Evaluation of Cytotoxic Effects and Underlying Mechanism of Phenolic Compounds on Breast Cancer Cell Lines

    Tutkimustuotos: VäitöskirjaCollection of Articles

    Abstrakti

    Breast cancer (BC) is the second most common cancer that causes higher mortality rates worldwide. It is a complex and heterogeneous disease with a median survival range of around three years. Breast cancer patients' overall survival has increased due to using chemotherapeutic medicines, namely anthracyclines and taxanes. However, drug resistance and subsequent progression of this disease were still observed in metastatic patients. Furthermore, the efficacy failure of even today's sophisticated chemotherapeutics negatively impacts breast cancer patients with side effects, highlighting the urgent need for the development of nontoxic medications, that have low side effects, and are patient-friendly. Tumor cell death has been associated with the activation of apoptotic signal transduction pathways in cancer cells, such as the intrinsic and/or extrinsic pathways. Thus, understanding the molecular mechanism of apoptosis opens the future perspective for drug development for breast cancer treatment. The present study focuses on the possibility of using newly synthesized indoline analogs as targeted therapy for breast cancer, which could selectively induce apoptosis in cancer cells. Advances in anticancer drug discovery using broad-spectrum drugs, such as substituted alkylamino phenolic rings or indoline rings, have emerged as promising molecules. Thus, investigating the effects of these compounds in inducing apoptosis would provide opportunities that directly evade the significant challenges in current breast cancer therapies.

    The present research work focuses on the in-vitro analysis of the anti-breast cancer activity of three novel indoline derivatives, 2-((1, 2, 3, 4-Tetrahydroquinolin-1-yl) (4 methoxyphenyl) methyl) phenol (THMPP), 2-((3,4-Dihydroquinolin-1(2H)-yl) (ptolyl)methyl)phenol)(THTMP) and N-(2-hydroxy-5-nitrophenyl (4’-methylphenyl)methyl) indoline (HNPMI). The present study’s findings have been published in four publications, each of which highlights the mechanism of action of each compound’s cytotoxic potential, its apoptotic induction potential, the regulation of the genes involved in the Epithelial Growth Factor Receptor (EGFR) signaling pathway, and the in-silico analysis to identify the compound’s interaction with the target receptor, EGFR. Absorption, distribution, metabolism, excretion, and toxicity (ADME/ T) analysis also confirms the drug likeliness of the compound to be used as a potent anticancer drug. The compounds' cytotoxicity was tested in breast cancer cells like MCF7 (ER+PR+/HER-), SkBr3 (ER-PR-/HER+), MDA MB-231, and non-tumorous cells like HEK293 and H9C2. The first compound we analyzed was 2-((1, 2, 3, 4-Tetrahydroquinolin-1-yl) (4 methoxyphenyl) methyl) phenol (THMPP). In human breast cancer cell lines MCF- 7 and SkBr3 and non-cancerous mouse myoblast cells, H9C2, it was evaluated for its potential cytotoxicity and method of action. THMPP induced cell death in MCF- 7 and SkBr3 cells at their IC50 concentration of 83.23 μM and 113.94 μM, respectively. The toxicity was 36.4% in MCF-7 cells and 18.86% in SkBr3 cells at 10μM concentrations. Interestingly, THMPP showed the lowest percentage of cytotoxicity to H9C2 cells (0.91%) than the other breast cancer cell line. The compound induced apoptosis through increased caspase three and caspase 9 with the fold level of 0.17-fold and 0.47-fold in MCF-7 cells and 0.07 and 0.25 in SkBr3 cells, respectively. THMPP also enhanced apoptosis of the breast cancer cells, causing inter-nucleosomal DNA fragmentation, thus leading to DNA strand breakage and cell death. FACS analysis has proved that THMPP improves breast cancer cells to enter various stages of apoptosis, especially in the late apoptotic stage. With a score of 5.79 kJ/mol, molecular docking validates THMPP's substantial interaction with EGFR, predicted to activate the downstream signaling pathway. The downregulation of PI3K and S6K1 genes involved in the Phosphatidylinositol 3-Kinase (PI3K)/AKT signaling pathway, which were considerably overexpressed in cancer cells, was validated by gene expression analysis. Quantitative Structure-Activity Relationship (QSAR) analysis confirmed the toxicity of THMPP against breast cancer cells. ADME/T analysis predicts the drug-likeliness of THMPP.

