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High-throughput, Label-free Proteomics Identifies Salient Proteins and Genes in MDA-MB-231 Cells Treated with Natural Neem-based Electrochemotherapy

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Abstract

With the absence of the three most common receptor targets, and with high vascularity and higher-grade tumors, triple-negative breast cancer (TNBC) is the most aggressive of all breast cancer subtypes and is in need of additional/alternative/novel treatment strategies. With ~ 15% of the over 2 million new cases each year, there is an unmet need to treat TNBC. MDA-MB-231, human TNBC cells, were treated with neem leaf extract (Neem) and eight, 1200 V/cm, 100 µs electric pulses (EP), and their viability and proteomic profiles were studied. With EP + Neem, a lower viability of 37% was observed after 24 h, compared to 85% in the neem-only samples, indicating the efficacy of the combinational treatment. The proteomics results indicated significant upregulation of 525 proteins and downregulation of 572 proteins, with a number of different pathways in each case. These include a diverse group of proteins, such as receptors, heat shock proteins, and many others. The upregulated TCA cycle and OXPHOS pathways and the downregulated DNA replication and ubiquitin-mediated proteolytic pathways were associated with effective cell death, demonstrating the potency of this treatment. Viability results reveal the efficacious anticancer effects of the EP + Neem combination, via growth inhibition, on TNBC cells. Proteomics studies could readily identify the effected protein pathways, and their corresponding genes, that are responsible for cell death. This represents a potential therapeutic strategy against TNBC when patients are refractory to standard treatments.

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Acknowledgements

Jeya Shree T is very grateful to the 2019-2020 SERB Fellowship that enabled her to visit Purdue and do the experiments. All the mass spec experiments were performed at the Purdue Proteomics Facility, Bindley Bioscience Center. We are very grateful to its Director, Dr. Uma Aryal, for his kind help. The Q Exactive Orbitrap HF and UltiMate 3000 HPLC system used for LC-MS/MS analysis were purchased from funding provided by the Purdue Office of the Executive Vice President for Research and Partnership. The authors are grateful to Dr. Laura W. Bower’s lab for the use of a spectrophotometer, and Drs. Emily C. Dykhuizen’s and Ourania M. Andrisani’s labs for their qPCR machines. The authors are grateful to Poompavai and Lakshya Mittal for their help with sample collection and data analysis.

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  1. Jeya Shree Thulasidas and Pragatheiswar Giri equally contributed

Authors and Affiliations

  1. Department of Electrical Engineering, Divison of High Voltage Engineering, Anna University, Guindy, India

    Gowrisree Varadarajan & Jeya Shree Thulasidas

  2. School of Engineering Technology, Purdue University, West Lafayette, IN, 47907, USA

    Pragatheiswar Giri & Raji Sundararajan

  3. Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907, USA

    Ignacio G. Camarillo

  4. Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, 47907, USA

    Ignacio G. Camarillo

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  1. Gowrisree Varadarajan
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  4. Ignacio G. Camarillo
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  5. Raji Sundararajan
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Contributions

Concept and design: Gowrisree V, R Sundararajan, IG Camarillo. Neem leaf collection and extract preparation: Gowrisree V. Viability and other Experiments: Jeya Shree T, IG Camarillo. Data Analysis: P Giri, Jeya Shree T, R Sundararajan. Manuscript: All.

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Correspondence to Raji Sundararajan.

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Varadarajan, G., Thulasidas, J.S., Giri, P. et al. High-throughput, Label-free Proteomics Identifies Salient Proteins and Genes in MDA-MB-231 Cells Treated with Natural Neem-based Electrochemotherapy. Appl Biochem Biotechnol 194, 148–166 (2022). https://doi.org/10.1007/s12010-021-03787-3

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