Figure 1.
Temsirolimus differentially regulates cell viability and Akt phosphorylation in a dose-dependent manner.
A, B, Endometrial cancer cells were treated with increasing doses of temsirolimus for 72 hrs. Results are separated by (A) sensitivity or (B) resistance as determined by cell viability. C, D, Phosphorylation of rS6 (P-rS6) and Akt (P-Akt T308 and P-Akt S473) after treatment with indicated doses of temsirolimus for 72 hrs in temsirolimus-sensitive (C) or temsirolimus-resistant (D) cells was determined by Western blotting. Expression of total rS6 and Akt protein serves as loading controls.
Figure 2.
Baseline expression of PTEN, phospho-Akt and phospho-PDK1.
Eight endometrial cancer cell lines were grown without treatment. Total protein extracts were analyzed by Western blotting for PTEN, phospho-Akt (P-Akt S473 and P-Akt T308), total Akt, phospho-PDK1 (P-PDK1 S241), and total PDK1. β-actin expression served as a loading control.
Figure 3.
Temsirolimus-induced Akt phosphorylation is decreased by BEZ235 and ZSTK474, but not by AZD6244.
A, Ishikawa H (upper panels) and Hec50co (lower panels) cells were grown for 24 hrs and treated overnight with the indicated inhibitors. Phospho-Akt (P-Akt S473) and total Akt were assessed by Western blotting. B, Endometrial cancer cell lines were treated with the indicated inhibitors overnight. Total protein extracts were analyzed by Western blotting for P-Akt S473 and total Akt (left panels) or phospho-p70S6K T389 (P-S6K) and total p70S6K (S6K, right panels). Phospho-Akt blots for Hec1A and KLE cells were subjected to a long exposure to visualize low levels of Akt phosphorylation.
Figure 4.
Combination treatment of BEZ235 or ZSTK474 with temsirolimus synergistically inhibits cell proliferation.
A, B, Cell viability was determined in the indicated endometrial cancer cell lines after treatment with increasing concentrations of (A) BEZ235 or (B) ZSTK474 alone or in the presence of 1 nM temsirolimus for 72 hrs.
Figure 5.
BEZ235 alone or in combination with temsirolimus induces G1 cell cycle arrest and p27 expression.
A–B, AN3CA (A) or Hec50co (B) cells were treated for 24 hrs or 72 hrs, respectively, with vehicle (DMSO) or BEZ235 (1 nM–100 nM) in the presence or absence of 1 nM temsirolimus. Cell cycle distribution was analyzed by flow cytometry and the percentage of cells in G1 determined. C, AN3CA cells were treated for 24 hrs with vehicle (DMSO) or ZSTK474 (1 µM) in the presence or absence of 1 nM temsirolimus. Cells were analyzed as in A. D, Expression of the cyclin-dependent kinase inhibitor p27 was assessed by Western blotting 24 hrs after the indicated treatments.
Figure 6.
Combination treatment induces autophagy and PARP cleavage.
(A) Expression of the autophagy marker LC3 was assessed by Western blotting after the indicated treatments for 24 hrs. Arrows denote full-length LC3-I (16 kDa) and cleaved LC3-II (14 kDa). (B) PARP cleavage was assessed by Western blotting after the indicated treatments for 48 hrs–72 hrs. Arrows denote full-length PARP (116 kDa) and cleaved PARP (89 kDa).
Figure 7.
Mechanism of synergistic effect for combination treatment of temsirolimus and BEZ235.
A, Phosphorylation of 4E-BP1 (P-4E-BP1 T37/T46) was assessed after incubating cells with the indicated treatments for 24 hrs. Total 4E-BP1 expression serves as a loading control. B, Cells were treated with 1 nM or 10 nM temsirolimus or BEZ235 for 72 hrs then phosphorylation of rS6 (P-rS6 S235/S236) determined by Western blotting. Total rS6 serves as a loading control. C, Proposed mechanism for synergistic effect. Temsirolimus blocks one arm of mTORC1 signaling as evidenced by lack of phosphorylation of rS6. As a dual inhibitor of PI3K and mTOR, BEZ235 acts both upstream as well as downstream of mTORC1 and mTORC2 through inhibition of Akt (upstream) and 4E-BP1 (downstream) phosphorylation. BEZ235 has minimal effects on rS6 phosphorylation, but combination with temsirolimus blocks both arms of mTORC1 signaling. As a specific PI3K inhibitor, ZSTK474 acts upstream of the PI3K/Akt/mTOR signaling pathway, and in combination with temsirolimus, can block rS6 but not 4E-BP1 activation.