Rare sugar L-sorbose exerts antitumor activity by impairing glucose metabolism

doi:10.21203/rs.3.rs-1694501/v1

Download PDF

Article

Rare sugar L-sorbose exerts antitumor activity by impairing glucose metabolism

https://doi.org/10.21203/rs.3.rs-1694501/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 11 Mar, 2023

Read the published version in Communications Biology →

Version 1

posted

You are reading this latest preprint version

Rare sugars are monosaccharides with low natural abundance. They are structural isomers of dietary sugars, but hardly be metabolized. Here, we report that rare sugar l-sorbose induces apoptosis in various cancer cells. As a C-3 epimer of d-fructose, l-sorbose is internalized via the transporter GLUT5 and phosphorylated by ketohexokinase (KHK) to produce l-sorbose-1-phosphate (S-1-P). Cellular S-1-P inactivates the glycolytic enzyme hexokinase resulting in attenuated glycolysis. Consequently, mitochondrial function is impaired and reactive oxygen species are produced. Moreover, l-sorbose downregulates the transcription of KHK-A, a splicing variant of KHK. Since KHK-A is a positive inducer of antioxidation genes, the antioxidant defense mechanism in cancer cells can be attenuated by l-sorbose-treatment. Thus, l-sorbose performs multiple anticancer activities to induce cell apoptosis. In mouse xenograft models, l-sorbose inhibits the proliferation of tumors either alone or in combination with other anticancer drugs. These results demonstrate l-sorbose as an attractive therapeutic reagent for cancer treatment.

Sugars, in particular glucose, serve as sources of energy and organic carbon in mammalian cells. In normal tissues, glucose is metabolized via glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. However, metabolic pathways are altered by cellular and environmental conditions. For example, many types of cancer cells are dependent on aerobic glycolysis in that glucose is metabolized via glycolysis and lactic acid fermentation; this phenomenon is known as the Warburg effect^1,2. Remodeling of the metabolic pathway is required for cancer cells to maintain their rapid proliferation and viability³. Therefore, in cancer cells, rate-limiting enzymes for glycolysis, including hexokinase (HK), phosphofructokinase, and pyruvate kinase, are often upregulated⁴.

Some sugars are known to modulate glycolytic processes and influence the proliferation of cancer cells. For example, mannose is known to inhibit the growth of cancer cells⁵. Mannose exhibits antiproliferative activity because mannose-6-phosphate, which is generated by HK, inhibits glycolytic enzymes including HK itself. Although mannose does not induce cancer cell death, it can enhance the effect of tumor chemotherapy⁵. In contrast to mannose, fructose is reported to activate the proliferation of cancer cells^6–8. Fructose is a monosaccharide widely used as a sweetener. After internalization in cells via the glucose/fructose transporter GLUT2 or the fructose-specific transporter GLUT5, the monosaccharide is phosphorylated to produce fructose-1-phosphate (F-1-P)⁹. Accumulation of F-1-P leads to inactivation of pyruvate kinase M2 (PKM2). The inhibitory effect of F-1-P is most likely mediated by its direct binding to PKM2. In cells under hypoxic conditions, such as small intestinal epithelial cells and tumors, inactivation of PKM2 is beneficial for their proliferation. Indeed, a previous study showed that feeding a high-fructose diet in mice caused intestinal tumor progression⁶.

Ketohexokinase (KHK) is the enzyme that produces F-1-P from fructose. Intriguingly, alternative splicing of the KHK gene can generate two distinctive functional isoforms termed KHK-A and KHK-C¹⁰. KHK-C has a much greater affinity for fructose than KHK-A; phosphorylation of fructose is primarily mediated by KHK-C¹¹. KHK-A can mediate the phosphorylation of proteins besides sugars^12,13. The selective autophagy receptor p62 is one of the substrates of the kinase. Phosphorylation of p62 at Ser28 results in the activation of nuclear factor erythroid 2–related factor 2 (Nrf2) via degradation of its inhibitor Kelch-like ECH-associated protein 1 (Keap1)¹⁴. Nrf2 is a transcription factor that induces antioxidant genes to counteract ROS. The expression of KHK-A is upregulated in various cancer cells^10,14,15. Thus, KHK-A is involved in the proliferation of cancer cells irrespective of the activity to produce F-1-P. The function of KHK-A in normal tissues remains elusive.

Rare sugars are defined as monosaccharides and their derivatives are rarely found in nature. Recent progress in the large-scale production of rare sugars enables us to analyze their biological activities¹⁶. Previous reports have shown that rare sugars exhibit beneficial activities including anti-obesity, anti-diabetic, anti-pathogenic microorganism, and antitumor effects^17–20. For example, d-allose exhibits antitumor activity, although its mechanism remains elusive^21–24. Here, we report that another rare sugar, l-sorbose, uniquely induces apoptotic death in cancer cells. Given that l-sorbose is harmless in humans, this rare sugar could be applied for cancer treatment.

l-Sorbose triggers mitochondrial dysfunction and enhances ROS accumulation in cancer cells to induce cell death

The rare sugar d-allose was previously reported to target several cancer cells. To determine whether other rare sugars have potential in cancer therapy, a cell viability screening employing six rare sugars was performed on six cancer cell lines (Fig. 1a). From this screening, we found that l-sorbose could kill various cancer cell lines. In particular, the viabilities of the liver cancer cell lines Huh7 and HepG2 were significantly decreased by treatment with 25 mM l-sorbose; however, their viabilities were not influenced by treatment with the same concentration of d-allose (Fig. 1a). l-Sorbose exhibited half-maximum inhibitory concentration (IC50) values of 33.82 mM (24 h), 27.32 mM (48 h) and 30.88 mM (72 h) in Huh7 cells and 27.68 mM (24 h), 34.89 mM (48 h) and 22.60 mM (72 h) in HepG2 cells (Fig. 1b). In addition, long-term colony formation assay showed that l-sorbose treatment impaired the proliferation of these two liver cancer cell lines (Fig. 1c).

