Carnosine inhibits the proliferation of human gastric cancer SGC-7901 cells through both of the mitochondrial respiration and glycolysis pathways

Abstract

Carnosine, a naturally occurring dipeptide, has been recently demonstrated to possess anti-tumor activity. However, its underlying mechanism is unclear. In this study, we investigated the effect and mechanism of carnosine on the cell viability and proliferation of the cultured human gastric cancer SGC-7901 cells. Carnosine treatment did not induce cell apoptosis or necrosis, but reduced the proliferative capacity of SGC-7901 cells. Seahorse analysis showed SGC-7901 cells cultured with pyruvate have active mitochondria, and depend on mitochondrial oxidative phosphorylation more than glycolysis pathway for generation of ATP. Carnosine markedly decreased the absolute value of mitochondrial ATP-linked respiration, and reduced the maximal oxygen consumption and spare respiratory capacity, which may reduce mitochondrial function correlated with proliferative potential. Simultaneously, carnosine also reduced the extracellular acidification rate and glycolysis of SGC-7901 cells. Our results suggested that carnosine is a potential regulator of energy metabolism of SGC-7901 cells both in the anaerobic and aerobic pathways, and provided a clue for preclinical and clinical evaluation of carnosine for gastric cancer therapy.

Figure 1. Effects of carnosine on SGC-7901 cell viability and proliferation.

(A) Cells were pre-treated with different concentrations of carnosine for 24 or 48 h, and then the cell viability was assayed using the MTT reduction assay. Results were expressed as percentage of control, and were showed mean ± SD. n = 10–12. **P<0.01 vs. control in 24 h group; ^## P<0.01 vs. control in 48 h group. (B) Cells were treated with 20 mM carnosine for 48 h, and then cell death was determined by PI and annexin V-FITC staining followed by flow cytometry. (C) SGC-7901 cells were treated with 20 mM carnosine and the total cell number was calculated after carnosine treatment for 2, 3, 4, 5, 6 days using cell counting plate. Data were expressed as mean ± SD. n = 6. **P<0.01 vs. control.

Figure 2. Effect of carnosine on SGC-7901 cells colony formation.

(A) Representative images of the cloning wells. Cells were seeded at low density in DMEM supplement with or without carnosine (20 mM) for 14 days. The colonies were subsequently fixed with 70% ethanol and stained with Coomassie Brilliant Blue for analysis of colony formation. (B) Quantitative image analysis of colonies in cultured SGC7901 cells. Data were expressed as mean ± SD. n = 6. **P<0.01 vs. control group.

Figure 3. Bioenergetic characterization of SGC-7901 cells cultured in DMEM supplemented with pyruvate.

(A) Real-time analysis of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of cultured SGC-7901 cells by perturbing them with small molecule metabolic modulators. Oligomycin (O; 1 µg/ml), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; F; 1 µM), oxamate (Ox; 100 mM), and rotenone (R; 1 µM) were injected sequentially at the indicated time points into each well containing SGC-7901 cells after baseline rate measurement. Each data represents mean ± SD. n = 17. (B) Quantitative image analysis of OCR on cultured SGC-7901 cells. Basal, endogenous rate; Oligomycin, ATP-synthase-inhibited rate; FCCP, maximal uncoupled rate; and Rotenone, rotenone-inhibited rate. (C) Basal ECAR and proton production rate from measurements taken in tandem with respiration rates. (D) ATP production rate from oxidative phosphorylation and glycolysis.

Figure 4. Regulation of mitochondrial respiration by carnosine in cultured SGC-7901 cells.

The cells were seeded in specialized microplates and cultured with or without carnosine (20 mM) for 48 h. Cells were then switched to unbuffered DMEM supplemented with 2 mM sodium pyruvate and 20 mM carnosine, and mitochondrial function was assessed using sequential injection of oligomycin, FCCP, and rotenone. (A) Basal OCR, (B) Basal ECAR, (C) ATP-linked OCR, (D) proton leak, (E) non-mitochondrial OCR (Non-Mito), (F) maximal OCR, and (G) spare capacity are shown. Results are means ± SD. n = 6–9. *P<0.05; **P<0.01 vs. control group.

Figure 5. Effect of carnosine on extracellular lactate acid level in cultured SGC-7901 cells.

The cells were cultured for 48 h in DMEM with or without carnosine (20 mM). The supernatant was collected for HPLC analysis of lactate acid. Data were expressed as mean ± SD. n = 6. **P<0.01 vs. control group.

Figure 6. Carnosine changed the relative contribution of glycolysis and OXPHOS to ATP charge in SGC-7901 cells cultured in DMEM free of pyruvate.

Intracellular ATP level was measured in response to glycolysis inhibitor 2-deoxyglucose (2-DG, 100 mM), mitochondrial uncoupler FCCP (1 µM), and complex I inhibitor rotenone (1 µM) treatment for 45 min, individually or in combination as shown, in cultured SGC7901 cells with or without carnosine (20 mM) treatment for 48 h. ATP level was expressed as % of control, which was defined as the baseline value in cells exposed only to vehicle. Each data represents mean ± SD. n = 6. **P<0.01 vs. vehicle in carnosine absent group, ^##P<0.01 vs. vehicle in carnosine absent group, ^&P<0.05, ^&&P<0.01 vs. vehicle in carnosine present group.

Figure 7. Changes of mitochondrial membrane potential and mtDNA copy number induced by carnosine in SGC-7901 cells.

(A) Changes in JC-1 fluorescence with carnosine treatment in cultured SGC-7901 cells. The cells were treated with carnosine (20 mM) for 48 h, and then were stained with JC-1. Red fluorescence indicates a polarized state and green fluorescence indicates a depolarized state. Scale bar: 20 µm. (B) MtDNA copy number in SGC-7901 cells treated with or without carnosine.

Figure 8. Effect of pyruvate on the inhibitive action of carnosine on SGC-7901 cells mitochondrial function.

SGC-7901 cells were cultured in DMEM supplemented with increasing doses of pyruvate (2, 4, 6 mM) in the presence or absence of carnosine (20 mM) for 48 h, and then the cells mitochondrial metabolism activity was measured by MTT reduction assay. Results are mean ± SD. n = 8–16. **P<0.01 vs. control group.