Relationship between anticancer sensitivities and cellular respiration properties in 5‐fluorouracil‐resistant HCT116 human colorectal cancer cells

5‐Fluorouracil (5‐FU) is widely used for colorectal cancer (CRC) treatment; however, continuous treatment of CRC cells with 5‐FU can result in acquired resistance, and the underlying mechanism of 5‐FU resistance remains unclear. We previously established an acquired 5‐FU‐resistant CRC cell line, HCT116RF10, and examined its biological features and 5‐FU resistance mechanisms. In this study, we evaluated the 5‐FU sensitivity and cellular respiration dependency of HCT116RF10 cells and parental HCT116 cells under conditions of high‐ and low‐glucose concentrations. Both HCT116RF10 and parental HCT116 cells were more sensitive to 5‐FU under low‐glucose conditions compared with high‐glucose conditions. Interestingly, HCT116RF10 and parental HCT116 cells exhibited altered cellular respiration dependence for glycolysis and mitochondrial respiration under high‐ and low‐glucose conditions. Additionally, HCT116RF10 cells showed a markedly decreased ATP production rate compared with HCT116 cells under both high‐ and low‐glucose conditions. Importantly, glucose restriction significantly reduced the ATP production rate for both glycolysis and mitochondrial respiration in HCT116RF10 cells compared with HCT116 cells. The ATP production rates in HCT116RF10 and HCT116 cells were reduced by approximately 64% and 23%, respectively, under glucose restriction, suggesting that glucose restriction may be effective at enhancing 5‐FU chemotherapy. Overall, these findings shed light on 5‐FU resistance mechanisms, which may lead to improvements in anticancer treatment strategies.

Edited by Ivana Novak 5-Fluorouracil (5-FU) is widely used for colorectal cancer (CRC) treatment; however, continuous treatment of CRC cells with 5-FU can result in acquired resistance, and the underlying mechanism of 5-FU resistance remains unclear. We previously established an acquired 5-FU-resistant CRC cell line, HCT116R F10 , and examined its biological features and 5-FU resistance mechanisms. In this study, we evaluated the 5-FU sensitivity and cellular respiration dependency of HCT116R F10 cells and parental HCT116 cells under conditions of high-and low-glucose concentrations. Both HCT116R F10 and parental HCT116 cells were more sensitive to 5-FU under low-glucose conditions compared with high-glucose conditions. Interestingly, HCT116R F10 and parental HCT116 cells exhibited altered cellular respiration dependence for glycolysis and mitochondrial respiration under high-and low-glucose conditions. Additionally, HCT116R F10 cells showed a markedly decreased ATP production rate compared with HCT116 cells under both high-and low-glucose conditions. Importantly, glucose restriction significantly reduced the ATP production rate for both glycolysis and mitochondrial respiration in HCT116R F10 cells compared with HCT116 cells. The ATP production rates in HCT116R F10 and HCT116 cells were reduced by approximately 64% and 23%, respectively, under glucose restriction, suggesting that glucose restriction may be effective at enhancing 5-FU chemotherapy. Overall, these findings shed light on 5-FU resistance mechanisms, which may lead to improvements in anticancer treatment strategies.
Colorectal cancer (CRC) is the third most deadly cancer in the world [1]. 5-Fluorouracil (5-FU) is the most important anticancer medicine for CRC treatment [2,3]. Following administration, 5-FU is converted to three active metabolites: fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP), and fluorouridine triphosphate (FUTP) [2][3][4][5]. Among these, FdUMP has been found to strongly inhibit thymidylate synthase (TYMS) by forming a covalent complex with TYMS and 5,10-methylenetetrahydrofolate [2,3,5]. This covalent ternary complex inhibits the TS enzyme, depletes the intracellular dTTP pool, and subsequently inhibits DNA synthesis and cell proliferation [2,3]. FdUTP and FUTP induce cytotoxicity through their incorporation into DNA and RNA, respectively [2][3][4]. However, previous clinical and laboratory studies indicate that continuous treatment and exposure of CRC cells to 5-FU can result in acquired resistance [3]. Many previous studies have described the various mechanisms of 5-FU resistance, revealing some of the characteristics of resistant cancer cells. As a resistance mechanism, TYMS gene amplification, which leads to mRNA overexpression and TYMS enzyme overproduction, is known as a major mechanism of resistance to fluoropyrimidines, including 5-FU and its derivatives [6]. Interestingly, the expression of TYMS undergoes translational autoregulation by interacting with the TYMS enzyme and TYMS mRNA [7][8][9][10][11]. This translational autoregulation of TYMS expression is disrupted by the TYMS ligand, resulting in TYMS translational derepression and TYMS enzyme upregulation [7,8,11]. However, treatments have not yet been developed to circumvent this resistance mechanism.
Abnormal metabolism is considered a hallmark of cancer cells; thus, understanding this process has become an important area of research [12][13][14]. Unlike normal cells, which derive most of their energy from mitochondrial oxidative phosphorylation, cancer cells are known to depend on aerobic glycolysis in the presence of abundant oxygen as their primary energy source [15][16][17]; this process is called the Warburg effect [15][16][17]. Abnormal energy metabolism is a promising target for cancer treatment [18]. Recent studies have demonstrated a relationship between the glucose concentration in the cellular environment and the effects of anticancer chemotherapeutic agents, including 5-FU, in various cancer cells [19][20][21]. Previous reports have indicated that high-glucose conditions can increase the proliferation of the human CRC cell lines, SW480, SW620, LoVo, and HCT116 [19]. In addition, high-glucose conditions attenuate cancer growth inhibition by 5-FU in these CRC cell lines [19]. Furthermore, in the pancreatic cancer cell lines, AsPC-1 and Panc-1, the anticancer effects of 5-FU have been shown to decrease in a dose-dependent manner at high-glucose concentrations [20]. Interestingly, high-glucose conditions suppress 5-FU-induced cell death [20]. Furthermore, highglucose conditions enhance cell proliferation and reduce the susceptibility of cells to chemotherapeutic drugs, including 5-FU, in gastric cancer cells [21].
In this study, we investigated the anticancer sensitivity to 5-FU and cellular respiration dependency of 5-FU-resistant HCT116R F10 cells and parental HCT116 cells under high (25 mM) and low (5.5 mM) glucose culture conditions. We also investigated the relationship between anticancer 5-FU activity, cellular respiration dependency, and glucose concentration in 5-FU-resistant HCT116R F10 cells and 5-FU-sensitive parental HCT116 cells.

