The Effect of Hypoxic and Normoxic Culturing Conditions in Different Breast Cancer 3D Model Systems

The field of 3D cell cultures is currently emerging, and material development is essential in striving toward mimicking the microenvironment of a native tissue. By using the response of reporter cells to a 3D environment, a comparison between materials can be assessed, allowing optimization of material composition and microenvironment. Of particular interest, the response can be different in a normoxic and hypoxic culturing conditions, which in turn may alter the conclusion regarding a successful recreation of the microenvironment. This study aimed at determining the role of such environments to the conclusion of a better resembling cell culture model to native tissue. Here, the breast cancer cell line MCF7 was cultured in normoxic and hypoxic conditions on patient-derived scaffolds and compared at mRNA and protein levels to cells cultured on 3D printed scaffolds, Matrigel, and conventional 2D plastics. Specifically, a wide range of mRNA targets (40), identified as being regulated upon hypoxia and traditional markers for cell traits (cancer stem cells, epithelial–mesenchymal transition, pluripotency, proliferation, and differentiation), were used together with a selection of corresponding protein targets. 3D cultured cells were vastly different to 2D cultured cells in gene expression and protein levels on the majority of the selected targets in both normoxic and hypoxic culturing conditions. By comparing Matrigel and 3DPS-cultured cells to cells cultured on patient-derived scffolds, differences were also noted along all categories of mRNA targets while specifically for the GLUT3 protein. Overall, cells cultured on patient-derived scaffolds closely resembled cells cultured on 3D printed scaffolds, contrasting 2D and Matrigel-cultured cells, regardless of a normoxic or hypoxic culturing condition. Thus, these data support the use of either a normoxic or hypoxic culturing condition in assays using native tissues as a blueprint to optimize material composition.

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Combined Luteolin and Indole-3-Carbinol Synergistically Constrains ERα-Positive Breast Cancer by Dual Inhibiting Estrogen Receptor Alpha and Cyclin-Dependent Kinase 4/6 Pathway in Cultured Cells and Xenograft Mice

Cancers ◽

10.3390/cancers13092116 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2116

Author(s):

Xiaoyong Wang ◽

Lijuan Zhang ◽

Qi Dai ◽

Hongzong Si ◽

Longyun Zhang ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cell ◽

Breast Cancer Cells ◽

Cultured Cells ◽

Inhibitory Effect ◽

Cyclin Dependent Kinase ◽

Xenograft Mice ◽

The Individual

The high concentrations of individual phytochemicals in vitro studies cannot be physiologically achieved in humans. Our solution for this concentration gap between in vitro and human studies is to combine two or more phytochemicals. We screened 12 phytochemicals by pairwise combining two compounds at a low level to select combinations exerting the synergistic inhibitory effect of breast cancer cell proliferation. A novel combination of luteolin at 30 μM (LUT30) and indole-3-carbinol 40 μM (I3C40) identified that this combination (L30I40) synergistically constrains ERα+ breast cancer cell (MCF7 and T47D) proliferation only, but not triple-negative breast cancer cells. At the same time, the individual LUT30 and I3C40 do not have this anti-proliferative effect in ERα+ breast cancer cells. Moreover, this combination L30I40 does not have toxicity on endothelial cells compared to the current commercial drugs. Similarly, the combination of LUT and I3C (LUT10 mg + I3C10 mg/kg/day) (IP injection) synergistically suppresses tumor growth in MCF7 cells-derived xenograft mice, but the individual LUT (10 mg/kg/day) and I3C (20 mg/kg/day) do not show an inhibitory effect. This combination synergistically downregulates two major therapeutic targets ERα and cyclin dependent kinase (CDK) 4/6/retinoblastoma (Rb) pathway, both in cultured cells and xenograft tumors. These results provide a solid foundation that a combination of LUT and I3C may be a practical approach to treat ERα+ breast cancer cells after clinical trials.

