2-hydroxy-3-methyl anthraquinone promotes apoptosis and inhibits invasion of human hepatocellular carcinoma cells by targeting nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1/cellular tumor antigen p53 signaling pathway

Abstract

OBJECTIVE:

To investigate the anti-liver cancer effect of 2-hydroxy-3-methyl anthraquinone (HMA) and the specific mechanism based on nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1 (SIRT1)/cellular tumor antigen p53 (p53) pathway.

METHODS:

Cell counting kit-8 method was used to observe the effect of HMA on the activity of human hepatocellular carcinoma cells (HepG2) cells. At 72 h and 80 μL HMA, the apoptosis rate of HepG2 cells in each group was measured by flow cytometry. Transwell was used to assay for cell invasion. The protein expression levels of SIRT1, p53, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), caspase-9 (CASP9) and caspase-3 (CASP3) were detected by Western Blot.

RESULTS:

HMA significantly inhibited the proliferation of HepG2 cells, The half inhibiting concentration (IC50) of the HMA at 24, 48 and 72 h were examined and it were 126.3, 98.6, and 80.55 μM, respectively. Compared with the control group, the apoptosis rate of HMA, Selisistat (EX527), and HMA+ EX527 groups enhanced, while the apoptosis rate of SRT1720 diminished, demonstrating that inhibition of SIRT1 can lead to apoptosis of HepG2 cells. HMA+ EX527 group had the highest apoptosis rate, the lowest expression of SIRT1 and Bcl-2, and the highest expression of p53, Bax, CASP9 and CASP3. The number of invasions of HepG2 was significantly reduced after HMA and EX527 intervened. Western blot shows HMA could inhibit SIRT1, promote the expression of p53, and decrease the ratio of Bcl-2/Bax.

CONCLUSIONS:

HMA induced apoptosis in HepG2 cells, while inhibiting proliferation and invasion. The mechanism of HMA against HCC may be related to the SIRT1/p53 pathway.

1. INTRODUCTION

Primary liver cancer has a high mortality rate and ranks among the top four of all cancers. Hepatocellular carcinoma (HCC) accounts for 75%-90% of primary liver cancers and remains the leading cause of death among patients with cirrhosis.¹ Persistent cancer cells survival and proliferation signals are hallmarks of HCC, triggered by an imbalance between oncogenes and tumor suppressor genes.² ​Due to the absence of specific symptoms in the early stage, the majority of HCC patients are often diagnosed in the advanced stage, missing the golden period of surgery. Sorafenib, a polytyrosine kinase inhibitor (TKI), has been the standard of care for patients with advanced HCC. Nevertheless, Sorafenib does not promote tumor shrinkage or necrosis and causes comparatively serious adverse events such as skin responses in the hands and feet. Consequently, the need for the development of novel molecularly targeted agents is well justified, or the use of an adjuvant second-line drug after sorafenib has failed to control disease progression or intolerance. Several promising drug candidates have failed in clinical trials, hindering prospects for expanding systemic therapy options for hepatocellular carcinoma.³​

The introduction of herb medicine and herbal monomer for anti-HCC has been heralded as one of the fruits of decades of remarkable progress. For instance, aloperine can induce apoptosis of liver cancer cells through the phosphatidylinositol 3-kinase/Serine threonine protein kinase (Akt) signaling pathway, accompanied by mitochondrial damage and G2/M cell cycle arrest.⁴ Dulcitol and Platycodin D were shown to suppress migration and invasion in human human hepatocellular carcinoma cells (HepG2) hepatocellular carcinoma cells.^5,6 Natural products and their derivatives tend to have good efficacy and stability, low toxicity, and are not susceptible to multidrug resistance, which may serve as potential alternatives to certain chemotherapies.​​​

