scholarly journals Truncated WT1 protein isoform expression is increased in MCF-7 cells in a long-term estrogen depletion

2019 ◽  
Author(s):  
Saavedra-Alonso Santiago ◽  
Zapata-Benavides Pablo ◽  
Mendoza-Gamboa Edgar ◽  
Chavez-Escamilla Ana Karina ◽  
Arellano-Rodríguez Mariela ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Breast Cancer Cells ◽  
Protein Isoforms ◽  
Breast Cancer Cell Lines ◽  
Rt Pcr ◽  
Estrogen Depletion ◽  
Wt1 Protein ◽  
Mcf 7

AbstractBackgroundThe WT1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer, however, its role during tumor progression is still unknown.MethodologyIn this work were analyzed the expression of WT1 isoforms (36-38 kDa and 52-54 kDa, and 17 AA (+/−) and KTS (+/−)) in breast cancer cells. On the other hand, with the purpose of mimicking the process of switch from a hormone-dependent to a hormone-independent neoplasm, an assay was performed using the MCF-7 cells cultured in long-term estrogen depletion (MCF-7 LTED cells) to determine the WT1 protein isoforms expression by western blot and RT-PCR, and Her2/neu and Estrogen receptor (ER) expression by quantitative RT-PCR assay. Growth kinetics and sensitivity to tamoxifen were performed in the MCF-7 LTED cells by trypan blue exclusion.ResultsThe western blot shows the presence of the 52-54 kDa WT1 isoform in the ER (+) breast cancer cells, but not in the ER (−) cells. The 36-38 kDa WT1 isoform was detected in all the breast cancer cell lines analyzed. Using specific primers was found that 17 AA (+) / KTS (−) WT1 isoform was the most frequent in four breast cancer cell lines. During the sampling of the MCF-7 cells in estrogen depletion, an increase in the short-term of 52-54 kDa WT1 isoform was observed and this was kept until week 13, thereafter, its expression was absent; alternately, the 36-38 kDa WT1 isoform was observed from week 1 and it remained constant until week 27. MCF-7 LTED cells growth kinetic decreased 1.4 folds and were not sensitive to tamoxifen antiproliferative effect (p ≤ 0.05). Finally, were observed an increase of expression of ER and Her2/neu in the MCF-7 LTED cells.ConclusionsThe 36-38 kDa WT1 isoform expression occurs during the modifications of the hormonal environment, suggesting that it may be playing an important role in its adaptation and tumor progression.

scholarly journals Truncated WT1 Protein Isoform Expression Is Increased in MCF-7 Cells with Long-Term Estrogen Depletion

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Saavedra-Alonso Santiago ◽  
Zapata-Benavides Pablo ◽  
Mendoza-Gamboa Edgar ◽  
Chavez-Escamilla Ana Karina ◽  
Arellano-Rodríguez Mariela ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Protein Isoforms ◽  
Protein Isoform ◽  
Estrogen Depletion ◽  
Wt1 Protein ◽  
Cells Cultured ◽  
Mcf 7

Background. The wt1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer; however, its role during tumor progression is still unknown. Methodology. In this work, we analyzed the expression of WT1 isoforms in several breast cancer cells with different tumor marker statuses and an in vitro assay using MCF-7 cells cultured with long-term estrogen depletion (MCF-7 LTED cells) with the finality to mimic the process of switching from hormone-dependent to hormone-independent. Moreover, growth kinetics, sensitivity to tamoxifen, and relative expression analysis of ER and Her2/neu were performed. Results. Initially, the expression of 52-54 kDa protein isoform of WT1 in the breast cancer cell line ER (+) was detected by western blot and was absent in ER (-), and the 36-38 kDa protein isoform of WT1 was detected in all cell lines analyzed. The analysis of alternative splicing by RT-PCR shows that the 17AA (+)/KTS (-) isoform of WT1 was the most frequent in the four cell lines analyzed. In vitro, the MCF-7 cells in the estrogen depletion assay show an increase in the expression of the 52-54 kDa isoform of WT1 in the first 48 hours, and this was maintained until week 13, and later, this expression was decreased, and the 36-38 kDa isoform of WT1 did not show change during the first 48 hours but from week 1 showed an increase of expression, and this remained until week 27. Growth kinetic analysis showed that MCF-7 LTED cells presented a 1.4-fold decrease in cellular proliferation compared to MCF-7 cells cultured under normal conditions. In addition, MCF-7 LTED cells showed a decrease in sensitivity to the antiproliferative effect of tamoxifen ( p ≤ 0.05 ). Samples collected until week 57 analyzed by qRT-PCR showed an increase in the relative expression of the Her2/neu and ER. Conclusions. Modulation of protein isoforms showed differential expression of WT1 isoforms dependent on estrogen receptor. The absence of 52-54 kDa and the presence of the 36-38 kDa protein isoform of WT1 were detected in ER-negative breast cancer cell lines classified as advanced stage cells. Long-term estrogen depletion assay in MCF-7 cells increased the expression of the 36-38 kDa isoform and reduced the 52-54 kDa isoform, and these cells show an increase in the expression of tumor markers of ER and Her2/neu. MCF-7 LTED cells showed low proliferation and insensitivity to tamoxifen compared to MCF-7 cells in normal conditions. These results support the theory about the relationship of the 36-38 kDa isoform of WT1 and the absence of ER function in advanced breast cancer.

