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Bioluminescence Imaging-Based Assessment of the Anti-Triple-Negative Breast Cancer and NF-Kappa B Pathway Inhibition Activity of Britanin.

ABSTRACT: Britanin has been reported to have therapeutic effects on neurodegenerative and inflammation-based diseases. However, whether it is involved in the regulation of triple-negative breast cancer development has not been elucidated. In this study, we investigated the anti-tumor activity against triple-negative breast cancer tumor of Britanin by bioluminescence imaging in vivo using athymic (nu/nu) mice implanted with MDA-MB-231 and SUM-159 cells expressing a luciferase reporter gene, and explored the anti-tumor mechanism of Britanin. The results showed that Britanin treatment inhibited triple-negative breast cancer cell proliferation in vivo, and Cell Counting Kit-8 (IC50 values are 4.27 and 5.05 ?M) and colony formation tests (P < 0.001) confirmed this result. Transwell assays were conducted to verify that Britanin treatment inhibited cell migration and invasion (P < 0.001). Apoptosis was determined by TdT-mediated dUTP nick-end labeling method. Western blot and qRT-PCR analysis showed that Britanin treatment caused a decrease in the member expression of NF-kappa B signaling pathway. Computational modeling showed that Britanin could directly bind to a p-65 core region composed of Cys38, Cys120, and Gln128 residues. The results showed that the inhibitory mechanisms of Britanin on cancer cells may be by ways of inhibiting the NF-kappa B pathway. In addition, bioluminescence imaging screening system is useful for accelerating the application of Britanin in the antitumor field, and provides a useful tool for evaluating the phytochemicals efficacy in inhibiting cancer cell proliferation in animal models.

SUBMITTER: Xu X

PROVIDER: S-EPMC7215071 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Bioluminescence Imaging-Based Assessment of the Anti-Triple-Negative Breast Cancer and NF-Kappa B Pathway Inhibition Activity of Britanin.

Xu Xinyi X Guo Yingying Y Du Getao G Liu Huifang H Wang Lin L Chen Dan D

Frontiers in pharmacology 20200505

Britanin has been reported to have therapeutic effects on neurodegenerative and inflammation-based diseases. However, whether it is involved in the regulation of triple-negative breast cancer development has not been elucidated. In this study, we investigated the anti-tumor activity against triple-negative breast cancer tumor of Britanin by bioluminescence imaging <i>in vivo</i> using athymic (nu/nu) mice implanted with MDA-MB-231 and SUM-159 cells expressing a luciferase reporter gene, and expl ...[more]

PMID: 32431613

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Project description:BACKGROUND: There are a lot of unmet needs in patients with triple-negative breast cancer (TNBC). Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPAR-?) agonist, has been used for decades to treat hypertriglyceridaemia and mixed dyslipidaemia. Recent studies show that it might have anti-tumor effects, however, the mechanism remains unclear. Here, we assessed the ability of fenofibrate to induce apoptosis of TNBC in vitro and in vivo and explored involved mechanisms. METHODS: MTT method was used to evaluate the anti-proliferation effect of fenofibrate, and invert microscope to observe the apoptotic morphological changes. The percentage of apoptotic cells and distribution ratios of cell cycle were determined by flow cytometric analysis. The related protein levels were measured by Western blot method. The changes of genes and pathways were detected by gene expression profiling. The tumor growth in vivo was assessed by MDA-MB-231 xenograft mouse model. Terminal deoxytransferase-catalyzed DNA nick-end labeling (TUNEL) assay was employed to estimate the percentage of apoptotic cells in vivo. In order to evaluate the safety of fenofibrate, blood sampled from rat eyes was detected. RESULTS: We found that fenofibrate had anti-proliferation effects on breast cancer cell lines, of which the first five most sensitive ones were all TNBC cell lines. Its induction of apoptosis was independent on PPAR-? status with the highest apoptosis percentage of 41.8 ± 8.8%, and it occurred in a time- and dose-dependent manner accompanied by up-regulation of Bad, down-regulation of Bcl-xl, Survivin and activation of caspase-3. Interestingly, activation of NF-?B pathway played an important role in the induction of apoptosis by fenofibtate and the effect could be almost totally blocked by a NF-?B specific inhibitor, pyrrolidine dithiocarbamate (PDTC). In addition, fenofibrate led to cell cycle arrest at G0/G1 phase accompanied by down-regulation of Cyclin D1, Cdk4 and up-regulation of p21, p27/Kip1. In vivo, fenofibrate slowed down tumor growth and induced apoptosis with a good safety profile in the MDA-MB-231 xengograft mouse model. CONCLUSIONS: It is concluded that fenofibrate induces apoptosis of TNBC via activation of NF-?B pathway in a PPAR-? independent way, and may serve as a novel therapeutic drug for TNBC therapy.

