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. 2023 Nov 14;29(1):52–62. doi: 10.1111/gtc.13084

The PKM2 inhibitor shikonin enhances piceatannol‐induced apoptosis of glyoxalase I‐dependent cancer cells

Manami Inoue 1, Yuki Nakagawa 1, Miku Azuma 1, Haruka Akahane 1, Ryusei Chimori 1, Yasunari Mano 1, Ryoko Takasawa 1,✉
PMCID: PMC11448369  PMID: 37963646

Abstract

Glyoxalase I (GLO I), a major enzyme involved in the detoxification of the anaerobic glycolytic byproduct methylglyoxal, is highly expressed in various tumors, and is regarded as a promising target for cancer therapy. We recently reported that piceatannol potently inhibits human GLO I and induces the death of GLO I‐dependent cancer cells. Pyruvate kinase M2 (PKM2) is also a potential therapeutic target for cancer treatment, so we evaluated the combined anticancer efficacy of piceatannol plus low‐dose shikonin, a potent and specific plant‐derived PKM2 inhibitor, in two GLO I‐dependent cancer cell lines, HL‐60 human myeloid leukemia cells and NCI‐H522 human non‐small‐cell lung cancer cells. Combined treatment with piceatannol and low‐dose shikonin for 48 h synergistically reduced cell viability, enhanced apoptosis rate, and increased extracellular methylglyoxal accumulation compared to single‐agent treatment, but did not alter PKM1, PKM2, or GLO I protein expression. Taken together, these results indicate that concomitant use of low‐dose shikonin potentiates piceatannol‐induced apoptosis of GLO I‐dependent cancer cells by augmenting methylglyoxal accumulation.

Keywords: anticancer, apoptosis, glyoxalase I, piceatannol, pyruvate kinase M2, shikonin

Shikonin potentiates piceatannol‐induced apoptosis of GLO I‐dependent cancer cells by augmenting methylglyoxal accumulation. Neither piceatannol alone, shikonin alone, nor combined treatment altered GLO I, PKM1, and PKM2 expression levels.

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1. INTRODUCTION

Glyoxalase I (GLO I) catalyzes the glutathione‐mediated detoxification of methylglyoxal, a byproduct of aerobic glycolysis (Thornalley, 1990). In rapidly growing tumors, cellular glucose metabolism is largely anaerobic, especially at the tumor core, suggesting that continued survival is dependent on efficient GLO I‐mediated elimination of methylglyoxal. Indeed, methylglyoxal induces apoptosis in tumor cells (Kang et al., 1996; Roy et al., 2017), and GLO I activity is upregulated in multiple human cancers, including lung (Sakamoto et al., 2001), colon (Ranganathan & Tew, 1993), pancreatic (Wang et al., 2012), stomach (Cheng et al., 2012), prostate (Burdelski et al., 2017), and breast (Tamori et al., 2018) cancers, as well as in melanoma (Bair III et al., 2010) and anticancer drug‐resistant human leukemia cells (Sakamoto et al., 2000). Hence, GLO I overexpression is considered essential to carcinogenesis and anticancer drug resistance, and specific GLO I inhibition may be an effective treatment strategy for tumors resistant to currently available chemotherapy drugs. We recently reported that piceatannol, a stilbene compound found in numerous plant species, potently inhibits human GLO I in vitro with an IC50 of 0.76 μM, induces the death of GLO I‐dependent cancer cells (Takasawa et al., 2017; Yamamoto et al., 2019), and suppresses tumor growth without body weight loss in mice (Yoshizawa et al., 2020).

The pyruvate kinase M2 (PKM2) isoenzyme (EC 2.7.1.40), a homotetramer that converts phosphoenolpyruvate to pyruvate, is also considered a potential therapeutic target for cancer treatment. In mammalian cells, there are four PK isoforms, PKL and PKR produced by alternative splicing of the PMLR gene, and PKM1 and PKM2 produced by alternative splicing of the PKM gene (Noguchi et al., 1986, 1987). PKM1 and PKM2 are major isoforms in normal adult and embryonic tissues, respectively, while PKL and PKR are exclusively expressed in hepatocytes and erythrocytes, respectively (Imamura & Tanaka, 1972; Nakashima et al., 1974). In contrast, tumor cells almost ubiquitously express PKM2 (Dombrauckas et al., 2005). The PK activity of PKM2 depends on its quaternary structure as the dimeric form is almost inactive (Mazurek et al., 2005). Cancer cell growth is suppressed by shifting the predominant PK expression profile from PKM2 to PKM1, indicating that high PKM2 expression is essential for cancer cell metabolism and proliferation (Christofk et al., 2008). Recently, shikonin, a natural product isolated from the medicinal plant Lithospermum erythrorhizon, was reported to potently and specifically inhibit PKM2 and exert antitumor effects (Chen et al., 2011; Thonsri et al., 2020).

In the present study, we investigated the combined anticancer efficacy of the GLO I inhibitor piceatannol and the PKM2 inhibitor shikonin. We report that low‐dose shikonin synergistically enhances piceatannol‐induced methylglyoxal accumulation in GLO I‐dependent cancer cells, thereby accelerating apoptosis.

2. RESULTS

2.1. Shikonin enhanced the antiproliferative efficacy of piceatannol on HL‐60 cells

The combined anticancer efficacy of piceatannol plus shikonin was tested using the human GLO I‐dependent promyelocytic leukemia cell line HL‐60 (Takasawa et al., 2008, 2010, 2016, 2022; Thornalley et al., 1996). We first examined the antiproliferative effects of single‐agent treatments to determine the optimal test concentration ranges. Piceatannol treatment for 48 h dose‐dependently suppressed the proliferation of HL‐60 cells (Figure 1a) with an IC50 value of about 20 μM, and shikonin treatment alone for 48 h dose‐dependently suppressed the proliferation of HL‐60 cells with an IC50 value of 1.9 μM (Figure 1b). Shikonin treatment for 48 h did not significantly influence cell viability at concentrations up to 0.5 μM (Figure 1b). From these results, we selected 0.25 μM as the shikonin test concentration for combination treatment and 0–20 μM as the piceatannol test range. Cells were then treated with vehicle (0 μM), 10 μM piceatannol, or 20 μM piceatannol with or without 0.25 μM shikonin for 24 or 48 h, and cell viability (% of control) measured using a WST‐based viable cell counting assay. From these results, EOBA scores were calculated to indicate the nature of the drug–drug interaction (antagonism, additivity, or synergy). Combined treatment with piceatannol plus shikonin for 24 h additively suppressed HL‐60 cell proliferation (Figure 2a), while combined treatment for 48 h produced a synergistic antiproliferative effect as indicated by an EOBA value >0.1 (Borisy et al., 2003; Rotin et al., 2016) (Figure 2b). Combined treatment for 48 h also reduced the IC50 value of piceatannol to 10.2 μM.

