BT474 Cells Research Articles

KLF14/miR-1283/TFAP2C axis inhibits HER2-positive breast cancer progression via declining tumor cell proliferation.

MiR-1283 has been identified as a tumor suppressor in some malignancies. Whereas, the role of miR-1283 in HER2-positive (HER2+) breast cancer, particularly its role in regulating cell proliferation, one of the most significant features of tumor progression, is unclear. The related microRNA screened by the breast cancer sample GSE131599 dataset were detected in HER2+ breast cancer tissues and cell lines. Then, the obtained miR-1283 was overexpressed in SKBR3 and BT-474 cells followed by relevant functional assays concerning cell proliferation and apoptosis. The xenograft mouse model was induced and the effect of miR-1283 on tumor growth and cell proliferation was examined. The target of miR-1283 and the transcription factor regulating miR-1283 were predicted and identified. Finally, the influence of transcription factor KLF14 on cell proliferation and apoptosis was investigated. An integrated analysis confirmed that miR-1283 expression was significantly decreased in HER2+ breast cancer tissues. Also, by q-RT-PCR detection, miR-1283 expression was markedly reduced in HER2+ breast cancer tissues and cell lines. The miR-1283 overexpression prevented the proliferation and enhanced apoptosis of HER2+ breast cancer cells, as well as inhibited tumor growth. Mechanistically, miR-1283 inhibited TFAP2C expression by targeting the 3'-untranslated regions of TFAP2C messenger RNA, and the KLF14 enhanced miR-1283 level via binding to its promoter. The result subsequently confirmed the KLF14/miR-1283 signaling suppressed cell proliferation in HER2+ breast cancer. Our results suggested that the KLF14/miR-1283/TFAP2C axis inhibited HER2+ breast cancer progression, which might provide novel insight into mechanical exploration for this disease.

Shallow- and Deep-Water Ophiura Species Produce a Panel of Chlorin Compounds with Potent Photodynamic Anticancer Activities.

A Pacific brittle star Ophiura sarsii has previously been shown to produce a chlorin (3S,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (ETPA) (1) with potent phototoxic activities, making it applicable to photodynamic therapy. Using extensive LC-MS metabolite profiling, molecular network analysis, and targeted isolation with de novo NMR structure elucidation, we herein identify five additional chlorin compounds from O. sarsii and its deep-sea relative O. ooplax: 10S-Hydroxypheophorbide a (2), Pheophorbide a (3), Pyropheophorbide a (4), (3S,4S,21R)-14-Ethyl-9-(hydroxymethyl)-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (5), and (3S,4S,21R)-14-Ethyl-21-hydroxy-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (6). Chlorins 5 and 6 have not been previously reported in natural sources. Interestingly, low amounts of chlorins 1-4 and 6 could also be identified in a distant species, the basket star Gorgonocephalus cf. eucnemis, demonstrating that chlorins are produced by a wide spectrum of marine invertebrates of the class Ophiuroidea. Following the purification of these major Ophiura chlorin metabolites, we discovered the significant singlet oxygen quantum yield upon their photoinduction and the resulting phototoxicity against triple-negative breast cancer BT-20 cells. These studies identify an arsenal of brittle star chlorins as natural photosensitizers with potential photodynamic therapy applications.

S100A9 promotes glycolytic activity in HER2-positive breast cancer to induce immunosuppression in the tumour microenvironment

PurposeThe purpose of this study was to investigate the correlation between S100 calcium binding protein A9 (S100A9), tumour glycolysis and tumour infiltrating lymphocytes (TIL) in human epidermal growth factor receptor 2 (HER2) - positive breast cancer (BRCA). Materials and methodsA total of 667 BRCA patients in Xiangya Hospital of Central South University were enrolled in this study. Haematoxylin and eosin (H&E) staining were used to count TIN in tissues. Human breast cancer cell lines (SK-BR-3 cells and BT474 cells) were transfected with S100A9 specific small interfering RNA (siRNA). The expressions of S100A9, glycolytic enzymes and lymphocyte markers were detected by immunohistochemistry (IHC) staining, Western blot and immunofluorescence. Lactate production, glucose consumption and the extracellular acidification rate (ECAR) were detected to assess glycolysis activity. ResultsS100A9 was significantly overexpressed in HER2+ cases. The expressions of phosphoglycerol kinase 1 (PGK1), lactate dehydrogenase A (LDHA) and enolase α (ENO1) were significantly up-regulated in S100A9 dominant tissues. The expressions of PGK1, LDHA and ENO1 detected in S100A9 silenced cell lines were significantly down-regulated. Moreover, S100A9 silencing significantly altered lactate production, glucose uptake and ECAR levels in HER2+ cell lines. Co-expression of S100A9 and c-Myc was detected in HER2+ tissues. The absence of S100A9 greatly hindered β-catenin expression in cell lines, which later induced the phosphorylation of c-Myc.The amount of TILs in cases with abundant S100A9 and LDHA was much greater than in cases with low S100A9 levels and poorer LDHA. TIL deficiency and elevated S100A9 intensity are factors affecting the survival rate of HER2+ BRCA cases. ConclusionsS100A9 overexpression upregulated the glycolysis activity of tumour cells through the c-Myc-related pathway, suppressing lymphocyte infiltration in the tumour stroma, affecting the efficacy of immune regulation and long-term survival of patients.

