Document Type
Dissertation
Date of Award
10-2025
School/College
College of Science, Engineering, and Technology (COSET)
Degree Name
Ph.D. in Environmental Toxicology
Committee Chairperson
Dr. Ayodotun Sodipe
Committee Member 1
Dr. Shodimu-Emmanuel Olufemi
Committee Member 2
Dr. Desiree Jackson
Committee Member 3
Dr. Tuan Phan
Abstract
Cancer is a complex pathological condition characterized mainly by uncontrolled rapid cell divisions caused by several factors, which include genetics and environmental factors. Of all cancer types, breast cancer (BC) remains the second most diagnosed among women worldwide; thus, BC burden has become a global health issue. Of all known breast cancer subtypes, triple-negative breast cancer (TNBC) is the only primary subtype without effective therapy as a result of their lack of the essential estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) common to other BCs and are the usual therapeutic targets for treatment. MDA-MB 231 cancer cell line is a typical triple negative breast cancer cell (TNBC) exhibiting the lack of these receptors, thus being a representative cancer cell line for TNBC. As confirmed by several studies, the lack of effective treatment for TNBC has attracted the use of phytochemicals due to their potential anticancer effects. Sulforaphane (SFN), an isothiocyanate phytochemical compound found in cruciferous vegetables such as broccoli, has gained attention for its possible role in the chemoprevention and chemotherapeutic treatment of various types of cancer, including TNBC. SFN has hormetic effects that favorably promote tumor growth at lower doses or induce toxicity at higher doses. It is an anticancer agent that kills cancer cells, and its efficacy or toxicity is sensitively dosage-dependent. On the other hand, microRNAs (miRNAs) are small noncoding RNA molecules, typically 15-25 nucleotides long, that regulate post-transcriptional gene silencing by binding partially to 3' untranslated region (3'-UTR) of target messenger RNAs (mRNAs) to inhibit translation and cause target mRNA degradation They are implicated in several human diseases and regulate mRNAs of genes in different biological pathways. In cancer, miRNAs can function as a tumor suppressor or an oncogene. The study rationale is to find alternative therapy for cancer drug treatment and deduce whether the alternatives, such as miRNAs, have similar potency as the cancer drugs. This study aims to demonstrate that transfection treatment of Green fluorescence protein (GFP) miR-DNA constructs of the non-clustered tumor suppressor miRNAs (i.e., miRNA-130b, -497, and -377), non-tumor suppressor miRNAs (i.e., miRNA-1277, -8089, and -4714) exhibits the same potency as SFN treatment to kill cancer cells, such as MDA-MB 231 cells, which exhibits triple-negative breast cancer (TNBC) properties. Therefore, the two hypotheses developed are to show at different time intervals, 24 h, 48 h, and 72 h, that 1. The miRNAs have similar effects as the SFN on the morphology of the MDA-MB 231 cancer cells, and 2.The miRNAs alter the AKT signaling pathway and its downstream regulatory proteins,including cell cycle genes, similar to the SFN on the MDA-MB 231 cancer cells. The MDA-MB 231 cancer cells were transfected or treated separately at different time intervals (i.e., 24 h, 48 h, and 72 h) with GFP miR-DNA constructs of the non-clustered tumor suppressor miRNAs (i.e., miRNA-130b, -497, and -377), non-tumor suppressor miRNAs (i.e., miRNA-1277, -8089, and -4714) and SFN. In addition, the MDA-MB 231 cancer cells were transfected separately at different intervals (i.e., 24 h, 48 h, and 72 h) with individual GFP miR-DNA construct of the non-clustered tumor and non-tumor suppressor miRNAs. This study's results shows that the non-clustered tumor and non-clustered non tumor suppressor miRNAs, similarly as SFN, altered the morphology of the MDA-MB 231 cancer cells at 24 h and 48 h but not 72 h since some cells that did not take up the GFP miR-DNA constructs continued to proliferate. SFN however, killed the cancer cells at 72 h.Observed morphological cell changes are apoptosis, necrosis, and blebbing. Westernblot analysis was used to detect downregulation expression of the proteins in the apoptosis AKT pathway (i.e., AKT, Bcl-xL, CASP9, and CASP3) and the cell cycle pathway (i.e., Cyclin D1 and CDK4), including beta-actin, at 24 h, 48 h and 72 h in the MDA-MB 231 cancer cells. Cells transfected with combined non-clustered tumor and non-clustered non tumor suppressor GFP miRNA-DNA constructs showed a synergistic downregulated expression of AKT and downstream genes at 24 and 48h while SFN downregulated genes in the AKT pathway (i.e., AKT, Bcl-xL, CASP9, and CASP3) and the cell cycle pathway (i.e., Cyclin D1 and CDK4) at 24 h, 48 h, and 72h precisely. The individual effect of the transfected non-clustered tumor and non-clustered tumor suppressor GFP miRNA-DNA constructs was negligible, compared to their combined transfected non-clustered tumor and combined non-clustered non tumor suppressor GFP miRNA-DNA constructs, suggesting that the MDA-MB 231 cancer cells responded favorably to combined treatment with non-clustered tumor and non-clustered tumor suppressor miRNAs treatment similarly as SFN, thus making it an excellent alternative therapy for treating for the MDA-MB 231 cancer cells, which do not respond normally to several available cancer drugs. This is a novel approach and the first report to show that non-clustered tumor and non-clustered non tumor suppressor miRNAs could kill MDA-MB 231 cancer cells, which exhibit triple-negative breast cancer (TNBC) properties.
Copyright
Copyright © for this work is retained by the author. Any documents and information presented are protected by copyright under US Copyright laws and are the property of the author. All Rights Reserved. For permission to use this content please contact the author or the Graduate School at Texas Southern University ([email protected]).
Recommended Citation
Garuba, Omobolanle David, "Sulforaphane and Non-Clustered Tumor/Non-Tumor Suppressor microRNAs Regulate AKT and Its Downstream Genes in MDA-MB-231 Cancer Cells" (2025). Dissertations (2016-Present). 106.
https://digitalscholarship.tsu.edu/dissertations/106