Baihuasheshecao (Herba Hedyotdis) is a type of herbal medicine containing a variety of active substances with rich pharmacological effects such as anti-cancer, anti-inflammatory, analgesic, antioxidant, etc. The anticancer effect of Baihuasheshecao (Herba Hedyotdis) is the most prominent, and 20 active components have been screened out by the network pharmacology method, acting on 276 cancer targets such as cellular tumor antigen p53 (TP53), RAC-alpha serine/threonine-protein kinase (AKT1), mitogen-activated protein kinase 1, and tumor necrosis factor. 2-hydroxy-3-methyl anthraquinone (HMA) is the main anticancer activity matter of Baihuasheshecao (Herba Hedyotdis). Recent studies have found that HMA inhibits the proliferation and invasion of lung carcinoma cells by regulating the interleukin-6-induced Janus Kinase 2/signal transducer and activator of transcription 3 pathway.⁷ In addition, the antitumor effect of HMA can be induced by stimulating apoptosis. In previous studies, the pro-apoptotic mechanisms of HMA on leukemia THP-1 and U937 cells have been revealed.^8,9 However, the possible mechanism of HAM against liver cancer cells has not been investigated. The aim of this study is to fill this gap.

2. METHODS

2.1. Materials and samples

HMA was purchased from Yuanye Biotechnology Co. Ltd., Shanghai, China. EX-527 (SIRT1 inhibitor) and SRT1720 (SIRT1 agonists) were from Sigma-Aldrich (Sigma-Aldrich: Merck KGaA, St. Louis, MO, USA). HepG2 was cultured in dulbecco's modified eagle medium (DMEM) (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) at 37 ℃ in a chamber with 5% CO₂. Cell counting kit-8 (CCK-8) was purchased from Beijing Solar-bio Science and Technology Co. Ltd. (Beijing, China). Primary antibodies, including anti-SIRT1, anti-p53, anti-B-cell lymphoma-2 (Bcl-2), anti-Bcl-2 associated X protein (Bax), anti-caspase3, anti-caspase 9 and anti-β-actin were bought from Cell Signaling Technology, Beverly, Beverly, MA, USA.

2.2. CCK-8 assay

2 × 10⁴ HepG2 cells were seeded in 96-well plates. Then treated with HMA at concentrations of 0, 20, 40, 60, 80, 100 and 120 μMol/L for 24, 48 and 72 h. The medium was discarded. A 10 μL of CCK-8 reagent was added to each well in the plate and incubated at 37 ℃ for 4 h and the absorbance was measured at 450 nm. Cell viability = (Treatment group OD - Blank OD)/(Control group OD- Blank OD). 0, 5, 10, 20, 40, 80, 160, 320, 640 and 1280 μMol/L for 24, 48 and 72 h were used to explore half maximal inhibitory concentration (IC50). Each experiment was repeated 3 times.

2.3. Cell grouping

The HMA Group, EX-527 group, and SRT1720 group were supplemented with the 80 μmol/L HMA, EX527 (1 µmol/L), and SRT1720 (1 µmol/L) based on the control group, respectively. HMA + EX527 group and HMA +SRT1720 group were supplemented with the EX527 (1 µmol/L) and SRT1720 (1 µmol/L) based on the HMA group, respectively. The optimal time chose 72 h.

2.4. Detection of apoptosis by flow cytometry assay

Apoptosis rates were tested using BD FACSCalibur flow cytometry (BD Bioscience, Franklin Lake, NJ, USA) with an Annexin V-fluorescein isothiocyanate (FITC) / propidium iodide (PI) kit (Sanjian Biological Technology Co. Ltd., Chongqing, China). Collect cells directly into one 10 mL centrifuge tube. Cells per sample were counted at 1 × 10⁶/mL. After centrifugation at 1000 rpm for 5 min, the medium was discarded. The incubation buffer was used to wash once, and centrifugation was performed again at 1000 r/min for 5 min. The cells were resuspended with 100 μL of tag solution and incubated at room temperature in the dark for 10 min. The cells were precipitated and incubated by centrifugation at 1000 rpm/min for 5 min. Furthermore, we mixed fluorescence solution and set it for 20 min in the dark at 4°. The excitation wavelength of the flow cytometer was 488 nm. A filter at 515 nm was used for FITC fluorescence detection, and a filter at 560 nm was used for PI detection. Apoptotic rate: Q2 + Q3.

