[Metformin inhibits proliferation and promotes apoptosis of HER-2 positive breast cancer cells possibly through the Hippo-YAP pathway].

OBJECTIVE: To investigate the effect of metformin on the proliferation and apoptosis of HER-2-positive breast cancer cell line SKBR3 and explore the possible mechanism of its action. METHODS: SKBR3 cells were treated with different concentrations (20-120 µmol/L) of metformin, and the changes in cell proliferation and colony formation ability were assessed using CCK-8 assay and crystal violet staining, respectively. Flow cytometry was performed to analyze cell apoptosis and cell cycle changes. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect mRNA expressions of YAP, TAZ, EGFR, CTGF, CYR61, E-cadherin, N-cadherin, vimentin and fibronectin in the treated cells, and the protein expressions of YAP and TAZ were detected using Western blotting; immunofluorescence assay was used to observe YAP/TAZ nuclear translocation in the cells. RESULTS: Metformin treatment significantly inhibited the proliferation of SKBR3 cells (P < 0.05) in a concentration- and time-dependent manner. The results of flow cytometry showed that metformin significantly promoted apoptosis and caused cell cycle arrest at G1 phase in SKBR3 cells. Metformin treatment significantly down-regulated the mRNA expressions of YAP, TAZ, EGFR, CTGF and CYR61, N-cadherin, vimentin and fibronectin (P < 0.05) and up-regulated the expression of E-cadherin (P < 0.05); Western blotting results showed that YAP and TAZ protein expressions were significantly down-regulated in the cells after metformin treatment (P < 0.05). Immunofluorescence assay revealed that metformin treatment caused the concentration of YAP and TAZ in the cytoplasm, and significantly reduced their amount in the cell nucleus. CONCLUSION: Metformin can inhibit proliferation and promote apoptosis and epithelal-mesenchymal transition of HER-2 positive breast cancer cells possibly by that inhibing YAP and TAZ expression and their nuclear localization.

Metformin inhibits proliferation and promotes apoptosis of HER-2 positive breast cancer cells possibly through the Hippo-YAP pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of metformin on the proliferation and apoptosis of HER-2-positive breast cancer cell line SKBR3 and explore the possible mechanism of its action.@*METHODS@#SKBR3 cells were treated with different concentrations (20-120 μmol/L) of metformin, and the changes in cell proliferation and colony formation ability were assessed using CCK-8 assay and crystal violet staining, respectively. Flow cytometry was performed to analyze cell apoptosis and cell cycle changes. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect mRNA expressions of YAP, TAZ, EGFR, CTGF, CYR61, E-cadherin, N-cadherin, vimentin and fibronectin in the treated cells, and the protein expressions of YAP and TAZ were detected using Western blotting; immunofluorescence assay was used to observe YAP/TAZ nuclear translocation in the cells.@*RESULTS@#Metformin treatment significantly inhibited the proliferation of SKBR3 cells (P < 0.05) in a concentration- and time-dependent manner. The results of flow cytometry showed that metformin significantly promoted apoptosis and caused cell cycle arrest at G1 phase in SKBR3 cells. Metformin treatment significantly down-regulated the mRNA expressions of YAP, TAZ, EGFR, CTGF and CYR61, N-cadherin, vimentin and fibronectin (P < 0.05) and up-regulated the expression of E-cadherin (P < 0.05); Western blotting results showed that YAP and TAZ protein expressions were significantly down-regulated in the cells after metformin treatment (P < 0.05). Immunofluorescence assay revealed that metformin treatment caused the concentration of YAP and TAZ in the cytoplasm, and significantly reduced their amount in the cell nucleus.@*CONCLUSION@#Metformin can inhibit proliferation and promote apoptosis and epithelal-mesenchymal transition of HER-2 positive breast cancer cells possibly by that inhibing YAP and TAZ expression and their nuclear localization.

A highly sensitive electrochemiluminescence cytosensor for detection of SKBR-3 cells as metastatic breast cancer cell line: A constructive phase in early and precise diagnosis.

