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GLUT1 inhibition by BAY-876 induces metabolic changes and cell death in human colorectal cancer cells

BMC Cancer volume 25, Article number: 716 (2025) Cite this article

Abstract

Background

Glucose transporter 1 (GLUT1) is known to play a crucial role in glucose uptake in malignant tumors. GLUT1 inhibitors reportedly exhibit anti-tumor effects by suppressing cancer cell proliferation. BAY-876, a selective GLUT1 inhibitor, has been shown to inhibit tumor growth in ovarian and breast cancers. In this study, we investigated the anti-proliferative effects of BAY-876 treatment in human colorectal cancer (CRC) cell lines.

Methods

We investigated the metabolic changes and effects on proliferation from BAY-876 treatment in HCT116, DLD1, COLO205, LoVo, and Caco-2 cells in vitro. Additionally, a mouse xenograft model was established using HCT116 cells to examine the tumor-inhibitory effects of BAY-876 treatment in vivo.

Results

BAY-876 treatment inhibited cell proliferation in HCT116, DLD1, COLO205, and LoVo cells. Reduced GLUT1 protein expression levels were observed through western blot analysis. Flux analysis indicated enhanced mitochondrial respiration, accompanied by increased reactive oxygen species levels and apoptosis rates. Tumor-inhibitory effects were also observed in the xenograft model, with the BAY-876-treated groups showing GLUT1 suppression.

Conclusions

BAY-876 treatment induced metabolic changes and inhibited cell proliferation in human CRC cell lines. Using BAY-876 is a potential novel approach for treating CRC.

Peer Review reports

Background

Colorectal cancer (CRC) is the third most common cancer and second leading cause of cancer-related deaths worldwide [1], making it a global health issue with an urgent unmet need for new therapeutic strategies [2]. Numerous novel treatment methods have been developed, which have been used in combination with advanced diagnostic methods and have led to a steady improvement in treatment results [3]. However, at advanced stages, the disease becomes challenging to treat. The tumors eventually develop resistance to most forms of combination therapy, making CRC metastasis a leading cause of cancer-related deaths [4].

The reprogramming of energy metabolism has recently emerged as a new hallmark of cancer [5]. Rather than relying on mitochondrial oxidative phosphorylation (OXPHOS), most cancer cells increase glucose consumption and convert it to lactate, a phenomenon known as the “Warburg effect” [5, 6]. Numerous studies have revealed that various human malignancies display increased glycolysis, even in the presence of an adequate oxygen supply to support aerobic respiration [5, 7, 8]. It is unclear how many components of the glycolytic pathway are involved in mediating active glycolysis in a malignant setting [9]. The glycolysis pathway includes many enzymes, including hexokinase 2, phosphofructokinase 2, lactate dehydrogenase A, and pyruvate dehydrogenase kinase-1 (PDK1), in various types of cancers. Many of them are coordinately regulated by common signaling mechanisms related to oncoprotein activation, tumor suppressor loss, or environmental changes [10]. Glucose transmembrane transport is the first step of glucose metabolism, as well as the rate-limiting step of glycolysis [11]. There are two classes of glucose transporters: sodium-dependent glucose transporter (SGLT) and facilitative glucose transporter (GLUT) [12]. As a member of the glucose transporter family, glucose transporter 1 (GLUT1) regulates glucose transport across the cell membrane [13]. The Warburg effect confers several advantages to cancer cells, particularly by providing intermediates for different biosynthetic pathways required for unrestrained proliferation. It also supports cancer cell adaptation to the hypoxic conditions often observed in solid tumors [14, 15]. In a hypoxic environment, hypoxia-inducible factor 1 (HIF-1) is activated, leading to the activation of its downstream genes, GLUT1 and PDK1. This results in increased glycolysis in response to hypoxic conditions. Another consequence of the Warburg effect is the decreased usage of the mitochondrial respiratory chain from reduced OXPHOS and oxygen consumption [14]. Under such circumstances, PDK1 plays a crucial role as a glycolytic enzyme by inhibiting the mitochondrial oxidation of pyruvate under hypoxic conditions, which blocks pyruvate from being converted into acetyl-CoA by phosphorylation of pyruvate dehydrogenase [16]. Therefore, PDK1 is a critical enzyme for attenuating mitochondrial reactive oxygen species (ROS) production and maintaining adenosine triphosphate (ATP) levels [17]. GLUT1 overexpression in cancer cells protects them from glucose deprivation-induced oxidative stress and enhances their resistance to apoptosis [18, 19]. Additionally, GLUT1 overexpression in CRC is reportedly independently associated with poor prognosis [20]. GLUT1 activity is regulated not only by its expression levels, but also by protein trafficking to the plasma membrane [9]. The activities of HIF-1 and c-Myc, as well as mutations in the tumor suppressor protein p53, have been linked to the overexpression or membrane localization of GLUT1 [21,22,23].

Recently, a potent GLUT1 inhibitor, BAY-876, was identified in a screening of approximately 3 million compounds [24]. Preclinical investigations of the efficacy of this GLUT1 inhibitor alone or in combination with other cancer therapeutics have shown promising results [25,26,27]. BAY-876 has been shown to have anti-tumor effects in esophageal cancer and ovarian cancer in vivo [9, 28]. These studies showed that BAY-876 treatment can alter the glycolytic enzyme expression levels and glycolytic rate in cells, indicating that a metabolic shift (Warburg change) was occurring. However, the effect of BAY-876 on CRC cell glycolytic metabolism is yet to be determined.