To examine whether the cell death induced by l-sorbose is apoptosis, cells treated with l-sorbose were stained with annexin V and propidium iodide (PI), and the ratio of apoptotic cells was measured. The ratio of early stages of apoptosis (annexin V-positive, PI-negative cells) and total apoptosis (both annexin V- and PI-positive cells) were increased in the cells treated with 25 mM and 50 mM l-sorbose (Fig. 1d). In addition, the levels of cleaved caspase 3 and the ratio of BAX/Bcl2 increased in Huh7 cells as the l-sorbose concentration increased (Fig. 1e).

The intrinsic apoptotic pathway can be induced by mitochondrial dysfunction and reactive oxygen species (ROS) production^25,26. Indeed, the mitochondrial membrane potential (MMP) decreased significantly when Huh7 and HepG2 cells were treated with 25 mM l-sorbose for 3 h (Figs. 2a, b). ROS levels were significantly elevated when the cells were treated with 25 mM l-sorbose for 6 h (Figs. 2c, d). Cell viability was significantly decreased after 25 mM l-sorbose treatment for 9 h (Fig. 2e). To determine whether l-sorbose-induced apoptosis is attributable to ROS production, Huh7 and HepG2 cells were treated with the antioxidant N-acetyl-L-cysteine (NAC). As shown in Fig. 2f, l-sorbose-induced cell death was abrogated by NAC treatment. To further verify that l-sorbose treatment causes an increase in ROS production in mitochondria, intracellular ROS were stained with dichlorodihydrofluorescein diacetate (DCFH-DA) in Huh7 cells incubated with l-sorbose for 6 h (Fig. 2g). Fluorescence-activated cell sorting (FACS) analysis showed that the levels of DCFH-DA staining were higher in cells incubated in l-sorbose (Fig. 2h). ROS production in mitochondria can be reduced by mitoquinone mesylate (mitoQ) treatment²⁷. As shown in Fig. 2h, in cells incubated with l-sorbose, the levels of DCFH-DA staining were decreased in the presence of mitoQ. The volume of mitochondria was not altered by l-sorbose treatment (Fig. 2i). These results suggest that l-sorbose can promote mitochondrial ROS production in cancer cells, which induces apoptotic cell death.

The combination of sorafenib with l-sorbose enhances cell death

To examine whether l-sorbose inhibits tumor growth in vivo, the efficacy of l-sorbose on Huh7 cells subcutaneously injected into nude mice was analyzed. The mice were orally gavaged with either normal drinking water or 20% l-sorbose for 4 weeks. l-Sorbose treatment resulted in growth inhibition of xenografts, as well as reductions in tumor weight (Figs. 3a-c, Extended Data Fig. 1a).

Next, we examined whether l-sorbose could enhance the anticancer effect of chemotherapy. Sorafenib is a multikinase inhibitor used to treat liver, kidney, and thyroid cancers^28,29. The IC50 values of sorafenib in Huh7 and HepG2 cells were 8.275 mM and 6.731 mM, respectively (Extended Data Fig. 1b). These cancer cells were then treated with sorafenib (2 or 4 mM) and l-sorbose (12.5 or 25 mM). As shown in Fig. 3d, the low-dose combination of l-sorbose (12.5 mM) and sorafenib (2 mM) markedly decreased the cell viability compared with either alone. l-Sorbose- and sorafenib-treated cells died due to the induction of apoptosis (Fig. 3e). In line with this notion, cell death induced by sorafenib and l-sorbose was alleviated by incubation with the pancaspase inhibitor z-VAD-FMK (Figs. 3e, f). Cell death was not induced when the cancer cells were incubated with 25 mM of other rare sugars and 2 µM sorafenib (Extended Data Fig. 1c). l-Sorbose also exhibited a synergistic effect to kill cancer cells with other chemotherapeutic drugs (Extended Data Fig. 1d). To examine whether l-sorbose can enhance the therapeutic effect of sorafenib in vivo, nude mice subcutaneously injected with Huh7 cells were treated with l-sorbose and sorafenib either alone or in combination. We found that tumor volume became less than half by the combined use of l-sorbose and sorafenib compared to l-sorbose or sorafenib treatment (Fig. 3b). However, we could not find significant differences in tumor weight between the combination treatment and sorafenib treatment alone (Fig. 3c). A greater inhibitory effect on tumor growth was observed when sorafenib was administered in combination with l-sorbose in mouse xenograft models (Figs. 3a-c, Extended Data Fig. 1a). No notable body weight loss was observed in mice treated with l-sorbose and/or sorafenib (Fig. 3g).

