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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.


Apoptosis induced by BTCI MDA-MB-231. Cells were incubated with BTCI (100 μM) for 24 h, and then stained with annexin V-FITC/PI and flow cytometry analysis. (a and b) representative images of flow cytometry analysis were displayed. (c) The percentage of apoptotic cells was statistically analyzed. Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.001 versus untreated cells.
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fig4: Apoptosis induced by BTCI MDA-MB-231. Cells were incubated with BTCI (100 μM) for 24 h, and then stained with annexin V-FITC/PI and flow cytometry analysis. (a and b) representative images of flow cytometry analysis were displayed. (c) The percentage of apoptotic cells was statistically analyzed. Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.001 versus untreated cells.

Mentions: We previously showed that BTCI significantly induces cell cycle arrest at G2/M phase, DNA fragmentation and enhanced numbers of cells marked by annexin-V in breast cancer MCF-7, suggesting apoptosis as a possible mechanism for the cell death process.13 To explore whether apoptosis is involved in BTCI-induced MDA.MB.231, the amount of sub-G1 DNA in cancer cells treated with BTCI was investigated. As shown in Figure 3, the treatment of MDA.MB.231 cells with BTCI resulted in a marked accumulation of cells in the sub-G1 phase, suggesting a massive apoptotic, but not necrotic, cell death. In addition, BTCI arrested cell cycle at G2/M phase after 24 h. The apoptotic effect of BTCI was confirmed by an annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining assay. BTCI at the concentration of 100 μM for 24 h induced apoptosis in MDA-MB-231 cells (Figure 4). Taken together, these results confirm that BTCI-induced apoptotic cell death in breast adenocarcinoma cells.


A Bowman – Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition
Apoptosis induced by BTCI MDA-MB-231. Cells were incubated with BTCI (100 μM) for 24 h, and then stained with annexin V-FITC/PI and flow cytometry analysis. (a and b) representative images of flow cytometry analysis were displayed. (c) The percentage of apoptotic cells was statistically analyzed. Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.001 versus untreated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979482&req=5

fig4: Apoptosis induced by BTCI MDA-MB-231. Cells were incubated with BTCI (100 μM) for 24 h, and then stained with annexin V-FITC/PI and flow cytometry analysis. (a and b) representative images of flow cytometry analysis were displayed. (c) The percentage of apoptotic cells was statistically analyzed. Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.001 versus untreated cells.
Mentions: We previously showed that BTCI significantly induces cell cycle arrest at G2/M phase, DNA fragmentation and enhanced numbers of cells marked by annexin-V in breast cancer MCF-7, suggesting apoptosis as a possible mechanism for the cell death process.13 To explore whether apoptosis is involved in BTCI-induced MDA.MB.231, the amount of sub-G1 DNA in cancer cells treated with BTCI was investigated. As shown in Figure 3, the treatment of MDA.MB.231 cells with BTCI resulted in a marked accumulation of cells in the sub-G1 phase, suggesting a massive apoptotic, but not necrotic, cell death. In addition, BTCI arrested cell cycle at G2/M phase after 24 h. The apoptotic effect of BTCI was confirmed by an annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining assay. BTCI at the concentration of 100 μM for 24 h induced apoptosis in MDA-MB-231 cells (Figure 4). Taken together, these results confirm that BTCI-induced apoptotic cell death in breast adenocarcinoma cells.

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.