Contributed equally
Licochalcone A (LicA) is a chalcone extracted from liquorice which has been used as a traditional Chinese medicine for many generations. Increased glucose consumption and glycolytic activity are important hallmarks of cancer cells, and hexokinase 2 (HK2) upregulation is a major contributor to the elevation of glycolysis. Recently, the antitumor activities of LicA have been reported in various cancers; however, its effect on tumor glycolysis in gastric cancer and the underlying mechanisms are completely unknown.
Glucose is the energy source and the important metabolic intermediate in mammalian cells. Under a condition of sufficient oxygen, glucose is metabolized into H2O and CO2 via the Krebs cycle to generate abundant ATP to satisfy energy requirements. Different from normal cells, in tumor cells glucose is converted to pyruvate and lactate even in the presence of oxygen, which is termed as aerobic glycolysis or the Warburg effect (
In contrast with small molecules developed through rational chemical design, natural products have more potential to be applied to tumor chemotherapy owing to their structural diversity. Recently, numerous natural compounds have been demonstrated to exert antitumor effects by inhibiting cell growth, promoting cell death and suppressing angiogenesis (
Although antitumor activities of LicA have been verified, its effect on tumor glycolysis remains largely unknown. In the present study, we investigated the effect of LicA on tumor glycolysis in gastric cancer cells as well as the underlying mechanisms. The results demonstrated that LicA exhibited substantial activities against tumor glycolysis by reducing HK2 expression. Further investigation illustrated that the decrease in HK2 by LicA was due to the blockade of the Akt signaling pathway.
Gastric cancer cell lines MKN45 and SGC7901 and the gastric epithelial cell GES-1 were purchased from the Chinese Committee of Type Culture Collection Cell Bank, Chinese Academy of Sciences (Shanghai, China). MKN45 and SGC7901 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 1% antibiotics. The GES-1 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% antibiotics. LicA and specific signaling pathway inhibitors including LY294002, PD98059, Bay11-7082 and parthenolide were purchased from Selleck (Shanghai, China). Primary anti-HK2 (#2867), Glut1 (#12939), PKM2 (#4053), LDHA (#3582), p-Akt (#4060), p-ERK1/2 (#4370), p-p65 (#3031), p-IκBα (#9246), p-S6 (#4858), p-GSK3β (#12456), Akt1 (#75692), MCL-1 (#5453), BCL2 (#2870), BCL-XL (#2764), cleaved caspase-3 (#9664), cleaved PARP (#5625) antibodies and secondary antibodies anti-rabbit IgG HRP (#7074) and anti-mouse IgG HRP (#7076) were products of Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-β-actin antibody (A5316) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Myr-Akt1 plasmid was purchased from Addgene (Cambridge, MA, USA). HK2 (ORF004940) construct was a product of Applied Biological Materials (ABM) Inc. (Richmond, BC, Canada). Lipofectamine® 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Annexin V-FITC and propidium iodide were products of Biolegend (San Diego, CA, USA).
MKN45 or SGC7901 cells in logarithmic growth phase (3×103/well) were seeded into 96-well plate. Twenty-four hours later, the cells were treated with various concentrations of LicA. At different time points (24, 48 or 72 h), CellTiter96 Aqueous One Solution (Promega Corp., Madison, WI, USA) (20 µl/well) was added to a 96-well plate and the cell viability was assessed according to the manufacturer's protocol.
Cells cultured in 10-cm plastic dishes were treated with different concentrations of LicA for 24 h, and the cells were harvested and reseeded into a 6-well plate in duplicate at the appropriate density. The cells were cultured for 1–3 weeks until the colonies with substantially good size (minimum 50 cells per colony) were formed in the control group. After washing with PBS, the colonies were fixed with the fixation solution (methanol:acetic acid 3:1) and then stained with 0.5% crystal violet for 2 h at room temperature. The crystal violet was carefully removed by immersing the plates in tap water repeatedly and the plates were air-dried. The number of colonies was photographed and counted.
After the treatment of LicA, gastric cancer cells were lysed with RIPA lysis buffer containing protease cocktail (Roche, Mannheim, Germany) on ice for 30 min. The cell lysate was harvested and centrifugated at 12,000 × g for 5 min, and the supernatant was collected. The protein concentrations were determined by the Bradford assay (Bio-Rad, Philadelphia, PA, USA). Twenty micrograms per sample was subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking the non-specific binding site on the membrane with 5% non-fat milk solution, the membranes were incubated with specific primary antibodies at a dilution of 1:1,000 at 4°C overnight. After washing three times with TBS-Tween-20, the membranes were incubated with HRP-conjugated secondary antibody at a dilution of 1:2,000 at room temperature for 1 h, and then the bands on the membrane were visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Gastric cancer cells (5×105/well) were plated in 6-well tissue culture plate and cultured overnight. Then, the culture medium was discarded and cells were incubated with fresh medium containing different concentrations of LicA for 12 h. The Automatic Biochemical Analyzer (7170A, Hitachi, Tokyo, Japan) was used for the measurement of the levels of glucose and lactate in the culture medium. Protein concentration per sample acted as the control to normalize the relative glucose consumption and lactate production rate.
