlated to cellular DNA damage. Normal cells showed higher levels of GSH than cancer cells and exhibited efficient DNA repair mechanisms, which can both prevent and repair potential DNA damage induced by 1, contributing to the selective cytotoxicity of the prodrug towards triple negative breast cancer cells.

Graphical Abstract

INTRODUCTION

Bifunctional DNA alkylating agents can react with nucleotides of two complementary oligodeoxynucleotide (ODN) strands, forming DNA interstrand cross-link (ICL), which prevents separation of DNA double helix, thus blocking replication and transcription and finally leading to the cell death.^1-2 Among various DNA ICL agents, compounds that can be activated by UV irradiation to alkylate DNA have received significant attention due to their low toxicity and high selectivity. Various photo-activated ICL agents have been developed, such as coumarin,^3-4 psoralen,^5-6 3-cyanovinylcarbazole analogs,^7-8 furan moiety,⁹ p-stilbazole,¹⁰ naphthalene compounds,^11-14 bifunctional benzene derivatives,^15-18 etc. Many showed various pharmacological activities, including anticancer, anti-microbial, antiviral, anti-inflammatory effects, etc. Among various photo-inducible DNA cross-linking agents, naphthalene derivatives have an extended π-conjugated ring system, which allows their activation by the lights (wavelength >350 nm) that are compatible to biological system. For example, the Freccero group has demonstrated that several 1,1’-binaphthalene derivatives induced DNA ICL formation via photo-generated binol-quinone methide capable of alkylating DNA.¹⁴ Some derivatives showed high photo efficiency and cytotoxicity towards cancer cells.¹⁹ Our group discovered that 350 nm irradiation of several bifunctional binaphthalene analogs could generate naphthalenylmethyl cations directly, producing DNA ICL formation via alkylation.^11-13 We further demonstrated that the efficiency for photo-induced DNA ICL formation by naphthalene analogs strongly depends on the leaving groups as well as aromatic substituents.^{11, 13, 20}

Selenium has attracted significant attention since it was first discovered in 1818.²¹ Organoselenium compounds play essential biochemical roles in living organisms. For example, selenium-containing amino acids act as direct and indirect antioxidants with great potential use in therapeutics.^22-23 Many selenium-based compounds showed selective anticancer effects without interfering with the normal cells, such as decreasing the angiogenesis that may induce the cancers, activating the tumor suppressor protein P53, and preventing tumor development.^24-26 The organoselenium compounds are more bioavailable and less toxic than inorganic selenium.^27-29 Selenide compounds can be selected as H₂O₂ acceptors to develop reactive oxygen species (ROS)-responsive prodrugs.^{21, 30} Mao group developed seven ROS-responsive theranostic prodrugs with allyl phenyl selenides which can be further oxidized to selenoxides to release active drug camptothecin (CPT).³¹ In addition, the research groups of Greenberg and Zhou discovered that phenyl selenide-modified nucleosides can be activated upon oxidation, resulting in DNA interstrand cross-linking.^32-34 Recently, Sun and his colleagues observed that phenyl selenide-based precursors and explored their activities as H₂O₂-inducible DNA ICL agents.³⁵ These selenide precursors are oxidized to the selenoxide, releasing benzeneselenenic acid leaving group and promoting the DNA ICL formation. They later synthesized DNA ICL precursors containing photo-responsive coumarin moiety and H₂O₂ inducible phenyl selenide group.³⁶ These precursors generated reactive quinone methides (QMs) cross-linking DNA and exhibited potent cytotoxicity towards cancer cells. In this work, we designed and synthesized binapthalene phenyl selenide 1, studied its photochemical reactivity towards DNA, investigated the mechanism of DNA ICL formation induced by 1 and determined its alkylation sites on DNA duplexes. Finally, we examined the cytotoxicity of phenyl selenide 1 toward MDA-MB-468 cancer cells with or without UV irradiation and evaluated the cellular DNA damage caused by 1.

EXPERIMENTAL PROCEDURES

General Information.

Reagents and chemicals were commercially purchased and used without further purification. H₂O₂ was purchased from Sigma-Aldrich^® and used directly with dilution. The ¹H and ¹³C spectra were obtained with Bruker DRX 300 or 500 MHZ spectrophotometer and the HRMS spectra was collected using LCMS-IT-TOF or QTOF mass spectrometer at the Shimadzu Analytical Instrumentation Laboratory and Research Center (SAILARC) at the University of Wisconsin-Milwaukee, Milwaukee, WI. The oligonucleotides (ODNs) were synthesized by DNA/RNA ABI synthesizer (Model. 394 from AZCO^® BioTech. Inc.), deprotected and further purified. ODNs were labeled with [γ-³²P] ATP following standard procedure³⁷ and hybridized under 90 °C for 5 min and cooled to r.t. to form 49-mer DNA duplex 3, 21-mer DNA duplex 4 and 24-mer duplex 5. The 350 nm irradiation was carried out with Model RPR-100 Rayonet^© irradiator, and the cross-linking yields were determined by denaturing polyacrylamide gel electrophoresis (PAGE) with phosphorimager analysis (Image Quant 5.2). The triple negative breast cancer cell line MDA-MB-468 and normal epithelial cell line MCF 10A were obtained from the American Type Culture Collection (ATCC). MDA-MB-468 cells were cultured in L-15 Leibovitz media (Thermo Scientific: 41300070) supplemented with non-essential amino acids (100X solution, Hyclone: SH30238.01, 5.0 mL), fetal bovine serum (Biowest: S1620, 50 mL), penicillin and streptomycin (100X solution, Hyclone: SV30010, 5.0 mL) at 37 °C with 100% air at 100% relative humidity. MCF 10A cells were maintained with Lonza media kit (MEGM: CC-3150) supplemented with 100 ng/mL Cholera toxin in 5% CO₂ incubator at 37 °C. The animal studies were conducted according to the Institutional Animal Care and Use Committee (IACUC) Protocol 22-23 #21 and Biosafety Protocol # B22.028, approved by the Institutional Biosafety Committee at the University of Wisconsin Milwaukee (UWM).

Synthesis of ((4,4'-dibromo-[1,1'-binaphthalene]-3,3'-diyl)bis(methylene))bis(phenylselane) (1).