l-Sorbose interferes with glucose metabolism by targeting hexokinase

To gain insight into the molecular basis for the anticancer activity of l-sorbose, we performed central carbon metabolomic analyses. The results showed that treatment with l-sorbose in cancer cells led to marked decreases in several intermediate metabolites in glycolysis and the tricarboxylic acid (TCA) cycle, including glucose-6-phosphate (G-6-P), 3-phosphoglyceric acid, l-lactic acid, and a-ketoglutarate (a-KG) (Fig. 4a). The decrease in G-6-P levels was of particular interest because the result suggested that the activity of HK is reduced by l-sorbose treatment. Indeed, the kinase activity in lysates of Huh7 and HepG2 cells was decreased by l-sorbose treatment (Fig. 4b). To further verify that the cytotoxic effect of l-sorbose is attributable to a decrease in HK activity, we assessed whether the effect of l-sorbose was abolished by culturing cells in glucose-free DMEM supplemented with galactose. Unlike glucose or other monosaccharides including mannose, galactose enters glycolysis without using HK^30,31. l-Sorbose treatment resulted in substantial decreases in cell viability in the cells cultured with glucose or mannose; however, the cytotoxicity was significantly alleviated by culturing cells in galactose (Fig. 4c). These results suggest that the apoptotic cell death induced by l-sorbose treatment is attributable to the reduced activity of HK. In many cancer cells, the Type II isoform of hexokinase (HK2) is overexpressed³². Thus, we analyzed whether HK2 levels were decreased in l-sorbose-treated cells. However, HK2 levels were not altered by l-sorbose treatment (Fig. 4d). This result suggests that l-sorbose treatment inactivates HK.

The expression level of KHK dictates l-sorbose sensitivity

Structurally, l-sorbose is the C-3 epimer of d-fructose³³. We found that the cytotoxicity of l-sorbose was alleviated by the addition of d-fructose (Fig. 5a), suggesting that l-sorbose and fructose are internalized by the same transporter. Thus, we examined whether GLUT5, which is the specific transporter for fructose, was required for the uptake of l-sorbose³⁴. SLC2A5 (the gene name of GLUT5) was knocked out in Huh7 and HepG2 cells; loss of GLUT5 protein in SLC2A5 KO cells was verified by western blotting (Fig. 5b). SLC2A5 KO cells showed greater tolerance to l-sorbose; these cells did not exhibit a marked decrease in cell viability even under 100 mM l-sorbose (Fig. 5c). These results indicate that the cellular uptake of l-sorbose is mediated by GLUT5.

Upon entering cells, monosaccharides are often phosphorylated in the first step of their metabolism. Accordingly, accumulation of l-sorbose-1-phosphate (S-1-P) was observed in cells incubated with l-sorbose (Fig. 5d). Since ketohexokinase (KHK) can phosphorylate furanoses including fructose, we examined whether phosphorylation of l-sorbose was mediated by this kinase. KHK has two isoforms KHK-A and KHK-C; KHK-C exhibits greater activity to phosphorylate fructose¹¹. Thus, we performed an in vitro phosphorylation assay using recombinant human KHK-C. The results demonstrated that KHK-C can catalyze the conversion of l-sorbose into S-1-P (Extended Data Fig. 2). In accordance with this result, S-1-P levels were increased by overexpression of KHK-C in Huh7 and HepG2 cells (Figs. 5d, e). By overexpression of KHK-C, Huh7 and HepG2 cells became more sensitive to l-sorbose treatment and the levels of apoptotic cell death were elevated (Figs. 5f, g). In contrast, the knockout of KHK in these cells prevented the cell lines from being affected by l-sorbose cytotoxicity (Figs. 5h, i). These results suggest that KHK is a critical enzyme to dictate sensitivity to l-sorbose. Thus, we analyzed the expression levels of KHK-C and viability after l-sorbose treatment in various cancer cell lines. The results showed that the expression levels of KHK-C in HeLa, MCF7, and T24 cells were relatively low (Fig. 6a). Although HeLa cells were killed by incubation with l-sorbose, MCF7 and T24 cells were resistant to the treatment (Fig. 6b). We speculated that the resistance of these cells to l-sorbose is attributable to lower expression levels of KHK-C. In line with this hypothesis, overexpression of KHK-C caused a decrease in cell viability and an increase in apoptosis levels in MCF7 and T24 cells (Figs. 6c-g).

l-Sorbose regulates the expression of the two KHK isoforms

We found that KHK levels were elevated by treatment with l-sorbose (Fig. 7a). In cancer cells, KHK-A is generally upregulated and supports survival by counteracting ROS production¹⁴. Intriguingly, we found that KHK-A levels were decreased in cancer cell lines treated with l-sorbose. KHK-C levels were elevated by l-sorbose treatment (Figs. 7b, c). Since KHK-A serves as a positive regulator to induce antioxidative genes via activation of Nrf2, a decrease in KHK-A levels may be another cause of apoptosis induction in l-sorbose-treated cancer cells. Thus, we assessed whether l-sorbose treatment affects the Nrf2 pathway. In cancer cell lines, Nrf2 is predominantly detected in the nuclear fraction. However, when the cells were treated with l-sorbose, the levels of Nrf2 detected in the nuclear fraction were significantly decreased (Fig. 7d). In addition, antioxidative genes regulated by Nrf2, including heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic (GCLC), quinone oxidoreductase 1 (NQO1), and phosphogluconate dehydrogenase (PGD), were downregulated in l-sorbose-treated cells (Fig. 7e). Notably, KHK-A levels were not decreased by l-sorbose treatment in the normal hepatocyte cell line WRL68 (Fig. 7c). Nrf2 levels detected in the nuclear fraction were not altered by l-sorbose treatment in WRL68 cells (Fig. 7d). These results suggest that l-sorbose modulates the splicing switch to generate KHK isoforms in cancer cells, which helps to induce apoptosis. A previous study showed that mannose exhibits anticancer activity via HK inactivation⁵. However, KHK-A levels were not altered by mannose and neither KHK-C (Extended Data Fig. 3). Thus, modulation of KHK splicing would be a unique activity for l-sorbose.

l-Sorbose is the intermediate of a fermentation process for manufacturing vitamin C³⁵. Although it is rare in nature, l-sorbose can be efficiently produced by the bioconversion from d-sorbitol in Gluconobacter or Acetobacter³⁶. Here, we show that the rare sugar l-sorbose induces apoptotic cell death in cancer cells. Our results show that l-sorbose exhibits two anticancer activities: ROS production induced by HK inactivation and attenuation of a cellular antioxidant defense mechanism by KHK-A downregulation. Our results suggest that l-sorbose-mediated apoptotic cell death is induced by the synergy of these effects (Fig. 8).