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PARP7 and Mono-ADP-Ribosylation Negatively Regulate Estrogen Receptor α Signaling in Human Breast Cancer Cells

Cells ◽

10.3390/cells10030623 ◽

2021 ◽

Vol 10 (3) ◽

pp. 623

Author(s):

Marit Rasmussen ◽

Susanna Tan ◽

Venkata S. Somisetty ◽

David Hutin ◽

Ninni Elise Olafsen ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Protein Modification ◽

Target Genes ◽

Estrogen Receptor Α ◽

Transactivation Domain ◽

Adp Ribosylation ◽

Protein Levels ◽

17Β Estradiol ◽

Mcf 7

ADP-ribosylation is a post-translational protein modification catalyzed by a family of proteins known as poly-ADP-ribose polymerases. PARP7 (TIPARP; ARTD14) is a mono-ADP-ribosyltransferase involved in several cellular processes, including responses to hypoxia, innate immunity and regulation of nuclear receptors. Since previous studies suggested that PARP7 was regulated by 17β-estradiol, we investigated whether PARP7 regulates estrogen receptor α signaling. We confirmed the 17β-estradiol-dependent increases of PARP7 mRNA and protein levels in MCF-7 cells, and observed recruitment of estrogen receptor α to the promoter of PARP7. Overexpression of PARP7 decreased ligand-dependent estrogen receptor α signaling, while treatment of PARP7 knockout MCF-7 cells with 17β-estradiol resulted in increased expression of and recruitment to estrogen receptor α target genes, in addition to increased proliferation. Co-immunoprecipitation assays revealed that PARP7 mono-ADP-ribosylated estrogen receptor α, and mass spectrometry mapped the modified peptides to the receptor’s ligand-independent transactivation domain. Co-immunoprecipitation with truncated estrogen receptor α variants identified that the hinge region of the receptor is required for PARP7-dependent mono-ADP-ribosylation. These results imply that PARP7-mediated mono-ADP-ribosylation may play an important role in estrogen receptor positive breast cancer.

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The Relevance of the SH2 Domain for c-Src Functionality in Triple-Negative Breast Cancer Cells

Cancers ◽

10.3390/cancers13030462 ◽

2021 ◽

Vol 13 (3) ◽

pp. 462

Author(s):

Víctor Mayoral-Varo ◽

María Pilar Sánchez-Bailón ◽

Annarica Calcabrini ◽

Marta García-Hernández ◽

Valerio Frezza ◽

...

Keyword(s):

Breast Cancer ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Sh2 Domain ◽

Model Systems ◽

System A ◽

Promising Therapeutic Target ◽

Relevant Role ◽

Inactivating Mutation

The role of Src family kinases (SFKs) in human tumors has been always associated with tyrosine kinase activity and much less attention has been given to the SH2 and SH3 adapter domains. Here, we studied the role of the c-Src-SH2 domain in triple-negative breast cancer (TNBC). To this end, SUM159PT and MDA-MB-231 human cell lines were employed as model systems. These cells conditionally expressed, under tetracycline control (Tet-On system), a c-Src variant with point-inactivating mutation of the SH2 adapter domain (R175L). The expression of this mutant reduced the self-renewal capability of the enriched population of breast cancer stem cells (BCSCs), demonstrating the importance of the SH2 adapter domain of c-Src in the mammary gland carcinogenesis. In addition, the analysis of anchorage-independent growth, proliferation, migration, and invasiveness, all processes associated with tumorigenesis, showed that the SH2 domain of c-Src plays a very relevant role in their regulation. Furthermore, the transfection of two different aptamers directed to SH2-c-Src in both SUM159PT and MDA-MB-231 cells induced inhibition of their proliferation, migration, and invasiveness, strengthening the hypothesis that this domain is highly involved in TNBC tumorigenesis. Therefore, the SH2 domain of c-Src could be a promising therapeutic target and combined treatments with inhibitors of c-Src kinase enzymatic activity may represent a new therapeutic strategy for patients with TNBC, whose prognosis is currently very negative.