In this study, we used five human CRC cell lines (HCT116, COLO205, DLD1, LoVo, and Caco-2) to investigate the anti-proliferative effects of BAY-876 treatment. We hypothesized that BAY-876 can downregulate glucose transport and glycolysis by inhibiting GLUT1, which should effectively inhibit cancer growth. Additionally, BAY-876 treatment can induce a metabolic shift, leading to increased ROS accumulation and cell death. Our results suggest a potential novel anti-tumor approach using BAY-876 for CRC treatment.

Methods

Cell lines and cell culture

Five human CRC cell lines were used in this study. HCT116, Caco-2, and LoVo cells were purchased from the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan), while DLD1 and COLO205 cells were purchased from the Cell Response Center for Biochemical Research Institute of Development, Aging and Cancer, Tohoku University (Sendai, Miyagi, Japan). The mutation statuses of the CRC cell lines are listed in Supplementary Table 1. HCT116 and LoVo cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS). Caco-2 cells were cultured in DMEM with 20% FBS. DLD1 and COLO205 cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco) supplemented with 10% FBS. HCT116, Caco-2, and COLO205 cells were cultured on plastic dishes, 2-well plates, 6-well plates, and 96-well plates coated with Type I collagen solution (0.5% Atelocollagen Acidic Solution IPC-50; Koken Co., Ltd., Tokyo, Japan) at 37°C in a 5% CO2 atmosphere.

Chemicals

BAY-876 was obtained from Selleck Chemical Ind. Co. (Kanagawa, Japan). BAY-876 was dissolved in dimethyl sulfoxide (DMSO), then diluted to the required concentrations in medium for the in vitro study or dissolved in carboxymethylcellulose (CMC) for the in vivo study. Cobalt chloride (CoCl2) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay

MTS assays were performed using the CellTiter96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA). HCT116, Caco-2, LoVo, DLD1, and COLO205 cells were seeded into 96-well plates (5,000 cells/well) and incubated for 24 h. The cells were treated with BAY-876 diluted to various concentrations, with DMSO used as a negative control. After 24 h, cell viability was measured according to the manufacturer’s instructions. Briefly, 20 μL of MTS reagent was added directly to the wells, and the plates were then incubated at 37°C for 2 h. Absorbance values were measured at 490 nm using a microplate spectrophotometer (Multiskan GO; Thermo Fisher Scientific), then normalized to and expressed as a relative percentage of the plate-averaged DMSO control value.

Cell growth assay

To assess the effect of BAY-876 treatment on proliferation, 1.0 × 105 cells/well were cultured in 6-well plates (VIOLAMO, Osaka, Japan) for 24 h, then treated with a wide range of BAY-876 concentrations for three days. After treatment, the cells were counted using the automated cell counter EVE (AR Brown Co., Ltd., Tokyo, Japan) after trypsinization.

GLUT1 knockdown assay

For cell counts, 3,000 cells were seeded per well in 96-well black/clear bottom plates and transfected with 0.5 μM of small interfering RNAs (siRNAs) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. The siRNA duplexes targeting GLUT1 (siRNA IDs: SASI_Hs01_00028732 and SASI_Hs02_000341745) and control siRNAs were purchased from Sigma-Aldrich Japan (Tokyo, Japan).

The cell numbers in each well were counted 72 h after transfection using a cell number normalization kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions.

Quantitative real-time RT-PCR (qRT-PCR) assay

The siRNA-transfected cells were harvested 72 h after transfection and the total RNA was extracted using ISOGEN2 according to the manufacturer’s protocol (NIPPON GENE, Tokyo, Japan). Total RNA (500 ng) was reverse transcribed into cDNA using PrimeScript RT Master Mix (TaKaRa, Shiga, Japan). Then, qRT-PCR was performed using PowerUp SYBR Green Master Mix for qPCR in Quant Studio 6 Pro according to the manufacturer’s instructions (Thermo Fisher Scientific). The specific qRT-PCR cycling conditions were 95°C for 3 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The relative mRNA expression levels of the target genes were calculated by the 2−ΔΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The relevant primer sequences are listed in Supplementary Table 2.

Western blot analysis

Cells were cultured in plastic dishes overnight and treated with the indicated BAY-876 concentrations for 24 h. The cells were also treated with or without CoCl2 (100 μM) and cultured for 24 h to mimic hypoxia. After treatment, the cells were washed with phosphate-buffered saline (PBS) and lysed with radio-immunoprecipitation assay (RIPA) buffer (FUJIFILM) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The lysates were sonicated with a Misonix Sonicator XL2020 Liquid Processor (Qsonica, Newtown, CT, USA), then centrifuged at 13,200 × g for 5 min at 4°C. For the in vivo study, mouse tissues were frozen in liquid nitrogen and homogenized with RIPA buffer. Protein concentrations were quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

The following primary antibodies were used for western blot analysis: rabbit anti-HIF1-α (#NB100-479, Novus Biologicals, Littleton, CO, USA, 1:50), rabbit anti-β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA, 1:100), rabbit anti-GAPDH (#2118, Cell Signaling Technology, 1:200), rabbit anti-GLUT1 (#12939, Cell Signaling Technology, 1:50), and rabbit anti-poly (ADP-ribose) polymerase (PARP) (#9542, Cell Signaling Technology, 1:50). The protein bands were detected with an anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Anti-Rabbit Detection Module, #DM-001A, Protein Simple, Bio-Techne, San Jose, CA, USA) according to the manufacturer’s protocol and analyzed using the capillary-based instrument Simple Western JESS using Compass for SW software (Protein Simple).

