Journal: Theranostics

Article Title: Targeted imaging and inhibition of triple-negative breast cancer metastases by a PDGFRβ aptamer

doi: 10.7150/thno.27798

Figure Lengend Snippet: PDGFRβ aptamer inhibits mesenchymal TNBC cell migration and invasion. (A) MDA-MB-231 and BT-549 (PDGFRβ-positive) cell migration toward 10% FBS was analyzed by transwell migration assay in the presence of 200 nM Gint4.T or Scr for 24 h. (B) Invasion of MDA-MB-231 and BT-549 cells toward 10% FBS through Matrigel was carried out in the presence of 200 nM Gint4.T or Scr for 72 h. (C) Migration and (D) invasion of control BT-474 (PDGFRβ-negative) cells were analyzed as in (A) and (B), respectively. (A-D) Photographs of a representative experiment are shown. Magnification 10 ×, scale bar = 200 μm. Data are presented as percentage of migrated or invaded cells in the presence of Gint4.T compared with Scr control. Bars depict mean ± SD of three independent experiments. *** P < 0.001.

Article Snippet: Growth conditions for human TNBC MDA-MB-231 and BT-549 cells, and HER2-positive breast cancer BT-474 cells (American Type Culture Collection, Manassas, VA) were previously reported .

Techniques: Migration, Transwell Migration Assay

Journal: Theranostics

Article Title: Targeted imaging and inhibition of triple-negative breast cancer metastases by a PDGFRβ aptamer

doi: 10.7150/thno.27798

Figure Lengend Snippet: NIR-Gint4.T aptamer specifically binds to human PDGFRβ-positive TNBC cells. (A) Left panel: Binding curve of NIR-Gint4.T on MDA-MB-231 cells for calculation of the apparent K d of aptamer-cell interaction. Binding was analyzed using flow cytometry. The nonspecific binding value for NIR-Scr sequence was subtracted from every data point. Bars depict mean ± SD of three independent experiments. Right panel: MDA-MB-231 cells were mock-treated or incubated with 25 nM NIR-Gint4.T prior to incubation with 50 nM EC-PDGFRβ for 30 min at RT, and binding was analyzed by flow cytometry. (B) Control BT-474 (PDGFRβ-negative) cells were mock-treated or incubated with 500 nM NIR-Gint4.T or NIR-Scr and binding was analyzed by flow cytometry. (C) Following 5 min incubation with 500 nM NIR-Gint4.T or NIR-Scr, MDA-MB-231 cells were fixed and labeled with anti-PDGFRβ antibody without permeabilization, visualized by confocal microscopy and photographed. Representative 2D and 3D images constructed using Z-scan are shown. NIR-aptamer, PDGFRβ and nuclei are visualized in red, green and blue, respectively. Co-localization results appear yellow in the merged images. (D) Following 5 min incubation with NIR-Gint4.T or NIR-Scr, BT-474 cells were visualized by confocal microscopy and photographed. (A-D) At least three independent experiments were performed. All digital images were captured at the same setting to allow direct comparison of staining patterns. Magnification 63 ×, scale bar = 20 μm. DAPI: 4′,6-Diamidino-2-phenylindole; K d : dissociation constant; MFI: mean fluorescence intensity; NIR: near-infrared; PDGFRβ: platelet-derived growth factor receptor β; 3D: three-dimensional; 2D: two-dimensional.

Article Snippet: Growth conditions for human TNBC MDA-MB-231 and BT-549 cells, and HER2-positive breast cancer BT-474 cells (American Type Culture Collection, Manassas, VA) were previously reported .

