Low-dose YC-1 combined with glucose and insulin selectively induces apoptosis in hypoxic gastric carcinoma cells by inhibiting anaerobic glycolysis

This study aimed to establish a therapeutic strategy targeting hypoxic cancer cells in gastric carcinoma (GC). YC-1 is a HIF-1α inhibitor, and we revealed that low-dose YC-1 (10 µM) suppressed HIF-1α expression, and induced hypoxia-dependent apoptosis in the GC cell line 58As9. This hypoxia-specific apoptosis induction by YC-1 involved excessive reactive oxygen species (ROS) generation. The apoptotic effect of 10 µM YC-1 was enhanced by additional glucose (G) and insulin (I) treatments. RT-PCR demonstrated that 10 µM YC-1 reduced hypoxia-induced expression of HIF-1α targets involved in anaerobic glycolysis. Metabolic analysis showed that YC-1 shifted glucose metabolism in hypoxic cells from anaerobic glycolysis to oxidative phosphorylation (OXPHOS). Additional GI accelerated membranous GLUT1 translocation, elevating glucose uptake, and increased acetyl-CoA levels, leading to more ROS generation in hypoxic YC-1-treated cells. Finally, we evaluated the anti-cancer effect of low-dose YC-1 (1 mg/kg) + G (2 g/kg) and I (1 unit/3 g G) treatment in xenograft models. YC-1 + GI therapy strongly inhibited tumour growth. Immunohistochemical analysis demonstrated that YC-1 + GI reduced HIF-1α expression and pimonidazole accumulation in tumours. Conversely, YC-1 + GI increased intra-tumoral 8-OHdG and levels of apoptosis markers. Low-dose YC-1 + GI is a unique therapy targeting hypoxic GC cells that generates lethal ROS via forced activation of OXPHOS.

control system including anaerobic glycolysis in 58As9 cells 16 . This study further revealed that hypoxia-induced apoptosis was accelerated by additional glucose (G) and insulin (I) treatments in the KD cells, as higher ROS generated via increased glucose uptake 16 . Based on this study, we attempted to establish a novel anti-cancer therapy using a specific HIF-1α inhibitor combined with GI to target hypoxic cancer cells in gastric tumours.
ROS are mainly generated in the mitochondria by oxidative phosphorylation (OXPHOS), a process performed by the electron transport chain (ETC) [17][18][19][20][21] . Excessive ROS generation is known to cause ROS-mediated damage to nucleic acids, proteins and lipids, leading to cell death [18][19][20][21] . It has been reported that ROS are increased in hypoxic cancer cells, and HIF-1α induction plays an adaptive mechanism in controlling ROS generation via up-regulating genes involved in anaerobic glycolysis 3,15,16,19 . In the anaerobic glycolysis pathway, HIF-1α first activates GLUT1 transcription to increase glucose uptake in cells 22 . Glucose is then metabolized to pyruvate by the actions of glycolytic members including ALDOC 23 . Under aerobic conditions, pyruvate is converted to acetyl-CoA by pyruvate dehydrogenase (PDH) for entry into the tricarboxylic acid (TCA) cycle 18 . Conversely, in cancer cells exposed to hypoxia, pyruvate is shunted away from the mitochondria by HIF-1α-mediated PDK1 upregulation, which inhibits PDH activity. Thereafter, LDHA alternatively converts pyruvate to lactate and MCT4 effluxes the lactate [24][25][26] . Together, these reports indicate that HIF-1α is a central molecule in suppressing excessive ROS production in hypoxic cells via up-regulating target genes involved in anaerobic glycolysis.
YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] was originally developed as a potential therapeutic agent for circulation disorders because of its inhibitory effect on platelet aggregation and vascular contraction 27 . Recent studies have found that YC-1 blocked HIF-1α expression at the post-transcriptional level and consequently inhibits the transcription factor activity of HIF-1 in cancer cells under hypoxia [28][29][30] . However, no study has assessed the anti-cancer effect of YC-1 on cancer metabolism under hypoxia.