Reagents
The anticancer drug, 5-FU, was obtained from FUJIFILM Wako Pure Chemical (Osaka, Japan). The drug was stored as a 100-mM stock in dimethyl sulfoxide (DMSO, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at À20°C.

Exome analysis
Genomic DNA extraction was performed as described previously [22]. Genomic DNA was extracted from both types of cells using a DNeasy Tissue Kit (QIAGEN, Venlo, The Netherlands). Exome sequencing analysis of parental HCT116 and HCT116R F10 cells was performed by APRO Life Science Institute Inc. (Tokushima, Japan) and Macrogen Global Headquarters (Seoul, Korea).

Colony formation assay
The colony formation assay was performed as previously described [22][23][24][25]. Cells were dissociated with Accutase and then suspended in the medium. Cells were then inoculated into 6-well plates (200 cells per well) in triplicate and incubated overnight. The cells were treated with various concentrations of the drug or with a solvent (DMSO) as a negative control. After 10 days of incubation, the cells were fixed with 4% formaldehyde solution and stained with 0.1% (w/v) crystal violet, and the number of colonies in each well was counted. The culture medium was not refreshed for 11 days.

Cellular respiration analysis
Cells were dissociated with Accutase and then suspended in high-glucose DMEM. Cells were then seeded in an Agilent Seahorse XF24 cell culture microplate (Agilent Technologies, Santa Clara, CA, USA; 6 9 10 4 cells per well), and the plates were incubated for 24 h. The culture medium was replaced with an analysis medium, high-or lowglucose XF DMEM (Cat#103575-100, Agilent Technologies), and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed using an Agilent Seahorse XFe24 analyzer (Agilent Technologies). The analysis medium contained 2 mM Seahorse XF glutamine solution (Agilent Technologies), 1 mM Seahorse XF pyruvate solution (Agilent Technologies), and 25 mM glucose in the high-glucose medium or 5.5 mM glucose in the low-glucose medium. The OCR and ECAR were analyzed at two time points, 1 h (short period) and 24 h (long period), after replacement with fresh high-and low-glucose DMEM. Additionally, the rate of ATP production was analyzed using a Seahorse XF Real-Time ATP Rate Assay Kit (Cat# 103592-100, Agilent Technologies), according to the manufacturer's protocol.