In cancer cells, various metabolites, including glucose-6-phosphate (G-6-P), 3-phosphoglyceric acid, l-lactic acid, and α-ketoglutarate (α-KG), are decreased by l-sorbose treatment (Fig. 4a). This result suggests that a number of enzymes required to produce the metabolites are inactivated by l-sorbose treatment. Nevertheless, we conclude that the anticancer activity is primarily attributable to inactivation of HK because cell death induced by l-sorbose treatment was significantly reduced by culturing cells in galactose-containing media (Fig. 4c). Galactose is converted to G-6-P without using HK^30,31. HK is a rate-limiting enzyme in glycolysis, and its activity is generally elevated to satisfy their high metabolic demands in cancer cells³⁷. Depletion of glucose or inactivation of critical glycolytic enzymes in cancer cells is known to induce oxidative stress³⁸. Thus, ROS production in l-sorbose-treated cells is most likely attributable to a decrease in HK activity and a subsequent reduction in glucose metabolism. In l-sorbose treated cells, levels of HK2 were not altered, while total activity of HK2 in the cells was decreased, indicating that enzymatic activity of HK is inhibited by l-sorbose treatment (Figs. 4b, d). KHK is required for l-sorbose to exhibit anticancer activity, showing that inhibition of HK is mediated by S-1-P or its derivatives. A previous report showed that S-1-P inhibited HK in dialyzed bovine brain extract³⁹. Although the detailed mechanism for HK inhibition by S-1-P remains unknown, S-1-P could serve as a direct inhibitor of the kinase. Given that KHK is a pivotal enzyme for l-sorbose to perform its anticancer activity, the expression levels of this kinase could be a marker to indicate sensitivity to l-sorbose. In support of this hypothesis, l-sorbose exhibits cytotoxicity in a manner dependent on the levels of KHK expression (Figs. 5e-i, Fig. 6).

Similar to l-sorbose, mannose suppresses the proliferation of cancer cells because phosphorylated mannose inhibits HK activity⁵. However, unlike l-sorbose, mannose does not induce apoptotic cell death. l-sorbose exhibits more severe anticancer activity than mannose, presumably because the rare sugar performs an additional function; that is, conversion of KHK isoforms. Incubation of cancer cells with l-sorbose results in elevation of KHK-C levels (Figs. 7b, c). Genes involved in fructose metabolism are known to be induced by the uptake of fructose. Thus, like fructose, l-sorbose may be able to induce fructolytic genes. In contrast to KHK-C, KHK-A is significantly decreased by the l-sorbose treatment (Figs. 7b, c). Downregulation of KHK-A causes attenuation of the Nrf2 antioxidation pathway, which should make cancer cells sensitive to ROS. Given that KHK-C and -A are generated via alternative splicing, these results indicate that the regulation of KHK splicing is modulated by l-sorbose treatment. c-Myc, and heterogeneous nuclear ribonucleoproteins H1 and H2 are known to be involved in the splicing switch from KHK-C to KHK-A; thus, this pathway may be affected in l-sorbose-treated cells¹⁰.

We demonstrate that l-sorbose alone or in combination with other chemotherapy reagents exhibits anticancer activity in vitro and in vivo (Figs. 3a-f, Extended Data Figs. 1a, c, d). It is notable that l-sorbose is reported to induce hemolysis in dogs due to inactivation of glycolysis in erythrocytes^40–42. However, in erythrocytes derived from other animals, including humans, hemolysis is not induced by inactivation of glycolysis⁴⁰. Indeed, no obvious health impact was observed in our mouse experiments. The toxicity of l-sorbose for humans has not been reported thus far. Thus, although further examinations are required regarding the safety and therapeutic effects of l-sorbose in vivo, the rare sugar would be an attractive reagent used for anticancer treatment.

Plasmids

The oligonucleotides and plasmids used in this study are listed in Supplementary Table 1 and Table 2. For gene overexpression, human KHK-C was cloned into pLVX-puro. For gene knockout, guide RNA sequences of KHK or SLC2A5 were cloned into the pLVX-CRISPR-v2-puro vector. Human KHK was cloned into the pET28a vector for recombinant KHK expression in E. coli. All plasmids were sequenced and verified.

Cell lines and cell culture conditions

The cell line WRL68 was purchased from Mingzhou Biotechnology. Other cell lines were obtained from the Cell Bank of the Type Culture Collection of Chinese Academy of Sciences. Huh7, HepG2, A549, HeLa, K562, MCF7 and HEK293T cells were cultured in DMEM high glucose medium (Biological Industries, 0023119), and T24 and WRL68 cells were cultured in RPMI 1640 medium (Biological Industries, 2035127) with 10% fetal bovine serum (FBS) (Biological Industries, 1841924) in 5% CO₂ at 37 ℃. All cells were identified without mycoplasma infection.