Diphenyl diselenide (0.62 g, 2 mmol) and sodium borohydride (0.15 g, 4 mmol) were dissolved in anhydrous DMF (5 mL) and the mixture was stirred at r.t. for 10 min under nitrogen. A solution of 2 prepared as previously reported¹¹ (0.50 g, 1 mmol) in DMF (10 mL) was added dropwise by syringe in the mixture, which was further stirred at r.t. overnight. The reaction mixture was quenched with water (20 mL) and then extracted with EtOAc (25 mL) three times. The organic phase was collected, washed with brine, and dried over Na₂SO₄. Next, the organic phase was concentrated by vacuum to get the crude product, which was further purified by column chromatography (Hexane/CH₂Cl₂ = 20/1) over silica gel to obtain 1 as a white solid with 83% yield. m.p. 99-101°C. ¹H NMR (500 MHz, CDCl₃): 8.46 (d, J = 8.5 Hz, 2H), 7.61 (t, J = 7.5 Hz, 2H), 7.55 (d, J = 7.0 Hz, 4H), 7.32 (t, J = 7.0 Hz, 2H), 7.25-7.18 (m, 8H), 7.03 (s, 2H), 4.50 (q, J = 11.5 Hz, 4H). ¹³C NMR (125 MHz, CDCl₃): δ 136.8, 136.3, 135.0, 132.6, 132.6, 129.8, 129.4, 129.1, 128.0, 127.7, 127.6, 126.8, 126.6, 124.3, 34.2. HRMS (ESI): m/z [M+H]⁺ calcd for C₃₄H₂₄Se₂Br²⁺: 750.86480, found: 750.85995.

DNA ICL Formation.

A certain amount of 1 was dissolved in CH₃CN to make the compound solutions with different concentrations (0.1/3 mM-10/3 mM). The ³²P-labeled ODN duplex (0.5 μM, 2 μL) was mixed with 1 M NaCl (2 μL), 100 mM potassium phosphate buffer (pH 8, 2 μL), autoclaved H₂O (8 mL), and certain concentrations of 1 in CH₃CN (6 μL) to make a reaction solution of 20 μL (final concentration range of 1: 0.01-1.0 mM). Then, the reaction mixture was irradiated upon 350 nm UV light, quenched by 20 μL of 90% formamide loading buffer, and subjected to 20% denaturing PAGE.

Free-Radical-Trapping Assay of DNA ICL Formation.

A certain amount of TEMPO was dissolved in CH₃CN and diluted to the solutions with different concentrations (10/3 mM-4000/3 mM). Then, 100 mM potassium buffer (pH 8, 2 μL), 1 M NaCl (2 μL), H₂O₂ (4 mM, 2 μL), TEMPO (3 μL) and 1 in CH₃CN (2/3 mM, 3 μL) were added to the ³²P-labeled ODN duplex (2 μL, 0.5 μM). 6 μL of autoclaved distilled water was added in the mixture to give a total volume of 20 μL (final TEMPO concentration: 0.5 mM-200 mM; final concentration of 1: 0.1 mM; final concentration of H₂O₂: 0.4 mM). The reaction mixture was irradiated with 350 nm light for 10 h, quenched with 20 μL of 90% formamide loading buffer, and subjected to 20% denaturing PAGE.

Carbocation Trapping Assay of DNA ICL Formation.

A solution of MeONH₂·HCl (2 M) was titrated with NaOH (5 M) to pH 7.0, which was diluted to the desired concentration (10/2 to 4000/ 2 mM). Next, 100 mM potassium buffer (pH 8, 2 μL), 1 M NaCl (2 μL), H₂O₂ (4 mM, 2 μL), methoxyamine solution (2 μL) and 1 in CH₃CN (1/3 mM, 6 μL) were added to the ³²P-labeled ODN duplex (2 μL, 0.5 μM). 4 μL of autoclaved distilled water was added in the mixture to give a total volume of 20 μL (final methoxyamine concentration: 0.5 mM-200 mM; final concentration of 1: 0.1 mM; final concentration of H₂O₂: 0.4 mM). The reaction mixture was irradiated with 350 nm light for 10 h, quenched with 20 μL of 90% formamide loading buffer, and subjected to 20% denaturing PAGE.

Determination of Alkylation Sites by Studying the Heat Stability of DNA ICL Products Formed with Phenyl Selenide 1.

The reaction mixture with ³²P-labeled ODN duplexes (0.5 μM, 60 μL), NaCl (1 M, 12 μL), pH 8 potassium phosphate (100 mM, 12 μL), and 1 in CH₃CN (5/3 mM, 36 μL) was irradiated with 350 nm light for optimal times. Then, the reaction mixture (120 μL) was coprecipitated with calf thymus DNA (25 μg/mL, 40 μL) in the presence of NaOAc (3 M, 20 μL) and ethanol (540 μL) at −80 °C for 30 min and centrifuged at 15000 rpm for 5 min by Eppendorf^® centrifuge. Next, the supernatant was removed, and the pellet was dissolved in autoclaved distilled water (60 μL), precipitated again with NaOAc (3 M, 10 μL) and ethanol (240 μL) at −80 °C for 30 min, and centrifuged for 5 min at 15000 rpm. After removal of the supernatant, the pellet was lyophilized, dissolved in autoclaved distilled water (25 μL), and aliquoted into two parts. One portion (20 μL) of the precipitated ODN adducts was incubated with 10 M piperidine (10 μL) and autoclaved distilled water (70 μL) at 90 °C for 30 min. The second portion (5 μL) was used as a control sample. Finally, two samples were dissolved in the 90% formamide loading buffer and H₂O (1:1) mixture and subjected to 20% denaturing PAGE analysis.

Heat Stability Study of the Isolated DNA Cross-Linking Products Formed with 1 for Determining Alkylation Sites.

A reaction mixture containing ³²P-labeled ODN duplex 3 (0.5 μM, 60 μL), NaCl (1 M, 12 μL), pH 8 potassium phosphate (100 mM, 12 μL), and 1 in CH₃CN (5/3 mM, 36 μL) was irradiated at 350 nm for 10 h. The ICL products and single-stranded ODNs were purified by 20% denaturing PAGE following standard procedures. The gel band containing cross-linked products or single-stranded ODNs was cut, crushed, extracted with a mixture of NaCl (200 mM) and EDTA (20 mM) (2.0 mL), and desalted with a C18 column (1 cc, 100 mg) eluting with H₂O (1.0 mL) for three times, followed by MeOH/H₂O mixture (3/2, 1.0 mL). The isolated ICL products and single-stranded ODNs were lyophilized by a Centrivap concentrator of LABCONCO^®, then dissolved in 25 μL of autoclaved distilled H₂O and aliquoted into two parts of 20 μL and 5 μL, respectively. One portion (20 μL) of the isolated ODNs was incubated with 10 M piperidine (10 μL) and autoclaved distilled water (70 μL) at 90 °C for 30 min. The second portion (5 μL) was used as a control sample. After removal of the solvent by vacuum, the reaction residue was dissolved in the mixture of 90% formamide loading buffer and H₂O (1:1), then subjected to 20% denaturing PAGE analysis.

