Determinants of CPT-11 and SN-38 activities in human lung cancer cells.

Ma Kedde,E Boven,Hm Pinedo,J Van Ark-Otte,A-Mc Dingemans,Wjf Van Der Vijgh,Wjm Jansen,G Giaccone

doi:10.1038/bjc.1998.362

Abstract

Irinotecan (CPT-11) is a semisynthetic camptothecin derivative with a broad spectrum of anti-tumour activity. Carboxylesterase (CE) catalyses the conversion of CPT-11 to SN-38 (7-ethyl-10-hydroxycamptothecin), the active form of CPT-11. The antiproliferative effects of CPT-11 and SN-38, CE-activity and topoisomerase I protein expression were investigated in five human small-cell lung cancer (SCLC) cell lines and four human non-small-cell lung cancer (NSCLC) cell lines. Antiproliferative activity, expressed as IC50 values, was determined using the MTT assay. CPT-11 was significantly more active in SCLC than in NSCLC cell lines (P = 0.0036), whereas no significant difference between histological types was observed with SN-38. A significant correlation (r2 = 0.52, P = 0.028) was observed between CE activity and chemosensitivity to CPT-11 but not to SN-38, and significantly higher CE activity was observed in SCLC compared with NSCLC cell lines (P = 0.025). Western blotting experiments showed topoisomerase I protein expressions within a factor of 2, and a granular nuclear staining was detectable in all cell lines by immunocytochemistry of cytospins. No correlation was observed between protein expression and sensitivity to CPT-11 or SN-38. Cellular and medium concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography (HPLC) in one SCLC cell line with high CE activity and high sensitivity to CPT-11, and one NSCLC cell line with low sensitivity to CPT-11 and CE activity. Intracellular concentrations of CPT-11 and SN-38 were higher in the SCLC cell line, and this was associated with an increase in cellular uptake of CPT-11 compared with the medium, and an increased intracellular formation of SN-38. In conclusion, CE activity appears to be associated with higher sensitivity to CPT-11 in human lung cancer cell lines and may partly explain the difference in the in vitro sensitivity to CPT-11 between SCLC and NSCLC cells. The assessment of CE activity in clinical material of lung cancer patients undergoing treatment with CPT-11 may be warranted. However, other mechanisms may influence sensitivity to CPT-11, possibly including drug transport.

Highlights

The non-small-cell lung cancer (NSCLC) cell lines were cultured in Nunc flasks (Roskilde, Denmark) as monolayers, whereas the small-cell lung cancer (SCLC) cells grew as floating clusters
The antiproliferative activities of CPT- 11 and SN-38 are summarized in Table 1: a wide range of sensitivities was found between CPT-1 1 and SN-38 in the different cell lines
A significantly higher activity of CPT-11 was observed in SCLC [mean IC50 (± s.d.) = 0.93 (+ 0.55) x 106 M] than in NSCLC [mean IC50 (+ s.d.) = 2.38 (± 1.54) x 106 M] (P = 0.0036)

Summary

Methods

CPT-1 1 and SN-38 were kindly provided by Yakult Honsha (Tokyo, Japan). Both compounds were solubilized in dimethyl sulphoxide and stored, protected from light, at 4°C. Para-nitrophenylacetate (p-NPA) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were from Sigma Chemical (Zwijndrecht, NL). All other chemicals were of standard analytical quality and were commercially available. 2172 J van Ark-Otte et al Cell lines. The culture medium was RPMI medium (Flow Labs, Irvine, UK) supplemented with 10% fetal calf serum (FCS, Gibco, Paisley, UK) and 1 mM L-glutamine, except for the NSCLC cell line SW-1573, which was cultured in Dulbecco's modified Eagle medium (DMEM, Flow Labs) supplemented with 10% FCS. The resistant cell line GLC4/ADR was cultured in the presence of doxorubicin until 7-14 days before experiments. All cell lines were free from Mycoplasma as tested with the mycoplasma T.C. rapid detection system with a 3Hlabelled DNA probe from Gen-probe (San Diego, CA, USA)

Results

Discussion

Conclusion