To overexpress genes in cultured cells, expression plasmids were stably transfected with a retrovirus-mediated transfection method. For this, HEK293T cells were transfected with pLVX-puro (empty plasmid) or pLVX-KHK-C-puro and the two packaging plasmids, psPAX.2 and pMD2.G. Two days later, the virus particles were collected, filtered, and used to infect the target cells. After 48 hours of infection, transfected cells were selected with 2 mg/mL puromycin. Cells stably transfected with pLVX-puro were used as a control cell line.

CRISPR–Cas9-mediated gene knockout

HEK293T cells were transfected with pLVX-CRISPR-v2-puro (empty plasmid), pLVX-CRISPR-v2-SLC2A5-puro or pLVX-CRISPR-v2-KHK-puro and the two packaging plasmids, psPAX.2 and pMD2.G. Two days later, the virus particles were collected, filtered, and used to infect the target cells. After 48 hours of infection, 2 mg/mL puromycin (InvivoGen, QLL-41-03) was used to select positive cells. The selected cells were subjected to limiting dilution to obtain knockout cells. Western blotting was used to verify the expression of the target genes. Selected knockout clones were analyzed and verified by DNA sequencing.

Metabolomic analysis

For central carbon metabolomic analyses, cells were seeded on 10 cm culture plates and cultured to a density sufficient for 70-80% confluence. After l-sorbose treatment for 12 h, the cells were collected and added to 400 mL water. Cells were vortexed for 1 min after the addition of 500 mL precooled chloroform/methanol (1/3, v/v) and then homogenized for 4 min at 35 Hz and sonicated for 10 min. The homogenization and sonication circles were repeated three times, vibrating at 4 ℃ for 15 min, and incubating at -80 ℃ for 1 h. The samples were centrifuged at 9,000 × g for 10 min. The supernatants were collected and dried under a gentle stream of nitrogen. Then, they were dissolved in 200 mL ultrapure water. Reconstituted samples were vortexed before filtration through a centrifuge tube filter and subjected to HPIC-MS/MS analysis.

The HPIC separation was performed using a Thermo Scientific Dionex ICS-6000 HPIC System (Thermo Scientific) equipped with Dionex IonPac AS11-HC (2 × 250 mm) and AG11-HC (2 mm × 50 mm) columns. Mobile phase A was 100 mM NaOH in water, and mobile phases C and D were methanol and water, respectively. Another pumping system was used to supply the solvent (2 mM acetic acid in methanol), and the solvent was mixed with the effluent before entering electrospray ionization (ESI) (flow rate of 0.15 mL/min). The column temperature was 30 ℃. The temperature of the autosampler was 4 ℃ and the injection volume was 5 mL. An AB SCIEX 6500 QTRAP+ triple quadrupole mass spectrometer (AB Sciex) equipped with an ESI interface was used for analysis and development. Typical ion source parameters were as follows: ion spray voltage = -4500 V, curtain gas = 30 psi, ion source gas 1 = 45 psi, ion source gas 2 = 45 psi, and temperature = 450 ℃. By injecting a standard solution of a single analyte into the API source of the mass spectrometer, flow injection analysis was used to optimize the MRM parameters for each target analyte. AB SCIEX Analyst Workstation Software (1.6.3 AB SCIEX), MultiQuant 3.0.3. software and Chromeleon 7 were employed for MRM data collection and processing.

Quantitative analysis of intracellular l-sorbose-1-phosphate

Cells were seeded on 35 mm culture plates and cultured to a density of approximately 70%. After l-sorbose treatment for 24 h, the medium was aspirated, and 1 mL of 80% methanol:20% water mixture was added to extract metabolites. The plate was placed at -20 degrees for 20 minutes. Then, the cell material was scraped into a 1.5 mL test tube prechilled on ice. The cell debris was pelleted by centrifugation at 15,000 × g at 4 ℃ for 10 min, and the supernatant was transferred to a new tube and stored at -20 ℃ until analysis.

HPLC separation was carried out using a WATERS ACQUITY UPLC System (WATERS) equipped with HILIC-Z 2.7 μm (2.1×100 mm) columns. The mobile phases were acetonitrile and 0.1% formic acid. The column temperature was 45 ℃. The injection volume was 5 mL. The mass spectrometric data were collected on a MALDI SYNAPT Q-TOF mass spectrometer (WATERS) connected with an electrospray ionization interface in positive ion mode (ESI+). Typical ion source parameters were as follows: capillary voltage of 3.5 kV, cone voltage of 30 V, source block temperature of 100 ℃, desolvation temperature of 400 ℃, desolvation gas flow of 700 l/h, cone gas flow of 50 l/h, and collision energy of 6/20 eV. The mass range of m/z 20–2000 was scanned. MassLynx V4.2 software (WATERS) was employed for data acquisition and processing.

Xenograft model

All procedures were conducted in compliance with all the relevant ethical regulations and were approved by the Jiangnan University Animal Welfare and Ethical Review Body. Male BALB/c nude mice aged 5 weeks were purchased from Charles River Laboratories and placed in the Animal Experiment Center of Jiangnan University. Mice were placed five per cage with free access to water and food (chow diet). After habituation for a week, mice were inoculated subcutaneously with Huh7 cells (1.0 ×10⁷ cells in 100 μL PBS per mouse). When the tumors reached 50-100 mm³, the mice were randomly assigned to different groups. Sorafenib was dissolved in dimethyl sulfoxide (DMSO) and diluted in 5% sodium carboxymethyl cellulose. Mice received 20% l-sorbose in water according to their body weights (200 mL/20 mg) by oral gavage once every day. Sorafenib was intragastrically administered to mice at a dose of 50 mg/kg every day. Mice were sacrificed when the xenograft tumor size reached 1,000 mm³. None of the mice showed severe weight loss or signs of infection or wounds. The tumor volume was measured every other day until the endpoint and calculated according to the equation: Volume = Length × Width² × 1/2.