FIGURE 1.

FIGURE 1

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The dose‐dependent antiproliferative effects of piceatannol and shikonin alone on HL‐60 cells. Cells were treated with (a) 0 (vehicle control), 5, 10, or 20 μM piceatannol or (b) 0 (vehicle control), 0.125, 0.25, 0.5, or 1 μM shikonin for 48 h. Cell viability (% of vehicle control) was measured using a WST‐based cell counting assay. Results are the averages of triplicate experiments and bars show the standard error of the mean (SEM). Asterisks (* and ***) indicate p value smaller than .05 (p < .05) and .001 (p < .001), respectively.

FIGURE 2.

FIGURE 2

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Shikonin enhances the antiproliferative efficacy of piceatannol on HL‐60 cells. Cells were treated with 0 (vehicle control), 5, 10, or 20 μM piceatannol with or without 0.25 μM shikonin for (a) 24 h or (b) 48 h. Cell viability (% of control) was measured using a WST assay, and EOBA scores were calculated to assess the nature of drug–drug interactions (antagonism, additivity, or synergism). Results are the averages of three independent experiments and bars show the SEM.

2.2. Shikonin increased piceatannol‐induced accumulation of methylglyoxal in the culture medium of HL‐60 cells

Treatment of HL‐60 cells with the GLO I inhibitor BBGD or TLSC702 was reported to significantly increase methylglyoxal concentration in the culture medium (Azuma et al., 2021). Therefore, we measured the methylglyoxal concentration in the culture medium of HL‐60 cells treated with vehicle (control), piceatannol alone, shikonin alone, piceatannol plus shikonin, or BBGD (positive control) as an indicator of GLO I inhibition. Combined treatment with 20 μM piceatannol plus 0.25 μM shikonin significantly increased medium methylglyoxal concentration compared to 20 μM piceatannol or 0.25 μM shikonin alone treatment. Treatment with 20 μM piceatannol alone for 48 h increased medium methylglyoxal by 12‐fold, while treatment with 0.25 μM shikonin alone increased medium methylglyoxal by only 2‐fold compared to vehicle treatment. However, combined treatment with 20 μM piceatannol plus 0.25 μM shikonin increased the medium methylglyoxal concentration by approximately 23‐fold relative to control cells (Figure 3), suggesting that shikonin acts with piceatannol to synergistically enhance intracellular methylglyoxal accumulation.

FIGURE 3.

FIGURE 3

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Shikonin enhances piceatannol‐induced methylglyoxal accumulation in the culture medium of HL‐60 cells. Cells were treated with vehicle (control), 20 μM piceatannol alone, 0.25 μM shikonin alone, 20 μM piceatannol plus 0.25 μM shikonin, or 15 μM BBGD (positive control) for 48 h. The amount of MG in the culture medium was measured by HPLC/UV as described in Section 4. Results are the averages of two independent experiments and bars show the SEM. Asterisks (*, **, and ***) indicate p value smaller than .05 (p < .05), 0.01 (p < .01), and .001 (p < .001), respectively.

2.3. Shikonin enhanced the antiproliferative effect of piceatannol on NCI‐H522 cells

The human non‐small‐cell lung cancer cell line NCI‐H522 also expresses high levels of GLO I, and proliferation can be substantially suppressed by both pharmacological GLO I inhibition using BBGD (Sakamoto et al., 2001), TLSC702 (Takasawa et al., 2016), triphenylbismuth dichloride (Takasawa et al., 2022), or piceatannol (Takasawa et al., 2017), as well as by siRNA‐mediated knockdown of GLO I (Santarius et al., 2010). Therefore, we also evaluated the antiproliferative effects of combined treatment with piceatannol plus shikonin on NCI‐H522 cells. Piceatannol treatment alone for 48 h dose‐dependently suppressed the proliferation of NCI‐H522 cells with an IC50 of 25.2 μM (Figure 4a), and shikonin treatment alone for 48 h dose‐dependently suppressed the proliferation of NCI‐H522 cells with an IC50 value of 0.5 μM (Figure 4b). From these results, we selected shikonin concentrations of 0.13, 0.25, and 0.40 μM, equivalent to about 0.25, 0.5, and 0.75 times the IC50, respectively, for examination of combined effects with 5, 10, 20, and 40 μM piceatannol. Cotreatment with piceatannol and shikonin produced a synergistic antiproliferative effect on NCI‐H522 cells, especially with 0.25 μM shikonin, as indicated by the EOBA values (Figure 5a). Cotreatment with 0.13 μM shikonin yielded a CI value of 0.95, indicating an additive effect, while 0.25 μM yielded a CI value of 0.75 (Table 1; Figure 5b), indicating a synergistic effect.

FIGURE 4.

FIGURE 4

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The antiproliferative effects of piceatannol and shikonin alone on NCI‐H522 cells. NCI‐H522 cells were treated with (a) 0 (vehicle control), 5, 10, 20, or 40 μM piceatannol or (b) 0 (vehicle control), 0.1, 0.2, 0.4, or 0.8 μM shikonin for 48 h. Cell viability (% of control) was measured using the WST assay. Results are the averages of triplicate experiments and bars show the SEM. Asterisks (***) indicate p value smaller than .001 (p < .001) versus control.

