Identification of 2′,4′,6′-Trihydroxyacetophenone as Promising Cysteine Conjugate Beta-Lyase Inhibitor for Preventing Cisplatin-Induced Nephrotoxicity

Cis-diamminedichloroplatinum(II) (cisplatin) is a standard treatment for many solid tumors, including bladder, ovarian, non–small cell lung, and esophageal cancers (1). Although cisplatin is associated with various side effects, nephrotoxicity is the major dose-limiting side effect (2, 3). Aggressive or short-duration intravenous administration of saline and mannitol over several hours before, during, and after cisplatin treatment is widely performed and is considered as the standard of care to reduce cisplatin-induced nephrotoxicity (4). The number of patients receiving outpatient cancer treatment has recently increased; however, cisplatin treatment requires hospitalization in many cases due to hydration regimens used to reduce nephrotoxicity. Furthermore, cisplatin-induced acute kidney injury reportedly occurs in up to 31.5% of patients, even after intravenous fluid administration (5, 6). Improving the quality of life in patients undergoing cisplatin-based cancer treatment is essential for preventing nephrotoxicity without compromising the drug's anticancer effects.

Cisplatin has a structure in which two chloride atoms are bound to platinum (7). Cisplatin cross-links double-stranded DNA, arrests its replication, and suppresses cancer growth. Most cisplatin is excreted in the urine, but some is metabolized in the kidneys (1). In the kidneys, cisplatin is metabolized to thiol-cisplatin, a highly active toxic metabolite, by cysteine conjugate-β lyase 1 (CCBL1), also named kynurenine aminotransferase 1 (KAT1) and glutamine transaminase K (GTK; refs. 1, 8–13). Thiol-cisplatin produces free radicals and damages mitochondria, leading to renal tubular cell apoptosis (1). Therefore, we hypothesized that CCBL1 inhibition could prevent nephrotoxicity associated with cisplatin treatment. Several studies attempted to inhibit CCBL1 using aminooxyacetic acid (AOAA) to protect C57BL/6 mice from cisplatin-induced renal toxicity (12, 14, 15). However, AOAA is a general pyridoxal phosphate (PLP)-dependent enzyme inhibitor, such as 4-aminobutyrate aminotransferase and aspartate aminotransferase (16–18). Therefore, it is unclear if AOAA protection against cisplatin-induced renal toxicity is mediated by CCBL1 inhibition.

This study sought to (i) identify effective CCBL1 inhibitors from the compound library using a high-throughput screening assay and (ii) evaluate the protective effects of newly identified CCBL1 inhibitor (2′,4′,6′-trihydroxyacetophenone, THA) on cisplatin-induced nephrotoxicity. We examine the protective in vitro and in vivo effects of the CCBL1 inhibitor on cisplatin-induced renal tubular cell damage. Furthermore, we used mice bearing the LLC subcutaneous syngeneic tumor to demonstrate that THA can prevent nephrotoxicity without compromising cisplatin's anticancer effects. This study's results may provide a new strategy reducing nephrotoxicity associated with cisplatin chemotherapy.

Human CCBL1 cDNA expression cloning and purification of recombinant human CCBL1 protein were performed as described elsewhere (19). In addition, high-throughput screening assays for CCBL1 inhibitors were conducted using a microplate fluorescence assay as measured by KAT1 activity (19). Sixteen microliters of a mixture containing 10 ng/mL of CCBL1, 1 mmol/L L-kynurenine, 1 mmol/L sodium pyruvate, 100 mmol/L PLP, 0.005% Tween 20 in 150 mmol/L 2-amino-2-methyl-1-propanol (AMP) buffer (pH9.5) was added to 384-well plates containing compounds using a Multidrop dispenser (Thermo Fisher Scientific). The Center for Supporting Drug Discovery and Life Science Research, Graduate School of Pharmaceutical Sciences, Osaka University, provided the compound library, which included 2,320 compounds comprising the United States and international drug collections and natural products (a part of The Spectrum Collection, MicroSource Discovery Systems). All compounds were dissolved and diluted in DMSO to a final concentration of 10 μmol/L. Reaction mixtures were incubated with 4 μL of compounds for 2 hours at room temperature, and 20 μL of 300 mmol/L zinc acetate (pH 5.5) were then added directly using a Multidrop dispenser. Fluorescence intensities of the reaction product, kynurenic acid, were measured using an Infinite M1000 plate reader (TECAN) at 340 nm excitation wavelength and 400 nm emission wavelength. Assay quality was validated by calculating signal background and Z′ factor.