After LicA treatment, MKN45 cells and the cell culture medium were collected and centrifugated. After washing with PBS, the cell pellets were resuspended and stained with Annexin V-FITC and propidium iodide according to the manufacturer's instructions, and then the stained cells were subjected to flow cytometry. The results were analyzed and quantified by a flow cytometer (BD Biosciences, San Jose, CA, USA).
For Myr-Akt1 or pORF-HK2 transfection, HCC cells were seeded into a 6-well culture plate and grown to 70–90% confluence. Before transfection, the culture medium was replaced with 1.5 ml fresh medium without fetal bovine serum. Plasmid DNA (2.5 µg) and Lipofectamine 2000 reagent (7.5 µl) were diluted with 150 µl Opti-MEM medium respectively and incubated at room temperature for 5 min. The diluted DNA and transfection reagent were mixed and incubated for 10 mins avoiding light, then 250 µl mixture was added per well and the culture medium was replaced with fresh medium with fetal bovine serum after 4–6 h. Forty-eight hours later, the transfected cells were used for further studies.
In accordance with the protocol approved by the Institutional Animal Care and Use Committee, BALB/ca nude mice maintained under specific pathogen-free (SPF) conditions were subcutaneously injected with MKN45 cells (2×106 cells/mice). Once the tumor was formed and the volume was ~50 mm3, the mice were randomly assigned into a vehicle and experimental group. The vehicle group received 0.2 ml sterile PBS solution, and the experimental group was treated with 10 mg/kg LicA by i.p. injection daily. The tumor volume (V) was measured twice per week with microcalipers and was calculated as V= (length × width2)/2. At the end of the experiment, the mice were sacrificed and the tumors were removed.
Tumor tissues isolated from the mice were embedded in paraffin and cut into 5-µm sections. After dewaxing in xylene and hydration in serial ethanol, the slides were boiled in sodium citrate buffer (10 mmol/l, pH 6.0) for 10 min to expose antigens. To block endogenous peroxidase, the slides were treated with 3% H2O2 for 10 min. After incubation with goat serum at room temperature for 1 h, the slides were incubated with the anti-HK2 (1:100) or anti-Ki-67 (1:200) antibody respectively at 4°C in a humidified chamber overnight. Following washing with PBS three times, the slides were hybridized with biotinylated goat anti-rabbit secondary antibody at room temperature for 30 min. After washing with PBS, the sections were probed with HRP-conjugated streptavidin and then visualized with DAB solution. After counterstaining with Harris' hematoxylin, the slides were dehydrated and mounted. The intensity was analyzed by Image-Pro PLUS (v.6) and ImageJ (NIH) software programs.
SPSS software (version 13.0; IBM SPSS, Armonk, NY, USA) was used for statistical analysis. Student's t-test or one-way ANOVA was adopted to evaluate statistical significance. P<0.05 indicated a statistically significant difference.
Firstly, we tested the effect of LicA on normal gastric epithelium cell line GES-1. The results showed that LicA had no obvious cytotoxicity at concentrations ≤180 µM (
In order to study the effect of LicA on tumor glycolysis, the amount of glucose consumption in gastric cancer cells was determined. As shown in
To confirm which signaling pathway is engaged in the regulation of HK2 expression, the effect of LicA on the main signaling pathways in gastric cancer cells was investigated. As demonstrated, several signaling pathways in gastric cancer, such as ERK, Akt and NF-κB were blocked by LicA dose-dependently (
Based on the results shown above, the HK2 decrease caused by LicA may be attributed to the blockade of Akt activation, thus, we tested the effect of LicA on the Akt signaling pathway. After LicA treatment, along with the inactivation of Akt, the phosphorylation of S6 and GSK3β, which are the main downstream signaling of Akt, were inhibited dose-dependently (
Except glycolysis suppression, LicA also induced apoptosis in gastric cancer cells. The expression of cleaved-caspase-3 and PARP, which are signals of cells undergoing apoptosis, were significantly increased (
The
Gastric cancer ranks fourth as the most most common malignancy and the second leading cause of cancer-associated mortality. In China, it was estimated the mortality reached ~498,000 in 2015 (
The potency of LicA in different tumor types is highly different. As reported, breast cancer cells demonstrated high sensitivity to LicA with an IC50 of 20 µM (
Tumor glycolysis is an important hallmark of cancer cells. Clinical observations verified the 18F-FDG uptake is an independent and significant prognostic indicator of tumor recurrence in gastric cancer (
The regulation of HK2 expression in tumor cells is complex. Like other glycolytic enzymes, its expression is primarily mediated by altered oncogenic pathways, such as PI3K-Akt, NF-κB, c-myc, HIF-1α and p53 (
Numerous studies had reported that LicA induced apoptosis in cancer cells via various mechanisms. In our study, the results demonstrated that HK2 plays a pivotal role in LicA-induced cell apoptosis in gastric cancer cells. Except the involvement of tumor glycolysis regulation, HK2 interacts with VDAC-1 on the outer mitochondria membrane to maintain membrane integrity under stressed condition and prevents cancer cells from apoptosis (
Taken together, to the best of our knowledge, the present study is the first to report the effect of LicA on tumor glycolysis. We found that the reduction in HK2 was an important underlying mechanism for LicA to display its effects on glycolysis suppression and apoptosis induction. Moreover, we also disclosed that the decrease in HK2 caused by LicA was mainly attributed to the inhibition of the Akt signaling pathway. Our studies provide a preclinical rationale for LicA, or its derivatives to be administered for gastric cancer therapy.
