Vol. 39, No. 5 869Biol. Pharm. Bull. 39, 869–873 (2016)

© 2016 The Pharmaceutical Society of Japan

Note

TLSC702, a Novel Inhibitor of Human Glyoxalase I, Induces Apoptosis in Tumor CellsRyoko Takasawa,*,a Nami Shimada,a Hiromi Uchiro,a Satoshi Takahashi,b Atsushi Yoshimori,b and Sei-ichi Tanumaa,c

a Faculty of Pharmaceutical Sciences, Tokyo University of Science; 2641 Yamazaki, Noda, Chiba 278–8510, Japan: b Institute for Theoretical Medicine, Inc.; Tokyo Institute of Technology Yokohama Venture Plaza W101, 4259–3 Nagatsuda, Midori-ku, Yokohama 226–8510, Japan: and c Genome & Drug Research Center, Tokyo University of Science; 2641 Yamazaki, Noda, Chiba 278–8510, Japan.Received September 15, 2015; accepted January 21, 2016

Human glyoxalase I (hGLO I) is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), which is the side product of tumor-specific aerobic glycolysis. GLO I has been reported to be overexpressed in various types of cancer cells, and has been expected as an attractive target for the development of new anticancer drugs. We previously discovered a novel inhibitor of hGLO I, named TLSC702, by our in silico screening method. Here, we show that TLSC702 inhibits the proliferation of human leukemia HL-60 cells and induces apoptosis in a dose-dependent manner. In addition, TLSC702 more significantly inhibits the proliferation of human lung cancer NCI-H522 cells, which highly express GLO I, than that of GLO I lower-expressing human lung cancer NCI-H460 cells. Furthermore, this antiproliferative effect of TLSC702 on NCI-H522 cells is in a dose- and time-dependent manner. These results suggest that TLSC702 can induce apoptosis in tumor cells by GLO I inhibition, which lead to accumulation of MG. Taken together, TLSC702 could become a unique seed compound for the generation of novel chemotherapeutic drugs targeting GLO I-dependent human tumors.

Key words glyoxalase I; inhibitor; anticancer drug

Glyoxalase I (GLO I) is a key enzyme in the pathways leading to glutathione (GSH)-mediated detoxification of meth-ylglyoxal (MG), the side product of tumor-specific aerobic gly-colysis (Warburg effect).1) MG is highly reactive with DNA/RNA and proteins and has been suggested to induce apoptosis in tumor cells.2) Furthermore, in many human tumors includ-ing colon,3) pancreatic,4) melanoma,5) prostate,6,7) breast8,9) and lung,10) and anticancer drug-resistant human leukemia cells,11) abnormal expression and higher activity of GLO I have been reported. These observations indicate that the increased ex-pression of GLO I is closely associated with carcinogenesis3–10) and anticancer drug resistance.9) So, specific inhibitors of GLO I have long been sought as possible effective anticancer drugs, which selectively kill GLO I-overexpressing and anti-cancer drug-resistant tumors.12,13)

Some GSH analogs, such as S-p-bromobenzylglutathione (BBG),14,15) S-(N-hydroxy-N-methylcarbamoyl) glutathione,16) and S-(N-aryl-N-hydroxycarbamoyl) glutathione derivatives,17) have been reported as GLO I inhibitors. However, such GSH analogs are likely to be readily degraded in vivo by ubiqui-tously distributed γ-glutamyltranspeptidase, a GSH cataboliz-ing enzyme. Probably, they also inhibit other GSH-dependent enzymes. Furthermore, for designing of small molecular GLO I inhibitors, it is not better to use the GSH peptide scaffold as a lead structure because of its flexibility. Therefore, the search of new types of scaffold for GLO I inhibitory small molecules is required to open a way for the development of novel anti-cancer drugs.

