Cryptotanshinone Prevents the Binding of S6K1 to mTOR/Raptor Leading to the Suppression of mTORC1-S6K1 Signaling Activity and Neoplastic Cell Transformation

Abstract

Cryptotanshinone is known for its inhibitory activity against tumorigenesis in various human cancer cells. However, exact mechanisms underlying the anticancer effects of cryptotanshinone are not fully elucidated. Here, we propose a plausible molecular mechanism, wherein cryptotanshinone represses rapamycin-sensitive mTORC1/S6K1 mediated cancer cell growth and cell transformation. We investigated the various effects of cryptotanshinone on the mTORC1/S6K1 axis, which is associated with the regulation of cell growth in response to nutritional and growth factor signals. We found that cryptotanshinone specifically inhibited the mTORC1-mediated phosphorylation of S6K1, which consequently suppressed the clonogenicity of SK-Hep1 cells and the neoplastic transformation of JB6 Cl41 cells induced by insulin-like growth factor-1. Finally, we observed that cryptotanshinone prevented S6K1 from binding to the Raptor/mTOR complex, rather than regulating mTOR and its upstream pathway. Overall, our findings provide a novel mechanism underlying anti-cancer effects cryptotanshinone targeting mTORC1 signaling, contributing to the development of anticancer agents involving metabolic cancer treatment.

Keywords: Raptor protein; cryptotanshinone; mTORC1; neoplastic cell transformation; p70S6K.

Figure 1. Cryptotanshinone inhibits mTORC1 signaling and cell growth in cancer cells.

(A) Effects of three tanshinones on cell viability in SK-Hep1 cells (hepatocellular carcinoma cells). Cell viability was estimated. The asterisk (*) indicates a significant (*P < 0.05) change compared to the untreated control. (B, C) Serum-starved SK-Hep1 cells were treated with three tanshinones (at 12 and 25 µM) for 6 hours, followed by immunoblotting with the corresponding antibodies. TI, Tanshinone I; TIIA, Tanshinone IIA; CT, Cryptotanshinone.

Figure 2. Cryptotanshinone inhibits the clonogenicity of SK-Hep1 cells.

(A) Effect of CT on the clonogenicity of human hepatocellular carcinoma cells. SK-Hep1 cells were subjected to a soft agar clonogenic assay with cryptotanshinone (CT) or rapamycin (Rapa). Data are represented as means ± SD from three experiments (*P < 0.05). (B, C) Cells were starved without serum for 24 hours and were either treated or not treated with the indicated concentration of CT and Rapa (0.1 µM) for an additional 6 hours. Next, the cells were stimulated with 10% FBS-DMEM. Cell lysates were subjected to immunoblotting assays with the indicated antibodies.

Figure 3. Cryptotanshinone inhibits the IGF-1-induced neoplastic transformation of JB6 Cl41 cells.

(A) JB6 Cl41 cells were pre-treated with cryptotanshinone (CT) or rapamycin (Rapa) at the indicated concentrations for 20 hours. The cells were then collected by trypsinization and subjected to the soft agar assay. Data are represented as means ± SD from three experiments (*P < 0.05). (B, C) JB6 Cl41 cells were starved without serum for 24 hours and were either treated or not treated with the indicated concentration of CT or Rapa (0.1 µM) for an additional 6 hours. Next, the cells were stimulated with IGF-1 (10 ng/mL). Cell lysates were subjected to immunoblotting assays with the indicated antibodies.

Figure 4. Cryptotanshinone can prevent the binding of S6K1 to mTOR/Raptor induced by IGF-1.

(A, B) Effect of cryptotanshinone (CT) on IGF-1-induced mTOR signaling in SK-Hep1 cells. Serum-starved SK-Hep1 cells were treated with the indicated concentrations of CT or rapamycin (Rapa; 0.1 µM) for 6 hours, followed by stimulation with IGF-1 (10 ng/mL). Cells were then disrupted in a 0.3% CHAPS buffer. Cell lysates were subjected to either an immunoblotting assay as indicated for mTOR signaling (panels A and B) or to an immunoprecipitation (IP) assay with anti-mTOR (IP: mTOR, panel C). (C) Effect of CT on IGF-1-induced mTORC1-S6K1 complex formation. Proteins (500 µg) were used for immunoprecipitation with anti-mTOR and analyzed via immunoblotting with Raptor, GβL, and S6K1 antibodies, as indicated. The cell lysates used in the immunoprecipitation reactions were loaded as a positive control (Lysate). (D) Schematic diagram of the model hypothesizing the CT effect on the regulation of S6K1 phosphorylation by mTORC1.