The present study was supported by the Foundation of the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Jiangsu Provincial Special Program of Medical Science (BL2014100), the Jiangsu Hospital of TCM (Y14074) and the National Natural Science Foundation of China (nos. 81202954 and 81473605).
LicA inhibits gastric cancer cell proliferation and clonogenic survival
LicA suppresses gastric cancer cell tumor glycolysis via reducing HK2 expression. (A) Glucose consumption (left panels) and lactate production (right panels) in cell culture medium were analyzed as described. The results of three independent experiments expressed as mean ± SD are shown, *P<0.05; **P<0.01; ***P<0.001 (Student's t-test) indicate a significant difference vs. the control. (B) The expression of key glycolytic enzymes after treatment with LicA. MKN45 (left) and SGC7901 (right) cells were treated with various concentrations of LicA and the change in the indicated proteins was examined by western blotting. LicA, licochalcone. HK2, hexokinase 2.
LicA decreases HK2 expression by blocking the Akt signaling pathway. (A) LicA inhibited the signaling pathways in gastric cancer cells. MKN45 cells were incubated with different concentrations of LicA for 24 h and the effect on phosphorylation of the indicated proteins was examined. (B and C) LicA blocked EGF-induced activation of ERK, Akt and S6 (B), and TNF-α-induced activation of the NF-κB signaling pathway (C). MKN45 cells were starved overnight and then treated with 30 µM LicA for 2 h. After stimulation with 100 ng/ml EGF or 30 ng/ml TNF-α for the indicated times, cell lysates were collected and western blotting was used to examine the indicated protein. (D) MKN45 cells were treated with specific PI3-K inhibitor (LY294002), MEK inhibitor (PD98059), NF-κB inhibitor (Bay11-7082) and parthenolide, respectively, for 12 h, and cell lysates were probed with anti-HK2 antibodies. LicA, licochalcone. HK2, hexokinase 2.
Hyperactivation of Akt attenuates LicA-mediated glycolysis suppression. (A and B) LicA inhibited the Akt signaling pathway and HK2 expression in MKN45 (A) and SGC7901 (B) cells. MKN45 or SGC7901 cells were incubated with LicA for 12 h and cell lysates were detected with the indicated antibodies. (C and D) Hyperactivation of Akt reversed HK2 suppression by LicA. MKN45 (C) and SGC7901 (D) cells were transfected with Myr-Akt1 plasmid and then treated with 60 µM LicA, and western blotting was performed to probe HK2 expression. (E and F) Hyperactivation of Akt impaired glycolysis suppression by LicA. MKN45 and SGC7901 cells were transfected with Myr-Akt1 and then exposed to 60 µM LicA. Glucose consumption (E) and lactate production (F) were examined at 12 h as described. The results of three independent experiments expressed as means ± SD are shown, *P<0.05; **P<0.01 (Student's t-test) indicates significant difference between the different groups. LicA, licochalcone. HK2, hexokinase 2.
Overexpression of HK2 impairs LicA-induced cell apoptosis. MKN45 (A) and SGC7901 (B) underwent apoptosis after LicA treatment. MKN45 or SGC7901 cells were treated with LicA for 24 h, and cell lysates were collected and examined by western blotting with the indicated antibodies. (C) MKN45 cells were treated with LicA for 24 h and collected for Annexin V-PI double staining, and then subjected to FACS analysis. (D-F) Overexpression of HK2 decreased cell apoptosis induced by LicA. MKN45 (D) and SGC7901 (E) cells were transfected with pORF-HK2 and then treated with LicA, and cell lysates were examined with the indicated antibodies. (F) MKN45 cells were transfected with pORF-HK2 and then treated with LicA. Then, the cells were collected and stained with Annexin V and PI, and analyzed with FACS. *P<0.05, **p<0.01 and ***p<0.001 (Student's t-test) indicates significant difference. HK2, hexokinase 2. LicA, licochalcone.
LicA inhibits the growth of an MKN45 xenograft model