In this study, we first determined the optimal dose of YC-1 that effectively inhibited HIF-1α expression and induced hypoxia-dependent apoptosis in GC cells. We next analyzed whether additional GI treatment enhanced this apoptotic effect. Metabolic analysis addressed the mechanism of YC-1 + GI-induced apoptosis in cells under hypoxia. Finally, we assessed whether this combination therapy selectively induced apoptosis in hypoxic cancer cells in vivo.

Results
Growth inhibition by YC-1 treatment in GC cells. The GC cell line 58As9 was treated with YC-1 at 1, 10 and 100 μM, and cell viability was evaluated by the MTS assay (Fig. 1a). The results showed that 1 μM YC-1 did not influence cell viability, while 10 μM YC-1-treated 58As9 and KD cells showed significantly decreased viability under hypoxia but not normoxia. At 100 μM, YC-1 decreased viability under both normoxia and hypoxia (Fig. 1a). YC-1 exhibited similar effects in another GC cell line, MKN74 (Supplemental Fig. 1). Cell death was next evaluated in 58As9 cells with or without 10 μM YC-1 (Fig. 1b). Under normoxia, there was no difference in the cell death rate between controls (no YC-1) and 10 μM YC-1. The cell death rate significantly increased under hypoxia with 10 μM YC-1, but not in controls (Fig. 1b). WB analysis showed that HIF-1α was elevated by hypoxia in control cells, while the hypoxic induction of HIF-1α was inhibited in YC-1-treated 58As9 and KD cells (Fig. 1c).

YC-1 plus GI treatment induced apoptosis in 58As9 cells under hypoxia.
We next evaluated the effect of additional GI treatment on hypoxia-dependent cell death in 10 μM YC-1-treated 58As9 cells. Cell viability was evaluated in control (no YC-1) and 10 μM YC-1-treated cells in the presence of glucose (G) and/or insulin (I) (Fig. 2a). In the control group, cell viability was unchanged between normoxia and hypoxia in control, G and I treatments, whereas viability decreased under hypoxia in GI treatment (fold change (FC) of hypoxia/normoxia: 0.9) (Fig. 2a). In the 10 μM YC-1 group, cell viability was more strongly inhibited under hypoxia than normoxia in all treatments (Fig. 2a). The lowest FC was found in the YC-1 + GI (FC: 0.5) treatment. Further, in the YC-1 group, cell viability under hypoxia was significantly lower in GI treatment than controls (Fig. 2a). The strong inhibitory effect of YC-1 + GI on cell viability was also observed in hypoxic MKN74 cells (Supplement Fig. 2). As shown in Fig. 2b, the cell death rate was not different among control, 10 μM YC-1, GI and YC-1 + GI under normoxia. However, the cell death rate was significantly increased in 10 μM YC-1 and YC-1 + GI compared with controls, and there was a higher death rate in YC-1 + GI than 10 μM YC-1 alone (Fig. 2b). WB analysis was used to evaluate apoptotic cell death in hypoxic 58As9 cells treated with YC-1 and/or GI (Fig. 2c). Expression of the apoptosis markers cleaved-caspase3 and cleaved-PARP was increased in hypoxic 58As9 cells by the four treatments, but not controls (Fig. 2c). The highest expression of apoptosis markers was observed in YC-1 + GI-treated cells (Fig. 2c).
Assessing ROS production after YC-1 + GI treatment under hypoxia. We analyzed ROS levels in 58As9 cells under hypoxia with or without 10 μM YC-1 (Fig. 3a). Compared with controls (no YC-1), 10 μM YC-1 more strongly elevated ROS production in a time-dependent manner in hypoxic 58As9 cells (Fig. 3a). ROS levels on day 3 were significantly higher in 10 μM YC-1-treated cells than control cells under hypoxia, while the highest ROS was produced in hypoxic KD cells. ROS levels in hypoxic 58As9 cells were significantly higher in YC-1 + GI than in YC-1 on day 3 (Fig. 3b). Moreover, elevated ROS was blocked by the antioxidant NAC in YC-1 + GI-treated cells under hypoxia (Fig. 3c). The cell death rate was assessed with or without NAC in YC-1 + GI-treated cells (Fig. 3d). Under normoxia, the cell death rate was not different between the NAC (−) and NAC (+). In contrast, the cell death rate was significantly higher in the NAC (−) than NAC (+) under hypoxia on day 3 (Fig. 3d).
