A Novel Androstenedione Derivative Identified As DSTD Induces Autophagy And Attenuates Drug Resistance In Osteosarcoma

BackgroundOsteosarcoma, a common primary bone tumor in children and adolescents, is prone to early metastasis via the blood. Treatment with a combination of surgery and aggressive adjuvant chemotherapy has improved the survival rate of osteosarcoma patients. The 5-year-survival rates of non-metastatic patients have reached a plateau of approximately 70%. However, patients with poor responses to chemotherapeutics will undergo local recurrence and metastasis, which reduce the 5-year-survival rates to only 20% despite additional doses or drugs. Drug resistance is responsible for the poor prognosis. Attenuating chemoresistance facilitates better treatment of osteosarcoma. Novel treatment strategies that combine anticancer drugs with adjuvant agents could improve the antitumor effects.In the 1960s, macrophage migration inhibitory factor (MIF) was identified as a pluripotent protein that modulates inflammation. Increasing evidence suggests that inflammation is closely related to tumorigenesis. MIF plays a bridging role between inflammation and tumorigenesis. MIF triggers the activation of the MAPK and PI3K pathways by binding its membrane receptor CD74, resulting in the inhibition of cell apoptosis. Recently, MIF was demonstrated to be involved in cell proliferation, differentiation, angiogenesis and tumorigenesis. Some evidence has indicated that MIF is abundantly expressed in various cancers and is significantly associated with tumor invasion and metastasis. MIF has been well established to be involved in the development of glioblastoma, breast cancer, bladder cancer and colon cancer. MIF was also up-regulated in osteosarcoma. The knockdown of MIF blocked osteosarcoma cell proliferation and invasion. However, the effect of MIF on drug resistance in osteosarcoma has not yet been investigated. Research have revealed that MIF knockdown promoted chemosensitivity by inducing autophagy in breast cancer. In contrast, autophagy reportedly contributed to chemoresistance in osteosarcoma. These controversial results prompted us to confirm the role of MIF in autophagy and drug resistance in osteosarcoma.In this study, we tested the role and mechanism of a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrost-1,3,5,7-tetraene-9,17-dione (DSTD) in autophagy and drug resistance in osteosarcoma. DSTD could inhibit MIF expression in MG-63 and U2OS cells. Both 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. Furthermore, MIF inhibition by DSTD enhances chemosensitivity by downregulating HMGB1 in osteosarcoma cells. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.Part 1 The effect of DSTD on the viability, cytotoxicity and MIF expression of the osteosarcoma cellsObjectiveExamine the effect of DSTD on the viability and cytotoxicity of osteosarcoma cells lines MG-63 and U2OS and the potential role of DSTD in the regulation of MIF expression.Methods1. The human osteosarcoma cell lines (MG-63 and U2OS) were cultured in Dulbecco’s Modified Eagle’s Medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone),100 U/ml penicillin,100 U/ml streptomycin and 0.03% L-glutamine at 37℃ in 5% CO2. MG-63 and U2OS cells were successfully cultured in vitro. Cells of ogarithmic growth phase were used for the following study.2. The cells were treated with DSTD(0,1μM,10μM,50λM,100μM) for 24h. MTT assay was used to determine DSTD influence on MG-63 and U2OS cells proliferation and their concentration-dependent relationship. Cell cytotoxicity were determined using LDH realease assay.3. MG-63 and U2OS osteosarcoma cells were treated with 100100μM DSTD for 24h, MIF expression level was detected by quantitative real-time reverse transcriptase polymerase chain reaction(qRT-PCR). Expression level of MIF protein was detected by Western blot.4. Statistical analysis:Analysis was performed by Statistical Package for Social Sciences Version 21.0 (SPSS, Chicago, IL, USA). All the experiments were repeated three times in the study, the experimental data are represented as mean±SD(Standard Deviation). A student’s t-test was used for statistical comparisons between two groups. P<0.05 was considered significant.Results1. The results of MTT assay and lactate dehydrogenase (LDH) release assay indicated that exposure to 100 μM DSTD for 24 h did not significantly affect the cell viabilities and cytotoxicity of the osteosarcoma cells lines MG-63 and U2OS.2. The results of real-time PCR revealed that MIF mRNA was decreased after treatment with 100 μM DSTD for 24 h.3. The results of Western Blot illustrated that exposing cells to 100 μM DSTD for 24 h resulted in a significant reduction of MIF protein level.ConclusionDSTD significantly inhibited the expression of MIF and did not significantly affect the the viability and cytotoxicity of osteosarcoma cells.Part 2 DSTD induced ROS-mediated autophagy in osteosarcoma by inhibiting MIF