Normal Breast Epithelial Cells Research Articles

Tubulin Carboxypeptidase Activity Promotes Focal Gelatin Degradation in Breast Tumor Cells and Induces Apoptosis in Breast Epithelial Cells That Is Overcome by Oncogenic Signaling.

Simple SummaryThe recent discovery of the genetic identity of the tubulin carboxypeptidase (TCP) provides a unique opportunity to study the role of the detyrosination of α-tubulin (deTyr-Tub), as performed by the TCP, in breast epithelial cells and breast cancer cells. Previous research has shown that elevated deTyr-Tub conveys a poor prognosis in breast cancer and is upregulated in a coordinated manner at the invasive margin of patient tumor samples. Using TCP expression constructs, we have shown that increased deTyr-Tub promotes apoptosis in normal breast epithelial cells, that does not occur in the same cells with an oncogenic KRas mutation or Bcl-2/Bcl-xL overexpression. Furthermore, the addition of the TCP to the breast cancer cell lines MDA-MB-231 and Hs578t, also harboring Ras mutations, leads to increased focal gelatin degradation.Post-translational modifications (PTMs) of the microtubule network impart differential functions across normal cell types and their cancerous counterparts. The removal of the C-terminal tyrosine of α-tubulin (deTyr-Tub) as performed by the tubulin carboxypeptidase (TCP) is of particular interest in breast epithelial and breast cancer cells. The recent discovery of the genetic identity of the TCP to be a vasohibin (VASH1/2) coupled with a small vasohibin-binding protein (SVBP) allows for the functional effect of this tubulin PTM to be directly tested for the first time. Our studies revealed the immortalized breast epithelial cell line MCF10A undergoes apoptosis following transfection with TCP constructs, but the addition of oncogenic KRas or Bcl-2/Bcl-xL overexpression prevents subsequent apoptotic induction in the MCF10A background. Functionally, an increase in deTyr-Tub via TCP transfection in MDA-MB-231 and Hs578t breast cancer cells leads to enhanced focal gelatin degradation. Given the elevated deTyr-Tub at invasive tumor fronts and the correlation with poor breast cancer survival, these new discoveries help clarify how the TCP synergizes with oncogene activation, increases focal gelatin degradation, and may correspond to increased tumor cell invasion. These connections could inform more specific microtubule-directed therapies to target deTyr-tubulin.

Exosomal microRNAs synergistically trigger stromal fibroblasts in breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. TNBC progression is sustained by recruitment of a strong tumor microenvironment (TME) mainly composed of cancer-associated fibroblasts (CAFs) able to endorse tumor hallmarks. Increasing evidences demonstrate that exosomes mediate the crosstalk between cancer cells and the TME. We examined TNBC-derived exosomes and their microRNA (miRNA) cargo in activation of normal fibroblasts (NFs) toward CAFs. We demonstrated that TNBC cell-derived exosomes increased NF collagen contraction and migration alongside CAF molecular markers. Exosome-activated fibroblasts promoted the invasion potential of normal breast epithelial cells, as assessed by an organotypic co-culture assay that resembled the in vivo context. We also investigated TNBC cell-derived exosome cargo in activating NFs to CAFs by performing small RNA sequencing. We found that the synergistic action of miR-185-5p, miR-652-5p, and miR-1246 boosted fibroblast migration and contraction, promoting specific CAF subspecialization toward a pro-migratory functional state. These data highlight the role of breast cancer cells in re-education of the TME and their contribution to tumor evolution.

Enhanced Anticancer Performance of Eco-Friendly-Prepared Mo-ZnO/RGO Nanocomposites: Role of Oxidative Stress and Apoptosis.

ZnO nanoparticles (NPs) have attracted great attention in cancer therapy because of their novel and tailorable physicochemical features. Pure ZnO NPs, molybdenum (Mo)-doped ZnO NPs, and Mo-ZnO/reduced graphene oxide nanocomposites (Mo-ZnO/RGO NCs) were prepared using a facile, inexpensive, and eco-friendly approach using date palm (Phoenix dactylifera L.) fruit extract. Anticancer efficacy of green synthesized NPs/NCs was examined in two different cancer cells. The potential mechanism of the anticancer activity of green synthesized NPs/NCs was explored through oxidative stress and apoptosis. The syntheses of pure ZnO NPs, Mo-ZnO NPs, and Mo-ZnO/RGO NCs were confirmed by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and photoluminescence (PL). Dynamic light scattering (DLS) study indicated the excellent colloidal stability of green prepared samples. Mo-ZnO/RGO NCs exhibited threefold higher anticancer activity in human colon (HCT116) and breast (MCF7) cancer cells as compared to pure ZnO NPs. The anticancer activity of Mo-ZnO/RGO NCs was mediated through reactive oxygen species, p53, and the caspase-3 pathway. Moreover, cytocompatibility of Mo-ZnO/RGO NCs in human normal colon epithelial (NCM460) and normal breast epithelial cells (MCF10A) was much better than those of pure ZnO NPs. Altogether, green stabilized Mo-ZnO/RGO NCs exhibited enhanced anticancer performance and improved cytocompatibility because of green mediated good synergism between ZnO, Mo, and RGO. This study suggested the high nutritional value fruit-based facile preparation of ZnO-based nanocomposites for cancer therapy.