Combination of Conventional Drugs with Biocompounds Derived from Cinnamic Acid: A Promising Option for Breast Cancer Therapy.

Despite the options available for breast cancer (BC) therapy, several adverse effects and resistance limit the success of the treatment. Furthermore, the use of a single drug is associated with a high failure rate. We investigated through a systematic review the in vitro effects of the combination between conventional drugs and bioactive compounds derived from cinnamic acid in BC treatment. The information was acquired from the following databases: PubMed, Web of Science, Embase, Scopus, Lilacs and Cochrane library. We focused on "Cinnamates", "Drug Combinations" and "Breast neoplasms" for publications dating between January 2012 and December 2022, based on the PRISMA statement. The references of the articles were carefully reviewed. Finally, nine eligible studies were included. The majority of these studies were performed using MCF-7, MDA-MB-231, MDA-MB-468 and BT-20 cell lines and the combination between cisplatin, paclitaxel, doxorubicin, tamoxifen, dactolisib and veliparib, with caffeic acid phenethyl ester, eugenol, 3-caffeoylquinic acid, salvianolic acid A, ferulic acid, caffeic acid, rosmarinic acid and ursolic acid. The combination improved overall conventional drug effects, with increased cytotoxicity, antimigratory effect and reversing resistance. Combining conventional drugs with bioactive compounds derived from cinnamic acid could emerge as a privileged scaffold for establishing new treatment options for different BC types.

Small-molecule inhibitor of Fam20C in combination with paclitaxel suppresses tumor growth by LIF-JAK2/STAT3-modulated apoptosis in triple-negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is a strongly proliferative, relapse prone, high-mortality subtype of breast cancer. Due to the lack of effective therapeutic targets, the current clinical treatment is still dominated by chemotherapeutic drugs, such as paclitaxel (PTX). Moreover, Fam20C kinase inhibitor (3R) has recently been reported to inhibit TNBC growth. Thus, the objectives of this study were to verify whether the combination therapy with 3R could enhance the efficacy of PTX treatment on TNBC, and to further elucidate their potential synergistic mechanisms. MethodsThe inhibitory effect on TNBC cell viability was verified by the MTT assay, and the effect of drug combination on apoptosis was analyzed by flow cytometry. The mechanism of action of the drug combination was validated based on transcriptome analyses and western blot (WB) analysis. Xenograft mouse models were used to verify the therapeutic efficacy of PTX+3R in vivo by immunohistochemistry and WB analysis. Significant findingsFam20C inhibitor (3R) enhanced the inhibitory effect of PTX on the viability and proliferation of MDA-MB-231 and BT-549 cells, and their combination significantly promoted cell apoptosis. In addition, we found that 3R reduced the expression of leukemia inhibitor factor (LIF). 3R and PTX limit the phosphorylation activation of signal transducer and activator of translation 3 (STAT3). Finally, immunohistochemical staining showed that combined treatment with 3R and PTX inhibited tumor growth in an ectopic tumor model of MDA-MB-231 cells in nude mice.

Dalpiciclib partially abrogates ER signaling activation induced by pyrotinib in HER2+HR+ breast cancer.

