METHODS: Semen specimens of 151 normozoospermic men were analyzed for ROS by chemiluminescence and classified according to seminal ROS levels [in relative light units (RLU)/s/106 sperm]: group 1 (n = 39): low (ROS DNA fragmentation index (DFI), chromatin maturation index (CMI), H19-Igf2 methylation status, and untargeted seminal metabolic profiling using nuclear magnetic resonance spectroscopy (1H-NMR), was carried out.

METHODS: MTT assay, DNA fragmentation, ELISA and cell cycle analysis were carried out.

RESULTS: Nordamnacanthal and damnacanthal at IC50 values of 1.7 μg/mL and10 μg/mL, respectively. At the molecular level, these compounds caused internucleosomal DNA cleavage producing multiple 180-200 bp fragments that are visible as a "ladder" on the agarose gel. This was due to the activation of the Mg2+/Ca2+-dependent endonuclease. The induction of apoptosis by nordamnacanthal was different from the one induced by damnacanthal, in a way that it occurs independently of ongoing transcription process. Nevertheless, in both cases, the process of dephosphorylation of protein phosphates 1 and 2A, the ongoing protein synthesis and the elevations of the cytosolic Ca2+ concentration were not needed for apoptosis to take place. Nordamnacanthal was found to have a cytotoxic effect by inducing apoptosis, while damnacanthal caused arrest at the G0/G1 phase of the cell cycle.

METHODS: The cytotoxicity activity was measured using MTS assay. The mode of cell death was analysed by early (phosphatidylserine externalization) and late apoptosis (DNA fragmentation). The caspases 8, 9, 3/7 and apoptotic proteins bax, bcl-2 study were done by western blot and ELISA method.

RESULTS: The methanol extract was found to inhibit 50% growth of T-47D cells at the concentration of 79.43µg/ml respectively after 72hr. From seven fractions, fraction F1, F2 and F3 produced cytotoxicity effects in T-47D cell line with IC50 (72hr) < 30µg/ml. The results obtained by Annexin V/PI apoptosis detection assay and TUNEL assay suggest that active fractions of  Vitex rotundifolia induced early and late apoptosis (DNA fragmentation) in T-47D cell line. Moreover, western blot analysis and Caspase GloTM luminescent assay demonstrated that fractions F2 and F3 triggered apoptotic cell death via activation of caspases -8, -9 and -3/7 and up-regulation of  Bax and down-regulation of Bcl-2 protein.  Furthermore, chemical profiling confirms the presence of potential metabolites (vitexicarpin) in fractions of Vitex rotundifolia.

MATERIALS AND METHODS: Anti-cancer activity of a tocotrienol-rich fraction (TRF) and a tocotrienol-enriched fraction (TEF) isolated from palm oil, as well as pure vitamin E analogues (α-tocopherol, α-, δ- and γ-tocotrienols) were studied using highly aggressive triple negative MDA-MB-231 cells and oestrogen-dependent MCF-7 cells, both of human breast cancer cell lines. Cell population growth was evaluated using a Coulter particle counter. Cell death mechanism, poly(ADP-ribose) polymerase cleavage and levels of NF-κB were determined using commercial ELISA kits.

RESULTS: Tocotrienols exerted potent anti-proliferative effects on both types of cell by inducing apoptosis, the underlying mechanism of cell death being ascertained using respective IC50 concentrations of all test compounds. There was marked induction of apoptosis in both cell lines by tocotrienols compared to treatment with Paclitaxel, which was used as positive control. This activity was found to be associated with cleavage of poly(ADP-ribose) polymerase (a DNA repair protein), demonstrating involvement of the apoptotic cell death signalling pathway. Tocotrienols also inhibited expression of nuclear factor kappa-B (NF-κB), which in turn can increase sensitivity of cancer cells to apoptosis.