RT-qPCR analysis of genes involved in anaerobic glycolysis. RT-qPCR was used to evaluate hypoxia-induced expression of HIF-1α target genes in 58As9 cells in the control, GI, 10 μM YC-1 and YC-1 + GI groups (Fig. 4). The FC of hypoxia/normoxia was significantly decreased compared with controls by YC-1 and YC-1 + GI for all genes, except MCT4 in YC-1 + GI. However, KD cells showed the lowest FC for all five genes (Fig. 4). Levels of GLUT1, ALDOC, PDK1 and MCT4 expression under hypoxia were significantly decreased in YC-1 and YC-1 + GI to similar degrees, compared with controls. Expression of all five genes was most strongly suppressed in hypoxic KD cells (Fig. 4).

Glucose uptake analysis.
To assess glucose uptake, the 2DG uptake test was performed in 58As9 cells ( Fig. 5a-b). As shown in Fig. 5a, 2DG uptake was elevated by insulin in 58As9 cells with and without YC-1 under normoxia. Under hypoxia, 2DG uptake was strongly increased in control (no YC-1) cells, and additional insulin treatment further elevated uptake (Fig. 5b). In 10 μM YC-1-treated cells, 2DG uptake was also promoted by insulin, but to a lower degree than in control cells (Fig. 5b). We next analyzed membranous GLUT1 expression in control and 10 μM YC-1 cells under hypoxia, in combination with G and/or I treatments (Fig. 5c). In the control group, membranous GLUT1 was increased with G or I, and most strongly elevated by GI. In the 10 μM YC-1 group, membranous GLUT1 was entirely reduced, compared with controls ( Fig. 5c). Among treatments, the highest GLUT1 expression was observed in GI (Fig. 5c).
YC-1 plus GI treatment suppressed xenograft tumour growth in nude mice. Finally, we evaluated the in vivo effect of YC-1 + GI treatment in tumour xenografts (Fig. 7). The four drugs were ip injected into mice from day 1 to day 14, as shown in Fig. 7a. On day 15, tumours were harvested and subjected to WB analysis. HIF-1α expression was observed in control and GI mice, while its expression was inhibited in YC-1 and YC-1 + GI (Fig. 7b). In contrast, cleaved-PARP and cleaved-caspase3 were present in YC-1 and YC-1 + GI, and the levels were higher in YC-1 + GI than YC-1 (Fig. 7b). Figure 7c shows the growth curves of xenograft tumours that underwent the four treatments. There was no significant difference in size between control and GI tumors on day 15 (Fig. 7c). In contrast, tumour sizes of YC-1 or YC-1 + GI were significantly smaller than control, and tumour growth in YC-1 + GI was more strongly inhibited than in YC-1 (Fig. 7c). In the representative images of tumor-bearing mice, tumours appeared to be smaller in order of control, YC-1 and YC-1 + GI (Fig. 7d). In this model, no mice died in any treatment. Immunohistochemistry evaluated levels of HIF-1α, pimonidazole, cleaved-caspase3 and an oxidized base, 8-OHdG, in xenograft tumours that underwent control or YC-1 + GI treatments (Fig. 7e). HIF-1α and pimonidazole staining appeared to be stronger in control than YC-1 + GI-treated tumours, whereas cleaved-caspase3 and 8-OHdG staining were stronger in YC-1 + GI (Fig. 7e). Statistical analysis demonstrated that the number of positive HIF-1α and pimonidazole cells was significantly higher in control than YC-1 + GI, while cleaved-caspase3 and 8-OHdG were higher in YC-1 + GI (Fig. 7f).

