title: Epoxy clerodane diterpene inhibits MCF-7 human breast cancer cell growth by regulating the expression of the functional apoptotic genes Cdkn2A, Rb1, mdm2 and p53.
abstract: Systematic analyses of plants that are used in traditional medicine may lead to the discovery of novel cytotoxic secondary metabolites. Diterpene possesses multiple bioactivities; here, epoxy clerodane diterpene (ECD) was isolated from Tinospora cordifolia (Willd.) stem and shown potential antiproliferative effect in MCF-7 human breast cancer cells. The antiproliferative effect of ECD on MCF-7 cells was systematically analyzed by cell and nuclear morphology, alterations in oxidative stress, and the expression of tumor suppressor and mitochondria-mediated apoptosis-related genes. We found that the IC50value of ECD was 3.2μM at 24h and 2.4μM at 48h. We observed that the cytotoxicity of ECD was specific to MCF-7 cells, whereas ECD was nontoxic to normal Vero and V79 cells. ECD significantly triggered intracellular ROS generation even from the lower doses of 0.6 and 1.2μM; and it is relative to higher dose of 2.4μM. Further, we used 0.6μM, 1.2μM and 2.4μM as experimental doses to analyze the relative dose-dependent effects. Nuclear staining revealed that cells treated with the 2.4μM dose exhibited characteristic apoptotic morphological changes and that 46% of the cells were apoptotic and 4% were necrotic after 48h. ECD significantly increased the expression of mitochondria-dependent apoptotic pathway-related genes after 48h; we observed significantly (p≤0.05) increased expression of CYP1A, GPX, GSK3β and TNF-α and downregulated expression of NF-κB. ECD also increased the expression of tumor suppressor genes such as Cdkn2A, Rb1 and p53. In addition, we observed that ECD treatment significantly (p≤0.001) upregulated the expression of apoptotic genes such as Bax, cas-3, cas-8, cas-9 and p21 and downregulated the expression of BCL-2, mdm2 and PCNA. In conclusion, ECD regulates the expression of Cdkn2A, p53 and mdm2 and induces apoptosis via the mitochondrial pathway in MCF-7 human breast cancer cells.
authors.affiliation: Cancer Molecular Biology Research Lab, Department of Food Sceince and Nutrition, College of Food Science and Agriculture, King Saud University, Riyadh - 11451, Saudi Arabia.
authors.forename: P
authors.initials: P
authors.lastname: Subash-Babu
authors.affiliation: Cancer Molecular Biology Research Lab, Department of Food Sceince and Nutrition, College of Food Science and Agriculture, King Saud University, Riyadh - 11451, Saudi Arabia.
authors.forename: Ghedeir M
authors.initials: GM
authors.lastname: Alshammari
authors.affiliation: Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai - 600 034, Tamil Nadu, India.
authors.forename: S
authors.initials: S
authors.lastname: Ignacimuthu
authors.affiliation: Cancer Molecular Biology Research Lab, Department of Food Sceince and Nutrition, College of Food Science and Agriculture, King Saud University, Riyadh - 11451, Saudi Arabia. Electronic address: alshatwi@ksu.edu.sa.
authors.forename: Ali A
authors.initials: AA
authors.lastname: Alshatwi
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects* / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18 / Q000235:genetics*; D045785:Diterpenes, Clerodane / Q000494:pharmacology*; D005260:Female; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006801:Humans; D061986:MCF-7 Cells; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics*; D056286:Retinoblastoma Binding Proteins / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
chemical_list: C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18; D045785:Diterpenes, Clerodane; D016328:NF-kappa B; C000606393:RB1 protein, human; D056286:Retinoblastoma Binding Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases
keywords: Apoptosis; Caspase; Epoxy clerodane diterpene; MCF-7; Tinospora cordifolia