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A Bowman – Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition

View Article: PubMed Central - PubMed

ABSTRACT

Proteasome inhibitors are emerging as a new class of chemopreventive agents and have gained huge importance as potential pharmacological tools in breast cancer treatment. Improved understanding of the role played by proteases and their specific inhibitors in humans offers novel and challenging opportunities for preventive and therapeutic intervention. In this study, we demonstrated that the Bowman–Birk protease inhibitor from Vigna unguiculata seeds, named black-eyed pea trypsin/chymotrypsin Inhibitor (BTCI), potently suppresses human breast adenocarcinoma cell viability by inhibiting the activity of proteasome 20S. BTCI induced a negative growth effect against a panel of breast cancer cells, with a concomitant cytostatic effect at the G2/M phase of the cell cycle and an increase in apoptosis, as observed by an augmented number of cells at the sub-G1 phase and annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining. In contrast, BTCI exhibited no cytotoxic effect on normal mammary epithelial cells. Moreover, the increased levels of intracellular reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in cells treated with BTCI indicated mitochondrial damage as a crucial cellular event responsible for the apoptotic process. The higher activity of caspase in tumoral cells treated with BTCI in comparison with untreated cells suggests that BTCI induces apoptosis in a caspase-dependent manner. BTCI affected NF-kB target gene expression in both non invasive and invasive breast cancer cell lines, with the effect highly pronounced in the invasive cells. An increased expression of interleukin-8 (IL-8) in both cell lines was also observed. Taken together, these results suggest that BTCI promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition. These findings highlight the pharmacological potential and benefit of BTCI in breast cancer treatment.

No MeSH data available.


Related in: MedlinePlus

BTCI-induced mitochondrial membrane depolarization. (a) MCF-7 and (b) MDA-MB-231 cells were incubated with BTCI at the concentration of 150 μM and 100 μM, respectively, for 24 h. Dye-loading solutions were added to cells and incubated for 30 min. The change of mitochondrial membrane potential was measured by using Mitochondrial Membrane Potential Kit (Sigma). Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.01 versus untreated cells.
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fig5: BTCI-induced mitochondrial membrane depolarization. (a) MCF-7 and (b) MDA-MB-231 cells were incubated with BTCI at the concentration of 150 μM and 100 μM, respectively, for 24 h. Dye-loading solutions were added to cells and incubated for 30 min. The change of mitochondrial membrane potential was measured by using Mitochondrial Membrane Potential Kit (Sigma). Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.01 versus untreated cells.

Mentions: Proteasome inhibition is known to induce apoptosis through mitochondrial cytochrome c release. As BTCI inhibits the proteasome function, its involvement in mitochondrial dysfunction in cancer cells was investigated. A sensitive cationic and lipophilic JC-10 fluorescent probe was used to monitor the mitochondrial membrane potential change in cells based on the presence of the JC-10 dye. JC-10 concentrates in the mitochondrial matrix where it forms red fluorescent aggregates. However, in apoptotic and necrotic cells, JC-10 diffuses out of mitochondria and changes from the aggregate to a monomeric form, causing a shift in fluorescence emission from ~590 to 515 nm, staining cells in green fluorescence. As shown in Figure 5, the mitochondrial membrane potential in both MCF-7 and MDA.MB.231 cells dropped, as indicated by increasing in monomer/aggregate ratio (P=0.016 and P=0.009, respectively). These data indicate that mitochondrial membrane depolarization and mitochondrial swelling precede an increase in oxidative stress and loss of cell viability in cells exposed to BTCI.


A Bowman – Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition
BTCI-induced mitochondrial membrane depolarization. (a) MCF-7 and (b) MDA-MB-231 cells were incubated with BTCI at the concentration of 150 μM and 100 μM, respectively, for 24 h. Dye-loading solutions were added to cells and incubated for 30 min. The change of mitochondrial membrane potential was measured by using Mitochondrial Membrane Potential Kit (Sigma). Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.01 versus untreated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979482&req=5

fig5: BTCI-induced mitochondrial membrane depolarization. (a) MCF-7 and (b) MDA-MB-231 cells were incubated with BTCI at the concentration of 150 μM and 100 μM, respectively, for 24 h. Dye-loading solutions were added to cells and incubated for 30 min. The change of mitochondrial membrane potential was measured by using Mitochondrial Membrane Potential Kit (Sigma). Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.01 versus untreated cells.
Mentions: Proteasome inhibition is known to induce apoptosis through mitochondrial cytochrome c release. As BTCI inhibits the proteasome function, its involvement in mitochondrial dysfunction in cancer cells was investigated. A sensitive cationic and lipophilic JC-10 fluorescent probe was used to monitor the mitochondrial membrane potential change in cells based on the presence of the JC-10 dye. JC-10 concentrates in the mitochondrial matrix where it forms red fluorescent aggregates. However, in apoptotic and necrotic cells, JC-10 diffuses out of mitochondria and changes from the aggregate to a monomeric form, causing a shift in fluorescence emission from ~590 to 515 nm, staining cells in green fluorescence. As shown in Figure 5, the mitochondrial membrane potential in both MCF-7 and MDA.MB.231 cells dropped, as indicated by increasing in monomer/aggregate ratio (P=0.016 and P=0.009, respectively). These data indicate that mitochondrial membrane depolarization and mitochondrial swelling precede an increase in oxidative stress and loss of cell viability in cells exposed to BTCI.

View Article: PubMed Central - PubMed

ABSTRACT

Proteasome inhibitors are emerging as a new class of chemopreventive agents and have gained huge importance as potential pharmacological tools in breast cancer treatment. Improved understanding of the role played by proteases and their specific inhibitors in humans offers novel and challenging opportunities for preventive and therapeutic intervention. In this study, we demonstrated that the Bowman&ndash;Birk protease inhibitor from Vigna unguiculata seeds, named black-eyed pea trypsin/chymotrypsin Inhibitor (BTCI), potently suppresses human breast adenocarcinoma cell viability by inhibiting the activity of proteasome 20S. BTCI induced a negative growth effect against a panel of breast cancer cells, with a concomitant cytostatic effect at the G2/M phase of the cell cycle and an increase in apoptosis, as observed by an augmented number of cells at the sub-G1 phase and annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining. In contrast, BTCI exhibited no cytotoxic effect on normal mammary epithelial cells. Moreover, the increased levels of intracellular reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in cells treated with BTCI indicated mitochondrial damage as a crucial cellular event responsible for the apoptotic process. The higher activity of caspase in tumoral cells treated with BTCI in comparison with untreated cells suggests that BTCI induces apoptosis in a caspase-dependent manner. BTCI affected NF-kB target gene expression in both non invasive and invasive breast cancer cell lines, with the effect highly pronounced in the invasive cells. An increased expression of interleukin-8 (IL-8) in both cell lines was also observed. Taken together, these results suggest that BTCI promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition. These findings highlight the pharmacological potential and benefit of BTCI in breast cancer treatment.

No MeSH data available.


Related in: MedlinePlus