2.5. Transwell chamber assay for invasion ability

The diluted Matrigel Matrigel was evenly spread onto the upper chamber membrane surface of the Transwell chamber with a precooled pipette tip. Add 40 μL Matrigel to each chamber and incubate for 30 min in the incubator. Adjust the concentration of treated HepG2 cells to 5 × 10⁴ cells/mL. 100 μL of serum-free cell suspension was added to the upper chamber of the Transwell chamber, and 500 μL of DMEM medium containing 20% FBS was added to each well of the lower chamber and placed in an incubator for 48 h. Then, the cells under the membrane were fixed with 4% tissue cell fixative for 30 min, rinsed twice with phosphate buffer solution, stained with 1% crystal violet solution for 15 min, and the residual crystal violet dye in the chamber was rinsed with running water. After the end, use a disposable cotton swab to gently wipe off the uninvaded cells in the upper layer of the chamber, and place it in a cool place to dry. Finally, penetrating cells were photographed and manually counted in 3 randomly selected areas using an inverted microscope.

2.6. Western blotting

HepG2 cells were intervened 72 h. Then lyse cells were in a buffer at 4 °C for 30 min, and the supernatant was harvested by centrifuging at 12 000 ×g for 5 min at 4 °C. The protein samples were loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. SIRT1, p53, Bcl-2, Bax, Caspase 3 and Caspase 9 protein levels were analyzed using β-actin as a loading control. Primary antibodies were performed using anti-SIRT1, anti-p53, anti-Bcl-2, anti-Bax, anti-caspase 3, anti-caspase 9 and anti-β-actin. Secondary blotting was performed using anti-goat or anti-rabbit IgG conjugated with horseradish peroxidase. The gel imaging analysis system was used to scan and analyze the images.

2.7. Statistical analysis

The data obtained from at least three independent experiments were presented as mean ± standard deviation. One-way analysis of variance followed by Tukey's multiple comparisons test was performed using GraphPad Prism version 9.0.0 for Windows, GraphPad Software, Boston, MA, USA, www.graphpad.com. P < 0.05 was considered to indicate a statistically significant difference.

3. RESULTS

3.1. HMA inhibits the proliferation of HepG2

The molecular formula was shown in Figure 1A. Different concentrations of HMA were intervened after 24, 48 and 72 h, respectively. The IC50 of the HMA at 24, 48 and 72 h were examined and it were 126.3, 98.6, and 80.55 μM, respectively (supplementary Figure 1). Then we performed the detection with a gradient of 20 μM. Compared with the control group, HMA can inhibit the proliferation of HepG2. Cell viability was inverse proportion to treatment time and dose (Figure 1B，P < 0.001). At the same dose, cell viability at 72 h was significantly lower than at 24 and 48 h (Figure 1B, P < 0.001). At the same time, 80 and 100 µmol/L HMA could play the best repression. Whether 24, 36, or 72 h, compared with 80, 100 µmol/L didn’t represent a better effect (Figure 1B, P > 0.05). So we chose 80 µmol/L HMA for the follow-up experiment at 72 h.

Figure 1. Effects of HMA on the viability of HepG2 cells.

A: molecular formula of HMA; B: cell viability at different HMA doses and administration times. HMA with 0, 20, 40, 60, 80, 100 and 120 μMol/L for 24, 48 and 72 h to treatment HepG2 cells. HepG2: human hepatocellular carcinoma cells; HMA: 2-hydroxy-3-methyl anthraquinone. One-way analysis of variance followed by Tukey's multiple comparisons test, Graphpad 9.0.0. Compare with 0 μM, ^aP < 0.001; compare with same time, ^bP < 0.001. Data were presented as mean ± standard deviation (n = 3).

3.2. HMA induces the apoptosis of HepG2 may relate with SIRT1

Compared with the control group, the apoptosis rate of HepG2 was significantly higher after HMA, HMA + EX527, HMA + SRT1720, and EX527 intervene (Figure 2, P < 0.001). Apoptosis was significantly reduced by treatment with SRT1720 (Figures 2D, 2G, P < 0.001). This illustrates that HMA and EX527 have pro-apoptotic effects while SRT1720 may inhibited apoptosis of HepG2. After HMA + EX527 treatment, the apoptosis rate of HepG2 was significantly higher than HMA group (Figures 2B, 2C, 2G). This suggests that EX527 exerts a synergistic effect on the pro-apoptotic effect of HMA. After HMA + SRT1720 intervene, the apoptosis rate of HepG2 was significantly lower than HMA (Figures 2B, 2D, 2G). It means HMA may though inhibit SIRT1 play in the pro-apoptosis function.