Long noncoding RNA CCAT2 reduces chemosensitivity to 5-fluorouracil in breast cancer cells by activating the mTOR axis.

Breast cancer (BC) is the most prevalent cancer in women and the second leading cause for cancer‐related death in women. LncRNA CCAT2 is involved in BC cell drug sensitivity. Drug resistance of BC cells after chemotherapy is the main obstacle to therapeutic effects. This study explored whether BC cell drug sensitivity to 5‐Fu was related to lncRNA CCAT2‐regulated mTOR pathway. Normal breast tissues and BC tissues before/after neoadjuvant chemotherapy were collected, and CCAT2 expression was detected by RT‐qPCR. Correlation between CCATA2 expression and neoadjuvant chemotherapy efficacy was analysed using the Kendall's tau‐b correlation analysis. Normal breast epithelial cells and BC cell lines were cultured. BC cell lines were treated with 5‐Fu, and CCAT2 mRNA level in cells was detected. The 5‐Fu‐resistant MCF‐7/5‐Fu and MDA‐MB‐231/5‐Fu cells were treated with CCAT2 overexpression/knockdown or CCI‐779 (the mTOR pathway inhibitor). The mTOR pathway levels were detected. Expression of apoptosis‐related factors was identified. A subcutaneous xenograft model was carried out. High CCAT2 expression was detected in BC tissues and BC drug‐resistant cells after neoadjuvant chemotherapy, and a negative link was revealed between CCAT2 expression and efficacy of neoadjuvant chemotherapy. p‐mTOR/mTOR in 5‐Fu‐resistant BC cells with inhibited CCAT2 was decreased, while CCAT2 overexpression activated the mTOR pathway. IC50 value, proliferation, cells in S phase increased and apoptosis reduced after CCAT2 overexpression. After si‐CCAT2 or CCI‐779 treatment, the growth rate of transplanted tumours was inhibited, while promoted after CCAT2 overexpression. CCAT2 may reduce BC cell chemosensitivity to 5‐Fu by activating the mTOR pathway.

Abstract P3-06-06: Pg5 promotes breast cancer metastasis by activatingpi3k/akt/mtor signaling pathways