Cell Culture.

The human tumor cell lines MDA-MB-468 (ATCC Cat# HTB-132) and normal cell line MCF 10A (ATCC Cat# CRL-10317) were purchased from the American Type Culture Collection. MDA-MB-468 cells were cultured in L-15 Leibovitz media (Thermo Scientific: 41300070) supplemented with 10 % fetal bovine serum (Biowest: S1620), 1% non-essential amino acids (NEAA 100X solution, HyClone: SH30238.01), and 1% penicillin and streptomycin (HyClone: SV30010) at 37 °C in 100% relative humidity. MCF 10A cells were maintained in Lonza media kit MEGM (Lonza: CC-3150) supplemented with 100 ng/mL Cholera toxin. MCF 10A cells were maintained in a 5% CO₂ incubator at 37 °C.

Cytotoxicity study.

MDA-MB-468 cells and MCF 10A cells were detached and plated into 96-Well Optical-Bottom White Plates (Thermo Scientific, Nunc: 165306) in a final volume of 40 μL at a density ranging from 3,000 to 5,000 cells/well. After incubation at 37 °C for 3 h before adding the compound. Compound 1, solubilized in dimethyl sulfoxide (DMSO) at 5 mM stock. IC₅₀ determination: 5 mM stock solution was serially diluted (2-fold) in Biotix clear, sterile 96-well assay plate (VWR: 89511-214). Subsequently, 800 nL of the serially diluted compound solution (1:50 dilution) was dispensed into the cell plates using a Tecan Freedom EVO liquid handling system. The cell plates were then subjected to incubation with or without UV irradiation at 350 nm, 64 W for 30 minutes, followed by additional incubation for 48 h at 37 °C with 100% air in 100% relative humidity. Viability was assessed by adding an equal volume (40 μL) of the Celltiter-Glo reagent (Promega: PAG7572) to the assay plates, followed by a 30-minute incubation at room temperature. The luminescence was measured with an Infinite M1000 (Tecan) plate reader. The viability was measured as a percentage against the control samples. Specific dose response: 5 mM stocks were further diluted in DMSO to obtain intermediate stocks (0 μM, 250 μM, 500 μM, 1000 μM, and 2000 μM). 100 nL of diluted stocks were added to the assay plates (1:400 dilution) using the Tecan Freedom EVO liquid handling system to reach final concentration of 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM, respectively. Plates were then incubated with/without UV irradiation at 350 nm, 64 W for 30 minutes, followed by additional incubation for 48 h at 37 °C with 100% air in 100% relative humidity. Viability was assessed by adding an equal volume (40 μL) of the Celltiter-Glo reagent (Promega: PAG7572) to the assay plates, followed by a 30-minute incubation at room temperature. The luminescence was measured with an Infinite M1000 (Tecan) plate reader. The viability was determined as a percentage against the control samples.

Evaluation of cellular DNA damage by alkaline comet assay.

The alkaline comet assay was conducted following the manufacturer’s instructions (Abcam: ab238544). Triple negative breast cancer cells (MDA-MB-468) and normal epithelial cells (MCF 10A) were seeded in 6-well Tissue culture, surface treated, sterile, clear plates (VWR: 10062-892) at a cell density of 10⁵ cells. Upon reaching 90% confluency (over 3 days), cells were treated under various conditions for 48 h (vehicle, vehicle + 350 nm irradiation, 1 alone or 1 + 350 nm irradiation). Subsequently, cells were detached using a cell scraper, washed with ice-cold PBS buffer (without Mg²⁺ and Ca²⁺), and diluted with ice-cold PBS buffer to obtain 1 x 10⁵ cells/mL concentration. The cell suspension (20 μL) was then gently mixed with warm agarose (180 μL, 37 °C) and spread onto pre-warmed glass slides (150 μL/well), which were kept at 4 °C for 30 minutes in the dark to solidify the agarose. The slides were then immersed in lysis buffer at 4 °C for 2 hours in the dark to disrupt the cell membrane, followed by incubation in pre-chilled alkaline unwinding solution (300 mM NaOH, 1 mM EDTA) at 4 °C for 30 minutes in the dark to denature DNA. Horizontal electrophoresis was conducted in pre-chilled alkaline electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH > 13) at 35 V for 30 minutes. The slides were removed and immersed in pre-chilled DI water twice for 2 mins then in 70% ethanol for 5 minutes. After air-drying in the dark for 1 h, 100 μL/well of the diluted Vista Green DNA dye was added to each well, and the slides were allowed to sit at rt for 15 mins. DNA damage was analyzed with an EVOS Digital Inverted Microscope at 20X magnification. DNA damage was quantified using Tritek CometScore Software.

Preparation of RNA Samples and RT-qPCR.