scholarly journals miR-17-5p Combined with Epirubicin Inhibits the Progression of Breast Cancer Cells

2020 ◽  
Author(s):  
Lingling Zhao ◽  
Zhongjian Zhu ◽  
Jiang Du ◽  
Yuanyu Zhao ◽  
Dandan Fan
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Mtt Assay ◽  
Breast Cancer Cells ◽  
Luciferase Reporter ◽  
Rt Pcr ◽  
Expression Levels ◽  
Cancer Tissues ◽  
Mcf 7

Abstract Background: Breast cancer is the most common malignant tumor for women, which has been ranked first in women’ s cancer-related death. The objectives of the study are to uncover the underlying mechanisms of combination therapy of epirubicin with miR-17-5p in breast cancer. Methods: The expression levels of miR-17-5p were determined by quantitative RT-PCR. The survival rate of MCF-7 cell was detected respectively by MTT assay. The expression levels of miR-17-5p in MCF-7 cell was tested respectively with Ep via quantitative RT-PCR. miR-17-5p was to be transected with miR-17-5p mimic and negative control of miR-17-5p mimic (NC). Quantitative RT-PCR, MTT assay, flow cytometry assay, western blot for the proliferation and apoptosis related proteins were performed to determine the function of miR-17-5p in breast cancer cells. The bioinformatic programs TargetScan was used to predict the targets for miR-17-5p. Luciferase reporter gene assay system was used to validate and determine the targets of miR-17-5p. The relation between targets protein levels in breast cancer cells was investigated by western blot. Results: The expression levels of miR-17-5p was associated with the breast cancer tissues. The levels of miR-17-5p was down-regulated in breast cancer tissues and cells. Ep could inhibit viability of cancer cells in a concentration dependent manner and promote the expression of miR-17-5p in breast cancer cell lines. Over-expression of miR-17-5p induced cell apoptosis and upregulated the expression of p53, p21 and p27. miR-17-5p co-cultured with Ep is better than the other groups. The relative luciferase activity revealed that STAT3 was a potential target gene of miR-17-5p. Conclusions: Our work will prove that epirubicin regulated the expression of miR-17-5p to strengthen this effect of epirubicin and inhibited the progression of breast cancer.

scholarly journals New Arylethanolimidazole Derivatives as HO-1 Inhibitors with Cytotoxicity against MCF-7 Breast Cancer Cells

2020 ◽  
Vol 21 (6) ◽  
pp. 1923 ◽  
Author(s):  
Valeria Ciaffaglione ◽  
Sebastiano Intagliata ◽  
Valeria Pittalà ◽  
Agostino Marrazzo ◽  
Valeria Sorrenti ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Cells ◽  
Docking Studies ◽  
Hydroxyl Group ◽  
Breast Cancer Cell Lines ◽  
Heme Oxygenase 1 ◽  
Hormone Sensitive ◽  
Hydrophobic Portion ◽  
Resistant Breast Cancer Cell ◽  
Mcf 7

In this paper, a novel series of imidazole-based heme oxygenase-1 (HO-1) inhibitors is reported. These compounds were obtained by modifications of previously described high potent and selective arylethanolimidazoles. In particular, simplification of the central linker and repositioning of the hydrophobic portion were carried out. Results indicate that a hydroxyl group in the central region is crucial for the potency as well as the spatial distribution of the hydrophobic portion. Docking studies revealed a similar interaction of the classical HO-1 inhibitors with the active site of the protein. The most potent and selective compound (5a) was tested for its potential cytotoxic activity against hormone-sensitive and hormone-resistant breast cancer cell lines (MCF-7 and MDA-MB-231).