Abstract Background: Breast cancer is the most common malignancy and the leading cause of cancer death among women. Glycoprotein V (GP V) is involved in thrombin-induced platelet activation and in other platelet responses. However, its roles and underlying mechanisms in the pathogenesis of breast cancer are still unclear. This study aimed at assessing GP V expression in breast cancer, its subsequent effects on cancer cell apoptosis, proliferation, invasion, and migration, and the underlying molecular mechanisms. Methods: GP V mRNA expression was analysed in total RNA samples obtained from tumor and adjacent normal tissue of 41 breast cancer patients and cell lines (MDA-MB-231, MCF-7 and MCF-10A). The correlation between the expression of GP V and clinicopathological features was analyzed by immunohistochemical (IHC) staining. Stable knockdown and overexpression MDA-MB-231 cell lines were generated by using the lentiviral system. Then, the cells were harvested for CCK-8 assay, flow cytometry, scratch healing experiments, and western blotting. Two samples each from the Stable knockdown and control groups were used for transcriptome sequencing and pathway analysis. Finally key molecules associated with the related signaling pathways were analyzed by RT-PCR and western blot. Results: The results showed that the mRNA (P<0.0001) and protein expression (P<0.001) of GP V in the breast cancer tissues was significantly increased compared with in the adjacent non-tumor tissues. The protein and mRNA expression of GP V were significantly higher in MDA-MB-231 and MCF-7cell lines compared with human normal breast epithelial cells (MCF-10A) (P<0.01). GP V expression was positively correlated with higher histological grade and advanced TNM stage. (P<0.05), but was not related to gender, age, menopausal status, tumor size, vascular invasion, ER, PR, Ki-67 and lymph node metastasis (P>0.05). CCK-8 assay showed that cell viability was enhanced after GP V upregulation compared to the control group (P<0.05). The in vitro scratch assay showed that the number of GP-V-overexpressing cells migrating to the scratch area was significantly higher than that of the control group at 24 h and 48 h, respectively (P<0.001). Flow cytometric analysis demonstrated there was no significant difference in cell cycle distribution and apoptosis. Differential gene expression analysis showed 13 significantly down- and 141 upregulated mRNAs. The mRNA expression levels of PI3K and AKT were significantly increased after GP V upregulation compared with those in the control group (P<0.001). N-cadherin, Vimentin and MMP2/9 were up-regulated after GP V upregulation compared with those in the control group (P<0.05).Conclusion: GP5 is highly expressed in breast cancer tissues and may play an important role as a cancer-promoting gene in breast cancer. The high expression of GP5 was significantly associated with higher nuclear grade, TNM stage and Her-2 negativity. GP5 can promote the proliferation, invasion, and metastasis of breast cancer cells. GP5 may promote the proliferation, invasion, and metastasis by activating PI3K/AKT signaling pathway to upregulate MET, and is expected to become a potential target for clinic diagnosis and treatment. Citation Format: Kui Xiang, Wang Yan, Hua Yanshan, Yi Xiaojia, Zhao Chunmei, Guo Minmin. Pg5 promotes breast cancer metastasis by activatingpi3k/akt/mtor signaling pathways [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-06.

Ajuba Overexpression Promotes Breast Cancer Chemoresistance and Glucose Uptake through TAZ-GLUT3/Survivin Pathway.

The LIM protein Ajuba has been implicated in the development of human cancers. To date, its expression pattern and biological significance in breast cancers (BC) have not been fully investigated. In the current study, we examined Ajuba protein levels in 93 invasive ductal carcinoma specimens by immunohistochemistry. The Ajuba expression level was elevated in breast cancer tissue compared with normal tissue. Ajuba overexpression is correlated with advanced tumor-node-metastasis (TNM) stage, positive node status, and adverse patient outcomes. The Ajuba protein level was also higher in BC cell lines compared to normal breast epithelial cell line MCF-10A. Ectopically expressed Ajuba in MCF-7 cells stimulated in vitro and in vivo cell growth, invasion, cell cycle progression, and decreased paclitaxel-induced apoptosis. RNA-sequencing (RNA-seq) followed by gene set enrichment analysis (GSEA) analysis showed that Ajuba overexpression regulated the Hippo signaling pathway. Ajuba overexpression also increased glucose uptake and increased expression of TAZ, GLUT3, and Survivin. TAZ knockdown abolished the role of Ajuba on GLUT3 and Survivin induction. The ChIP assay showed that TEAD4, a major TAZ binding transcription factor, could bind to the GLUT3 and Survivin promoter regions. In conclusion, our data demonstrated that elevated Ajuba expression is correlated with poor BC prognosis and regulated malignant behavior through TAZ-GLUT3/Survivin signaling in BC cells.

Snail Mucus Enhances Chemosensitivity of Triple-negative Breast Cancer Via Activation of the Fas Pathway.

The poor prognosis and chemoresistance of patients with triple-negative breast cancer (TNBC) urge the development of new therapeutic strategies. Snail mucus has shown its ability against inflammation, a process closely related to tumorigenesis, suggesting a potential anti-cancer activity. The effect and mechanisms of snail mucus on cell viability were determined by IncuCyte Live-cell analysis and molecular biological methods. The anti-cancer fractions of snail mucus were isolated and identified by medium pressure liquid chromatography (MPLC) and nuclear magnetic resonance (NMR) spectrometry analysis. Snail mucus significantly decreased the viability of TNBC cells with relatively lower cytotoxicity to normal breast epithelial cells and enhanced their response to chemotherapy through activation of Fas signaling by suppressing nucleolin. Two peptide fractions have been identified as the anti-cancer ingredients of the snail mucus. Snail mucus can induce programmed cell death via the extrinsic apoptotic pathway and has therapeutic potential by achieving a chemo-sensitizing effect in TNBCs.

SSX2 promotes the formation of a novel type of intranuclear lamin bodies.

