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Biotransformed soybean extract (BSE) inhibits melanoma cell growth and viability in vitro: involvement of nuclear factor-kappa B signaling.

Vilela FM, Syed DN, Chamcheu JC, Calvo-Castro LA, Fortes VS, Fonseca MJ, Mukhtar H - PLoS ONE (2014)

Bottom Line: Further studies in A375 cells showed that decrease in cell viability with BSE treatment (1.5-1.9 mg/ml; 24 h) was associated with induction of apoptosis.Immunoblot analysis revealed that BSE treatment resulted in induction of PARP cleavage, activation of caspase-3, -7, and -8 and increased expression of TRAIL and its receptor DR4.BSE did not activate the intrinsic apoptotic pathway in A375 cells, as no change was observed in caspase-9 expression.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil; Department of Dermatology, University of Wisconsin, Madison, Wisconsin, United States of America.

ABSTRACT
Melanoma is recognized as one of the most aggressive cancers with a relatively high propensity for metastasis. The prognosis of melanoma remains poor in spite of treatment advances, emphasizing the importance of additional preventive measures. Isoflavonoids have become not only potential chemopreventive, but also important therapeutic natural agents. We evaluated the antiproliferative and proapoptotic properties of biotransformed soybean extract (BSE) in A375 melanoma cells. Previous analyses demonstrated that the concentration of daidzein, genistein and aminoacids/peptides present in BSE, fermented by Aspergillus awamori is much higher than in the non biotransformed extract (NBSE). Experiments comparing the efficacy of the extracts in preventing cancer cell growth showed that treatment (24 h) of aggressive melanoma cells (A375 and 451Lu) with BSE resulted in a dose-dependent inhibition of growth and viability. In contrast, treatment with similar doses of NBSE failed to inhibit melanoma cell viability. Further studies in A375 cells showed that decrease in cell viability with BSE treatment (1.5-1.9 mg/ml; 24 h) was associated with induction of apoptosis. Immunoblot analysis revealed that BSE treatment resulted in induction of PARP cleavage, activation of caspase-3, -7, and -8 and increased expression of TRAIL and its receptor DR4. BSE did not activate the intrinsic apoptotic pathway in A375 cells, as no change was observed in caspase-9 expression. The expression of Bcl-2 apoptotic proteins such as Bid and Bax remained unaffected with BSE treated cells. Interestingly, we also showed that BSE treatment increased the phosphorylation and activation of IKK, IκBα degradation and p65/NF-κB translocation to the nucleus, and that stimulation of the NF-???B pathway was required for BSE-induced apoptosis of A375 cells. Our findings indicate that the biotransformation of soybean plays a crucial role in the extract anti-cancer effect observed in melanoma cells. However, further studies are warranted to define the active anti-cancer agent(s) present in BSE.

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BSE treatment induced apoptosis in A375 melanoma cells.(A) Phase contrast microscopy: representative pictures of A375 cells treated with BSE (0–1.9 mg/mL) for 24 h. (B) Effect of BSE treatment (0–1.9 mg/mL) on apoptosis in A375 cells as demonstrated by annexin-V staining (green fluorescence). (C) Effect of BSE treatment (0–1.9 mg/mL) on activation of caspase-3 and PARP cleavage. Whole cell lysates were analyzed by immunoblot analysis. Equal loading was confirmed by reprobing for β-actin. The values above the figures represent relative density of the bands (cleaved caspase 3 and PARP) normalized to β-actin. Data shown are representative of three independent experiments. (D) Effect of BSE (1.7 mg/mL) treatment on the activation of caspase-3 as detected by immunocytochemistry. A representative picture from three independent experiments with similar results is shown.
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pone-0103248-g002: BSE treatment induced apoptosis in A375 melanoma cells.(A) Phase contrast microscopy: representative pictures of A375 cells treated with BSE (0–1.9 mg/mL) for 24 h. (B) Effect of BSE treatment (0–1.9 mg/mL) on apoptosis in A375 cells as demonstrated by annexin-V staining (green fluorescence). (C) Effect of BSE treatment (0–1.9 mg/mL) on activation of caspase-3 and PARP cleavage. Whole cell lysates were analyzed by immunoblot analysis. Equal loading was confirmed by reprobing for β-actin. The values above the figures represent relative density of the bands (cleaved caspase 3 and PARP) normalized to β-actin. Data shown are representative of three independent experiments. (D) Effect of BSE (1.7 mg/mL) treatment on the activation of caspase-3 as detected by immunocytochemistry. A representative picture from three independent experiments with similar results is shown.

Mentions: A375 cells treated with BSE (1.5–1.9 mg/mL; 24 h) showed dose-dependent alterations in cell morphology (Figure 2A) and induction of apoptosis which was evident from the enhancement in fluorescent green Annexin V staining (Figure 2B). To further assess the molecular mechanism involved in BSE-induced apoptosis, we examined whether BSE treatment would result in activation of caspase-3 and PARP cleavage, which are considered important indicators of apoptotic cell death [17], [18]. Accordingly western blot analysis and immunocytochemistry showed that BSE treatment resulted in an increase in both caspase-3 activation (Figure 2C and D) and PARP cleavage (Figure 2C).