FIGURE 5.

FIGURE 5

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Shikonin enhances the antiproliferative efficacy of piceatannol on NCI‐H522 cells. Cells were treated with 0 (vehicle control), 5, 10, 20, or 40 μM piceatannol plus 0 (vehicle), 0.13, 0.25, or 0.40 μM shikonin for 48 h. (a) Cell viability (% of control) was measured using a WST assay and EOBA scores were calculated. Results are the averages of three independent experiments and bars show the SEM. (b) Isobologram analysis to determine the half maximal inhibitory concentrations (IC50 values) of piceatannol plus shikonin on NCI‐H522 cell proliferation. The shikonin dose was fixed at 0.13 or 0.25 μM, corresponding to 0.26 (●) and 0.5 (▲) IC50 equivalents, respectively. Different concentrations of piceatannol (5–40 μM) were combined at fixed doses of shikonin. The line of additivity on the isobologram represents the 50% effect level for each drug. Synergy is indicated by values below the line of additivity (CI < 0.9), additivity by values near the line (CI = 0.9–1.1), and antagonism by values above the line (CI > 1.1).

TABLE 1.

Effects of combination piceatannol plus shikonin treatment on NCI‐H522 cell viability.

Concentration, IC50 equivalent Combination index at 50% effect level Evaluation at 50% effect level IC50 piceatannol IC50 shikonin
Piceatannol Shikonin
0.69 0.26 0.95 Additive 17.41 0.13
0.25 0.50 0.75 Moderate synergism 6.28 0.25
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2.4. Shikonin enhanced the pro‐apoptotic effects of piceatannol on NCI‐H522 cells

Multiple studies have been reported that GLO I inhibitors can induce cancer cell apoptosis, including NCI‐H522 cell apoptosis (Sakamoto et al., 2001; Shimada et al., 2018; Thornalley et al., 1996). To investigate if shikonin can enhance piceatannol‐induced apoptosis of NCI‐H522 cells, we measured poly(ADP‐ribose) polymerase (PARP) cleavage following treatment. Neither piceatannol alone (5, 10, or 20 μM) nor 0.25 μM shikonin alone induced detectable PARP cleavage (Figure 6a, lane 2–5), but cotreatment with 0.25 μM shikonin induced substantial piceatannol dose‐dependent PARP cleavage (Figure 6a, lane 6–8). Cotreatment with 0.25 μM shikonin significantly increased PARP cleavage in 20 μM piceatannol‐treated NCI‐H522 cells (Figure 6b).

FIGURE 6.

FIGURE 6

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Shikonin enhances piceatannol‐induced apoptosis of NCI‐H522 cells. (a) Cells were treated with 0 (vehicle control), 5, 10, or 20 μM piceatannol with or without 0.25 μM shikonin for 48 h. Equal amounts of protein from whole cell lysates (equivalent to 2 × 105 cells) were analyzed by Western blotting using an antibody specific for PARP (see Section 4). β‐actin expression was measured as the gel loading control. The results shown are representative of three independent experiments. (b) Relative level of cleaved PARP normalized to β‐actin (means ± SEM) was determined using Image J software (n = 3 experiments, *p < .05 compared with (−) Shikonin).

2.5. Neither piceatannol alone, shikonin alone, nor combined treatment altered GLO I, PKM1, and PKM2 expression levels in NCI‐H522 cells

Finally, we examined the effects of these treatments on the expression levels of GLO I, PKM1, and PKM2 proteins in NCI‐H522 cells. As shown in Figure 7a, PKM1 protein was below the limit of detection in NCI‐H522 cells, and not changed by treatment with 0.25 μM shikonin alone, 20 μM piceatannol alone, or combined treatment for 48 h. Furthermore, the expression levels of GLO I (Figure 7a,b) and PKM2 (Figure 7a,c) were not significantly altered by treatment with 0.25 μM shikonin alone, 20 μM piceatannol alone, or combined treatment for 48 h.

FIGURE 7.

FIGURE 7

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Effects of piceatannol and shikonin on GLO I, PKM1, and PKM2 expression levels in NCI‐H522 cells. (a) Cells were treated with 20 μM piceatannol with or without 0.25 μM shikonin for 48 h. Equal amounts of protein from whole cell lysates (equivalent to 2 × 105 cells) were analyzed by Western blotting using antibodies specific for GLO I, PKM1, and PKM2 (see Section 4). β‐actin expression was measured as the gel loading control. The results shown are representative of three independent experiments. (b) Relative level of GLO I normalized to β‐actin (means ± SEM) was determined using Image J software (n = 3 experiments). There is no significant difference among samples (p < .05) from Tukey's test. (c) Relative level of PKM2 normalized to β‐actin (means ± SEM) was determined using Image J software (n = 3 experiments). There is no significant difference among samples (p < .05) from Tukey's test.

3. DISCUSSION

We demonstrate that low‐dose (0.25 μM) shikonin, a natural PKM2 inhibitor, can enhance the dose‐dependent antiproliferative effect of the GLO I inhibitor piceatannol on cultured cancer cell lines. This drug–drug interaction was synergistic when piceatannol and shikonin were coapplied for 48 h but only additive after 24 h. These interactive effects on proliferation were also mirrored by the extent of methylglyoxal accumulation in the culture medium, as 48‐h treatment with 20 μM piceatannol alone increased methylglyoxal in the HL‐60 cell culture medium by 12‐fold and 0.25 μM shikonin alone increased methylglyoxal by only about twofold, while combined treatment increased methylglyoxal in the culture medium by 23‐fold. The GLO I substrate methylglyoxal is freely membrane permeable (Thornalley, 1988), so the observed increase in extracellular free methylglyoxal likely reflects augmented intracellular methylglyoxal accumulation. This further suggests that PKM2 inhibition by shikonin complements piceatannol‐induced GLO I blockade by facilitating a rise in intracellular methylglyoxal.