SSX proteins are normally restricted to spermatogenic cells, but ectopic expression can be observed in many types of human cancer. We recently demonstrated that SSX family members may contribute to tumorigenesis by modifying chromatin structure and, in specific settings, compromise chromatin stability. Here, we used normal and tumorigenic breast epithelial cell line models to further study the effect of ectopic expression of SSX2 on nuclear organization. We show that SSX2 induces the formation of a novel type of nucleoplasmic lamin bodies. Ectopic expression of SSX2 in various breast epithelial cell lines led to the formation of a previously undescribed type of intranuclear bodies containing both A and B type lamins but no other components of the nuclear lamina. SSX2-expressing cells contained a highly variable number of lamin bodies distributed throughout the nuclear space. SSX2-mediated establishment of intranuclear lamin bodies could not be linked to previous molecular interactions of SSX proteins, including polycomb proteins and the Mediator complex, but was, however, dependent on S-phase progression. These results reveal a novel interaction between SSX2 and lamins in the nucleoplasmic space. They further suggest that SSX2 promotes the formation of chromatin neighborhoods supporting the organization of lamins into nuclear bodies. We speculate that this may have implications for the organization and functional regulation of chromatin in cancer cells. Our study contributes to the further understanding of the biology of SSX proteins in tumorigenesis.

Circular RNA circPRMT5 is upregulated in breast cancer and is required for cell proliferation and migration.

To evaluate the role of cyclic protein arginine methyltransferase 5 (circPRMT5) in the occurrence and development of breast cancer (BC). A total of 90 BC patients who underwent radical mastectomy and 40 age-matched healthy female controls were recruited in the Second People's Hospital of China Three Gorges University, Yichang Second People's Hospital from 2017 to 2020. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expression levels of circPRMT5 in BC tissues, serum, normal breast cell line (MCF-10A), and BC cell line (T47D, MCF-7, BT549, Hs-578T, and MDA-MB-231, MDAMB-468). The associations between circPRMT5 expression level and age, tumor size, degree of differentiation, TNM stage, distant metastasis, estrogen receptor (ER) or progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status were analyzed. BC cell lines with circPRMT5 knockdown or overexpression were subject to CCK-8 cell proliferation assay, and transwell cell invasion/migration assay. CircPRMT5 expression in BC tissue was higher than that in adjacent normal breast tissue. Consistently, the expression level of circPRMT5 was also elevated in serum samples collected from BC patients when compared with healthy controls. And in multiple breast cancer cell lines, circPRMT5 was upregulated as compared to normal breast epithelial MCF-10A cells. CircPRMT5 expression level was correlated with tumor size, TNM stage, lymph node metastasis distant metastasis, but no correlation was observed with ER, PR, HER2 status. Overexpression of circPRMT5 promoted the proliferation, invasion, and migration of MCF7 cells; while the knockdown of circPRMT5 inhibited cell proliferation, invasion, and migration. CircPRMT5 seems to act as an oncogene in the progression of BC. Our data suggest that CircPRMT5 may be used as a biomarker for the diagnosis, prognosis evaluation, and targeted therapy of breast cancer.

Artesunate-induced Cellular Effects Are Mediated by Specific EPH Receptors and Ephrin Ligands in Breast Carcinoma Cells

The aberrant regulation of erythropoietin-producing hepatocellular carcinoma (EPH) receptors and ephrin ligands has been implicated in breast carcinoma, and artesunate has been shown to have anticancer effects. The aim of this study was to characterize the involvement of EPH receptors and ephrin ligands in mediating artesunate (ART)-induced growth suppression of normal breast cells and breast carcinoma cell lines. The normal breast epithelial cells (MCF10A), non-invasive ductal breast carcinoma cells (MCF7), and invasive triple-negative breast carcinoma cells (MDA-MB-231) were grown in the absence or the presence of different concentrations of artesunate. The cells were counted, and total RNA was isolated. The abundance of transcripts corresponding to EPH receptors and ephrin ligands was determined by quantitative polymerase chain reaction. Cell viability was significantly reduced when cells were treated with artesunate, with MDA-MB-231 cells having the highest sensitivity. Artesunate had no significant effect on transcription of EPH/ephrins in MCF10A cells, but markedly increased EPHA8, EPHA10, EPHB6 and ephrin-A2 expression in MCF7 cells, and significantly increased EPHA3 and EPHA10 expression while reducing that of EPHA7 and ephrin-A3 in MDA-MB-231 cells. The relative changes in artesunate-treated MCF7 and MDA-MB-231 cells as compared to similarly treated MCF10A cells allow us to implicate combinatorial expression and receptor interactions for EPH receptor-mediated signal transduction that converges into pathways responsible for cell growth, proliferation, and apoptosis. Specifically, the alterations in EPHA7, EPHA8, EPHA10 and EPHB6 transcripts appear to be important participants in artesunate-mediated cellular effects.