Recent evidences from clinical trials (NCT04486911) revealed that the combination of pyrotinib, letrozole, and dalpiciclib exerted optimistic therapeutic effect in treating HER2+HR+ breast cancer; however, the underlying molecular mechanism remained elusive. Through the drug sensitivity test, the drug combination efficacy of pyrotinib, tamoxifen, and dalpiciclib to BT474 cells was tested. The underlying molecular mechanisms were investigated using immunofluorescence, Western blot analysis, immunohistochemical staining, and cell cycle analysis. Potential risk factor that may indicate the responsiveness to drug treatment in HER2+/HR+ breast cancer was identified using RNA-sequence and evaluated using immunohistochemical staining and in vivo drug susceptibility test. We found that pyrotinib combined with dalpiciclib exerted better cytotoxic efficacy than pyrotinib combined with tamoxifen in BT474 cells. Degradation of HER2 could enhance ER nuclear transportation, activating ER signaling pathway in BT474 cells, whereas dalpiciclib could partially abrogate this process. This may be the underlying mechanism by which combination of pyrotinib, tamoxifen, and dalpiciclib exerted best cytotoxic effect. Furthermore, CALML5 was revealed to be a risk factor in the treatment of HER2+/HR+ breast cancer and the usage of dalpiciclib might overcome the drug resistance to pyrotinib + tamoxifen due to CALML5 expression. Our study provided evidence that the usage of dalpiciclib in the treatment of HER2+/HR+ breast cancer could partially abrogate the estrogen signaling pathway activation caused by anti-HER2 therapy and revealed that CALML5 could serve as a risk factor in the treatment of HER2+/HR+ breast cancer.

The combined effect and mechanism of antiangiogenic drugs and PD-L1 inhibitor on cell apoptosis in triple negative breast cancer.

Breast cancer is the most common cancer worldwide, and triple-negative breast cancer (TNBC) has the worst prognosis. Standard systemic treatment includes chemotherapy and immunotherapy. Poly ADP-ribose polymerase (PARP) inhibitors are considered in breast cancer (BRCA) susceptibility genes mutated tumors. The role of antiangiogenic drugs is controversial. Immunotherapy with immune checkpoint inhibitor is now a standard of care for TNBC in the US, but its use in combination with anlotinib, an inhibitor of angiogenesis, on TNBC cells was never investigated. We tested the effects of anlotinib and programmed cell death-ligand 1 (PD-L1) inhibitor on the proliferation, apoptosis, migration, and invasion of MDA-MB-468 and BT-549 TNBC cells through 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assays, cell apoptosis assay, wound healing and transwell matrix assays, and verified whether the combination of the two drugs had synergistic effect. Western blotting was used to detect the effect of anlotinib and PD-L1 inhibitor on the protein expression levels of PI3K, p-PI3K, AKT, p-AKT, Bcl-xl in MDA-MB-468 and BT-549 cells. The effects of anlotinib, PD-L1 inhibitor and the combination of the two drugs on the transplanted tumor of TNBC mice were tested by animal experiments. Anlotinib and PD-L1 inhibitor inhibited the proliferation and promote cell apoptosis of MDA-MB-468 and BT-549 cells, and the combination demonstrated the synergetic effect. Anlotinib and PD-L1 inhibitor inhibited cell migration and invasion, and the effect was strongest in the combination group. Both anlotinib and PD-L1 inhibitor reduced the expression of p-PI3K, p-AKT and Bcl-xl proteins in cells and the effects were the strongest in the combination group. Both anlotinib and PD-L1 inhibitor inhibited the growth of transplanted tumors in mice, and the combined group demonstrated the strongest growth suppression. Anlotinib and PD-L1 inhibitor can inhibit cell proliferation, migration, and invasion of TNBC and promote cell apoptosis, and the two drugs show combined anti-tumor effects in vivo and in vitro. The combination of anlotinib and PD-L1 inhibitor may promote apoptosis of TNBC cells through PI3K/AKT/Bcl-xl signaling pathways, which might offer potential clinical treatment roles for these.

ENST00000535926 is an unfavorable prognosis-related and tumor-promoting transcript of the CHPF gene in luminal A and B breast cancer

Chondroitin sulfate synthase 2 (CHPF) is characterized as an oncogenic and poor prognosis-related gene in breast cancer. However, this gene has alternative splicing products encoding proteins of different lengths. Breast cancer is a group of heterogeneous tumors with distinct clinical and genomic characteristics. In this study, we explored the expression profile and prognostic value of the two transcripts of CHPF using data from The Cancer Genome Atlas (TCGA)-BRCA. The functional regulation of the two transcripts was also studied in MCF-7 and BT-474 cells. Among the two transcripts of CHPF, ENST00000535926 expression was significantly upregulated in the tumor samples and was the dominant isoform. ENST00000535926, but not ENST00000243776 upregulation, was associated with significantly worse progression-free survival (PFS) and disease-specific survival (DSS) in luminal A/B cases. However, no significant association was observed in PFS or DSS in other Prediction Analysis of Microarray 50 (PAM50) subgroups. CHPF isoform 2 protein (encoded by ENST00000535926) significantly elevated the expression of P3H1 and RCN3 at the mRNA and protein levels in MCF-7 and BT-474 cells. The effect of ENST00000535926 was significantly stronger than ENST00000243776 in promoting tumor cell colony formation. The expression of P3H1 and RCN3 was negatively correlated with CD8+ T cell infiltration but was positively correlated with cancer-associated fibroblast infiltration in luminal A/B tumors. In summary, this study revealed that ENST00000535926 is an unfavorable prognosis-related and tumor-promoting transcript of the CHPF gene in luminal A/B breast cancer.

