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Apoptosis

, Volume 16, Issue 2, pp 184–197 | Cite as

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

  • Byeong Mo Kim
  • Sung Hee Hong
Original Paper

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.

Keywords

Saikosaponin a (SSa) Human colon carcinoma (HCC) Apoptosis Caspase-2 Caspase-8 Cell death mechanisms 

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

Notes

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.

References

  1. 1.
    Just MJ, Recio MC, Giner RM, Cuéllar MJ, Máñez S, Bilia AR et al (1998) Anti-inflammatory activity of unusual lupane saponins from Bupleurum fruticescens. Planta Med 64:404–407CrossRefPubMedGoogle Scholar
  2. 2.
    Wang H, Gao J, Kou J, Zhu D, Yu B (2008) Anti-inflammatory activities of triterpenoid saponins from Polygala japonica. Phytomedicine 15:321–326CrossRefPubMedGoogle Scholar
  3. 3.
    Yadava RN, Chakravarti N (2008) Anti-inflammatory activity of a new triterpenoid saponin from carthamus tinctorius linn. J Enzyme Inhib Med Chem 23:543–548CrossRefPubMedGoogle Scholar
  4. 4.
    Hattori T, Maruyama H, Nishimura H, Nakai Y, Sakakibara I, Kase Y et al (2006) Effects of Saireito, a Japanese herbal medicine, on edema via antagonistic actions against aldosterone in anti-GBM nephritic rats. Clin Exp Nephrol 10:13–18CrossRefPubMedGoogle Scholar
  5. 5.
    Ai P, Yong G, Dingkun G, Qiuyu Z, Kaiyuan Z, Shanyan L (2008) Aqueous extract of Astragali Radix induces human natriuresis through enhancement of renal response to atrial natriuretic peptide. J Ethnopharmacol 116:413–421CrossRefPubMedGoogle Scholar
  6. 6.
    Gallo D, Zannoni GF, De Stefano I, Mosca M, Ferlini C, Mantuano E (2008) Soy phytochemicals decrease nonsmall cell lung cancer growth in female athymic mice. J Nutr 138:1360–1364PubMedGoogle Scholar
  7. 7.
    Toshkova RA, Krasteva IN, Wesselinova DW, Nikolov SD (2007) Influence of purified saponin mixture from Astragalus corniculatus Bieb. on phagocytic cells in Graffi-tumor bearing hamsters. J Ethnopharmacol 109:394–399CrossRefPubMedGoogle Scholar
  8. 8.
    Motto Y, Sawabu N (1994) Antitumor effects of saikosaponins, baicalin and baicalein on human hepatoma cell lines. Cancer Lett 86:91–95CrossRefGoogle Scholar
  9. 9.
    Qian L, Murakami T, Kimura Y, Takahashi M, Okita K (1995) Saikosaponin A-induced cell death of a human hepatoma cell line (HuH-7): the significance of the ‘sub-G1 peak’ in a DNA histogram. Pathol Int 45:207–214CrossRefPubMedGoogle Scholar
  10. 10.
    Chiang LC, Ng LT, Liu LT, Shieh DE, Lin CC (2003) Cytotoxicity and anti-hepatitis B virus activities of saikosaponins from Bupleurum species. Planta Med 69:705–709CrossRefPubMedGoogle Scholar
  11. 11.
    Hsu YL, Kuo PL, Chiang LC, Lin CC (2004) Involvement of p53, nuclear factor kappaB and Fas/Fas ligand in induction of apoptosis and cell cycle arrest by saikosaponin d in human hepatoma cell lines. Cancer Lett 213:213–221CrossRefPubMedGoogle Scholar
  12. 12.
    Yano H, Mizoguchi A, Fukuda K, Haramaki M, Ogasawara S, Momosaki S et al (1994) The herbal medicine sho-saiko-to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the G0/G1 phase. Cancer Res 54:448–454PubMedGoogle Scholar
  13. 13.
    Kato M, Pu MY, Isobe K, Hattori T, Yanagita N, Nakashima I (1995) Cell type-oriented differential modulatory actions of saikosaponin-d on growth responses and DNA fragmentation of lymphocytes triggered by receptor-mediated and receptor-bypassed pathway. Immunopharmacology 29:207–213CrossRefPubMedGoogle Scholar
  14. 14.
    Zong Z, Fujikawa-Yamamoto K, Tanino M, Teraoka K, Yamagishi H, Odashima S (1996) Saikosaponin b2-induced apoptosis of cultured B16 melanoma cell line through down-regulation of PKC activity. Biochem Biophys Res Commun 219:480–485CrossRefPubMedGoogle Scholar
  15. 15.
    Hsu YL, Kuo PL, Lin CC (2004) The proliferative inhibition and apoptotic mechanism of Saikosaponin D in human non-small cell lung cancer A549 cells. Life Sci 75:1231–1242CrossRefPubMedGoogle Scholar
  16. 16.
    Shyu KG, Tsai SC, Wang BW, Liu YC, Lee CC (2004) Saikosaponin C induces endothelial cells growth, migration and capillary tube formation. Life Sci 76:813–826CrossRefPubMedGoogle Scholar
  17. 17.
    Wu SJ, Lin YH, Chu CC, Tsai YH, Chao JC (2008) Curcumin or saikosaponin a improves hepatic antioxidant capacity and protects against CCl4-induced liver injury in rats. J Med Food 11:224–229CrossRefPubMedGoogle Scholar
  18. 18.
    Chen JC, Chang NW, Chung JG, Chen KC (2003) Saikosaponin-A induces apoptotic mechanism in human breast MDA-MB-231 and MCF-7 cancer cells. Am J Chin Med 31:363–377CrossRefPubMedGoogle Scholar
