Effects of melatonin on the global proteomic profile of breast carcinoma cells (MCF-7 and MDA-MB-468 strains)

• Grant number: 22/09434-2
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: February 01, 2023
• End date: January 31, 2027
• Field of knowledge: Biological Sciences - Biology
• Principal Investigator: Luiz Gustavo de Almeida Chuffa
• Grantee: Roberta Carvalho Cesário
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Breast cancer (BC) is the most incident tumor and the leading cause of death among women worldwide. In Brazil, the estimate is around 66,280 new cases by the end of 2021. BC is stratified into molecular subtypes depending on markers that may have a better or worse prognosis. Cell lines expressing the estrogen receptor (ER+) and progesterone receptor (PR+) account for 70% of all BC cases. However, they have a better prognosis since they respond to hormonal therapies. On the other hand, triple-negative strains that do not express ER, PR and HER-2 receptors have lower incidence (about 10 to 20% of cases), but the treatment is challenging with worst prognosis. Melatonin (Mel) is a neurohormone secreted by the pineal gland in the dark phase and has shown in vitro and in vivo antitumor properties. Therefore, the present study will evaluate the role of melatonin on the global proteomic profile using total and mitochondrial cell extracts from breast carcinoma cells with good (MCF-7) and poor (MDA-MB-468) prognosis. For this experiment, the groups will be divided into: 1) Control Group of MCF-7 cells receiving vehicle only; 2) Control Group of MDA-MB-468 cells receiving vehicle only; 3) Group of MCF-7 cells treated with melatonin; 4) Group of MDA-MB-468 cells treated with melatonin; 5) Control Group of MCF-7 cells receiving vehicle only with mitochondrial proteome extraction; 6) Control group of MDA-MB-468 cells receiving vehicle only with the extraction of the mitochondrial proteome; 7) Group of MCF-7 cells treated with melatonin with the extraction of the mitochondrial proteome; 8) Group of MDA-MB-468 cells treated with melatonin with the extraction of the mitochondrial proteome. The concentration and exposure time to melatonin will be established through the cell viability assay. The migratory capacity of the cells will be evaluated after the treatments. After melatonin exposure, the extraction and quantification of the total and mitochondrial proteins will be performed. According to the manufacturer's recommendations, mitochondria fractionation will be performed using a Mitochondrial Isolation Kit for Cell Culture (ThermoScientific). The proteomic profile of the strains will be identified by mass spectrometry using an Ultimate 3000 LC liquid nanochromatography equipment coupled to Q-Exactive mass spectrometry equipment. The raw data in.RAW format will be submitted to the PatternLab software (version 4.0.0.84) to obtain the identification of proteins. Data will be statistically studied based on fold-change e 1.5, false discovery rate (FDR) d 1 %, and presented using bioinformatics tools. We hope to understand the effects of melatonin on the proteome of breast carcinoma cells presenting good and poor prognosis in an attempt to elucidate possible mechanisms by which melatonin affects survival and mitochondrial energy metabolism in the tumor cell, providing the basis for potential personalized therapies for BC.