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DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

Biochemical Journal ◽

10.1042/bj20121085 ◽

2013 ◽

Vol 451 (3) ◽

pp. 453-461 ◽

Cited By ~ 26

Author(s):

Claudia C. S. Chini ◽

Carlos Escande ◽

Veronica Nin ◽

Eduardo N. Chini

Keyword(s):

Breast Cancer ◽

Nuclear Receptor ◽

Clock Genes ◽

Mrna Levels ◽

Protein Levels ◽

The Stability ◽

And Function ◽

Interfering Rna ◽

In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

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Elevated serum acute phase protein levels as predictors of disseminated breast cancer

Cancer ◽

10.1002/1097-0142(19830601)51:11<2100::aid-cncr2820511124>3.0.co;2-j ◽

1983 ◽

Vol 51 (11) ◽

pp. 2100-2104 ◽

Cited By ~ 30

Author(s):

Donna K. Thompson ◽

James E. Haddow ◽

Dwight E. Smith ◽

Robert F. Ritchie

Keyword(s):

Breast Cancer ◽

Acute Phase ◽

Acute Phase Protein ◽

Elevated Serum ◽

Protein Levels ◽

Phase Protein

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Ionic Dissolution Products of NovaBone® Promote Osteoblastic Proliferation via Influences on the Cell Cycle

Journal of International Medical Research ◽

10.1177/147323000903700317 ◽

2009 ◽

Vol 37 (3) ◽

pp. 737-745 ◽

Cited By ~ 4

Author(s):

Z Qiu ◽

H Yang ◽

J Wu ◽

L Wei ◽

J Li

Keyword(s):

Cell Cycle ◽

Cell Cycle Regulation ◽

Cultured Cells ◽

Bone Morphogenic Protein ◽

Mg63 Cells ◽

Silicon Ions ◽

Cycle Regulation ◽

Bone Morphogenic Protein 2 ◽

Cells Cultured

This study investigated the effects of the ionic dissolution products of NovaBone® on osteoblastic proliferation and cell cycle regulation. MG63 osteoblast-like cells were cultured in NovaBone®-conditioned Dulbecco's Modified Eagle's Medium (DMEM) or control DMEM for 10 days. The concentration of silicon ions was significantly higher in NovaBone®-conditioned DMEM than control DMEM. MG63 cells cultured in NovaBone®-conditioned DMEM exhibited greater proliferation on days 1 and 4 than control cells. There were increased proportions of Novabone®-conditioned DMEM-cultured cells in the S and G2/M phases, and decreased proportions in the G0/G1 phase on days 1 and 4 versus control cells, while no differences were observed on days 7 and 10 between the two groups. Bone morphogenic protein 2 production increased in both groups, but was significantly higher for the NovaBone®-conditioned DMEM-cultured cells on day 10 compared with the controls. In conclusion, the NovaBone® ionic dissolution products, particularly the silicon ions, promoted proliferation of MG63 osteoblast-like cells in vitro via influences on the cell cycle.

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Discussion On The Mechanism of Que In The Treatment of Breast Neoplasms And Immune Prevention And Treatment

10.21203/rs.3.rs-819024/v1 ◽

2021 ◽

Author(s):