Recently, we identified myricetin as a substrate transition-state mimetic inhibitor of hGLO I.18) Based on the binding mode of myricetin to hGLO I, which was simulated by the computational Autodock program,19) we constructed a hGLO

I/inhibitor 4-point pharmacophore.20) By using the pharmaco-phore, we discovered TLSC702, which has a unique scaffold and inhibits hGLO I more effectively than BBG20) (Fig. 1). Previous report has demonstrated that S-p-bromobenzyl-glutathione cyclopentyl diester (BBGC), a cell-permeable prodrug of BBG, sensitizes drug-resistant leukemia cells to Etoposide.11) Also, BBGC itself has been reported to have anti-proliferative activity and induce apoptosis in human leukemia HL-60 cells.15) Moreover, BBGC suppressed more strongly the

* To whom correspondence should be addressed. e-mail: takasawa@rs.noda.tus.ac.jp

Fig. 1. Structures of TLSC702 and S-p-Bromobenzylglutathione (BBG)In vitro IC50 values of each compound are shown.

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proliferation of GLO I higher-expressing human lung cancer cell line, NCI-H522 cells, as compared with that of GLO I lower-expressing human lung cancer cell line, NCI-H460 cells.10) These results indicate that the human GLO I inhibitors have potential as therapeutic agents against GLO I-overex-pressing human tumors.

In this study, we investigated the antiproliferative activity and apoptosis inducibility of TLSC702 in HL-60 cells and compared the antiproliferative effect between the human lung cancer cell lines, NCI-H522 and NCI-H460 cells. As a result, TLSC702 could induce apoptosis in HL-60 cells in a dose-dependent manner. Futhermore, TLSC702 more preferentially suppressed the proliferation of NCI-H522 cells than that of NCI-H460 cells. This antiproliferative effect of TLSC702 on NCI-H522 cells was in a dose- and time-dependent manner. Thus, TLSC702 could become a unique seed compound for the generation of novel chemotherapeutic drugs targeting GLO I-overexpressing human tumors.

MATERIALS AND METHODS

Chemicals and Reagents 3-(1,3-Benzothiazol-2-yl)-4-(4-methoxyphenyl) but-3-enoic acid, named TLSC702, was purchased from Namiki Shoji Co., Ltd. (Japan). Mouse anti-β-actin monoclonal antibody (clone AC-15), mouse anti-poly(ADP-ribose) polymerase (PARP)-1 monoclonal antibody (clone C2-10), mouse anti-GLO I monoclonal antibody (clone Glo1a) and anti-argpyrimidine (mAb6B) monoclonal antibody were from Sigma (U.S.A.), Wako Pure Chemical Industries, Ltd. (Japan), Novus Biologicals (U.S.A.) and Cosmobio Co., Ltd. (Japan), respectively. Goat anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugate was from Jack-son Immunological Research (U.S.A.). All other chemicals were of reagent grade.

Cell Lines and Cell Culture Human myeloid leukemia HL-60 cells, human lung cancer NCI-H522 and NCI-H460 cells were maintained in RPMI1640 (Wako Pure Chemical Industries, Ltd.) supplemented with 100 U/mL of penicil-lin/100 µg/mL of streptomycin (Gibco, Japan), and 10% heat-inactivated fetal bovine serum. The cells were grown at 37°C in a humidified atmosphere of 5% CO2.

Expression Vector Human GLO I (hGLO I) cDNA frag-ment was subcloned into pcDNA3.1 myc-His B (Invitrogen, U.S.A.). Transfection of expression vectors was performed using FuGENE 6 reagent (Roche Applied Science, Germany).

Measurements of Cell Growth Inhibition The sensi-tivities of HL-60 cells, NCI-H522 and NCI-H460 cells to TLSC702 were evaluated by the inhibition of cell growth. The number of viable cells was estimated by WST-8 assay using a Cell Counting Kit-8 (Dojindo Laboratories, Japan).

