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Styrylpyrone Derivative (SPD) induces apoptosis in a caspase-7-dependent manner in the human breast cancer cell line MCF-7.

Lee AT, Azimahtol HL, Tan AN - Cancer Cell Int. (2003)

Bottom Line: RESULTS: We found that the intrinsic apoptotic pathway was invoked, with the accumulation of cytosolic cytochrome c and processing of the initiator caspase-9.To confirm that apoptosis was induced following caspase-7 activation, the caspase inhibitor Ac-DEVD-CHO was used.CONCLUSIONS: Taken together, these results suggest SPD as a potent antiproliferative agent on MCF-7 cells by inducing apoptosis in a caspase-7-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences & Biotechnology, Faculty of Science & Technology, National University of Malaysia, 43600 Bangi, Selangor, MALAYSIA. alvinlee@email.com

ABSTRACT
BACKGROUND: Styrylpyrone derivative (SPD) is a plant-derived pharmacologically active compound extracted from Goniothalamus sp. Previously, we have reported that SPD inhibited the proliferation of MCF-7 human breast cancer cells by inducing apoptotic cell death, while having minimal effects on non-malignant cells. Here, we attempt to further elucidate the mode of action of SPD. RESULTS: We found that the intrinsic apoptotic pathway was invoked, with the accumulation of cytosolic cytochrome c and processing of the initiator caspase-9. Cleaved products of procaspase-8 were not detected. Next, the executioner caspase-7 was cleaved and activated in response to SPD treatment. To confirm that apoptosis was induced following caspase-7 activation, the caspase inhibitor Ac-DEVD-CHO was used. Pre-incubation of cells with this inhibitor reversed apoptosis levels and caspase-7 activity in SPD-treated cells to untreated levels. CONCLUSIONS: Taken together, these results suggest SPD as a potent antiproliferative agent on MCF-7 cells by inducing apoptosis in a caspase-7-dependent manner.

No MeSH data available.


Related in: MedlinePlus

Western Blot analysis of apoptotic proteins in SPD-treated cells. Proteins from MCF-7 cells treated with 10-6 M SPD for the indicated times were resolved on 12% SDS-PAGE and submitted to Western Blotting with an anti-procaspase-8 antibody. Two bands were observed, corresponding to the uncleaved 55/50-kDa procaspase-8 isoforms. The active p18 subunit was not detected. Samples were also detected for procaspase-9 with an anti-procaspase-9 antibody (Clone B40). The anti-caspase-9 antibodies recognized the proenzyme; and the decrease of this band indicated activation of caspase-9. When proteins from cytosolic fractions of MCF-7 cells treated with 10-6 M SPD were resolved on 15% SDS-PAGE and submitted to immunoblotting with the cytochrome c antibody (Clone 7H8.2C12), increasing amounts of cytochrome c were detected in the cytosol in a time-dependent manner. All blots were then washed and reprobed with β-actin to confirm equal loading.
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Figure 2: Western Blot analysis of apoptotic proteins in SPD-treated cells. Proteins from MCF-7 cells treated with 10-6 M SPD for the indicated times were resolved on 12% SDS-PAGE and submitted to Western Blotting with an anti-procaspase-8 antibody. Two bands were observed, corresponding to the uncleaved 55/50-kDa procaspase-8 isoforms. The active p18 subunit was not detected. Samples were also detected for procaspase-9 with an anti-procaspase-9 antibody (Clone B40). The anti-caspase-9 antibodies recognized the proenzyme; and the decrease of this band indicated activation of caspase-9. When proteins from cytosolic fractions of MCF-7 cells treated with 10-6 M SPD were resolved on 15% SDS-PAGE and submitted to immunoblotting with the cytochrome c antibody (Clone 7H8.2C12), increasing amounts of cytochrome c were detected in the cytosol in a time-dependent manner. All blots were then washed and reprobed with β-actin to confirm equal loading.

Mentions: During apoptosis, initiator caspases are activated in response to proapoptotic signals [23]. By SDS-PAGE and subsequent Western blot analysis with a caspase-8 specific antibody, it was found that SPD treatment did not lead to the activation of the initiator caspase-8. Procaspase-8, expressed in two functionally active isoforms, caspase-8a and caspase-8b [24] was not processed. From immunoblotting, the two bands observed were the 55/50-kDa procaspase-8 isoforms (Figure 2), similarly reported by Sun et al., [25] and Dirsch et al., [26] and the active p18 subunit could not be detected. As processing of this caspase did not occur, it is possible that the other initiator caspase, caspase-9 may be involved in SPD-induced apoptosis.