The resulting area of each specific molecular weight peak was divided by either the β-actin or GAPDH value in the same capillary or the total protein value of the respective antibody for normalization.

Mitochondrial function analysis

The extracellular acidification rate (ECAR), oxygen consumption rate (OCR), and ATP production rate of cells treated with or without BAY-876 were measured using the Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA). Assays were performed using HCT116 (2.0 × 104 cells/well), Caco-2 (0.75 × 104 cells/well), DLD1 (1.6 × 104 cells/well), and COLO205 (1.6 × 104 cells/well) cells seeded in XF96 microplates (HCT116, Caco-2, and COLO205 cells were seeded on collagen-coated wells) and cultured for 24 h until they were ~ 80% confluent. The medium was removed, then fresh medium was added that included BAY-876 at the indicated concentrations. After 24 h, the medium was switched to the seahorse assay medium supplemented with 10 mM glucose and 2 mM glutamine 2 h before assay measurements. Testing of mitochondrial function was initiated by three baseline OCR measurements. Additional OCR measurements were acquired after sequential injections of 1.5 μM oligomycin, 1.0–2.0 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (the concentration that resulted in the highest OCR in each cell line), and 0.5 μM rotenone and antimycin A.

ROS assay

ROS activity was determined using a ROS assay kit (Dojindo) following the manufacturer’s instructions. Briefly, HCT116, COLO205, DLD1, and Caco-2 cells were cultured in 6-well plates (VIOLAMO, 1 × 105 cells/well) for 24 h, then treated with BAY-876 at the indicated concentrations for 48 to 72 h. Subsequently, the cells were washed with PBS (FUJIFILM, 0.01 mol/L, pH 7.2–7.4) and the highly sensitive 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) dye working solution was added. The cells were incubated at 37°C for 30 min. As a positive control for ROS, HCT116 cells were treated with H2O2. The cells were observed with a BZ-X700 microscope (Keyence, Osaka, Japan), then separated with Accutase (Innovative Cell Technologies, San Diego, CA, USA) and resuspended in PBS with 1% FBS. Any changes in ROS levels were detected using a BD FACSCanto II system and analyzed with FlowJo Academic software (Becton Dickinson, Ashland, OR, USA).

Apoptosis assay

Briefly, HCT116, COLO205, DLD1, LoVo, and Caco-2 cells were cultured in 6-well plates (1 × 105 cells/well) for 24 h, then treated with the indicated concentrations of BAY-876 for 72 h. After treatment, the cells were trypsinized, then washed with cold PBS and resuspended in 85 μL of binding buffer (MEBCYTO, Tokyo, Japan). Afterwards, 10 μL of annexin V-FITC (MEBCYTO) was added and incubated for 15 min in the dark, then 300 μL of binding buffer was added. Immediately before flow cytometry analysis, the cells were stained with 0.4 μL propidium iodide (Invitrogen, Thermo Fisher Scientific, P3566) and analyzed using an Attune Flow Cytometer (Thermo Fisher Scientific). Apoptosis data were analyzed using FlowJo academic software.

Mouse xenograft model

All animal experiments were performed according to Kanazawa University’s standard guidelines. Female immunocompromised BALB/c-nu/nu mice (Charles River Laboratories Inc., Yokohama, Japan) at 4–6 weeks of age were maintained in a sterile environment. HCT116 cells were used to generate a xenograft model. HCT116 cells were cultured as described previously for four days. After the mice were anesthetized with a combination of medetomidine, midazolam, and butorphanol, 5.0 × 106 cells in 100 μL of DMEM were subcutaneously implanted into the right dorsal side on day 0. The tumors were monitored and measured with a caliper, with the tumor volume calculated as ½ × (large diameter) × (smaller diameter)2. The tumor volume reached an average volume of 150 mm3 on day 7. The mice were divided into three groups (five mice/group): 1) control group, 2) low-dose BAY-876 group (2 mg/kg), and 3) high-dose BAY-876 group (4 mg/kg). BAY-876 was suspended in CMC, then the mice were treated by gavage feeding once daily for 16 days. Tumor volumes and body weights were measured three times weekly. On day 23, the mice were euthanized using cervical dislocation under anesthesia with a combination of medetomidine (0.75 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg). The tumors were then removed for western blot, immunohistochemistry (IHC), and immunofluorescence analyses.

Histological and IHC examinations

Tumor specimens were fixed with 10% neutral buffered formalin (NBF) for 24 h at room temperature and embedded in paraffin at 60°C. Then, 4 μm-thick sections were cut. The sections were deparaffinized, then pretreated by autoclaving in 10% citric acid buffer at 120°C for 15 min. After being treated with protein block serum (Dako, Carpinteria, CA, USA) for 10 min and incubated with goat serum (ImmunoBioScience, Mukilteo, WA, USA, IHR-8136) for 45 min to block nonspecific interactions, the sections were incubated with an anti-Ki-67 primary antibody (Cell Signaling Technology, #12202, 1:400) at 4°C overnight. After the sections were washed in tris-buffered saline with Tween 20 (TBST), they were incubated with an anti-rabbit HRP-conjugated secondary antibody (Dako) for 1 h at room temperature. Following TBST washes, the slides were developed with diaminobenzidine and counterstained with hematoxylin.

All sections were examined using a BZ-X700 microscope (Keyence). The number of stained cells was counted in at least five randomly selected fields at 40 × magnification.