Statistical analysis
Statistical analyses were performed using the GRAPHPAD PRISM 9 software (GraphPad Software, Boston, MA, USA). Data were presented as the mean AE standard error. Significant differences among the groups were evaluated using Student's t-test, one-way analysis of variance (ANOVA), and two-way ANOVA followed by Tukey's multiple comparisons test. The two-way ANOVA was performed using a general linear model to analyze the interaction between the two factors, including 'cell line' and 'glucose concentration'. The P-values of < 0.05 were considered statistically significant.

Anticancer sensitivity of parental HCT116 cells and resistant HCT116R F10 cells to 5 FU under high-and low-glucose culture conditions
We investigated the resistance mechanisms of 5-FU in HCT116R F10 and parental HCT116 cells, wherein we had previously revealed the genetic background through genomic analysis [22]. We examined the effect of 5-FU on the proliferation of parental HCT116 and 5-FU-resistant HCT116R F10 cells under high and lowglucose culture conditions using clonogenic assays. The high-and low-glucose culture media contained 25 and 5.5 mM glucose, respectively. As shown in Fig. 1A,B, and Table 1, HCT116R F10 cells were 0.6 times (EC 50 = 24 lM) more sensitive to 5-FU under the low-glucose conditions compared with the high-glucose conditions (EC 50 = 38 lM). Similarly, the parental HCT116 cells were 0.6 times (EC 50 = 3.4 lM) more sensitive under the low-glucose condition compared with the high-glucose condition (EC 50 = 5.5 lM; Fig. 1A,B, and Table 1). After treatment with 30 lM 5-FU, the colony formation (%) of the HCT116R F10 cells was significantly decreased in the low-glucose condition (41.7%) compared with the high-glucose condition (60.9%; Fig. 1C). Similarly, the colony formation (%) of the parental HCT116 cells after treatment with 10 lM 5-FU was decreased in the low-glucose condition (2.1%) compared with the high-glucose condition (20.9%; Fig. 1C). Furthermore, parental HCT116 cells were more resistant to 5-FU (EC 50 = 5.2 lM) at highglucose conditions than low-glucose conditions (EC 50 = 3.2 lM; Fig. 1D and Table 1). Alternatively, HCT116R F10 cells were similarly sensitive to 5-FU in either culture condition (EC 50 ≥ 85 lM in high glucose, EC 50 > 100 lM in low glucose). Notably, HCT116R F10 cells treated with low 5-FU concentrations of 3-10 lM had increased sensitivity to 5-FU under low-glucose conditions compared with high-glucose conditions (Fig. 1D). These results suggest that glucose restriction is effective for 5-FU sensitivity in both parental HCT116 cells and HCT116R F10 cells.

Cellular respiration dependency of parental HCT116 cells and HCT116R F10 cells under highand low-glucose conditions
To elucidate the association between cellular respiration dependency and 5-FU sensitivity, we analyzed the OCR and ECAR in both the parental HCT116 cells and the 5-FU-resistant HCT116R F10 cells under high-and lowglucose culture conditions using an extracellular flux analyzer. The OCR and ECAR have been identified as key indicators of mitochondrial respiration and glycolysis [26]. The OCR and ECAR of parental HCT116 and 5-FU-resistant HCT116R F10 cells were analyzed at two time points (1 and 24 h after replacement in a lowglucose medium). As shown in Fig. 2A Fig. 2A,B). In addition, the OCR/ ECAR ratio was found to be slightly higher in HCT116R F10 cells compared with the parental HCT116 cells under high-glucose conditions. In contrast, the OCR/ECAR ratio was slightly lower in the HCT116R F10 cells compared with parental HCT116 cells under low-glucose culture conditions. Interestingly, the OCR/ECAR ratio in the parental HCT116 cells was slightly higher under the low-glucose conditions (ratio = 9.4) compared with the high-glucose conditions (ratio = 9.0). Meanwhile, the OCR/ECAR ratio in HCT116R F10 cells was lower in the low-glucose conditions (ratio = 8.8) compared with the high-glucose conditions (ratio = 9.7). These data suggest that HCT116R F10 cells are more dependent on mitochondrial respiration in high-glucose conditions and on glycolysis under low-glucose culture conditions compared with the parental cells. Furthermore, parental HCT116 cells exhibited a higher cellular respiratory activity of glycolysis and mitochondrial respiration during lowglucose culture conditions compared with high-glucose culture conditions after 1 h.