Techniques: Binding Assay, Flow Cytometry, Sequencing, Incubation, Labeling, Confocal Microscopy, Construct, Staining, Fluorescence, Derivative Assay

Journal: Theranostics

Article Title: Targeted imaging and inhibition of triple-negative breast cancer metastases by a PDGFRβ aptamer

doi: 10.7150/thno.27798

Figure Lengend Snippet: Selective imaging of PDGFRβ-positive tumors by NIR-Gint4.T. (A) Nude mice bearing MDA-MB-231 xenografts were i.v. injected with 1 nmol of either NIR-Gint4.T or NIR-Scr and analyzed with FMT at the indicated times. Top: Representative volume renderings taken at the same color gating for NIR-Gint4.T and NIR-Scr (n = 5)-injected mice are shown. Bottom, left: The amount of fluorescence (pmol) was quantified in specific VOIs encompassing the tumor in the animal. Bars depict mean ± SD. *** P < 0.001; ** P < 0.01; * P < 0.05 (n = 5). Bottom, right: Shown are representative images of the tumors excised from mice 24 h post NIR-Gint4.T and NIR-Scr injection. (B) Mice bearing PDGFRβ-positive (left flank) or PDGFRβ-negative (right flank) tumors were i.v. injected with 1 nmol of either NIR-Gint4.T (MDA-MB-231 and BT-474 tumors) or NIR-Scr (MDA-MB-231 tumors) and analyzed with FMT at 2 h following injection. (C) Representative images of FMT obtained 2 h after injection of 100 pmol NIR-Gint4.T in the absence (left) or in the presence (right) of pretreatment with a large excess of unlabeled Gint4.T. Notably, the ten-fold reduced amount of NIR-Gint4.T is efficacious to detect the tumor. (D) Analyses of the in vitro serum stability of the NIR-RNAs. Denaturing PAGE of NIR-Gint4.T and NIR-Scr following incubation with 80% human serum at the indicated times. Depicted results represent one of three typical experiments performed. Gint4.T and Scr contain 2´F-Py in all the sequence to increase nuclease resistance. (E) Plasma pharmacokinetic profile of NIR-Gint4.T aptamer. Concentration of aptamer is shown as a function of time following a single i.v. injection in Balb/c mice. Data are presented as the mean ± SD (n = 3 for each administration). Note that we obtained a pharmacokinetic profile of unlabeled Gint4.T aptamer superimposable to that of NIR-Gint4.T. Em: emission; Ex: excitation; NIR: near-infrared; PDGFRβ: platelet-derived growth factor receptor β.

Article Snippet: Growth conditions for human TNBC MDA-MB-231 and BT-549 cells, and HER2-positive breast cancer BT-474 cells (American Type Culture Collection, Manassas, VA) were previously reported .

Techniques: Imaging, Injection, Fluorescence, In Vitro, Incubation, Sequencing, Concentration Assay, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: miR-135b-5p enhances the sensitivity of HER-2 positive breast cancer to trastuzumab via binding to cyclin D2

doi: 10.3892/ijmm.2020.4681

Figure Lengend Snippet: miR-135b-5pagomirenhancesthe anti-proliferation effect oftrastuzumab in HER-2 positive breast cancer cells. (A) The levels of miR-135b-5p in BT-474, SK-BR-3 and MCF-10A cells were evaluated using RT-qPCR. ** P<0.01 vs. MCF-10A. (B) The cells were transfected with either agomir or NC for 48 h. The levels of miR-135b-5p in BT-474 and SK-BR-3 cells after transfection was detected using RT-qPCR. ** P<0.01 vs. blank. Breast cancer cells (C) BT-474 and (D) SK-BR-3 were treated with trastuzumab (0, 0.5, 1, 2 or 4 µ g/ml) and miR-135b-5p agomirfor 48 h. Cell viability was detected using a Cell-Counting Kit 8 assay. * P<0.05 and ** P<0.01 vs. blank. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; OD, optical density.

Article Snippet: HER-2-positive breast cancer cells BT-474 and SK-BR-3 and the normal breast epithelial cell line MCF-10A were obtained from the American Type Culture Collection.

Techniques: Quantitative RT-PCR, Transfection, Cell Counting, Real-time Polymerase Chain Reaction, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: miR-135b-5p enhances the sensitivity of HER-2 positive breast cancer to trastuzumab via binding to cyclin D2

doi: 10.3892/ijmm.2020.4681

Figure Lengend Snippet: miR-135b-5p agomir enhances the anti-proliferation effects of trastuzumab via inducing apoptosis. (A) Annexin V/PI staining in BT-474 cells was detected by flow cytometry. (B) Expression levels of Bax, cleaved caspase 3 and Bcl-2 in BT-474 cells were detected using western blotting. The relative expression levels of (C) Bax, (D) cleaved caspase-3 and (E) Bcl-2 were quantifiedby normalizing to β-actin levels. * P<0.05 and ** P<0.01 vs. control; ## P<0.01 vs. trastuzumab. miR, microRNA; PI, propidium iodide.

