Identification of thymidylate synthase as a potential therapeutic target for lung cancer

Abstract

Background: Thymidylate synthase (TS), a key enzyme in the de novo synthesis of thymidine, is an important chemotherapeutic target for malignant tumours including lung cancer. Although inhibition of TS has an antiproliferative effect in cancer cells, the precise mechanism of this effect has remained unclear.

Methods: We examined the effects of TS inhibition with an RNA interference-based approach. The effect of TS depletion on the growth of lung cancer cells was examined using colorimetric assay and flow cytometry.

Results: Measurement of the enzymatic activity of TS in 30 human lung cancer cell lines revealed that such activity differs among tumour histotypes. Almost complete elimination of TS activity by RNA interference resulted in inhibition of cell proliferation in all tested cell lines, suggestive of a pivotal role for TS in cell proliferation independent of the original level of enzyme activity. The antiproliferative effect of TS depletion was accompanied by arrest of cells in S phase of the cell cycle and the induction of caspase-dependent apoptosis as well as by changes in the expression levels of cyclin E and c-Myc. Moreover, TS depletion induced downregulation of the antiapoptotic protein X-linked inhibitor of apoptosis (XIAP), and it seemed to activate the mitochondrial pathway of apoptosis.

Conclusion: Our data provide insight into the biological relevance of TS as well as a basis for clinical development of TS-targeted therapy for lung cancer.

Figure 1

Thymidylate synthase (TS) activity in lung cancer cell lines stratified according to histotype. Central horizontal lines represent median values, with the upper and lower bars representing the 95% confidence interval. ^*P<0.05 for squamous cell carcinoma vs non-squamous cell carcinoma; ^**P<0.05 for SCLC vs either squamous cell or non-squamous cell carcinoma.

Figure 2

Effects of transient depletion of TS on TS activity and the proliferation of lung cancer cell lines. (A) The indicated cell lines were transfected with a nonspecific (NS) siRNA or with either of three different siRNAs specific for TS mRNA (TS-1, TS-2, and TS-3) for 48 h, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to TS and β-actin (loading control). (B) The indicated cell lines were left untreated (NT) or were transfected with nonspecific or TS-1 siRNAs for 48 h, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to TS and β-actin. (C) Cells were left untreated or were transfected with NS or TS-1 siRNAs for 72 h, after which cell lysates were prepared and assayed for TS activity. Data are expressed as a percentage of the value for untreated cells and are means±s.d. of triplicates from experiments that were repeated on at least one additional occasion with similar results. (D) Cells were left untreated or were transfected with NS or TS-1 siRNAs for 72 h, after which cell viability was assessed with the MTT assay. Data are expressed as a percentage of the value for untreated cells and are means of triplicates from experiments that were repeated on two additional occasions with similar results. (E) Cells were left untreated or were transfected with NS or TS-1 siRNAs for the indicated times, after which cell viability was assessed with the MTT assay. Data are means±s.d. of triplicates from experiments that were repeated on two additional occasions with similar results.

Figure 3

Effects of TS depletion on cell cycle distribution and on cyclin E and c-Myc expression in lung cancer cells. (A) The indicated cell lines were transfected with nonspecific (NS) or TS-1 siRNAs for 48 or 72 h and were then fixed, stained with propidium iodide, and analysed for cell cycle distribution by flow cytometry. Data are means±s.d. of triplicates from experiments that were repeated on two additional occasions with similar results. (B) Cells were transfected with NS or TS-1 siRNAs for 72 h, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to TS, cyclin E, c-Myc, and β-actin. Transfection with the NS siRNA had no substantial effect on cell cycle distribution or on the expression of cyclin E or c-Myc compared with untreated cells.

Figure 4

Effect of TS depletion on apoptosis in lung cancer cells. (A) The indicated cell lines were transfected with nonspecific (NS) or TS-1 siRNAs for 48 or 72 h and were then fixed, stained with propidium iodide, and subjected to flow cytometry for quantitation of the sub-G₁ population. Data are means±s.d. of triplicates from experiments that were repeated on two additional occasions with similar results. (B) Cells were transfected with NS or TS-1 siRNAs for 72 h, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to TS, PARP, and β-actin. The positions of intact and cleaved forms of PARP are indicated. (C) Cells were transfected with NS or TS-1 siRNAs for 48 or 72 h, lysed, and assayed for caspase-3 activity. Data are expressed relative to the value for cells transfected with NS siRNA and are means±s.d. from three independent experiments. (D) Cells were incubated for 2 h with or without ZVAD-FMK (50 μM), transfected with NS or TS-1 siRNAs for 48 or 72 h (in the continued absence or presence of ZVAD-FMK), and then evaluated for the size of the sub-G₁ population as in (A). Data are means±s.d. of triplicates from experiments that were repeated on two additional occasions with similar results. ^*P<0.05 for the indicated comparisons. Transfection with the NS siRNA had no substantial effects on these markers of apoptosis compared with untreated cells.

Figure 5

Effects of TS depletion on the expression of Bcl-2 and IAP family members and on the release of mitochondrial proteins into the cytosol in lung cancer cells. (A) The indicated cell lines were transfected with nonspecific (NS) or TS-1 siRNAs for 72 h, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the indicated proteins. (B) SBC-3 cells were transfected with NS or TS-1 siRNAs for 24, 48, or 72 h, after which a cytosolic fraction was prepared and subjected to immunoblot analysis with antibodies to cytochrome c, Smac/Diablo, Omi/HtrA2, and β-actin. Transfection with the NS siRNA had no substantial effects on the abundance of Bcl-2 or IAP family proteins or on the release of mitochondrial proteins into the cytosol, compared with untreated cells.