Top

Published in:

01-09-2015 | Research Article

The M30 assay does not detect apoptosis in epithelial-derived cancer cells expressing low levels of cytokeratin 18

Authors: Buse Cevatemre, Engin Ulukaya, Mehmet Sarimahmut, Arzu Yilmaztepe Oral, Fiona M. Frame

Published in: Tumor Biology | Issue 9/2015

Abstract

The primary aim of this study was to compare measurement of apoptosis by M30 immunoreactivity (a biomarker for apoptosis) to other apoptosis assays (morphological assessment of nuclei, Annexin-V-FITC staining, DNA fragmentation and PARP cleavage) in vitro. Caspase-cleaved cytokeratin 18 (M30, ccK18) is only produced in epithelial cells and is regarded as a pharmacodynamic biomarker of apoptotic cell death because it is released from cells during apoptosis induced by chemotherapeutic agents. However, we have observed false negative results using this assay in clinical samples. Therefore, we tested its ability to accurately detect apoptosis in a panel of lung cancer cell lines with a range of clinically approved chemotherapeutic drugs. Three different non-small cell lung cancer (NSCLC) cell lines (A549, H1299, PC3) were used to correlate M30 levels with alternate apoptosis assays. Following successful induction of apoptosis, the A549 cell line showed an increase in M30 levels along with other well-known features of apoptosis, whilst H1299 and PC3 cell lines did not show an increase in M30 levels, even when apoptosis was detected by other means. Further analysis showed that H1299 and PC3 cell lines expressed much lower levels of cytokeratin 18 protein compared to the A549 cell line. Our results suggest that reliable detection of apoptosis via the M30 assay only works when sufficient levels of cytokeratin 18 are present in the cells. This means that the M30 assay may result in false negative results for apoptosis, and as such, the ELISA should be used in conjunction with other assays.

Chou CF, Riopel CL, Rott LS, Omary MB. A significant soluble keratin fraction in ‘simple’ epithelial cells. Lack of an apparent phosphorylation and glycosylation role in keratin solubility. J Cell Sci. 1993;105:433–44.PubMed

Fuchs E, Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem. 1994;63:345–82.CrossRefPubMed

Kramer G, Erdal H, Mertens HJ, Nap M, Mauermann J, Steiner G, et al. Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer Res. 2004;64:1751–6.CrossRefPubMed

Cummings J, Ward TH, Greystoke A, Ranson M, Dive C. Biomarker method validation in anticancer drug development. Br J Pharmacol. 2008;153:646–56.CrossRefPubMed

Leers MP, Kölgen W, Björklund V, Bergman T, Tribbick G, Persson B, et al. Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol. 1999;187:567–72.CrossRefPubMed

Hägg M, Bivén K, Ueno T, Rydlander L, Björklund P, Wiman KG, et al. A novel high-through-put assay for screening of pro-apoptotic drugs. Investig New Drugs. 2002;20:253–9.CrossRef

Schutte B, Henfling M, Kolgen W, Bouman M, Meex S, Leers MP, et al. Keratin 8/18 breakdown and reorganization during apoptosis. Exp Cell Res. 2004;297:11–26.CrossRefPubMed

Steele NL, Plumb JA, Vidal L, Tjørnelund J, Knoblauch P, Rasmussen A, et al. A phase 1 pharmacokinetic and pharmacodynamic study of the histone deacetylase inhibitor belinostat in patients with advanced solid tumors. Clin Cancer Res. 2008;14:804–10.CrossRefPubMed

Scott LC, Evans TR, Cassidy J, Harden S, Paul J, Ullah R, et al. Cytokeratin 18 in plasma of patients with gastrointestinal adenocarcinoma as a biomarker of tumour response. Br J Cancer. 2009;101:410–7.CrossRefPubMedPubMedCentral

10.

Hou JM, Greystoke A, Lancashire L, Cummings J, Ward T, Board R, et al. Evaluation of circulating tumor cells and serological cell death biomarkers in small cell lung cancer patients undergoing chemotherapy. Am J Pathol. 2009;175:808–16.CrossRefPubMedPubMedCentral

11.

Olofsson MH, Ueno T, Pan Y, Xu R, Cai F, van der Kuip H, et al. Cytokeratin-18 is a useful serum biomarker for early determination of response of breast carcinomas to chemotherapy. Clin Cancer Res. 2007;13:3198–206.CrossRefPubMed

12.

