Novel lipid metabolism factor HIBCH inhibitor synergizes with doxorubicin to suppress osteosarcoma growth and impacts clinical prognosis in osteosarcoma patients

Fig. 1. Identification of LMRGs and development of a prognostic RM in OS. (A) Heatmap showing the expression patterns of 176 differentially expressed LMRGs between OS patients and normal controls. Data were obtained from the TARGET and GTEx databases. (B) Volcano plot illustrating the differential expression analysis results of LMRGs. Red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent non-significantly changed genes. (C) Forest plot of UCR analysis results for the six significant LMRGs (ALDH2, HSD17B3, SLC25A1, HSD17B8, SCD5, and HIBCH) associated with OS prognosis (p < 0.05). (D) MCR analysis results for the six genes, showing their coefficients in the final RM. (E) KM survival curves comparing the HRG and LRG based on the developed RM in the training dataset. (F) Time-responsive ROC curves for the prognostic performance of the RM at different time points in the training dataset. (G) PCA plot comparing patient clustering based on the RM (left) versus clustering based on the mean expression of all LMRGs (right) in 85 patients with OS. OS, osteosarcoma; RM, risk model; LMRG, lipid metabolism-related gene; UCR, univariate Cox regression;…

Fig. 2. Prognostic validation of the lipid metabolism-related RM in OS patients. (A) KM survival curves comparing the HRG and LRG based on the median risk score in the training dataset. (B) KM survival curves comparing the HRG and LRG based on the median risk score in the validation dataset. (C) ROC curve analysis of the RM for predicting overall survival in the combined training and validation datasets. (D) Time-responsive ROC curves for the prognostic performance of the RM for 5YSR. (E) Heatmap (top) showing the expression distribution of prognostic factors in the HRG and LRG. Scatter plot (bottom) displaying the distribution of patient survival status and risk scores. (F) Nomogram integrating the prognostic RM and various clinical indicators for predicting 3YSRs and 5YSRs in OS patients. OS, osteosarcoma; RM, risk model; LMRG, lipid metabolism-related gene; ROC, receiver operating characteristic; AUC, area under the ROC curve; 5YSR, 5-year survival rate; 3YSR, 3-year survival rate; HRG, high-risk group; LRG, low-risk group.

Fig. 3. HIBCH regulates proliferation, colony formation, and apoptosis of OS cells. (A and B) Western blots (A) and quantification (B) of HIBCH levels in OS cell lines and normal cells. (C and D) Verification of the efficiency of HIBCH knockdown in U2OS cells (C) and HIBCH overexpression in U2R cells (D) by Western blot assays. (E) Cell viability assay results of OS cells in response to HIBCH knockdown and overexpression compared to their respective controls. (F) Representative images and quantification of colony formation assays of OS cells in response to HIBCH knockdown and overexpression. (G) Transwell migration and invasion assay results of OS cells in response to HIBCH knockdown and overexpression. (H) Western blot analysis of EMT-related markers (E-cadherin, N-cadherin, and vimentin) of OS cells in response to HIBCH knockdown and overexpression. (I) Flow cytometric analysis of apoptosis of OS cells in response to HIBCH knockdown and overexpression compared to their respective controls. (J) Western blot analysis of apoptosis-related proteins (cleaved-PARP and cleaved-CASP-9) of OS cells in response to HIBCH knockdown and overexpression. Data are presented as mean ± SD from three…

Fig. 4. HIBCH knockdown inhibits the TCA cycle and OXPHOS in OS cells. (A and B) Quantification of TCA cycle metabolites using UFLC-MS in U2OS or U2R cells with knockdown or overexpression of HIBCH. (C and D) Measurement of NAD+ (left) and NADH (right) levels using EnVision multimode plate reader in U2OS or U2R cells with knockdown or overexpression of HIBCH. (E and F) Assessment of cell viability using CCK-8 assay in U2OS and U2R cells treated with varying concentrations of HIBCH inhibitor SBF-1 for 24 and 48 h. (G and H) Quantification of TCA cycle metabolites using UFLC-MS in U2OS or U2R cells under indicated treatments. OS, osteosarcoma; TCA, tricarboxylic acid; OXPHOS, oxidative phosphorylation; HIBCH, 3-hydroxyisobutyryl-CoA hydrolase.

Fig. 5. Synergistic effects of HIBCH inhibitor SBF-1 and DOX on OS cells. (A) Dose-responsive curves of SBF-1 and DOX in U2OS and U2R cells after 48 h of treatment. IC50 values are indicated. (B) CI plots for SBF-1 and DOX in U2OS and U2R cells, as computed using the Chou-Talalay method. CI < 1 indicates synergism. (C and D) Representative images (C) and quantification (D) of colony formation assays in U2OS and U2R cells treated as indicated. (E and F) Representative images (E) and quantification (F) of Transwell migration and invasion assays in U2OS and U2R cells treated as indicated. (G and H) Western blots (G) and densitometric quantification (H) of the levels of EMT-related markers (E-cadherin, N-cadherin, and vimentin) in U2OS and U2R cells treated as indicated. Cells were treated with DMSO (control), SBF-1 (40 nM), DOX (120 nM), or their combination for 48 h in all experiments unless otherwise stated. Data in panels D, F, and H are presented as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control. OS, osteosarcoma; DOX, doxorubicin; CI, combination index; HIBCH, 3-hydroxyisobutyryl-CoA hydrolase.

Fig. 6. Combination of SBF-1 and DOX enhances apoptosis and inhibits the Akt-mTOR pathway in OS cells. Flow cytometry plots showing apoptosis of U2OS and U2R cells treated as indicated. (B) Quantification of apoptosis rates from (A). (C) Western blot analysis of apoptosis-related proteins (cleaved PARP and cleaved CASP-9) in U2OS and U2R cells treated as indicated. (D) Densitometric quantification of protein levels from (C), as normalized to β-actin. (E) Western blot analysis of Akt-mTOR pathway-related proteins (p-Akt, Akt, p-mTOR, mTOR) in U2OS and U2R cells treated as indicated. (F) Densitometric quantification of protein levels from (E), as normalized to total protein levels or β-actin as appropriate. Data in panels B, D, and F are presented as mean ± SD from three independent experiments. Cells were treated with DMSO (control), SBF-1 (40 nM), DOX (120 nM), or their combination for 48 h in all experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control. OS, osteosarcoma; DOX, doxorubicin; CASP-9, caspase-9.

Fig. 7. SBF-1 and DOX synergistically inhibit OS cell xenograft growth in vivo. (A) Changes in tumor volumes over time in immunodeficient mice bearing OS cell xenografts treated with control (DMSO), SBF-1 alone, DOX alone, or their combination. (B) Tumor weights at the endpoint of the experiment for each treatment group. (C) Tumor growth inhibition rates for SBF-1, DOX, and their combination, computed as the percentage reduction in tumor volume compared to the control group. OS, osteosarcoma; DOX, doxorubicin.