MATERIALS AND METHODS: The inhibitory effects of hexane (LHXN), dichloromethane (LDCM), ethyl acetate (LEA) and methanol (LMEOH) extracts from leaves of PS on Aβ-induced production and mRNA expression of pro-inflammatory mediators in BV-2 microglial cells were assessed using colorimetric assay with Griess reagent, ELISA kit and real-time RT-PCR respectively. Subsequently, MTT reduction assay was used to evaluate the neuroprotective effects of PS leaf extracts against Aβ-induced microglia-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. The levels of tau proteins phosphorylated at threonine 231 (pT231) and total tau proteins (T-tau) were determined using ELISA kits.
RESULTS: Polar extracts of PS leaves (LEA and LMEOH) reduced the Aβ-induced secretion of pro-inflammatory cytokines (IL-1β and TNF-α) in BV-2 cells by downregulating the mRNA expressions of pro-inflammatory cytokines. The inhibition of nitric oxide (NO) production could be due to the free radical scavenging activity of the extracts. In addition, conditioned media from Aβ-induced BV-2 cells pre-treated with LEA and LMEOH protected SH-SY5Y cells against microglia-mediated neurotoxicity. Further mechanistic study suggested that the neuroprotective effects were associated with the downregulation of phosphorylated tau proteins.
CONCLUSIONS: The present study suggests that polar extracts of PS leaves confer neuroprotection against Aβ-induced microglia-mediated neurotoxicity in SH-SY5Y cells by attenuating tau hyperphosphorylation through their anti-inflammatory actions and could be a potential therapeutic agent for Alzheimer's disease.
PURPOSE: Here, we investigated whether OSCC cells were sensitive towards zerumbone treatment and further determined the molecular pathways involved in the mechanism of action.
METHODS: Cytotoxicity, anti-proliferative, anti-migratory and anti-invasive effects of zerumbone were tested on a panel of OSCC cell lines. The mechanism of action of zerumbone was investigated by analysing the effects on the CXCR4-RhoA and PI3K-mTOR pathways by western blotting.
RESULTS: Our panel of OSCC cells was broadly sensitive towards zerumbone with IC50 values of less than 5 µM whereas normal keratinocyte cells were less responsive with IC50 values of more than 25 µM. Representative OSCC cells revealed that zerumbone inhibited OSCC proliferation and induced cell cycle arrest and apoptosis. In addition, zerumbone treatment inhibited migration and invasion of OSCC cells, with concurrent suppression of endogenous CXCR4 protein expression in a time and dose-dependent manner. RhoA-pull down assay showed reduction in the expression of RhoA-GTP, suggesting the inactivation of RhoA by zerumbone. In association with this, zerumbone also inhibited the PI3K-mTOR pathway through the inactivation of Akt and S6 proteins.
CONCLUSION: We provide evidence that zerumbone could inhibit the activation of CXCR4-RhoA and PI3K-mTOR signaling pathways leading to the reduced cell viability of OSCC cells. Our results suggest that zerumbone is a promising phytoagent for development of new therapeutics for OSCC treatment.
METHODS: The effect of AMEAE on cell proliferation of different cell lines was analyzed by MTT assay. High content screening (HCS) was applied to investigate the suppression of NF-κB translocation, cell membrane permeability, mitochondrial membrane potential (MMP) and cytochrome c translocation from mitochondria to cytosol. Reactive oxygen species (ROS) formation, lactate dehydrogenase (LDH) release and activation of caspase-3/7, -8 and -9 were measured while treatment. The western blot analysis also carried out to determine the protein expression of cleaved caspase-3 and -9. Flow cytometry analysis was used to determine the cell cycle distribution and phosphatidylserine externalization. Quantitative PCR analysis was performed to measure the gene expression of Bax and Bcl-2 proteins.
RESULTS: Cell viability analysis revealed the selective cytotoxic effect of AMEAE towards lung cancer cells, A549, with an IC50 value of 5.09 ± 0.41 μg/mL after 72 h of treatment. Significant LDH leakage and phosphatidylserine externalization were observed in AMEAE treated cells by fluorescence analysis. Treatment of A549 cells with AMEAE significantly elevated ROS formation, followed by attenuation of MMP via upregulation of Bax and downregulation of Bcl-2, accompanied by cytochrome c release to the cytosol. The incubation of A549 cells with superoxide dismutase and catalase significantly attenuated the cytotoxicity caused by AMEAE, indicating that intracellular ROS plays a pivotal role in cell death. The released cytochrome c triggered the activation of caspase-9 followed by caspase-3. In addition, AMEAE-induced apoptosis was accompanied by cell cycle arrest at G0/G1 phase. Moreover, AMEAE suppressed the induced translocation of NF-κB from cytoplasm to nucleus.
CONCLUSIONS: Our data showed for the first time that the ethyl acetate extract of Annona muricata inhibited the proliferation of A549 cells, leading to cell cycle arrest and programmed cell death through activation of the mitochondrial-mediated signaling pathway with the involvement of the NF-kB signalling pathway.