Label-free, Quantitative proteomics study of human triple negative cancer MDA-MB-231 cells treated by Electrochemotherapy with tomato lipophilic extract - Supplementary data

Objective: To analyze the effect of electrical pulses (EP) with tomato lipophilic extract (TLE) on human, triple negative breast cancer (TNBC) MDA-MB-231 cells and non-cancerous breast MCF-10A cells and to gain insights into the mechanisms of action. Methods: We prepared TLE using solvent extraction method. We identified the constituents of TLE using LC-MS analysis. TLE with the dose of 250μg/ml was applied with eight, 800V/cm, 100μs pulses to MDA-MB-231 and MCF-10A cells and their viability was studied. We studied the proteins involved in the anticancer activity of TLE in MDA-MB-231 cells using label-free, quantitative proteomics. To understand the correlation between protein level and transcription level changes, we performed real time quantitative PCR (RT-qPCR). We detected Hydrogen Peroxide (H2O2) levels of untreated and treated cells to determine how oxidative stress regulates various intracellular pathways. Results: TLE comprises fatty acids which heighten MDA-MB-231 cell proliferation. However, the application of electrical pulses nullifies these cell proliferative effects of fatty acids, as evident by the significant MDA-MB-231 cells viability reduction for TLE+EP treatment. Tomato extract, electrical pulses, and combinational treatment did not affect normal breast MCF-10A cells. Proteomic analysis identified a total of 1877 proteins. Conclusion: Applying EP in combination with TLE suppresses and reverses back the proliferative effects caused by TLE. EP and TLE+EP treatments increase H2O2 ROS production in TNBC cells to inflict cell death due to excessive oxidative stress. Proteins, such as PCNA, LDHB and ENO1 were downregulated in EP treatment and it was confirmed with the mRNA levels by qPCR results. Our results provide novel insights into the mechanisms of electrical pulses with TLE. Significance: Our research serves as a technological platform capable of identifying the set of proteins responsible for the minimizing TNBC cell proliferation when treated with TLE+EP.