  19. 19.
    Nuñez G, Benedict MA, Hu Y, Inohara N (1998) Caspases: the proteases of the apoptotic pathway. Oncogene 17:3237–3245CrossRefPubMedGoogle Scholar
  20. 20.
    Salvesen GS (1999) Caspase 8: igniting the death machine. Structure 7:R225–R229CrossRefPubMedGoogle Scholar
  21. 21.
    Zhivotovsky B, Orrenius S (2005) Caspase-2 function in response to DNA damage. Biochem Biophys Res Commun 331:859–867CrossRefPubMedGoogle Scholar
  22. 22.
    Lin CF, Chen CL, Chang WT, Jan MS, Hsu LJ, Wu RH et al (2004) Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramide and etoposide-induced apoptosis. J Biol Chem 279:40755–40761CrossRefPubMedGoogle Scholar
  23. 23.
    Yamaguchi H, Wang HG (2001) The protein kinase PKB/Akt regulates cell survival and apoptosis by inhibiting Bax conformational change. Oncogene 20:7779–7786CrossRefPubMedGoogle Scholar
  24. 24.
    Wu CA, Yang YW (2004) Induction of cell death by saponin and antigen delivery. Pharm Res 21:271–277CrossRefPubMedGoogle Scholar
  25. 25.
    Gerkens PC, Dobson R, Tabatadze N, Mshviladzade V, Elias R, Peulen OJ et al (2007) Apoptosis and cytolysis induced by giganteosides and hederacolchisides in HL-60 cells. Anticancer Res 27:2529–2534PubMedGoogle Scholar
  26. 26.
    Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssière JL, Petit PX et al (1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med 181:1661–1672CrossRefPubMedGoogle Scholar
  27. 27.
    Zhang D, Berry MD, Paterson IA, Boulton AA (1999) Loss of mitochondrial membrane potential is dependent on the apoptotic program activated: prevention by R-2HMP. J Neurosci Res 58:284–292CrossRefPubMedGoogle Scholar
  28. 28.
    Düssmann H, Rehm M, Kögel D, Prehn JH (2003) Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis. J Cell Sci 116:525–536CrossRefPubMedGoogle Scholar
  29. 29.
    Mehmet H (2000) Caspases find a new place to hide. Nature 403:29–30CrossRefPubMedGoogle Scholar
  30. 30.
    Troy CM, Shelanski ML (2003) Caspase-2 redux. Cell Death Differ 10:101–107CrossRefPubMedGoogle Scholar
  31. 31.
    Chandra D, Choy G, Deng X, Bhatia B, Daniel P, Tang DG (2004) Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death. Mol Cell Biol 24:6592–6607CrossRefPubMedGoogle Scholar
  32. 32.
    Li H, Zhu H, Xu CJ, Yuan J (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94:491–501CrossRefPubMedGoogle Scholar
  33. 33.
    Sun XM, MacFarlane M, Zhuang J, Wolf BB, Green DR, Cohen GM (1999) Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J Biol Chem 274:5053–5060CrossRefPubMedGoogle Scholar
  34. 34.
    Kruidering M, Evan GI (2000) Caspase-8 in apoptosis: the beginning of “the end”? IUBMB Life 50:85–90PubMedGoogle Scholar
  35. 35.
    Luo X, Budihardjo I, Zou H, Slaughter C, Wang X (1998) Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cells 94:481–490CrossRefGoogle Scholar
  36. 36.
    Esposti MD (2002) The roles of Bid. Apoptosis 7:433–440CrossRefPubMedGoogle Scholar
  37. 37.
    Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S et al (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 144:891–901CrossRefPubMedGoogle Scholar
  38. 38.
    Eskes R, Desagher S, Antonsson B, Martinou JC (2000) Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol 20:929–935CrossRefPubMedGoogle Scholar
  39. 39.
    Guo Y, Srinivasula SM, Druilhe A, Fernandes-Alnemri T, Alnemri ES (2002) Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria. J Biol Chem 277:13430–13437CrossRefPubMedGoogle Scholar
  40. 40.
    Robertson JD, Enoksson M, Suomela M, Zhivotovsky B, Orrenius S (2002) Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis. J Biol Chem 277:29803–29809CrossRefPubMedGoogle Scholar
  41. 41.
    Panaretakis T, Laane E, Pokrovskaja K, Björklund AC, Moustakas A, Zhivotovsky B et al (2005) Doxorubicin requires the sequential activation of caspase-2, protein kinase Cdelta, and c-Jun NH2-terminal kinase to induce apoptosis. Mol Biol Cell 16:3821–3831CrossRefPubMedGoogle Scholar
  42. 42.
    Kuwana T, Smith JJ, Muzio M, Dixit V, Newmeyer DD, Kornbluth S (1998) Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c. J Biol Chem 273:16589–16594CrossRefPubMedGoogle Scholar
  43. 43.
    Krumschnabel G, Sohm B, Bock F, Manzl C, Villunger A (2009) The enigma of caspase-2: the laymen’s view. Cell Death Differ 16:195–207CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Division of Radiation Cancer ResearchKorea Institute of Radiological and Medical SciencesSeoulKorea

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