Short- and long-term cytotoxic effects of doxorubicin conjugates with dendrimers and vector protein on MCF-7/MDR1 chemoresistant breast cancer cells

2017 ◽  
Author(s):  
I. A. Zamulaeva ◽  
O. N. Matchuk ◽  
K. A. Churyukina ◽  
V. A. Kudryavtzev ◽  
N. G. Yabbarov ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Cytotoxic Effects ◽  
Short And Long Term ◽  
Mcf 7

scholarly journals ARTEMISININ MODULATING EFFECT ON HUMAN BREAST CANCER CELL LINES WITH DIFFERENT SENSITIVITY TO CYTOSTATICS

2017 ◽  
Vol 39 (1) ◽  
pp. 25-29 ◽  
Author(s):  
V F Chekhun ◽  
N Yu Lukianova ◽  
T Borikun ◽  
T Zadvornyi ◽  
A Mokhir
Keyword(s):  
Breast Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Iron Homeostasis ◽  
Chemotherapeutic Agents ◽  
Fold Change ◽  
Breast Cancer Cell Lines ◽  
Cellular Iron ◽  
Mcf 7

Aim: To explore effects of Artemisinin on a series of breast cancer cells with different sensitivity to typical cytotoxic drugs (doxorubicin — Dox; cisplatin — DDP) and to investigate possible artemisinin-induced modification of the mechanisms of drug resistance. Materials and Methods: The study was performed on wild-type breast cancer MCF-7 cell line (MCF-7/S) and its two sublines MCF-7/Dox and MCF-7/DDP resistant to Dox and DDP, respectively. The cells were treated with artemisinin and iron-containing magnetic fluid. The latter was added to modulate iron levels in the cells and explore its role in artemisinin-induced effects. The MTT assay was used to monitor cell viability, whereas changes of expression of selected proteins participating in regulation of cellular iron homeostasis were estimated using immunocytochemical methods. Finally, relative expression levels of miRNA-200b, -320a, and -34a were examined by using qRT-PCR. Results: Artemisinin affects mechanisms of the resistance of breast cancer cells towards both Dox and DDP at sub-toxic doses. The former drug induces changes of expression of iron-regulating proteins via different mechanisms, including epigenetic regulation. Particularly, the disturbances in ferritin heavy chain 1, lactoferrin, hepcidin (decrease) and ferroportin (increase) expression (р ≤ 0.05) were established. The most enhanced increase of miRNA expression under artemisinin influence were found for miRNA-200b in MCF-7/DDP cells (7.1 ± 0.98 fold change), miRNA-320a in MCF-7/Dox cells (2.9 ± 0.45 fold change) and miRNA-34a (1.7 ± 0.15 fold change) in MCF-7/S cells. It was observed that the sensitivity to artemisinin can be influenced by changing iron levels in cells. Conclusions: Artemisinin can modify iron metabolism of breast cancer cells by its cytotoxic effect, but also by inducing changes in expression of iron-regulating proteins and microRNAs (miRNAs), involved in their regulation. This modification affects the mechanisms that are implicated in drug-resistance, that makes artemisinin a perspective modulator of cell sensitivity towards chemotherapeutic agents in cancer treatment.

scholarly journals The role of miRNAs 34a, 146a, 320a and 542 in the synergistic anticancer effects of methyl 2-(5-fluoro-2-hydroxyphenyl)-1H- benzo[d]imidazole-5-carboxylate (MBIC) with doxorubicin in breast cancer cells

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5577 ◽  
Author(s):  
Mohadeseh Hasanpourghadi ◽  
Nazia Abdul Majid ◽  
Mohd Rais Mustafa
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Breast Cancer Cells ◽  
Cancer Cell Lines ◽  
Human Breast Cancer Cell ◽  
Breast Cancer Cell Lines ◽  
Human Breast ◽  
Mcf 7

