Study On The Mechanism And Regulation Of P21^Waf1/Cip1 And PI3K/Akt Signaling Pathway In Furazolidone-induced Apoptosis In Hepg2Cells

Furazolidone (FZD) is an artificial synthetic nitrofuran drug, with a broad spectrum of antimicrobial activities. FZD was widely used as a growth promoter as feed additive for livestock. With the intensive study of its toxicity in vitro and in vivo, FZD has been exhibited genetic toxicity and potential carcinogenicity. But its toxic mechanism remains unclear. Our previous studies found that FZD can inhibit HepG2cell proliferation, induce S phase arrest and cause cell DNA oxidative damage. The JNK/p38MAPK signaling pathway is involved in the process of FZD-induced HepG2cell S phase arrest. However, it is still unknown whether apoptosis is involved in FZD toxic effects on HepG2cells. Therefore, the present study focused on the molecular mechanism of FZD-induced apoptosis in HepG2cells, the regulatory effects of PI3K/Akt signaling pathway and p21Wafl/Cipl (p21) gene during the apoptotic process, which may help to provide a theoretical basis for further elucidate the toxic mechanism of FZD.In this study, HepG2cells were treated with0,12.5,25and50μg/ml of FZD for24h, respectively, then cell proliferation, apoptotic morphological changes and apoptotic rate were detected by MTT assay, Hoechst/PI staining and flow cytometry. The results showed that cell proliferation was blocked by FZD, typical apoptotic morphological changes were observed, and the rates of apoptosis were dose-dependently increased. Using specific fluorescent dyes DCFH-DA and Rhodamine123to measure the changes of intracellular ROS level and mitochondrial membrane potential (MMP), the results showed that FZD dramatically increased intracellular ROS levels and decreased the MMP in a dose-dependent manner. Detection of caspases activities and apoptosis-related genes mRNA and protein expression showed that FZD dose-dependently increased activities of caspase-9and caspase-3, upregulated mRNA levels of caspase-9, caspase-3, Bax and Cyt c, downregulated mRNA levels of Bcl-2. Western blot indicated that FZD induced PAPR cleavage, decreased the protein expression of procaspase-9and procaspase-3, increased Bax/Bcl-2ratio, and promoted Cyt c release to cytosol. Moreover, the inhibitor of caspase-9, caspase-3and pan caspase significantly suppressed FZD-induced apoptosis. Antioxidant NAC attenuated FZD-induced ROS generation, blocked the collapse of MMP, regulated apoptosis-related proteins expression and caspases activities, finally suppressed FZD-induced apoptosis. These data demonstrated that FZD could induce apoptosis in HepG2cells, which might be associated with ROS-and caspase-dependent mitochondrial pathway.In order to investigate the mechanism of PI3K/Akt signaling pathway in FZD-induced apoptosis in HepG2cells, firstly, the effects of FZD on mRNA and protein levels of Akt were measured. Then after activated or inhibited the PI3K/Akt pathway by the activator IGF-1or inhibitor LY294002, cell proliferation, apoptosis rate, ROS levels, MMP, activities of caspase and apoptosis related protein expression of FZD-treated HepG2cells were detected. The results showed that FZD downregulated the mRNA levels of Akt and protein levels of total Akt and p-Akt. Interestingly, NAC increased the expression of p-Akt without affecting the total Akt expression.50ng/ml IGF-1and20μM LY294002 effectively enhanced or inhibited the phosphorylation of Akt. The activation of PI3K/Akt obviously reduced FZD-induced apoptosis and inhibition of cell proliferation, while the inhibition of PI3K/Akt further enhanced the apoptosis and inhibition of cell proliferation. Activation or inhibition of PI3K/Akt did not affect the ROS levels in HepG2cells. The MMP, expression of apoptosis-related proteins and caspase activities showed that IGF-1inhibited FZD-induced cleavage of PARP, activation of caspase-9and caspase-3and release of Cyt c. Consistently, MMP collapse and increase of Bax/Bcl-2ratio were both blocked by IGF-1. After LY294002inhibited PI3K/Akt, the above-mentioned results showed the opposite trend. These data indicated that PI3K/Akt signaling pathway was involved in FZD-induced mitochondrial dysfunction and apoptosis. Moreover, FZD inhibited PI3K/Akt pathway through elevation the intracellular ROS levels.To investigate the role of p21in FZD-induced apoptosis, firstly, the regulation of p21expression by FZD was detected FZD increased the mRNA level of p21, but the protein expression of p21was reduced dramatically. The western blot and immnuoflourescence staining results showed that FZD decreased p21protein stability, shorten the half-life, increased ubiquitination of p21and induced p21translocated from cytoplasm to nucleus in HepG2cells. Further study found that IGF-1stabilized p21protein and blocked FZD-induced p21translocation. On the contrary, LY294002enhanced the effect of FZD on p21stability and translocation. These results suggested that the change of p21stability and nucleus translocation by FZD might be related to inhibition of P13K/Akt signaling pathway.Next, after transfected p21expression plasmid or siRNA into HepG2cells, the effects of different p21expression levels on FZD-induced apoptosis were investigated. The MTT assay and flow cytometry showed that the over expression of p21increased HepG2cells viability and decreased apoptosis rate, the low expression of p21enhanced FZD-induced apoptosis. The western blot analysis showed that different expression levels of p21affected activation of caspase-3and cleavage of PARP by FZD. The intracellular ROS detection showed that over expression of p21reduced ROS generation and low expression of p21had opposite effects. Meanwhile, p21regulated Nrf2/HO-1signaling pathway which is related to oxidative stress. Over expression of p21further activated Nrf2/HO-1pathway to reduce oxidative damage. In contrast, low expression of p21inhibited FZD-induced activation of Nrf2/HO-l pathway to reduce antioxidant ability of cell. These above data indicated that p21participated FZD-induced apoptosis via activating oxidative protective pathway, reducing ROS generation and suppressing activation of caspase.In conclusion, FZD can cause apoptosis in HepG2cells. The possible mechanism is as follows:the elevation of ROS induced by FZD could activate mitochondrial apoptotic pathway and inhibit PI3K/Akt signaling pathway, causing apoptosis. Meanwhile, suppression of PI3K/Akt by FZD causes decreased expression of p21, which blocks the activation of Nrf2/HO-l signaling pathway under oxidative stress, leading to promoting the occurrence of apoptosis.