Immunofluorescence examination of mouse tissues

Tumor specimens were fixed with 10% NBF for 24 h at room temperature and embedded in paraffin. Then, 4 μm-thick sections were cut. The sections were deparaffinized, then pretreated by autoclaving in 10% citric acid buffer at 120°C for 15 min. After being treated with protein block serum (Dako) for 10 min and incubated with goat serum (ImmunoBioScience, IHR-8136) for 45 min to block nonspecific interactions, the sections were incubated with an anti-GLUT1 primary antibody (Abcam, Cambridge, UK, ab115730, 1:500) at 4°C overnight. The sections were then washed in TBST and incubated with an anti-rabbit Alexa Fluor 594-conjugated IgG antibody (Abcam, ab150080, 1:1000) for 1 h at room temperature in the dark. Following TBST washes, 4',6-diamidino-2-phenylindole (DAPI, Abcam, ab228549 1:1000 dilution) in TBST was used for nuclear counterstaining for 20 min at room temperature.

All sections were examined using a BZ-X700 microscope (Keyence), with cells counted in at least five randomly selected fields at 40 × magnification.

TdT-mediated dUTP nick end labeling (TUNEL) assay

TUNEL assays were conducted using a TUNEL assay kit (Abcam, ab66110) following the manufacturer’s instructions. The nuclei were stained blue with DAPI and the apoptotic cells were stained red. Under a BZ-X700 microscope (Keyence), five randomly selected fields at 40 × magnification were used to count the number of apoptotic cells.

Statistical analyses

Statistical analyses were conducted using SPSS statistical software, version 23 (IBM Corp., Armonk, NY, USA). Statistical significance was determined using Student’s t-tests (two-tailed) and set at P < 0.05. The data are presented as the mean ± standard deviation (SD) of three replicate assays.

Results

Inhibiting GLUT1 suppresses CRC cell viability and proliferation

First, we evaluated the effects of 24 h of BAY-876 treatment on CRC cell viability and proliferation (Fig. 1). The BAY-876 concentrations, which ranged up to 1.25 μM, resulted in varying sensitivity levels. Cell viability was measured using the MTS assay. HCT116, COLO205, DLD1, and LoVo cells exhibited sensitivity to BAY-876 treatment (Fig. 1A). COLO205 cells were the most responsive, showing a half maximal inhibitory concentration (IC50) value of approximately 4 nM. Conversely, Caco-2 cells remained insensitive to BAY-876, even at the highest concentration of 1.25 μM (Fig. 1A).

Fig. 1
figure 1

Inhibiting glucose transporter 1 (GLUT1) suppresses colorectal cancer (CRC) cell viability and proliferation. CRC cell lines were treated with the indicated concentrations of BAY-876 for 24 h before MTS assays to determine any cytotoxic effects (A). The effects of BAY-876 treatment on cell proliferation were analyzed by determining the cell numbers after 24 h (B). The values represent the mean ± SD of three independent experiments performed in triplicate; * P < 0.05; **P < 0.01

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Cell proliferation assessments showed a dose-dependent decrease in the number of HCT116, COLO205, and LoVo cells following BAY-876 treatment (Fig. 1B, P < 0.05). Although DLD1 cells were significantly inhibited by BAY-876 treatment (P < 0.05), no dose-dependent decrease was observed, likely because of their high proliferation rate [29]. As shown in Supplementary Fig. 1A, GLUT1 mRNA levels were significantly suppressed by GLUT1-specific siRNA in HCT116, COLO205, DLD1, LoVo, and Caco-2 cells, with the most pronounced suppression in Caco-2 cells. We then examined the effect of siRNA-mediated GLUT1 knockdown on cell proliferation (Supplementary Fig. 1B). GLUT1 knockdown significantly reduced cell proliferation rates in HCT116, COLO205, DLD1, and LoVo cells, similar to the effects of BAY-876 treatment. Although GLUT1 suppression was evident in Caco-2 cells, no inhibition of cell proliferation was observed. These results suggest that GLUT1 is essential for cell survival in CRC cell lines where BAY-876 treatment effectively inhibited proliferation, whereas alternative glucose transport mechanisms may compensate for GLUT1 suppression in Caco-2 cells. Therefore, Caco-2 cells were used as a negative control in the subsequent experiments.

GLUT1 inhibition by BAY-876 treatment reduces GLUT1 and HIF1-α expression levels in hypoxic conditions

CoCl2 was used to stabilize HIF-1α to mimic hypoxia. We then examined GLUT1 inducibility in CRC cells, reflecting the in vivo hypoxic environment. Western blot analysis revealed upregulated HIF-1α and GLUT1 protein expression levels with mimicked hypoxia (Fig. 2A, B). BAY-876 treatment effectively reduced both the basal and CoCl2-induced upregulation of GLUT1 and HIF-1α expression in HCT116, COLO205, LoVo, and DLD1 cells. Notably, in Caco-2 cells, the GLUT1 expression levels remained low under both normoxic and hypoxic conditions (Fig. 2B), suggesting that GLUT1-independent mechanisms drive HIF-1α-mediated glycolysis in Caco-2 cells.

Fig. 2
figure 2

Glucose transporter 1 (GLUT1) activity and stability in hypoxic conditions are reduced by BAY-876 treatment. Colorectal cancer cell lines were treated for 24 h with CoCl2 (100 μM) in the presence or absence of BAY-876. Hypoxia-inducible factor 1 (HIF-1α) (A) and GLUT1 (B) protein expression levels were compared among cell lines. GLUT1 normalization was performed using GAPDH protein expression levels. The full-length blots are presented in Supplementary Fig. 6A–E

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These findings suggest that the antineoplastic effects of BAY-876 treatment may be associated with GLUT1 expression regulation, leading to Warburg effect reversal.