expressionObjectiveTo investigate the expression of autophagy related proteins after MG-63 and U2OS cells were treated with DSTD, trying to find the molecular mechanism of the autophagy of human osteosarcoma cell MG-63 and U2OS induced by DSTD.Methods1. MG-63 and U2OS cells were successfully cultured in vitro. Cells of ogarithmic growth phase were used for the following study.2. Cells were treated with 100 μM DSTD for 24h or transfected with shRNA targeted for MIF for 48h. The cytoplasmic and nucleic fractions were separated. Bcl-2, Bax, p-ERK, HMGB1 and Histone H3 were analyzed by western blot.3. Detecting the expression of LC3B-Ⅰ and LC3B-Ⅱ protein in MG-63 and U2OS cells by Western blot after the treatment with 100μM DSTD on Oh,4h,8h,12h,24h, 48h.4. Detecting the expression of MIF, Bcl-2, Bax, pERK, HMGB1, LC3B-Ⅰ and LC3B-Ⅱ protein of U2OS and MG-63 cells by Western blot after the treatment with 0μM,50μM and 100μM DSTD for 24h.5. After the cultured cells were treated with 100μM DSTD in the presence or absence of 15mM NAC for the indicated time, the cytoplasmic and nucleic fractions were separated. Subsequently, MIF, Bcl-2, Bax, pERK, PARP, c-PARP, LC3B-I, LC3B-II, Histone H3, Cyto HMGB1, Nucleic HMGB1 were anaylzed by western blot.The U2OS and MG-63 cells were pretreated with 1mM 3-MA for lh, and was subsequently treated with 100100μM DSTD for another 24h. LC3 conversion and PARP cleavage were analyzed by Western blot.6. After the cells were treated with 100μM DSTD in the presence or absence of 15mM NAC for the indicated time. LC3 conversion and PARP cleavage were analyzed by Western blot. Subsequently, the Viabilityand cytotoxicity of U2OS and MG-63 were tested by MTT and LDH rease assay, respectively. U2OS and MG-63 cells were pretreated with 1mM 3-MA for 1h, and was subsequently treated with 100 μM DSTD for another 24h. Values are means of at least three independent experiments. Subsequently,the Viabilityand cytotoxicity of U2OS and MG-63 were tested by MTT and LDH rease assay, respectively.7. The cell apoptosis rates were measured by Annexin V-FITC fluorescence staining assay. Cells were treated with 100μM DSTD in the presence or absence of 15mM NAC for indicated time. Subsequently,the apoptosiswas analyzed using aflow cytometer (Beckman Coulter, Chicago, USA). Treated cells were harvested with trypsin without EDTA and phenol red. After washing three times with PBS, the treated cells were incubated with annexinV-FITC (5 μl,20 μg/ml) and propidiumiodide (PI,5 μl,20 μg/ml) in binding buffer at room temperature for 10 min in the dark. Subsequently, the labeled cells were analyzed by aflow cytometer and the data were processed using WinMDI 2.9 software.8. Determination of ROS levels:Cells were treated with 100μM DSTD in the presence or absence of 15mM NAC for the indicated time. After treatment, the generated ROS was probed using an oxidation-sensitive fluorescent reagent (DCF-DA). The treated cells were rinsed with cold PBS and then incubated in 10μM DCF-DA for 30 min at 37℃. After rinsing three times with PBS, the fluorescence excitation was performed at 488 nm and the emission was detected at 525 nm using a fluorescence microscope (LEICA DMIRE2).9. Immunofluorescence:Cells were treated with 1000μM DSTD in the presence or absence of 15mM NAC for the indicated time. After treatment, The cells were fixed with 4% paraformaldehyde in PBS for 30 minutes and permeabilized with 0.2% Triton X-100 in PBS for 15 min. Subsequently, the cells were blocked for one hour with 10% normal goat serum at room temperature. After being rinsed with PBST for three times, the cells were incubated with MAP1-LC3B overnight at 4℃ and then incubated with FITC-conjugated secondary antibody IgG for 2 h. After being rinsed five times with PBST, the cells were visualized using a fluorescence microscope (LEICA DMIRE2).10. Statistical analysis:Analysis was performed by Statistical Package for Social Sciences Version 21.0 (SPSS, Chicago, IL, USA). All the experiments were repeated three times in the study, the experimental data are represented as mean±SD(Standard Deviation). A student’s t-test was used for statistical comparisons between two groups. One-way ANOVA was used for statistical comparisons among multiple groups. P <0.05 was considered significant.Results1.100μM DSTD and the target-specific shRNA of MIF significantly suppressed MIF expression. MIF knockdown led to a large decrease of Bcl-2 and an increase of Bax. MIF knockdown also inhibited ERK phosphorylation. Intriguingly, MIF knockdown decreased the nucleic HMGB1 level but increased the cytosolic HMGB1 level, which suggested that MIF down-regulation led to the translocation of HMGB1 from the nucleus to the cytoplasm.2. A significant conversion of LC3-I to LC3-II was detected after exposing MG-63 and U2OS cells to 100μM DSTD for 24 h. The accumulation of LC3-Ⅱ positively correlated with the treatment time, which suggested that DSTD induced autophagy in a time-dependent manner.3. After