Discussion
In this study, we first attempted to isolate a compound that inhibited cell viability in 58As9 cells specifically under hypoxia, similar to what we had previously shown for HIF-1α KD 58As9 cells 16 . Many small molecules have been reported to be HIF-1α inhibitors [12][13][14][15] . We explored a desirable drug that reduced cell viability specifically in hypoxia, but not normoxia among the 15 known HIF-1α inhibitors [12][13][14][15] . The MTS assay determined that only In this study, we did not assess whether YC-1 treatment inhibits HIF-2α expression, or whether HIF-2α KD affects cell growth in hypoxic 58As9 cells. Further investigation may solve this question.
We next demonstrated that additional GI to 10 μM YC-1 induced hypoxia-dependent apoptosis more strongly than YC-1 mono-treatment. Low-dose YC-1 or YC-1 + GI exhibited similar effects in another GC cell line, MKN74, indicating hypoxia-dependent cell death by these treatments was not restricted to one cell line. A previous study reported that 100 μM YC-1 inhibited HIF-1α expression and induced apoptosis in PC3 cells under hypoxia 29 . However, this study did not evaluate YC-1 under normoxia. Another study reported that 1 μM YC-1 inhibited Hep3B proliferation under both normoxia and hypoxia 30 . Therefore, we showed for the first time that hypoxia-dependent apoptosis is induced by 10 μM YC-1 in GC cells.
Thereafter, we showed YC-1 or YC-1 + GI time-dependently accumulated ROS in hypoxic 58As9 cells, and higher ROS was generated in YC-1 + GI than YC-1. NAC reversed cell death by YC-1 + GI in hypoxic cells, indicating apoptosis was induced by excessive ROS generation. RT-qPCR analysis revealed hypoxic induction of the HIF-1α targets GLUT1, ALDOC, PDK1 and MCT4 was attenuated by YC-1 and YC-1 + GI to similar degrees. These results implied HIF-1α inhibition by YC-1 suppressed anaerobic glycolysis via reducing expression of these genes. However, the inhibitory effect of YC-1 or YC-1 + GI on HIF-1α targets was weaker than hypoxic KD cells. Additionally, hypoxic LDHA induction was not affected by 10 μM YC-1 treatment. Higher doses of YC-1 than 10 μM may be necessary for stronger HIF-1α inhibition, thereby hypoxic induction of the HIF-1α targets GLUT1, ALDOC, LDHA, PDK1 and MCT4 may be more strongly suppressed.
We further investigated the biological effect of YC-1 + GI on glucose metabolism. To assess the effect of YC-1 or YC-1 + GI on glucose uptake, the 2DG uptake test was performed with or without YC-1 ± I. 2DG uptake was strongly accelerated by hypoxic stimuli in control cells, which was further elevated by insulin. The promotion of 2DG uptake by hypoxia was smaller in YC-1 (10 μM) compared with controls. WB analysis showed that membranous GLUT1 expression was elevated by GI treatment in control cells under hypoxia, while it was entirely reduced by YC-1 under hypoxia; however, membranous GLUT1 was increased by GI treatment. This suggested that the strong elevation of glucose uptake in hypoxic control cells was due to GLUT1 up-regulation via HIF-1α activation, and the uptake was further promoted by insulin, which enhanced GLUT1 membrane translocation. Insulin signalling may stimulate GLUT1 translocation from intracellular storage vesicles to the plasma membrane as was reported for GLUT4 in adipocytes 31 . Conversely, the hypoxic stimulation of glucose uptake was smaller in 10 μM YC-1 than controls. RT-qPCR showed that the hypoxic induction of GLUT1 mRNA was attenuated by YC-1, which may lead to weaker stimulation of glucose uptake by hypoxia in YC-1-treated cells. However, additional GI sustained increased GLUT1 translocation, and may contribute to promotion of glucose uptake.