Novel Combinatorial Strategy Using Thermal Inkjet Bioprinting, Chemotherapy, and Radiation on Human Breast Cancer Cells; an In-Vitro Cell Viability Assessment

Background: Breast cancer (BC) continues to have the second highest mortality amongst women in the United States after lung cancer. For 2021, the American Cancer Association predicted 281,550 new invasive breast cancer cases besides 49,290 new cases of non-invasive breast cancer and 43,600 deaths from the metastatic disease. A treatment modality is radiation therapy, which is given for local control as well as palliation of patient symptoms. The initial step of new drug development is in-vitro cell studies, which help describe new drug properties and toxicities. However, these models are not optimal, and better ones have yet to be determined. This study uses bioprinting technology to elucidate the sensitivity of tumor cells to the combination of palbociclib (PD) and letrozole (Let) treatment. We hypothesize that this technology could serve as a model to predict treatment outcomes more efficiently. Methods: The breast cancer cell lines MCF7 and MDA-MB-231 as well as the normal breast epithelial cell line, MCF-10A, were treated with PD-Let with and without radiotherapy (RT), and cell viability was compared in pairwise fashion for thermally inkjet bioprinted (TIB) and manually seeded (MS) cells. Results: In absence of radiation, the TIB MCF7 cells have 2.5 times higher viability than manually seeded (MS) cells when treated with 100 µM palbociclib and 10 µM letrozole, a 36% higher viability when treated with 50 µM palbociclib and 10 µM letrozole, and an 8% higher viability when treated with 10 µM palbociclib and 10 µM letrozole. With 10 Gy of radiation, TIB cells had a 45% higher survival rate than MS cells at the lowest palbociclib concentration and a 29% higher survival rate at the intermediate palbociclib concentration. Without radiation treatment, at a concentration of 10 μM PD-Let, TIB MDA-MB-231 cells show a 8% higher viability than MS cells when treated with 10 µM PD and 10 µM Let; at higher drug concentrations, the differences disappeared, but some 1.7% of the TIB MDA-MB-231 cells survived exposure to 150 μM of PD + 10 μM letrozole vs. none of the MS cells. These cells are more radiation sensitive than the other cell lines tested and less sensitive to the combo drug treatments. We observed an 18% higher survival of TIB MCF-10A cells without radiation treatment when exposed to 10 μM PD + 10 μM Let but no difference in cell survival between the two groups when radiation was applied. Independent of growth conditions, TIB cells did not show more resistance to radiation treatment than MS cells, but a higher resistance to the combo treatment was observed, which was most pronounced in the MCF-7 cell line. Conclusion: Based on these results, we suggest that TIB used in in-vitro models could be a feasible strategy to develop and/or test new anticancer drugs.

Defining the landscape of metabolic dysregulations in cancer metastasis

Metastasis is the primary cause of cancer related deaths due to the limited number of efficient druggable targets. Signatures of dysregulated cancer metabolism could serve as a roadmap for the determination of new treatment strategies. However, the metabolic signatures of metastatic cells remain vastly elusive. Our aim was to determine metabolic dysregulations associated with high metastatic potential in breast cancer cell lines. We have selected 5 triple negative breast cancer (TNBC) cell lines including three with high metastatic potential (HMP) (MDA-MB-231, MDA-MB-436, MDA-MB-468) and two with low metastatic potential (LMP) (BT549, HCC1143). The normal epithelial breast cell line (hTERT-HME1) was also investigated. The untargeted metabolic profiling of cells and growth media was conducted and total of 479 metabolites were quantified. First we characterized metabolic features differentiating TNBC cell lines from normal cells as well as identified cell line specific metabolic fingerprints. Next, we determined 92 metabolites in cells and 22 in growth medium that display significant differences between LMP and HMP. The HMP cell lines had elevated level of molecules involved in glycolysis, TCA cycle and lipid metabolism. We identified metabolic advantages of cell lines with HMP beyond enhanced glycolysis by pinpointing the role of branched chain amino acids (BCAA) catabolism as well as molecules supporting coagulation and platelet activation as important contributors to the metastatic cascade. The landscape of metabolic dysregulations, characterized in our study, could serve as a roadmap for the identification of treatment strategies targeting cancer cells with enhanced metastatic potential.