Anti-tumor effects of low-dose metronomic vinorelbine in combination with alpelisib in breast cancer cells.

In metastatic breast cancer (MBC), PIK3CA mutations, activating the phosphatidylinositol 3-kinase (PI3K) signaling pathway seem to be associated with chemotherapy resistance and poor outcome. Inhibition of the PI3K signaling pathway may lead to sensitization and prevention of the development of resistance to cytotoxic drugs. The present study aimed to investigate the anti-tumor activity of low-dose vinorelbine (VRL) combined with alpelisib, an α-selective PI3K inhibitor and degrader, in breast cancer (BC) cells. Human BC cell lines MCF-7, T-47D [both hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, PIK3CA-mutated], MDA-MB-231 and BT-549 (both triple-negative, wild-type PIK3CA) were exposed to a combination of low-dose VRL and alpelisib for 3 and 7 days. Cell viability was detected by the Alamar blue assay, and cell proliferation was determined by the BrdU incorporation. The effect of the substances on the p110α protein expression that is encoded by PIK3CA gene was investigated by Western blot. Low-dose VRL plus alpelisib showed synergistic anti-tumor effects and significantly inhibited cell viability and proliferation of MCF-7 and T-47D cells. Even lower alpelisib concentrations (10 ng/ml and 100 ng/ml) combined with low-dose metronomic VRL led to a significant reduction of cell viability of PIK3CA-mutated cells, and the anti-tumor activity was comparable with the effects at 1000 ng/ml alpelisib. Cell viability and proliferation of MDA-MB-231 and BT-549 cells were inhibited by VRL but not by alpelisib alone. This indicates that alpelisib did not significantly affect the cell growth of triple-negative, PIK3CA wild-type BC cells. The p110α expression was downregulated or not affected in PIK3CA-mutated cell lines, and not significantly upregulated in PIK3CA wild-type cell lines. In conclusion, combination of low-dose metronomic VRL and alpelisib showed synergistic anti-tumor effects and significantly inhibited the growth of HR-positive, HER2-negative, PIK3CA-mutated BC cells, providing a rationale for further efforts to evaluate this combination in vivo.

Metabolic Pathway of Monounsaturated Lipids Revealed by In-Depth Structural Lipidomics by Mass Spectrometry.

The study of lipid metabolism relies on the characterization of the lipidome, which is quite complex due to the structure variations of the lipid species. New analytical tools have been developed recently for characterizing fine structures of lipids, with C=C location identification as one of the major improvements. In this study, we studied the lipid metabolism reprograming by analyzing glycerol phospholipid compositions in breast cancer cell lines with structural specification extended to the C=C location level. Inhibition of the lipid desaturase, stearoyl-CoA desaturase 1, increased the proportion of n-10 isomers that are produced via an alternative fatty acid desaturase 2 pathway. However, there were different variations of the ratio of n-9/n-7 isomers in C18:1-containing glycerol phospholipids after stearoyl-CoA desaturase 1 inhibition, showing increased tendency in MCF-7 cells, MDA-MB-468 cells, and BT-474 cells, but decreased tendency in MDA-MB-231 cells. No consistent change of the ratio of n-9/n-7 isomers was observed in SK-BR-3 cells. This type of heterogeneity in reprogrammed lipid metabolism can be rationalized by considering both lipid desaturation and fatty acid oxidation, highlighting the critical roles of comprehensive lipid analysis in both fundamental and biomedical applications.

Hesperetin effect on MLH1 and MSH2 expression on breast cancer cells BT-549.

