OBJECTIVE: To entrap carvacrol (CAR) in bovine serum albumin nanoparticles (BSANPs) to form CAR-loaded BSANPs (CAR@BSANPs) and to explore the anti-cancer effects in breast adenocarcinoma cells (MCF-7 cells) treated with CAR and CAR@BSANPs. METHODS: A desolvation method was used to synthesize BSANPs and CAR@BSANPs. The BSANPs and CAR@BSANPs were characterized by several physicochemical methods, including visual observation, high-resolution field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and high-performance liquid chromatography. MCF-7 cells were used and analyzed after 24 h of exposure to CAR and CAR@BSANPs at half-maximal inhibitory concentration. The anti-proliferative, apoptotic, reactive oxygen species (ROS), and nitric oxide (NO) scavenging activity as well as gene expression analysis were investigated by the cell viability assay, phase-contrast microscopy, 2',7'-dichlorofluorescein-diacetate assay, Griess-Illosvoy colorimetric assay, and quantitative real-time polymerase chain reaction, respectively. RESULTS: CAR and CAR@BSANPs showed anti-proliferative, apoptotic, ROS generation, and NO scavenging effects on MCF-7 cells. Expression profile of B-cell lymphoma 2-like 11 (BCL2L11), vascular endothelial growth factor A (VEGFA), hypoxia inducible factor factor-1α (HIF1A), BCL2L11/apoptosis regulator (BAX), and BCL2L11/Bcl2 homologous antagonist/killer 1 (BAK1) ratios revealed downregulated genes; and BAX, BAK1, and CASP8 were upregulated by CAR and CAR@BSANPs treatment. In vitro anticancer assays of the CAR and CAR@BSANPs showed that CAR@BSANPs demonstrated higher therapeutic efficacy in the MCF-7 cells than CAR. CONCLUSIONS CAR and CAR@BSANPs affect gene expression and may subsequently reduce the growth and proliferation of the MCF-7 cells. Molecular targeting of regulatory genes of the MCF-7 cells with CAR and CAR@BSANPs may be an effective therapeutic strategy against breast cancer.
Humans ; Cymenes ; Nanoparticles/ultrastructure* ; MCF-7 Cells ; Breast Neoplasms/genetics* ; Apoptosis/drug effects* ; Serum Albumin, Bovine/chemistry* ; Monoterpenes/therapeutic use* ; Adenocarcinoma/genetics* ; Cell Proliferation/drug effects* ; Reactive Oxygen Species/metabolism* ; Female ; Cell Survival/drug effects* ; Animals ; Gene Expression Regulation, Neoplastic/drug effects* ; Nitric Oxide/metabolism* ; Cattle

OBJECTIVES: To investigate the effect of downregulation of medium-chain acyl-coenzyme A dehydrogenase (ACADM) on invasion and migration of estrogen receptor-positive breast cancer cells and the underlying mechanism. METHODS: The Kaplan-Meier Plotter database was used to analyze the ACADM expression levels in breast cancer and normal tissues and their association with patient prognosis. Human breast cancer MCF-7 and T47D cell lines with lentivirus-mediated ACADM knockdown were established, and their in situ tumor formation and metastasis after tail vein injection were evaluated in nude mice. The MCF-7 and T47D cells with ACADM knockdown and their unmodified parental cells were examined with oil-red O staining assay, ROS assay, mitochondrial respiratory chain function assay before and after treatments with ROS scavenger, Elamipretide (a cardiolipin oxidation inhibitor) or SC79 (an AKT activator), and the changes in migration and invasion abilities of the treated cells were analyzed with Transwell invasion assay and Boyden chamber assay. Western blotting was used to detect protein expression levels of related signaling pathways in the treated cells. RESULTS: ACADM overexpression was associated with a significantly shorter overall survival of breast cancer patients. In MCF-7 and T47D cells, ACADM knockdown resulted in downregulation of N calnexin, vimentin, p-P13K and p-AKT proteins, increased levels of free fatty acids and reactive oxygen species, lowered activities of mitochondrial respiratory chain complex III and V, and reduced mitochondrial inner phospholipids. ACADM knockdown significantly decreased the invasive capacity of the cells, which were obviously reversed by treatment with ROS scavenger, Elamipretide, and SC79. CONCLUSIONS Down-regulation of ACADM inhibits migration and invasion ability of estrogen receptor-positive breast cancer cells by lowering lipotoxicity and impairing mitochondrial function through the ROS/PI3K/AKT pathway.
Humans ; Breast Neoplasms/metabolism* ; Female ; Mice, Nude ; Down-Regulation ; Neoplasm Invasiveness ; Animals ; Mice ; Receptors, Estrogen/metabolism* ; MCF-7 Cells ; Cell Movement ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism* ; Acyl-CoA Dehydrogenase/genetics* ; Signal Transduction ; Neoplasm Metastasis ; Proto-Oncogene Proteins c-akt/metabolism*