Effect of microrna-138 on epithelial-Mesenchymal transition and invasion of breast cancer cells by targeting semaphorin 4C

ABSTRACT In view of the role of miR-138 in cancer cells, we predicted the target of miR-138 and its targeting to SEMA4C by bioinformatics software and luciferase experiment. The expression levels of miR-138 in human normal breast epithelial cells and two kinds of BC cells were compared, and the transfection cells were selected. MiR-138 mimetic negative control (miR-NC), miR-138 mimic and miR-138 inhibitor were designed for cell transfection. The results showed that the expression level of miR-138 in MCF-7 cells was the lowest. The up regulation of miR-138 would lead to the high expression of E-cad and the low expression of N-cad, vim and SEMA4C, and the vitality and invasion of BC cells would decrease. The down regulation of miR-138 would lead to the low expression of E-cad and the high expression of N-cad, vim and SEMA4C, and the vitality and invasion of BC cells would increase. miR-138 targeted regulation of SEMA4C can promote the expression of N-cad, inhibit the expression of E-cad, vim and SEMA4C, reverse the EMT of BC cells, and inhibit the activity and invasion of BC cells. MiR-138 has clinical potential as a tumor marker of BC.

Tumorigenic activity of alternative per- and polyfluoroalkyl substances (PFAS): Mechanistic in vitro studies

Environmental contaminants including long-chain per- and polyfluoroalkyl substances (PFAS) have been linked to cancer, which is a central cause of mortality in humans and many wildlife species. Today shorter-chain PFAS are extensively used as replacement compounds and commonly found in the environment. Mechanistic studies are important for a better understanding of their toxicological potential and possible role in cancer etiology. Here, we treated normal human breast epithelial cells (MCF-10A) with 500 pM to 500 μM of perfluorohexane sulfonate (PFHxS), undecafluorohexanoic acid (PFHxA), hexafluoropropylene oxide-dimer acid (GenX), perfluoro 3,6 dioxaoctanoic acid (PFO2OA), heptafluorobutyric acid (HFBA) and perfluorobutanesulfonic acid (PFBS) for 72 h to investigate potential effects on cell proliferation and neoplastic transformation. PFHxA, GenX, PFO2OA, HFBA and PFBS induced no alterations compared to controls at any of the concentrations tested. Exposure to 100 μM PFHxS on the other hand was shown to affect important regulatory cell-cycle proteins (cyclin D1, CDK6, p27, p53 and ERK) and induced cell proliferation, at least in part through activation of the constitutive androstane receptor (CAR) and the peroxisome proliferator-activated receptor alpha (PPARα). PFHxS also altered histone modifications and induced cell malignance by reducing the levels of adhesion proteins (E-cadherin and β-integrin) and promoting cell migration and invasion. These results demonstrate that five out of six alternative PFAS tested are clearly less harmful to MCF-10A cells than previously studied PFOS and PFOA, but raise concerns about PFHxS that also has been associated with breast cancer in epidemiological studies.

Differential Collective Cell Migratory Behaviors Modulated by Phospholipid Nanocarriers.

Phospholipid nanocarriers have been widely explored for theranostic and nanomedicine applications. These amphiphilic nanocarriers possess outstanding cargo encapsulation efficiency, high water dispersibility, and excellent biocompatibility, which render them promising for drug delivery and bioimaging applications. While the biological applications of phospholipid nanocarriers have been well documented, the fundamental aspects of the phospholipid-cell interactions beyond cytotoxicity have been less investigated. In particular, the effect of phospholipid nanocarriers on collective cell behaviors has not been elucidated. Herein, we evaluate the interactions of phospholipid nanocarriers possessing different functional groups and sizes with normal and cancerous immortalized breast epithelial cell sheets with varying metastatic potential. Specifically, we examine the impact of nanocarrier treatments on the collective migratory dynamics of these cell sheets. We observe that phospholipid nanocarriers induce differential collective cell migratory behaviors, where the migration speed of normal and cancerous breast epithelial cell sheets is retarded and accelerated, respectively. To a certain extent, the nanocarriers are able to alter the migration trajectory of the cancerous breast epithelial cells. Furthermore, phospholipid nanocarriers could modulate the stiffness of the nuclei, cytoplasm, and cell-cell junctions of the breast epithelial cell sheets, remodel their actin filament arrangement, and regulate the expressions of the actin-related proteins. We anticipate that this work will further shed light on nanomaterial-cell interactions and provide guidelines for rational and safer designs and applications of phospholipid nanocarriers for cancer theranostics and nanomedicine.

