Mechanism of juglone-induced apoptosis of MCF-7 cells by the mitochondrial pathway

Abstract

ABSTRACT. This study investigated the nature and mechanism of juglone-induced apoptosis in the human breast cancer cell line MCF-7. The inhibitory effect of juglone on MCF-7 cell growth was evaluated by the dimethylthiazol tetrazolium assay. Morphological apoptotic changes were characterized using an inverted microscope, Hoechst 33258 fluorescence staining, and Giemsa staining. The rate of cell apoptosis, intracellular levels of reactive oxygen species (ROS), and mitochondrial membrane potential were detected using flow cytometry. Intracellular Ca 2+ concentrations were detected using laser scanning confocal fluorescence microscopy. Expression of the proteins Bcl-2, Bax, and cytochrome C was assessed by western blotting. Caspase-3 activity was quantified using a caspase-3 activity kit. Juglone inhibited the growth of MCF-7 cell line with an IC50 of 11.99 µM. The rates of MCF-7 cell apoptosis at 24 h after exposure to 5, 10, and 20 µM juglone were 9.29, 20.67, and 28.39%, respectively; compared to unexposed cells, juglone-exposed cells exhibited significant elevation in intracellular ROS level, decrease in mitochondrial membrane potential, and increase in intracellular Ca 2+ concentration. Juglone upregulated the expression of Bax, and downregulated the expression of Bcl-2, promoting the release of cytochrome C, thereby upregulating the activity of caspase-3. The results suggest that the mechanism of juglone-induced apoptosis in MCF-7 cells is characterized by elevated ROS levels, reduced Bcl-2 expression, increased Bax expression, decreased mitochondrial membrane potential, increased intracellular Ca 2+ concentration, outer mitochondrial-membrane rupture, cytochrome C release, and caspase-3 activation.

Indomethacin and juglone inhibit inflammatory molecules to induce apoptosis in colon cancer cells

“…[36] Overexpression of Bcl2 causes the loss of proapoptotic molecules Bax, Bad, cytochrome c, and PUMA in cancer. [38][39][40][41] The results obtained in this study show that indomethacin, juglone, and cotreatment of these two drugs, exhibits their action, presumably, by downregulating the inflammatory mediators, inhibiting the activation of antiapoptotic molecules and increasing the levels of proapoptotic molecules to induce apoptosis in HT29 cells. Cells treated with drugs (indomethacin, juglone, and combination of indomethacin and juglone) not only exhibited decreased expression of antiapoptotic Bcl2, but also showed an increased expression of proapoptotic proteins, which is in agreement with previous reports.…”

Notch3 contributes to T-cell leukemia growth via regulation of the unfolded protein response

“…In order to evaluate the potential anti-leukemic effects of the Notch3 downregulation under ER stress conditions previously observed (Figs. 2 and 3, siN3+Tun) also in in vivo studies, we chose the natural compound Juglone for the subsequent experiments, since several literature's data 11,[33][34][35] , including ours 36 , showed its ability to induce different molecular mechanisms potentially resulting in the simultaneous ER stress induction and Notch3 blocking.…”

Natural Products to Fight Cancer: A Focus on Juglans regia

“…Juglone ( Figure 1 ) is a member of this family, acting as a growth-stunting agent [ 136 ] and apoptosis inducer. It exhibits antitumour activity on many types of tumours, such as breast [ 92 , 93 , 94 , 95 , 137 ], skin [ 99 , 100 , 137 ], glial cells [ 109 , 110 , 111 , 135 , 138 ], lung [ 137 , 138 ], prostate [ 105 , 106 , 107 , 137 ], pancreas [ 97 ], bladder [ 108 ], stomach [ 98 ], cervix [ 102 , 103 , 139 , 140 ], ovary [ 104 ], and blood [ 113 , 114 ]. For some of them, the mechanism of action has been investigated.…”

“…Juglone exhibited anticancer effects on different breast cancer models. On MCF-7, doxo-resistant MCF-7 (MCF-7Adr) and transtuzumab-resistant SKBR3, juglone promoted G1 cell-cycle arrest [ 94 , 95 ] and ROS-driven apoptosis [ 92 , 93 , 95 ]. An exhaustive study on MCF-7 proved that juglone increased Bax/Bcl2 ratio , intracellular calcium (Ca 2+ ) levels, ΔΨ disruption, cytochrome c (Cyt-c) release and caspase 3 activation, demonstrating the activation of the intrinsic apoptotic pathway [ 92 ].…”

“…On MCF-7, doxo-resistant MCF-7 (MCF-7Adr) and transtuzumab-resistant SKBR3, juglone promoted G1 cell-cycle arrest [ 94 , 95 ] and ROS-driven apoptosis [ 92 , 93 , 95 ]. An exhaustive study on MCF-7 proved that juglone increased Bax/Bcl2 ratio , intracellular calcium (Ca 2+ ) levels, ΔΨ disruption, cytochrome c (Cyt-c) release and caspase 3 activation, demonstrating the activation of the intrinsic apoptotic pathway [ 92 ]. On MCF-7 and SKBR3, it inhibited cell proliferation, colony formation, and migration capability [ 93 , 94 ], while on MCF-7Adr, angiogenesis was inhibited by decreased levels of VEGF-A, -B and -C [ 94 ].…”