OBJECTIVES: To study the molecular mechanisms of LDH-loaded si-NEAT1 for regulating paclitaxel resistance and tumor-associated macrophage (TAM) polarization in breast cancer. METHODS: qRT-PCR and Western blotting were used to detect the expression of lncRNA NEAT1, miR-133b, and PD-L1 in breast cancer SKBR3 cells and paclitaxel-resistant SKBR3 cells (SKBR3-PR). The effects of transfection with si-NEAT1 and miR-133b mimics on MRP, MCRP and PD-L1 expressions and cell proliferation, migration and apoptosis were investigated using qRT-PCR, Western blotting, scratch and Transwell assays, and flow cytometry. Rescue experiments were conducted using si-NEAT1 and miR-133b inhibitor. Human THP-1 macrophages were cultured in the presence of conditioned media (CM) derived from SKBR3 and SKBR3-PR cells with or with si-NEAT1 transfection for comparison of IL-4-induced macrophage polarization by detecting the surface markers. LDH@si-NEAT1 nanocarriers were constructed, and their effects on MRP, MCRP and PD-L1 expressions and cell behaviors of the tumor cells were examined. THP-1 cells were treated with the CM from LDH@si-NEAT1-treated tumor cells, and the changes in their polarization were assessed. RESULTS: SKBR3-PR cells showered significantly upregulated NEAT1 and PD-L1 expressions and lowered miR-133b expression as compared with their parental cells. Transfection with si-NEAT1 and miR-133b mimics inhibited viability, promoted apoptosis and enhanced MRP and BCRP expressions in SKBR3-PR cells. NEAT1 knockdown obvious upregulated miR-133b and downregulated PD-L1, MRP and BCRP expressions. The CM from SKBR3-PR cells obviously promoted M2 polarization of THP-1 macrophages, which was significantly inhibited by CM from si-NEAT1-transfected cells. Treatment with LDH@si-NEAT1 effectively inhibited migration and invasion, promoted apoptosis, and reduced MRP, BCRP and PD-L1 expressions in the tumor cells. The CM from LDH@si-NEAT1-treated SKBR3-PR cells significantly downregulated Arg-1, CD163, IL-10, and PD-L1 and upregulated miR-133b expression in THP-1 macrophages. CONCLUSIONS LDH@si-NEAT1 reduces paclitaxel resistance of breast cancer cells and inhibits TAM polarization by targeting the miR-133b/PD-L1 axis.
Humans ; MicroRNAs/genetics* ; RNA, Long Noncoding/genetics* ; Paclitaxel/pharmacology* ; Breast Neoplasms/metabolism* ; Drug Resistance, Neoplasm ; B7-H1 Antigen/metabolism* ; Cell Line, Tumor ; Female ; Tumor-Associated Macrophages ; Apoptosis ; Cell Proliferation ; Macrophages ; Cell Movement

The inhibition of cyclin-dependent kinases (CDKs) is considered a promising strategy for cancer treatment due to their role in cell cycle regulation. However, CDK inhibitors with no selectivity among CDK families have not been approved. A CDK inhibitor with high selectivity for CDK4/6 exhibited significant treatment effects on breast cancer and has become a heavy bomb on the market. Subsequently, resistance gradually decreased the efficacy of selective CDK4/6 inhibitors in breast cancer treatment. In this review, we first introduce the development of selective CDK4/6 inhibitors and then explain the role of CDK2 activation in inducing resistance to CDK4/6 inhibitors. Moreover, we focused on the development of CDK2/4/6 inhibitors and selective CDK2 inhibitors, which will aid in the discovery of novel CDK inhibitors targeting the cell cycle in the future.
Humans ; Cell Cycle/drug effects* ; Protein Kinase Inhibitors/chemistry* ; Cyclin-Dependent Kinases/metabolism* ; Neoplasms/genetics* ; Antineoplastic Agents/pharmacology* ; Animals ; Breast Neoplasms/enzymology* ; Cyclin-Dependent Kinase 4/metabolism*