Figure 2. Apoptosis rate of HepG2 after treatment by flow cytometry.

A-F: flow cytometry; A: Control group: Solvent treatment 72 h; B: HMA group: 80 µmol/L HMA treatment 72 h; C: HMA+EX527 group: 80 µmol/L HMA and 1 µmol/L EX527 treatment 72 h; D: HMA+SRT1720 group: 80 µmol/L HMA and 1 µmol/L SRT1720 treatment 72 h; E: EX527 group: 1 µmol/L EX527 treatment 72 h; F: SRT1720 group: 1 µmol/L SRT1720 treatment 72 h; G: comparison of apoptosis rates. HepG2: human hepatocellular carcinoma cells; HMA: 2-hydroxy-3-methyl anthraquinone; Selisistat: EX527. One-way analysis of variance followed by Tukey's multiple comparisons test, Graphpad 9.0.0. ^aP < 0.001, compare with Control group. Data were presented as mean ± standard deviation (n = 3).

3.3. HMA inhibits the invasion of HepG2 and may relate with SIRT1

Compared with the control group, the number of invasions of HepG2 was significantly reduced after HMA, HMA + EX527, and EX527 intervene (Figure 3, P < 0.01, P < 0.001). The number of invasions of HepG2 was significantly increased by treatment with SRT1720 (Figure 3, P < 0.05). The number of invasions of HepG2 in the HMA + SRT1720 group was unchanged compared with the normal group (Figure 3). This illustrates that HMA and EX527 could inhibit invasion while SRT1720 may promote invasion of HepG2. The number of invasion of HepG2 in the HMA + SRT1720 group were higher than HMA group. It shows the diminished ability of HMA to inhibit invasion after SIRT1 agonizes. HMA may inhibit the invasion of HepG2 through inhibit SIRT1.

Figure 3. Number of invasion of HepG2 after intervention by Transwell.

A-F: the number of invasion; A: Control group: Solvent treatment 72 h; B: HMA group: 80 µmol/L HMA treatment 72 h; C: HMA + EX527 group: 80 µmol/L HMA and 1 µmol/L EX527 treatment 72 h; D: HMA+SRT1720 group: 80 µmol/L HMA and 1 µmol/L SRT1720 treatment 72 h; E: EX527 group: 1 µmol/L EX527 treatment 72 h; F: SRT1720 group: 1 µmol/L SRT1720 treatment 72 h. Bar = 100 μm. G: comparison of number of invasion. HepG2: human hepatocellular carcinoma cells; HMA: 2-hydroxy-3-methyl anthraquinone; Selisistat: EX527. One-way analysis of variance followed by Tukey's multiple comparisons test, Graphpad 9.0.0. ^aP < 0.001, ^bP < 0.01, ^cP < 0.05, compare with Control group. Data were presented as mean ± standard deviation (n = 3).

3.4. HMA via SIRT1 / p53 activates Bcl-2 / Bax pathway to induce the apoptosis and inhibit the invasion of HepG2

After HMA intervention, the expression of SIRT1 decreased, and the presentation of p53 increased (Figures 4A-4C). p53 expression was elevated after SIRT1 inhibition by EX527(Figures 4A, 4C); SIRT1 activation by SRT1720 decreased p53 expression (Figures 4A-4C), indicating that SIRT1 can repress p53 expression. After intervention in HMA + EX527 group, the SIRT1 term decreased and was lower than that in the EX527 group (Figures 4A-4C); The agonistic effect of SRT1720 on SIRT1 was counteracted after the addition of HMA + SRT1720, indicating that HMA acted to inhibit SIRT1(Figures 4A-4C). p53 plays an important role in apoptosis. It could inhibit the function of Bcl-2. Compared with the control group, the Bcl-2 expression decreased and Bax expression increased after HMA intervention (Figures 4A, 4E, 4F). It illustrates that HMA may activate Bcl-2/Bax apoptotic pathway. Meanwhile, CASP3 and CASP9 expressions were increased (Figures 4A, 4G, 4H). After SIRT1 inhibition by EX527, p53 expression was elevated, and p53 could inhibit Bcl-2 expression and promote Bax, CASP3 and CASP9. Activation of SIRT1 by SRT1720 decreased the expression of p53, increased the expression of Bcl-2, and decreased the expression of Bax, CASP3 and CASP9. In conclusion, HMA can via SIRT1/p53 activates apoptotic pathway, and enable apoptosis to be promoted.

