Abstract
Background
This study assesses the chemotherapeutic drug gemcitabine in the human non-small cell lung cancer (NSCLC) cell line KNS62 in relation to the CD95-induced apoptotic pathway, and the role of the anti-apoptotic protein Bcl-xL in vitro and in vivo.
Materials and methods
Apoptosis was determined by JAM assay and DAPI staining analysis. Activation of key apoptotic proteins, including caspases 3, 8 and 9 and BID, as well as cytochrome c release and mitochondrial transmembrane potential (MTP), were measured. The impact of the caspase inhibitor zVAD on gemcitabine-induced apoptosis was quantified. The in vitro results were verified in vivo in an orthotopic murine xenotransplantation model.
Results
Gemcitabine treatment, as well as stimulation of CD95, resulted in cleavage of effector caspase 3 as well as its substrate PARP and caspase 9, followed by DNA fragmentation. Cleavage of caspase 8 was demonstrated after CD95 activation but not after the application of gemcitabine. In KNS62-Bcl-xL clones, release of cytochrome c and loss of mitochondrial transmembrane potential were suppressed. Consequently, apoptosis after gemcitabine therapy, as well as CD95-induced apoptosis, were significantly inhibited. Caspase inhibitor zVAD only partly reversed gemcitabine-induced DNA fragmentation. In vivo, there was a significant reduction in tumour volume under gemcitabine therapy. Bcl-xL over-expressing tumours were completely resistant to gemcitabine therapy.
Conclusions
In NSCLC cell line KNS62 gemcitabine activated the mitochondrial apoptotic pathway downstream of mitochondria without activation of initiator caspases. Bcl-xL over-expression induced significant resistance to gemcitabine. In vivo, the anti-apoptotic effect of Bcl-xL was more pronounced than in vitro. Gemcitabine also induced caspase-independent DNA fragmentation in KNS62 cells.
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References
Hickman JA (1992) Apoptosis induced by anticancer drugs. Cancer Metastasis Rev 11:121–139
Holzman D (1996) Apoptosis provides new targets for chemotherapy. J Natl Cancer Inst 88:1098–1100
Hinz S, Trauzold A, Boenicke L, Sandberg C, Beckmann S, Bayer E, Walczak H, Kalthoff H, Ungefroren H (2000) Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis. Oncogene 19:5477–5486
Kurdow R, Boehle AS, Haye S, Boenicke L, Schniewind B, Dohrmann P, Kalthoff H (2002) Ganciclovir prodrug therapy is effective in a murine xenotransplant model of human lung cancer. Ann Thorac Surg 73:905–910
Tomayko MM, Reynolds CP (1989) Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 24:148–154
Friesen C, Herr I, Krammer PH, Debatin KM (1996) Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug-induced apoptosis in leukemia cells. Nat Med 2:574–577
Shao RG, Cao CX, Nieves-Neira W, Dimanche-Boitrel MT, Solary E, Pommier Y (2001) Activation of the Fas pathway independently of Fas ligand during apoptosis induced by camptothecin in p53 mutant human colon carcinoma cells. Oncogene 20:1852–1859
Fulda S, Meyer E, Friesen C, Susin SA, Kroemer G, Debatin KM (2001) Cell type specific involvement of death receptor and mitochondrial pathways in drug-induced apoptosis. Oncogene 20:1063–1075
Wu XX, Mizutani Y, Kakehi Y, Yoshida O, Ogawa O (2000) Enhancement of Fas-mediated apoptosis in renal cell carcinoma cells by adriamycin. Cancer Res 60:2912–2918
Martins LM, Mesner PW, Kottke TJ, Basi GS, Sinha S, Tung JS, Svingen PA, Madden BJ, Takahashi A, McCormick DJ, Earnshaw WC, Kaufmann SH (1997) Comparison of caspase activation and subcellular localization in HL-60 and K562 cells undergoing etoposide-induced apoptosis. Blood 90:4283–4296
Perkins CL, Fang G, Kim CN, Bhalla KN (2000) The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis. Cancer Res 60:1645–1653
Ferreira CG, Span SW, Peters GJ, Kruyt FA, Giaccone G (2000) Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460. Cancer Res 60:7133–7141
Nabhan C, Gajria D, Krett NL, Gandhi V, Ghias K, Rosen ST (2002) Caspase activation is required for gemcitabine activity in multiple myeloma cell lines. Mol Cancer Ther 1:1221–1227
Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME (1998) Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17:1675–1687
Gross A, McDonnell JM, Korsmeyer SJ (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13:1899–1911
Narita M, Shimizu S, Ito T, Chittenden T, Lutz RJ, Matsuda H, Tsujimoto Y (1998) Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci U S A 95:14681–14686
Leech SH, Olie RA, Gautschi O, Simoes-Wust AP, Tschopp S, Haner R, Hall J, Stahel RA, Zangemeister-Wittke U (2000) Induction of apoptosis in lung-cancer cells following Bcl-xL anti-sense treatment. Int J Cancer 86:570–576
Dallaporta B, Pablo M, Maisse C, Daugas E, Loeffler M, Zamzami N, Kroemer G (2000) Proteasome activation as a critical event of thymocyte apoptosis. Cell Death Differ 7:368–373
Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397:441–446
Liu T, Brouha B, Grossman D (2004) Rapid induction of mitochondrial events and caspase-independent apoptosis in Survivin-targeted melanoma cells. Oncogene 23:39–48
Nicotera P, Melino G (2004) Regulation of the apoptosis–necrosis switch. Oncogene 23:2757–2765
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R. Kurdow and B. Schniewind contributed equally to this work
The data were presented at the 121st Deutscher Chirurgenkongress, Berlin, April 2004
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Kurdow, R., Schniewind, B., Zoefelt, S. et al. Apoptosis by gemcitabine in non-small cell lung cancer cell line KNS62 is induced downstream of caspase 8 and is profoundly blocked by Bcl-xL over-expression. Langenbecks Arch Surg 390, 243–248 (2005). https://doi.org/10.1007/s00423-004-0531-6
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DOI: https://doi.org/10.1007/s00423-004-0531-6