Triple-negative breast cancer (TNBC) represents an aggressive breast cancer subtype with poor prognosis and limited targeted treatment options. This investigation examined the anti-cancer potential of Caerulomycin A (Cae A), a natural compound derived from marine actinomycetes, against TNBC. Cae A demonstrated selective inhibition of viability and proliferation in TNBC cell lines, including 4T1, MDA-MB-231, and MDA-MB-468, through apoptosis induction. Mechanistic analyses revealed that the compound induced sustained endoplasmic reticulum (ER) stress and subsequent upregulation of C/EBP homologous protein (CHOP) expression, resulting in mitochondrial damage-mediated apoptosis. Inhibition of ER stress or CHOP expression knockdown reversed mitochondrial damage and apoptosis, highlighting the essential role of ER stress and CHOP in Cae A's anti-tumor mechanism. Both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) decreased in TNBC cells following Cae A treatment, indicating reduced mitochondrial respiratory and glycolytic capacities. This diminished energy metabolism potentially triggers ER stress and subsequent apoptosis. Furthermore, Cae A exhibited significant anti-tumor effects in the 4T1 tumor model in vivo without apparent toxicity. The compound also effectively inhibited human TNBC organoid growth. These results indicate that Cae A may serve as a potential therapeutic agent for TNBC, with its efficacy likely mediated through the disruption of glucose metabolism and the induction of ER stress-associated apoptosis.
Humans ; Endoplasmic Reticulum Stress/drug effects* ; Triple Negative Breast Neoplasms/genetics* ; Apoptosis/drug effects* ; Cell Line, Tumor ; Female ; Animals ; Glucose/metabolism* ; Mice ; Cell Proliferation/drug effects* ; Transcription Factor CHOP/genetics* ; Antineoplastic Agents/pharmacology* ; Mitochondria/metabolism* ; Mice, Inbred BALB C

The characteristic genetic abnormality of neuroendocrine neoplasms (NENs), a heterogeneous group of tumors found in various organs, remains to be identified. Here, based on the analysis of the splicing variants of an oncogene Focal Adhesion Kinase (FAK) in The Cancer Genome Atlas datasets that contain 9193 patients of 33 cancer subtypes, we found that Box 6/Box 7-containing FAK variants (FAK^6/7) were observed in 7 (87.5%) of 8 pancreatic neuroendocrine carcinomas and 20 (11.76%) of 170 pancreatic ductal adenocarcinomas (PDACs). We tested FAK variants in 157 tumor samples collected from Chinese patients with pancreatic tumors, and found that FAK^6/7 was positive in 34 (75.6%) of 45 pancreatic NENs, 19 (47.5%) of 40 pancreatic solid pseudopapillary neoplasms, and 2 (2.9%) of 69 PDACs. We further tested FAK splicing variants in breast neuroendocrine carcinoma (BrNECs), and found that FAK^6/7 was positive in 14 (93.3%) of 15 BrNECs but 0 in 23 non-NEC breast cancers. We explored the underlying mechanisms and found that a splicing factor serine/arginine repetitive matrix protein 4 (SRRM4) was overexpressed in FAK^6/7-positive pancreatic tumors and breast tumors, which promoted the formation of FAK^6/7 in cells. These results suggested that FAK^6/7 could be a biomarker of NENs and represent a potential therapeutic target for these orphan diseases.
Female ; Humans ; Alternative Splicing ; Breast Neoplasms/metabolism* ; Carcinoma, Pancreatic Ductal/pathology* ; Focal Adhesion Protein-Tyrosine Kinases/therapeutic use* ; Nerve Tissue Proteins/genetics* ; Neuroendocrine Tumors/genetics* ; Oncogenes ; Pancreatic Neoplasms/metabolism*

Humans ; Female ; Breast Neoplasms/genetics* ; Prognosis ; Gene Expression Profiling ; Biomarkers, Tumor/genetics* ; Gene Expression Regulation, Neoplastic/genetics* ; Receptor, ErbB-2/metabolism* ; Gene Regulatory Networks/genetics*