Metabolic analysis showed that the OCR/ECAR ratio was significantly elevated by YC-1 in 58As9 cells under hypoxia-mimetic conditions. These results suggested that YC-1 switched glucose metabolism from anaerobic glycolysis to OXPHOS in hypoxic cells. Assessments of glucose metabolites further revealed that 10 μM YC-1 elevated intracellular acetyl-CoA in hypoxic 58As9 cells, while the treatment decreased extracellular lactate. These results suggested that reduced PDK1 expression allows the conversion of pyruvate to acetyl-CoA but not lactate in YC-1-treated cells under hypoxia, which results in the elevation of intracellular acetyl-CoA instead of lactate; furthermore reduced MCT4 expression may decrease lactate efflux. This metabolic reprograming, derived from HIF-1α inhibition by YC-1, may generate excessive ROS and induce hypoxia-dependent apoptosis in YC-1-treated cells. Moreover, additional GI may elevate glucose uptake through membranous GLUT1 translocation in YC-1 treated cells under hypoxia. Thereafter, larger amounts of acetyl-CoA may be produced through glycolysis, inducing further ROS production in the OXPHOS pathway, causing more apoptosis than YC-1 mono-treatment.
Finally, we analyzed the in vivo effect of YC-1 + GI treatment using a tumour xenograft model. YC-1 is known to prevent intravascular thrombus formation by inhibiting platelet aggregation 27 . A previous study reported that YC-1 ip injections (10, 30 mg/kg) prolonged tail bleeding-time in mice 27,28 . Hence, we determined 1 mg/ kg of YC-1 as the optimal dose for preclinical study. Doses of GI at glucose (2 g/kg) and insulin (1 unit/3 g glucose) were determined, because higher doses of glucose (4 or 8 g/kg) resulted in serious hyperglycemia (supplement Fig. 3). The results demonstrated that YC-1 + GI strongly suppressed xenograft tumour growth, while YC-1 mono-treatment incompletely blocked growth. WB analysis showed HIF-1α expression in control 58As9 tumours, suggesting hypoxic regions persisted. In contrast, HIF-1α expression was inhibited in YC-1or YC-1 + GI-treated tumours, but apoptosis markers were more strongly induced by YC-1 + GI than YC-1. Immunohistochemistry findings indicated that YC-1 + GI inhibited HIF-1α expression in xenograft tumours, and this combination coincidently decreased pimonidazole expression. Conversely, YC-1 + GI increased cleaved-caspase3 and 8-OHdG levels in tumours. Thus, YC-1 + GI treatment selectively inhibited hypoxic cancer cell growth in xenograft tumours. In these hypoxic cells, YC-1 inhibits anaerobic glycolysis via HIF-1α suppression, and additional GI promotes glucose uptake. These dual effects of YC-1 + GI synergistically elevate acetyl-CoA through glycolysis, and induce lethal ROS production.  In summary, in vitro apoptotic mechanism of low-dose YC-1 + GI treatment in hypoxic 58As9 cells is illustrated in Fig. 8. This study revealed that low-dose YC-1 + GI therapy targets hypoxic cancer cells, where a metabolic switch from anaerobic glycolysis to OXPHOS is induced under hypoxia, resulting in ROS-mediated apoptosis. Additionally, this treatment may supply glucose to normal cells that live under normoxic environments. Therefore, low-dose YC-1 + GI may be an attractive anti-cancer therapy because hypoxic cancer cells with malignant behaviors may be selectively killed, while normal cells will survive and receive energy from the GI treatment.

Methods
Cell culture conditions and reagents. The GC cell line 58As9 was established and kindly provided by Dr.
Detecting intracellular ROS by flow cytometry. Intracellular ROS levels were evaluated using the Total ROS Detection Kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA) according to the manufacturer's instructions as previously described 16 . ROS fluorescence was detected using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA) and analyzed by the Cell Quest program to determine mean fluorescence.
Total RNA extraction and real-time qPCR (RT-qPCR). Total RNA extraction, followed by cDNA conversion was done as previously described 11 . RT-qPCR was performed using the Light Cycler instrument system (Roche Diagnostics GmbH, Mannheim, Germany) as previously described 16 . Five genes were analyzed by RT-qPCR: glucose transporter 1 (GLUT1), aldolase C (ALDOC), pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase A (LDHA) and monocarboxylate transporter 4 (MCT4). Primers were designed according to the reported cDNA sequences (GenBank, Bethesda, MD, USA), and the sequences are shown in previous study 16 . All experiments were performed in triplicate, and mean values were calculated.