In our previous study, we have reported that the GLO I protein levels of HL‐60 cells and NCI‐H522 cells are higher than that of NCI‐H460 cells, and antiproliferative effect of TLSC702, one of the GLO I inhibitors that we have identified, on HL‐60 cells (IC50 = 404 μM) is almost as same as on NCI‐H522 cells (IC50 = 340 μM), whereas NCI‐H460 cells are resistant to TLSC702 (Takasawa et al., 2016). In this study, the effect of piceatannol in HL‐60 cells ((IC50 ≈ 20 μM) is also almost the same as that in NCI‐H522 cells (IC50 = 25.2 μM). We previously reported that piceatannol more significantly inhibited the proliferation of NCI‐H522 cells than that of NCI‐H460 cells (Takasawa et al., 2017). Given this, the higher expression of GLO I is suggested to be significantly contribute to the sensitivity of cancer cells to GLO I inhibitors, such as piceatannol and TLSC702.

We previously reported that the GLO I inhibitors BBGD, TLSC702, and triphenylbismuth dichloride also increased methylglyoxal in the culture medium of HL‐60 cells. The PK activity of PKM2 depends on its quaternary structure as dimers are largely inactive (Mazurek et al., 2005). In tumor cells, PKM2 predominantly exists in a dimeric form, which provides a metabolic advantage by facilitating anabolic reactions (Kroemer & Pouyssegur, 2008; Spoden et al., 2009). On the other hand, it has been reported that peptide aptamers specifically bind to PKM2 and create dimers, which inhibit tumor cell growth (Spoden et al., 2008, 2009). Furthermore, shikonin has been reported to inhibit the PK activity of PKM2 and induce cancer cell death (Chen et al., 2011). These reports indicate that the PK activity of tetrameric PKM2 supports cancer cell metabolism and survival. Since PK converts its substrate phosphoenolpyruvate into pyruvate during glycolysis, inhibition of tetrameric PKM2‐mediated PK activity may cause accumulation of glucose metabolic intermediates, including the triose phosphate intermediates dihydroxyacetone phosphate and glyceraldehyde‐3‐phosphate, which are subsequently converted to methylglyoxal via a nonenzymatic pathway (Phillips & Thornalley, 1993). Thus, our results suggest that low‐dose shikonin may gradually induce the accumulation of dihydroxyacetone phosphate and glyceraldehyde‐3‐phosphate, leading to progressive accumulation of methylglyoxal. Together with direct GLO I inhibition by piceatannol, shikonin‐mediated PKM2 blockade synergistically enhances cytotoxic methylglyoxal accumulation in GLO I‐dependent cancer cells.

We also found that low‐dose shikonin augmented the antiproliferative effect of piceatannol on the GLO I‐dependent cancer cell line NCI‐H522, and again the interaction was synergistic. Cotreatment with 0.25 μM shikonin also facilitated piceatannol dose‐dependent cleavage of PARP, a substrate of the apoptosis effector caspase‐3. In contrast, neither cotreatment nor single‐agent treatment altered GLO I, PKM1, and PKM2 expression levels. These results provide additional evidence that the antiproliferative and proapoptotic effects of combined piceatannol plus shikonin treatment are due simultaneous inhibition of GLO I and PKM2 enzymatic activities and ensuing methylglyoxal elevation.

In this study, concomitant use of low‐dose shikonin, a PKM2 inhibitor, potentiates GLO I inhibitor piceatannol‐induced apoptosis of GLO I‐dependent cancer cells by augmenting methylglyoxal accumulation at normal O2 condition. Therefore, we consider that inhibition of PKM2 activity can complement GLO I inhibition to enhance apoptosis induction in cancer cells even at normal O2 condition. However, anaerobic culture condition must induce HIF‐1α and promote glycolytic metabolism. Recently, it has been reported that in anaerobic culture condition, the flux of glucose consumption and formation of D‐lactate were increased and the expression of GLO I was decreased, associated with an increase in potency of the antiproliferative activity of a GLO I inhibitor BBGD (Alhujaily et al., 2021). PKM2 has been reported to physically interact with HIF‐1α in the nucleus and activate the transcription of glycolysis‐related genes such as glucose transporter 1, lactate dehydrogenase A, and pyruvate dehydrogenase kinase 1 in cancer cells (Luo et al., 2011). Considering these points, it is important to evaluate the effects of piceatannol and shikonin on viability and apoptotic activities in glycolytic metabolism‐promoted tumor cells in anaerobic culture condition and the issue should be addressed in future studies.

In conclusion, we demonstrate that low‐dose shikonin can synergistically augment the cytotoxicity of piceatannol against GLO I‐dependent cancer cells. This combined treatment was associated with greatly enhanced accumulation of the GLO I substrate methylglyoxal but not with altered expression of PKM1, PKM2, and GLO I proteins, suggesting that shikonin‐mediated inhibition of PKM2 activity complements piceatannol‐mediated inhibition of GLO I by further enhancing methylglyoxal accumulation, leading to synergistically greater apoptosis induction. We previously reported that the combination of TLSC702, another GLO I inhibitor that we identified, with shikonin also suppressed the proliferation of GLO I‐dependent cancer cells and induced apoptosis (Shimada et al., 2018). Therefore, the current study provides further experimental support for the anticancer efficacy of combination GLO I and PKM2 inhibition.

4. EXPERIMENTAL PROCEDURES

4.1. Materials

Piceatannol, o‐phenylene diamine (o‐PD), and 5‐methylquinoxaline (5‐MQ) were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan), while shikonin and 2‐methylquinoxaline (2‐MQ) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). S‐p‐bromobenzylglutathione cyclopentyl diester (BBGD) was a gift from Taiho Pharmaceutical Co., Ltd. (Tokyo, Japan). Primary antibodies against PARP, PKM1, and PKM2, as well as horseradish peroxidase‐conjugated antirabbit and antimouse immunoglobulin G (IgG) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). An antibody against GLO I was purchased from Novus Biologicals (Centennial, CO, USA) and an antibody against β‐actin was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). All other chemicals used were of reagent grade.

