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Thymidylate synthase as a determinant of pemetrexed sensitivity in non-small cell lung cancer.

Takezawa K, Okamoto I, Okamoto W, Takeda M, Sakai K, Tsukioka S, Kuwata K, Yamaguchi H, Nishio K, Nakagawa K - Br. J. Cancer (2011)

Bottom Line: The sensitivity of NSCLC cells overexpressing TS to the antiproliferative effect of pemetrexed was markedly reduced compared with that of control cells.Finally, the level of TS expression in tumours of non-responding patients was significantly higher than that in those of responders, suggestive of an inverse correlation between TS expression and tumour response to pemetrexed.A high level of TS expression confers a reduced sensitivity to pemetrexed.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511, Japan.

ABSTRACT

Background: Although a high level of thymidylate synthase (TS) expression in malignant tumours has been suggested to be related to a reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in non-small cell lung cancer (NSCLC). We have now investigated the effect of TS overexpression on pemetrexed sensitivity in NSCLC cells.

Methods: We established NSCLC cell lines that stably overexpress TS and examined the effects of such overexpression on the cytotoxicity of pemetrexed both in vitro and in xenograft models. We further examined the relation between TS expression in tumour specimens from NSCLC patients and the tumour response to pemetrexed by immunohistochemical analysis.

Results: The sensitivity of NSCLC cells overexpressing TS to the antiproliferative effect of pemetrexed was markedly reduced compared with that of control cells. The inhibition of DNA synthesis and induction of apoptosis by pemetrexed were also greatly attenuated by forced expression of TS. Furthermore, tumours formed by TS-overexpressing NSCLC cells in nude mice were resistant to the growth-inhibitory effect of pemetrexed observed with control tumours. Finally, the level of TS expression in tumours of non-responding patients was significantly higher than that in those of responders, suggestive of an inverse correlation between TS expression and tumour response to pemetrexed.

Conclusion: A high level of TS expression confers a reduced sensitivity to pemetrexed. TS expression is thus a potential predictive marker for response to pemetrexed-based chemotherapy in NSCLC patients.

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Effect of TS depletion on pemetrexed sensitivity in TS-overexpressing NSCLC cells. (A) Cells of the indicated lines were transfected or not (−) with nonspecific (Con) or TS siRNAs for 48 h, after which cell lysates were subjected to immunoblot analysis with antibodies to TS and to β-actin. (B) Cells transfected as in A were cultured for 72 h in complete medium containing various concentrations of pemetrexed, after which cell viability was assessed as described in Materials and methods section, and the median inhibitory concentration (IC50) of pemetrexed was determined. Data are means of triplicates from experiments that were repeated on two additional occasions with similar results. *P<0.05 (Student's two-tailed t-test).
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fig2: Effect of TS depletion on pemetrexed sensitivity in TS-overexpressing NSCLC cells. (A) Cells of the indicated lines were transfected or not (−) with nonspecific (Con) or TS siRNAs for 48 h, after which cell lysates were subjected to immunoblot analysis with antibodies to TS and to β-actin. (B) Cells transfected as in A were cultured for 72 h in complete medium containing various concentrations of pemetrexed, after which cell viability was assessed as described in Materials and methods section, and the median inhibitory concentration (IC50) of pemetrexed was determined. Data are means of triplicates from experiments that were repeated on two additional occasions with similar results. *P<0.05 (Student's two-tailed t-test).

Mentions: To investigate whether the level of TS expression affects the sensitivity of NSCLC cells to pemetrexed, we first established A549 (A549/TS1 and A549/TS2), H1299 (H1299/TS1 and H1299/TS2), and PC9 (PC9/TS1 and PC9/TS2) cells that stably overexpress TS. Cells that stably harbour the corresponding empty vector (A549/Mock, H1299/Mock, and PC9/Mock) were established as controls. Immunoblot analysis showed that the abundance of TS was markedly increased in the TS-overexpressing lines compared with the parental or Mock cells (Figure 1A). The enzymatic activity of TS was also substantially higher in the TS-overexpressing cells than in the parental or Mock cells (Figure 1B). We then examined the effect of forced expression of TS on the cytotoxicity of anticancer drugs as determined with the MTT assay. The median inhibitory concentration of pemetrexed for the TS-overexpressing cells was about three to six times that for the corresponding Mock cells for all three lung cancer lines, whereas cisplatin and docetaxel inhibited the growth of the TS-overexpressing cells in a manner similar to that observed with the corresponding Mock cells (Table 1). To exclude the possibility that these results were because of nonspecific effects of transfection, we depleted A549/TS1, H1299/TS1, and PC9/TS1 cells of TS by RNA interference. Immunoblot analysis revealed that transfection of these cells with an siRNA specific for TS mRNA resulted in downregulation of the corresponding protein (Figure 2A). This reduction in the abundance of TS restored the sensitivity of the cells to the inhibitory effect of pemetrexed on cell growth (Figure 2B). These data thus indicated that high TS expression levels reduce the sensitivity of NSCLC cells to pemetrexed.