Inhibiting GLUT1 causes a bioenergetic source conversion of glycolysis

We next investigated whether BAY-876 serves as an effective glycolysis inhibitor by measuring the ECAR and OCR values in HCT116, DLD1, COLO205, and Caco-2 cells. Additionally, we explored how BAY-876 treatment can modulate ATP production rates through both glycolysis and OXPHOS.

BAY-876 treatment inhibited the ECAR in all cell lines examined. In contrast, it increased the OCR in HCT116, DLD1, and COLO205 cells (Fig. 3A–C). Significantly enhanced maximal respiratory capacity was observed in these three cell lines (P < 0.01), with increases in basal mitochondrial respiration (basal OCR) and ATP-linked OCR in BAY-876-treated DLD1 cells (P < 0.01). Conversely, no significant change in OCR was observed in Caco-2 cells (Fig. 3D).

Fig. 3
figure 3

BAY-876 treatment induced a bioenergetic source conversion of glycolysis. The oxygen consumption rate (OCR) and adenosine triphosphate (ATP) production rate were measured using the Seahorse XF96 Extracellular Flux Analyzer, as mentioned in the Materials and Methods. The real-time OCRs (pmol/min, mean ± SD) of the assay (A–D) and basal mitochondrial respiration (Basal), ATP-linked respiration (ATP-linked), maximal respiratory capacity (Maximum), and spare respiratory capacity were calculated and statistically analyzed to examine the effects of BAY-876 treatment. The ATP production rate was measured to determine if BAY876 administration affected the mitochondrial ATP production ratio (E, F). The values represent the mean ± SD of three independent experiments performed in triplicate; * P < 0.05; **P < 0.01

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BAY-876 treatment reduced the total ATP production rate in DLD1, COLO205, and Caco-2 cells, and was inversely correlated with an increased ATP production rate in HCT116 cells (Fig. 3E). This seemed to reflect the different rates of total ATP production by glycolysis and OXPHOS in each cell line. In HCT116, DLD1, and COLO205 cells, BAY-876 treatment increased the contribution of OXPHOS to the total ATP production rate, accompanied by a decreased glycolytic rate (Fig. 3F, P < 0.01). In Caco-2 cells, the ratio of ATP production derived from OXPHOS and glycolysis remained unchanged, with an overall decrease in the total ATP production rate.

Considering the absence of this effect in Caco-2 cells, these data suggest that the impact of BAY-876 treatment on the OCR in HCT116, DLD1, and COLO205 cells is GLUT1-dependent, indicating that BAY-876 may suppress glycolysis and promote mitochondrial respiration (Warburg shift).

BAY-876 treatment induces cellular oxidative stress and cell death

We hypothesized that BAY-876 can promote mitochondrial respiration, activating the tricarboxylic acid cycle and increasing ROS production, which thereby induces apoptosis.

Intracellular ROS levels measured by flow cytometry were significantly increased in BAY-876-treated HCT116, DLD1, and COLO205 cells (Fig. 4A–C, P < 0.01). Conversely, ROS levels in Caco-2 cells remained unchanged after BAY-876 treatment (Fig. 4D). Microscopic observations also revealed elevated ROS levels in HCT116 cells, but not in Caco-2 cells (Supplementary Fig. 2). Subsequently, we examined whether BAY-876 could induce apoptosis through ROS production. In HCT116, DLD1, LoVo, and COLO205 cells, BAY-876 treatment increased the apoptosis rates in a dose-dependent manner (Fig. 5A–D, P < 0.05). As shown in Supplementary Fig. 3, western blot analysis of cleaved-PARP expression exhibited a pattern similar to that observed in the Annexin-V assay, with increased expression levels following BAY-876 administration in the HCT116, COLO205, DLD1, and LoVo cell lines. However, apoptosis did not increase in Caco-2 cells following BAY-876 treatment (Fig. 5E).

Fig. 4
figure 4

BAY-876 treatment induces oxidative stress. The intracellular reactive oxygen species (ROS) levels were measured using the oxidation-sensitive probe, DCFH-DA, as detailed in the Materials and Methods. These cells were treated with 50 or 100 nM and 1000 nM of BAY-876. DCFH-DA was then added and the ROS levels were compared with those in the control group (A–D). The values represent the mean ± SD of three independent experiments performed in triplicate; * P < 0.05; **P < 0.01

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Fig. 5
figure 5

BAY-876 treatment results in colorectal cancer (CRC) cell apoptosis. CRC cell line apoptosis rates were analyzed using flow cytometry (A–E), with FITC-conjugated annexin V and PI staining. The flow cytometry results are presented as the percentage of cells in the different apoptosis stages. The values represent the mean ± SD of three independent experiments performed in triplicate; * P < 0.05; **P < 0.01

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These findings suggest that the antineoplastic effects of BAY-876 treatment may be associated with increased intracellular ROS production, which can lead to apoptosis.

BAY-876 treatment inhibits xenograft tumor growth

To confirm the effect of GLUT1 downregulation on CRC cell proliferation rates, we established a nude mouse model by subcutaneous injection of HCT116 cells. When the tumor volumes reached 100 mm3, the mice were randomly assigned to three groups and then orally administered either DMSO (control), low-dose BAY-876 (2.0 mg/kg/day), or high-dose BAY-876 (4.0 mg/kg/day) for 16 days. The tumor growth curves and body weight changes were monitored. In the BAY-876 treatment groups, the tumor volumes significantly decreased (Fig. 6A, B, P < 0.05), with a trend of decreased body weight (Fig. 6C, P > 0.05). Other than weight loss, these mice exhibited no other noticeable health issues.