4.2. Cell culture

The human myeloid leukemia cell line HL‐60 and the human non‐small‐cell lung cancer cell line NCI‐H522 were maintained in RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2 in air (~20% O2).

4.3. Measurements of cell viability

Cells were seeded in 96‐well plates and cultured for 24 h. In single‐agent treatment experiments, HL‐60 cells were incubated with piceatannol (5, 10, and 20 μM) or shikonin (0.125, 0.25, 0.5, or 1 μM) for 48 h. These concentrations were adjusted slightly for single‐agent treatment of NCI‐H522 cells (piceatannol: 5, 10, 20, or 40 μM); shikonin: 0.1, 0.2, 0.4, or 0.8 μM). In all cases, vehicle‐treated cultures in the same 96‐well plates served as controls. To investigate the combined effects of piceatannol plus shikonin, HL‐60 cells were treated with 0 (vehicle control), 5, 10, or 20 μM piceatannol plus 0.25 μM shikonin for 24 or 48 h, while NCI‐H522 cells were treated with 0 (vehicle control), 5, 10, 20, or 40 μM piceatannol plus 0.13, 0.25, or 0.40 μM shikonin for 48 h. The number of viable cells remaining in each well post‐treatment was estimated using a Cell Counting Kit‐8 according to the manufacturer's instructions (Dojindo Laboratories, Kumamoto, Japan). The absorbance of formazan dye formed from WST‐8 by viable cells was measured at 450 nm using a SpectraMax Microplate Reader (Molecular Devices, San Jose, CA, USA).

4.4. Evaluation of drug combinatorial effects

The combinatorial effects of piceatannol and shikonin were evaluated using an excess‐over‐Bliss additivism (EOBA) model (Borisy et al., 2003; Rotin et al., 2016). The model is defined by the formula

EOBA=C−A+B−A×B,

where C is the fractional inhibition of both drugs simultaneously, A is the fractional inhibition of drug A, B is the fractional inhibition of drug B, and fractional inhibition is equal to 1.0 minus the viability (expressed as a value from 0.0 to 1.0). Positive, negative, and near‐zero EOBA values reflect synergistic, antagonistic, and additive drug combinations, respectively.

4.5. Combination index analysis and construction of a normalized isobologram

Quantitative evaluation of nonconstant ratio combinations (fixed‐dose combinations) of piceatannol and shikonin was performed to measure drug interactions at the 50% effect level. Combination index (CI) values representing pharmacological drug interactions were determined for combination treatment using the Chou–Talalay method (Chou & Talalay, 1984). Based on single‐agent IC50 measurements, NCI‐H522 cells were treated for 48 h with 5–40 μM piceatannol and either 0.13 or 0.25 μM shikonin, corresponding to IC50 equivalents of 0.26 and 0.5, respectively. These IC50 equivalent concentrations of shikonin were calculated as the actual concentration used in combination treatment divided by the corresponding single‐agent IC50 value. Similarly, the IC50 equivalent concentrations of piceatannol were calculated as the IC50 value of piceatannol used in combination with shikonin divided by the corresponding single‐agent IC50 value. The CI values at the 50% effect level were calculated using the CI formula by adding the corresponding IC50 equivalent values of shikonin and piceatannol. The line of additivity in the isobologram represents the 50% effect level of each drug. Accordingly, CI >1.1 indicates antagonism, CI = 0.9–1.1 an additive effect, CI = 0.8–0.9 slight synergism, CI = 0.6–0.8 moderate synergism, CI = 0.4–0.6 synergism, and CI = 0.2–0.4 strong synergism.

4.6. Determination of methylglyoxal concentration in the culture medium

Methylglyoxal concentration was measured in the culture medium of HL‐60 cells according to the method of Hikita et al. (2015), which is based on the derivatization of methylglyoxal with o‐PD and subsequent measurement of the product 2‐MQ by HPLC/UV. Briefly, HL‐60 cells were seeded at 3.0 × 106 per 6‐cm dish and treated with vehicle (control), 20 μM piceatannol alone, 0.25 μM shikonin alone, 20 μM piceatannol plus 0.25 μM shikonin, or 15 μM BBGD as the positive control for 48 h in a CO2 incubator maintained at 37°C. After treatment, the culture medium was retrieved, centrifuged at 1300 rpm for 10 min, and mixed in 4.5‐mL aliquots with perchloric acid for deproteinization. The mixture was centrifuged at 8000 rpm for 10 min and the supernatant applied directly to a Sep‐Pak Light tC18 cartridge (Waters Corporation, Milford, MA, USA). The flow‐through fraction was collected and supplemented with 125 nmol o‐PD and 12.5 nmol 5‐MQ as an internal standard. After standing for 3.5 h at room temperature, the reaction mixture was applied to another Sep‐Pak Light tC18 cartridge. The cartridge was washed with 10 mM KH2PO4 (pH 2.5), and then 2‐MQ and 5‐MQ were eluted using 2 mL acetonitrile. The eluate was evaporated to dryness under nitrogen flow and the residue reconstituted in 100 μL of mobile phase. A 20‐μL sample was injected into an Extrema HPLC instrument (JASCO Corporation, Tokyo, Japan) equipped with a UV‐4075 UV/VIS detector (JASCO Corporation) and reverse‐phase TSKgel ODS‐80Ts column (4.6 × 250, 5 μm; Tosoh Corporation, Tokyo, Japan). The 2‐MQ product and internal standard 5‐MQ were separated using 68 vol% 10 mM KH2PO4 (pH 2.5) and 32 vol% acetonitrile as mobile phases and concentrations measured by the UV detector at 315 nm.

4.7. Western blotting

Cells treated as indicated were homogenized in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA). Total cellular proteins were then separated by SDS‐PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk and 0.25% bovine serum albumin in Tris‐buffered saline containing 0.1% Tween 20 (TTBS) for 1 h at room temperature, and then probed with the indicated primary antibodies overnight at 4°C. Membranes were washed with TTBS, incubated with the appropriate secondary antibody for 1 h at room temperature, and washed again in TTBS. Immunolabeled proteins were visualized using ImmunoStar LD reagent (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and an iBright Western Blot Imaging System (Thermo Fisher Scientific K.K., Tokyo, Japan).