Ultrasensitive monitoring of cancer cells, especially metastatic ones, has a great interest in human medicine. Despite the early diagnosis of diseases, there is an essential need for any prediction in the severity of side effects for therapeutic outcomes like metastasis. Therefore, the inhibition of cancer cells metastasis to other organs is of utmost importance for cancer suffering patients. In this regard, we developed an electrochemiluminescence (ECL)-based cytosensor for the quantification of metastatic breast cancer cells, namely SKBR-3. Silica-based mesoporous materials have a great potential for application in ECL biosensors due to their high loading capacity and mechanical strength. Herein, a silica-based electrode was prepared via in situ electrosyntheses of mesoporous silica as an environmentally friendly approach. In this protocol, luminol (as luminophore) was combined with chitosan (as attachment biomolecule) to produce a stable lumino-composite film on the electrode surface. At the optimum experimental conditions, the lower limit of quantitation (LLOQ) and linear dynamic range (LDR) were obtained as 20 cells/mL and 20 to 2000 cells/mL, individually. The specificity was desirably examined in the presence of other breast cancer cell lines such as MCF-7 and MDA-MB-231, as a model of early-stage and invasive phases of breast cancer cells. The repeatability was successfully examined for five repetitive measurements and the acceptable relative standard deviation (RSD) was calculated as about 1.6% for 500 cells/mL. As a proof of concept, the presented cytosensor has a high ability to use in clinical laboratories for the detection and separation of metastatic cells via the combination with microfluidic systems.

Molecular mechanism study of BPAF-induced proliferation of ERα-negative SKBR-3 human breast cancer cells in vitro/in vivo.

Bisphenol AF (BPAF) is a known estrogen disruptor of the ERα pathway. The aim of the present study was to characterize the proliferation effects of BPAF on ERα-negative SKBR-3 breast cancer cells with mechanistic insights. BPAF at low concentrations (0.001-0.1 µM) significantly induced the proliferation of SKBR-3 cells. In a SKBR-3 tumor model in BALB/c nude mice, BPAF at 100 mg/kg body weight/day also significantly promoted the growth of SKBR-3 tumors. Low concentrations of BPAF markedly increased the expression of G protein-coupled estrogen receptor (GPER1), c-Myc, CyclinD1 and c-Fos proteins, and enhanced phosphorylation of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in SKBR-3 cells. Further, BPAF significantly upregulated mRNA levels of related target genes in SKBR-3 cells and SKBR-3 tumor tissues in nude mice. The GPER1 inhibitor G15 and phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin (WM) inhibited phosphorylation of Erk and Akt. The specific signal inhibitors also markedly decreased the expression of target genes and weakened the cell proliferation induced by low-concentration BPAF. The findings showed that GPER1 could independently regulate BPAF-induced proliferation of SKBR-3 cells without requiring ERα. These results provide mechanistic insights into the effects of BPAF regarding ERα-negative human breast cancer development.

Studies on polyethylene glycol-monoclonal antibody conjugates for fabrication of nanoparticles for biomedical applications.

OBJECTIVE: The objective of this work is to synthesize and characterize PEGylated monoclonal antibody using the reactivity of oligosaccharide residues in the Fc region of trastuzumab and pertuzumab with a view to preserving their activities. METHODS: The hydrazide-functionalized PEG monomethacrylate was synthesized and reacted with NaIO4-generated aldehyde groups on glycans in the Fc-domain of trastuzumab and pertuzumab. The conjugates were purified by HPLC. SAMSA-fluorescein substitution method and MALDI MS spectroscopy were used to determine the number of PEG per antibody. Preliminary biological studies involved antiproliferative studies and binding (flow cytometry) following treatments with SKBR3 (HER2-overexpressing) cells and the control. RESULTS: 1H NMR and 13C NMR confirmed the formation of hydrazide-functionalized PEG monomethacrylate. MALDI mass-spectrometry showed that there are two PEGs per each antibody and it appears more reliable than the degree of SAMSA-fluorescein substitution method. HER-2 binding assay showed that PEGylated monoclonal antibody bound less efficiently to SKBR3 (high HER-2 expressing) cells than unmodified trastuzumab and pertuzumab. In vitro growth inhibitory effects of unmodified monoclonal antibodies increased with increase in concentration; while the in vitro growth inhibitory effects of PEGylated monoclonal antibodies also increased (but less than the pure antibody) with concentration and it appeared to be more active than unmodified mAbs at higher concentration. CONCLUSION: The results indicate that PEG can be site-specifically attached via the oxidized glycans in the Fc domain of monoclonal antibodies but the process needs further optimization in terms of PEG size and biological testing at each stage of development.