Fig. 6
figure 6

The anti-tumor effects of BAY-876 treatment in the HCT116 mice tumor model. Tumor growth curve after BAY-876 treatment and macroscopic views of the tumors (A–B). Effects on nude mice body weight (C). Western blot analysis of glucose transporter 1 (GLUT1) protein expression levels in tumors from vehicle- and BAY-876-treated mice (D–E, n = 3). Representative GLUT1 immunohistochemistry images (F; magnification: 40 ×). The full-length blots are presented in Supplementary Fig. 7. The values represent the mean ± SD of three independent experiments performed in triplicate; *P < 0.05; **P < 0.01

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Western blot analysis showed a decrease in GLUT1 protein expression levels in the tumor tissues (Fig. 6D, E, P< 0.05). IHC analysis of the tumor tissues revealed that BAY-876 treatment downregulated the GLUT1 and Ki-67 protein expression levels (Figs. 6F and 7A, P < 0.01). Furthermore, TUNEL assay evaluation of the excised tissues indicated an increased number of apoptotic cells in the BAY-876 treatment groups (Fig. 7B, P < 0.01). As shown in Supplementary Fig. 4, western blot analysis of cleaved-PARP expression showed a large expression difference between the samples.

Fig. 7
figure 7

Growth inhibition and increased apoptosis in tumor tissues. Effects of BAY-876 treatment on Ki-67 protein expression levels in tumor tissues analyzed by immunohistochemistry (A; magnification: 40 ×) and apoptosis analysis using TUNEL assays (B; magnification: 40 ×). The number of positive cells were measured in each as the average count of five non-overlapping tumor areas. The values represent the mean ± SD of three independent experiments performed in triplicate; *P < 0.05; **P < 0.01

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These results demonstrate that BAY-876 treatment can suppress tumor growth and increase apoptosis rates in vivo, suggesting that it has potential benefits for treating CRC.

Discussion

In this study, we reported the anti-proliferative effects of BAY-876 treatment on CRC cell growth both in vitro and in vivo. Our data demonstrated that BAY-876 treatment could suppress GLUT1 expression levels, thereby reducing glycolysis and shifting ATP production towards OXPHOS to maintain the ATP levels required for survival (Fig. 3F). This enhanced OXPHOS activity leads to elevated mitochondrial respiration, which generates ROS as a byproduct. ROS are toxic molecules that accumulate within the cell as a result of increased OXPHOS activity, leading to oxidative damage to cellular components, such as lipids, proteins, and DNA, which triggers cellular stress responses [30, 31]. If the damage becomes overwhelming or irreparable, then apoptosis (programmed cell death) is activated [32]. ROS are known to be a major contributor to oxidative stress, which plays a critical role in inducing apoptosis, particularly in cancer cells [33].

GLUT1 expression is regulated by the activity of many proteins, including HIF-1 and MYC [34]. PDK1 is also a direct target of HIF-1, and its overexpression in CRC is associated with poor prognosis and liver metastasis [17, 35]. Cancer cell glycolytic activity is further increased in response to various stressors, such as hypoxia, nutrient limitation, or other detrimental factors associated with the tumor microenvironment [36]. In most solid tumor types, GLUT1 and PDK1 expression patterns have been associated with increased HIF1 stabilization [37, 38]. Aiora et al. [39] reported that GLUT1 and PDK1 expression levels were increased in NiCl2 hypoxia-mimicking conditions, with vitamin C inhibiting GLUT1 and PDK1 expression in CRC cell lines. In our study, we also used CoCl2 to enhance the Warburg effect. Our results demonstrate that the GLUT1 expression levels increased when CoCl2 was used to mimic hypoxia, with BAY-876 treatment suppressing glycolytic enzymes. We believe that the difference in sensitivity to BAY-876 is caused by variations in GLUT1 expression levels among these cell lines. As shown in Supplementary Fig. 5, western blot analysis indicated that GLUT1 protein expression levels differed among the cell lines. Suzuki et al. [40] have reported that the combination of gefitinib and the GLUT1 inhibitor WZB-117 demonstrated differences in GLUT1 protein expression patterns among multiple lung cancer cell lines, which correlated with varying inhibitory effects of WZB-117 treatment on cell proliferation. Furthermore, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations are reportedly associated with GLUT1 expression and the promotion of glucose metabolism [41]. This study also indicated that glucose metabolism inhibitors exerted stronger inhibitory effects on mutant cell lines. Supplementary Table 1 shows the presence or absence of KRAS and BRAF mutations in the five CRC cell lines used in this study [42, 43]. These results suggest that more pronounced growth-inhibitory effects were observed in CRC cell lines harboring KRAS or BRAF mutations. Interestingly, BAY-876 treatment also exhibited significant inhibitory effects on HIF-1α expression. High glucose concentrations can reportedly upregulate HIF-1α expression levels [44]. As reported by Temre et al. [45], the low glucose uptake resulting from GLUT inhibition would have triggered the observed downregulation in HIF-1α expression patterns. These findings indicate that BAY-876 treatment can suppress the protein expression levels of glycolytic enzymes in CRC cells.