Objective: PD-1/PD-L1 immune checkpoint treatment is effective for some triple-negative breast cancer populations with PD-L1 expression, but the response rate is still not satisfactory. This study aims to explore the mechanism of drug resistance to breast cancer anti-PD-1 therapies and the strategies for overcoming the resistance to PD-1therapies. Methods: By constructing a human triple-negative breast cancer drug-resistant cell line called BT-549R5 and a mouse breast cancer drug-resistant cell line called 4T1R3, and applying the whole-gene shRNA library screening, candidate drug resistance-associated molecules were obtained and verified by cytological experiments. The expression of Tyro3, Axl and MerTK of the TAM family in the 4T1R3 group was tested using the Western blot method. The down-regulation of CDK9 on the effect of T cells killing the BT-549R5 cells was observed through T cell killing tests, while the down-regulation of Tyro3 and CDK9 on the effect of anti-PD-1 therapies for transplanted breast tumors was observed in mouse tumor formation experiments. Results: The cell lines and animal models of breast cancer resistant to PD-1 treatment were successfully constructed. Tyro3, Axl and MerTK were highly expressed in 4T1R3 cells. Whole genome sequencing showed that Tyro3 and CDK9 were highly expressed in BT-549R5 cells. T cell killing experiment showed that the survival rate of BT-549R5 cells in the CDK9 down-regulated group and the control group decreased gradually with the increase of T cells, but the survival rate of BT-549R5 cells in the CDK9 down-regulated group decreased rapidly. Tumor formation experiment in mice showed that under anti-PD-1 treatment, the transplanted tumor in the 4T1R3 cell group grew rapidly compared with the 4T1 cell group (P<0.05), and the tumor volume of the 4T1R3 group was larger than that of the 4T1 group on Day 20. Nevertheless, the tumor growth rates in the CDK9-knockdown 4T1R3 cell group and the Tyro3-knockdown 4T1R3 cell group were similar to that of the 4T1 cell group, and the tumor volumes at day 20 were signiference lower than that of 4T1R3 cell group(P<0.05). Conclusions: Tyro3 and CDK9 are associated with the drug resistance to anti-PD-1 therapies for breast cancer. Inhibiting the expression of Tyro3 and CDK9 can reverse the drug resistance to breast cancer treatment.
Humans ; Animals ; Mice ; c-Mer Tyrosine Kinase/metabolism* ; Receptor Protein-Tyrosine Kinases/genetics* ; Axl Receptor Tyrosine Kinase ; Proto-Oncogene Proteins/metabolism* ; B7-H1 Antigen/genetics* ; Triple Negative Breast Neoplasms/genetics* ; Drug Resistance, Neoplasm ; Biomarkers ; Cell Line, Tumor ; Cyclin-Dependent Kinase 9

Objective: PD-1/PD-L1 immune checkpoint treatment is effective for some triple-negative breast cancer populations with PD-L1 expression, but the response rate is still not satisfactory. This study aims to explore the mechanism of drug resistance to breast cancer anti-PD-1 therapies and the strategies for overcoming the resistance to PD-1therapies. Methods: By constructing a human triple-negative breast cancer drug-resistant cell line called BT-549R5 and a mouse breast cancer drug-resistant cell line called 4T1R3, and applying the whole-gene shRNA library screening, candidate drug resistance-associated molecules were obtained and verified by cytological experiments. The expression of Tyro3, Axl and MerTK of the TAM family in the 4T1R3 group was tested using the Western blot method. The down-regulation of CDK9 on the effect of T cells killing the BT-549R5 cells was observed through T cell killing tests, while the down-regulation of Tyro3 and CDK9 on the effect of anti-PD-1 therapies for transplanted breast tumors was observed in mouse tumor formation experiments. Results: The cell lines and animal models of breast cancer resistant to PD-1 treatment were successfully constructed. Tyro3, Axl and MerTK were highly expressed in 4T1R3 cells. Whole genome sequencing showed that Tyro3 and CDK9 were highly expressed in BT-549R5 cells. T cell killing experiment showed that the survival rate of BT-549R5 cells in the CDK9 down-regulated group and the control group decreased gradually with the increase of T cells, but the survival rate of BT-549R5 cells in the CDK9 down-regulated group decreased rapidly. Tumor formation experiment in mice showed that under anti-PD-1 treatment, the transplanted tumor in the 4T1R3 cell group grew rapidly compared with the 4T1 cell group (P<0.05), and the tumor volume of the 4T1R3 group was larger than that of the 4T1 group on Day 20. Nevertheless, the tumor growth rates in the CDK9-knockdown 4T1R3 cell group and the Tyro3-knockdown 4T1R3 cell group were similar to that of the 4T1 cell group, and the tumor volumes at day 20 were signiference lower than that of 4T1R3 cell group(P<0.05). Conclusions: Tyro3 and CDK9 are associated with the drug resistance to anti-PD-1 therapies for breast cancer. Inhibiting the expression of Tyro3 and CDK9 can reverse the drug resistance to breast cancer treatment.
Humans ; Animals ; Mice ; c-Mer Tyrosine Kinase/metabolism* ; Receptor Protein-Tyrosine Kinases/genetics* ; Axl Receptor Tyrosine Kinase ; Proto-Oncogene Proteins/metabolism* ; B7-H1 Antigen/genetics* ; Triple Negative Breast Neoplasms/genetics* ; Drug Resistance, Neoplasm ; Biomarkers ; Cell Line, Tumor ; Cyclin-Dependent Kinase 9

OBJECTIVE: To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells. METHODS: Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated. RESULTS: Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing. CONCLUSION RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.
Humans ; rab GTP-Binding Proteins/metabolism* ; Triple Negative Breast Neoplasms ; Cell Line, Tumor ; rab27 GTP-Binding Proteins/metabolism* ; RNA, Small Interfering/genetics* ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic