| Macrothelypteris torresiana (Gaud.) Ching, which belongs to Thelypteridaceae family, is widely distributed in the areas to the south of the Yangtze River and has been used as folk medicine usually for the treatment of diseases such as hydropsy and traumatic bleeding. According to literatures, protoapigenone, which is a main component extracted form the plant, exhibited significant antitumor potential. In the present study, the cytotoxic effect of a new flavonoid,2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5,7-dihydroxy-chromone (DEDC), which is a protoapigenone analog, was investigated in human hepatoma HepG2 cells.Plant flavonoids, which are a group of about 4000 naturally occurring compounds, have stimulated much interest in the biochemical and physiological activities of these chemicals. There are some reports about the mechanism for the cancer-preventive actions of flavonoids, but it remains largely unclear about the mechanism of cancer chemoprevention and inhibition. In recent years, great progress has been made in the research on antitumor effects of plant flavonoids, and it is broadly supported that the apoptosis induction activity of representative flavonoids are closely associated with antioxidant activities, membrane protection, mitochondrial toxicity, the inhibition of proteasome, the regulation of signaling pathways, and so on.The most abundantly occurring polyphenols in plants are flavonoids and many studies about flavonoids have focused on their antioxidant properties. In fact, these phenolic compounds have two different characteristics of anti- and pro-oxidant at the same time. It is largely believed that the chemopreventive effects of flavonoids are attributed to its antioxidant capacity and removal of free radicals, while the pro-oxidant activities of these substances may play a much more important role in preventing the formation of malignant tumors or inducing cell apoptosis. Research has shown that the phenolic compounds could be oxidized to phenol radicals by peroxidase and produce ROS by oxidation of GSH or Coenzyme I. A lot of chemopreventive agents can trigger DNA fragmentation and apoptosis in cancer cells in a ROS-dependent fashion.It has been shown that flavonoids can trigger apoptosis through the modulation of a number of key elements in cell signal transduction pathways linked to apoptosis. However, how DEDC regulate and induce the apoptotic process remains to be elucidated. And there are at least two main aspects being considered as below:1. Oxidative stress owing to intracellular pro-oxidation damaged biomembrane, DNA, and other biological macromolecules, which finally resulted in apoptosis of tumor cells; 2. ROS produced at higher concentration modulated some signal transduction pathways such as MAPKs, STAT3, and NF-?B, and then regulated cell physiological responses including growth, differentiation and apoptosis.In the present study, whether DEDC induces hepatoma cell and neuroblastoma cell apoptosis through above signaling pathways, and then initiates a series of cytotoxic effect, which is the main content of this study. The research experiment will go on in vitro, and trypan blue exclusion assay,3-(4,5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT), flow cytometry (FCM), electrophoretic mobility-shift assay (EMSA), real time fluorescent quantitation reverse transcriptase polymerase chain reaction (real time RT-PCR), and Western blot and so on will be used to study the cytotoxic effects and the apoptosis mechanism induced by DEDC. To further investigate the effects of ROS and signal molecules on DEDC-induced apoptosis, ROS scavenger NAC, SP600125, a JNK-specific inhibitor, and SB203580, a p38-specific inhibitor were recruited in HepG2 cells; NAC and PDTC, a NF-?B-specific inhibitor, were used in SH-SY5Y cells. Part?Experimental study on apoptosis of human hepatoma HepG2 cells induced by DEDCIn light of earlier reports suggesting that protoapigenone showed remarkable anticancer activities. In the present study, the cytotoxic effect of a new flavonoid, 2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5,7-dihydroxy-chromone (DEDC), which is a protoapigenone analog, was investigated in human hepatoma HepG2 cells. To further investigate the mechanisms of the DEDC-induced cell death, we examined the effects of reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) and selective inhibitors for MAPK pathways on the cell death.HepG2 cells and LO2 cells were treated by DEDC or protoapigenone with same concentration to detect the cell viability and cell apoptosis rate by Trypan blue exclusion assay and flow cytometry, respectively. HepG2 cells were treated with different concentrations of DEDC (2.5,5, 10?g/ml) for 24h alone, or pretreated with NAC for 2 h then co-treated with DEDC together for another 24 h to determine intracellular ROS accumulation and mitochondrial transmembrane potential. Protein expression levels of Cytochrome c?Procaspase-3,-8,-9, Bcl-2, Bax, ERK, JNK, and P38 MAPK in DEDC-treated hepatoma cells were investigated by Western blot in the presence or absence of respective inhibitor (NAC, SB203580, SP600125). The results were as follows:DEDC could cause obvious growth inhibition of HepG2 cells at the tested range of concentration as well as show much less cytotoxic to the hepatocyte LO2 cells while protoapigenone showed significant cytotoxicity to both HepG2 cells and LO2 cells. Compared to protoapigenone, DEDC has distinct apoptosis inducing effect on tumors, but no influence on normal cells. The sustainable and rapid generation of ROS and the collapse of mitochondrial membrane potential were observed in DEDC-induced cell death, while the effects could be antagonized by pretreatment with NAC. Western blot analysis confirmed that reduced Bcl-2 expression, increased cytochrome c release, and activated caspase-3,-8, and -9, phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK) were triggered by treatment with DEDC. When cells were preincubated with specific inhibitor, the DEDC-induced cell death was significantly inhibited by both NAC and JNK inhibitor SP600125, but promoted by p38 MAPK inhibitor, SB203580.In summary for this part, DEDC has the obvious toxic effect on HepG2 cell and inhibited hepatoma cell proliferation via the induction of apoptosis. DEDC-induced apoptosis might be associated with ROS-mediated regulation of JNK and p38 MAPK, at the same time involve Bcl-2- and caspase-dependent mitochondrial dysfunction.Part?Experimental study on apoptosis of human neuroblastoma SH-SY5Y cells induced by DEDCThe poor prognosis of neuroblastoma and lack of effective remedies have necessitated the application of new therapeutic scheme. Over the past few years, it has been found that flavonoids could exert specific cytotoxic activity towards cancer cells. 2-(cis-1,2-dihydroxy-4-oxo-cyclohex-5-enyl)-5,7-dihydroxy-chromone (DEDC) is a plant-derived flavonoid extracted from the aerial part of Macrothelypteris torresiana. The present study investigated the cytotoxic effects and underlying biochemical pathway leading to cell death on the response of DEDC treatment in human neuroblastoma cells.Cytotoxic effects of the treatments on four tumor cell lines (SH-SY5Y, A549, PC-3, and MCF-7) were determined according to the MTT assay. Electrophoretic mobility-shift assay and western blot were respectively used to detect DNA-protein interactions and protein expression levels of p-I?B and I?B when SH-SY5Y cells were exposed to DEDC (7.5?g/ml) at indicated time intervals (3 h,6 h,12 h, and 24 h). And changes of expression of STAT3, NF-?B, p53, p21, Bcl-2, and Bax proteins in SH-SY5Y cells after treated with different dosages of DEDC (2.5,5,7.5, 10?g/ml) western blot analysis. To further elucidate the roles of ROS and NF-?B in SH-SY5Y cell apoptosis, we investigated the cell viability, apoptosis rate, cell cycle distribution, intracellular ROS accumulation, and the expression levels of mRNA and proteins of NF-?B, STAT3/p-STAT3, P53, and p21 in the presence and absence of NAC or PDTC (a potent NF-?B inhibitor) by MTT, flow cytometry, real time fluorescent quantitation RT-PCR, and Western blot.The growth inhibition effect of DEDC on cancer cells was studied using the MTT assay and the IC50 values were ranged from 5.2?g/ml to 11.5?g/ml (Fig.2). Among the four tumor cell lines, SH-SY5Y showed the lowest IC50 value and therefore was selected for further analysis. We found that:(a) DEDC induced SH-SY5Y cells apoptosis by elevating reactive oxygen species (ROS) generation, quenching of ROS by the antioxidants N-acetyl cystein abolishes the pro-apoptotic activity of DEDC; (b) The signal transducer and activator of transcription 3 (STAT3) played a crucial role in DEDC-triggered cell death; (c) Nuclear factor Kappa B (NF-?B) was activated following exposure to DEDC, and suppressing NF-?B pathway by pyrrolidine dithiocarbamate (PDTC, a potent NF-?B inhibitor) significantly increased neuroblastoma cell sensitivity to the pro-apoptotic effect of DEDC.In summary for this part, DEDC could induce apoptosis via negative regulation of STAT3 signal transduction and following up-regulation of p53 protein and its downstream effects in a ROS-dependent fashion. Additionally, our results confirmed a role for NF-?B in DEDC-induced apoptosis exhibited by neuroblastoma SH-SY5Y cells, and the combination therapy targeting NF-?B may be a potential strategy for treating patients with neuroblastoma. |
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