The reliance of tumor cells on elevated glycolysis for rapid energy demands and uncontrolled proliferation has been known for almost 100 years [46]. The sensitivity of cancer cells to the Warburg change induced by GLUT1 inhibitors has been reported as a valuable target for anticancer therapy. Work by Ma et al. [9] showed that BAY-876 treatment increased mitochondrial respiration (basal OCR, ATP-linked OCR, and maximal respiratory capacity) in ovarian cancer cells. Another GLUT1 inhibitor, resveratrol, was also reported to promote OCR and suppress ECAR in CRC cells, with drug administration increasing apoptotic cell death rates [47]. Our results show that BAY-876 treatment increased maximal respiration in HCT116, DLD1, and COLO205 cells, demonstrating its ability to promote mitochondrial respiration in CRC cells.

Cancer cells display high rates of glucose metabolism to generate ATP and biosynthesize substrates [48]. Because inhibiting glucose metabolism promotes OXPHOS to maintain the necessary ATP production for survival, glucose deprivation can increase energy stress and enhance ROS-induced selective cell death in cancer cells compared with normal cells [49]. ROS are a toxic byproduct of OXPHOS, which play major roles in the progressive accumulation of damage in cancer cells and their efficient elimination through apoptosis [30]. Wang et al. [49] reported that treatment with STF31, a GLUT1 inhibitor, could suppress prostate cancer cell proliferation in vitro and in vivo, with IHC analysis of tumor tissues showing that STF31 treatment downregulated the GLUT1 and Ki-67 protein expression levels. Another study described that apigenin, a common flavone in fruits and vegetables, can decrease glycolysis by suppressing GLUT1 expression patterns and consequently decreasing pentose phosphate pathway activity and nicotinamide adenine dinucleotide phosphate production, resulting in cell apoptosis [50]. Therefore, we hypothesized that inhibiting glucose metabolism would result in oxidative stress induction and ROS accumulation in CRC cells that lead to selective cell death, which could be useful as an anticancer therapeutic approach. This study shows that BAY-876 treatment can induce a significant increase in ROS levels in CRC cells (HCT116, DLD1, and COLO205), leading to apoptotic cell death. We therefore propose that glucose metabolism inhibition by BAY-876 and the subsequent ROS accumulation are crucially involved in promoting cell death in CRC cells. Furthermore, we observed that BAY-876 treatment induced apoptosis in these CRC cell lines; however, the apoptosis rate differed, likely because of variations in GLUT1 protein expression levels among the cell lines. Furthermore, BAY-876 was administered for 72 h in the Annexin-V assay, but only the adhered cells were analyzed; cells that had already undergone apoptosis or detached were not included in this analysis, potentially explaining why the measured apoptosis rates were lower than expected. In addition, as shown in Supplementary Fig. 4, cleaved-PARP expression in in vivo samples differed among the samples, as shown by western blot analysis. This discrepancy may be attributed to variations in apoptosis detection timing, resulting from heterogeneity of the cell populations within tumor tissues [51].

BAY-876 could potentially serve as a selective anticancer treatment by suppressing GLUT1-induced cancer growth in vivo. However, GLUT1 is needed for basal glucose uptake in normal cells, such as normal erythrocytes and endothelial cells of the blood–brain barrier [52]. GLUT1 inhibition has been suggested to also affect normal cells, with some reports showing toxicity at certain doses in vivo [9, 53]. To date, no clinical trials have been performed because of the uncertain effects of GLUT1 inhibition in humans. For BAY-876, Ma et al. [9] reported that 4.5 mg/kg/day administration in mice resulted in weight loss, while 7.5 mg/kg/day resulted in dose-limiting toxicity for 28 to 30 days. In this study, the mice were treated with 2 mg/kg/day or 4 mg/kg/day of BAY-876 for 16 days. As shown in Fig. 6A, the average tumor volumes were decreased in the BAY-876-treated groups compared with the vehicle control group. Weight loss was also observed in the BAY-876-treated groups, but these decreases were not statistically significant (Fig. 6B). However, for potential clinical application, careful investigation of the effects on the human body is necessary.

In this study, BAY-876 treatment inhibited the growth of HCT116, DLD1, and COLO205 cells, but not of Caco-2 cells. Caco-2 cells are derived from a human colorectal adenocarcinoma tumor and closely mimic the human absorptive intestinal epithelium [54]. Intestinal glucose uptake is mainly performed by its specific transporters, such as SGLT1, GLUT2, and GLUT5, which are expressed in intestinal epithelial cells [55]. Therefore, glucose transporters other than GLUT1 may play an important role in glucose uptake in Caco-2 cells. Because BAY-876 is a GLUT1-selective inhibitor, we hypothesized that it would have no growth inhibitory effect on Caco-2 cells. Furthermore, Caco-2 cells, which exhibit WT status for both KRAS and BRAF (Supplementary Table 1), showed resistance to BAY-876, suggesting that the lack of these mutations may have contributed to the reduced efficacy of the treatment.

Our study has some limitations. First, the sample size was small and the experimental period was short. Although no significant side effects were observed in the in vivo study, extending the administration period is necessary to carefully examine any potential side effects for future clinical applications. Second, the drug administration time was not thoroughly examined in vitro. Cellular glucose metabolism involves complex pathways, and it is possible that we did not pinpoint the time when the effects of GLUT1 inhibition were most pronounced. Third, not all CRC cell lines were tested, and factors such as KRAS status and BRAF status were being considered. Because the KRAS status can affect anticancer drug efficacy, it is possible that glucose metabolism changes can depend on the molecular subtype. Further investigation is necessary to explore the potential differences in BAY-876-mediated GLUT1 inhibitory effects among different molecular subtypes of CRC cells.

