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.

Objectives:To investigate the enhancement of tumor penetration and photodynamic therapy (PDT) efficacy in triple-negative breast cancer by hyaluronidase (HAase) using a novel pH-responsive IR780-loaded photosensitive micelle.Methods:The pH-responsive IR780-loaded photosensitive micelles were prepared using the nanoprecipitation method, and their morphology, size, and encapsulation efficiency were characterized. The in vitro stability and pH-responsive drug release of the micelles were also evaluated. The cytotoxicity of the micelles on triple-negative breast cancer cells (MDA-MB-231) was assessed using a cell counting kit. A nude mouse breast cancer model was established, and HAase was injected intratumorally 24 hours before intravenous injection of the photosensitive micelles. The effect of HAase on the biodistribution and tumor uptake of the micelles was detected using small animal in vivo imaging. CD31 and HIF-1α immunofluorescence staining were performed to investigate the mechanism of HAase-enhanced tumor penetration. The body weight and tumor volume of the mice were measured, and necrosis and apoptosis of tumor tissues were assessed using HE staining and TUNEL staining, respectively. Results:Transmission electron microscopy showed that the micelles had a uniform particle size of approximately 60-70 nm, with a hydrated particle size of (98.03±0.22) nm. The IR780 encapsulation efficiency was 74.15%, with a drug loading content of 2.07%. After 7 days at 4 ℃, there was no significant change in hydrated particle size ( P=0.062). The 24-hour release rates of the micelles in PBS at pH 7.4 and 6.5 were (2.41±0.21)% and (43.69±2.09)%, respectively, showing a significant difference ( P＜0.000 1). The cytotoxicity assay revealed that the cell viability in the micelles group without light exposure was significantly higer than that in the micelles group under light exposure [(97.00±5.38)% vs. (53.27±9.00)%, P=0.000 2]. The micelles were able to target and accumulate in the tumor tissue, and this accumulation increased significantly with HAase treatment. CD31 and HIF-1α immunofluorescence staining indicated that the CD31 signal was enhanced [(0.27±0.05)% vs. (4.57±0.27)%, P＜0.000 1] and the HIF-1α signal was reduced [(5.14±0.38)% vs. (0.08±0.04)%, P＜0.000 1] in the HAase-treated group compared to that in the micelle-only group. After 11 days of treatment with HAase combined with photosensitive micelles, there was no statistically significant difference in mouse body weight ( P＞0.05). However, the tumor volume inhibition rate in the HAase-micelle-mediated PDT group was significantly higher than that in the micelle-mediated PDT group [(87.66±6.37)% vs. (25.34±12.63)%, P=0.002]. Histological staining showed a significant increase in tumor cell necrosis and apoptosis in the HAase-micelle-mediated PDT group. Conclusion:HAase enhances the deep tumor penetration and targeted accumulation of pH-responsive IR780-loaded photosensitive micelles, significantly improves the efficacy of photodynamic therapy in triple-negative breast cancer.

Objectives:To investigate the enhancement of tumor penetration and photodynamic therapy (PDT) efficacy in triple-negative breast cancer by hyaluronidase (HAase) using a novel pH-responsive IR780-loaded photosensitive micelle.Methods:The pH-responsive IR780-loaded photosensitive micelles were prepared using the nanoprecipitation method, and their morphology, size, and encapsulation efficiency were characterized. The in vitro stability and pH-responsive drug release of the micelles were also evaluated. The cytotoxicity of the micelles on triple-negative breast cancer cells (MDA-MB-231) was assessed using a cell counting kit. A nude mouse breast cancer model was established, and HAase was injected intratumorally 24 hours before intravenous injection of the photosensitive micelles. The effect of HAase on the biodistribution and tumor uptake of the micelles was detected using small animal in vivo imaging. CD31 and HIF-1α immunofluorescence staining were performed to investigate the mechanism of HAase-enhanced tumor penetration. The body weight and tumor volume of the mice were measured, and necrosis and apoptosis of tumor tissues were assessed using HE staining and TUNEL staining, respectively. Results:Transmission electron microscopy showed that the micelles had a uniform particle size of approximately 60-70 nm, with a hydrated particle size of (98.03±0.22) nm. The IR780 encapsulation efficiency was 74.15%, with a drug loading content of 2.07%. After 7 days at 4 ℃, there was no significant change in hydrated particle size ( P=0.062). The 24-hour release rates of the micelles in PBS at pH 7.4 and 6.5 were (2.41±0.21)% and (43.69±2.09)%, respectively, showing a significant difference ( P＜0.000 1). The cytotoxicity assay revealed that the cell viability in the micelles group without light exposure was significantly higer than that in the micelles group under light exposure [(97.00±5.38)% vs. (53.27±9.00)%, P=0.000 2]. The micelles were able to target and accumulate in the tumor tissue, and this accumulation increased significantly with HAase treatment. CD31 and HIF-1α immunofluorescence staining indicated that the CD31 signal was enhanced [(0.27±0.05)% vs. (4.57±0.27)%, P＜0.000 1] and the HIF-1α signal was reduced [(5.14±0.38)% vs. (0.08±0.04)%, P＜0.000 1] in the HAase-treated group compared to that in the micelle-only group. After 11 days of treatment with HAase combined with photosensitive micelles, there was no statistically significant difference in mouse body weight ( P＞0.05). However, the tumor volume inhibition rate in the HAase-micelle-mediated PDT group was significantly higher than that in the micelle-mediated PDT group [(87.66±6.37)% vs. (25.34±12.63)%, P=0.002]. Histological staining showed a significant increase in tumor cell necrosis and apoptosis in the HAase-micelle-mediated PDT group. Conclusion:HAase enhances the deep tumor penetration and targeted accumulation of pH-responsive IR780-loaded photosensitive micelles, significantly improves the efficacy of photodynamic therapy in triple-negative breast cancer.

