scholarly journals Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion

2021 ◽  
Vol 22 (12) ◽  
pp. 6483
Author(s):  
Sangsu Park ◽  
Minh Quan Nguyen ◽  
Huynh Kim Khanh Ta ◽  
Minh Tan Nguyen ◽  
Gunsup Lee ◽  
...  
Keyword(s):  
Cell Lines ◽  
Binding Protein ◽  
Metastatic Breast ◽  
Maltose Binding Protein ◽  
Soluble Form ◽  
Breast Cancer Cell Lines ◽  
Protein Fusion ◽  
Single Chain ◽  
Expression And Purification ◽  
Her2 Positive

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.

scholarly journals Response of Breast Cancer Cells to PARP Inhibitors Is Independent of BRCA Status

2020 ◽  
Vol 9 (4) ◽  
pp. 940 ◽  
Author(s):  
Man Yee Keung ◽  
Yanyuan Wu ◽  
Francesca Badar ◽  
Jaydutt V. Vadgama
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Breast Cancer Cells ◽  
Metastatic Breast ◽  
Breast Cancer Cell Lines ◽  
Cancer Type ◽  
Her2 Positive ◽  
Inhibitory Potency ◽  
Brca Mutations

Poly (ADP-ribose) polymerase inhibitors (PARPi) have proven to be beneficial to patients with metastatic breast cancer with BRCA1/2 (BReast CAncer type 1 and type 2 genes) mutations. However, certain PARPi in pre-clinical studies have been shown to inhibit cell growth and promote the death of breast cancer cells lacking mutations in BRCA1/2. Here, we examined the inhibitory potency of 13 different PARPi in 12 breast cancer cell lines with and without BRCA-mutations using cell viability assays. The results showed that 5 of the 8 triple-negative breast cancer (TNBC) cell lines were susceptible to PARPi regardless of the BRCA-status. The estrogen receptor (ER) negative/ human epidermal growth factor receptor 2 (HER2) positive (ER-/HER2+) cells, SKBR3 and JIMT1, showed high sensitivity to Talazoparib. Especially JIMT1, which is known to be resistant to trastuzumab, was responsive to Talazoparib at 0.002 µM. Niraparib, Olaparib, and Rucaparib also demonstrated effective inhibitory potency in both advanced TNBC and ER-/HER2+ cells with and without BRCA-mutations. In contrast, a BRCA-mutant TNBC line, HCC1937, was less sensitive to Talazoparib, Niraparib, Rucaparib, and not responsive to Olaparib. Other PARPi such as UPF1069, NU1025, AZD2461, and PJ34HCl also showed potent inhibitory activity in specific breast cancer cells. Our data suggest that the benefit of PARPi therapy in breast cancer is beyond the BRCA-mutations, and equally effective on metastatic TNBC and ER-/HER2+ breast cancers.

scholarly journals Evaluation of cytotoxic profile of hydroalcoholic extract of fruit rinds of Garcinia pedunculata on human embryonic kidney and breast carcinoma cells

2020 ◽  
Vol 9 (2) ◽  
pp. 259
Author(s):  
Sukrant Sharma ◽  
Ravi Mundugaru ◽  
Pradeepa H. Dakappa ◽  
Pundalik R. Naik
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cytotoxic Effect ◽  
Metastatic Breast ◽  
Cancer Cell Line ◽  
Cancer Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Alcoholic Extract ◽  
Human Embryonic Kidney ◽  
Hek 293

Background: The fruit rinds of Garcinia pedunculata has potential medicinal properties and used in many chronic ailments. It has been demonstrated that cytoprotective effects in various experimental research works. But its cytotoxic effect has not been evaluated. The present study was aimed to screen its relative cytotoxic effect on normal and cancer cell lines.Methods: In the present study, the cytotoxic effect of hydro alcoholic extract of Garcinia pedunculata was evaluated on normal human embryonic kidney (HEK-293) and M.D. Anderson metastatic breast cancer cell lines (MDA-MB 231) using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.Results: Higher dose level of hydro alcoholic extract of Garcinia pedunculata (HAGP) (500 μg/ml) has shown considerable increase (112.503) in the percentage viability of HEK-29 whereas; there is a remarkable decrease in the viable cell population (77.490) in MDA-MB 231.Conclusions: Based on the observed results we could conclude that HAGP has potential cytotoxic effect on the cancer cell line without altering the normal cell growth and proliferation. Thus it has potential to develop as a safer chemotherapeutic agent. Further detailed exploration is required to confirm its therapeutic efficacy in different cancer cell lines.

scholarly journals Differential Response to 1,25-dihydroxyvitamin D in Metastatic and Non-Metastatic Breast Cancer Cell Lines in Hypoxia (P05-006-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Violet Kiesel ◽  
Stephen Hursting ◽  
Dorothy Teegarden
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Mammary Cancer ◽  
Metastatic Breast ◽  
Cancer Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Dihydroxyvitamin D ◽  
1,25 Dihydroxyvitamin D

