Non-immunosuppressive triazole-based small molecule induces anticancer activity against human hormone-refractory prostate cancers: the role in inhibition of PI3K/AKT/mTOR and c-Myc signaling pathways

Abstract

A series of triazole-based small molecules that mimic FTY720-mediated anticancer activity but minimize its immunosuppressive effect have been produced. SPS-7 is the most effective derivative displaying higher activity than FTY720 in anti-proliferation against human hormone-refractory prostate cancer (HRPC). It induced G1 arrest of cell cycle and subsequent apoptosis in thymidine block-mediated synchronization model. The data were supported by a decrease of cyclin D1 expression, a dramatic increase of p21 expression and an associated decrease in RB phosphorylation. c-Myc overexpression replenished protein levels of cyclin D1 indicating that c-Myc was responsible for cell cycle regulation. PI3K/Akt/mTOR signaling pathways through p70S6K- and 4EBP1-mediated translational regulation are critical to cell proliferation and survival. SPS-7 significantly inhibited this translational pathway. Overexpression of Myr-Akt (constitutively active Akt) completely abolished SPS-7-induced inhibitory effect on mTOR/p70S6K/4EBP1 signaling and c-Myc protein expression, suggesting that PI3K/Akt serves as a key upstream regulator. SPS-7 also demonstrated substantial anti-tumor efficacy in an in vivo xenograft study using PC-3 mouse model. Notably, FTY720 but not SPS-7 induced a significant immunosuppressive effect as evidenced by depletion of marginal zone B cells, down-regulation of sphingosine-1-phosphate receptors and a decrease in peripheral blood lymphocytes. In conclusion, the data suggest that SPS-7 is not an immunosuppressant while induces anticancer effect against HRPC through inhibition of Akt/mTOR/p70S6K pathwaysthat down-regulate protein levels of both c-Myc and cyclin D1, leading to G1 arrest of cell cycle and subsequent apoptosis. The data also indicate the potential of SPS-7 since PI3K/Akt signalingis responsive for the genomic alterations in prostate cancer.

Keywords: Myc; PI3K/Akt/mTOR signaling; autophagy; non-immunosuppression; triazole-base.

Figure 1. SPS-7 inhibits cell proliferation in human prostate cancer cells

(A) Cells were incubated in the absence or presence of SPS-7 for 48 h in PC-3 cells and 72 h in DU145, LNCaP and primary prostate cells. After the treatment, cell proliferation was determined using sulforhodamine B assay. Basal, seeding cell numbers. (B) PC-3 cells were incubated in the absence or presence of SPS-7 (10 μM) for the indicated time. After treatment, the cells were harvested for flow cytometric analysis of CFSE staining. The cell proliferation was to be followed by monitoring decrease in label intensity in successive daughter cell generations. The proliferation index and the cell populations of parent or different generations were calculated by Modfit LT Version 3.2 and WinList Version 5.0 software. Quantitative data are expressed as mean ± SEM of three to four independent experiments. *P < 0.05 and ***P < 0.001 compared with the control.

Figure 2. SPS-7 induces G1 arrest of the cell cycle and apoptosis

(A) Synchronization of PC-3 cells was performed by thymidine block as described in the Materials and Methods section. Then, the cells were released in the absence or presence of 10 μM SPS-7 or FTY720 for the indicated times. Data are representative of three independent experiments. (B) Quantitative data are expressed as mean ± SEM of three independent experiments. (C) PC-3 or DU-145 cells were incubated in the absence or presence of the compound (10 μM) for 24 or 48 hours. After the treatment, the cells were harvested for the determination of sub-G1 population using the detection of DNA content analyzed with FACScan and CellQuest software. Data are expressed as mean ± SEM of three independent determinations. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the respective control.

Figure 3. SPS-7 induces changes of expression levels of several proteins

PC-3 cells were incubated in the absence or presence of SPS-7 (10 μM) for the indicated time. After the treatment, the cells were harvested and lysed for the detection of protein expressions of cell cycle regulators (A), and Akt/mTOR pathway signals (B) by Western blot analysis. The data are representative of three independent experiments. Data are expressed as mean ± SEM of three determinations. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control of 100%.

Figure 4. c-Myc and Akt involve in SPS-7-induced signaling pathways

PC-3 cells were transfected with c-myc plasmid (A) or Myr-Akt plasmid (B). Then, the cells were incubated in the absence or presence of SPS-7 (10 μM) for nine hours. The cells were harvested and lysed for the detection of the indicated protein by Western blot analysis. The expression was quantified using the computerized image analysis system Image Quant (Amersham Biosciences). The data are expressed as mean ± SEM of three to five independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5. SPS-7 induces autophagy which counteracts apoptotic cell death

PC-3 cells were incubated in the absence or presence of (A) 10 μM SPS-7 or (B, C) 10 μM SPS-7 and/or 10 μM chloroquine for the indicated time (A, B) or 24 hours (C). Cells were harvested and lysed for the detection of the indicated protein expression by Western blot analysis (A), or the cells were harvested for the detection of hypodiploid DNA content (apoptotic sub-G1 population) (B) or mitochondrial membrane potential (C) by flow cytometric analysis. Protein expression was quantified using computerized image analysis system ImageQuant (Amersham Biosciences, NJ, USA). Data are expressed as mean ± SEM of three to five independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control of 100%.

Figure 6. FTY720 but not SPS-7 induces immunosuppressive effect

CD2F1 mice were treated with or without intraperitoneal FTY720 or SPS-7 for 6 hours. Whole blood was collected and the spleens were dissected. (A) Splenic sections were fixed, embedded with paraffin and de-paraffinized for H&E staining. IHC staining was used for detecting S1PR expression and performed by using UltraVision^™ Quanto Detection System HRP DAB (Thermo Fisher Scientific, Waltham, MA, USA) (A, figure inset). (B) Blood lymphocyte count was performed using specific CD marker antibody conjugation and removal of erythrocytesas described in the Materials and Methods section. Then, lymphocyte count was determined by flow cytometric analysis (Becton Dickinson, Mountain View, CA). Data are expressed as mean ± SEM of five independent experiments. *P < 0.05 compared with the control.

Figure 7. SPS-7 induces in vivo anti-tumor effect

The nude mice were subcutaneously injected with PC-3 cells (10⁷ cell/mouse). The tumors were measured every two days. When the tumors had reached a volume of 200 to 300 mm³, the mice were divided into five groups (n = 6), and vehicle, SPS-7 or FTY720 was given intraperitoneally every day. Data are expressed as mean ± SEM of tumor size at indicated times post treatment with the compound. Weight change during treatment was also measured.

Figure 8. Schematic figure for SPS-7-mediated signaling pathways on the inhibition of cell proliferation and apoptosis induction

SPS-7 inhibits the activation of PI3K/Akt, which in turn inhibits mTOR signaling pathway and activation of c-Myc-cyclin D1-Rb axis, leading to G1 arrest of the cell cycle and apoptosis. SPS-7 also induces the activation of cytoprotective autophagy that partly blunts the apoptosis. Therefore, combination of autophagy inhibitors has been suggested on developing anticancer agents targeting PI3K/Akt/mTOR pathways.