Due to its genetic and phenotypic heterogeneity, breast cancer is very difficult to eliminate. The harmful consequences of conventional therapies like radiation and chemotherapy have prompted the search for organic-based alternatives. Hesperetin (HSP), a flavonoid, has been discovered to possess the ability to hinder the proliferation of cell associated with breast cancer by acting as an epigenetic agent and modifying gene expression. In this investigation, breast cancer cells (BT-549) and normal cells (MCF-10a) were subjected to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test and three different doses (200, 400, and 600 μM/mL) of HSP for real-time polymerase chain reaction and flow cytometry to examine its cytotoxic and anti-malignant potential. HSP was shown to be cytotoxic to both normal and breast cancer cells, but had a more pronounced effect on the cancer cell lines. After 48 h of treatment, the half-maximal inhibitory concentration (IC50) for BT-549 was 279.2 μM/mL, whereas the IC50 for MCF-10a was 855.4 μM/mL. At high HSP concentrations, upregulation of the MLH1 and MSH2 genes was observed in both cell lines. The influence of HSP on MLH1 gene expression was concentration dependent. Moreover, HSP had a concentration-dependent effect on MSH2 gene expression in the BT-549 cell line but not in the MCF-10a cell line. Cell death and early apoptosis were shown to be concentration dependent upon the application of HSP, as determined by flow cytometric analysis. HSP's capacity to cause apoptosis and its stronger impact on the malignant cell line when analyzed with the normal cell line imply that it might be useful as an effective therapeutic approach for combating breast cancer.

Circular RNA_0000326 accelerates breast cancer development via modulation of the miR-9-3p/YAP1 axis.

Circular RNA (circ)_0000326 has been reported in bladder cancer and cervical cancer and is concerned to be involved with the development of cancerous cells. Whereas, there have been no reports concentrating on the influences of circ_0000326 in breast cancer (BC). Therefore, the latent modulatory mechanisms of circ_0000326 in BC are researched. circ_0000326 expression in BC tissues and correlative cells was evaluated via RT-qPCR, and the relevance between circ_0000326 expression and overall survival and the clinicopathological feature was also investigated. After a series of transfection, the effects of circ_0000326, microRNA-9-3p (miR-9-3p), and Yes-associated protein 1 (YAP1) in BC cell growth, invasion, and stemness were studied by CCK-8, flow cytometry, Transwell, and sphere-forming assays. The binding sites and correlation of circ_0000326, miR-9-3p, and YAP1 were certified via starBase website, luciferase reporter assay, and Pearson's χ2 test. The in vivo experiment was evaluated by establishing a subcutaneous tumorigenesis model. High-expressed circ_0000326 in BC tissues and cells was discovered, which was connected with an undesirable prognosis. Silencing of circ_0000326 visibly inhibited MCF-7 and BT549 cell growth, invasion, stemness, meanwhile declining the protein levels of SRY-related high-mobility group box gene 2 (SOX2) and octamer binding transcription factor 4 (OCT4). miR-9-3p was a sponger of circ_0000326, which was negatively regulated by circ_0000326. Moreover, YAP1 was confirmed as a target gene of miR-9-3p. circ_0000326 affected BC cell behaviors via mediating miR-9-3p and YAP1. Furthermore, circ_0000326 silencing prohibited tumor growth of BC in vivo. The research uncovered that circ_0000326 facilitated BC development via mediating the miR-9-3p/YAP1 axis.

Development and evaluation of 89Zr-trastuzumab for clinical applications.

Due to the suitable physical characteristics of 89Zr as a PET radionuclide and affinity of Trastuzumab monoclonal antibody against HER2, [89Zr]Zr-Trastuzumab was prepared and went through preclinical evaluations for ultimate human applications. 89Zr was produced by using 89Y(p,n)89Zr reaction at a 30 MeV cyclotron (radionuclide purity>99.9%, specific activity of 17 GBq/µg). p-SCN-Bn-Deferoxamine (DFO); was conjugated to trastuzumab, followed by labeling with 89Zr in oxalate form at optimized condition. Cell binding, internalization and, radioimmuno-activity assays were studied using HER2+ BT474 and HER2- CHO cell lines. Finally, the biodistribution of the radioimmunoconjugate was assessed in normal and HER2+ BT474 tumor-bearing mice using tissue counting and imaging at different intervals after injection. Also, a woman with HER2-positive metastatic breast cancer under treatment with Herceptin underwent both [89Zr]Zr-Trastuzumab and, [18F]FDG PET/CTs. 89Zr was produced with high radionuclidic and radiochemical purities (>99%) and [89Zr]Zr-DFO-Trastuzumab was prepared with radiochemical purity of >98% and specific activity of 9.85 GBq/µmol. The radioimmunoconjugate was stable both in PBS buffer and in human serum for at least 48 h. The radioimmunoactivity assay demonstrated about 70% of [89Zr]Zr-DFO-Trastuzumab is bound to the BT474 cells at the number of 250×106 cells. Cell binding studies showed that about 28% of radioimmunoconjugate is attached to BT474 cells after 90 min. Internalization studies showed that 50% of [89Zr]Zr-Trastuzumab is internalized to BT474 cells only in 6 h. The biodistribution study of the labeled compound in normal mice demonstrated the same pattern of the monoclonal antibodies which is entirely different from the biodistribution of free 89Zr. Biodistribution and imaging studies in tumor-bearing mice showed the significant uptake values of [89Zr]Zr-Trastuzumab in tumor sites. [89Zr]Zr-Trastuzumab PET/CT revealed metastatic lesions documented previously with [18F]FDG PET/CT scan in a woman with breast cancer who was under treatment with Herceptin. Although the [18F]FDG PET/CT scan had better quality images, the valuable and unique advantage of [89Zr]Zr-Trastuzumab PET/CT is delineating HER2+ metastasis, which is essential in diagnosis and HER2-based treatments. The prepared [89Zr]Zr-Trastuzumab has a high potential radio-pharmaceutical for immune-PET imaging of the patients with HER2+ tumors.

