Synergism of ursolic acid derivative US597 with 2-deoxy-D-glucose to preferentially induce tumor cell death by dual-targeting of apoptosis and glycolysis

Abstract

Ursolic acid (UA) is a naturally bioactive product that exhibits potential anticancer effects. The relatively safe and effective molecule intrigued us to explore a way to further improve its anti-cancer activity and tumor-targeting specificity. In the present study, a series of structural modifications of UA was achieved, which resulted in significant increase in growth inhibition on various cancer cell lines with minimal effects on normal cells. The leading molecule US597 (UA-4) caused depolarization of mitochondrial membrane potential, cell arrest in G0/G1 phase and apoptosis/necrosis in a dose-dependent manner. Structural docking suggested that the carbon chains of the modified UA derivatives compete strongly with glucose for binding to glucokinase, the key glycolysis enzyme presumably active in cancer cells. The combination of 2-deoxy-D-glucose (2-DG) and UA-4 induced cell cycle arrest in G2/M phase, promoted caspase-dependent cell death, reduced hexokinase activity, aggravated depletion of intracellular ATP, decreased lactate production and synergistically inhibited cancer cell growth in vitro (HepG2) and in vivo (H22). Collectively, our findings suggest that the structural modification enhances efficacy and selectivity of UA, and the combination of UA-4 with 2-DG produces synergistic inhibition on hepatoma cell proliferation by dual targeting of apoptosis and glycolysis.

Figure 1. Synthesis of di-amine derivatives of UA.

Reagents and conditions include (a) anhydride/pyr/DMAP, R.T.; (b) (CO)₂Cl, CH₂Cl₂, R.T.; (c) di-amine, CH₂Cl₂, R.T.; and (d) NaOH, CH₃OH/THF, R.T.

Figure 2. Structure and pharmacological effects of UA-4.

a, the structural formula of UA-4; b, dose-response of anti-proliferative effect of UA-4 on A-375, HepG2 and HELF cells; c, effects of UA-4 on cell cycle distribution in A-375 cells; d, effects of UA-4 on ΔΨm in A-375 cells. Results are expressed as percentage change in UA-4 treatment compared to the vehicle-treated control (*P < 0.05, **P < 0.01). e, A-375 cells were treated with UA-4 (1, 5, 10 μM) for 12 h, and 24 h (f), respectively. Apoptosis was determined by Annxin-V-FITC/PI labeling. g, One step TUNEL apoptosis was tested on A-375 cells treated with UA-4 (1, 5, 10 μM) for 24 h.

Figure 3. The binding simulation between hexokinase and 2-DG, 4C, 6C and 8C of the UA derivatives.

a, 2-DG binds with amino acid residues Glu 331, Lys 296 and Gly 295 of hexokinase via hydrophobic interaction, and binds with Thr 332 via hydrogen bonds; b, 4C binds with amino acid residue Lys 296 as 2-DG binds to one of the four residues of hexokinase; c, the docking and binding between 6C of UA-4 or UA-8 and hexokinase are the same as those between 2-DG and hexokinase. Furthermore, 6C can bind the amino acid residues Phe 330, Glu 300 and Thr 228 via hydrogen bond, the reaction that could not be simulated between 2-DG and hexokinase. d, 8C binds with amino acid residues Thr 332, Gly 295, and Lys 296 via hydrophobic interaction.

Figure 4. UA-4 elicited synergistic inhibition on cancer cells with glycolysis inhibitors and affects HepG2 cancer cell metabolism induced by 2-DG.

HepG2 (a) and L02 (b) cells were treated for 24 h with UA-4 at 4 μM and four glycolysis inhibitors (L-Cys: 15 mM; 2-DG: 50 mM; LM: 2.5 mM and 3-BrPA: 64 mM) at IC₈₀. HepG2 (c) and L02 (d) cells were treated with compound UA-4 in the presence and absence of 2-DG followed by cell viability assay (*p < 0.05, **p < 0.01, ^#p < 0.05 and ^##p < 0.01, compared to the combination groups).

Figure 5. The mechanism of anti-proliferation by the combination of UA-4 and 2-DG in HepG2 cells.

HepG2 cells were treated with 2-DG, UA-4 and 2-DG + UA-4 for 24 h. The cell cyle stages (a) and apoptosis (b) were determined by flow cytometry analysis. The activities of caspase (c, d, e), hexokinase activity (f) and ATP level (g) were determined by microplate reader, and lactate concentrations in medium (h) were detected by lactate assay kit (*p < 0.05, **p < 0.01, compared to the control group).

Figure 6. The combination of 2-DG and UA-4 effectively inhibits tumor growth in H22 tumor-bearing mice.

a, photographs of vehicle-treated or drug-treated H22 tumor-bearing mice with representative tumors. Photographs were taken at 7 and 14 days after compounds treatment. The bottom images show tumor volume of the groups at 14 days. Tumor volume (b) and mice weight (c) were measured every other day. After 14 days, inhibition rate (d) and tumor weight (e) were quantitatively analyzed. f, histological examinations; tumors were analyzed by hematoxylin and eosin staining for histological features. (*p < 0.05, **p < 0.01, compared to the control group).

Figure 7. Proposed mechanisms by which the leading UA derivative UA-4 and 2-DG produce synergistic inhibition on cancer cell proliferation.

After entering the cells, UA-4 is hydrolyzed by amidase to be broken down to the carbon chain 6C and UA metabolite. The former competes with glucose for binding to hexokinase to inhibit cancer glycolysis pathway, while the latter produces depolarization in ΔΨm, cell arrest, ATP consumption, necrosis/apoptosis via caspase-independent pathway. 2-DG also inhibits glycolysis of cancer cells by targeting hexokinase. The dual targeting of cell necrosis/apoptosis and glycolysis pathways by using the safe and effective molecules represents a novel anticancer agent.