Bioconjugated graphene oxide-based Raman probe for selective identification of SKBR3 breast cancer cells.

In this article, we demonstrate the use of bio-conjugated 2D graphene oxide (bio-GO) nanostructure to probe breast cancer cell (SKBR3) with excellent discrimination over other types of circulating tumor cells. We distinctly observed that bio-GO nanostructure targets and bind SKBR3 cell selectively in the cell mixture. Longer incubation of SKBR3 cell with bio-GO causes Raman signal "turn off" when excited with 532 nm laser. This is attributed to penetration of the bio-GO through the plasma membrane of the cell by generating transient hole. Extraction of GO after cell digestion also support the internalization rubric of 2D graphene through cell membrane. Our experimental data with the HaCaT healthy cell line, as well as with LNCaP prostate cancer cell line clearly demonstrated that this Raman scattering assay is highly selective to SKBR3. The mechanism of selectivity and the assay's response change have been verified and discussed utilizing fluorescence properties of GO and various other techniques. The experimental results open up a possibility of new label free Raman scattering assay, for reliable diagnosis of cancer cell lines by monitoring "turn-off" of the Raman signal from Bio-GO nanostructure.

Sensitizing effect of juglone is mediated by down regulation of Notch1 signaling pathway in trastuzumab-resistant SKBR3 cells.

Trastuzumab (Herceptin) monoclonal antibody directed against HER2 receptor has been administered as a treatment for metastatic HER2 positive breast cancer. The problematic issue in treatment of HER2 positive breast cancer cells is commonly the induction of resistance to trastuzumab which might be due to modulation of some vital signaling elements such as Notch1 and Pin1. In this study, we were aimed to investigate whether the cross talk between pin1 and Notch1 has a role in this event. Our results indicated that the expression level of Pin1 in resistant SKBR3 cells increased by about twofold relative to sensitive SKBR3 cells. Besides, Pin1 inhibition via juglone reduced the extent of proliferation, colony formation and migration capacity of resistant SKBR3 cells. In addition, despite a feed forward loop between Notch1 and Pin1 in sensitive SKBR3 cells, inhibition of Notch1 cleavage in resistant SKBR3 cells did not affect pin1 level whereas pin1 inhibition by juglone reduced the level of Hes1, p-Akt and increased the cellular content of Numb. Therefore, we concluded that pin1 inhibition could be considered as a promising sensitizing strategy to weaken trastuzumab resistance.

Inhibitory effect of curcumin on human breast cancer cell line SKBR3 and its mechanism / 中国生化药物杂志

Objective To observe the effect of curcumin on proliferation, apoptosis, Caspase-3 activity and telomerase activity of chronic granulocytic breast cancer SKBR3 cell line, and to investigate the mechanism of curcumin. Methods The cell growth curve was drawn by MTT;the apoptotic cells were detected by Annexin, V and PI double staining; the expression of Caspase-3 was detected by Westernblot; the activity of telomerase was detected by TRAP-PCR silver staining. Results Curcumin could significantly inhibit the proliferation of SKBR3 cells in a time and concentration dependent, has a certain effect on reducing the telomerase activity of SKBR3 cells can enhance the expression of Caspase-3 induced apoptosis. Conclusion Curcumin can effectively inhibit the proliferation of SKBR3 cells, increase the synthesis of Caspase-3 protein, inhibit the activity of telomerase and mediate the apoptosis of SKBR3 cells.

Inhibitory effect of curcumin on human breast cancer cell line SKBR3 and its mechanism / 中国生化药物杂志

Objective To observe the effect of curcumin on proliferation, apoptosis, Caspase-3 activity and telomerase activity of chronic granulocytic breast cancer SKBR3 cell line, and to investigate the mechanism of curcumin. Methods The cell growth curve was drawn by MTT;the apoptotic cells were detected by Annexin, V and PI double staining; the expression of Caspase-3 was detected by Westernblot; the activity of telomerase was detected by TRAP-PCR silver staining. Results Curcumin could significantly inhibit the proliferation of SKBR3 cells in a time and concentration dependent, has a certain effect on reducing the telomerase activity of SKBR3 cells can enhance the expression of Caspase-3 induced apoptosis. Conclusion Curcumin can effectively inhibit the proliferation of SKBR3 cells, increase the synthesis of Caspase-3 protein, inhibit the activity of telomerase and mediate the apoptosis of SKBR3 cells.