Combination Index (CI) analysis suggested that MBIC and doxorubicin synergistically inhibited up to 97% of cell proliferation in ER+/PR+MCF-7 and triple negative MDA-MB-231 breast cancer cell lines. Moreover, treatment of the breast cancer cells with the combined drugs resulted in lower IC50 values in contrast to the individual drug treatment. Small noncoding microRNAs (miRNA) may function as non-mutational gene regulators at post-transcriptional level of protein synthesis. In the present study, the effect of the combined treatment of MBIC and doxorubicin on the expression level of several miRNAs including miR-34a, miR-146a, miR-320a and miR-542 were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. These miRNAs have the potential to alter the protein level of survivin, the anti-apoptotic protein and reduce the metastatic activity in human breast cancer cell lines by interfering with the nuclear accumulation of NF-κB. Our results demonstrated the several fold changes in expression of miRNAs, which is drug and cell line dependent. This finding demonstrated a functional synergistic network between miR-34a, miR-320a and miR-542 that are negatively involved in post-transcriptional regulation of survivin in MCF-7 cells. While in MDA-MB-231 cells, changes in expression level of miR-146a was correlated with inhibition of the nuclear translocation of NF-κB. The overall result suggested that alteration in protein level and location of survivin and NF-κB by miR-34a, miR-320a, miR-146a and miR-542, remarkably influenced the synergistic enhancement of combined MBIC and doxorubicin in treatment of aggressive and less aggressive human breast cancer cell lines.

scholarly journals HOXD3 Plays a Critical Role in Breast Cancer Stemness and Drug Resistance

2018 ◽  
Vol 46 (4) ◽  
pp. 1737-1747 ◽  
Author(s):  
Yue Zhang ◽  
Qingyuan Zhang ◽  
Zhongru Cao ◽  
Yuanxi Huang ◽  
Shaoqiang Cheng ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Drug Resistance ◽  
Western Blot ◽  
Cell Viability ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Breast Cancer Cells ◽  
Rt Pcr ◽  
Integrin Β3

Background/Aims: Homeobox D3 (HOXD3) is a member of the homeobox family of genes that is known primarily for its transcriptional regulation of morphogenesis in all multicellular organisms. In this study, we sought to explore the role that HOXD3 plays in the stem-like capacity, or stemness, and drug resistance of breast cancer cells. Methods: Expression of HOXD3 in clinical breast samples were examined by RT-PCR and immunohistochemistry. HOXD3 expression in breast cancer cell lines were analyzed by RT-PCR and western blot. Ability of drug resistance in breast cancer cells were elevated by MTT cell viability and colony formation assays. We examined stemness using cell fluorescent staining, RT-PCR and western blot for stem cell marker expression. Finally, activity of wnt signaling was analyzed by FOPflash luciferase assays. RT-PCR and western blot were performed for downstream genes of wnt signaling. Results: We demonstrated that HOXD3 is overexpressed in breast cancer tissue as compared to normal breast tissue. HOXD3 overexpression enhances breast cancer cell drug resistance. Furthermore, HOXD3 upregulation in the same cell lines increased sphere formation as well as the expression levels of stem cell biomarkers, suggesting that HOXD3 does indeed increase breast cancer cell stemness. Because we had previously shown that HOXD3 expression is closely associated with integrin β3 expression in breast cancer patients, we hypothesized that HOXD3 may regulate breast cancer cell stemness and drug resistance through integrin β 3. Cell viability assays showed that integrin β 3 knockdown increased cell viability and that HOXD3 could not restore cancer cell stemness or drug resistance. Given integrin β 3’s relationship with Wnt/β-catenin signaling, we determine whether HOXD3 regulates integrin β 3 activity through Wnt/β-catenin signaling. We found that, even though HOXD3 increased the expression of Wnt/β-catenin downstream genes, it did not restore Wnt/β-catenin signaling activity, which was inhibited in integrin β3 knockdown breast cancer cells. Conclusion: We demonstrate that HOXD3 plays a critical role in breast cancer stemness and drug resistance via integrin β3-mediated Wnt/β-catenin signaling. Our findings open the possibility for improving the current standard of care for breast cancer patients by designing targeted molecular therapies that overcome the barriers of cancer cell stemness and drug resistance.