4.8. Statistical analysis

Data are presented as means ± standard errors (SEs). Statistical analyses were performed using Microsoft Excel or EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (R Foundation for Statistical Computing, Vienna, Austria). EZR is a modified version of R commander designed to add frequently used statistical functions in biostatistics (Kanda, 2013). Significant differences between two groups, among more than two groups, and for multiple comparisons versus the control (untreated) group were evaluated using Student's t‐test, one‐way ANOVA (Tukey's test), and one‐way ANOVA (Dunnett's test), respectively. Results with p < 0.05 were considered significant.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This work was supported by a Grant‐in‐Aid for Scientific Research (C) from the Japan Society for the Promotion of Science to Ryoko Takasawa (JSPS KAKENHI Grant Number JP19K05737). We would like to thank Dr Sei‐ichi Tanuma for discussion.

Inoue, M. , Nakagawa, Y. , Azuma, M. , Akahane, H. , Chimori, R. , Mano, Y. , & Takasawa, R. (2024). The PKM2 inhibitor shikonin enhances piceatannol‐induced apoptosis of glyoxalase I‐dependent cancer cells. Genes to Cells, 29(1), 52–62. 10.1111/gtc.13084

Communicated by: Hidenori Ichijo

REFERENCES

  1. Alhujaily, M. , Abbas, H. , Xue, M. , de la Fuente, A. , Rabbani, N. , & Thornalley, P. J. (2021). Studies of glyoxalase 1‐linked multidrug resistance reveal glycolysis‐derived reactive metabolite, methylglyoxal, is a common contributor in cancer chemotherapy targeting the spliceosome. Frontiers in Oncology, 11, 748698. 10.3389/fonc.2021.748698 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Azuma, M. , Inoue, M. , Nishida, A. , Akahane, H. , Kitajima, M. , Natani, S. , Chimori, R. , Yoshimori, A. , Mano, Y. , Uchiro, H. , Tanuma, S.‐I. , & Takasawa, R. (2021). Addition of hydrophobic side chains improve the apoptosis inducibility of the human glyoxalase I inhibitor, TLSC702. Bioorganic & Medicinal Chemistry Letters, 40, 127918. 10.1016/j.bmcl.2021.127918 [DOI] [PubMed] [Google Scholar]
  3. Bair, W. B., III , Cabello, C. M. , Uchida, K. , Bause, A. S. , & Wondrak, G. T. (2010). GLO1 overexpression in human malignant melanoma. Melanoma Research, 20(2), 85–96. 10.1097/CMR.0b013e3283364903 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borisy, A. A. , Elliott, P. J. , Hurst, N. W. , Lee, M. S. , Lehar, J. , Price, E. R. , Serbedzija, G. , Zimmermann, G. R. , Foley, M. A. , Stockwell, B. R. , & Keith, C. T. (2003). Systematic discovery of multicomponent therapeutics. Proceedings of the National Academy of Sciences of the United States of America, 100(13), 7977–7982. 10.1073/pnas.1337088100 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burdelski, C. , Shihada, R. , Hinsch, A. , Angerer, A. , Göbel, C. , Friedrich, E. , Hube‐Magg, C. , Burdak‐Rothkamm, S. , Kluth, M. , Simon, R. , Möller‐Koop, C. , Sauter, G. , Büscheck, F. , Wittmer, C. , Clauditz, T. S. , Krech, T. , Tsourlakis, M. C. , Minner, S. , Graefen, M. , … Jacobsen, F. (2017). High‐level glyoxalase 1 (GLO1) expression is linked to poor prognosis in prostate cancer. The Prostate, 77(15), 1528–1538. 10.1002/pros.23431 [DOI] [PubMed] [Google Scholar]
  6. Chen, J. , Xie, J. , Jiang, Z. , Wang, B. , Wang, Y. , & Hu, X. (2011). Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase‐M2. Oncogene, 30(42), 4297–4306. 10.1038/onc.2011.137 [DOI] [PubMed] [Google Scholar]
  7. Cheng, W. L. , Tsai, M. M. , Tsai, C. Y. , Huang, Y. H. , Chen, C. Y. , Chi, H. C. , Tseng, Y.‐H. , Chao, I.‐W. , Lin, W.‐C. , Wu, S.‐M. , Liang, Y. , Liao, C.‐J. , Lin, Y.‐H. , Chung, I.‐H. , Chen, W.‐J. , Lin, P. Y. , Wang, C.‐S. , & Lin, K. H. (2012). Glyoxalase‐I is a novel prognosis factor associated with gastric cancer progression. PLoS One, 7(3), e34352. 10.1371/journal.pone.0034352 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chou, T. C. , & Talalay, P. (1984). Quantitative analysis of dose‐effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Advances in Enzyme Regulation, 22, 27–55. 10.1016/0065-2571(84)90007-4 [DOI] [PubMed] [Google Scholar]
  9. Christofk, H. R. , Vander Heiden, M. G. , Harris, M. H. , Ramanathan, A. , Gerszten, R. E. , Wei, R. , Fleming, M. D. , Schreiber, S. L. , & Cantley, L. C. (2008). The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature, 452(7184), 230–233. 10.1038/nature06734 [DOI] [PubMed] [Google Scholar]
  10. Dombrauckas, J. D. , Santarsiero, B. D. , & Mesecar, A. D. (2005). Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry, 44(27), 9417–9429. 10.1021/bi0474923 [DOI] [PubMed] [Google Scholar]
  11. Hikita, K. , Yamada, S. , Shibata, R. , Katoh, M. , Murata, T. , Kato, K. , Tanaka, H. , & Kaneda, N. (2015). Inhibitory effect of isoflavones from Erythrina poeppigiana on the growth of HL‐60 human leukemia cells through inhibition of glyoxalase I. Natural Product Communications, 10(9), 1581–1584. [PubMed] [Google Scholar]