U2OS and MG-63 cells were treated with different concentrations of DSTD for 24h, Western Blot shows that MIF, Bcl-2, pERK, HMGB1 protein gradually reduced with the increase of DSTD concentration, while the expression of Bax protein level increase gradually. The accumulation of LC3-II was also DSTD dose-dependent. This suggests that DSTD also induced autophagy in a dose-dependent manner.4 we tested the effect of N-acetyl-L-cysteine (NAC) on the DSTD-induced signaling response. NAC co-treatment reversed DSTD-induced signaling responses. Notably, DSTD induced the accumulation of PARP and formed cleaved PARP, which could not be reversed by NAC treatment.5. MTT showed that NAC treatment significantly decreased the cell viability, LDH release assay showed that NAC treatment increased cytotoxicity.6. The result of flow cytometry shows that NAC co-treatment attenuated DSTD-induced autophagy.7. we used an oxidation-sensitive fluorescent reagent (DCF-DA) to track ROS generation in U2OS and MG-63 cells. DSTD induced a time-related increase in the ROS level, which was reversed by NAC treatment8. We examined DSTD-induced autophagy by monitoring the formation of LC3 puncta using immunofluorescent staining. The results indicated that the formation of LC3 puncta was distinct and time-dependent, NAC co-treatment attenuated DSTD-induced autophagy.9. To examine whether DSTD-induced autophagy delayed cell death, we investigated effect of 3-methyladenine (3-MA, an inhibitor of type III phosphatidylinositol 3-kinase that needed for initiation of autophagy) on cell viability and cytotoxicity. The results demonstrated that 3-MA reduced the level of LC3B-II induced by DSTD in U2OS and MG-63 cells. Pharmacological inhibition of autophagy by 3-MA also suppressed cell viability and increased cell cytotoxicity, suggesting that DSTD-induced autophagy did contribute to delay cell death.ConclusionDSTD induced autophagy of U2OS and MG-63 cells by inhibiting MIF expression in a time- and dose-dependent manner. DSTD induced ROS-mediated autophagy, autophagy contribute to delay cell death.Part 3 The role and mechanism of DSTD in drug resistance of osteosarcomaObjectiveTo explore the role of DSTD and its molecular mechanism in chemotherapy resistance of Osteosarcoma cells.Methods1. The human osteosarcoma cell lines (MG-63 and U2OS) were cultured in Dulbecco’s Modified Eagle’s Medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone),100 U/ml penicillin,100 U/ml streptomycin and 0.03% L-glutamine at 37℃ in 5% CO2. MG-63 and U2OS cells were successfully cultured in vitro. Cells of ogarithmic growth phase were used for the following study.2.Cells were treated with 0.5μM doxorubicin (Dox) or 500μM cisplatin(Cis) in the presence or absence of 100μM DSTD for 24h, then the microscopicimages of U2OS and MG-63 cells were observed with a microscope.3.Cells were treated with a different concentration of doxorubicin (Dox) and cisplatin (Cis) in the presence or absence of 100μM DSTD for 24h. Viability of U2OS and MG-63 cells were determined by MTT assay.4. MG-63 and U2OS osteosarcoma cells were treated with 0.5μM doxorubicin (Dox) and 50μM cisplatin (Cis) in the presence or absence of 100μM DSTD for 24h. Subsequently, MIF, HMGB1 and ERK protein level was analyzed by western blot and HMGB1 mRNA level was analyzed by real-time PCR.5. Cells were transfected with control plasmid and GV230-HMGB1 cDNA for 72h. HMGB1, pERK, LC3 conversion and PARP cleavage were measured by western blot. Cell death ratio were tested by LDH release assay.6. Statistical analysis:Analysis was performed by Statistical Package for Social Sciences Version 21.0. All the experiments were repeated three times in the study, the experimental data are represented as mean±D(Standard Deviation). A student’s t-test was used for statistical comparisons between two groups. One-way ANOVA was used for statistical comparisons among multiple groups. P<0.05 was considered statistically significant.Results1. DSTD resulted in slight cell shrinkage, and doxorubicin or cisplatin partly caused cell shrinkage compared with control cells. Notably, large-scale cell death occurred in the presence of DSTD combined with chemotherapy.2. Doxorubicin and cisplatin reduced the cell viability in a dose-dependent manner. DSTD treatment sensitized osteosarcoma cells to doxorubicin and cisplatin, which was evidenced by the further decrease in the cell viability compared to chemotherapy alone.3. Western blot and Real-time PCR shows that DSTD combined with chemotherapy drugs suppressed HMGB1 expression.4. Overexpression of HMGB1 blocked DSTD-induced signal transduction during chemotherapy.5. Overexpression of HMGB1 decreased DSTD-induced cell death during chemotherapy.ConclusionAutophagy induced by the overexpression of HMGB1 rescued cell death, and MIF inhibition by DSTD facilitated cell death by decreasing the HMGB1 level, which likely explains how DSTD contributed to chemosensitivity in osteosarcoma cells.