Effet radiosensibilisateur de l'acide linoléique conjugué chez les cellules cancéreuses du sein MCF-7 et MDA-MB-231

2004 ◽  
Vol 82 (2) ◽  
pp. 94-102 ◽  
Author(s):  
Geneviève Drouin ◽  
Annie Douillette ◽  
Pierre Lacasse ◽  
Benoit Paquette
Keyword(s):  
Breast Cancer ◽  
Linoleic Acid ◽  
Cancer Cells ◽  
Conjugated Linoleic Acid ◽  
Breast Cancer Cells ◽  
Fold Increase ◽  
Breast Cancer Cell Lines ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
Mcf 7

Apoptotic pathways in breast cancer cells are frequently altered, reducing the efficiency of radiotherapy. Conjugated linoleic acid (CLA), known to trigger apoptosis, was tested as radiosensitizer in breast cancer cells MCF-7 and MDA-MB-231. The CLA-mix, made up of the isomers CLA-9cis 11trans and CLA-10trans 12cis, was compared to three purified isomers, i.e., the CLA-9cis 11cis, CLA-9cis 11trans, and CLA-10trans 12cis. Using the apoptotic marker YO-PRO®-1, the CLA-9cis 11cis at 50 µmol/L turned out to be the best apoptotic inducer leading to a 10-fold increase in MCF-7 cells and a 2,5-fold increase in MDA-MB-231 cells, comparatively to the CLA-mix. Contrary to previous studies on colorectal and prostate cancer cells, CLA-10trans 12cis does not lead to an apoptotic response on breast cancer cell lines MCF-7 and MDA-MB-231. Our results also suggest that the main components of the CLA-mix (CLA-9cis 11trans and CLA-10trans 12cis) are not involved in the induction of apoptosis in the breast cancer cells studied. A dose of 5 Gy did not induce apoptosis in MCF-7 and MDA-MB-231 cells. The addition of CLA-9cis 11cis or CLA-mix has allowed us to observe a radiation-induced apoptosis, with the CLA-9cis 11cis being about 8-fold better than the CLA-mix. CLA-9cis 11cis turned out to be the best radiosensitizer, although the isomers CLA-9cis 11trans and CLA-10trans 12cis have also reduced the cell survival following irradiation, but using a mechanism not related to apoptosis. In conclusion, the radiosensitizing property of CLA-9cis 11cis supports its potential as an agent to improve radiotherapy against breast carcinoma.Key words: breast cancer, conjugated linoleic acid (CLA), radiotherapy, apoptosis.

scholarly journals Molecular and chemical characterization by Fourier transform infrared spectroscopy of human breast cancer cells with estrogen receptor expressed and not expressed

2010 ◽  
Vol 24 (5) ◽  
pp. 501-510 ◽  
Author(s):  
Leila Büttner Mostaço-Guidolin ◽  
Luciana Sayuri Murakami ◽  
Marina Ribeiro Batistuti ◽  
Auro Nomizo ◽  
Luciano Bachmann
Keyword(s):  
Breast Cancer ◽  
Fatty Acids ◽  
Estrogen Receptor ◽  
Cell Lines ◽  
Human Breast Cancer ◽  
Breast Cancer Cells ◽  
Chemical Characterization ◽  
Breast Cancer Cell Lines ◽  
Human Breast ◽  
Mcf 7

The present study was designed to identify and compare the infrared absorption spectra of two human breast cancer cell lines: MCF-7 (estrogen receptor expressed, ER+) and SKBr3 (estrogen receptor non-expressed, ER–). Comparison between SKBr3 and MCF-7 cells revealed differences in the following absorption band areas: 1087 cm–1(DNA), 1397 cm–1(CH3), 1543 cm–1(amide II), 1651 cm–1(amide I), 2924 cm–1(fatty acids). Additionally, peak shifts were observed at 1122 cm–1(RNA), 1397 cm–1(CH3), 1651 cm–1(amide I), 2851 cm–1(fatty acids) and 2962 cm–1(fatty acids). An analysis of the ratio between band areas was conducted, in order to obtain an index that could effectively distinguish between these two cell lines. The following ratios were found: 1650 cm–1/1540 cm–1, 1650 cm–1/1740 cm–1, 1650 cm–1/1084 cm–1and 1120 cm–1/1084 cm–1. This work demonstrates that it is possible to distinguish between MCF-7 and SKBr3 cells through differences in their FTIR spectra. This work enables distinction between two cell lines from the same breast cancer.

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