RETROSPECTIVE STUDY CORRELATION BETWEEN GRADE AND AMOUNT OF MITOSIS WITH ER, PR, AND HER2 EXPRESSION IN INVASIVE BREAST CARCINOMA OF NO SPECIAL TYPE

Background: In 2018 there were about 24.2% new cases of breast carcinoma worldwide. Molecular breast carcinoma, like expression estrogen receptor (ER), progesterone receptor (PR), and Human Epidermal Growth Factor Receptor2 (HER2) have a higher sensitivity than morphological feature, like tumour grade and mitotic count as prognostic factors. The grade of the tumor reflects how similar these cells are to normal breast epithelial cells. The dominant growth pathway in normal breasts predominantly involves ER and PR, so tumours with ER and PR positivity show lower grade and proliferation activity. In contrast, tumours with ER and PR negativity often have more severe mutation, like overexpression of HER2. Objective: To analyze the correlation between grade and mitotic count with ER, PR, and HER2 expression. Methods: Analytical observational study with a retrospective design. This study used medical record data from ER, PR, and HER2 immunohistochemical examinations as well as histopathological grade and mitotic data for invasive breast carcinoma of no special type in the Anatomical Pathology Laboratory of Dr. Soetomo Surabaya for the period January 1, 2017 - December 31, 2019. Furthermore, the analysis of the relationship between grade and mitotic count was carried out with the expression of ER, PR, and HER2. Results: There was a significant negative relationship between grade and ER with rs = -0.170 (p = 0.000), the number of mitosis and ER with rs = -0.010 (p = 0.001), grade and PR with rs = -0.168 (p = 0.000), and the number of mitosis and PR with rs = -0.110 (p = 0.000). There was no significant relationship between grade and HER2 (p = 0.470) and the number of mitosis and HER2 (p = 0.279). Conclusion: There is a negative significant relationship between grade and amount of mitosis with ER and PR expression. There is no significant relationship between grade and amount of mitosis with HER2 expression.

Christia vespertilionis extract induced antiproliferation and apoptosis in breast cancer (MCF7) cells.

C. vespertiliomis extracts were evaluated for antiproliferative and apoptosis effect on breast cancer (MCF7) cells. The leaves extracts were analysed for its antiproliferative effect on breast cancer (MCF7) cells and normal epithelial breast (MCF 10A) cells using Sulforhodamine B (SRB) assay. The selective extract was evaluated for its ability to induce apoptosis using Annexin V-FITC apoptosis staining and the expression of molecular genes using qualitative reverse transcription-polymerase chain reaction (RT-PCR) against MCF7 cells. Gas chromatography-mass spectrometry (GC-MS) was used to identify the compounds from the selective extract. The findings showed that dichloromethane fraction (CV-Dcm) extract had high antiproliferative effect against MCF7 cells (IC50 = 24µg/mL, selective index (SI) = 8.17). The percentages of apoptosis cells in CV-Dcm-treated MCF7 cells was 58.8%. The CV-Dcm extract induced downregulation of PCNA level. The apoptotic genes were also triggered in both extrinsic and intrinsic signaling pathways, affecting a 1.5-fold increase in BAX, 1.4-fold increase in cytochrome c, 1.3-fold increase in caspase-8, 1.7-fold increase in caspase-3 and 0.5-fold-decrease in BCL-2. Treated MCF7 cells also activated P53-dependent apoptotic death pathway. The present work strongly suggests that high efficacy of CV-Dcm extract was attributed to its antiproliferative and apoptosis-inducing activation in MCF7 cells, most likely due to its favourable compounds.

Palmatine, a Bioactive Protoberberine Alkaloid Isolated from Berberis cretica, Inhibits the Growth of Human Estrogen Receptor-Positive Breast Cancer Cells and Acts Synergistically and Additively with Doxorubicin.

