BACKGROUND:Gambogic acid is highly cytotoxic to breast cancer and can effectively kill triple negative breast cancer stem cells,but the underlying mechanism is still unclear.OBJECTIVE:To investigate the lethal effect of gambogic acid on triple negative breast cancer stem cells as well as the possible mechanisms.METHODS:PharmMapper database was used to predict the target protein of gambogic acid.String website was used to construct the protein interaction network of various drug targets.Active ingredient-target network was constructed by Cytoscape software.KEGG signal pathway enrichment analysis was performed on potential targets by R language software.The effect of different concentrations of gambogic acid on the activity of human breast cancer cell line MDA-MB-231 was detected by CCK-8 assay.The appropriate concentration was screened.MDA-MB-231 stem cells were enriched by cell ball culture method and treated with gambogic acid at different concentrations(0,0.5,1.0,and 2.0 μmol/L)for 24 hours.TUNEL fluorescence staining and flow cytometry were used to detect apoptosis of stem cells.qPCR and western blot assay were used to detect protein C receptor expression.The expression levels of p-PI3K,p-AKT,Caspase-3,and cleaved Caspase-3 were detected by western blot assay.Stem cells were cultured in four groups:Blank control group(stem cells were not treated),siRNA-NC group,siRNA-protein C receptor group,and siRNA-protein C receptor+PI3K agonist group.After culture for 36 hours,the expression levels of p-PI3K,p-AKT,Caspase-3,and cleaved Caspase-3 were detected by western blot assay.RESULTS AND CONCLUSION:(1)Network pharmacology exhibited that the protein C receptor,a marker of triple negative breast cancer stem cells,was one of the targets of gambogic acid.KEGG enrichment analysis involved apoptosis,epithelial growth factor receptor,RAS,and PI3K-AKT signaling pathways.(2)CCK-8 assay results showed that gambogic acid could inhibit the viability of MDA-MB-231 cells,and the median inhibitory concentration IC50 value was(1.18±0.34)μmol/L,so the concentrations of 0.5,1.0,and 2.0 μmol/L were selected for subsequent experiments.(3)TUNEL fluorescence staining and flow cytometry showed that gambogic acid induced apoptosis of triple negative breast cancer stem cells in a dose-dependent manner(P<0.05).qPCR and western blot assay confirmed that gambogic acid down-regulated mRNA and protein expression of protein C receptor,down-regulated Caspase-3,p-PI3K,and p-Akt protein expression,and up-regulated cleaved Caspase-3 protein expression(P<0.05).siRNA-protein C receptor transfection experiments further confirmed that knockdown of protein C receptor expression in triple negative breast cancer stem cells increased cleaved Caspase-3 protein expression(P<0.05),and down-regulated phosphorylation of PI3K/AKT signaling pathway(P<0.05).Application of PI3K agonist 740 Y-P decreased cleaved Caspase-3 protein expression(P<0.05),increased phosphorylation levels of p-PI3K and p-AKT(P<0.05),and improved apoptosis to a certain extent.(4)The results show that gambogic acid may play a role in killing and inducing apoptosis of triple negative breast cancer stem cells by down-regulating protein C receptor,and the further molecular mechanism may be related to the inhibition of PI3K/AKT signaling pathway.

Objective:To conduct enrichment and biological behavior studies on CD44 + CD24 - triple-negative breast cancer (TNBC) stem-like cells, and to construct 68Ga-labeled CD44 peptide ( 68Ga-1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA)-CD44p) and evaluate its targeting ability towards the surface marker CD44 of TNBC stem-like cells. Methods:Suspension sphere culture method was utilized to enrich and cultivate CD44 + CD24 - cell subpopulations from TNBC cell line MDA-MB-231 and non-TNBC cell line MCF-7. Flow cytometry was used to detect the expression of stem cell markers of different groups, cell scratch assay was performed to assess the migration ability of CD44 + CD24 - cell subpopulations, and Transwell invasion assay was performed to evaluate the invasion ability of CD44 + CD24 - cell subpopulations. 68Ga-NOTA-CD44p was prepared, followed by purification and identification with high-performance liquid chromatography (HPLC). The targeting ability of 68Ga-NOTA-CD44p towards CD44 + TNBC cells was evaluated through cellular uptake and blocking experiments. Data were analyzed by independent-sample t test, one-way analysis of variance and the least significant difference t test. Results:Suspension sphere culture successfully enriched CD44 + CD24 - TNBC stem-like cell spheres. Compared to the non-TNBC cell line MCF-7, TNBC cell line MDA-MB-231 exhibited better sphere-forming ability (18.50±3.73 vs 31.83±4.92; t=5.29, P<0.001) and a higher proportion of CD44 + CD24 - cell subset ((24.97±8.12)% vs (90.93±4.46)%; F=170.10, t=14.93, both P<0.001). The wound healing rate ((71.00±11.00)% vs (28.33±4.16)%; F=42.91, t=8.02, both P<0.001) and invasion rate ((60.60±16.87)% vs (24.16±8.15)%; F=11.83, t=4.40, both P<0.01) of CD44 + CD24 - MDA-MB-231 group cells were significantly increased compared to the CD44 + CD24 - MCF-7 group. MDA-MB-231 cells showed strong uptake ability of 68Ga-NOTA-CD44p, which decreased after CD44p blocking. Conclusions:Compared to CD44 + CD24 - MCF-7 cells, CD44 + CD24 - MDA-MB-231 cells exhibit higher malignant biological behavior. 68Ga-NOTA-CD44p targets the surface marker CD44 of TNBC stem-like cells, laying the research foundation for targeted therapy against TNBC with tumor stem cells as targets.