Styrylpyrone Derivative (SPD) induces apoptosis in a caspase-7-dependent manner in the human breast cancer cell line MCF-7.

Lee AT, Azimahtol HL, Tan AN - Cancer Cell Int. (2003)

Western Blot analysis of apoptotic proteins in SPD-treated cells. Proteins from MCF-7 cells treated with 10-6 M SPD for the indicated times were resolved on 12% SDS-PAGE and submitted to Western Blotting with an anti-procaspase-8 antibody. Two bands were observed, corresponding to the uncleaved 55/50-kDa procaspase-8 isoforms. The active p18 subunit was not detected. Samples were also detected for procaspase-9 with an anti-procaspase-9 antibody (Clone B40). The anti-caspase-9 antibodies recognized the proenzyme; and the decrease of this band indicated activation of caspase-9. When proteins from cytosolic fractions of MCF-7 cells treated with 10-6 M SPD were resolved on 15% SDS-PAGE and submitted to immunoblotting with the cytochrome c antibody (Clone 7H8.2C12), increasing amounts of cytochrome c were detected in the cytosol in a time-dependent manner. All blots were then washed and reprobed with β-actin to confirm equal loading.
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Related In: Results  -  Collection

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Figure 2: Western Blot analysis of apoptotic proteins in SPD-treated cells. Proteins from MCF-7 cells treated with 10-6 M SPD for the indicated times were resolved on 12% SDS-PAGE and submitted to Western Blotting with an anti-procaspase-8 antibody. Two bands were observed, corresponding to the uncleaved 55/50-kDa procaspase-8 isoforms. The active p18 subunit was not detected. Samples were also detected for procaspase-9 with an anti-procaspase-9 antibody (Clone B40). The anti-caspase-9 antibodies recognized the proenzyme; and the decrease of this band indicated activation of caspase-9. When proteins from cytosolic fractions of MCF-7 cells treated with 10-6 M SPD were resolved on 15% SDS-PAGE and submitted to immunoblotting with the cytochrome c antibody (Clone 7H8.2C12), increasing amounts of cytochrome c were detected in the cytosol in a time-dependent manner. All blots were then washed and reprobed with β-actin to confirm equal loading.
Mentions: During apoptosis, initiator caspases are activated in response to proapoptotic signals [23]. By SDS-PAGE and subsequent Western blot analysis with a caspase-8 specific antibody, it was found that SPD treatment did not lead to the activation of the initiator caspase-8. Procaspase-8, expressed in two functionally active isoforms, caspase-8a and caspase-8b [24] was not processed. From immunoblotting, the two bands observed were the 55/50-kDa procaspase-8 isoforms (Figure 2), similarly reported by Sun et al., [25] and Dirsch et al., [26] and the active p18 subunit could not be detected. As processing of this caspase did not occur, it is possible that the other initiator caspase, caspase-9 may be involved in SPD-induced apoptosis.

Bottom Line: RESULTS: We found that the intrinsic apoptotic pathway was invoked, with the accumulation of cytosolic cytochrome c and processing of the initiator caspase-9.To confirm that apoptosis was induced following caspase-7 activation, the caspase inhibitor Ac-DEVD-CHO was used.CONCLUSIONS: Taken together, these results suggest SPD as a potent antiproliferative agent on MCF-7 cells by inducing apoptosis in a caspase-7-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences & Biotechnology, Faculty of Science & Technology, National University of Malaysia, 43600 Bangi, Selangor, MALAYSIA. alvinlee@email.com

ABSTRACT
BACKGROUND: Styrylpyrone derivative (SPD) is a plant-derived pharmacologically active compound extracted from Goniothalamus sp. Previously, we have reported that SPD inhibited the proliferation of MCF-7 human breast cancer cells by inducing apoptotic cell death, while having minimal effects on non-malignant cells. Here, we attempt to further elucidate the mode of action of SPD. RESULTS: We found that the intrinsic apoptotic pathway was invoked, with the accumulation of cytosolic cytochrome c and processing of the initiator caspase-9. Cleaved products of procaspase-8 were not detected. Next, the executioner caspase-7 was cleaved and activated in response to SPD treatment. To confirm that apoptosis was induced following caspase-7 activation, the caspase inhibitor Ac-DEVD-CHO was used. Pre-incubation of cells with this inhibitor reversed apoptosis levels and caspase-7 activity in SPD-treated cells to untreated levels. CONCLUSIONS: Taken together, these results suggest SPD as a potent antiproliferative agent on MCF-7 cells by inducing apoptosis in a caspase-7-dependent manner.

No MeSH data available.


Related in: MedlinePlus