Efficacy of luteolin on the human gastric cancer cell line MKN45 and underlying mechanism

Abstract

Objective: To investigate the antitumour efficacy of luteolin on gastric cancer (GC) and study the mechanism underpinning the action.

Methods: Effects of luteolin on cell growth inhibition, apoptosis, and cell cycle arrest in MKN45 cells were investigated using the cell counting kit-8 assay. Changes in the mitochondrial membrane potential after luteolin treatment were assessed using 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanineiodi-de (JC-1) staining. To investigate whether apoptotic effect by luteolin is related to the phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene (PI3K/Akt) pathway, cells were additionally treated with LY294002, a PI3K/Akt pathway inhibitor. Moreover, the expressions of apoptosis-related proteins, namely B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein (Bax), Akt, p-Akt, caspase-3, and cytochrome C, were detected after luteolin treatment.

Results: The study revealed that in MKN45 cells, luteolin could inhibit the cell proliferation in a time- and dose-dependent manner; block the cell cycle in the S-phase; induce apoptosis; reduce the mitochondrial membrane potential; increase the expression of Bax, caspase-3, and cytochrome C; and decrease the expression of Bcl-2 and p-Akt. Luteolin might be involved in the PI3K/Akt signalling pathway, indicating that this pathway could be a therapeutic target for GC treatment.

Conclusion: Luteolin could inhibit the proliferation of GC cells and block the cell cycle in the S-phase. The mechanism of inducing apoptosis in these cells was related to the PI3K/Akt signalling pathway.

Keywords: Akt; apoptosis; luteolin; phosphoinositide 3-kinase; signal transduction; stomach neoplasms.

Figure 1. Effect of luteolin on cell cycle of gastric cancer MKN45

A: evaluation of cell cycle, using flow cytometry and PI staining, in MKN45 cells treated with varying concentrations of luteolin (0, 10, 30, and 50 μmol/L) for 48 h. B: the ratio of cells per cell cycle phase of MKN45 cells treated with varying concentrations of luteolin (0, 10, 30, and 50 μmol/L). Compared with the control, ^aP < 0.001. G: Gap phase. S: Synthesis phase. M: mitotic. Con: control.

Figure 2. Luteolin induces apoptosis of gastric cancer MKN45 cells

A: Evaluation of cell apoptosis, using flow cytometry and DAPI staining, in MKN45 cells treated with varying concentrations of luteolin (0, 10, 30, and 50 μmol/L) for 48 h. B: The percentage of total apoptotic MKN45 cells for varying concentrations of luteolin (0, 10, 30, and 50 μmol/L). Compared with the control, ^aP < 0.001. Con: control.

Figure 3. Effect of luteolin on apoptotic morphology of MKN45 gastric cancer cells

MKN45 cells treated with varying concentrations of luteolin (0, 10, 30, and 50 μmol/L) for 48 h and stained by DAPI, and observed the changes of cell morphology under a fluorescence microscope (× 200).

Figure 4. Effect of luteolin on mitochondrial membrane potential of MKN45 gastric cancer

A: evaluation of mitochondrial membrane potential, using flow cytometry and JC-l staining, in MKN45 cells treated with varying concentrations of luteolin (0, 10, 30, and 50 μmol/L) for 48 h. B: The percentage of JC-l positive MKN45 cells for varying concentrations of luteolin (0, 10, 30, and 50 μmol/L). Compared with the control, ^aP < 0.001. Con: control.

Figure 5. Effect of luteolin on apoptosis of MKN45 cells

A: Evaluation of cell apoptosis, using flow cytometry and DAPI staining, in MKN45 cells treated with varying concentrations of Luteolin (20 μmol/L), LY294002 inhibitor (20 μmol/L), and luteolin (20 μmol/L) + LY294002 inhibitor (20 μ mol/L) for 48 h. B: The percentage of total apoptotic MKN45 cells for varying concentrations of Luteolin (20 μmol/L), LY294002 inhibitor (20 μmol/L), and luteolin (20 μmol/L) + LY294002 inhibitor (20 μ mol/L). Compared with the control, ^aP < 0.001. Con: control.

Figure 6. Effect of luteolin on apoptotic protein of MKN45 gastric cancer cells

1: Con; 2: Luteolin; 3: LY294002; 4: LY294002 + Luteolin. Expression of PI3K/Akt signalling pathway-related and apoptosis-related proteins was determined using western blot, in MKN45 cells treated with varying concentrations of Luteolin (20 μmol/L), LY294002 inhibitor (20 μmol/L), and luteolin (20 μmol/L) + LY294002 inhibitor (20 μmol/L) for 48 h. Con: control. Bcl: B-cell lymphoma. Bax: Bcl-2 associated X protein.