MDA-MB- 468 and MCF10A cells were seeded in 6-well tissue culture plates (VWR: 10062-892). Once the cells reached 90% confluency (~1 million cells/well), they were treated with either vehicle or compound 1 at concentrations of 3 μM and 10 μM, with or without 30 mins of 350 nm irradiation, followed by 48 hours of incubation. After treatment, the cells were harvested and resuspended in 350 μL of RLT buffer in the presence of 1% β-mercaptoethanol. Cell lysis was performed with QIAshredder (Qiagen) spin columns, and the total RNA was isolated and purified using an RNAeasy kit (Qiagen). The RNA concentration and purity were assessed by measuring absorbance (260 nm/280 nm) using an Infinite M1000 (Tecan) plate reader. Quantified RNA was reverse transcribed into cDNA using qScript One-Step SYBR Green qRT-PCR Kit (QuantaBio: 95087-200). The primers used in this study were as follows: GAPDH forward primer (FP) 5′-ACCACAGTCCATGCCATCAC-3′, GAPDH reverse primer (RP) 5′-TCCACCACCCTGTTGCTGTA-3′; XPC FP 5′-TAAAGGGGTCCATGAGGACACA-3′, XPC RP 5′-CTGGCTGGCTGCAGATGTTA-3′; RAD51 FP 5′-GGGAAGACCCAGATCTGTCA-3′, RAD51 RP 5′-ATGTACATGGCCTTTCCTTCAC-3′, BRCA1 FP 5′-GCTTGACACAGGTTTGGAGTATGC-3′, BRCA1 RP 5′-GAGAGTTGGACACTGAGACTGGTT-3′, ATM FP 5′-TACCAAGCAGCATGGAGGAA-3′, ATM RP 5′-GATTCATGGTAACTGGTTCCTTCTAC-3′; ATR FP 5′-CATTCCAAAGCGCCACTGAA-3′, ATR RP 5′- CGCTGCTCAATGTCAAGAACA-3′; POLB FP 5′-AAGAAATTGCCTGGAGTAGGAACA-3′, POLB RP 5′-CAGATGGACCAATGCCACTAACT-3′; POLD1 FP 5′-AGCTGGTGGAGTCTAAGTACAC-3′, POLD1 RP 5′-GACGGAGTCAGTGTCACCATA-3′; MSH2 FP 5′-CCAGCAGCAAAGAAGTGCTA-3′, MSH2 RP 5′-TGTTTCACCTTGGACAGGAAC-3′; FANCG FP 5′-GGCCAGCTTCACCCTTCCTA-3′, FANCG RP 5′-CACCAATCTCACCAGTCCAG-3′; The cDNA synthesis and amplification was carried out using a Eppendorf Mastercycler in a 96-well twin.tec real-time PCR plate (Eppendorf, Cat. No. 951022015) with a total reaction volume of 20 μL. Each reaction contained 5.75 μL of 2x SybrGreen Mix, 10 μL of RNase-free water, 1 μL of each primer, 0.25 μL of Quantifast RT, and 3 μL of RNA (50 ng). The specificity of the target genes was confirmed through melting curve analysis. Gene expression levels were normalized to GAPDH for each sample, and relative gene expression was determined using the 2^−ΔΔCt method (Livak and Schmittgen).³⁸ The standard deviations were calculated based on three independent biological replicates, each performed in triplicate.

GSH/GSSG assay.

GSH/GSSG ratios were determined using a GSH/GSSG-Glo Assay (Promega: V6611) based on the manufacturer’s protocol. Briefly, MDA-MB- 468 and MCF10A cells were plated into 96-Well Optical Bottom White plates (Thermo Scientific, Nunc, 165306) in a final volume of 100 μL at a cell density 10000 cells/well, and allowed to grow overnight at 37 °C. A solution of 1 in DMSO (1 μL, desired concentrations) was added to each wells to reach final concentrations of 0 μM, 2 μM and 10 μM. The cells were irradiated with 350 nm wavelength for 30 minutes, followed by 48 h incubation at 37 °C. Then, the media was removed, and cells were lysed using 50 μL of freshly prepared Total Glutathione Lysis Reagent or Oxidized Glutathione Lysis Reagent. The cell plates were shaken for 10 minutes on a plate shaker, then 50 μL of Luciferin Generation Reagent was added. After 30 min incubation at rt, 100 μL of Luciferin Detection Reagent was added, followed by 15 min incubation at rt. The luminescence was then measured with an Infinite M1000 (Tecan) plate reader. The GSH/GSSG ratios were determined using net luminescence values against vehicle treated cells. Glutathione standard curve: The 5 mM glutathione solution was diluted in water to get 320 μM stock solution. A series of GSH standards were prepared by serial two-fold dilutions of 320 μM glutathione. 5 μL of each dilution was added to duplicate wells containing 50 μL of freshly prepared Total Glutathione Lysis Reagent. After shaking, 50 μL of Luciferin Generation Reagent was added. After 30 min incubation at rt, 100 μL of Luciferin Detection Reagent was added to each well, followed by 15 min incubation at rt. Then luminescence was measured with an Infinite M1000 (Tecan) plate reader. 5 μL of 320 μM GSH corresponds to a final concentration of 16 μM. The linear portion of this curve (0-4 μM GSH) was used for calculations.

In vivo Safety Study.

Female CD1 mice (Charles River Laboratory) were used for safety study. The animals were housed in a pathogen-free environment with regulated humidity and temperature and exposed to a controlled 12-hour light and dark cycle with free access to food and water. The study adhered to the guidelines set by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin–Milwaukee. To evaluate the safety of 1, repeated dose safety study was performed with female CD1 mice (three mice per group). Compound 1 was formulated in a mixture of DMSO (10%), Ethanol (10%), PEG 400 (50%), and Olive oil (30%) and was administered intraperitoneally (IP).³⁹ The maximum volume for IP injection was 300 μL. The prodrug (administered at doses of 0 mg/kg, 5 mg/kg, 10 mg/kg, and 20 mg/kg via IP injection) was given to female CD-1 mice five days a week for three weeks, with two days of monitoring each week (no treatment). The mice were then observed for an additional week to assess post-treatment toxicity effects.

RESULTS AND DISCUSSION

Synthesis of Phenyl Selenide Analogue.

The binaphthalene phenyl selenide analog 1 was synthesized from 4,4’-dibromo-3,3’-bis(bromomethyl)-1,1'-binaphthalene (2), which was prepared as we previously described.¹¹ Compound 2 was treated with diphenyl diselenide ((PhSe)₂) in the presence of sodium borohydride (NaBH₄) in dimethylformamide (DMF), yielding the phenyl selenide analog 1 in an 83% yield (Scheme 1). The compound was confirmed by ¹H, ¹³C NMR, and HRMS (Figures S6 and S7).

Scheme 1.

Synthesis of binaphthalene phenyl selenide analog 1 (compound 2 was prepared as previously reported^{11, 20}).

Photo-Induced DNA Interstrand Cross-link Formation.