Western blot

Cells were lysed in RIPA buffer (Solarbio, R0020) supplemented with protease inhibitor cocktail (MCE, HY-K0010) on ice for 30 min. After centrifugation at 15,000 × g for 10 min at 4 ℃, the protein lysates were collected. Then the protein lysates were separated by SDS–PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat powdered milk for 1 h and incubated with primary antibodies against b-actin (Proteintech, 23660-1-AP, 1:1000), Bax (CST, 2772T, 1:1000), Bcl-2 (CST, 3498T, 1:1000), b-tubulin (Proteintech, 10068-1-AP, 1:1000), PCNA (BBI, D220014, 1:1000), Nrf2 (Abcam, ab137550, 1:1000), HK2 (Proteintech, 66974-1-lg, 1:5000), GLUT5 (Santa Cruz, sc-271005, 1:1000), KHK (Santa Cruz, sc-377411, 1:1000), KHK-A (SAB, 21709, 1:500), and KHK-C (SAB, 21708, 1:500) at 4 ℃ overnight and then probed with the appropriate secondary antibodies for 1 h at room temperature. The bands were visualized with western ELC substrate (BIO-RAD, 1705060), and the images were captured on a Tanon-5200Multi visualization instrument.

Quantitative real-time PCR

Total RNA was isolated from cells using CellAmp^TM Direct RNA Prep Kit for RT–PCR (TaKaRa, 3732), and complementary DNA was synthesized from 5 mg total RNA using PrimeScript^TM RT Master Mix (TaKaRa, RR036A). qPCR was performed using TB Green® Premix Ex Taq^TM II (Tli RNaseH Plus) (TaKaRa, RR420A) on a Prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions. Primer sequences for qPCR are listed in Supplementary Table 3.

Cell viability assay

Cells were seeded in 96-well culture plates at a density of 5.0 × 10³ cells/well and grown overnight. After treatment with l-sorbose or other reagents, the cells were incubated with CCK-8 (Cell Counting Kit-8, DOJINDO Laboratories, CK04) solution and cultured at 37 ℃ for another 1 h. The absorbance was detected at 450 nm wavelength using a microplate reader (BIO-RAD).

Colony formation assay

Cells were seeded in 6-well plates at a density of 1000 cells/well. Cells were incubated for 24 h to allow attachment to the plates, after which l-sorbose was added to the cells and incubated for 24 h. The cells were cultured for 14 days in the absence of l-sorbose and fixed with a 4% paraformaldehyde fix solution (Beyotime Biotechnology, P0099) for 20 min. Then, the cells were stained with crystal violet (Beyotime Biotechnology, C0121) solution diluted in water. After 10 minutes, the plate was washed with water left to dry and scanned.

Cell apoptosis assay

The cellular apoptosis rate was determined using the Annexin V/PI double-staining Kit (Dojindo Laboratories, AD10). Cells were seeded in 6-well culture plates at a density of 2.0 × 10⁵ cells/well. After incubation with different concentrations of l-sorbose for 24 h, the cells were washed twice with annexin V binding buffer and double-stained with annexin V and PI. Cell apoptosis was examined on a FACSCalibur flow cytometer (BD Accuri C6) and analyzed by FlowJo software.

Measurement of ROS and mitochondrial volume

The Intracellular ROS levels were detected by DCFH-DA (Beyotime Biotechnology, S0033S). Cells were cultured in 6-well plates at a density of 2.0 × 10⁵ cells/well. After being treated with l-sorbose, the cells were gently washed with D-PBS (BBI, E607009) followed by incubation with DCFH-DA at 37 ℃ for 30 min.

To obtain microscopy images, cells were seeded on a confocal dish overnight and treated with l-sorbose for 6 h. After being treated with l-sorbose, the cells were gently washed with D-PBS followed by incubation with MitoBright LT (Dojindo Laboratories, MT11) and DCFH-DA (Dojindo Laboratories, CK04) at 37 ℃ for 30 min. Then the cells were washed with D-PBS twice. Images were obtained with a Nikon C2 Eclipse Ti-E inverted confocal microscope equipped with NIS-Element AR software.

To measure the volume of mitochondria, cells were cultured in 6-well plates at a density of 2.0 × 10⁵ cells/well. After being treated with l-sorbose, the cells were gently washed with D-PBS followed by incubation with MitoBright at 37 ℃ for 30 min. Then the cells were washed twice with D-PBS. The fluorescence of the cells was measured immediately on a FACSCalibur flow cytometer and analyzed by FlowJo software.

Measurement of mitochondrial membrane potential

Cells were cultured in 6-well plates at a density of 2.0 × 10⁵ cells/well. After being treated with l-sorbose, the cells were gently washed with D-PBS. The changes in mitochondrial membrane potential were detected by staining with JC-1 dye (Beyotime Biotechnology, C2006). After incubating with 10 mM JC-1 staining solution in a cell incubator at 37 ℃ for 15 minutes, the cells were washed twice with JC-1 staining buffer and then analyzed by flow cytometry.

