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Sequential caspase-2 and caspase-8 activation is essential for saikosaponin a-induced apoptosis of human colon carcinoma cell lines

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Abstract

In this study, we investigated the signaling pathways implicated in SSa-induced apoptosis of human colon carcinoma (HCC) cell lines. SSa-induced apoptosis of HCC cells was associated with proteolytic activation of caspase-9, caspase-3, and PARP cleavages and decreased levels of IAP family members, such as XIAP and c-IAP-2, but not of survivin. The fluorescence intensity of DiOC6 was significantly reduced after SSa treatment. CsA significantly inhibited SSa-induced loss of mitochondrial transmembrane potential and moderately inhibited SSa-induced cell death. SSa treatment also enhanced the activities of caspase-2 and caspase-8, Bid cleavage, and the conformational activation of Bax. Additionally, SSa-induced apoptosis was inhibited by both the selective caspase-2 inhibitor z-VDVAD-fmk and the selective caspase-8 inhibitor z-IETD-fmk and also by si-RNAs against caspase-2 and caspase-8. The selective caspase-9 inhibitor, z-LEHD-fmk, also inhibited SSa-induced apoptosis, albeit to a lesser extent compared to z-VDVAD-fmk and z-IETD-fmk, indicating that both mitochondria-dependent and mitochondria-independent pathways are associated with SSa-induced apoptosis. Both z-VDVAD-fmk and z-IETD-fmk significantly attenuated the colony-inhibiting effect of SSa. Moreover, inhibition of caspase-2 activation by the pharmacological inhibitor z-VDVAD-fmk, or by knockdown of protein levels using a si-RNA, suppressed SSa-induced caspase-8 activation, Bid cleavage, and the conformational activation of Bax. Although caspase-8 is an initiator caspase like caspase-2, the inhibition of caspase-8 activation by knockdown using a si-RNA did not suppress SSa-induced caspase-2 activation. Altogether, our results suggest that sequential activation of caspase-2 and caspase-8 is a critical step in SSa-induced apoptosis.

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Abbreviations

SSa:

Saikosaponin a

HCC:

Human colon carcinoma

z-DEVD-fmk:

N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fmk

z-LEHD-fmk:

N-benzyloxycarbonyl-Leu-Glu-His-Asp-fmk

z-VDVAD-fmk:

N-benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fmk

z-IETD-fmk:

N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fmk

PI:

Propidium iodide

FACS:

Fluorescence-activated cell sorter

p-NA:

p-nitroanilide

DiOC6:

3,3′-dihexyloxacarbocyanine

MMP:

Mitochondrial membrane potential

CsA:

Cyclosporine A

MPT:

Mitochondrial permeability transition

IAP:

Inhibitors of apoptosis proteins

ANOVA:

Analysis of variance

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Acknowledgments

This work was supported by the National Nuclear R&D Program of the Ministry of Education, Science and Technology (MEST) of the Republic of Korea.

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  1. Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-Gu, Seoul, 139-706, Korea

    Byeong Mo Kim & Sung Hee Hong

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Correspondence to Sung Hee Hong.

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Kim, B.M., Hong, S.H. Sequential caspase-2 and caspase-8 activation is essential for saikosaponin a-induced apoptosis of human colon carcinoma cell lines. Apoptosis 16, 184–197 (2011). https://doi.org/10.1007/s10495-010-0557-x

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