Project description:Simple Summary The cyclin E/CDK2 complex may present a promising target axis for the treatment of triple-negative breast cancers (TNBC); however, therapeutically relevant doses of CDK2 inhibitors have been associated with toxicities. Here, we report that the suboptimal dosing of the CDK 2, 7 and 9 inhibitor SNS-032 reduced the viability of TNBC cells and upregulated the checkpoint ligand PD-L1 expression in surviving cancer cells in vitro and in human orthotopic MDA-MB-231 TNBC xenografts grown in immunodeficient mice. Moreover, in immunodeficient, TNBC xenograft-bearing mice engrafted with human immune cells, SNS-032 treatment was associated with the infiltration of CD45+ human immune cells in tumors. In these orthotopic MDA-MB-231 TNBC-bearing mice, suboptimal SNS-032 doses given sequentially ahead of dosing with the anti-PD-L1 antibody avelumab significantly restricted tumor growth compared with monotherapy. These findings suggest that surviving cancer cells following suboptimal CDK inhibitor treatment may be responsive to checkpoint immunotherapy. Abstract Triple-negative breast cancers (TNBC) expressing PD-L1 qualify for checkpoint inhibitor immunotherapy. Cyclin E/CDK2 is a potential target axis in TNBC; however, small-molecule drugs at efficacious doses may be associated with toxicity, and treatment alongside immunotherapy requires investigation. We evaluated CDK inhibition at suboptimal levels and its anti-tumor and immunomodulatory effects. Transcriptomic analyses of primary breast cancers confirmed higher cyclin E/CDK2 expression in TNBC compared with non-TNBC. Out of the three CDK2-targeting inhibitors tested, the CDK 2, 7 and 9 inhibitor SNS-032 was the most potent in reducing TNBC cell viability and exerted cytotoxicity against all eight TNBC cell lines evaluated in vitro. Suboptimal SNS-032 dosing elevated cell surface PD-L1 expression in surviving TNBC cells. In mice engrafted with human immune cells and challenged with human MDA-MB-231 TNBC xenografts in mammary fat pads, suboptimal SNS-032 dosing partially restricted tumor growth, enhanced the tumor infiltration of human CD45+ immune cells and elevated cell surface PD-L1 expression in surviving cancer cells. In tumor-bearing mice engrafted with human immune cells, the anti-PD-L1 antibody avelumab, given sequentially following suboptimal SNS-032 dosing, reduced tumor growth compared with SNS-032 alone or with avelumab without prior SNS-032 priming. CDK inhibition at suboptimal doses promotes immune cell recruitment to tumors, PD-L1 expression by surviving TNBC cells and may complement immunotherapy.

Project description:While the overall mortality for breast cancer has recently declined, management of triple-negative breast cancer (TNBC) is still challenging because of its aggressive clinical behavior and the lack of targeted therapies. Genomic profiling studies highlighted the high level of heterogeneity of this cancer, which comprises different subtypes with unique phenotypes and response to treatment. Platelet-derived growth factor receptor ? (PDGFR?) is an established mesenchymal/stem cell-specific marker in human glioblastoma and, as recently suggested, it may uniquely mark breast cancer cells with stem-like characteristics and/or that have undergone epithelial-mesenchymal transition. Methods: Immunohistochemical analysis for PDGFR? expression was performed on a human TNBC tissue microarray. Functional assays were conducted on mesenchymal-like TNBC cells to investigate the effect of a previously validated PDGFR? aptamer on invasive cell growth in three-dimensional culture conditions, migration, invasion and tube formation. The aptamer was labeled with a near-infrared (NIR) dye and its binding specificity to PDGFR? was assessed both in vitro (confocal microscopy and flow cytometry analyses) and in vivo (fluorescence molecular tomography in mice bearing TNBC xenografts). A mouse model of TNBC lung metastases formation was established and NIR-labeled PDGFR? aptamer was used to detect lung metastases in mice untreated or intravenously injected with unlabeled aptamer. Results: Here, we present novel data showing that tumor cell expression of PDGFR? identifies a subgroup of mesenchymal tumors with invasive and stem-like phenotype, and propose a previously unappreciated role for PDGFR? in driving TNBC cell invasiveness and metastases formation. We show that the PDGFR? aptamer blocked invasive growth and migration/invasion of mesenchymal TNBC cell lines and prevented TNBC lung metastases formation. Further, upon NIR-labeling, the aptamer specifically bound to TNBC xenografts and detected lung metastases. Conclusions: We propose PDGFR? as a reliable biomarker of a subgroup of mesenchymal TNBCs with invasive and stem-like phenotype as well as the use of the PDGFR? aptamer as a high efficacious tool for imaging and suppression of TNBC lung metastases. This study will allow for the significant expansion of the current repertoire of strategies for managing patients with more aggressive TNBC.

Dataset Information

Bioluminescence Imaging-Based Assessment of the Anti-Triple-Negative Breast Cancer and NF-Kappa B Pathway Inhibition Activity of Britanin.

Publications

Bioluminescence Imaging-Based Assessment of the Anti-Triple-Negative Breast Cancer and NF-Kappa B Pathway Inhibition Activity of Britanin.

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