CCBL1 β-elimination activity was measured according to the method described by Selvam and colleagues (20). Briefly, 500 ng of recombinant CCBL1 protein in 5 and 1 μL of serially diluted THA (Tokyo Chemical Industry) were mixed and incubated at room temperature for 30 minutes. After incubation, 44 μL of the reaction mixture contained 100 mmol/L of potassium phosphate buffer (pH 7.4), 5 mmol/L of Se-methylselenocysteine, 100 μmol/L of dimethyl-2-oxoglutarate, 100 μmol/L of α-Keto-γ-methylthiobutyric acid sodium salt, 10 μmol/L of 2-amino-2-methyl-1,3-propanediol, and 10 μmol/L of PLP was added. Next, the reaction mixture was incubated at 37°C, and the reaction was terminated by adding 20 μL of 5 mmol/L 2,4-dinitrophenylhydrazine in 2M of HCl. Then, 70 μL of the assay mixture was incubated at 37°C for 10 minutes, 130 μL of 1M NaOH was added, and increases in absorbance were measured at 520 nm using a Nivo multimode plate reader (PerkinElmer). The assay mixture without enzymes served as a blank.

LLC-PK1 (pig kidney epithelial), LLC (murine Lewis lung carcinoma), and MDA-MB-231 (human breast cancer) cells were obtained from the JCRB Cell Bank. All cells were cultured at 37°C under a humidified atmosphere with 5% CO₂. Human CCBL1 cDNA was synthesized by RT-PCR from blood peripheral leukocytes total RNA (TaKaRa) using a ReverTra Ace Kit (Toyobo). The synthesized cDNA was cloned into the pcDNA3.1 mammalian expression vector (Thermo Fisher Scientific). To obtain human CCBL1 stably expressing LLC-PK1 cells, the CCBL1/pcDNA3.1 and empty pcDNA3.1 were linearized and transfected into LLC-PK1 cells using the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) following the manufacturer's instructions. After 2 days of transfections, cells were grown in a complete 199/EBSS medium (10% FBS, 1% penicillin/streptomycin) supplemented with 400 mg/mL of G418 for 7 days. After 7 days of the selection, single clones were obtained by limiting dilution. As described below, CCBL1 expression was verified by Western blotting, and the highest-expressing clones were selected for further experiments. LLC and MDA-MB-231 cells were grown in a DMEM medium (10% FBS and 1% penicillin/streptomycin).

An assay kit based on a tetrazolium compound (MTS) was used to determine cell viability. Cisplatin and THA were purchased from Tokyo Chemical Industry. Fresh stock solutions of 10 mmol/L cisplatin and 100 mmol/L THA were prepared in 0.9% NaCl and in DMSO, respectively, on the day of the experiment. Confluent LLC-PK1 cells were used to investigate the effects of THA on cisplatin toxicity in quiescent proximal tubule cells, as described previously (9). LLC-PK1 cells were seeded in 96-well plates (1 × 10⁴ cells/well). On the third day after plating, confluent monolayers were confirmed by microscopy and cell media was replaced with fresh media. On day 7, cells were treated with varying concentrations of either cisplatin or THA for 24 hours at 37°C in 5% CO₂. The stock solutions of cisplatin and THA were diluted in a complete 199/EBSS medium. Actively proliferating LLC and MDA-MB-231 cells were used to investigate the effects of THA on cisplatin toxicity in cancer cells. LLC and MDA-MB-231 cells were seeded in 96-well plates (2 × 10⁴ cells/well). The following day, 60% to 70% cell confluency was confirmed by microscopy and cells were treated with varying concentrations of cisplatin and/or THA. The stock solutions of cisplatin and THA were diluted in complete DMEM and cells were cultured for 24 hours. After treatment of LLC, MDA-MB-231, or LLC-PK1 cells with cisplatin or THA, 20 μL of CellTiter 96 Aqueous One Solution (Promega) was added to each well and cells were cultured for an additional hour. The absorbance at 490 nm was measured using a microplate reader (Molecular Devices). Cells incubated in complete medium only were used as controls. Cell viability was calculated as the absorbance of treated cells relative to that of untreated cells.