Palmatine (PLT) is a natural isoquinoline alkaloid that belongs to the class of protoberberines and exhibits a wide spectrum of pharmacological and biological properties, including anti-cancer activity. The aim of our study was to isolate PLT from the roots of Berberis cretica and investigate its cytotoxic and anti-proliferative effects in vitro alone and in combination with doxorubicine (DOX) using human ER+/HER2− breast cancer cell lines. The alkaloid was purified by column chromatography filled with silica gel NP and Sephadex LH-20 resin developed in the mixture of methanol: water (50:50 v/v) that provided high-purity alkaloid for bioactivity studies. The purity of the alkaloid was confirmed by high resolution mass measurement and MS/MS fragmentation analysis in the HPLC-ESI-QTOF-MS/MS-based analysis. It was found that PLT treatment inhibited the viability and proliferation of breast cancer cells in a dose-dependent manner as demonstrated by MTT and BrdU assays. PLT showed a quite similar growth inhibition on breast cancer cells with IC50 values ranging from 5.126 to 5.805 µg/mL. In contrast, growth of normal human breast epithelial cells was not affected by PLT. The growth inhibitory activity of PLT was related to the induction of apoptosis, as determined by Annexin V/PI staining. Moreover, PLT sensitized breast cancer cells to DOX. Isobolographic analysis revealed synergistic and additive interactions between studied agents. Our studies suggest that PLT can be a potential candidate agent for preventing and treating breast cancer.

MiR‑302d‑3p regulates the viability, migration and apoptosis of breast cancer cells through regulating the TMBIM6‑mediated ERK signaling pathway.

MicroRNAs (miRs/miRNAs) play important roles in the occurrence, metastasis and prognosis of multiple types of cancers. However, the specific role of miR-302d-3p and its underlying mechanism in breast cancer (BC) have not yet been reported. The present study aimed to identify the role of miR-302D-3p in BC and its potential mechanism using BC cell lines MCF7 and MDA-MB-231 and normal breast epithelial cell MCF-10A. Cancer and paracancerous tissue from patients with BC were also used. Reverse transcription-quantitative PCR was performed to detect the expression of miR-302d-3p and transmembrane Bax inhibitor motif containing 6 (TMBIM6). Dual-luciferase reporter assays verified the binding sites of miR-302d-3p and TMBIM6. Immunohistochemistry was used to measure the expression of TMBIM6. Cell transfection techniques were used to overexpress or interfere with miR-302d-3p and TMBIM6. A Cell Counting Kit-8 assay was performed to detect cell viability, and migration was measured using a wound healing assay. Apoptosis was detected by flow cytometry. The expression levels of apoptosis-related proteins and pathway-related proteins were detected by western blotting. The expression of miR-302d-3p in BC cell lines was found to be downregulated. It was also demonstrated that miR-302d-3p could inhibit cell viability and migration and promote apoptosis. The expression of TMBIM6 in BC cell lines and tissues was upregulated. Upregulated miR-302d-3p was shown to inhibit viability and migration, and promote apoptosis by targeting TMBIM6, during which extracellular signal-regulated kinase (ERK) and its phosphorylation were inhibited in the ERK signaling pathway in cells. Overall, the present study demonstrated that miR-302d-3p could regulate the viability, migration and apoptosis of BC cells through regulating TMBIM6-mediated ERK signaling pathway.

A Novel Green Preparation of Ag/RGO Nanocomposites with Highly Effective Anticancer Performance.

The efficacy of current cancer therapies is limited due to several factors, including drug resistance and non-specific toxic effects. Due to their tuneable properties, silver nanoparticles (Ag NPs) and graphene derivative-based nanomaterials are now providing new hope to treat cancer with minimum side effects. Here, we report a simple, inexpensive, and eco-friendly protocol for the preparation of silver-reduced graphene oxide nanocomposites (Ag/RGO NCs) using orange peel extract. This work was planned to curtail the use of toxic chemicals, and improve the anticancer performance and cytocompatibility of Ag/RGO NCs. Aqueous extract of orange peels is abundant in phytochemicals that act as reducing and stabilizing agents for the green synthesis of Ag NPs and Ag/RGO NCs from silver nitrate and graphene oxide (GO). Moreover, the flavonoid present in orange peel is a potent anticancer agent. Green-prepared Ag NPs and Ag/RGO NCs were characterized by UV-visible spectrophotometry, transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and dynamic light scattering (DLS). The results of the anticancer study demonstrated that the killing potential of Ag/RGO NCs against human breast cancer (MCF7) and lung cancer (A549) cells was two-fold that of pure Ag NPs. Moreover, the cytocompatibility of Ag/RGO NCs in human normal breast epithelial (MCF10A) cells and normal lung fibroblasts (IMR90) was higher than that of pure Ag NPs. This mechanistic study indicated that Ag/RGO NCs induce toxicity in cancer cells through pro-oxidant reactive oxygen species generation and antioxidant glutathione depletion and provided a novel green synthesis of Ag/RGO NCs with highly effective anticancer performance and better cytocompatibility.