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 explore the effects of low centrifugal force on sperm morphology analysis of normal and non-liquefied semen samples.Methods A total of 169 patients who underwent semen routine examination and sperm morphology analysis at the Reproduc-tive Center of the 904th Hospital of the Joint Logistics Support Force of PLA from January 2021 to December 2021 were collected as the study subjects.Among them,126 were the patients with normal semen routine parameters,and 43 with non-liquefied semen.The mor-phological analysis results of 126 normal semen samples treated by the direct smear method and centrifugation methods under two differ-ent of low centrifugal force were compared.The results of morphological analysis for the 43 non-liquefied semen samples treated by the direct smear method after promoting liquefaction and centrifugation were compared.Results The percentage of normal morphological sperm[(9.39±0.50)％]obtained by centrifugation of 340xg centrifugal force for 10 min was significantly lower than that[(11.08± 0.41)％]obtained by the direct smear method,and the abnormal rate of sperm in the middle segment[(62.05±1.44)％]obtained by centrifugation of 340xg centrifugal force was significantly higher than that[(57.17±0.82)％]obtained by the direct smear method.The percentage of normal morphological sperm[(9.41±0.57)％]obtained by centrifugation at 151×g centrifugal force was significantly lower than that obtained by the direct smear method,and the abnormal rate of sperm in the middle segment[(60.95±1.17)％]ob-tained by centrifugation at 151×g centrifugal force was significantly higher than that obtained by the direct smear method.For non-lique-fied semen samples,the percentage of normal morphological sperm obtained by centrifugation at 340xg centrifugal force for 10 min was[(9.61±0.60)％],while that obtained by the direct smear method after promoting liquefaction was[(11.10±0.73)％].The difference was not statistically significant between the two methods.The abnormal rate of sperm in the middle segment[(60.21±1.51)％]ob-tained by the centrifugation method was also not significantly different from that[(57.36±1.34)％]obtained by the direct smear meth-od after promoting liquefaction.Conclusion Both kinds of low centrifugal force affected the sperm morphology.However,the low cen-trifugal force has no significant effect on the non-liquefied semen.In the clinical application of sperm morphology analysis,the nature of male semen should be referred to determine whether the centrifugation is need or not and the magnitude of centrifugal force.

Objective To synthesize 18F-AlF-1,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA)-c (CGRRAGGSC),which could specifically bind to the α chain of interleukin (IL)-11 receptor (IL-11 R),and evaluate its targeting potential to IL-11 R-positive tumors.Methods Polypeptide c (CGRRAGGSC) was first coupled with NOTA and then labeled with 18F by AlF labeling method.The radiochemical purity and radiochemical yield of 18F-AlF-NOTA-c(CGRRAGGSC) were analyzed by high performance liquid chromatography,and the stability in vitro was evaluated.The tracer biodistribution in tumor-bearing mice (cell line SKOV3) was evaluated by the dynamic imaging with microPET 30 min,1 h,2 h after injection of 18F-AlF-NOTA-c (CGRRAGGSC).The tracer kinetics was performed in normal mice.Pharmacokinetics parameters were calculated using DAS2.0 software.Results The radiochemical purity of 18F-AlF-NOTA-c(CGRRAGGSC) was higher than 95％ and the radiochemical yield was (30.0±7.4)％.It could be stably maintained in phosphatebuffered solution and plasma for at least 2 h.MicroPET imaging showed that 18F-AlF-NOTA-c(CGRRAGGSC)had a good affinity to SKOV3 tumor.The tumor/muscle ratios at 30 min,1 h,2 h after the injection of 18F-AlF-NOTA-c(CGRRAGGSC) were 6.26±2.98,7.19±3.63 and 9.05±4.30,respectively.The tracer was cleared rapidly in blood and mainly excreted by the liver and kidneys.The T1/2α and T1/2β were (0.38±0.14) h and (2.64±0.28) h,respectively.Conclusions 18F-AlF-NOTA-c(CGRRAGGSC) is easy to be synthesized and has a good affinity to IL-11R-positive tumors.It will be a potential IL-11R-targeting imaging agent.

Objective To prepare 188 Re-c ( CGRRAGGSC) by different labeling methods, and compare the stability and targeting ability for triple-negative breast cancer (TNBC). Methods 188 Re la-beled c( CGRRAGGSC) was prepared by direct method ( pre-tinning method) and indirect method (the hydrazinonicotinamide (HYNIC) conjugated c(CGRRAGGSC) peptide) respectively. Quality control and sta-bilities of radiolabeled probes were measured. Nude mice bearing TNBC cells (MDA-MB-468) were estab-lished and used for detecting the distribution in vivo. Tumor/ non-tumor (T/ NT) ratios of radiolabeled probes prepared by direct method and indirect method were compared to certify the targeting ability and biological activi-ty. Two-sample t test was used to analyze the data. Results The labeling rate of 188Re-c(CGRRAGGSC) by di-rect method was >87％, but the serum stability was poor (the degradation rate was about 40％ at 2 h), and the T/ NT ratio was 2.82±0.23 (n=3) at 2 h. On the other hand, the labeling rate of 188Re-HYNIC-c(CGRRAGGSC) by indirect method was 61％, while the serum stability was about 10％ at 2 h, and T/ NT ratio was about 6. 27±0.51 (n= 3) at 2 h, which was significantly higher (t = 2.13, P<0.05). Conclusions The direct method may have higher labeling rate than the indirect method, but the radiolabeled compound by the direct method has poor stability and tumor targeting ability. 188 Re-HYNIC-c(CGRRAGGSC) prepared by indirect method may have remarkable advantages on stability and biological activity.