All mice were housed in a plastic cage, maintained on a 12-hour light/dark cycle (lights on at 8:00 a.m.), and given food and water ad libitum. All experiments were performed following the guidelines established by the Japanese Pharmacological Society and the Institute for Experimental Animals at Fujita Health University. In addition, the study protocols were approved by the Ethics Committee of Animal Experiments at the Institute for Experimental Animals at Fujita Health University (Permit No.: AP20004).

To create a cisplatin-induced renal injury model, male C57BL/6J mice (Japan SLC) weighing approximately 20 to 25 g were given a single intraperitoneal dose of cisplatin in 100 μL (20 mg/kg). Cisplatin was dissolved in 0.9% NaCl at 2 mg/mL. The mice administered the same volume of 0.9% NaCl were served as controls. To investigate the nephroprotective effect of CCBL1 inhibitors in cisplatin-induced renal injury, THA dissolved in 100 μL of corn oil (FUJIFILM Wako Chemicals) was administered intraperitoneally at doses of 0, 25, 50, and 100 mg/kg 30 minutes before cisplatin injection. The mice administered the same volume of corn oil were served as controls. The mice were sacrificed on day 3 after cisplatin injection. Blood and kidney samples were collected and stored at −80°C for further analysis.

To create a syngeneic tumor model, male C57BL/6J mice (Japan SLC) weighing approximately 20 to 25 g were injected dorsally subcutaneously with LLC cells (1 × 10⁶ cells in 50 μL of PBS). Tumor volume was measured using calipers, and volume was calculated as [(length × width × width)/2; ref. 21]. After the tumor reached a certain size, THA (100 mg/kg) was administered intraperitoneally 30 minutes before administering 20 mg/kg of cisplatin. The mice were sacrificed on the third day after cisplatin injection. Blood, tumor, and kidney samples were collected and stored at −80°C for further analysis.

Blood urea nitrogen (BUN) and creatinine (Cre) levels in serum were measured using a fully automatic blood biochemical analyzer according to the manufacturer's methods (BioMajesty JCA-BM9130; JEOL).

Body weight change (%), relative kidney weight (%), and tumor inhibition (%) were calculated using the following formulas:

Body weight change (%) = [(cisplatin plus THA administration)/(before cisplatin plus THA administration)] × 100

Relative kidney weight (%) = [kidney weight (g)/body weight at administration of cisplatin and compound X (g)] × 100

Tumor inhibition (%) = Before administration of cisplatin and compound X – [(administration of cisplatin and compound X)/(before administration of cisplatin and compound X)].

Histopathologic kidney changes were examined by periodate-Schiff (PAS) staining. Half of the kidneys were immersion fixed in 10% buffered formalin, embedded in paraffin, and sections were thinly sliced to 4 μmol/L thickness. After staining, the sections were observed under an optical microscope. Each section was observed in six fields of view, and we calculated the average damaged renal tubule percentage. The renal tubular injury results were converted into a renal tubular necrosis index, with <10% injury assigned a 0 index, 10% to 25% injury assigned a 1 index, 25% to 50% injury assigned a 2 index, 51% to 75% injury assigned a 3 index, and >75% injury assigned a 4 index as described previously (22, 23).

The human adult normal kidney section slide was obtained from the BioChain Institute. Human and mouse kidney paraffin sections were first dewaxed in xylene for 5 minutes three times, then replaced with fresh xylene. The resulting sections were twice deparaffinized with anhydrous ethanol for 2 minutes, 90% ethanol for 2 minutes, 80% ethanol for 2 minutes, and 70% ethanol for 2 minutes. After the deparaffinized sections were washed with water for 2 minutes, endogenous peroxidase was blocked with 3% H₂O₂ for 20 minutes and washed with distilled water for 2 minutes (twice) and 1× PBS for 3 minutes (thrice). Afterward, the sections were blocked with 10% goat serum in 1× PBS for 20 minutes. Next, the sections were incubated at 4°C overnight with the following primary antibodies: anti-CCBL1 (1:500, GTX32492; GeneTex) and Rabbit IgG isotype control antibody (1:500, R&D Systems). The next day, they were washed three times with 1 × PBS for 3 minutes. The slides were then incubated with the secondary antibody (Histofine Simple Stain MAX PO; Nichirei Biosciences) for 30 minutes and washed three times with 1 × PBS for 3 minutes. The slides were then incubated with a chromogen/substrate reagent for 30 seconds and counter-stained with hematoxylin. After staining, the sections were observed under an optical microscope.

