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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.


Cytotoxic effects induced by BTCI. (a) MCF-7, (b) MDA-MB-231 and (c) MCF-10 cells were incubated with BTCI (0-300 μM) for 24 h. Cell viability was determined by MTT assay. Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.05 versus untreated cells.
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fig2: Cytotoxic effects induced by BTCI. (a) MCF-7, (b) MDA-MB-231 and (c) MCF-10 cells were incubated with BTCI (0-300 μM) for 24 h. Cell viability was determined by MTT assay. Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.05 versus untreated cells.

Mentions: The effect of BTCI on viability of MDA.MB.231 (highly invasive human breast cells), MCF-7 (human breast adenocarcinoma cells) and MCF-10A (normal mammary epithelial cells) were determined using the MTT assay as described in the Methods section. Cells were incubated with BTCI at concentrations ranging from 0 to 300 μM for 24 h. Results showed that BTCI exerts its cytotoxic effect in a dose-dependent manner. BTCI at the concentration of 150 μM markedly induced MCF-7 death (P=0.014) (Figure 2a), meanwhile, the same effect on MDA.MB.231 was observed at a concentration of 100 μM (P<0.001; Figure 2b). In contrast, BTCI did not induce a cytotoxic effect on MCF-10A cells. The results of the MTT assay demonstrated a cytotoxic effect of BTCI on breast cancer cell lines in a dose-dependent manner, absent in normal mammary epithelial cells (Figure 2c).


A Bowman – Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition
Cytotoxic effects induced by BTCI. (a) MCF-7, (b) MDA-MB-231 and (c) MCF-10 cells were incubated with BTCI (0-300 μM) for 24 h. Cell viability was determined by MTT assay. Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.05 versus untreated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979482&req=5

fig2: Cytotoxic effects induced by BTCI. (a) MCF-7, (b) MDA-MB-231 and (c) MCF-10 cells were incubated with BTCI (0-300 μM) for 24 h. Cell viability was determined by MTT assay. Results are presented as mean±S.D. of two separate experiments conducted in triplicate, *P<0.05 versus untreated cells.
Mentions: The effect of BTCI on viability of MDA.MB.231 (highly invasive human breast cells), MCF-7 (human breast adenocarcinoma cells) and MCF-10A (normal mammary epithelial cells) were determined using the MTT assay as described in the Methods section. Cells were incubated with BTCI at concentrations ranging from 0 to 300 μM for 24 h. Results showed that BTCI exerts its cytotoxic effect in a dose-dependent manner. BTCI at the concentration of 150 μM markedly induced MCF-7 death (P=0.014) (Figure 2a), meanwhile, the same effect on MDA.MB.231 was observed at a concentration of 100 μM (P<0.001; Figure 2b). In contrast, BTCI did not induce a cytotoxic effect on MCF-10A cells. The results of the MTT assay demonstrated a cytotoxic effect of BTCI on breast cancer cell lines in a dose-dependent manner, absent in normal mammary epithelial cells (Figure 2c).

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.