PI3K/AKT/mTOR pathway promotes progestin resistance in endometrial cancer cells by inhibition of autophagy

Figures & data

Figure 1 Progestin-resistant cells show increased cell growth and decreased autophagy.

Notes: (A) The cell growth curves of Ishikawa parental- and progestin-resistant cells are shown by MTT assay. Data are expressed as the mean results from three independent experiments. ***P<0.001. (B) Western blot showed expression of PR. (C) Western blot showed expression of autophagy markers EIG121, Beclin-1, P62, LC3B, ATG3, and ATG5 and the expression of β-actin as internal control.
Abbreviations: MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; PR, progesterone receptor; PR-A, progesterone receptor isoform A; PR-B, progesterone isoform B; OD, optical density; h, hours.

Figure 2 Progestin activates the PI3K/AKT/mTOR pathway in progestin-resistant endometrial cancer cells.

Notes: (A) Western blot assay was used to characterize the activation of PI3K/AKT/mTOR pathway. (B) Western blot showed expression of AKT1 in control and AKT1-knockdown cells. (C) The cell growth curves of control and AKT1 knockdown resistant cells are shown by MTT assay. Data are expressed as the mean numbers of independent triplicate experiments. ***P<0.001. (D) Western blot showed expression of autophagy markers LC3B, ATG3 and ATG5 and the expression of β-actin as internal control.
Abbreviations: PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; mTOR, mechanistic target of rapamycin; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; OD, optical density; Ctrl, control; h, hours; pAKT, phosphor-AKT; pmTOR, phosphor-mTOR; siAKT1, short interfering RNA targeting AKT1; siCtrl, short interfering RNA control with scrambled sequence.

Figure 3 Phosphorylation of mTOR is sufficient and necessary for cell growth in endometrial cancer cells by regulating autophagy.

Notes: (A) Western blot showed expression of PR and mTOR in control and mTOR-knockdown cells. (B) The cell growth curves of control and mTOR-knockdown cells are shown by MTT assay. Data are expressed as the mean numbers of independent triplicate experiments. ***P<0.001. (C) Western blot showed expression of autophagy markers LC3B, ATG3, and ATG5 and the expression of β-actin as internal control. (D) Western blot showed expression and phosphorylation of mTOR after restoration of mutant mTOR. (E) The cell growth curves are shown by MTT assay. Data are expressed as the mean numbers of independent triplicate experiments. ***P<0.001. (F) Western blot showed expression of autophagy markers LC3B, ATG3, and ATG5 and the expression of β-actin as internal control.
Abbreviations: mTOR, mechanistic target of rapamycin; PR, progesterone receptor; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; OD, optical density; Ctrl, control; h, hours; pmTOR, phosphor-mTOR; siCtrl, short interfering RNA control with scrambled sequence; mut, mutant; simTOR, short interfering RNA targeting mTOR.

Figure 4 RAD001 inhibits proliferation in progestin-resistant endometrial cancer cells by inducing autophagy.

Notes: (A) Western blot showed phosphorylation of mTOR and expression of autophagy markers with or without RAD001 treatment. (B) The cell growth curves with or without RAD001 treatment are shown by MTT assay. Data are expressed as the mean numbers of independent triplicate experiments. ***P<0.001.
Abbreviations: mTOR, mechanistic target of rapamycin; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; Ctrl, control; OD, optical density; h, hours.