The role of activated androgen receptor in cofilin phospho-regulation depends on the molecular subtype of TNBC cell line and actin assembly dynamics.

Triple negative breast cancer (TNBC) is highly metastatic and of poor prognosis. Metastasis involves coordinated actin filament dynamics mediated by cofilin and associated proteins. Activated androgen receptor (AR) is believed to contribute to TNBC tumorigenesis. Our current work studied roles of activated AR and cofilin phospho-regulation during migration of three AR+ TNBC cell lines to determine if altered cofilin regulation can explain their migratory differences. Untreated or AR agonist-treated BT549, MDA-MB-453, and SUM159PT cells were compared to cells silenced for cofilin (KD) or AR expression/function (bicalutamide). Cofilin-1 was found to be the only ADF/cofilin isoform expressed in each TNBC line. Despite a significant increase in cofilin kinase caused by androgens, the ratio of cofilin:p-cofilin (1:1) did not change in SUM159PT cells. BT549 and MDA-MB-453 cells contain high p-cofilin levels which underwent androgen-induced dephosphorylation through increased cofilin phosphatase expression, but surprisingly maintain a leading-edge with high p-cofilin/total cofilin not found in SUM159PT cells. Androgens enhanced cell polarization in all lines, stimulated wound healing and transwell migration rates and increased N/E-cadherin mRNA ratios while reducing cell adhesion in BT549 and MDA-MB-453 cells. Cofilin KD negated androgen effects in MDA-MB-453 except for cell adhesion, while in BT549 cells it abrogated androgen-reduced cell adhesion. In SUM159PT cells, cofilin KD with and without androgens had similar effects in almost all processes studied. AR dependency of the processes were confirmed. In conclusion, cofilin regulation downstream of active AR is dependent on which actin-mediated process is being examined in addition to being cell line-specific. Although MDA-MB-453 cells demonstrated some control of cofilin through an AR-dependent mechanism, other AR-dependent pathways need to be further studied. Non-cofilin-dependent mechanisms that modulate migration of SUM159PT cells need to be investigated. Categorizing TNBC behavior as AR responsive and/or cofilin dependent can inform on decisions for therapeutic treatment.

In Vitro Evidence of Differential Immunoregulatory Response between MDA-MB-231 and BT-474 Breast Cancer Cells Induced by Bone Marrow-Derived Mesenchymal Stromal Cells Conditioned Medium.

Inside tumors, cancer cells display several mechanisms to create an immunosuppressive environment. On the other hand, by migration processes, mesenchymal stromal cells (MSCs) can be recruited by different cancer tumor types from tissues as distant as bone marrow and contribute to tumor pathogenesis. However, the impact of the immunoregulatory role of MSCs associated with the aggressiveness of breast cancer cells by soluble molecules has not been fully elucidated. Therefore, this in vitro work aimed to study the effect of the conditioned medium of human bone marrow-derived-MSCs (hBM-MSC-cm) on the immunoregulatory capability of MDA-MB-231 and BT-474 breast cancer cells. The hBM-MSC-cm on MDA-MB-231 cells induced the overexpression of TGF-β, IDO, and IL-10 genes. Additionally, immunoregulation assays of mononuclear cells (MNCs) in co-culture with MDA-MB-231 and hBM-MSC-cm decreased lymphocyte proliferation, and increased proteins IL-10, TGF-β, and IDO while also reducing TNF levels, shooting the proportion of regulatory T cells. Conversely, the hBM-MSC-cm did not affect the immunomodulatory capacity of BT-474 cells. Thus, a differential immunoregulatory effect was observed between both representative breast cancer cell lines from different origins. Thus, understanding the immune response in a broader tumor context could help to design therapeutic strategies based on the aggressive behavior of tumor cells.

