Metapristone (RU486 metabolite) suppresses NSCLC by targeting EGFR-mediated PI3K/AKT pathway

Abstract

Therapies targeting epidermal growth factor receptor (EGFR) can effectively treat with non-small cell lung cancer (NSCLC), but NSCLC's drug resistance makes it intractable. Herein, we showed that RU486 metabolite metapristone inhibited the proliferation of various NSCLC cell lines with either wild (A549, H1299, H520) or mutated EGFR (H1975, HCC827). The suppression was resulted from inhibition by metapristone of EGFR signaling pathways through down-regulating the EGFR, PTEN, as well as AKT and ERK proteins. In addition, metapristone inhibited anti-apoptotic marker Bcl-2, and activated pro-apoptotic key signaling proteins caspase-3, and poly (ADP-ribose) polymerase. Metapristone induced A549 and H1975 cell cycle via arrest at the G0-G1 stage. What's more, metapristone inhibited the growth of NSCLC xenografts in BALB/c nude mice through decreasing the expression of tumor growth biomarkers PCNA and EGFR. Taken together, the present study demonstrated that metapristone suppressed NSCLC proliferation by promoting apoptosis via decrease the cellular EGFR-mediated PI3K/AKT pathways. The results suggest metapristone a new treatment for EGFR-overexpressed NSCLC.

Keywords: EGFR; cell apoptosis; cell proliferation; metapristone; non-small cell lung cancer.

Figure 1. Effects of metapristone on non-small cell lung cancer cell viability

(A and B) Cells were incubated with different concentration of metapristone for 24, 48 or 72 h. Flow cytometry analyzed the cell cycle distribution in A549 (C) and H1975 (D) cells. (E) Western blotting (upper) and related quantitative analysis (lower) showed the changes of P-EGFR and EGFR in A549/H1975 cells induced by metapristone treatment. Date were the mean ± SEM (n=3). * P<0.05, ** P<0.01 and *** P<0.001, compared with the control.

Figure 2. Effects of metapristone on non-small lung cancer apoptosis in A549 and H1975 cells

(A) A549 and H1975 cell morphological changes and nuclear alterations after metapristone treatment. The condensed and fragmented fluorescent nuclei of apoptotic cells are visible in the treated cells, but not in the control cells. (B) Flow cytometric analyzed cell apoptosis in A549 and H1975 cells using the Annexin V-FITC/PI dual-labeling technique. Quantified values were shown on the right. (C) The levels of the typical apoptosis-related proteins total PARP were determined by Western blotting (upper). The expression of total PARP was quantified by using Image Lab analysis software. The condensed and fragmented fluorescent nuclel (white arrows) of apoptotic cells are visible in the treated cells, but not in the control cells. Date were the mean ± SEM (n=3). * P<0.05, ** P<0.01 and *** P<0.001, compared with the control.

Figure 3. Mechanism of metapristone induced apoptosis on non-small lung cancer

(A) Detection of caspase-3 activity by flow cytometric analysis of apoptosis in A549 cells. A549 cells were pre-incubated with the following: no inhibitor, 20 μM of a negative control inhibitor Z-FA-FMK or 20 μM of caspase-3 inhibitor Z-DEVD-FMK for 30 min, and then either left untreated (bottom row) or treated with 40 μM of metapristone for 24 h (top row). (B and C) The expression caspase-3(B), Bcl-2 and Bax (C and D) were determined by western blotting in A549 and H1975 cells after metapristone treatment. The quantitative analysis of the expressions of Bcl-2 in A549 or H1975 cells under different concentrations of metapristone were detected by RT-PCR assay. (E) Each bar represents the mean ± SEM (n=3). * P<0.05, ** P<0.01 and *** P<0.001, compared with the control.

Figure 4. Effects of metapristone treatment on PTEN/AKT, and ERKs signal pathway in NSCLC cells

(A) A549 and H1975 cells treated with 10-40 μM metapristone for 24 h was to determine its effect on the expression PTEN proteins. (B) Related quantitative analysis showed changes PTEN in A549 and H1975 cells induced by metapristone treatment. (C and D) Western blotting (C) and related quantitative (D) analysis showed changes in expression of P-AKT, T-AKT, P-ERK and T-ERK in A549 cells, and of P-AKT, T-AKT, P-ERK and T-ERK in H1975 cells induced by metapristone. The date expressed as the mean ± SEM (n=3). * P<0.05, ** P<0.01 and *** P<0.001, compared with the control.

Figure 5

Effect of metapristone treatment on the inherent and EGF-induced phosphorylation of EGFR in A549 and H1975 cells (A) A549 and H1975 cells were treated with metapristone (10, 20, 40 μM) for 24 h in serum-free cell medium. (B) Serum starved A549 and H1975 cells were treated with metapristone (10, 20, 40 μM) for 24 h and then incubated without or with EGF (50 ng/mL) for 30 min. (C) Related quantitative analysis showed changes in EGFR and phosphorylated-EGFR of A549 and H1975 induced by EGF treatment. Each bar represents the mean ± SEM (n=3). * P<0.05, ** P<0.01 and *** P<0.001, compared with the control.

Figure 6. Effect of metapristone in non-small lung cancer xenograft model

A549 cells were injected subcutaneously to the athymic nude mice. Two week after the injection, metapristone (20 mg/kg/day) was given to the mice for 18 days (n = 7) (A) Non-small lung cancer tumor volume results after treatment with metapristone. (B) Body weight change during the treatment periods. (C) Hematoxylin and Eosin (H&E) staining of paraffin-embedded non-small cell lung cancer tumors; Cell proliferation was detected by PCNA staining. Cell survival signaling was detected by EGFR staining. PCNA or EGFR-positive tumor cells are brown and the arrows indicate PCNA or EGFR-positive tumor cells.