LLC-PK1 cells and kidney samples were homogenized in the TNE buffer containing 10 mmol/L Tris-HCl, 0.15 M NaCl, 0.5M EDTA, 1% NP40, and a complete mini protease inhibitor cocktail (Roche). The lysate supernatants were obtained by centrifugation at 14,000 rpm for 10 minutes at 4°C. Total protein concentrations in the lysate were determined using Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific). The protein extracts were separated by SDS-PAGE (10%–12.5% gels) and blotted onto PVDF membranes (ATTO). After blocking with 5% fat-free milk, the membranes were incubated at 4°C overnight with the following primary antibodies: anti-CCBL1 (1:1,000, GTX32492; GeneTex), anti-Cleaved Caspase-3 (1:2,000, #9661; Abcam), and anti-β-actin (1:5,000, A5441; Sigma-Aldrich). After washing with TBS-T, the membranes were incubated with a horseradish peroxidase-conjugated anti-IgG (Sigma-Aldrich), and the protein bands were visualized with enhanced chemiluminescence (ECL) reagents according to the manufacturer's instructions (Cytiva). Protein band intensities, relative to β-actin bands, were analyzed using LuminoGraph (ATTO).

All statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software Inc.). Significant between-group differences were assessed using one-way ANOVA. The criterion for significance was ****P < 0.0001, ***P < 0.001 **P < 0.01, and *P < 0.05. All data are expressed as mean ± SE.

The data generated in this study are available upon request from the corresponding author.

After screening the compound libraries, we identified THA, a naturally occurring compound obtained from the Curcuma comosa rhizome (Family Zingiberaceae/ginger), inhibits CCBL1 aminotransferase activity (Fig. 1A). We further examined THA inhibition of CCBL1 β-elimination activity using recombinant human CCBL1. As shown in Fig. 1B, CCBL1 activity was inhibited by THA in a concentration-dependent manner. IC₅₀ of THA on CCBL1 was 13.20 μmol/L (Fig. 1B). These results indicate that THA inhibits CCBL1 enzyme activity.

To investigate whether THA attenuates cisplatin-induced renal tubular cell death, human CCBL1 overexpressing LLC-PK1 cells were treated with different cisplatin concentrations and THA for 24 hours; numbers of viable cells were then assessed by an MTS assay. Human CCBL1 expression in LLC-PK1 cells was confirmed by immunoblotting (Supplementary Fig. S1). THA attenuated cisplatin-induced reductions in the numbers of viable LLC-PK1 cells in a concentration-dependent manner (Fig. 2A). Next, we examined whether THA attenuated the antiproliferative effects of cisplatin in murine LLC and MDA-MB-231 cancer cell lines. In contrast to LLC-PK1 cells, THA did not affect cisplatin-induced reduction of numbers of viable cancer cells (Fig. 2B and C).

To investigate whether THA prevents renal tubular cell death in vivo, C57BL/6J mice were intraperitoneally injected with THA 30 minutes before cisplatin injection (Fig. 3A). First, we examined renal CCBL1 protein expression levels in each group using IHC. CCBL1 was expressed mainly in renal tubular cells. Cisplatin plus THA administration did not affect CCBL1 protein expression levels (Fig. 3B–E). We also confirmed that CCBL1 was expressed in renal tubular cells in the human kidney section (Supplementary Fig. S2).