Microscopic Analysis Apoptotic morphological changes were evaluated by phase-contrast microscopy. HL-60 cells were treated with 0 (control), 100, 300 and 1000 µM of TLSC702 for 24 h, and then observed under a phase-contrast microscope.21)

Detection of Apoptotic DNA Fragmentation HL-60 cells were lysed in lysis buffer (50 mM Tris–HCl (pH 7.8), 10 mM ethylenediaminetetraacetic acid (EDTA) and 0.5% (w/v) sodium N-lauroylsarcosinate) and incubated with 0.5 mg/mL RNase A at 50°C for 1 h. Then 0.5 mg/mL Proteinase K was added, and the lysates were incubated for 1 h.21) DNA thus

prepared was subjected to 1.8% agarose gel electrophoresis. The DNA was visualized by UV illumination after ethidium bromide staining.

Western Blot Analysis Harvested cell samples were lysed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 5% glycerol, 1% 2-mercaptoethanol and 0.1% bromophenol blue). Cell lysates were separated by SDS-PAGE, and then transferred onto nitrocellulose membranes. The membranes were blocked with 2.5% skim milk and 0.25% bovine serum albumin (BSA) in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 (TTBS) for 1 h at room temperature, and then probed with appropriate primary antibodies for overnight at 4°C. The membranes were washed with TTBS, and then incubated with the appropriate secondary antibody for 1 h at room temperature. After washing the membrane with TTBS, the blotted proteins were visualized with Light Capture Sys-tem (ATTO, Japan) by using ImmunoStar® Basic (Wako Pure Chemical Industries, Ltd.).

RESULTS

Antiproliferative Effect of TLSC702 on HL-60 Cells Previous report has been shown that a glutathione analog inhibitor of glyoxalase I, BBGC, could induce apoptosis in human leukemia HL-60 cells.15) In order to test whether TLSC702, a novel human GLO I inhibitor discovered by us, is effective in suppressing growth of HL-60 cells, we examined its cellular antiproliferative effect on HL-60 cells by using WST assay. As shown in Fig. 2, the TLSC702 treatment for 24 h is revealed to suppress the proliferation of HL-60 cells in a dose-dependent manner. The EC50 value was calculated to be approximately 400 µM.

Induction of Apoptosis in HL-60 Cells by TLSC702 Treatment To prove the antiproliferative effect of TLSC702 is due to induction of apoptosis, we examined the hallmarks of apoptosis, that is, apoptotic morphological changes, nucleo-somal DNA fragmentation and PARP cleavage. As shown in Fig. 3A, apoptotic cells and apoptotic bodies are observed by

Fig. 2. The Antiproliferative Effect of TLSC702 on HL-60 CellsHL-60 cells were treated with indicated concentrations of TLSC702 for 24 h.

Cell viability (% of control) was measured by using WST assay. Data are the aver-ages of three independent experiments and bars show the standard deviation (S.D.) values.

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phase-contrast microscopy in HL-60 cells treated with up to 300 µM TLSC702 for 24 h. At 1000 µM, necrotic cell death was also detected. Analysis of the DNA from TLSC702-treated HL-60 cells by agarose gel electrophoresis showed a typical pattern of internucleosomal cleavage characteristic of apopto-sis (DNA ladder) at 300 µM TLSC702 (Fig. 3B). The ladder formation was in a dose-dependent manner. Furthermore, a dose-dependent cleavage of PARP, one of the substrates of caspase-3, was detected by Western blot analysis (Fig. 3C).