Effects of Shenling Baizhu powder on pyrotinib-induced diarrhea: analysis of gut microbiota, metabonomics, and network pharmacology

BackgroundShenling Baizhu Powder (SBP) is a traditional Chinese medicine (TCM) prescription, which has the good efficacy on gastrointestinal toxicity. In this study, we used gut microbiota analysis, metabonomics and network pharmacology to investigate the therapeutic effect of SBP on pyrotinib-induced diarrhea.Methods24 Rats were randomly divided into 4 groups: control group, SBP group (3.6 g/kg /bid SBP for 10 days), pyrotinib model group (80 mg/kg/qd pyrotinib) and pyrotinib + SBP treatment group. A 16S rRNA sequencing was used to detect the microbiome of rat fecal bowel. Metabolic profiles were collected by non-targeted metabolomics and key metabolic pathways were identified using MetaboAnalyst 5.0. The antitumor effect of SBP on cells treated with pyrotinib was measured using a CCK-8 assay. Network pharmacology was used to predict the target and action pathway of SBP in treating pyrotinib-related diarrhea.ResultsIn vivo study indicated that SBP could significantly alleviate pyrotinib-induced diarrhea, reaching a therapeutic effect of 66.7%. SBP could regulate pyrotinib-induced microbiota disorder. LEfSe research revealed that the SBP could potentially decrease the relative abundance of Escherichia, Helicobacter and Enterobacteriaceae and increase the relative abundance of Lachnospiraceae, Bacilli, Lactobacillales etc. In addition, 25-Hydroxycholesterol, Guanidinosuccinic acid, 5-Hydroxyindolepyruvate and cAMP were selected as potential biomarkers of SBP for pyrotinib-induced diarrhea. Moreover, Spearman's analysis showed a correlation between gut microbiota and metabolite: the decreased 25-hydroxycholesterol in the pyrotinib + SBP treatment group was negatively correlated with Lachnospiraceae while positively correlated with Escherichia and Helicobacter. Meanwhile, SBP did not affect the inhibitory effect of pyrotinib on BT-474 cells and Calu-3 cells in vitro. Also, the network analysis further revealed that SBP treated pyrotinib-induced diarrhea through multiple pathways, including inflammatory bowel disease, IL-17 signaling pathway, pathogenic Escherichia coli infection and cAMP signaling pathway.ConclusionsSBP could effectively relieve pyrotinib-induced diarrhea, revealing that intestinal flora and its metabolites may be involved in this process.

A Novel Mechanism Underlying the Inhibitory Effects of Trastuzumab on the Growth of HER2-Positive Breast Cancer Cells.

To improve the efficacy of trastuzumab, it is essential to understand its mechanism of action. One of the significant issues that makes it difficult to determine the precise mechanism of trastuzumab action is the formation of various HER receptor dimers in HER2-positive breast cancer cells. So far, studies have focused on the role of HER2-HER3 heterodimers, and little is known regarding EGFR-HER2 heterodimers. Here, we study the role of trastuzumab on the cell signaling and cell proliferation mediated by EGFR-HER2 heterodimers in BT474 and SRBR3 cells. EGF stimulates the formation of both EGFR homodimer and EGFR-HER2 heterodimer. Trastuzumab only binds to HER2, not EGFR. Therefore, any effects of trastuzumab on EGF-induced activation of EGFR, HER2, and downstream signaling proteins, as well as cell proliferation, are through its effects on EGFR-HER2 heterodimers. We show that trastuzumab inhibits EGF-induced cell proliferation and cell cycle progression in BT474 and SKBR3 cells. Interestingly trastuzumab strongly inhibits EGF-induced Akt phosphorylation and slightly inhibits EGF-induced Erk activation, in both BT474 and SKBR3 cells. These data suggest the presence of a novel mechanism that allows trastuzumab to inhibit EGR-induced Akt activation and cell proliferation, without blocking EGF-induced EGFR-HER2 heterodimerization and activation. We show that trastuzumab inhibits EGF-induced lipid raft localization of the EGFR-HER2 heterodimer. Disruption of the lipid raft with MβCD blocks HER2-mediated AKT activation in a similar way to trastuzumab. MβCD and trastuzumab synergically inhibit AKT activation. We conclude that trastuzumab inhibits EGF-induced lipid raft localization of EGFR-HER2 heterodimer, which leads to the inhibition of Akt phosphorylation and cell proliferation, without blocking the formation and phosphorylation of the EGFR-HER2 heterodimer.

