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  • br Moreover As shown in


    Moreover, As shown in the Fig. 6, the cytosolic cytochrome c in 56-75-7 treated with BHAE increased remarkably campared to the control cells (**P ≤ .01).
    BHAE induced elevation of lipid peroxidation rate, a marker of reac-tive oxygen species (ROS) and an increase of cytosolic cytochrome c level, suggesting that the treated cells go to cell death via the mitochon-drial pathway of apoptosis.
    Consistent with our results, Meeran et al. (2008) reported that treat-ment of the human prostate cancer cells PC3 with Berberine inhibits the proliferation, increases the level of ROS, cytochrome c, Smac/DIABLO, cleaved of pro-caspase-9, -3 and PARP (Poly (ADP-Ribose) polymerase). The treatment also induces disruption of mitochondrial membrane po-tential leading to activation of the mitochondrial pathway of apoptosis. Moreover, they demonstrated that treatment of these cells with a ROS scavenger, N-acetylcysteine (NAC) inhibits the mitochondria swelling after berberine treatment and confirmed that berberine triggered apo-ptosis through ROS-generation (Meeran et al., 2008).
    The activity of BHAE on such stress markers was also investigated in this study. Thus, as presented in Fig. 6 and Table 1. Results obtained showed that BHAE did not induced significant change in Akt and Erk1/ 2 levels (Fig. 6 a and b respectively), however, we noted that the p38MAPK rates in the control and the BHAE treated cells were 60 pg/ mL and 170 pg/mL, respectively (**P ≤ 0.01) (Fig. 6 c). We also noted significant increase in the NF-κB rates in cells submitted to BHAE com-pared with the control cells (**P ≤ 0.01) (Fig. 6 d).
    3.6. The BHAE enhanced the activity ofp53 in Hep-2 cancer cells
    In order to provide a little more clarification and determinate if the p53 was involved in the cells death and in the increase of the Sub G0 fractions, the expression of p53 wasanalyzed by western blot in bothcontrol and subjected cells 56-75-7 and results are illustrated in Fig. 7. The data reveal that the expression of p53 increased in cells submitted to the BHAE compared to the control.
    Taken together, these finding suggest that the plausible mechanism by which treatment induced cell death occurs through the ROS-genera-tion which causes DNA damage and triggers the activation of p38-MAPK, NF-κB and p53 (Guo et al., 2016; Johnson et al., 1996; Schneider et al., 1997; Seger and Krebs, 1995; Waskiewicz and Cooper, 1995; Zarubin and Han, 2005).
    Fig. 7. Effect of Hep-2 cells subjected to BHAE on the p53 expression. Hep-2 cells were submitted to 75 μg/mL of BHAE. After 24 h of incubation, proteins were extracted from culture cells, and p53 and GAPDH protein levels were evaluate by Western blotting.
    In another hand, Chaudhary et al. (1997) and Schneider et al. (1997) demonstrated that high stress induction after anti-cancer drugs treat-ment could activate NF-κB pathway, leading to the activation of numer-ous signaling pathways and can promote, under certain circumstances the activation of apoptosis (Ravi et al., 2001). In fact, the involvement of NF-κB has been demonstrated in human melanoma cell death A375-S2 treated with the evodiamine via the Fas-L/NF-κB pathway (Wang et al., 2010). Further, it is widely accepted that p53 triggered the cell death, either by cell cycle arrest through the induction of genes encoding for the death receptors DR5, Fas and PERP (Nagata, 1997; Pan, 1997) or via the mitochondrial pathway by transcriptional up-regulation of pro-apoptotic genes such as Bax or PUMA (Mantena, 2006). r> Although generation of ROS is essential to p53-expression, cells un-dergoing p53-mediated apoptosis also generated ROS (Johnson et al., 1996). In fact, it has been reported that aberrant ROS-generation medi-ates p53-activation and apoptosis in HeLa cells. Interestingly, treatment of those cells with two antioxidants, N-acetylcysteine (NAC) and 4,5-di-hydroxy-1,3-benzenedisulfonic acid (tiron) significantly reduced the expression of p53 (Li, 1999).
    Taken together, these results indicate that the treatment induced both extrinsic and intrinsic apoptosis pathways and are inaccordance with previous study on KB human oral cancer cells treated with berber-ine (Kim et al., 2015).
    4. Conclusion
    Many studies showed that the use of medicinal plant extracts to con-trol or stop cancer progression should be an alternative to conventional treatment for this disease. In this work we demonstrated the antiprolif-erative effect of Algerian Berberis hispanica alkaloids extract on Hep-2 cancer cells and its ability to induce cell death through ROS-generation leading to morphological and molecular modifications, cell cycle arrest and induction of apoptosis. The results obtained open up prospects for a possible combination of this compound with existent anticancer drug already existing.