BACKGROUND:Gambogic acid is highly cytotoxic to breast cancer and can effectively kill triple negative breast cancer stem cells,but the underlying mechanism is still unclear.OBJECTIVE:To investigate the lethal effect of gambogic acid on triple negative breast cancer stem cells as well as the possible mechanisms.METHODS:PharmMapper database was used to predict the target protein of gambogic acid.String website was used to construct the protein interaction network of various drug targets.Active ingredient-target network was constructed by Cytoscape software.KEGG signal pathway enrichment analysis was performed on potential targets by R language software.The effect of different concentrations of gambogic acid on the activity of human breast cancer cell line MDA-MB-231 was detected by CCK-8 assay.The appropriate concentration was screened.MDA-MB-231 stem cells were enriched by cell ball culture method and treated with gambogic acid at different concentrations(0,0.5,1.0,and 2.0 μmol/L)for 24 hours.TUNEL fluorescence staining and flow cytometry were used to detect apoptosis of stem cells.qPCR and western blot assay were used to detect protein C receptor expression.The expression levels of p-PI3K,p-AKT,Caspase-3,and cleaved Caspase-3 were detected by western blot assay.Stem cells were cultured in four groups:Blank control group(stem cells were not treated),siRNA-NC group,siRNA-protein C receptor group,and siRNA-protein C receptor+PI3K agonist group.After culture for 36 hours,the expression levels of p-PI3K,p-AKT,Caspase-3,and cleaved Caspase-3 were detected by western blot assay.RESULTS AND CONCLUSION:(1)Network pharmacology exhibited that the protein C receptor,a marker of triple negative breast cancer stem cells,was one of the targets of gambogic acid.KEGG enrichment analysis involved apoptosis,epithelial growth factor receptor,RAS,and PI3K-AKT signaling pathways.(2)CCK-8 assay results showed that gambogic acid could inhibit the viability of MDA-MB-231 cells,and the median inhibitory concentration IC50 value was(1.18±0.34)μmol/L,so the concentrations of 0.5,1.0,and 2.0 μmol/L were selected for subsequent experiments.(3)TUNEL fluorescence staining and flow cytometry showed that gambogic acid induced apoptosis of triple negative breast cancer stem cells in a dose-dependent manner(P<0.05).qPCR and western blot assay confirmed that gambogic acid down-regulated mRNA and protein expression of protein C receptor,down-regulated Caspase-3,p-PI3K,and p-Akt protein expression,and up-regulated cleaved Caspase-3 protein expression(P<0.05).siRNA-protein C receptor transfection experiments further confirmed that knockdown of protein C receptor expression in triple negative breast cancer stem cells increased cleaved Caspase-3 protein expression(P<0.05),and down-regulated phosphorylation of PI3K/AKT signaling pathway(P<0.05).Application of PI3K agonist 740 Y-P decreased cleaved Caspase-3 protein expression(P<0.05),increased phosphorylation levels of p-PI3K and p-AKT(P<0.05),and improved apoptosis to a certain extent.(4)The results show that gambogic acid may play a role in killing and inducing apoptosis of triple negative breast cancer stem cells by down-regulating protein C receptor,and the further molecular mechanism may be related to the inhibition of PI3K/AKT signaling pathway.