    The second important compound that was analyzed for its anti-breast cancer activity was 2-((3,4-Dihydroquinolin-1(2H)-yl) (p-tolyl) methyl) phenol) (THTMP), the derivative of THMPP. A methyl group replaced the 4-OMe substituent of the aryl ring in THMPP to synthesize THMPP. The compound exhibits a cytotoxic effect against MCF7 and SK-BR3 cells, with IC50 values of 87.92 μM and 172.51 μM, respectively. THTMP caused cell death in breast cancer cells by regulating critical apoptosis enzymes, caspase-3 and-9, with 33 percent of cells in the late apoptotic stage after 24 hours of treatment. The significant interaction of THTMP with EGFR inhibits PI3K/S6K1 gene expression, thus enhancing the apoptotic response of the breast cancer cells. Structural validation of QSAR also confirms the anticancer property of THTMP. ADME/T screening suggested the compound’s oral availability and better intestinal absorption with acceptable metabolism and toxicity parameters. Furthermore, seven N-substituted indoline derivatives have been assessed for their ability to interact with the EGFR protein. Among the seven compounds analyzed by molecular docking, it was confirmed that the N-(2-hydroxy-5-nitrophenyl (4’- methyl phenyl) methyl) indoline (HNPMI) possesses a stronger affinity with EGFR active sites. As a result, the EGFR signaling pathway was activated, which reduced the expression of PI3K and S6K1 to about 0.4-fold and 0.3-fold, respectively, thereby inducing cell death via inter-nucleosomal DNA fragmentation. The IC50 value of HNPMI was found to be 64.10 μM in MCF-7 cells and 119.99 μM in SkBr3 cells. Furthermore, HNPMI stimulated DNA fragmentation, which was validated by FACS analysis, resulting in caspase-mediated apoptosis. Structural elucidation also revealed the bi-molecular interaction of HNPMI-EGFR, relating its activity to the anti-proliferative and apoptotic activity.

    Finally, a combined computational analysis was performed to predict the compounds’ interaction with the tyrosine kinase receptor of EGFR. The study revealed that the HNPMI, THMPP, and THTMP interact with the active site region of the EGFR structure (PDB ID: 1M17). The interactions include hydrogen bonds, hydrophobic interactions, pi stacking, and salt bridges. HNPMI was found to have the lowest Glide docking score among the three compounds, reflecting that it can be a better inhibitor than the other two compounds. The investigation of the MMGB/ SA and QM/MM analysis also showed a coherent pattern. It was also found that the protein-ligand complex was stable when it was simulated for 100ns. The Molecular dynamics results also revealed that the ligand interacted with the protein for more than 30% of the simulation time. The compounds also possessed good pharmacokinetic properties, which were predicted by ADME/T analysis.

    Overall, the study demonstrates the effect of cytotoxicity and apoptotic induction of indoline derivatives THMPP, THTMP, and HNPMI. Furthermore, our results revealed the anti-breast cancer property of all three phenolic compounds with HNPMI as the lead molecule. HNPMI was also observed to be a potent EGFR pathway inhibitor, inhibiting the PI3K/ S6K1 signaling pathway and causing cell death in breast cancer cells. Thus, HNPMI can be subjected to further clinical testing and developed as a promising therapeutic medication for the treatment of breast cancer.
    AlkuperäiskieliEnglanti
    JulkaisupaikkaTampere
    KustantajaTampere University
    ISBN (elektroninen)978-952-03-2447-6
    ISBN (painettu)978-952-03-2446-9
    TilaJulkaistu - 2022
    OKM-julkaisutyyppiG5 Artikkeliväitöskirja

    Julkaisusarja

    NimiTampere University Dissertations - Tampereen yliopiston väitöskirjat
    Vuosikerta619
    ISSN (painettu)2489-9860
    ISSN (elektroninen)2490-0028

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