A novel androstenedione derivative induces ROS-mediated autophagy and attenuates drug resistance in osteosarcoma by inhibiting macrophage migration inhibitory factor (MIF)

Abstract

Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.

Figure 1

Effect of DSTD on cell viability, cell cytotoxicity and MIF expression in osteosarcoma cells. (a) TLC analysis of androstenedione derivatives transformed by Bordetella sp. B4, lane1-5, androstenediones were transformed for 12, 24, 4872 h, respectively; lane 6, purified HSTD; lane 7, purified DSTD. (b) Transformation pathway of androstenediones by Bordetella sp. B4. (c) Effect of DSTD on viability and cytotoxicity of MG-63 and U2OS osteosarcoma cells. Cells were treated with indicated concentration of DSTD (dissolved in DMSO) for 24 h. Cell viability and cytotoxicity were determined respectively using MTT assay and LDH release assay. (d) Effect of DSTD on MIF expression. Cells were treated with 100 μM DSTD for 24 h. MIF protein level and mRNA level were analyzed respectively by western blot and real-time PCR. These experiments were repeated at least three times. *P<0.01 versus control group

Figure 2

Effect of blocking MIF on signaling components and autophagy. (a) Cells were treated with 100 μM DSTD for 24 h or transfected with shRNA targeted for MIF for 48 h. The cytoplasmic and nucleic fractions were separated. Bcl-2, Bax, p-ERK, HMGB1 and Histone H3 were analyzed by western blot. (b) DSTD induced time-dependent autophagy. Cells were treated with 100 μM DSTD for indicated time. LC3 conversion was detected by western blot. These experiments were repeated at least three times

Figure 3

DSTD induces autophagy in U2OS and MG-63 osteosarcoma cells. (a) DSTD induced dose-dependent autophagy. Cells were treated with the indicated concentration of DSTD for 24 h. MIF, Bcl-2, Bax, p-ERK, HMGB1 and LC3 were analyzed by western blot. (b) DSTD induced ROS-mediated autophagy in time-dependent manners. Cells were treated with 100 μM DSTD for indicated time. To determine the effect of NAC on autophagy, after treated with 100 μM DSTD for 24 h, cells continued to be treated with 100 μM DSTD combined with 15 mM NAC (pH 7.4) for 24 h. The cytoplasmic and nucleic fractions were separated. MIF, Bcl-2, Bax, HMGB1, p-ERK, PARP and LC3 were analyzed by western blot. (c) Viability of U2OS and MG-63 cells treated with 100 μM DSTD in the presence or absence of NAC for the indicated time. (d) Cytotoxicity induced by 100 μM DSTD in the presence or absence of NAC for the indicated time. Values are means of at least three independent experiments. *P<0.05 versus control group; ^#P<0.05 versus group treated with DSTD for 48 h; **P<0.01 versus control group

Figure 4

DSTD induces ROS generation, LC3 puncta formation and apoptosis in U2OS and MG-63 osteosarcoma cells. (a) Effect of DSTD on ROS generation and LC3 puncta formation. Cells were treated with 100 μM DSTD for 24 h in the presence or absence of 15 mM NAC. After treatment, cells were stained by DCF-DA or LC3 antibody to determine ROS generation and LC3 puncta formation, respectively. (b) Effect of DSTD on cell apoptosis. After treatment, apoptosis was determined by flow cytometry

Figure 5

DSTD-mediated autophagy delays cell death. The U2OS (a) and MG-63(b) cells were pretreated with 1 mM 3-MA for 1 h, and was subsequently treated with 100 μM DSTD for another 24 h. LC3 conversion and PARP cleavage were analyzed by Western blot. Cell viability and cytotoxicity in U2OS (c) and MG-63 (d) cells were measured by MTT assay and LDH release assay. **P<0.01 versus control group; *P<0.05 versus group treated with DSTD; #P<0.01 versus group treated with 3-MA

Figure 6

DSTD attenuated drug resistance in osteosarcoma cells. Cells were treated with 0.5 μM doxorubicin (Dox) and 50 μM cisplatin (Cis) in the presence or absence of 100 μM DSTD for 24 h. Viability of U2OS and MG-63 cells were determined by MTT assay

Figure 7

DSTD reversed the upregulation of HMGB1 during chemotherapy in osteosarcoma cells. (a) DSTD reversed Dox-induced HMGB1 expression. Cells were treated with 0.5 μM Dox in the presence or absence of 100 μM DSTD for 24 h. Western blot was used to analyze MIF, HMGB1 and ERK expression using their corresponding antibodies. (b) DSTD decreased mRNA level of HMGB1 during chemotherapy. mRNA level was analyzed by real-time PCR. (c) DSTD reversed Cis-induced HMGB1 expression. Cells were treated with 50 μM Cis in the presence or absence of 100 μM DSTD for 24 h. Western blot was used to analyze MIF, HMGB1 and ERK expression using their corresponding antibodies. (d) DSTD decreased mRNA level of HMGB1 during chemotherapy. mRNA level was analyzed by real-time PCR. (e) Overexpression of HMGB1 blocked DSTD-induced signal transduction during chemotherapy. Cells were transfected with control plasmid and GV230-HMGB1 cDNA for 72 h. HMGB1, pERK, LC3 conversion and PARP cleavage were measured by western blot. (f) Overexpression of HMGB1 decreased DSTD-induced cell death during chemotherapy. Values are means of at least 3 independent experiments. *P<0.05; ^#P<0.01