Preferential Suppression of Proliferation in GLO I Higher-Expressing Human Lung Cancer Cells by TLSC702 Treatment Abnormal expression and higher activity of GLO I have been detected more in various tumor cells than in normal cell samples.3–10) Among cancer cell lines, lung carcinoma cells frequently showed higher GLO I activity. BBGC has been reported to suppress more strongly prolifera-tion of human lung cancer NCI-H522 cells, which has higher GLO I activity, than that of GLO I lower-expressing human lung cancer NCI-H460 cells.10) Furthermore, it has been re-ported that RNA interference (RNAi) knockdown of GLO I in NCI-H522 cells significantly reduces cell growth and induces apoptosis.22) In order to elucidate whether or not the sup-pression of cell growth by TLSC702 is correlated with GLO I expression levels, we compared the effect of TLSC702 on proliferation between NCI-H522 and NCI-H460 cells. Western blot analyses showed that the GLO I protein expression level

in NCI-H522 cells is approximately 18-fold higher than that in NCI-H460 cells (Fig. 4A). Importantly, TLSC702 preferen-tially suppressed growth of NCI-H522 cells, as compared with that of NCI-H460 cells (Fig. 4B). This suppressive effect was found to be a time-dependent manner (Fig. 4C). The EC50 val-ues of TLSC702 for NCI-H522 cells at 24, 48 and 72 h treat-ments were about >300, 202 and 145 µM, respectively.

Accumulation of Argpyrimidine Adducts in TLSC702-Treated NCI-H522 Cells In order to elucidate that TLSC702 actually inhibits GLO I and accumulates MG in the cells, we evaluated the accumulation of argpyrimidine ad-ducts, that is, methylglyoxal-derived advanced glycation end-products in TLSC702-treated NCI-H522 cells by Western blot analysis. As shown in Fig. 5, argpyrimidine adducts were ac-cumulated in TLSC702-treated NCI-H522 cells in comparison with the non-treated cells.

The Restoration of Cell Viability by Overexpression of GLO I in TLSC702-Treated NCI-H522 Cells In order to ascertain the contribution of GLO I inhibition by TLSC702 for its ability to suppress cell proliferation, we investigated the effect of transient overexpression of GLO I on TLSC702 treat-ment in NCI-H522 cells. As shown in Fig. 6, overexpression of GLO I partially restore cell viability in TLSC702-treated NCI-H522 cells. This result will provide the evidence that inhibition of GLO I by TLSC702 treatment contributes to the inhibition of the cell proliferation in NCI-H522 cells.

Fig. 3. Apoptotic Morphological and Biochemical Changes in HL-60 Cells Treated with TLSC702HL-60 cells were treated with indicated concentrations of TLSC702 for 24 h. (A) The morphological changes of HL-60 cells treated with (a) 0 (control), (b) 100, (c) 300

and (d) 1000 µM of TLSC702 were analyzed by phase-contrast microscopy. Original magnification ×200. White arrows indicate apoptotic bodies. (B) Dose-dependent DNA fragmentation in HL-60 cells treated with TLSC702. Agarose gel analyses of DNA from TLSC702 treated-HL-60 cells show a pattern of internucleosomal cleavage characteristic of apoptosis (DNA ladder) in a dose-dependent manner. (C) Dose-dependent PARP cleavage in HL-60 cells treated with TLSC702. Equal amount of protein from the whole cell lysate samples (equivalent to 2×105 cells) were analyzed by Western blot analysis using antibodies specific for PARP as described under “Materials and Methods.”

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DISCUSSION

Accumulating MG by blocking the activity of GLO I is considered to be an attractive therapeutic strategy for target-ing tumor-specific aerobic glycolysis metabolism. In this study, we showed the antiproliferative activity and apoptosis inducibility of TLSC702, a novel human GLO I inhibitor dis-covered by us, in GLO I-dependent cancer cell lines.

TLSC702 suppressed cell proliferation in HL-60 cells in a dose-dependent manner. Furthermore, the apoptotic morpho-logical changes, DNA ladder formation and PARP cleavage observed in TLSC702-treated HL-60 cells strongly suggested the apoptosis inducibility of TLSC702. HL-60 cells have been

reported to undergo apoptosis by treatment with a GSH ana-log, BBGC.15) Our previous study has also shown that human GLO I inhibitory flavonoid compounds suppress the growth of HL-60 cells, and their degrees of antiproliferative activity are correlated to the in vitro human GLO I inhibitory activities.18) Furthermore, delphinidin, the major anthocyanidin present in berry fruits, has been found to have potent inhibitory effect on human GLO I and to induce apoptosis in HL-60 cells in a dose- and time-dependent manner.23) Taken together, GLO I inhibition is suggested to be an effective contribution to the antiproliferative and proapoptotic activities of TLSC702.