Objective:To conduct enrichment and biological behavior studies on CD44 + CD24 - triple-negative breast cancer (TNBC) stem-like cells, and to construct 68Ga-labeled CD44 peptide ( 68Ga-1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA)-CD44p) and evaluate its targeting ability towards the surface marker CD44 of TNBC stem-like cells. Methods:Suspension sphere culture method was utilized to enrich and cultivate CD44 + CD24 - cell subpopulations from TNBC cell line MDA-MB-231 and non-TNBC cell line MCF-7. Flow cytometry was used to detect the expression of stem cell markers of different groups, cell scratch assay was performed to assess the migration ability of CD44 + CD24 - cell subpopulations, and Transwell invasion assay was performed to evaluate the invasion ability of CD44 + CD24 - cell subpopulations. 68Ga-NOTA-CD44p was prepared, followed by purification and identification with high-performance liquid chromatography (HPLC). The targeting ability of 68Ga-NOTA-CD44p towards CD44 + TNBC cells was evaluated through cellular uptake and blocking experiments. Data were analyzed by independent-sample t test, one-way analysis of variance and the least significant difference t test. Results:Suspension sphere culture successfully enriched CD44 + CD24 - TNBC stem-like cell spheres. Compared to the non-TNBC cell line MCF-7, TNBC cell line MDA-MB-231 exhibited better sphere-forming ability (18.50±3.73 vs 31.83±4.92; t=5.29, P<0.001) and a higher proportion of CD44 + CD24 - cell subset ((24.97±8.12)% vs (90.93±4.46)%; F=170.10, t=14.93, both P<0.001). The wound healing rate ((71.00±11.00)% vs (28.33±4.16)%; F=42.91, t=8.02, both P<0.001) and invasion rate ((60.60±16.87)% vs (24.16±8.15)%; F=11.83, t=4.40, both P<0.01) of CD44 + CD24 - MDA-MB-231 group cells were significantly increased compared to the CD44 + CD24 - MCF-7 group. MDA-MB-231 cells showed strong uptake ability of 68Ga-NOTA-CD44p, which decreased after CD44p blocking. Conclusions:Compared to CD44 + CD24 - MCF-7 cells, CD44 + CD24 - MDA-MB-231 cells exhibit higher malignant biological behavior. 68Ga-NOTA-CD44p targets the surface marker CD44 of TNBC stem-like cells, laying the research foundation for targeted therapy against TNBC with tumor stem cells as targets.

Objective:To investigate the effects and mechanism of gut metabolite sodium butyrate on the proliferation and apoptosis of steatosis HepG2 cells in vitro. Methods:The in vitro steatosis hepatocyte model was established with human liver cell line HepG2 and free fatty acid (FFA; the concentration ratio of oleic acid to palmitic acid was 2∶1). Normal control group, model group and intervention groups with different concentration (1, 2, 5, 10, 20 and 50 mmol/L) of sodium butyrate were set up. The inhibition of sodium butyrate on the proliferation of steatosis HepG2 cells was detected by cell counting kit (CCK-8). The proportion of apoptotic cells of normal control group, model group and sodium butyrate 5 mmol/L (sodium butyrate intervention) group was detected by flow cytometry. Normal control group, model group, intervention group with different concentration (1, 2, 5 and 10 mmol/L) of sodium butyrate, negative small interfering RNA (siRNA) control group, G protein-coupled receptor (GPR) 43-siRNA group, GPR109a-siRNA group, GPR43+ GPR109 a double knockout group were set up. The change of the levels of phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR) before and after transfection were detected by Western blotting. One-way analysis of varivance, SNK- q test and logistic regression analysis were used for statistical analysis. Results:The results of CCK-8 test indicated that sodium butyrate inhibited the proliferation of steatosis HepG2 cells in a dose-dependent and time-dependent manner. The results of flow cytometry showed that the proportion of apoptotic cells of the sodium butyrate intervention group was higher than that of the model group and normal control group ((3.400±0.100)% vs. (1.800±0.400)% and(1.067±0.451)%), and the differences were statistically significant ( t=6.721 and 8.705, both P<0.01). There was no significant difference in the proportion of apoptotic cells between the model group and the normal control group ( P>0.05). Before transfection, the expressions of p-AKT and p-mTOR at protein level of the model group were both higher than those of the normal control group (2.300±0.058 vs. 1.000±0.012, 2.160±0.125 vs. 1.000±0.052), and the differences were statistically significant ( t=22.080 and 8.575, both P<0.05). The expressions of p-AKT and p-mTOR at protein level of sodium butyrate intervention groups at 1, 2, 5 and 10 mmol/L were all lower than those of the model group (1.530±0.085, 1.407±0.096, 1.032±0.035 and 1.036±0.099 vs. 2.300±0.058; 1.483±0.073, 1.297±0.048, 1.067±0.035 and 0.970±0.072 vs. 2.160±0.125), and the differences were statistically significant ( t=7.491, 7.997, 19.790, and 11.020; 4.683, 6.445, 8.424, and 8.245; all P<0.05). After transfection, the expressions of p-AKT and p-mTOR at protein level of GPR43-siRNA group, GPR109a-siRNA group and GPR43/ GPR109 a double knockout group were all higher than those of the negative siRNA control group and 5 mmol/L sodium butyrate group (1.474±0.045, 1.471±0.058 and 2.067±0.120 vs. 1.158±0.030 and 1.139±0.031; 1.850±0.082, 1.683±0.058 and 2.160±0.091 vs. 1.469±0.037 and 1.490±0.116), and the differences were statistically significant ( tp-AKT=5.807, 4.816, 7.322, 6.109, 5.080 and 7.463; tp-mTOR=4.235, 3.113, 7.044, 2.542, 1.497 and 4.562; all P<0.05). Conclusions:The effect of sodium butyrate on the proliferation and apoptosis of steatosis HepG2 cells is associated with the GPR43/GPR109a-pAKT-mTOR signaling pathway.