Grb7 and Hax1 may colocalize partially to mitochondria in EGF-treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1.

Growth factor receptor bound protein 7 (Grb7) is a signal-transducing adaptor protein that mediates specific protein-protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein-binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras-GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti-apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor-treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time-dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright © 2016 John Wiley & Sons, Ltd.

Involvement of Numb-mediated HIF-1α inhibition in anti-proliferative effect of PNA-antimiR-182 in trastuzumab-sensitive and -resistant SKBR3 cells.

Trastuzumab is a humanized monoclonal antibody against the human epidermal growth factor receptor 2 (HER2) that is overexpressed in about 25 % of breast cancer patients. However, primary and/or acquired resistance to trastuzumab develops in most affected persons. In this study, we explored the functional role of miR-182 inhibition with aiming the sensitization of SKBR3 cells to trastuzumab. Cell viability, apoptosis, colony formation, and migration capacities of SKBR3(S) (sensitive) and SKBR3(R) (resistant) cells were assessed to determine the anti-proliferative effects of PNA-antimiR-182. In addition, the expression levels of miR-182, mRNA of FOXO1, and Bim as well as the protein levels of HER2 and Notch1 signaling factors were evaluated by stem-loop RT-qPCR, RT-qPCR, and Western blot, respectively. The results indicated that miR-182 might play a causal role in the mechanism of trastuzumab. In line with that, PNA-antimiR-182 inhibited synergistically the viability of both the sensitive and resistant cell groups. Furthermore, the inhibitory effect of PNA-anitmiR-182 on migration in SKBR3 cells was more than the induction of apoptosis. In addition, PNA-antimiR-182 reduced the levels of NICD, Hes1, HIF-1α, and p-Akt in both cell groups, while it augmented the intracellular content of FOXO1 and Numb suppressor proteins. In other words, PNA-antimiR-182-mediated upregulation of Numb was associated with downregulation of HIF-1α and Hes1. Consequently, downregulation of miR-182 might find therapeutical value for overcoming trastuzumab resistance. Graphical Abstract The crosstalk between HER2 and Notch1 signaling pathway is mediated by miR-182.

Local variations of HER2 dimerization in breast cancer cells discovered by correlative fluorescence and liquid electron microscopy.

The formation of HER2 homodimers plays an important role in breast cancer aggressiveness and progression; however, little is known about its localization. We have studied the intra- and intercellular variation of HER2 at the single-molecule level in intact SKBR3 breast cancer cells. Whole cells were visualized in hydrated state with correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM). The locations of individual HER2 receptors were detected using an anti-HER2 affibody in combination with a quantum dot (QD), a fluorescent nanoparticle. Fluorescence microscopy revealed considerable differences of HER2 membrane expression between individual cells and between different membrane regions of the same cell (that is, membrane ruffles and flat areas). Subsequent ESEM of the corresponding cellular regions provided images of individually labeled HER2 receptors. The high spatial resolution of 3 nm and the close proximity between the QD and the receptor allowed quantifying the stoichiometry of HER2 complexes, distinguishing between monomers, dimers, and higher-order clusters. Downstream data analysis based on calculating the pair correlation function from receptor positions showed that cellular regions exhibiting membrane ruffles contained a substantial fraction of HER2 in homodimeric state. Larger-order clusters were also present. Membrane areas with homogeneous membrane topography, on the contrary, displayed HER2 in random distribution. Second, HER2 homodimers appeared to be absent from a small subpopulation of cells exhibiting a flat membrane topography, possibly resting cells. Local differences in homodimer presence may point toward functional differences with possible relevance for studying metastasis and drug response.

Lead Optimization of 2-Cyclohexyl-N-[(Z)-(3-methoxyphenyl/3-hydroxyphenyl) methylidene]hydrazinecarbothioamides for Targeting the HER-2 Overexpressed Breast Cancer Cell Line SKBr-3.