It has been reported that cisplatin administration reduces body weight and increases relative kidney weight (24). The cisplatin administration alone reduced the body weight (15.5% reduction compared with cisplatin, 0 mg/kg; THA, 0 mg/kg group) and increased relative kidney weight (20.8% increase compared with cisplatin, 0 mg/kg; THA, 0 mg/kg group; Fig. 4A and B). The administration of cisplatin plus THA did not attenuate body weight loss; however, the administration of cisplatin plus THA significantly attenuated cisplatin-induced increases in relative kidney weight. Next, we examined nephrotoxicity by measuring BUN and Cre in serum from cisplatin plus THA-injected mice. Cisplatin administration alone increased BUN and Cre levels; however, THA administration attenuated cisplatin-induced increases in BUN and Cre levels at doses between 25 and 100 μmol/L, with the greatest attenuating effect observed at 100 μmol/L (Fig. 4C and D). Furthermore, we investigated renal tubular cell damage, such as tubular cell death, cell shedding, interstitial edema, and renal cortical tubular damage, by histochemical examination. Histochemical analysis showed that cisplatin plus THA administration had lower tubular damage scores than the cisplatin-only group (Fig. 5A–G). Next, we examined the effects of THA administration on apoptosis markers and cleaved caspase-3 by immunoblotting in kidney samples after cisplatin plus THA administration. THA reduced the cisplatin-induced increase of cleaved caspase-3 (Fig. 5H). These results suggest that CCBL1 inhibition by THA reduces apoptosis of renal tubular cells, thereby inhibiting cisplatin-induced nephrotoxicity.

We investigated whether THA could reduce renal tubular cell damage without compromising cisplatin's anticancer effects in tumor-bearing mice. First, LLC cells were administered subcutaneously, and tumor size was measured every few days (Fig. 6A). When the tumors were clearly visualized at day 10, THA was intraperitoneally administered 30 minutes before cisplatin administration. Cisplatin administration reduced tumor volume, and this effect was unchanged when THA was administered before cisplatin (Fig. 6B and C). Like the cisplatin-induced nephrotoxicity model, administration of cisplatin alone reduced the body weight (18.8% reduction compared with cisplatin, 0 mg/kg; THA, 0 mg/kg group) and increased relative kidney weight (26.0% increase compared with cisplatin, 0 mg/kg; THA, 0 mg/kg group; Fig. 6D and E). However, cisplatin plus THA administration reduced the relative kidney weight. The administration of cisplatin plus THA did not attenuate body weight loss; however, the administration of cisplatin plus THA significantly attenuated cisplatin-induced increases in relative kidney weight. Next, BUN and Cre in the serum were measured to investigate the nephrotoxicity in each group. THA administration attenuated cisplatin-induced increases in BUN and Cre (Fig. 6F and G). Furthermore, the histochemical analysis showed that THA reduced cisplatin-induced increases in renal tubular damage scores in tumor-bearing mice (Supplementary Figs. S3A–S3E). Also, cleaved caspase-3 was decreased in the THA and cisplatin groups compared with cisplatin alone (Supplementary Fig. S3F). These results suggest that THA prevents cisplatin-induced nephrotoxicity without compromising the anticancer effects of cisplatin in tumor-bearing mice.

Cisplatin is widely used as an antitumor drug to treat solid tumors. However, irrespective of its potent anticancer properties and efficacy, its clinical use is limited due to the potential for severe, adverse effects—principally nephrotoxicity—which is a dose-limiting factor. This study revealed that THA prevents cisplatin-induced renal tubular cell damage both in vitro and in vivo. Using a mouse tumor model, we also found that THA protects against cisplatin-induced renal cell injury without affecting its anticancer effects. Therefore, THA is a potential adjuvant for clinical cisplatin therapy.

Cisplatin accumulates in terminal proximal and distal tubule cells, where it causes either apoptosis or necrosis, depending on the exposure time and concentration (25). Current evidence suggests that the organic cation transporter 2 (OCT2), highly expressed in the proximal tubular cell basolateral membrane, is the predominant transporter mediating cisplatin uptake from the circulation into the kidneys (26–28). Therefore, OCT2 might play a crucial role in cisplatin-induced nephrotoxicity. Indeed, past efforts have attempted to attenuate cisplatin-induced nephrotoxicity by inhibiting OCT2 (29). For example, Li and colleagues demonstrated that OCT2 inhibition by L-tetrahydropalmatine attenuated cisplatin-induced nephrotoxicity in healthy and tumor-bearing nude mice without impairing antitumor efficacy (30). In addition, Hamano and colleagues found that diphenhydramine, a previously developed drug, potentially prevented cisplatin-induced nephrotoxicity based on a Food and Drug Administration Adverse Events Reporting System repositioning analysis (31). Diphenhydramine pre-administration reduced cisplatin-induced nephrotoxicity by inhibiting increases in inflammatory cytokines, apoptosis, and oxidative stress in a mouse model.