Dan Qiu ◽

Xianxin Yan ◽

Xinqin Xiao ◽

Guijuan Zhang ◽

Yanqiu Wang ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

T Cell ◽

Breast Neoplasms ◽

Signaling Pathway ◽

Immune Regulation ◽

Γδ T Cells ◽

Protein Levels ◽

Stat1 Signaling ◽

Mcf 7

Abstract Background: The precancerous disease of breast cancer is an inevitable stage in the emergence and development of breast neoplasms. Breast cancer (BC) is a common malignant tumor in female worldwide. A large number of literatures have proved that, as antitumor drugs, flavonoid compounds can promote proliferation and immune regulation of T cell. Many researchers believe that Quercetin (Que) has great potential in the field of anti-breast cancer. Besides that, γδ T cells are a class of non-traditional T cells, which have long attracted attention due to their potential in immunotherapy. Above all, JAK/STAT1 signaling pathway is closely related to the immunity.MethodsIn the experiment designed in this paper, we first used Que, one of the flavonoids, to screen the target gene. Then, MCF-10A, MCF-10AT, MCF-7 and MDA-MB231 BC cells were co-cultured with Que for 24h and 48h, apoptosis was found in some the cells. We then cultured Que with γδ T cells and found that Que can promote the proliferation of Vδ2 T cell subsets of γδ T cells, thus enhancing the killing effect of γδ T cells. Western blot was use to showed the change of JAK/STAT1 signaling pathway related proteins after the Que was co-cultured with MCF-10AT and MCF-7 for 48h.ResultsNetwork pharmacology has shown that Que related pathways include the JAK/STAT1 signaling pathway and are associated with precancerous breast cancers. Que induced apoptosis of MCF-10AT, MCF-7 and MDA-MB-231 in a time and concentration-dependent manner. Most importantly, Que can promote the differentiation of γδ T cells into the Vδ2 T cell subpopulation, this means that Que and γδ T cells may play a synergistic role in killing tumor cells and cellular immune regulation. In addition, our results showed that Que can increase in protein levels of IFNγ-R, p-JAK2 and p-STAT1, while the concomitant decrease protein levels of PD-L1.ConclusionsIn conclusion, Que plays a synergistic role in killing BC cells and promoting apoptosis by regulating the expression of IFNγ-R, p-JAK2, p-STAT1, and PD-L1 in the JAK/STAT1 signaling pathway and promoting the regulation of γδ T cells. Que may be a potential drug for the prevention of precancerous breast cancer and adjuvant treatment of BC.

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Dihydrotestosterone induces chemo-resistance of triple-negative breast MDA-MB-231 cancer cells towards doxorubicin independent of ABCG2 and miR-328-3p

Current Molecular Pharmacology ◽

10.2174/1874467214666210531170355 ◽

2021 ◽

Vol 14 ◽

Author(s):

Bayan Al-Momany ◽

Hana Hammad ◽

Mamoun Ahram

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Drug Response ◽

Therapeutic Strategy ◽

Triple Negative ◽

Model System ◽

Cell Resistance ◽

Protein Levels ◽

Abcg2 Protein ◽

Mcf 7

Background: Androgens potentially have an important role in the biology of breast cancer, particularly triple-negative breast cancer (TNBC). Androgen receptor (AR) may offer a novel therapeutic strategy including the use of microRNA (miRNA) molecules. We have previously shown that AR agonist, dihydrotestosterone (DHT), increases the expression of miR-328-3p in the TNBC MDA-MB-231 cells. One target of the latter miRNA is ATP-binding cassette subfamily G member 2 (ABCG2), which modulates the chemo-response of cancer cells by pumping out xenobiotics. Objective: Using MDA-MB-231 cells as a model system for TNBC, we hypothesized that DHT would induce cell sensitivity towards doxorubicin via increasing levels of miR-328-3p and, consequently, reducing ABCG2 levels. Methods: Chemo-response of cells towards doxorubicin, tamoxifen, and mitoxantrone was evaluated using cell viability MTT assay. Cells were transfected with both miR-328-3p mimic or antisense molecules. Real-time PCR was utilized to assess RNA levels and immunoblotting was performed to investigate levels of ABCG2 protein. PCR arrays were used to assess changes in the expression of drug response regulatory genes. Results: Contrary to our hypothesis, treating MDA-MB-231 cells with DHT, no effect towards tamoxifen or mitoxantrone and increased cell resistance towards doxorubicin were noted, concomitant with decreased expression of ABCG2. This under-expression of ABCG2 was also found in MCF-7 and MDA-MB-453 cells treated with DHT. Although miR-328-3p decreased ABCG2 mRNA and protein levels, the miRNA did not alter the chemo-response of cells towards doxorubicin and did not affect DHT-induced chemo-resistance. AR activation slightly decreased the expression of 5 genes, including insulin-like growth factor 1 receptor, that may explain the mechanism of DHT-induced chemo-resistance of cells. Conclusion: DHT regulates chemo-response via a mechanism independent of ABCG2 and miR-328-3p.

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