The photochemical reactivity of phenyl selenide 1 was investigated with a 49-mer DNA duplex (3) by evaluating DNA interstrand cross-link (ICL) formation. The reaction mixture containing selenide 1 and duplex 3 was irradiated with 350 nm light in a Rayonet^® Model RPR-100 irradiator. The DNA ICL efficiency was determined by denaturing polyacrylamide gel electrophoresis (PAGE) with phosphorimager analysis (Image Quant 5.2). No ICL products were observed with 1 alone, suggesting that 1 is not reactive toward DNA, while 350 nm irradiation of 1 (0.1 mM) led to ~16% DNA ICL product (Figure 1(A)). These data suggested that 1 can act as a photo-inducible DNA cross-linking agent. Time-dependent DNA cross-linking study indicated that the optimal reaction time was 22 h when the highest ICL yield (17 ± 3%) was achieved (Figure 1B). We also observed that the concentration greatly affected the DNA ICL formation induced by 1 upon photoirradiation. The highest ICL yield was observed with 0.5 mM 1 (~26%) (Figures S2). However, 1 with > 0.1 mM concentrations precipitated out in aqueous solution, causing a non-homogenous phase of the reaction mixture, which lead to erratic yields of DNA ICL formation. Precipitated 1 might re-dissolve in the reaction mixture during long-time irradiation, resulting in more DNA ICL formation. In addition, DNA damage was observed when the concentrations were higher than 0.5 mM. Therefore, we concluded that 0.1 mM was the optimal concentration of selenide 1, which photo-induced 17 ± 3% DNA ICL formation (error was determined with several repetitive trials).

Figure 1.

DNA interstrand cross-link study of phenyl selenide 1. (A) Photo-induced DNA ICL formation upon ~22 h irradiation. Lane 1: DNA without 1; lane 2: DNA with 0.1 mM 1 without UV irradiation; DNA with 0.1 mM 1 (~16 %); (B) time-dependent ICL formation of duplex 3 with 0.1 mM 1. The study was carried out with 0.05 μM DNA duplex 3 in a 10 mM pH = 8 phosphate buffer upon 350 nm irradiation. All ICL yield was presented as average ± deviation from duplicate experiment samples.

Sun’s group recently showed that H₂O₂ significantly enhanced DNA ICL formation induced by several bisphenylselanylmethyl benzene analogs that can act as light and H₂O₂ dual-responsive DNA cross-linkers.^35-36 However, such a phenomenon was not observed with selenide 1. The yields of DNA ICL formation in the presence of H₂O₂ were close to those obtained without H₂O₂ (Figure S3). In order to test the chemical reactivity of selenide 1 with H₂O₂, we performed a monomer reaction of selenide 1 and H₂O₂, which was monitored by NMR spectroscopy. No change was observed with ¹H and ¹³C NMR peaks of 1 during 3 h incubation of 1 with H₂O₂ (Figure S4). The monomer reaction was further monitored by ¹³C NMR spectroscopy for another 19 h, which suggested no reaction occurred with 1 in the presence of H₂O₂ (Figure S4). We propose that the different effects of H₂O₂ on 1 from those described by Sun may be correlated with different structures of phenyl selenide precursors and ICL formation mechanisms. The precursors developed by Sun induced ICL formation via a quinone methide while selenide 1 may undergo a carbocation formation upon UV irradiation.

Investigation of DNA ICL Formation Mechanism.

Previous studies have demonstrated that most bifunctional aromatic compounds induce DNA ICL formation via photogenerated carbocations.^17-18 Two pathways were proposed for the carbocation generation: homolysis of C-X bond (X: leaving group) to generate free radicals spontaneously oxidized to carbocation or direct heterolysis of C-X bond.^17-18 In this work, we investigated the mechanism for the photo-induced DNA ICL formation induced by selenide 1. As previously described,¹¹ 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) was used as a radical trapper and methoxyamine (MeONH₂) as a carbocation trapper. Both TEMPO and MeONH₂ suppressed photo-induced DNA ICL formation induced by 1, as shown by greatly decreased ICL yields (Figure 2 and S5). The yields finally dropped to ~3.6% with 200 mM TEMPO and ~ 3.5% with 200 mM MeONH₂, indicating the inhibition of radical or carbocation formation. Similar to the previous work,¹¹ the decreased ICL yields caused by the trappers suggested a mechanism that the photo-irradiation of 1 generated the carbocation via homolysis of C-X bond transfer (Scheme 2).

Figure 2.

The effect of TEMPO and MeONH₂ on DNA interstrand cross-linking induced by 1. A mixture of 3 (50 nM) and 1 (0.1 mM) in a pH 8 phosphate buffer was irradiated with 350 nm light for 10 h.

Scheme 2.

Proposed mechanism for photo-induced DNA ICL formation induced by 1.

Determination of the ICL Reaction Sites of Selenide 1.

Upon heating in basic condition, the N7-alkylated purine nucleosides undergo deglycosylation, leading to DNA strand break and revealing the alkylation site on purines.^40-41 In the current work, we first irradiated duplex 3 with 0.5 mM 1 (at 350 nm) for 10 h to obtain ICL products and the alkylated single-stranded oligonucleotides (ssODN) 3a′. Then, we isolated the ICL products and ssODN 3a′ by denaturing PAGE and heated them in 1 M piperidine at 90°C for 30 min. The phosphor-image autoradiogram of 20% denaturing PAGE analysis showed that DNA cleavage occurred at A14, A15, A18, G22, A24, A25, and G27 for ssODN 3a′, suggesting that the monoalkylation induced by 1 occurred at both dAs and dGs (Figure 3, lane 4). However, for DNA ICL products, strand cleavage only occurred at dG sites, not at dA sites (Figure 3, lanes 2), indicating that DNA ICL formation induced by 1 mainly occurred at dGs but not at dAs.

Figure 3.

Determination of the cross-linking sites of 1 with DNA duplex 3. Phosphorimage of a 20% denaturing PAGE analysis of the isolated DNA interstrand cross-linking products and single-stranded ODN 3a' induced by 1: lane 1, isolated ICL product without piperidine treatment; lane 2: DNA ICL product in 1.0 M piperidine at 90 °C for 30 min; lane 3: isolated single-stranded ODN 3a' without piperidine treatment; lane 4: single-stranded ODN 3a' in 1.0 M piperidine at 90 °C for 30 min; lane 5: G/A sequencing.

