Nobiletin Inhibits Breast Cancer Stem Cell by Regulating the Cell Cycle: A Comprehensive Bioinformatics Analysis and In Vitro Experiments

Abstract

Inhibiting breast cancer stem cell (BCSC) signaling pathways is a strategic method for successfully treating breast cancer. Nobiletin (NOB) is a compound widely found in orange peel that exhibits a toxic effect on various types of cancer cells, and inhibits the signaling pathways that regulate the properties of BCSCs; however, the effects of NOB on BCSCs remain elusive. The purpose of this study was to determine the target genes of NOB for inhibiting BCSCs using in vitro three-dimensional breast cancer cell culture (mammospheres) and in silico approaches. We combined in vitro experiments to develop mammospheres and conducted cytotoxicity, next-generation sequencing, and bioinformatics analyses, such as gene ontology, the Reactome pathway enrichment, network topology, gene set enrichment analysis, hub genes selection, genetic alterations, prognostic value related to the mRNA expression, and mRNA and protein expression of potential NOB target genes that inhibit BCSCs. Here, we show that NOB inhibited BCSCs in mammospheres from MCF-7 cells. We also identified CDC6, CHEK1, BRCA1, UCHL5, TOP2A, MTMR4, and EXO1 as potential NOB targets inhibiting BCSCs. NOB decreased G0/G1, but increased the G2/M cell population. These findings showed that NOB is a potential therapeutic candidate for BCSCs treatment by regulating cell cycle.

Acknowledgments

The authors thank PT. Genetika Science for their assistance in performing the next-generation sequencing and Ms. Ririn Widarti and Ms. Dian Anita for their administrative support.

Availability of Data and Materials

RNA sequencing data are available in the public Gene Expression Omnibus database (Accession number GSE220298).

Disclosure Statement

The authors declare no conflicts of interest.

Additional information

Funding

This study was supported by a Research Grant from the Ministry of Education, Culture, Research, and Technology through the Research Project PDUPT (contract nos. 1669/UN1/DITLIT/DIT-LIT/PT/2020 and 1619/UN1/DITLIT/DIT-LIT/PT/2021).