RESUMEN
Epoxyazadiradione is an important limonoid with immense pharmacological potential. We have reported previously that epoxyazadiradione (EAD) induces apoptosis in triple negative breast cancer cells (MDA-MB 231) by modulating diverse cellular targets. Here, we identify the key genes/pathways responsible for this effect through next-generation sequencing of the transcriptome from EAD treated cells and integrated molecular data analysis using bioinformatics. In silico analysis indicated that EAD displayed favourable drug-like properties and could target multiple macromolecules relevant to TNBC. RNA sequencing revealed that EAD treatment results in the differential expression of 1838 genes in MDA-MB 231 cells, with 752 downregulated and 1086 upregulated. Gene set enrichment analysis of these genes suggested that EAD disrupts protein folding in the endoplasmic reticulum, triggering the unfolded protein response (UPR) and potentially leading to cell death. EAD also induced oxidative stress and DNA damage, downregulated pathways linked to metabolism, cell cycle progression, pro-survival signalling, cell adhesion, motility and inflammatory response. The identification of protein cluster and hub genes were also done. The validation of the identified hub genes gave an inverse correlation between their expression in EAD treated cells and TNBC patient samples. Thus, the identified hub genes could be explored as therapeutic or diagnostic markers for TNBC. Hence, EAD appears to be a promising therapeutic candidate for TNBC by targeting various hallmarks of cancer, including cell death resistance, uncontrolled proliferation and metastasis. To conclude, the identified pathways and validated targets for EAD will provide a roadmap for further in vivo studies and preclinical/clinical validation required for potential drug development.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Apoptosis/efectos de los fármacos , Limoninas/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Biología Computacional/métodosRESUMEN
Diindolylmethane (DIM) is a metabolic product of indole-3-carbinol (I3C), the major phytochemicals present in cruciferous vegetables, which can modulate multiple signalling pathways in cancer. The present study deals with the mechanism of action of two synthetic biaryl conjugates of DIM in triple negative breast cancer cells. Out of 12 DIM derivatives tested, two compounds, DIM-1 and DIM-4, exhibit cytotoxicity with GI50 values of 9.83 ± 0.2195 µM and 8.726 ± 0.5234 µM, respectively, in 2D culture. In 3D culture, DIM-1 and DIM-4 show GI50 values of 24.000 ± 0.7240 µM and 19.230 ± 0.3754 µM, respectively. The non-toxic nature of the compounds was also established by the toxicity studies using the zebrafish model system. The two compounds induced apoptosis and anoikis in the cancer cells, which was confirmed by morphological analysis, nuclear fragmentation, membrane integrity assay, caspase activity measurements and modulation of pro/anti-apoptotic proteins. The compounds inhibited cell migration and MMP-2 and MMP-9 activities indicating their anti-metastatic property. They also reduced the expression of active Ras, phosphorylated forms of PI3K, Akt and mTOR. Immunofluorescence studies revealed the reduced expression of EGFR and pEGFR in treated cells. To conclude, DIM-1 and DIM-4 induced anti-breast cancer effects by blocking EGF receptor and subsequently inhibiting Ras-mediated PI3K-Akt-mTOR signalling pathway.
Asunto(s)
Metaloproteinasa 2 de la Matriz , Neoplasias de la Mama Triple Negativas , Animales , Proteínas Reguladoras de la Apoptosis , Caspasas/metabolismo , Línea Celular Tumoral , Receptores ErbB , Humanos , Indoles , Metaloproteinasa 9 de la Matriz , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Serina-Treonina Quinasas TOR , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Pez Cebra/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) is one of the most aggressive forms of its kind, which accounts for 15-20% of all breast cancers. As this cancer form lacks hormone receptors, targeted chemotherapy remains the best treatment option. Apoptosis and anoikis (detachment-induced cell death) induction by small molecules can prevent TNBC metastasis to a greater extent. Epoxyazadiradione (EAD) is a limonoid from the neem plant with an anticancer property. Here, we demonstrate that EAD induced mitochondria-mediated apoptosis and anoikis in TNBC cells (MDA-MB-231). Apart from this, it promotes antimigration, inhibition of colony formation, downregulation of MMP-9 and fibronectin, induction of G2/M phase arrest with downregulation of cyclin A2/cdk2, interference in cellular metabolism, and inhibition of nuclear factor kappa-B (NF-kB) nuclear translocation. Moreover, a significant reduction is observed in the expression of EGFR on the plasma membrane and nucleus upon treatment with EAD. Among the diverse cellular effects, anoikis induction, metabolic interference, and downregulation of membrane/nuclear EGFR expression by EAD are reported here for the first time. To summarize, EAD targets multiple cellular events to induce growth arrest in TNBC, and hence can be developed into the best antineoplastic agent in the future.
Asunto(s)
Anoicis/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Limoninas/farmacología , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Mama Triple Negativas/metabolismo , Femenino , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Improved treatment of breast cancer, the world's second most common cancer, requires identification of new sensitive prognostic and diagnostic biomarkers. Exosomes are lipid-bilayer extracellular vesicles of size 30-150 nm, released by all cell types, including breast cancer cells. Cellular communication is the primary function attributed to them. This review discusses the potential utility of exosomes and exosomal RNAs (microRNAs [miRNAs]/long non-coding RNAs [LncRNAs]) in breast cancer biology and treatment. The existing literature shows that exosomes play a significant role in breast tumorigenesis and progression through transfer miRNAs and LncRNAs. These miRNAs and LncRNAs function by post-transcriptionally regulating their target mRNAs, eventually leading to modulation of expression/repression. Over the past two decades, numerous publications point towards diagnostic and therapeutic applications of exosomal miRNAs/LncRNAs. Until now, we do not have clinically approved exosome-based therapeutics. Therefore, it is high time that clinicians and cancer researchers utilise exosome's benefits through randomised clinical trials for better management of breast cancer.