  12. Imamura, K. , & Tanaka, T. (1972). Multimolecular forms of pyruvate kinase from rat and other mammalian tissues. I. Electrophoretic studies. Journal of Biochemistry, 71(6), 1043–1051. 10.1093/oxfordjournals.jbchem.a129852 [DOI] [PubMed] [Google Scholar]
  13. Kanda, Y. (2013). Investigation of the freely available easy‐to‐use software “EZR” for medical statistics. Bone Marrow Transplantation, 48(3), 452–458. 10.1038/bmt.2012.244 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kang, Y. , Edwards, L. G. , & Thornalley, P. J. (1996). Effect of methylglyoxal on human leukaemia 60 cell growth: Modification of DNA, G1 growth arrest and induction of apoptosis. Leukemia Research, 20(5), 397–405. 10.1016/0145-2126(95)00162-x [DOI] [PubMed] [Google Scholar]
  15. Kroemer, G. , & Pouyssegur, J. (2008). Tumor cell metabolism: cancer's Achilles' heel. Cancer Cell, 13(6), 472–482. 10.1016/j.ccr.2008.05.005 [DOI] [PubMed] [Google Scholar]
  16. Luo, W. , Hu, H. , Chang, R. , Zhong, J. , Knabel, M. , O'Meally, R. , Cole, R. N. , Pandey, A. , & Semenza, G. L. (2011). Pyruvate kinase M2 is a PHD3‐stimulated coactivator for hypoxia‐inducible factor 1. Cell, 145(5), 732–744. 10.1016/j.cell.2011.03.054 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mazurek, S. , Boschek, C. B. , Hugo, F. , & Eigenbrodt, E. (2005). Pyruvate kinase type M2 and its role in tumor growth and spreading. Seminars in Cancer Biology, 15(4), 300–308. 10.1016/j.semcancer.2005.04.009 [DOI] [PubMed] [Google Scholar]
  18. Nakashima, K. , Miwa, S. , Oda, S. , Tanaka, T. , & Imamura, K. (1974). Electrophoretic and kinetic studies of mutant erythrocyte pyruvate kinases. Blood, 43(4), 537–548. [PubMed] [Google Scholar]
  19. Noguchi, T. , Inoue, H. , & Tanaka, T. (1986). The M1‐ and M2‐type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. The Journal of Biological Chemistry, 261(29), 13807–13812. [PubMed] [Google Scholar]
  20. Noguchi, T. , Yamada, K. , Inoue, H. , Matsuda, T. , & Tanaka, T. (1987). The L‐ and R‐type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. The Journal of Biological Chemistry, 262(29), 14366–14371. [PubMed] [Google Scholar]
  21. Ranganathan, S. , & Tew, K. D. (1993). Analysis of glyoxalase‐I from normal and tumor tissue from human colon. Biochimica et Biophysica Acta, 1182(3), 311–316. 10.1016/0925-4439(93)90074-b [DOI] [PubMed] [Google Scholar]
  22. Rotin, L. E. , Gronda, M. , MacLean, N. , Hurren, R. , Wang, X. , Lin, F.‐H. , Wrana, J. , Datti, A. , Barber, D. L. , Minden, M. D. , Slassi, M. , & Schimmer, A. D. (2016). Ibrutinib synergizes with poly(ADP‐ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK‐independent mechanism. Oncotarget, 7(3), 2765–2779. 10.18632/oncotarget.6409 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Roy, A. , Ahir, M. , Bhattacharya, S. , Parida, P. K. , Adhikary, A. , Jana, K. , & Ray, M. (2017). Induction of mitochondrial apoptotic pathway in triple negative breast carcinoma cells by methylglyoxal via generation of reactive oxygen species. Molecular Carcinogenesis, 56(9), 2086–2103. 10.1002/mc.22665 [DOI] [PubMed] [Google Scholar]
  24. Sakamoto, H. , Mashima, T. , Kizaki, A. , Dan, S. , Hashimoto, Y. , Naito, M. , & Tsuruo, T. (2000). Glyoxalase I is involved in resistance of human leukemia cells to antitumor agent‐induced apoptosis. Blood, 95(10), 3214–3218. [PubMed] [Google Scholar]
  25. Sakamoto, H. , Mashima, T. , Sato, S. , Hashimoto, Y. , Yamori, T. , & Tsuruo, T. (2001). Selective activation of apoptosis program by S‐p‐bromobenzylglutathione cyclopentyl diester in glyoxalase I‐overexpressing human lung cancer cells. Clinical Cancer Research, 7(8), 2513–2518. [PubMed] [Google Scholar]
  26. Santarius, T. , Bignell, G. R. , Greenman, C. D. , Widaa, S. , Chen, L. , Mahoney, C. L. , Butler, A. , Edkins, S. , Waris, S. , Thornalley, P. J. , Futreal, P. A. , & Stratton, M. R. (2010). GLO1—A novel amplified gene in human cancer. Genes, Chromosomes & Cancer, 49(8), 711–725. 10.1002/gcc.20784 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shimada, N. , Takasawa, R. , & Tanuma, S. (2018). Interdependence of GLO I and PKM2 in the metabolic shift to escape apoptosis in GLO I‐dependent cancer cells. Archives of Biochemistry and Biophysics, 638, 1–7. 10.1016/j.abb.2017.12.008 [DOI] [PubMed] [Google Scholar]
  28. Spoden, G. A. , Mazurek, S. , Morandell, D. , Bacher, N. , Ausserlechner, M. J. , Jansen‐Dürr, P. , Eigenbrodt, E. , & Zwerschke, W. (2008). Isotype‐specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation. International Journal of Cancer, 123(2), 312–321. 10.1002/ijc.23512 [DOI] [PubMed] [Google Scholar]