We studied the sequence effect on the ICL efficiency induced by 1 to provide further evidence for the cross-linking reaction sites. DNA duplexes 4 and 5 were designed to determine if the ICL reaction takes place with AT sequences or only with duplexes containing GC sequences. Duplex 4 contains dCs/dTs in one strand 4a and dAs/dGs in the other strand 4b. Duplex 5 is a self-complementary duplex containing dAs and dTs only. The DNA cross-linking reactions with duplexes 4 or 5 were performed under optimized conditions. As expected, DNA ICL product was observed with duplex 4 (Figure 4, lanes 1 and 2) but not 5 (Figure 4, lanes 5 and 6), suggesting that DNA cross-linking reaction did not occur with AT base pair but only with GC base pair. Heat stability study indicated that alkylation took place at the dG sites but not at dA sites for duplex 4 (Figure 4 lane 3). Although no ICL products were observed with self-complementary duplex 5, the strand cleavages were detected with both dA and dT sites upon heating in 1 M piperidine at 90 °C for 30 min (Figure 4, lane 7). These data suggested that mono alkylation could take place with dA sites. The cleavage at dTs might be caused by free radicals that induce the formation of alkaline-labile lesions,⁴² which was consistent with the proposed mechanism. This phenomenon was not observed with 49-mer duplex 3, suggesting the photo-reactivity of binaphthalene phenyl selenide 1 is sequence-dependent.

Figure 4.

The determination of the alkylation sites of 1 with DNA duplexes 4 and 5. A mixture of 4 (10 μM) or 5 (10 μM) and 1 (0.5 mM) in phosphate buffer (pH 8) was irradiated with 350 nm light for 10 h. Lane 1: DNA duplex 4 without treatment; lane 2: DNA duplex 5 with precipitation; lane 3: DNA duplex 4 with precipitation and piperidine treatments; lane 4, G/A sequencing of DNA duplex 4; lane 5, DNA duplex 5 without treatment; lane 6: DNA duplex 5 with precipitation; lane 7: DNA duplex 5 with precipitation and piperidine treatments; lane 8: A sequencing of DNA duplex 5.

Cell cytotoxicity assay.

After demonstrating that 1 is responsive to photo-irradiation, we investigated its cytotoxicity towards normal epithelial cellss (MCF 10A) and triple-negative breast cancer cells (MDA-MB-468), two cell lines used for our previous study.²⁰ The cells were treated with or without 350 nm irradiation (64 W) for 30 min followed by 48 h incubation at 37 °C. Without 350 nm irradiation, 1 did not show any cytotoxicity towards both normal cells and cancer cells, even at a concentration of 100 μM (Figure 5). However, UV irradiation significantly enhanced the cytotoxicity of 1 towards cancer cells but spared normal cells, with an IC₅₀ of 1.7 μM and 170 μM for MDA-MB-468 and MCF 10A, respectively, upon 350 nm irradiation for 30 min (Figure 5A). For example, 100% cancer cell death was observed with 5.0 μM 1 or higher concentration and 90% cancer cell death with 2.5 μM 1 upon 350 nm irradiation. In comparison, a 100% survival rate was observed with 1 (all concentrations tested, 0-100 μM) without UV irradiation (Figure 5B). An IC₅₀ of 1.7 μM was observed with selenide 1 against cancer cells upon UV irradiation, which is significantly lower than without UV irradiation (> 200 μM). These data demonstrated that 1 is a potent photo-activated anticancer agent with reasonable control over toxicity, activity, and selectivity.

Figure 5.

Cytotoxicity of 1 in MDA-MB-468 and MCF 10A with or without 350 nm irradiation. Normalized data were fitted to a concentration-response equation with the indicated IC₅₀ values. Dose-response screening of 1 at concentrations of 0 - 100 μM was performed in MDA-MB-468 cancer cells and normal MCF10A cells, with or without UV irradiation for 30 minutes, followed by a 48 h incubation at 37°C. Each data point represents 3 independent replicate experiments, and the data are presented as the mean ± SD (n = 3).

Evaluation of cellular DNA damage by alkaline comet assay.

Having established that photo-irradiation greatly enhanced the anticancer activity of 1 and also induced DNA ICL formation, we propose that the photo-induced cytotoxicity of 1 may be correlated with cellular DNA damage induced by 1 upon 350 nm irradiation, driving extensive cell death. To determine cellular DNA damage, we performed an alkaline comet assay with MCF 10A cells and MDA-MB-468 cells using OxiSelect Comet Assay Kit (Cell Biolabs, Inc. Cat. STA-350). Cells were treated with 350 nm irradiation alone, 5 μM 1 alone, and 5 μM 1 plus 350 nm irradiation. We chose 5 μM of 1, a dose higher than the IC₅₀ value (1.7 μM) for this study because we observed that a concentration higher than 2X IC₅₀ value is needed to obtain detectable DNA damage (data not included). Severe DNA damage was observed with cells treated with a combination of 1 (5 μM) and 350 nm irradiation, which was indicated by the longer and more intense comet tail (60% Tail DNA) (Figure 6A). However, comet tails were not detected with MDA-MB-468 control samples (vehicle, vehicle + UV, and 1 alone) and in all MCF 10A samples, including those treated with 1 irrespective of UV irradiation, indicating no cellular DNA damage. Collectively, these results suggested that 350 nm irradiation increased cellular DNA damage induced by 1, which led to improved cytotoxicity of 1 toward cancer cells.

Figure 6.

Evaluation of cellular DNA damage induced by 1 in MCF 10A cells and MDA-MB-468 cells using alkaline comet assay. (A) The images compare comets between vehicle-treated and 1-treated (5 μM) MCF 10A and MDA-MB-468 cells with or without 350 nm irradiation (scalebar = 200 μm); (B and C): Alkaline comet assay quantification is presented as % DNA in tail (B) and tail olive moment (C). Comet images were analyzed using TriTek Cometscore Software. Each data point represents three independent replicate experiments, and the results are expressed as the mean ± SD (n = 3). The cells were incubated with selenide 1 at 37 °C for 48 h with/without UV irradiation (350 nm, 64 w, 30 min) in 6-well Tissue culture, surface treated, sterile, clear plates (VWR: 10062-892).

Molecular basis for selective cytotoxicity of 1 towards MDA-MB-468 cancer cells.

Having established that 1 showed a selective killing effect on cancer cells upon photoirradiation without harm to normal cells, we investigated the potential mechanisms for this selectivity. Because photo-irradiation of 1 induced oxidative DNA damage and DNA ICL formation, leading to base-labile cellular DNA damage selectively in cancer cells but not in normal cells, we propose that this selective action may be caused by several unique characteristics of cancer cells, such as aberrant metabolism, high oxidative stress, malfunctioning antioxidant system, and impaired DNA repair. To explore this further, we assessed oxidative stress responses and DNA repair gene expression in MDA-MB-468 cancer cells and MCF 10A normal cells.