Glucose uptake assay. Glucose uptake in cultured cells was determined using a 2-Deoxyglucose (2-DG) Uptake Measurement Kit (COSMO BIO Co. Ltd., Tokyo, Japan) as previously described 16 . Briefly, cells were cultured under a serum-starved condition for 6 h, followed by further culture for 18 h in regular medium supplemented with 10% FBS. The cells were then incubated for 24 h under normoxia or hypoxia. Thereafter, the cells were treated with or without 500 ng/ml insulin for 18 h. Finally, the cells were treated with 2DG for 20 min and subjected to 2DG uptake measurements according to the manufacturer's instruction. All experiments were performed in triplicate, and mean values were calculated.
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurement. The mitochondrial OCR and ECAR were measured using Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA) as described previously 33 . Briefly, 3 × 104 cells were seeded in each well of Seahorse XFp Cell Culture Miniplates coated with poly-L-lysine solution (Sigma-Aldrich, St. Louis, MO) two days prior to the assay. The cells were exposed to cobalt chloride (CoCl2) (Sigma-Aldrich) at 100 μM final concentration for 16hr to create the mimic hypoxia. Subsequently, the cells were treated with or without YC-1 (10 μM) for 24 h. Before performing the glycolysis stress test, the culture medium was removed from each well, and then the cells were washed 2 times and filled by the assay medium for Seahorse adjusted the pH to 7.4 ( ± 0.02). Thereafter, the glycolysis stress test was employed by sequential injections of glucose (10 mM), oligomycin (2.5 μM) and 2-deoxy-glucose (2-DG) (50 mM). OCR and ECAR under basal and glucose-stimulated conditions were evaluated as means of values at the three time points before and after the addition of glucose, respectively.
Evaluation of acetyl-CoA and lactate levels. Intracellular acetyl-CoA was measured using a Pico Probe ™ Acetyl-CoA Fluorometric Assay Kit (BioVision Inc.). Extracellular lactate was measured using a Lactate Colorimetric/Fluorometric Assay Kit (BioVision Inc.).
Animal experiments. All methods were performed in accordance with the relevant guidelines and regulations. Further, all animal protocols were approved by the Animal Care Committee of Saga University. Female athymic BALB/cA Jcl mice (nu/nu, 4-weeks-old) were obtained from Nihon Crea Co. (Osaka, Japan), kept under specific-pathogen-free conditions and given sterile food and autoclaved water. To establish the tumour models, 3 × 10 6 58As9 cells were subcutaneously injected into the back of the mice. One week after inoculation, xenografts became palpable. The 20 xenograft-bearing mice were divided into four treatment groups as follows: Control [phosphate-buffered saline (PBS)], GI (glucose: 4 g/kg/day, insulin: 1 unit per 3 g-glucose/day), YC-1 (1 mg/kg) and YC-1 + GI (above treatments combined). All drugs were intraperitoneally (ip) administered every 24 h from day 1 to day 14. During treatment, tumours sizes were measured along 2 perpendicular dimensions with a caliper every 4 d. Tumour size (T) was evaluated as the maximum cut area and determined by the formula: T = π/4 × a × b, where a (mm) is the shorter axis and b (mm) is the longer axis.
Evaluation of immunohistochemical staining. The proportion of positively-stained nuclei for HIF-1α and 8-OHdG, or cytoplasm for pimonidazole and cleaved caspase-3 were assessed in the central region of tumours, and semi-quantitatively scored by a pathologist. The proportion of stained cells was evaluated in three fields of hot-spot areas at high power (200× ) and scored from 0-100%. Statistical Analysis. Data were analyzed by ANOVA using Prism 5 software (GraphPad Software, La Jolla, CA, USA). For comparisons between two groups, the differences in mean values were evaluated by Student's t-test and Mann-Whitney U test. For comparisons among three or more groups, Bonferroni post-hoc tests were performed for One-way ANOVA. A value of p < 0.05 was considered statistically significant. All values are expressed as means ± SEM.