Lead derivatives of 2-cyclohexyl-N-[(Z)-(3-methoxyphenyl/3-hydroxyphenyl) methylidene]hydrazinecarbothioamides 1-18 were synthesized, characterized and evaluated in vitro against HER-2 overexpressed breast cancer cell line SKBr-3. All the compounds showed activity against HER-2 overexpressed SKBr-3 cells with IC50 = 17.44 ± 0.01 µM to 53.29 ± 0.33 µM. (2Z)-2-(3-Hydroxybenzylidene)-N-(3-methoxyphenyl)hydrazinecarbothioamide (12, IC50 = 17.44 ± 0.01 µM) was found to be most potent compound of this series targeting HER-2 overexpressed breast cancer cells compared to the standard drug 5-fluorouracil (5-FU) (IC50 = 38.58 ± 0.04 µM). Compound 12 inhibited the cellular proliferation via DNA degradation.

Inhibitory effects of O-methylated isoflavone glycitein on human breast cancer SKBR-3 cells.

Glycitein is an O-methylated isoflavone which accounts for 5-10% of the total isoflavones in soy food products. Cell proliferation studies on the dietary phytoestrogen, glycitein against human breast carcinoma SKBR-3 cells showed that glycitein exhibits biphasic regulation on SKBR-3 cells. At concentrations of less than 10 mg/mL, cells respond to glycitein by increasing cell growth and de novo DNA synthesis whereas the addition of glycitein at concentrations greater than 30 mg/mL significantly inhibited cell growth and DNA synthesis in a dose-dependent manner. Cells treated with 60 mg/mL of glycitein did not regain normal growth after treatment was stopped. Glycitein was found to be cytostatic at low concentrations and cytotoxic at higher concentrations. Treatment with 100 mg/mL of glycitein severely altered the cell morphology. Collective results showed that glycitein damaged the cell membranes by increasing membrane permeability and suggested possible mechanisms of the action of dietary phytoestrogens on human breast carcinoma SKBR-3 cells.

Differential behaviors of trastuzumab-sensitive and -resistant SKBR3 cells treated with menadione reveal the involvement of Notch1/Akt/FOXO1 signaling elements.

Given that HER2 serves as a putative target for therapy in HER2-positive breast cancer, intrinsic and/or acquired resistance to trastuzumab (T) has been proposed to be the major obstacle in treatments. In addition, chemoresistance is commonly attributed to increased antioxidant capacity. In that regard, we evaluated the effect of menadione (M) alone and/or its combination with trastuzumab on proliferation, intracellular GSH and ROS contents as well as HER2 and Notch1 signaling pathways in both trastuzumab-resistant (SKBR3(R)) and -sensitive SKBR3 (SKBR3(S)) cells. In spite of increased level of ROS and reduced level of GSH in M-treated SKBR3(S) cells, M-treated SKBR3(R) cells showed a decreased content of ROS and GSH compared to untreated cells. However, M/T co-treatment of SKBR3 cells indicated no effect on ROS content, while decreased the level of GSH compared to untreated control cells. Based on the extent of apoptosis, colony formation and wound healing assays, M alone, and/or in combination with T had a stronger inhibitory effect on proliferation of SKBR3(R) cells relative to SKBR3(S) cells. These effects might be due to the stronger effects of M and/or M/T on downregulation of p-Akt, Hes1, NICD, and upregulation of FOXO1 among SKBR3(R) cells relative to the sensitive SKBR3 cells. These findings would certainly shed light on some of the signaling factors involved in induction of trastuzumab resistance and would be of value in designing more efficient chemosensitization strategies.

Activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs in a human cell line: difference in specificity between human and sea urchin NAADP receptors.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger that has been identified. We have previously shown that NAADP analogs substituted at the 5-position of nicotinic acid were recognized by the sea urchin receptor at low concentration, whereas the 4- substituted analogs were not as potent. However, to date the structure-activity relationship (SAR) of these analogs has not been addressed in mammalian systems. Thus, we asked whether these structurally modified analogs behave similarly in an NAADP-responsive mammalian cell line (SKBR3) using microinjection and single cell fluorescent imaging methods. Novel "caged" 4- and 5-substituted NAADP analogs that were activated inside the cell by flash photolysis resulted in Ca2+ mobilizing activity in SKBR3 cells in a concentration dependent manner, but with reduced effectiveness compared to unmodified NAADP. The SAR in mammalian SKBR3 cells was quite different from that of sea urchin and may suggest that there are differences between NAADP receptors in different species or tissues. Importantly, these data indicate that modifications at the 4- and 5-position of the nicotinic acid ring may lead to the development of functional photoaffinity labels that could be used for receptor localization and isolation in mammalian systems.