Higher oxidative stress and reduced antioxidant defenses in MDA-MB-468 cancer cells than normal cells contribute to the selective anticancer effects of 1.

Glutathione (GSH), a natural antioxidant, plays a dual role in preventing oxidative damage and detoxifying reactive electrophile species. GSH can be oxidized to GSSG in response to ROS.⁴³ Thus, the GSH/GSSG ratio can serve as an oxidative stress marker that is useful for elucidating the prodrug’s cellular selectivity mechanism. Healthy cells typically have a high GSH/GSSG ratio, while a reduced ratio signifies elevated oxidative stress and potential cellular damage. Thus, we evaluated the GSH/GSSG ratio and GSH levels in both MDA-MB-468 and MCF10A cell lines using the GSH/GSSG-Glo Assay (Promega). MDA-MB-468 and MCF10A cells were treated with 2 μM and 10 μM of 1 upon 350 nm irradiation for 30 mins, followed by 48 h incubation at 37 °C and the addition of GSH/GSSG-Glo assay reagents. MCF10A cells showed much higher GSH/GSSG ratios and higher GSH concentration than MDA-MB-468 cells in control samples (vehicle-treated cells), which suggests that normal cells have higher basal levels of antioxidants than MDA-MB-468 cancer cells, protecting normal cells from oxidative damage (Figure 7A & 7E). The high GSH levels in normal cells may effectively neutralize the reactive intermediates generated by photo-irradiation of 1, such as free radicals and electrophiles, preventing DNA cross-linking and/or oxidative DNA damage in normal cells. This could explain why normal cells are resistant to the cytotoxic effects of 1. On the other hand, the low basal level of GSH in MDA-MB-468 cells (Figure 7E) makes these cancer cells more susceptible to 1-induced cytotoxicity. GSH/GSSG ratio further decreased in MDA-MB-468 cancer cells after treatment with 1, suggesting depletion of GSH and/or accumulation of GSSG in cancer cells in response to elevated oxidative stress and potential cellular damage caused by photoirradiation of 1 (Figure 7B). In contrast, GSH/GSSG ratio of MCF10A cells only slightly decreased (Figure 7B) and remains high after treatment with 1 (Figure 7A). These data indicate that normal cells have the capability to maintain redox homeostasis, preventing 1-induced oxidative damage to normal cells and leading to the selective anticancer effect of 1.

Fig.7.

GSH/GSSG ratios in MDA-MB-468 cancer cells and MCF 10A normal cells measured by GSH/GSSG-Glo Assay (Promega): (A) GSH/GSSG ratios in MDA-MB-468 and MCF10A cells treated under different conditions, vehicle or 1 at 2 μM and 10 μM concentrations, followed by 350 nm UV irradiation for 30 mins (n=3) and 48 h incubation at 37 °C; (B) The percentage reduction in GSH/GSSG ratio was calculated using the formula: $[(\text{Vehicle}\text{GSH}∕\text{GSSG}\text{ratio}-\text{Treated}\text{GSH}∕\text{GSSG}\text{ratio})∕\text{Vehicle}\text{GSH}∕\text{GSSG}\text{ratio}]\times 100$. (C) GSH standards were prepared by serial two-fold dilutions of 320 μM glutathione solution. Luminescence values were measured in duplicates and a linear trendline (R² = 0.9987) was fitted. Error bars represent the mean ± SD (n = 2). (D) GSSG standard curve was plotted by dividing x axis values of the GSH standard curve by two to reflect the μM GSSG concentration; Two moles of GSH are generated per 1 mole of GSSG. (E) GSH concentrations in MDA-MB-468 and MCF10A cells treated with vehicle or 1 (2 μM and 10 μM). Absolute GSH concentrations were calculated from net RLU values using standard curves, subtracting the GSSG contribution as: $(\mu \mathrm{M}\text{total GSH}\text{-}(\mu \mathrm{M}\text{GSSG}\times 2))$. Results are expressed as mean ± SD (n = 3).

Evaluation of DNA repair gene expression by RT-qPCR.

Another potential mechanism for the selective anticancer effect of 1 may lie in the differences in DNA repair machinery between MDA-MB-468 cancer cells and normal cells. The DNA damage response (DDR) plays a pivotal role in determining the effectiveness and selectivity of DNA-damaging agents used in cancer treatment, as it directly impacts how effectively cancer and normal cells can repair DNA damage induced by chemotherapy and photodynamic treatments. Photo-irradiation of 1 induces DNA damage, including ICL products and alkaline-labile DNA lesions. These lesions activate DDR pathways essential for both the cytotoxicity and selectivity of 1.⁴⁴ To explore this, we investigated the regulation of DNA repair genes in MCF10A (normal) and MDA-MB-468 (cancer) cells after treatment with 1 upon 350 nm irradiation. DNA damage can be repaired through multiple pathways, including nucleotide excision repair (NER), homologous recombination (HR), base excision repair (BER), and the Fanconi Anemia (FA), pathways that work together to remove ICLs. In contrast, mismatch repair (MMR) can correct DNA mispairing caused by alkylated bases.⁴⁵ Thus, we evaluated regulation of DNA repair genes functioning through these pathways, such as POLB, POLD1, and XPC (NER); RAD51 and BRCA1 (HR)⁴⁶; POLB and POLD1 (BER); FANCG (FA); MSH2 (MMR); and ATM/ATR (DNA damage checkpoint proteins) by quantitative reverse transcription polymerase chain reaction (RT-qPCR). MDA-MB-468 and MCF10A cell lines were treated with 30 min UV irradiation of 1, followed by 48 h incubation at 37 °C. Then, RNA was extracted and used for real-time RT-qPCR to measure the expression levels of selected DNA repair genes. Most DNA repair genes in MDA-MB-468 (cancer) cells, including RAD51, BRCA1, POLB, POLD1, MSH2, and XPC, were strongly upregulated after treatment with 1.