Kramer G, Schwarz S, Hagg M, Havelka AM, Linder S. Docetaxel induces apoptosis in hormone refractory prostate carcinomas during multiple treatment cycles. Br J Cancer. 2006;94:1592–8.PubMedPubMedCentral

13.

Ozturk B, Coskun U, Sancak B, Yaman E, Buyukberber S, Benekli M. Elevated serum levels of M30 and M65 in patients with locally advanced head and neck tumors. Int Immunopharmacol. 2009;9:645–8.CrossRefPubMed

14.

Ausch C, Buxhofer-Ausch V, Olszewski U, Hinterberger W, Ogris E, Schiessel R, et al. Caspase-cleaved cytokeratin 18 fragment (M30) as marker of postoperative residual tumor load in colon cancer patients. Eur J Surg Oncol. 2009;35:1164–8.CrossRefPubMed

15.

Koelink PJ, Lamers CB, Hommes DW, Verspaget HW. Circulating cell death products predict clinical outcome of colorectal cancer patients. BMC Cancer. 2009;9:88.CrossRefPubMedPubMedCentral

16.

de Haas EC, di Pietro A, Simpson KL, Meijer C, Suurmeijer AJ, Lancashire LJ, et al. Clinical evaluation of M30 and M65 ELISA cell death assays as circulating biomarkers in a drug-sensitive tumor, testicular cancer. Neoplasia. 2008;10:1041–8.CrossRefPubMedPubMedCentral

17.

Bühler H, Schaller G. Transfection of keratin 18 gene in human breast cancer cells causes induction of adhesion proteins and dramatic regression of malignancy in vitro and in vivo. Mol Cancer Res. 2005;3:365–71.CrossRefPubMed

18.

Schaller G, Fuchs I, Pritze W, Ebert A, Herbst H, Pantel K, et al. Elevated keratin 18 protein expression indicates a favorable prognosis in patients with breast cancer. Clin Cancer Res. 1996;2:1879–85.PubMed

19.

Ulukaya E, Yilmaztepe A, Akgoz S, Linder S, Karadag M. The levels of caspase-cleaved cytokeratin 18 are elevated in serum from patients with lung cancer and helpful to predict the survival. Lung Cancer. 2007;56:399–404.CrossRefPubMed

20.

Cevatemre B, Ari F, Sarimahmut M, Oral AY, Dere E, Kacar O, et al. Combination of fenretinide and indole-3-carbinol results in synergistic cytotoxic activity inducing apoptosis against human breast cancer cells in vitro. Anticancer Drugs. 2013;24:577–86.PubMed

21.

Andreotti PE, Cree IA, Kurbacher CM, Hartmann DM, Linder D, Harel G. Chemosensitivity testing of human tumors using a microplate adenosine triphosphate luminescence assay: clinical correlation for cisplatin resistance of ovarian carcinoma. Cancer Res. 1995;55:5276–82.PubMed

22.

Ulukaya E, Sarimahmut M, Cevatemre B, Ari F, Yerlikaya A, Dimas K. Additive enhancement of apoptosis by TRAIL and fenretinide in metastatic breast cancer cells in vitro. Biomed Pharmacother. 2014;68:477–82.CrossRefPubMed

23.

Ulukaya E, Frame FM, Cevatemre B, Pellacani D, Walker H, Mann VM, et al. Differential cytotoxic activity of a novel palladium-based compound on prostate cell lines, primary prostate epithelial cells and prostate stem cells. PLoS ONE. 2013;8(5):e64278.CrossRefPubMedPubMedCentral

24.

Ueno T, Toi M, Linder S. Detection of epithelial cell death in the body by cytokeratin 18 measurement. Biomed Pharmacother. 2005;59 Suppl 2:S359–362.CrossRefPubMed

25.

Demiray M, Ulukaya EE, Arslan M, Gokgoz S, Saraydaroglu O, Ercan I, et al. Response to neoadjuvant chemotherapy in breast cancer could be predictable by measuring a novel serum apoptosis product, caspase-cleaved cytokeratin 18: a prospective pilot study. Cancer Investig. 2006;24:669–76.CrossRef

26.

Bilici A, Ustaalioglu BB, Ercan S, Seker M, Yilmaz BE, Orcun A, et al. The prognostic significance of the increase in the serum M30 and M65 values after chemotherapy and relationship between these values and clinicopathological factors in patients with advanced gastric cancer. Tumour Biol. 2012;33:2201–8.CrossRefPubMed

27.

Yaman E, Coskun U, Sancak B, Buyukberber S, Ozturk B, Benekli M. Serum M30 levels are associated with survival in advanced gastric carcinoma patients. Int Immunopharmacol. 2010;10:719–22.CrossRefPubMed

28.