  29. Spoden, G. A. , Rostek, U. , Lechner, S. , Mitterberger, M. , Mazurek, S. , & Zwerschke, W. (2009). Pyruvate kinase isoenzyme M2 is a glycolytic sensor differentially regulating cell proliferation, cell size and apoptotic cell death dependent on glucose supply. Experimental Cell Research, 315(16), 2765–2774. 10.1016/j.yexcr.2009.06.024 [DOI] [PubMed] [Google Scholar]
  30. Takasawa, R. , Akahane, H. , Tanaka, H. , Shimada, N. , Yamamoto, T. , Uchida‐Maruki, H. , Sai, M. , Yoshimori, A. , & Tanuma, S. (2017). Piceatannol, a natural trans‐stilbene compound, inhibits human glyoxalase I. Bioorganic & Medicinal Chemistry Letters, 27(5), 1169–1174. 10.1016/j.bmcl.2017.01.070 [DOI] [PubMed] [Google Scholar]
  31. Takasawa, R. , Jona, A. , Inoue, M. , Azuma, M. , Akahane, H. , Ueno, Y. , Nakagawa, Y. , Chimori, R. , Mano, Y. , Murata, Y. , Yasuike, S. , & Kaji, T. (2022). Triphenylbismuth dichloride inhibits human glyoxalase I and induces cytotoxicity in cultured cancer cell lines. The Journal of Toxicological Sciences, 47(12), 539–546. 10.2131/jts.47.539 [DOI] [PubMed] [Google Scholar]
  32. Takasawa, R. , Saeki, K. , Tao, A. , Yoshimori, A. , Uchiro, H. , Fujiwara, M. , & Tanuma, S. (2010). Delphinidin, a dietary anthocyanidin in berry fruits, inhibits human glyoxalase I. Bioorganic & Medicinal Chemistry, 18(19), 7029–7033. 10.1016/j.bmc.2010.08.012 [DOI] [PubMed] [Google Scholar]
  33. Takasawa, R. , Shimada, N. , Uchiro, H. , Takahashi, S. , Yoshimori, A. , & Tanuma, S. (2016). TLSC702, a novel inhibitor of human glyoxalase I, induces apoptosis in tumor cells. Biological & Pharmaceutical Bulletin, 39(5), 869–873. 10.1248/bpb.b15-00710 [DOI] [PubMed] [Google Scholar]
  34. Takasawa, R. , Takahashi, S. , Saeki, K. , Sunaga, S. , Yoshimori, A. , & Tanuma, S. (2008). Structure–activity relationship of human GLO I inhibitory natural flavonoids and their growth inhibitory effects. Bioorganic & Medicinal Chemistry, 16(7), 3969–3975. 10.1016/j.bmc.2008.01.031 [DOI] [PubMed] [Google Scholar]
  35. Tamori, S. , Nozaki, Y. , Motomura, H. , Nakane, H. , Katayama, R. , Onaga, C. , Kikuchi, E. , Shimada, N. , Suzuki, Y. , Noike, M. , Hara, Y. , Sato, K. , Sato, T. , Yamamoto, K. , Hanawa, T. , Imai, M. , Abe, R. , Yoshimori, A. , Takasawa, R. , … Akimoto, K. (2018). Glyoxalase 1 gene is highly expressed in basal‐like human breast cancers and contributes to survival of ALDH1‐positive breast cancer stem cells. Oncotarget, 9(92), 36515–36529. 10.18632/oncotarget.26369 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Thonsri, U. , Seubwai, W. , Waraasawapati, S. , Wongkham, S. , Boonmars, T. , Cha'on, U. , & Wongkham, C. (2020). Antitumor effect of shikonin, a PKM2 inhibitor, in cholangiocarcinoma cell lines. Anticancer Research, 40(9), 5115–5124. 10.21873/anticanres.14515 [DOI] [PubMed] [Google Scholar]
  37. Thornalley, P. J. (1988). Modification of the glyoxalase system in human red blood cells by glucose in vitro. The Biochemical Journal, 254(3), 751–755. 10.1042/bj2540751 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Thornalley, P. J. (1990). The glyoxalase system: New developments towards functional characterization of a metabolic pathway fundamental to biological life. The Biochemical Journal, 269(1), 1–11. 10.1042/bj2690001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Phillips, S. A. , & Thornalley, P. J. (1993). The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal. European Journal of Biochemistry, 212(1), 101–105. 10.1111/j.1432-1033.1993.tb17638.x [DOI] [PubMed] [Google Scholar]
  40. Thornalley, P. J. , Edwards, L. G. , Kang, Y. , Wyatt, C. , Davies, N. , Ladan, M. J. , & Double, J. (1996). Antitumour activity of S‐p‐bromobenzylglutathione cyclopentyl diester in vitro and in vivo: Inhibition of glyoxalase I and induction of apoptosis. Biochemical Pharmacology, 51(10), 1365–1372. 10.1016/0006-2952(96)00059-7 [DOI] [PubMed] [Google Scholar]
  41. Wang, Y. , Kuramitsu, Y. , Ueno, T. , Suzuki, N. , Yoshino, S. , Iizuka, N. , Akada, J. , Kitagawa, T. , Oka, M. , & Nakamura, K. (2012). Glyoxalase I (GLO1) is up‐regulated in pancreatic cancerous tissues compared with related non‐cancerous tissues. Anticancer Research, 32(8), 3219–3222. [PubMed] [Google Scholar]
  42. Yamamoto, T. , Sato, A. , Takai, Y. , Yoshimori, A. , Umehara, M. , Ogino, Y. , Inada, M. , Shimada, N. , Nishida, A. , Ichida, R. , Takasawa, R. , Maruki‐Uchida, H. , Mori, S. , Sai, M. , Morita, M. , & Tanuma, S. (2019). Effect of piceatannol‐rich passion fruit seed extract on human glyoxalase I‐mediated cancer cell growth. Biochemistry and Biophysics Reports, 20, 100684. 10.1016/j.bbrep.2019.100684 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yoshizawa, K. , Tabuchi, M. , Ukai, S. , Suzuki, H. , Kawano, Y. , & Takasawa, R. (2020). The putative glyoxalase 1 inhibitor piceatannol exhibits both anxiolytic‐like and antitumor effects in mice. Anticancer Research, 40(6), 3271–3276. 10.21873/anticanres.14309 [DOI] [PubMed] [Google Scholar]

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