ONC201-Induced Mitochondrial Dysfunction, Senescence-like Phenotype, and Sensitization of Cultured BT474 Human Breast Cancer Cells to TRAIL.

ONC201, the anticancer drug, targets and activates mitochondrial ATP-dependent caseinolytic peptidase P (ClpP), a serine protease located in the mitochondrial matrix. Given the promise of ONC201 in cancer treatment, we evaluated its effects on the breast ductal carcinoma cell line (BT474). We showed that the transient single-dose treatment of BT474 cells by 10 µM ONC201 for a period of less than 48 h induced a reversible growth arrest and a transient activation of an integrated stress response indicated by an increased expression of CHOP, ATF4, and GDF-15, and a reduced number of mtDNA nucleoids. A prolonged exposure to the drug (>48 h), however, initiated an irreversible loss of mtDNA, persistent activation of integrated stress response proteins, as well as cell cycle arrest, inhibition of proliferation, and suppression of the intrinsic apoptosis pathway. Since Natural Killer (NK) cells are quickly gaining momentum in cellular anti-cancer therapies, we evaluated the effect of ONC201 on the activity of the peripheral blood derived NK cells. We showed that following the ONC 201 exposure BT474 cells demonstrated enhanced sensitivity toward human NK cells that mediated killing. Together our data revealed that the effects of a single dose of ONC201 are dependent on the duration of exposure, specifically, while short-term exposure led to reversible changes; long-term exposure resulted in irreversible transformation of cells associated with the senescent phenotype. Our data further demonstrated that when used in combination with NK cells, ONC201 created a synergistic anti-cancer effect, thus suggesting its possible benefit in NK-cell based cellular immunotherapies for cancer treatment.

Grafting, characterization and enhancement of therapeutic activity of berberine loaded PEGylated PAMAM dendrimer for cancerous cell

Berberine is an anticancer medication that generates side effects due to its hydrophobicity and low cellular promiscuity as well as high dose requirement. Thus, have to prepare PEGylated dendrimer conjugates which increases the targeting and release of chemotherapeutic drugs at the tumor site although falling the adverse side effects. The circulation time of drug is enhanced by PEGylation. It is the covalent attachment of PEG to therapeutic protein or any molecule. PEGylated berberine dendrimer was prepared by biotinylation cross linking method and characterized by particle size, zeta potential, entrapment efficiency, in vitro release and stability study. The Structure validation of berberine before and after grafting was confirmed by FTIR and NMR spectroscopy. Further prepared PEGylated complex were proceeded for the cellular uptake study in AMJ-13, and BT-20 cells line by fluorescent microscopy study and MTT assay cytotoxicity study in MCF-7 cell line. The prepared PEGylated formulation showed nanometric size, desired zeta potential, and 69.56 ± 23% entrapment efficiency. The prepared PEGylated particle showed 70.23% release at 72 h with good stability at 90 days. The cellular uptake of formulation was highly appreciable which is clearly observed in AMJ-13 and BT-20 cells line. In comparison to pure drug, developed formulation has 10.8 M high efficiency for breast cancer cell line. PEGylation is easy and reasonable way, as it requires lesser time and is proved to be superior technique for treatment of cancer.

Apoptosis, Proliferation, and Autophagy Are Involved in Local Anesthetic-Induced Cytotoxicity of Human Breast Cancer Cells.

Breast cancer accounts for almost one quarter of all female cancers worldwide, and more than 90% of those who are diagnosed with breast cancer undergo mastectomy or breast conservation surgery. Local anesthetics effectively inhibit the invasion of cancer cells at concentrations that are used in surgical procedures. The limited treatment options for triple-negative breast cancer (TNBC) demonstrate unmet clinical needs. In this study, four local anesthetics, lidocaine, levobupivacaine, bupivacaine, and ropivacaine, were applied to two breast tumor cell types, TNBC MDA-MB-231 cells and triple-positive breast cancer BT-474 cells. In addition to the induction of apoptosis and the suppression of the cellular proliferation rate, the four local anesthetics decreased the levels of reactive oxygen species and increased the autophagy elongation indicator in both cell types. Our combination index analysis with doxorubicin showed that ropivacaine had a synergistic effect on the two cell types, and lidocaine had a synergistic effect only in MDA-MB-231 cells; the others had no synergistic effects on doxorubicin. Lidocaine contributed significantly to the formation of autophagolysosomes in a dose-dependent manner in MDA-MB-231 cells but not in BT-474 cells. Our study demonstrated that the four local anesthetics can reduce tumor growth and proliferation and promote apoptosis and autophagy.