Conclusions

In conclusion, our data suggest that GLUT1 is an important factor contributing to cell survival in human CRC. Inhibiting GLUT1 with BAY-876 treatment induced metabolic changes and inhibited cell proliferation in vitro and in vivo. Therefore, BAY-876 treatment is a potential novel glycolysis-targeted therapeutic method for CRC.

Data availability

All the data supporting the findings of the present study are available within the article and its supplementary files.

Abbreviations

ATP:

Adenosine triphosphate

CMC:

Carboxymethylcellulose

CRC:

Colorectal cancer

DAPI:

4',6-Diamidino-2-phenylindole

DCFH-DA:

2',7'-Dichlorodihydrofluorescein diacetate

DMEM:

Dulbecco’s Modified Eagle Medium

DMSO:

Dimethyl sulfoxide

ECAR:

Extracellular acidification rate

FBS:

Fetal bovine serum

GLUT1:

Glucose transporter 1

HIF-1:

Hypoxia-inducible factor 1

HRP:

Horseradish peroxidase

IC50:

Half maximal inhibitory concentration

IHC:

Immunohistochemistry

KRAS:

V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog

MTS:

3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

NBF:

Neutral buffered formalin

OCR:

Oxygen consumption rate

OXPHOS:

Oxidative phosphorylation

PARP:

Poly (ADP-ribose) polymerase

PBS:

Phosphate-buffered saline

PDK1:

Pyruvate dehydrogenase kinase-1

PI:

Propidium iodide

RIPA:

Radio-immunoprecipitation assay

ROS:

Reactive oxygen species

SGLT:

Sodium-dependent glucose transporter

TBST:

Tris-buffered saline with Tween 20

TUNEL:

TdT-mediated dUTP nick end labeling

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Acknowledgements

We thank J. Iacona, Ph.D., from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

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Authors and Affiliations

  1. Department of Gastroenterological Surgery, Division of Cancer Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa, 920-8641, Japan

    Masato Hayashi, Hiroto Saito, Toshikatsu Tsuji, Daisuke Yamamoto, Hideki Moriyama, Jun Kinoshita & Noriyuki Inaki

  2. Department of Surgery, Public Central Hospital of Matto Ishikawa, 3-8 Kuramitsu, Hakusan, Ishikawa, 924-8588, Japan

    Keishi Nakamura

  3. Center for Biomedical Research and Education, School of Medicine, Kanazawa University, Kanazawa, Ishikawa, 920-8640, Japan

    Shinichi Harada, Mariko Tanaka & Akiko Kobayashi

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  1. Masato Hayashi
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Contributions

Conceptualization, M.H. and K.N.; methodology, M.H., K.N., J.K., and N.I.; investigation, M.H., S.H., M.T., A.K., H.S., T.T., D.Y., and H.M.; formal analysis, M.H., S.H., M.T., A.K., H.S., T.T., D.Y., and H.M.; data curation, M.H. and K.N.; writing–original draft, M.H.; writing–review and editing, K.N.; visualization, M.H.; supervision, N.I.; funding acquisition, M.H. and K.N.; All authors have read and agreed with the final version of the manuscript.

Corresponding author

Correspondence to Keishi Nakamura.

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All institutional and national guidelines for the care and use of laboratory animals were followed. Animals were treated in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions, under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan. All animal experiments were approved by the Committee on Animal Experimentation of Kanazawa University (approval number AP-214257).

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Supplementary Information

12885_2025_14141_MOESM1_ESM.pptx

Additional file 1: Supplementary Figure 1. Cell proliferation inhibition assay using small interfering RNA transfection in colorectal cancer cell lines. qRT-PCR analysis of GLUT1 mRNA levels (A) and cell proliferation assay (B) after transfection with control or GLUT1-targeting siRNA. The expression pattern in control cells was used as a reference value. Supplementary Figure 2. Increased reactive oxygen species (ROS) levels were visually confirmed by fluorescence microscopy. ROS-positive cells labeled with DCFH-DA were observed using a fluorescence microscope in HCT116 and Caco-2 cells (magnification: 40´). Supplementary Figure 3. Western blot analysis of cleaved-PARP expression showed a similar pattern to that observed in the Annexin-V assay. The full-length blots are presented in Supplementary Figure 8. Supplementary Figure 4. Western blot analysis of cleaved-PARP expression showed a large expression difference between the samples, with no clear trend. The full-length blots are presented in Supplementary Figure 9. Supplementary Figure 5. Comparison of glucose transporter 1 (GLUT1) protein expression levels among cell lines. The expression pattern in HCT116 cells was used as a reference value. Supplementary Figure 6. Full-length blots of the samples in Figure 2. HCT116 (A), COLO205 (B), DLD1 (C), LoVo (D) and Caco-2 (E) samples. The red lines indicate cropped areas. Supplementary Figure 7. Full-length blots of the samples in Figure 6D. The red lines indicate cropped areas. Supplementary Figure 8. Full-length blots of samples in Supplementary Figure 3. The red lines indicate cropped areas. Supplementary Figure 9. Full-length blots of samples in Supplementary Figure 4. The red lines indicate cropped areas. Supplementary Table 1 Mutation status of colorectal cancer cell lines (WT: wild-type, Mut: mutated). Supplementary Table 2 Sequences of quantitative RT-PCR primers.

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Hayashi, M., Nakamura, K., Harada, S. et al. GLUT1 inhibition by BAY-876 induces metabolic changes and cell death in human colorectal cancer cells. BMC Cancer 25, 716 (2025). https://doi.org/10.1186/s12885-025-14141-9

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