Biotransformed soybean extract (BSE) inhibits melanoma cell growth and viability in vitro: involvement of nuclear factor-kappa B signaling.

Vilela FM, Syed DN, Chamcheu JC, Calvo-Castro LA, Fortes VS, Fonseca MJ, Mukhtar H - PLoS ONE (2014)

BSE treatment induced apoptosis in A375 melanoma cells.(A) Phase contrast microscopy: representative pictures of A375 cells treated with BSE (0–1.9 mg/mL) for 24 h. (B) Effect of BSE treatment (0–1.9 mg/mL) on apoptosis in A375 cells as demonstrated by annexin-V staining (green fluorescence). (C) Effect of BSE treatment (0–1.9 mg/mL) on activation of caspase-3 and PARP cleavage. Whole cell lysates were analyzed by immunoblot analysis. Equal loading was confirmed by reprobing for β-actin. The values above the figures represent relative density of the bands (cleaved caspase 3 and PARP) normalized to β-actin. Data shown are representative of three independent experiments. (D) Effect of BSE (1.7 mg/mL) treatment on the activation of caspase-3 as detected by immunocytochemistry. A representative picture from three independent experiments with similar results is shown.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4114525&req=5

pone-0103248-g002: BSE treatment induced apoptosis in A375 melanoma cells.(A) Phase contrast microscopy: representative pictures of A375 cells treated with BSE (0–1.9 mg/mL) for 24 h. (B) Effect of BSE treatment (0–1.9 mg/mL) on apoptosis in A375 cells as demonstrated by annexin-V staining (green fluorescence). (C) Effect of BSE treatment (0–1.9 mg/mL) on activation of caspase-3 and PARP cleavage. Whole cell lysates were analyzed by immunoblot analysis. Equal loading was confirmed by reprobing for β-actin. The values above the figures represent relative density of the bands (cleaved caspase 3 and PARP) normalized to β-actin. Data shown are representative of three independent experiments. (D) Effect of BSE (1.7 mg/mL) treatment on the activation of caspase-3 as detected by immunocytochemistry. A representative picture from three independent experiments with similar results is shown.
Mentions: A375 cells treated with BSE (1.5–1.9 mg/mL; 24 h) showed dose-dependent alterations in cell morphology (Figure 2A) and induction of apoptosis which was evident from the enhancement in fluorescent green Annexin V staining (Figure 2B). To further assess the molecular mechanism involved in BSE-induced apoptosis, we examined whether BSE treatment would result in activation of caspase-3 and PARP cleavage, which are considered important indicators of apoptotic cell death [17], [18]. Accordingly western blot analysis and immunocytochemistry showed that BSE treatment resulted in an increase in both caspase-3 activation (Figure 2C and D) and PARP cleavage (Figure 2C).

Bottom Line: Further studies in A375 cells showed that decrease in cell viability with BSE treatment (1.5-1.9 mg/ml; 24 h) was associated with induction of apoptosis.Immunoblot analysis revealed that BSE treatment resulted in induction of PARP cleavage, activation of caspase-3, -7, and -8 and increased expression of TRAIL and its receptor DR4.BSE did not activate the intrinsic apoptotic pathway in A375 cells, as no change was observed in caspase-9 expression.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil; Department of Dermatology, University of Wisconsin, Madison, Wisconsin, United States of America.

ABSTRACT
Melanoma is recognized as one of the most aggressive cancers with a relatively high propensity for metastasis. The prognosis of melanoma remains poor in spite of treatment advances, emphasizing the importance of additional preventive measures. Isoflavonoids have become not only potential chemopreventive, but also important therapeutic natural agents. We evaluated the antiproliferative and proapoptotic properties of biotransformed soybean extract (BSE) in A375 melanoma cells. Previous analyses demonstrated that the concentration of daidzein, genistein and aminoacids/peptides present in BSE, fermented by Aspergillus awamori is much higher than in the non biotransformed extract (NBSE). Experiments comparing the efficacy of the extracts in preventing cancer cell growth showed that treatment (24 h) of aggressive melanoma cells (A375 and 451Lu) with BSE resulted in a dose-dependent inhibition of growth and viability. In contrast, treatment with similar doses of NBSE failed to inhibit melanoma cell viability. Further studies in A375 cells showed that decrease in cell viability with BSE treatment (1.5-1.9 mg/ml; 24 h) was associated with induction of apoptosis. Immunoblot analysis revealed that BSE treatment resulted in induction of PARP cleavage, activation of caspase-3, -7, and -8 and increased expression of TRAIL and its receptor DR4. BSE did not activate the intrinsic apoptotic pathway in A375 cells, as no change was observed in caspase-9 expression. The expression of Bcl-2 apoptotic proteins such as Bid and Bax remained unaffected with BSE treated cells. Interestingly, we also showed that BSE treatment increased the phosphorylation and activation of IKK, IκBα degradation and p65/NF-κB translocation to the nucleus, and that stimulation of the NF-???B pathway was required for BSE-induced apoptosis of A375 cells. Our findings indicate that the biotransformation of soybean plays a crucial role in the extract anti-cancer effect observed in melanoma cells. However, further studies are warranted to define the active anti-cancer agent(s) present in BSE.

Show MeSH
Related in: MedlinePlus