Intracellular HK activity measurement

Cells were seeded into 6-well dishes at a density of 2.0 × 10⁵ cells/well. Cells were treated with l-sorbose for 24 h and then collected for the measurement of HK activity using a Hexokinase Activity Detection Kit (Solarbio, BC0740) according to the manufacturer’s protocol. HK activity was calculated and normalized to the cell number.

Production of recombinant ketohexokinase

The plasmid pET28a-KHK was transformed into Escherichia coli BL21 cells. BL21 cells were grown to an absorbance of 0.6-0.8 at 600 nm and then induced with 0.1 mM IPTG for 20 h at 16 ℃. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5), and the cell lysate was precipitated by centrifugation. The enzyme was purified by batch binding to Ni-NTA resin (Qiagen, 30721). The resin was then washed with lysis buffer contain ing 250 mM imidazole, and 2His-tagged KHK was eluted with 500 mM imidazole. The purified enzyme was concentrated and desalted with an Amicon Ultra centrifugal filter (10 kDa) using 50 mM Tris-HCl (pH 7.5). The amounts of purified protein were determined by BCA protein assay kit (Beyotime Biotechnology, P0011).

Statistics and reproducibility

GraphPad Prism8 was used for statistical analysis. At least three independent or parallel experiments were performed for statistical analysis. Data were analyzed by the unpaired Student's t test or one-way ANOVA with Dunnett’s test. Three levels of significance were determined: *P < 0.05, **P < 0.01, ***P < 0.001.

Liberti, M. V. & Locasale, J. W. The warburg effect: How does it benefit cancer cells? Trends Biochem. Sci. 41, 211–218 (2016).
Warburg, O. On the origin of cancer cells. Science 123, 309–314 (1956).
Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033 (2009).
Abdel-Wahab, A. F., Mahmoud, W. & Al-Harizy, R. M. Targeting glucose metabolism to suppress cancer progression: prospective of anti-glycolytic cancer therapy. Pharmacol. Res. 150, 104511 (2019).
Gonzalez, P. S. et al. Mannose impairs tumour growth and enhances chemotherapy. Nature 563, 719–723 (2018).
Taylor, S. R. et al. Dietary fructose improves intestinal cell survival and nutrient absorption. Nature 597, 263–267 (2021).
Jeong, S. et al. High fructose drives the serine synthesis pathway in acute myeloid leukemic cells. Cell Metab. 33, 145-159e6 (2021).
Goncalves, M. D. et al. High-fructose corn syrup enhances intestinal tumor growth in mice. Science 363, 1345–1349 (2019).
Herman, M. A. & Birnbaum, M. J. Molecular aspects of fructose metabolism and metabolic disease. Cell Metab. 33, 2329–2354 (2021).
Li, X. et al. A splicing switch from ketohexokinase-C to ketohexokinase-A drives hepatocellular carcinoma formation. Nat. Cell Biol. 18, 561–571 (2016).
Asipu, A., Hayward, B. E., O'Reilly, J. & Bonthron, D. T. Properties of normal and mutant recombinant human ketohexokinases and implications for the pathogenesis of essential fructosuria. Diabetes 52, 2426–2432 (2003).
Lu, Z. & Hunter, T. Metabolic kinases moonlighting as protein kinases. Trends Biochem. Sci. 43, 301–310 (2018).
Kim, J. et al. Ketohexokinase-A acts as a nuclear protein kinase that mediates fructose-induced metastasis in breast cancer. Nat. Commun. 11, 5436 (2020).
Xu, D. et al. The protein kinase activity of fructokinase A specifies the antioxidant responses of tumor cells by phosphorylating p62. Sci. Adv. 5, eaav4570 (2019).
Yang, X. Y. et al. Prognostic impact of metabolism reprogramming markers acetyl-CoA synthetase 2 phosphorylation and ketohexokinase-A expression in non-small-cell lung carcinoma. Front. Oncol. 9, 1123 (2019).
Bilal, M., Iqbal, H. M. N., Hu, H. B., Wang, W. & Zhang, X. H. Metabolic engineering pathways for rare sugars biosynthesis, physiological functionalities, and applications-a review. Crit. Rev. Food Sci. 58, 2768–2778 (2018).
Hossain, A. et al. Rare sugar d-allulose: Potential role and therapeutic monitoring in maintaining obesity and type 2 diabetes mellitus. Pharmacol. Therapeut. 155, 49–59 (2015).
Perazzolli, M. et al. Ecological impact of a rare sugar on grapevine phyllosphere microbial communities. Microbiol Res 232, 126387 (2020).
Liang, Y. X. et al. The constipation-relieving property of d-tagatose by modulating the composition of gut microbiota. Int. J. Mol. Sci. 20, 5721 (2019).
Chen, Z. W. et al. Recent research on the physiological functions, applications, and biotechnological production of d-allose. Appl Microbiol Biot 102, 4269–4278 (2018).
Kanaji, N. et al. Additive antitumour effect of d-allose in combination with cisplatin in non-small cell lung cancer cells. Oncol. Rep. 39, 1292–1298 (2018).
Hirata, Y. et al. Analysis of the inhibitory mechanism of d-allose on MOLT-4F leukemia cell proliferation. J. Biosci. Bioeng. 107, 562–568 (2009).
Ishiyama, H. et al. Development of a d-allose-6-phosphate derivative with anti-proliferative activity against a human leukemia MOLT-4F cell line. Carbohyd. Res. 487, 107859 (2020).
Naha, N. et al. Rare sugar d-allose induces programmed cell death in hormone refractory prostate cancer cells. Apoptosis 13, 1121–1134 (2008).
Bock, F. J. & Tait, S. W. G. Mitochondria as multifaceted regulators of cell death. Nat. Rev. Mol. Cell Bio. 21, 85–100 (2020).
Sinha, K., Das, J., Pal, P. B. & Sil, P. C. Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis. Arch. Toxicol. 87, 1157–1180 (2013).
Kelso, G. F. et al. Selective targeting of a redox-active ubiquinone to mitochondria within cells-Antioxidant and antiapoptotic properties. J. Biol. Chem. 276, 4588–4596 (2001).
Flaherty, K. T. Sorafenib in renal cell carcinoma. Clin. Cancer Res. 13, 747s-752s (2007).
Cheng, A. L. et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet. Oncol. 10, 25–34 (2009).
Holden, H. M., Rayment, I. & Thoden, J. B. Structure and function of enzymes of the Leloir pathway for galactose metabolism. J. Biol. Chem. 278, 43885–43888 (2003).
Sanders, R. D., Sefton, J. M., Moberg, K. H. & Fridovich-Keil, J. L. UDP-galactose 4' epimerase (GALE) is essential for development of drosophila melanogaster. Dis. Model. Mech. 3, 628–638 (2010).
Feng, J. et al. Emerging roles and the regulation of aerobic glycolysis in hepatocellular carcinoma. J. Exp. Clin. Canc. Res. 39, 126 (2020).
Cordon, M. J., Vega-Vila, J. C., Casper, A., Huang, Z. & Gounder, R. Tighter confinement increases selectivity of d-glucose isomerization toward l-sorbose in titanium zeolites. Angew. Chem. Int. Ed. Engl. 59, 19102–19107 (2020).
Nomura, N. et al. Structure and mechanism of the mammalian fructose transporter GLUT5. Nature 526, 397–401 (2015).
Zou, W., Liu, L. M. & Chen, J. Structure, mechanism and regulation of an artificial microbial ecosystem for vitamin C production. Crit. Rev. Microbiol. 39, 247–255 (2013).
Liu, L., Chen, Y., Yu, S. Q., Chen, J. & Zhou, J. W. Enhanced production of l-sorbose by systematic engineering of dehydrogenases in Gluconobacter oxydans. Syn. Syst. Biotechno. 7, 730–737, (2022).
DeWaal, D. et al. Hexokinase-2 depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular carcinoma and sensitizes to metformin. Nat. Commun. 9, 446 (2018).
Ganapathy-Kanniappan, S. & Geschwind, J. F. H. Tumor glycolysis as a target for cancer therapy: progress and prospects. Mol. Cancer. 12, 152 (2013).
Lardy, H. A., Wiebelhaus, V. D. & Mann, K. M. The mechanism by which glyceraldehyde inhibits glycolysis. J. Biol. Chem. 187, 325–337 (1950).
Kistler, A. & Keller, P. Inhibition of glycolysis by l-sorbose in dog erythrocytes. Experientia. 34, 800–802 (1978).
Goto, I., Shimizu, T. & Maede, Y. l-Sorbose does not cause hemolysis in dog erythrocytes with inherited high Na, K-ATPase activity. Comp. Biochem. Physiol. C. Comp. Pharmacol. Toxicol. 101, 657–660 (1992).
Bar, A. & Leeman, W. R. l-Sorbose but not d-tagatose induces hemolysis of dog erythrocytes in vitro. Regul. Toxicol. Pharmacol. 29, S43-45 (1999).