Importantly, TLSC702 preferentially suppressed growth of NCI-H522 cells, which overexpress GLO I, as compared with that of GLO I lower-expressing NCI-H460 cells. The

Fig. 4. The Antiproliferative Effects of TLSC702 on NCI-H522 and NCI-H460 Cells

(A) Western blot analysis of GLO I protein levels in HL-60 cells, NCI-H522 and NCI-H460 cells. The values were the ratios of GLO I to β-actin band intensities calculated by densitometric analysis. (B) NCI-H522 (—■—) and NCI-H460 cells (—▲—) were treated with indicated concentrations of TLSC702 for 24 h. Cell vi-ability (% of control) was measured by using WST assay. Data are the averages of three independent experiments and bars show the S.D. values. (C) Time-dependen-cy of antiproliferative effect of TLSC702 on NCI-H522 cells. NCI-H522 cells were treated with indicated concentrations of TLSC702 for 24, 48 and 72 h. Cell viability (% of control) was measured by using WST assay. Data are the averages of three independent experiments and bars show the S.D. values.

Fig. 5. Accumulation of Argpyrimidine Adducts in TLSC702-Treated NCI-H522 Cells

NCI-H522 cells were treated with 300 µM of TLSC702 for 24, 48 and 72 h. Accu-mulation of argpyrimidine adducts was detected by Western blot analysis.

Fig. 6. The Effect of Transient Overexpression of GLO I on TLSC702 Treatment in NCI-H522 Cells

NCI-H522 cells were transfected with expression vector for human GLO I (GLO I) or mock-transfected with empty vector (mock). 24 h post-transfection, the cells were treated with indicated concentrations of TLSC702 for 48 h. Cell viability (% of control) was measured by using WST assay. Data are the averages of three inde-pendent experiments and bars show the S.D. values.

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suppressive effect of TLSC702 on NCI-H522 cells showed a time-dependent manner, suggesting the involvement of MG accumulation in its apoptosis inducibility. Indeed, accumula-tion of argpyrimidine adducts was detected in TLSC702-treated NCI-H522 cells. Furthermore, overexpression of GLO I partially restore cell viability in TLSC702-treated NCI-H522 cells. These results strongly support that the antiproliferative and proapoptotic activities of TLSC702 are mediated by inhi-bition of GLO I activity in the cells. It should be emphasized that more experiments are necessary to elucidate whether TLSC702 induces apoptosis in GLO I-dependent tumor cells without harming healthy cells. These issues should be ad-dressed in future studies using more potent GLO I inhibitors derived from TLSC702.

To sum up, a novel GLO I inhibitory compound TLSC702, which has been discovered by using a myricetin-based hGLO I/inhibitor 4-point pharmacophore,20) could induce apoptosis in GLO I-dependent tumor cells. However, there remains a problem that higher doses of TLSC702 are required to induce apoptosis in tumor cells, compared with doses needed to in-hibit GLO I activity in vitro. If a reason of the problem is its poor permeability to cell membranes, structural improvements of TLSC702 by adding some hydrophobic groups at adequate positions are required. The purpose of our study is to create ultimately the human GLO I specific inhibitors, which are capable to use as anticancer drugs in vivo. Now, by using the unique pharmacophore of TLSC702 and in silico methods for structure-based drug design (SBDD), creation of novel lead small molecules for cancer therapeutics that have more potent and specific inhibitory activities on the human GLO I is under study.

Conflict of Interest The authors declare no conflict of interest.

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