We attempted to prevent cisplatin-induced nephrotoxicity using a different approach from previously reported ones. Once it enters renal epithelial cells, cisplatin becomes a potent nephrotoxin via gamma-glutamyl transpeptidase (GGT)-dependent metabolic activation. Cisplatin is metabolized by GGT and aminopeptidases, which are expressed on the surface of proximal tubule cells, and transported into proximal tubule cells where they are further metabolized by CCBL1 to form a highly reactive thiol-cisplatin (1, 9, 11). Therefore, molecularly targeted drugs for CCBL1 might prevent cisplatin-induced nephrotoxicity. Indeed, we found that CCBL1 inhibition by THA attenuated cisplatin-induced proximal tubule cell death both in vitro and in vivo. In addition, THA administration attenuated cisplatin-induced nephrotoxicity without affecting cisplatin's antitumor efficacy in tumor-bearing mice. Therefore, THA pretreatment might prevent cisplatin-induced nephrotoxicity. Furthermore, administration of THA may facilitate the use of cisplatin in ineligible patients with compromised renal function as THA may attenuate the nephrotoxicity of cisplatin by inhibiting CCBL1.

Although this study clearly demonstrated that THA prevented cisplatin-induced nephrotoxicity, it is not ascertained if this occurs because THA inhibits thiol-cisplatin synthesis in renal cells. However, we could not determine if renal thiol-cisplatin concentrations were decreased in THA and cisplatin-injected mice. It may be possible to measure thiol-cisplatin using mass spectrometry; however, we did not attempt this as thiol-cisplatin is highly reactive and unstable, and a standard of thiol-cisplatin was not available. In addition, THA is known to exert antioxidant and cholesterol-lowering effects which may be related to the protective effects of THA on cisplatin-induced nephrotoxicity (32–34). Further research must be carried out to elucidate the molecular mechanisms governing the protective effects of THA.

Furthermore, this study had several limitations in demonstrating the potential clinical utility of THA. This study only evaluated the effects of THA in a limited number of tumor cell lines in vitro and the effects of a single-dose administration of cisplatin in a murine LLC isograft model in vivo. The administration of a single dose of cisplatin may avoid cumulative nephrotoxicity and treatment resistance that may emerge after the use of more clinically relevant dosing regimens. GGT has been reported to be overexpressed in a range of human tumor types, which is associated with the development of resistance to chemotherapeutic drugs (35). Accordingly, expression levels of GGT may have confounded the observed effects of THA on the antitumor efficacy of cisplatin. Further studies using different tumor models, such as human tumor xenografts or other orthotopic models, are required to strengthen the conclusions of this study.

In conclusion, pre-administration of THA attenuated cisplatin-induced nephrotoxicity without affecting its antitumor efficacy. These effects might be attributed to inhibition of the CCBL1-mediated formation of thiol-cisplatin. Our results suggest THA might prevent cisplatin-induced nephrotoxicity and potentially provide a new strategy for patients receiving cisplatin-based cancer treatments.

No author disclosures were reported.

N. Sukeda: Data curation, formal analysis, validation, investigation, visualization, writing–original draft. H. Fujigaki: Conceptualization, resources, data curation, supervision, funding acquisition, validation, investigation, writing–original draft, writing–review and editing. T. Ando: Data curation, investigation, methodology. H. Ando: Data curation, investigation. Y. Yamamoto: Investigation, methodology, writing–review and editing. K. Saito: Resources, supervision, funding acquisition, writing–review and editing.

We gratefully acknowledge the technical assistance of Kyoka Yamazaki, Yui Ito, Miku Tsuchikawa, Moe Nojima, and Kenta Nagasaka in animal experiments. This study was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research [Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)] from AMED under Grant No. JP21am0101001. This study was also supported by JSPS KAKENHI Grant No. 20K05757.

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