Dive C, Smith RA, Garner E, Ward T, George-Smith SS, Campbell F, et al. Considerations for the use of plasma cytokeratin 18 as a biomarker in pancreatic cancer. Br J Cancer. 2010;102:577–82.CrossRefPubMedPubMedCentral

29.

Oven Ustaalioglu B, Bilici A, Ercan S, Orcun A, Seker M, Ozkan A, et al. Serum M30 and M65 values in patients with advanced stage non-small-cell lung cancer compared with controls. Clin Transl Oncol. 2012;14:356–61.CrossRefPubMed

30.

Oyama K, Fushida S, Kinoshita J, Okamoto K, Makino I, Nakamura K, et al. Serum cytokeratin 18 as a biomarker for gastric cancer. Clin Exp Med. 2013;13:289–95.CrossRefPubMed

31.

van der Velden LA, Schaafsma HE, Manni JJ, Ruiter DJ, Ramaekers FC, Kuijpers W. Cytokeratin and vimentin expression in normal epithelium and squamous cell carcinomas of the larynx. Eur Arch Otorhinolaryngol. 1997;254:376–83.CrossRefPubMed

32.

Proby CM, Churchill L, Purkis PE, Glover MT, Sexton CJ, Leigh IM. Keratin 17 expression as a marker for epithelial transformation in viral warts. Am J Pathol. 1993;143:1667–78.PubMedPubMedCentral

33.

Hendrix MJ, Seftor EA, Chu YW, Seftor RE, Nagle RB, McDaniel KM, et al. Coexpression of vimentin and keratins by human melanoma tumor cells: correlation with invasive and metastatic potential. J Natl Cancer Inst. 1992;84:165–74.CrossRefPubMed

34.

Iyer SV, Dange PP, Alam H, Sawant SS, Ingle AD, Borges AM, et al. Understanding the role of keratins 8 and 18 in neoplastic potential of breast cancer derived cell lines. PLoS ONE. 2013;8(1):e53532.CrossRefPubMedPubMedCentral

35.

Caulin C, Salvesen GS, Oshima RG. Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol. 1997;138:1379–94.CrossRefPubMedPubMedCentral

36.

MacFarlane M, Merrison W, Dinsdale D, Cohen GM. Active caspases and cleaved cytokeratins are sequestered into cytoplasmic inclusions in TRAIL-induced apoptosis. J Cell Biol. 2000;148:1239–54.CrossRefPubMedPubMedCentral

37.

Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature. 1998;391:96–9.CrossRefPubMed

38.

Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 1998;391:43–50.CrossRefPubMed

39.

Liu X, Zou H, Slaughter C, Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell. 1997;89:175–84.CrossRefPubMed

40.

Linder S, Olofsson MH, Herrmann R, Ulukaya E. Utilization of cytokeratin-based biomarkers for pharmacodynamic studies. Expert Rev Mol Diagn. 2010;10:353–9.CrossRefPubMed

Title: The M30 assay does not detect apoptosis in epithelial-derived cancer cells expressing low levels of cytokeratin 18
Authors: Buse Cevatemre
Engin Ulukaya
Mehmet Sarimahmut
Arzu Yilmaztepe Oral
Fiona M. Frame
Publication date: 01-09-2015
Publisher: Springer Netherlands
Published in: Tumor Biology / Issue 9/2015
Print ISSN: 1010-4283
Electronic ISSN: 1423-0380
DOI: https://doi.org/10.1007/s13277-015-3367-5

Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras

Prof. Fabrice Barlesi

Developed by: Springer Medicine

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Please log in to get access to this content

Other articles of this Issue 9/2015

miR-335 inhibits the proliferation and invasion of clear cell renal cell carcinoma cells through direct suppression of BCL-W

A novel component from citrus, ginger, and mushroom family exhibits antitumor activity on human meningioma cells through suppressing the Wnt/β-catenin signaling pathway

Retraction Note to: A translocator protein ligand PK11195 shows antigrowth activity in human choriocarcinoma cells

Akt2/ZEB2 may be a biomarker for exfoliant cells in ascitic fluid in advanced grades of serous ovarian carcinoma

MicroRNA signatures in total peripheral blood of gallbladder cancer patients

Retraction Note to: Prognostic value of CD133 expression in cancer patients treated with chemoradiotherapy: a meta-analysis

Keynote webinar | Spotlight on antibody–drug conjugates in cancer