Thymidylate synthase as a determinant of pemetrexed sensitivity in non-small cell lung cancer.

Takezawa K, Okamoto I, Okamoto W, Takeda M, Sakai K, Tsukioka S, Kuwata K, Yamaguchi H, Nishio K, Nakagawa K - Br. J. Cancer (2011)

Effect of TS depletion on pemetrexed sensitivity in TS-overexpressing NSCLC cells. (A) Cells of the indicated lines were transfected or not (−) with nonspecific (Con) or TS siRNAs for 48 h, after which cell lysates were subjected to immunoblot analysis with antibodies to TS and to β-actin. (B) Cells transfected as in A were cultured for 72 h in complete medium containing various concentrations of pemetrexed, after which cell viability was assessed as described in Materials and methods section, and the median inhibitory concentration (IC50) of pemetrexed was determined. Data are means of triplicates from experiments that were repeated on two additional occasions with similar results. *P<0.05 (Student's two-tailed t-test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101907&req=5

fig2: Effect of TS depletion on pemetrexed sensitivity in TS-overexpressing NSCLC cells. (A) Cells of the indicated lines were transfected or not (−) with nonspecific (Con) or TS siRNAs for 48 h, after which cell lysates were subjected to immunoblot analysis with antibodies to TS and to β-actin. (B) Cells transfected as in A were cultured for 72 h in complete medium containing various concentrations of pemetrexed, after which cell viability was assessed as described in Materials and methods section, and the median inhibitory concentration (IC50) of pemetrexed was determined. Data are means of triplicates from experiments that were repeated on two additional occasions with similar results. *P<0.05 (Student's two-tailed t-test).
Mentions: To investigate whether the level of TS expression affects the sensitivity of NSCLC cells to pemetrexed, we first established A549 (A549/TS1 and A549/TS2), H1299 (H1299/TS1 and H1299/TS2), and PC9 (PC9/TS1 and PC9/TS2) cells that stably overexpress TS. Cells that stably harbour the corresponding empty vector (A549/Mock, H1299/Mock, and PC9/Mock) were established as controls. Immunoblot analysis showed that the abundance of TS was markedly increased in the TS-overexpressing lines compared with the parental or Mock cells (Figure 1A). The enzymatic activity of TS was also substantially higher in the TS-overexpressing cells than in the parental or Mock cells (Figure 1B). We then examined the effect of forced expression of TS on the cytotoxicity of anticancer drugs as determined with the MTT assay. The median inhibitory concentration of pemetrexed for the TS-overexpressing cells was about three to six times that for the corresponding Mock cells for all three lung cancer lines, whereas cisplatin and docetaxel inhibited the growth of the TS-overexpressing cells in a manner similar to that observed with the corresponding Mock cells (Table 1). To exclude the possibility that these results were because of nonspecific effects of transfection, we depleted A549/TS1, H1299/TS1, and PC9/TS1 cells of TS by RNA interference. Immunoblot analysis revealed that transfection of these cells with an siRNA specific for TS mRNA resulted in downregulation of the corresponding protein (Figure 2A). This reduction in the abundance of TS restored the sensitivity of the cells to the inhibitory effect of pemetrexed on cell growth (Figure 2B). These data thus indicated that high TS expression levels reduce the sensitivity of NSCLC cells to pemetrexed.

Bottom Line: The sensitivity of NSCLC cells overexpressing TS to the antiproliferative effect of pemetrexed was markedly reduced compared with that of control cells.Finally, the level of TS expression in tumours of non-responding patients was significantly higher than that in those of responders, suggestive of an inverse correlation between TS expression and tumour response to pemetrexed.A high level of TS expression confers a reduced sensitivity to pemetrexed.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511, Japan.

ABSTRACT

Background: Although a high level of thymidylate synthase (TS) expression in malignant tumours has been suggested to be related to a reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in non-small cell lung cancer (NSCLC). We have now investigated the effect of TS overexpression on pemetrexed sensitivity in NSCLC cells.

Methods: We established NSCLC cell lines that stably overexpress TS and examined the effects of such overexpression on the cytotoxicity of pemetrexed both in vitro and in xenograft models. We further examined the relation between TS expression in tumour specimens from NSCLC patients and the tumour response to pemetrexed by immunohistochemical analysis.

Results: The sensitivity of NSCLC cells overexpressing TS to the antiproliferative effect of pemetrexed was markedly reduced compared with that of control cells. The inhibition of DNA synthesis and induction of apoptosis by pemetrexed were also greatly attenuated by forced expression of TS. Furthermore, tumours formed by TS-overexpressing NSCLC cells in nude mice were resistant to the growth-inhibitory effect of pemetrexed observed with control tumours. Finally, the level of TS expression in tumours of non-responding patients was significantly higher than that in those of responders, suggestive of an inverse correlation between TS expression and tumour response to pemetrexed.

Conclusion: A high level of TS expression confers a reduced sensitivity to pemetrexed. TS expression is thus a potential predictive marker for response to pemetrexed-based chemotherapy in NSCLC patients.

Show MeSH
Related in: MedlinePlus