Article Snippet: HER-2-positive breast cancer cells BT-474 and SK-BR-3 and the normal breast epithelial cell line MCF-10A were obtained from the American Type Culture Collection.

Techniques: Staining, Flow Cytometry, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: miR-135b-5p enhances the sensitivity of HER-2 positive breast cancer to trastuzumab via binding to cyclin D2

doi: 10.3892/ijmm.2020.4681

Figure Lengend Snippet: Anti-migration and anti-invasion effects of trastuzumab on breast cancer cells is enhanced by miR-135b-5p agomir. BT-474 and SK-BR-3 cells were transfected with 1 µ g/ml trastuzumab, 50 nM miR-135b-5p agomir or a combination of trastuzumab and miR-135b-5p agomir for 24 h. Cell migration assay was performed to evaluate the migration ability of (A) BT-474 and (B) SK-BR-3 cells. Magnification ×200. Cell invasion assay was performed to evaluate the invasion ability of (C) BT-474 and (D) SK-BR-3 cells. Magnification, ×200. * P<0.05 and ** P<0.01 vs. control; ## P<0.01 vs. trastuzumab. miR, microRNA.

Article Snippet: HER-2-positive breast cancer cells BT-474 and SK-BR-3 and the normal breast epithelial cell line MCF-10A were obtained from the American Type Culture Collection.

Techniques: Migration, Transfection, Cell Migration Assay, Invasion Assay

Journal: International Journal of Molecular Medicine

Article Title: miR-135b-5p enhances the sensitivity of HER-2 positive breast cancer to trastuzumab via binding to cyclin D2

doi: 10.3892/ijmm.2020.4681

Figure Lengend Snippet: miR-135b-5pinduces G 0 /G 1 cell cycle arrest in BT-474 cells via directly targeting cyclin D2. (A) The target sequences for miR-135b-5p within the 3′-UTR of cyclin D2. (B) Comparison of luciferase activity in cells of different groups that were co-transfected with wild-type or mutant 3′-UTR cyclin D2. ** P<0.01 vs. control. (C) The levels of cyclin D2 mRNA was evaluated using reverse transcription-quantitative PCR. ** P<0.01 vs. 0 h. (D) Cell cycle analysis was performed to determine the effect of miR-135b-5p agomir on cell cycle distribution. (E) Quantification of cell cycle distribution ratios. * P<0.05 and ** P<0.01 vs. control. 3′UTR, 3′-untranslated region; miR, microRNA.

Article Snippet: HER-2-positive breast cancer cells BT-474 and SK-BR-3 and the normal breast epithelial cell line MCF-10A were obtained from the American Type Culture Collection.

Techniques: Luciferase, Activity Assay, Transfection, Mutagenesis, Real-time Polymerase Chain Reaction, Cell Cycle Assay

Journal: International Journal of Molecular Medicine

Article Title: miR-135b-5p enhances the sensitivity of HER-2 positive breast cancer to trastuzumab via binding to cyclin D2

doi: 10.3892/ijmm.2020.4681

Figure Lengend Snippet: miR-135b-5p agomir regulates cyclin D2, p27 Kip1 and cyclin E1 signaling pathways. BT-474 cells were transfected with 1 µ g/ml trastuzumab, 50 nM miR-135b-5p agomir or a combination of trastuzumab and miR-135b-5p agomir for 48 h. (A) Expression levels of cyclin D2, p27 Kip1 , cyclin E1 and p-Akt in BT-474 cells were detected with western blotting. The relative levels of (B) cyclin D2, (C) p27 Kip1 , (D) cyclin E1 and (E) p-Akt were quantified by normalizing to β-actin levels. (F) BT-474 cells were transfected with miR-135b-5p agomir or NC for 48 h. The levels of cyclin D1 in BT-474 cells was detected using western blotting. (G) Quantification of cyclin D1 expression. (H) BT-474 cells were transfected with NC or pcDNA 3.1-cyclin D2 for 48 h. (I) The level of cyclin D2 in BT-474 cells was detected with western blotting. (J) BT-474 cells were transfected with pcDNA 3.1-cyclin D2 and miR-135b-5p agomir for 48 h in the presence of trastuzumab. The cell viability was detected using a Cell Counting Kit-8 assay. ** P<0.01 vs. control; # P<0.05 and ## P<0.01 vs. trastuzumab; ^^ P<0.01 vs. trastuzumab + miR-125b-3p agomir. p-Akt, phosphorylated Akt; miR, microRNA; OD, optical density; NC, negative control.

