Anti-tumor effect of AZD8055 against bladder cancer and bladder cancer-associated macrophages

Abstract

The increased activity of the mTOR pathway in bladder cancer has been extensively studied, but no satisfactory mTOR inhibitor has been found in bladder cancer. The role of AZD8055, a second-generation mTOR inhibitor, has not been reported in bladder cancer. Herein, we investigated the effects of AZD8055 on bladder cells and their interaction with macrophages in vivo and in vitro. In four bladder cancer cell lines, the phosphorylation of mTOR, AKT and S6K1 was suppressed by AZD8055. AZD8055 inhibited proliferation and induced G1 cell-cycle arrest and apoptosis of bladder cancer cells in a concentration-dependent manner. AZD8055 also inhibits the migration and invasion of bladder cancer cells by blocking EMT and MMP9. In addition, AZD8055 inhibited chemotaxis and M2 phenotype of macrophage after co-culture with bladder cancer cells. These anti-tumor effects of AZD8055 were verified in vivo. Our findings collectively demonstrated that low-dose AZD8055 induces cytotoxicity and apoptosis, and inhibits the Akt/mTOR activation, invasion and migration of bladder cancer. These findings also demonstrate that AZD8055 partially blocked the interactions of bladder cancer cells and macrophages. In conclusion, AZD8055 is a promising mTOR inhibitor for bladder cancer.

Keywords: AZD8055; Bladder cancer; Macrophage; mTOR.

Fig. 1

Effects of AZD8055 on the proliferation of bladder cancer cells. (A) AZD8055 induces cytotoxicity in a concentration-dependent manner. T24, 5637, SCaBER and UM-UC-3 cells were treated with different concentrations of AZD8055 for 48 or 72 h. Cell viability was quantified by the CCK-8 assay, and the relative viability and the IC50 were calculated; (B) AZD8055 induces cytotoxicity in a time-dependent manner. The four cells were treated with different concentrations of AZD8055 for 24,48,72 or 96 h; (C) morphological changes of four bladder cancer cells after treatment with different concentrations of AZD8055 under optical microscope; (D) colony formation of four bladder cancer cells after treatment with different concentrations of AZD8055.

Fig. 2

Effects of AZD8055 on the migration and invasion of bladder cancer cells. (A and B) Cell scratch test result of four bladder cancer cells after treatment with AZD8055. Experimental images were taken with light microscope at 0 h, 24 h and 48 h; (C and D) after treatment of T24, 5637, SCaBER and UM-UC-3 cells with different concentrations of AZD8055 for 24 h, the invaded cells were observed using light microscopy; (E) Western blot results demonstrate the expression of EMT-related proteins and MMP9 in T24, 5637, SCaBER and UM-UC-3 cells after treatment with different concentrations of AZD8055.

Fig. 3

AZD8055 regulates the AKT/mTOR signaling and induces cell-cycle arrest in bladder cancer cells. (A) Four bladder cancer cell lines were treated with different concentrations of AZD8055 for 48 h. The levels of p-AKT, p-S6K1, p-mTOR and GAPDH were analyzed by Western blot; (B) effect of AZD8055 on cell-cycle distribution in bladder cancer cells. Four bladder cancer cells were treated with different concentrations of AZD8055 for 48 h. The histograms show the percentage of cells in the different phases of the cell cycle and the stacked bar plot represents the collective data from the histograms.

Fig. 4

AZD8055 induces apoptosis in bladder cancer cells. (A) Four bladder cancer cells were treated with different concentrations of AZD8055 for 48 h, after which the cells were stained with propidium iodide/annexin V, and analyzed by flow cytometry. The percentage of cells in early apoptosis (right lower phase) and late apoptosis (right upper phase) are shown; (B) Western blot results showed that the expression of apoptosis marker protein Bax and anti-apoptosis marker protein BCL-2 after AZD8055 treatment; (C) two bladder cancer cells were treated with AZD8055 for 48 h, after which the cells were stained with JC-1, and analyzed by fluorescent microscope. The shift from red to green fluorescence is indicative of depolarization of mitochondrial membrane potential and early apoptosis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

AZD8055 inhibits the chemotaxis of macrophage. (A) After 48 h of PMA induction, light microscopic images show that THP-1 cells have successfully adhered to the wall and changed their morphology; (B) flow cytometry results showed the expression of CD68, a marker of mature macrophage, in cells before and after induction; (C) macrophages were treated with different concentrations of AZD8055 for 24 h; D. Optical microscopy images showed the effect of AZD8055 on macrophage chemotaxis after co-culture with T24 and UM-UC-3 bladder cancer cells. The number of macrophages stained with crystalline violet in the image represents the chemotaxis of macrophages to bladder cancer cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

AZD8055 blocks the induction of M2 macrophage polarization by bladder cancer cells. (A) Flow cytometry results show the effect of AZD8055 on the expression of the M1 marker CD86 and the M2 marker CD206 in macrophage after co-culture with T24 and UM-UC-3 cells for 48 h. Histogram results showed the proportion of macrophages expressing CD86 and CD206; (B) qPCR results showing the effect of AZD8055 on the expression of mRNA for the M1 marker NOS2 and TNF-α and the M2 marker Arg1 and TGF-β in macrophages after co-culture with bladder cancer cells.

Fig. 7

In vivo anti-tumor effects of AZD8055 in the subcutaneous xenograft tumor of human bladder cancer in nude mice. (A) Body weight of mice after starting AZD8055 treatment; (B) body weights of mice in the experimental and control groups at the end of treatment; (C) pictures of subcutaneous xenograft tumors in mice after 2 weeks of AZD8055 treatment; (D) pictures of HE staining, tunel staining, and immunohistochemistry for p-mtor, E-cadherin, and F4-80 on subcutaneous xenograft tumors; (E) volume of subcutaneous graft tumor at the end of treatment; (F) weight of subcutaneous graft tumor at the end of treatment; (G–J) Analysis of immunohistochemistry results. (K and L) Images and analysis of double immunofluorescence staining of F4-80 and CD206 on mouse transplanted tumor sections.