In contrast, these genes remain unchanged or slightly downregulated in MCF10A (normal) cells (Figure 8). Upregulation of repair genes in cancer cells may suggest a response to severe DNA damage induced by photoactivation of 1, including ICL formation and alkaline-labile DNA lesions. This finding is consistent with the results obtained with comet assay and GSH/GSSG evaluation, which revealed significant DNA damage and lower GSH levels in MDA-MB-468 cancer cells. These changes likely activate DNA repair pathways to mitigate the damage induced by 1. In contrast, these repair genes were not upregulated in normal cells after treatment with 1, suggesting less severe DNA damages were induced by 1. This may be due to the high basal levels of GSH in normal cells, which effectively neutralize reactive species produced by photo-irradiation of 1, thus preventing extensive DNA damage and avoiding activation of certain DNA repair pathways.

Fig. 8.

DNA repair gene expression level (n = 2). MDA-MB-468 and MCF10A cells were treated with vehicle and 1 (3 μM) with 350 nm irradiation; Data was normalized with relative GAPDH mRNA levels, and fold change was calculated using the 2^−ΔΔCt method of Lovak and Schmittgen.

However, different patterns were observed in the regulation of ATM, ATR, and FANCG genes between normal and cancer cells. Normal cells exhibited higher expression levels for these genes than MDA-MB-468cells. ATM and ATR are key DNA damage sensors and repair initiators, while FANCG plays a crucial role in recognizing and repairing DNA ICLs through the FA pathway. These data suggests that normal cells can efficiently sense DNA damage and activate DNA repair pathways, ensuring prompt repair of any ICLs formed. Collectively, our mechanism investigation demonstrated that normal cells have strong protective mechanisms that not only protect normal cells from DNA damage induced by photo-irradiation of 1 due to high levels of GSH but also efficiently activate DNA repair pathways, allowing repair of any DNA damage induced. This enhanced DNA repair response helps normal cells maintain genomic integrity and protect against drug-induced DNA damage, therefore mitigating the cytotoxic effects of 1.

Safety study.

We have demonstrated that the selenium analogue 1 showed low toxicity without UV irradiation, which is safe to normal cells. To further evaluate its safety, we tested its in vivo toxicity in mice. A repeated dose safety study was conducted in female CD1 mice using groups of three mice per group. To address the issue of poor aqueous solubility of 1 that brings a challenge for formulation, we used cosolvents to make an injectable solution of 1 following the formulation decision tree proposed by Lee et al.³⁹. Compound 1 was successfully solubilized in a cosolvent mixture consisting of DMSO (10%), Ethanol (10%), PEG400 (50%), and Olive oil (30%), a formulation that has been proved safe.³⁹ Toxicity study was then performed with single daily intraperitoneal (IP) injections at doses of 0, 5, 10, and 20 mg/kg over three weeks, followed by one week of post-monitoring. A lower dosage range (≤ 20 mg/kg) was selected due to the drug’s limited solubility. Mice were monitored closely for signs of illness or mortality. No mouse death was observed in any treatment group, and the pattern of mice weight change was similar to the control group over three weeks. Slight weight loss was noted after day 1 in all groups, including control mice, possibly due to the adoption of the formulation. A Mouse Intervention Scoring System from Koch et. al and Paster et. al was used to assess the health and welfare of mice in the experiments.^47-48 A scoring chart was generated based on daily observations of distress, illness, or abnormal behavior in mice, which was assigned a score for each parameter and averaged into an overall terminal score with a value of >10 being normal, 9-6 being likely morbid, and <6 being moribund (Table S1). Individual terminal scores of each mouse are plotted in Figure 9D. No obvious signs of toxicity were observed with 1 dosage up to 20 mg/kg for three weeks, as shown in the mice scoring chart. These results demonstrate that 1 does not induce sub-chronic toxicity, supporting the organoselenium compound’s viability as a drug.

Fig. 9.

In vivo safety study. (A) Repeated-dose safety study schedule; (B) The percentage of mice weight change (%) observed during 3 weeks of period of doses of 0, 5, 10 and 20 mg/kg of prodrug; (C) Mice weights during 3 weeks of treatment of 0, 5, 10 and 20 mg/kg of prodrug; (D) Mouse Intervention Scores for repeated-dose study.

CONCLUSIONS

In this work, we characterized the photochemical reactivity of binaphthalene phenyl selenide 1 toward DNA and investigated its cytotoxicity, selectivity, and mechanism of function. Upon 350 nm irradiation, 1 induced DNA ICL formation and various alkaline labile lesions. Further study showed that free radical and cation trapper (TEMPO and MeONH₂) prevented DNA ICL formation, providing evidence that free radicals and carbocations were produced during the photo-induced DNA cross-linking process. The free radicals were formed via the homolysis of the C-X bond, which were further oxidized to the carbocation via one-electron transfer. Additionally, we figured out that the photochemical reactivity of 1 was sequence-dependent. DNA ICLs occurred mainly at dGs/dCs, while mono-alkylation occurred at dGs and dAs. The cytotoxicity of 1 toward triple negative breast cancer cells was significantly increased with 350 nm irradiation, while 1 spared normal MCF 10A cells under all conditions tested. The photo-induced anticancer efficacy of 1 was correlated with the increased cellular DNA damage observed only in MDA-MB-468 breast cancer cells upon 350 nm irradiation.

Further mechanistic investigation revealed that normal MCF10A cells have higher levels of GSH than MDA-MB-468 breast cancer cells, which protect normal cells from oxidative DNA damage induced by photo-irradiation of 1, leading to selective cytotoxicity towards breast cancer cells. RT-qPCR analysis of DNA repair genes demonstrated that distinct DNA repair pathways were activated in MCF10A normal cells and MDA-MB-468 cancer cells in response to UV irradiation of 1. MCF10A cells showed efficient DNA repair mechanisms, such as high expression levels of ATM, ATR, and FANCG genes that act as DNA damage sensors and repair initiators, allowing normal cells to recognize and repair DNA damage quickly. Thus, normal cells can efficiently prevent potential DNA damage induced by 1 due to a high GSH level and enhanced DNA repair response. Although MDA-MB-468 cancer cells showed upregulation of several DNA repair genes, DNA damage induced by photo-irradiation 1 was too great to repair effectively, possibly due to low levels of GSH in breast cancer cells, as evidenced by comet assays. The selective redox disruption and DNA damage induced by photoirradiation of 1 in MDA-MB-468 breast cancer cells highlight its potential as a targeted cancer therapy and provide new insights into drug development.

Supplementary Material

Gel electrophoresis image of DNA ICL formation, NMR, and IT-TOF analysis.