Abstract Objectives Prevention of metastasis is of utmost importance for increasing survival in breast cancer patients. Oxygen tension is variable throughout tumors, creating regions of hypoxia that have been linked with poor cancer prognosis. Hypoxia increases glycolytic flux via hypoxia-inducible factor-1α (HIF1α), and can therefore alter growth and survival of cancer cells. Our objectives are to (1) characterize changes in metabolism and survival that occur when metastatic and non-metastatic mammary cancer cell lines are cultured in hypoxia, and (2) determine whether 1,25-dihydroxyvitamin D (1,25(OH)2D) reduces overall survival in hypoxia. Methods We utilized Wnt oncogene-driven murine mammary cancer cells that are non-metastatic (M-Wnt) or that preferentially metastasize to the lung in vivo (metM-Wntlung). Viability of M-Wnt and metM-Wntlung cells treated with 10 nM 1,25(OH)2D and/or 20 mM 2-deoxyglucose (2DG, an inhibitor of glycolysis) was measured with MTT. Expression of HIF1α protein was determined by Western blotting. Results We show that 1,25(OH)2D treatment significantly decreased viability of metastatic metM-Wntlung cells grown in hypoxia by 41%, whereas viability of M-Wnt cells was not significantly impacted by 1,25(OH)2D treatment. Furthermore, treating cells with 2DG significantly decreased viability of both cells lines in hypoxia, with metM-Wntlung cells being more sensitive to 2DG. Interestingly, 1,25(OH)2D treatment partially rescued M-Wnt cells by 22% and metM-Wntlung cells by 24% when treated with 2DG in hypoxia. Finally, we show that M-Wnt cells have 1.9-fold increased expression of HIF1α protein compared to metM-Wntlung cells when grown in hypoxia. Conclusions Our results collectively suggest that non-metastatic M-Wnt cells are less sensitive to treatment with 1,25(OH)2D and 2DG in hypoxia than metastatic metM-Wntlungcells. These data may be explained, in part, by elevated expression of HIF1α in M-Wnt cells, which may contribute to their improved survival in hypoxia. Funding Sources National Institute of Health and USDA.

scholarly journals miR-342-5p as a Potential Regulator of HER2 Breast Cancer Cell Growth

MicroRNA ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 155-165 ◽  
Author(s):  
Evita Maria Lindholm ◽  
Suvi-Katri Leivonen ◽  
Eldri Undlien ◽  
Daniel Nebdal ◽  
Anna Git ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Breast Cancer Cell Lines ◽  
Nucleotide Exchange ◽  
Her2 Positive ◽  
Her2 Breast Cancer

Background: HER2 positive Breast Cancers (BC) have aggressive behavior and poor prognosis. Previously, we have identified miR-342-5p as an upstream regulator of HER2 signaling, as well as inhibitor of HER2 positive BC cell line growth. Objective: Here, we aimed to further investigate the molecular mechanisms behind miR-342-5pinduced HER2 pathway deregulation. </P><P> Method: Two HER2 amplified breast cancer cell lines were transiently transfected with miR-342-5p mimic or negative control, and gene expression was analyzed by Agilent microarrays. Three clinical datasets with BC patients were used to identify correlations between candidate genes and miR-342- 5p, and associations with survival. Results: Pathway analyses of all deregulated genes revealed a significant suppression of the HER2 downstream pathways ERK/MAPK and SAPK/JNK, whereas the miR-342-5p predicted target genes were enriched for pathways associated with cell motility.Biological functions linked to mitochondrial stability were ranked among the top toxicological functions in both gene lists. Among the most deregulated genes, Cytochrome B5 Reductase 3 (CYB5R3) and Rap Guanine Nucleotide Exchange Factor 6 (RAPGEF6) significantly anticorrelated and correlated, respectively, with miR-342-5p in all three clinical BC datasets. Low CYB5R3 levels and high RAPGEF6 levels were significantly associated with survival, although this was not directly associated with HER2 expression. Conclusion: Our data suggest that miR-342-5p overexpression in HER2 positive BC cell lines elicits broad effects on HER2 downstream signaling, cell motility and mitochondrial stability. Together these effects may render cells less proliferative and more sensitive to cellular stress.

scholarly journals Trastuzumab in combination with AT-101 induces cytotoxicity and apoptosis in Her2 positive breast cancer cells

2020 ◽  
Vol 16 (3) ◽  
pp. 4485-4495
Author(s):  
Gulcan Bulut ◽  
Harika Atmaca ◽  
Burcak Karaca
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Breast Cancer Cells ◽  
Breast Cancer Cell Lines ◽  
Her2 Positive ◽  
Protein Levels ◽  
Her2 Positive Breast Cancer ◽  
Synergistic Cytotoxicity ◽  
Positive Breast Cancer

Aim: AT-101 is a polyphenolic compound with potent anti-apoptotic effects in various cancers. In this study, the possible synergistic cytotoxic and apoptotic effect of trastuzumab/AT-101 combination was investigated in HER2-positive breast cancer cell lines. Materials & methods: SKBR-3, MDA-MB-453 and MCF-10A cell lines were treated with a trastuzumab/AT-101 combination. Synergistic cytotoxicity and apoptosis effects were shown and then PI3K and Akt protein levels were studied. Result: The trastuzumab/AT-101 combination induced synergistic cytotoxicity and apoptosis in both breast cancer cells but not in MCF-10A cells. Combination treatment induced cytotoxicity via inhibiting PI3K/AKT but not the MAPK/ERK pathway. Conclusion: The trastuzumab/AT-101 combination may be a good candidate for patients with trastuzumab-resistant Her2-positive breast cancer and inhibition of the PI3K/AKT pathway may be one of the underlying mechanisms.

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