Figure 4. HMA via SIRT1 / p53 activates apoptotic pathway.

A: protein levels by Western blot; ‘+’ represent treatment, ‘+’ represent no-treatment; B: SIRT1 protein expression; C: p53 protein expression; D: Bcl-2/Bax protein expression; E: Bcl-2 protein expression; F: Bax protein expression; G: CASP3 protein expression; H: CASP9 protein expression. Control: Solvent treatment 72 h; HMA: 80 µmol/L HMA treatment 72 h; HMA+EX527: 80 µmol/L HMA and 1 µmol/L EX527 treatment 72 h; HMA+SRT1720: 80 µmol/L HMA and 1 µmol/L SRT1720 treatment 72 h; EX527: 1 µmol/L EX527 treatment 72 h; SRT1720: 1 µmol/L SRT1720 treatment 72 h. HepG2: human hepatocellular carcinoma cells; HMA: 2-hydroxy-3-methyl anthraquinone; Selisistat: EX527. SIRT1: nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1; p53: cellular tumor antigen p53; Bcl-2: B-cell lymphoma-2; Bax: anti-Bcl-2 associated X protein; CASP9: caspase-9; CASP3: caspase-3. One-way analysis of variance followed by Tukey's multiple comparisons test, Graphpad 9.0.0. ^aP < 0.001, ^bP < 0.05, compare with Control group. Data were presented as mean ± standard deviation (n = 3).

4. DISCUSSION

HCC is caused by multiple etiologies and becomes the second most common cancer worldwide due to multifocal recurrence.¹⁰ In the United States, the age-adjusted mortality rate of liver cancer increased 43% between 2000 and 2016¹¹. Although the age-standardized incidence of liver cancer has declined in most Asian countries, the prognosis remains poor, with a 5-year relative survival rate reported in China as low as 12.1%.^12,13 In the context of chronic liver disease, underlying cirrhosis, and malignancy, the prognosis of HCC requires close monitoring.​It has been reported that the 5-year recurrence rate after liver resection is close to 70%, and the recurrence of 2/3 of patients within 2 years is caused by intrahepatic spread.¹⁴​ So far, no effective adjuvant therapy has been reported for advanced liver cancer, including sorafenib and regorafenib. It is urgent to discover late-model targeted drugs to prevent metastasis of hepatocellular carcinoma. HMA, a natural anthraquinone compound in Baihuasheshecao (Herba Hedyotdis), may be one of the characterized anticancer drugs.​Anthraquinones and their derivatives have demonstrated their leading value in the development of anticancer drugs.¹⁵ As a JAK inhibitor, HMA is involved in many essential biological processes such as cell proliferation, differentiation, apoptosis and immune regulation.¹⁶ Previous studies have found that HMA promotes apoptosis of leukemia THP-1 cells by inducing the activation of the apoptosis components Fas/FasL and caspase-8.¹⁷

This research was to investigate the function of HMA on the proliferation, survival and invasion of the hepatoma cell line HepG2. The results specified that HMA could significantly inhibit the proliferation of HepG2 cells in a concentration and time-dependent manner. In a previous study, the HMA of 72 h, 80 μM had an obvious inhibitory effect on the cell viability of Hep G2 cells detected by the CCK method. Combined with gradient concentration data, 80 μM was used to perform subsequent experiments. At 24, 48 and 72 h, the IC50 were 126.3, 98.6, and 80.55 μM. At the optimal time and concentration, the apoptosis rate was detected by flow cytometry, the invasion was detected by Transwell chamber, and the expressions of SIRT1/p53 and downstream Bcl-2, Bax, CASP9 and CASP3 proteins were detected by Western Blot. The experiment proved for the first time that HMA promotes p53 expression by inhibiting SIRT1 and activates the Bcl-2/Bax/CASP9/CASP3 apoptotic signal. In addition, in vitro, HMA inhibited the invasion of HepG2, which may also be related to the inhibition of SIRT1.​​

