[Combined inhibition of PI3K and MEK has synergistic inhibitory effect on the proliferation of cisplatin-resistant ovarian cancer cells]

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Jun;30(6):592-6.
[Article in Chinese]


Objective: To investigate the effect of phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002 combined with mitogen activated protein kinase kinase (MEK) inhibitor AZD6244 on the proliferation of cisplatin-resistant SKOV3/DDP ovarian cancer cell line.

Methods: The alteration in the cell proliferation of SKOV3/DDP cells treated with 5, 10, 20, 40, 80 μmol/L LY294002 and 1, 2, 4, 8, 16 μmol/L AZD6244 alone or together, was detected by MTT assay. The 50% inhibitory concentration (IC50) and combined index (CI) were calculated. The concentration of the combination was obtained based on the MTT assay, and the cells were divided into four groups: the control group, LY294002 group (5 μmol/L), AZD6244 group (7 μmol/L) and combination group (LY294002 5 μmol/L and AZD6244 7 μmol/L); Forty-eight hours later, MTT assay was used to detect the cell proliferation and calculate cell doubling time; colony formation assay was performed to detect colony formation efficiency; Annexin V-PE/7-ADD staining combined with flow cytometry (FCM) was used to detect the apoptotic rates and cell cycle; Western blotting was employed to detect the levels of AKT, phosphorylated AKT (p-AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), cyclin D1 and cleaved caspase-3 protein.

Results: Cell growth was inhibited by LY294002 or AZD6244 alone, and the effect was strengthened when LY294002 was combined with AZD6244 (CI<1), indicating that the two inhibitors showed synergistic effect. The proliferation of cells was significantly slower, the cell doubling time was significantly prolonged, and colony number was reduced in the combined treatment group (P<0.01) compared with the single inhibitor treatment group and the control group. Meanwhile, FCM demonstrated that the apoptotic rate of the combination group was significantly higher than that of the other groups, and the cells significantly increased in G1 phase and decreased in S phase (P<0.05). As Western blotting showed, there were no differences in the expressions of AKT and ERK1/2 protein among the four groups (P>0.05); in the LY294002 treatment group, the level of p-AKT protein decreased and p-ERK increased; in the AZD6244 group, the level of p-ERK1/2 protein decreased and p-AKT increased; in the combination group, the levels of p-AKT, p-ERK1/2 and cyclin D1 protein were significantly inhibited, while cleaved caspase-3 protein was up-regulated.

Conclusion: Combined PI3K inhibitor LY294002 and MEK inhibitor AZD6244 has synergistic effect on inhibition of SKOV3/DDP cell growth by inducing apoptosis and blocking cell cycle.

MeSH terms

  • Antineoplastic Agents / pharmacology
  • Apoptosis / drug effects
  • Benzimidazoles / pharmacology
  • Blotting, Western
  • Caspase 3 / metabolism
  • Cell Cycle / drug effects
  • Cell Line, Tumor
  • Cell Proliferation / drug effects*
  • Cell Survival / drug effects
  • Chromones / pharmacology
  • Cisplatin / pharmacology
  • Dose-Response Relationship, Drug
  • Drug Resistance, Neoplasm / drug effects
  • Drug Synergism
  • Enzyme Inhibitors / pharmacology*
  • Female
  • Flow Cytometry
  • Humans
  • Inhibitory Concentration 50
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Mitogen-Activated Protein Kinase Kinases / antagonists & inhibitors*
  • Mitogen-Activated Protein Kinase Kinases / metabolism
  • Morpholines / pharmacology
  • Ovarian Neoplasms / enzymology
  • Ovarian Neoplasms / pathology
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoinositide-3 Kinase Inhibitors*
  • Proto-Oncogene Proteins c-akt / metabolism


  • AZD 6244
  • Antineoplastic Agents
  • Benzimidazoles
  • Chromones
  • Enzyme Inhibitors
  • Morpholines
  • Phosphoinositide-3 Kinase Inhibitors
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • Proto-Oncogene Proteins c-akt
  • MAPK1 protein, human
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinase Kinases
  • Caspase 3
  • Cisplatin