Genetic determinants of pemetrexed responsiveness and nonresponsiveness in non-small cell lung cancer cells.
BACKGROUND
Pemetrexed disodium (Alimta), LY231514, is an antifolate that is able to simultaneously inhibit the synthesis of purines and pyrimidines. Pemetrexed has been approved for first- and second-line treatment in patients with non-small cell lung cancer (NSCLC). However, there is still a lack of clinical biomarkers for predicting the therapeutic response to pemetrexed. The aim of this study is to establish new biomarkers for pemetrexed treatment in NSCLC.


METHODS
Human NSCLC cell lines were exposed to pemetrexed. The antitumor effect was measured by growth inhibition with MTT assay and expression of cell cycle mediators with immunoblots. Using the Superarray cancer pathway gene array, 482 genes were screened for differential expression in A549 cells that were untreated or treated with pemetrexed.


RESULTS
A549 cells exhibited sensitivity but H1355 cells showed resistance to pemetrexed. To investigate the mechanisms of responsiveness and nonresponsiveness to pemetrexed in these cell lines, we measured the expression levels of thymidylate synthase (TS), dihydrofolate reductase (DHFR), reduced folate carrier, and folylpoly-gamma-glutamate synthetase genes. TS, DHFR, and reduced folate carrier gene expressions were significantly reduced in A549 and H1355 cells. Pemetrexed caused cell cycle arrest in the G1 phase and S phase in H1355 and A549 cells, respectively. Significantly higher expressions of many genes, especially lipocalin-2 (Lcn-2) and nm23-H1 proteins, were noted in A549 cells treated with pemetrexed in comparison with untreated cells. Furthermore, reverse transcriptase polymerase chain reaction and Western blot showed that Lcn-2 and nm23-H1 expressions increase in response to pemetrexed treatment in a dose-responsive manner in pemetrexed-sensitive A549 cells but not in resistant H1355 cells.


CONCLUSIONS
Our results indicated that downregulation of TS and DHFR genes and upregulation of p21, p27, Lcn-2, and nm23-H1 genes may serve as new biomarkers for predicting responsiveness to pemetrexed.
Wu
Ming-Fang
MF
Departments of Chest Medicine and Medical Oncology, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC.
Hsiao
Yi-Min
YM
Huang
Chuan-Fu
CF
Huang
Yu-Hsin
YH
Yang
Wan-Jung
WJ
Chan
Hsiu-Wen
HW
Chang
Jinghua Tsai
JT
Ko
Jiunn-Liang
JL
D000209:Acute-Phase Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D005971:Glutamates / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D000071068:Lipocalin-2; D054834:Lipocalins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D054778:NM23 Nucleoside Diphosphate Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D000068437:Pemetrexed; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000209:Acute-Phase Proteins; D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D005971:Glutamates; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D054778:NM23 Nucleoside Diphosphate Kinases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000068437:Pemetrexed; D006147:Guanine; C517938:NME1 protein, human