HepG2, the most commonly used human liver cancer cell line, is used as an in vitro model to assess liver disease and drug interaction potential due to its protein expression closer to that of the human liver.¹⁶ Genetic alterations in TP53 (p53) appear relatively early in HCC development and play a driver role in hepato-carcinogenesis.¹⁷ It is well known that inflammatory response, oxidative stress, and genotoxic stress can lead to DNA damage and therefore activate the p53 gene to counteract this damage. In the vast majority of cancer cases, the p53 gene is inactivated or mutated, or its function is interrupted due to changes in regulatory factors and targets.¹⁸ The C-terminal domain of p53 can be modified by acetylation to induce cell cycle arrest, DNA repair, apoptosis, ferroptosis, and other pathways.¹⁹ ​Mammalian sirtuins are a highly conserved family of NAD-dependent histone deacetylases. SIRT1 can regulate chromatin silencing through histone deacetylation, and promote H1 aggregation near the promoter and h3-k79 hypomethylation, resulting in transcriptional inhibition.²⁰ The expression of wild-type SIRT1 in human cells reduces the transcriptional activity of p53 through deacetylation.²¹ Compared with the control group, the EX527 (SIRT1 inhibitor) group raised p53 expression and apoptosis rate, while the SRT1720 (SIRT1 activator) group revealed the opposite trend, convincing that inhibiting SIRT1 can up-regulate p53, thereby promoting HepG2 apoptosis. The apoptosis rate of the HMA group was higher than that of the Control group, and at the same time, it was lower than that of the HMA + SRT1720 group, indicating that HMA promoted apoptosis by inhibiting the expression of SIRT1.​After SIRT1 was inhibited, the number of cell invasions in the EX527 group, HMA group, and HMA + EX527 group was lower than that in the Control group, which implied that the effect of HMA on inhibiting cell invasion may be related to SIRT1.​​​​​

Apoptotic signaling contributes to defending the integrity of the cellular genome, and apoptotic defects are one of the hallmarks of tumors. Tumor cells can utilize several molecular mechanisms to inhibit apoptosis and gain resistance to apoptotic agents, for example, by inducing amplification of anti-apoptotic proteins such as Bcl-2 or down-regulation or mutation of pro-apoptotic proteins such as Bax. Bax and Bcl-2 both belong to the Bcl-2 family and are downstream transcribers of the p53 gene. Bax can alter the transmembrane potential by enhancing mitochondrial membrane permeability. This induces the release of cytochrome C from the mitochondria into the cytoplasm, causing a cascade reaction of cysteine aspartate lyase, which eventually leads to the activation of caspase-3 and triggers apoptosis.²² Under normal circumstances, Bax and Bcl-2 are in equilibrium in the cell. Bcl-2 inhibits the release of cytochrome C from mitochondria initiated by Bax, and the Bcl-2/Bax ratio determines whether cells survive or die.²³ With the increase in the apoptosis rate of HepG2, the ratio of Bcl-2/Bax decreased. Our study demonstrated that HMA participates in HepG2 apoptosis by regulating the intracellular Bcl-2/Bax ratio through the SIRT1-p53 signaling pathway.​​

In conclusion, this study confirmed that HMA can inhibit the proliferation, invasion, and induce apoptosis of hepatocellular carcinoma HepG2 cells in vitro, and the mechanism is related to the SIRT1/p53 signaling pathway. And the toxicity data of the HMA was presented. This provides participation data for drug application. Further, the in vivo efficacy and safety of HMA on HCC need to be considered.​​

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.cn.

Funding Statement

Supported by Natural Science Basic Research Plan in Shannxi Province of China, the Intervention and Mechanism of Developmenta-lendotheliallocus-1 Regulating Macrophage Efferocytosis on Airway Inflammation in Chronic Obstructive Pulmonary Disease (No. 2020JQ-543); the Function and Mechanism of New Tumor Suppressor TSC22D2 in Cancer Glycometabolism (No. 2020JQ-927); Fundamental Research Funds for The Central University: the Role of TSC22D2-centered Protein Interaction Map in Cancer Glycometabolism and Cell Growth (No. xzy012019129)