Discussion
Cancer cells usually exhibit aberrant metabolism resulting from metabolic reprogramming [15][16][17]27]. This results in aerobic glycolysis as a priority over mitochondrial oxidative phosphorylation, which, in turn, provides continuous energy and nutrients to support uncontrolled proliferation. This reprogramming is known as the Warburg effect [15][16][17]. Increasing evidence has indicated that the glucose concentration modulates 5-FU sensitivity in various cancer cell lines [19][20][21]. However, the relationship between 5-FU resistance and respiratory dependence under glucose concentrations in CRC cells remains unclear. Our results revealed that glucose restriction enhances the anticancer effects of 5-FU in parental HCT116 and 5-FUresistant HCT116R F10 cells. In particular, our experiments showed that the respiration dependency of glycolysis and mitochondrial respiration on glucose restriction at short (1 h) and long (24 h) periods differs between 5-FU-sensitive and 5-FU-resistant cells. Glucose restriction at a short period in parental Table 1. Sensitivity of parental HCT116 and HCT116R F10 cells to 5fluorouracil (5-FU) under high-and low-glucose culture conditions. CFA, colony formation assay; EC 50 , 50% effective concentration; HG, high-glucose condition; LG, low-glucose condition; RI, resistance index; SI, sensitivity index; WST-8, cell viability WST-8 assay. RI shows the ratio of EC 50 values between the resistant and parental cell lines; RI indicates the ratio of EC 50 in HCT116R F10 /EC 50 in HCT116. SI shows the ratio of EC 50 values between high-and lowglucose conditions in parental HCT116 and resistant HCT116R F10 cells, respectively. SI indicates the ratio of EC 50 in LG/EC 50 in highglucose condition.   HCT116 cells resulted in enhanced metabolic activity for both glycolysis and mitochondrial respiration. In contrast, glucose restriction for long periods in parental HCT116 cells or at short and long periods in 5-FU-resistant HCT116R F10 cells resulted in reduced metabolic activity for both glycolysis and mitochondrial respiration. In addition, our results indicated that parental HCT116 and 5-FU-resistant HCT116R F10 cells remain dependent on mitochondrial respiration during 24-h glucose restriction. Moreover, the ATP production rate in the long period of glucose restriction was more decreased in the 5-FU-resistant HCT116R F10 cells than in the parental HCT116 cells, indicating that 5-FU-resistant HCT116R F10 cells suppress ATP synthesis from glycolysis and mitochondrial respiration more effectively than parental HCT116 cells under both high-and low-glucose culture conditions. Importantly, cellular respiration dependence under high-and low-glucose conditions was reversed in both the sensitive parental HCT116 and the 5-FUresistant HCT116R F10 cells. We considered that the metabolic properties of these resistance cells contribute to 5-FU resistance. Previous studies have suggested that fasting exerts extensive anticancer effects in various cancers, including CRC [14,28]. Weng et al. [14] reported that fasting negatively regulates glucose metabolism and proliferation in CRC via the upregulation of cholesterogenic FDFT1 mediated the suppression of AKT-mTOR-HIF1 signaling. In addition, the authors indicated in clinical significance that patients with high expression levels of FDFT1 and low expression levels of AKT1, mTOR, HIF1a, GLUT1, and HK2 exhibited longer survival than those with low expression levels of FDFT1 and high expression levels of the AKT1-mTOR-HIF1a pathway and glycolytic genes [14]. Our findings further indicate that glucose restriction is effective in sensitizing 5-FU-resistant CRC cells to chemotherapy. Interestingly, we demonstrated that several cluster genes related to glucose metabolism (including glycolysis, the citric acid cycle, and oxidative phosphorylation) were differentially altered in HCT116 and HCT116R F10 cells. We further investigated the associations between cellular respiratory dependence, mitochondrial function, glucose metabolism-related genes, and 5-FU resistance mechanisms. Collectively, our findings provide a better understanding of 5-FU sensitivity and resistance mechanisms and may lead to strategies to circumvent resistance to 5-FU and its derivatives.

Conclusion
We demonstrated that glucose restriction enhances the sensitivity to 5-FU in both 5-FU-resistant HCT116R F10 cells and parental HCT116 cells. In addition, we revealed that 5-FU-resistant HCT116R F10 cells suppress the ATP synthesis rate of glycolysis and mitochondrial respiration more effectively than

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Fig. S1. Cellular respiration property of 5-FU-resistant HCT116R F10 and parental HCT116 cells to 5-FU under high-and low-glucose culture conditions.