Data availability

The data supporting the findings of this study are available within the Article and its Supplementary Information. Further relevant data are available from corresponding authors upon reasonable request.

Acknowledgements

We are grateful to members of the Gao laboratory for reagents, comments and other contributions to this project. This work was supported by grants-in-aid from the National Natural Science Foundation of China (31971216; 32071467; 32171475), Natural Science Foundation of Jiangsu Province (No. BK20210465), Collaborative Innovation Center of Jiangsu Modern Industrial Fermentation, Top-notch Academic Programs Project of Jiangsu Higher Education Institutions, National first-class discipline program of Light Industry Technology and Engineering (LITE2018-015), Shandong Provincial Major Scientific and Technological Innovation Project (2019JZZY011006), Special fund for Zaozhuang Excellence agglomeration project to X-D Gao.

Author contributions

Conceptualization, X.-D.G., Z.L., H.N., and H.-L.X.; methodology, H.-L.X., X.Z., S.C., and Z.L.; validation, H.-L.X. and S.X.; formal analysis, H.-L.X., X.Z, S.C., and H.N.; investigation, H.-L.X., X.-D.G., and Z.L.; writing—original draft preparation, H.-L.X. and H.N.; writing—review and editing, H.N., H.-L.X., Z.L., and X.-D.G.; supervision, X.-D.G., H.N., and Z.L.; funding acquisition, X.-D.G. and Z.L. All authors have read and agreed to the published version of the manuscript.

Competing interests

The authors declare no conflict of interest.

There is NO Competing Interest.

SupplementaryInformation.pdf
supplementary Information
ExtendedDataFigures.pdf

Download PDF

Journal Publication

published 11 Mar, 2023

Read the published version in Communications Biology →

Version 1

posted

You are reading this latest preprint version

Rare sugar L-sorbose exerts antitumor activity by impairing glucose metabolism

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Discussion

Methods

References

Declarations

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1