Article Snippet: HER-2-positive breast cancer cells BT-474 and SK-BR-3 and the normal breast epithelial cell line MCF-10A were obtained from the American Type Culture Collection.

Techniques: Transfection, Expressing, Western Blot, Cell Counting, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Development and Properties of Valine-Alanine based Antibody-Drug Conjugates with Monomethyl Auristatin E as the Potent Payload

doi: 10.3390/ijms18091860

Figure Lengend Snippet: Cytotoxicity and selectivity of ADCs in tumor cells in vitro. Cytotoxicity assay performed in HER2-positive, weakly positive and negative cell lines. Breast cancer cell lines BT-474 and SK-BR-3, gastric cancer cell line NCI-N87, and ovarian cancer cell line SK-OV-3 were HER2-positive. Breast cancer cell lines MCF-7 and MDA-MB-468 were HER2-weakly positive and -negative, respectively. Each group was established three holes and results are shown as mean ± SD. IC 50 is presented on .

Article Snippet: The HER2-positive breast cancer cell line BT-474, SK-BR-3, gastric cancer cell line NCI-N87, ovarian cancer cell line SK-OV-3, weakly positive breast cancer cell line MCF-7 and HER2-negative breast cancer cell line MDA-MB-468 were obtained from ATCC (Manassas, VA, USA) and maintained in RPMI-1640 medium (Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and Glutamax (Invitrogen).

Techniques: In Vitro, Cytotoxicity Assay

Journal: International Journal of Molecular Sciences

Article Title: Development and Properties of Valine-Alanine based Antibody-Drug Conjugates with Monomethyl Auristatin E as the Potent Payload

doi: 10.3390/ijms18091860

Figure Lengend Snippet: Cytotoxicity of ADCs, antibodies and MMAE in cancer cell lines in vitro.

Article Snippet: The HER2-positive breast cancer cell line BT-474, SK-BR-3, gastric cancer cell line NCI-N87, ovarian cancer cell line SK-OV-3, weakly positive breast cancer cell line MCF-7 and HER2-negative breast cancer cell line MDA-MB-468 were obtained from ATCC (Manassas, VA, USA) and maintained in RPMI-1640 medium (Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and Glutamax (Invitrogen).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Development and Properties of Valine-Alanine based Antibody-Drug Conjugates with Monomethyl Auristatin E as the Potent Payload

doi: 10.3390/ijms18091860

Figure Lengend Snippet: Xenograft studies of VA-based ADC (mil40- 12b ). NOD/SCID mice were implanted subcutaneously with BT-474 cells. When the tumors reached ~150 mm 3 , the animals were given vehicle, the mixture of mil40 and MMAE, and ADCs on: Days 0, 7, 14, and 21 ( A – C ); or Days 0, 7, and 14 ( D ). Results are shown as mean ± SD, n = 6/group. ( A ) Comparison of the efficacy of ADC in the middle dose group with the control group; ( B ) dose-efficacy dependence of the ADC treatment groups; ( C ) changes in body weight of the mice during the observation period; ( D ) comparison of the efficacy of ADCs based on VA and VC dipeptides.

Article Snippet: The HER2-positive breast cancer cell line BT-474, SK-BR-3, gastric cancer cell line NCI-N87, ovarian cancer cell line SK-OV-3, weakly positive breast cancer cell line MCF-7 and HER2-negative breast cancer cell line MDA-MB-468 were obtained from ATCC (Manassas, VA, USA) and maintained in RPMI-1640 medium (Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and Glutamax (Invitrogen).

Techniques: