Tetsuya Oguri

Determinants of sensitivity and resistance to gemcitabine: The roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non‐small cell lung cancer

Gemcitabine is one of the most commonly used agents for lung cancer chemotherapy, but the determinants of sensitivity and/or resistance to this agent are not yet fully understood. In this study we used quantitative RT‐PCR to examine the expression levels of human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) genes in non‐small cell lung cancer (NSCLC) cell lines in relation to sensitivity and resistance to gemcitabine. The basal expression levels of hENT1 were significantly correlated with the IC50 values for gemcitabine (r=‐0.6769, P=0.0005), whereas dCK expression levels were not. In a highly gemcitabine‐sensitive cell line, NCI‐H23, the sensitivity to gemcitabine was inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of hENT1, without significant modulation of hENT1 expression. These data suggest that hENT1 is associated with gemcitabine sensitivity in lung cancer. We also continuously exposed NCI‐H23 cells to gemcitabine and subsequently established the drug‐resistant clone H23/GEM‐R, which showed a significant decrease of dCK expression; however, hENT1 expression was not altered in the continuously exposed sublines or in the resistant clone. We conclude that increased hENT1 expression is a determinant of gemcitabine sensitivity, while decreased dCK expression is associated with acquired resistance to gemcitabine in NSCLC cells. Thus, hENT1 and dCK might be useful as predictive markers for efficacy of gemcitabine therapy in NSCLC.

Significance of thymidylate synthase for resistance to pemetrexed in lung cancer

Pemetrexed (MTA) is a multitargeted antifolate with promising clinical activity in lung cancer. We exposed the small cell lung cancer cell line PC6 to stepwise‐increasing pemetrexed concentrations of 0.4, 1.6, and 4.0 μm, and established three pemetrexed‐resistant lung cancer cell lines: PC6/MTA‐0.4, PC6/MTA‐1.6, and PC6/MTA‐4.0 cells. To investigate the mechanisms of acquired resistance to pemetrexed, we measured the expression levels of the thymidylate synthase (TS), reduced folate carrier (RFC), and folylpoly‐gamma‐glutamate synthetase (FPGS) genes. TS gene expression was significantly increased in PC6/MTA‐1.6 and PC6/MTA‐4.0 cells relative to parental cells in a pemetrexed dose‐dependent manner. In contrast, the levels of RFC gene expression in PC6/MTA‐0.4 cells and FPGS in PC6/MTA‐1.6 cells were significantly decreased, whereas the levels of both genes were restored in PC6/MTA‐4.0 cells. Knockdown of TS expression using siRNA enhanced pemetrexed cytotoxicity in PC6/MTA‐4.0 cells. The expression level of the TS gene was significantly correlated with the concentration of pemetrexed for 50% cell survival (IC50) in 11 non‐small cell lung cancer cell lines. These results suggest that the alteration of molecular pharmacological factors in relation with pemetrexed resistance is dose‐dependent, and that up‐regulation of the expression of the TS gene may have an important role in the acquired resistance to pemetrexed. In addition, TS may be a predictive marker for pemetrexed sensitivity in lung cancer. (Cancer Sci 2009)

MRP8/ABCC11 directly confers resistance to 5-fluorouracil

Multidrug-resistance–associated protein, MRP8/ABCC11 (ABCC11), is an efflux pump for nucleotide analogues and 5-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP). To test whether ABCC11 directly confers 5-fluorouracil (5-FU) resistance, we used the 5-FU–resistant subline PC-6/FU23-26 selected from PC-6 human small-cell lung cancer cells by 5-FU and found that it increases the resistance by ∼25-fold. The intracellular FdUMP accumulation was reduced in PC-6/FU23-26 cells concomitant with the overexpression of the ABCC11 gene. These findings suggest that ABCC11 confers 5-FU resistance in the sublines by enhancing the efflux for the active metabolite FdUMP. Previously, methotrexate also increased the efflux by ABCC11, and we found cross-resistance to methotrexate in PC-6/FU23-26 cells. To confirm our hypothesis, we examined whether decreasing the expression of ABCC11 in PC-6/FU23-26 cells by small interfering RNA altered the cytotoxicity to 5-FU and methotrexate and found that this enhanced 5-FU and methotrexate cytotoxicity in PC-6/FU23-26 cells. These data indicate that expression of the ABCC11 gene is induced by 5-FU, and that ABCC11 is directly involved in 5-FU resistance by the efflux transport of the active metabolite FdUMP. [Mol Cancer Ther 2007;6(1):122–7]

Cyclooxygenase‐2 (COX‐2) mRNA Expression Levels in Normal Lung Tissues and Non‐small Cell Lung Cancers

One of the cyclooxygenase (COX) isoforms, COX‐2, is over expressed in various human cancers. In this study, we examined the gene expression levels of COX‐2 in primary non‐small cell lung cancers (NSCLC), metastatic lymph nodes, and normal lung tissues. The expression levels of the COX‐2 gene were assessed by means of the reverse transcription polymerase chain reaction in 76 autopsy samples (29 primary NSCLC, 29 corresponding normal lung tissues, and 9 metastatic lymph nodes). The expression levels in NSCLC (both adenocarcinomas and squamous cell carcinomas) were significantly higher than in normal lung tissues and were significantly higher in adenocarcinomas than in squamous cell carcinomas. Differences between the levels of expression of COX‐2 in primary tumors and their corresponding metastatic lymph nodes in 9 adenocarcinoma patients were not significant. Our results indicate that COX‐2 may be associated with carcinogenesis of NSCLC, and that it may be a target for the treatment of NSCLC.

SGLT Gene Expression in Primary Lung Cancers and Their Metastatic Lesions

Cancer cells show increased glucose uptake and utilization in comparison with their normal counterparts. Glucose transporters play an important role in glucose uptake. We previously reported the differential gene expression of the GLUT family in primary and metastatic lesions of lung cancer. To investigate the role of Na+/glucose cotransporter (SGLT) genes in cancers, we examined the levels of expression of SGLT1 and SGLT2 genes in primary lung cancers and their metastatic lesions. Ninety‐six autopsy samples (35 primary lung cancers, 35 corresponding normal lung tissues, 10 metastatic liver lesions, and 16 metastatic lymph nodes) from 35 patients were analyzed for SGLT1 and SGLT2 expression by reverse transcription (RT)‐polymerase chain reaction (PCR). There were no significant differences in the level of expression of either gene between the primary lung cancers and normal lung tissues. The level of SGLT1 expression in the metastatic lesions and primary lung cancers did not differ significantly. The level of SGLT2 expression was, however, significantly higher in the metastatic lesions of both the liver and lymph node than in the primary lung cancers. These results suggest that SGLT2 plays a role in glucose uptake in the metastatic lesions of lung cancer.

The determinants of sensitivity and acquired resistance to gemcitabine differ in non-small cell lung cancer: a role of ABCC5 in gemcitabine sensitivity

We examined the expression levels of the multidrug resistance protein 5 (ABCC5) gene in non–small cell lung cancer (NSCLC) cell lines to clarify the relationship with the sensitivity to gemcitabine. The expression levels of ABCC5 were inversely correlated with gemcitabine sensitivity significantly (r = 0.628; P < 0.01) in 17 NSCLC cells, whereas the expression of ABCC5 in the gemcitabine-resistant NSCLC cell line H23/GEM-R was the same as that in parental NCI-H23 cells. Treatment with the ABCC5 inhibitor zaprinast altered the sensitivity to gemcitabine in ABCC5-expressing NSCLC cells. In addition, decreasing the expression of ABCC5 by small interfering RNA altered the cytotoxicity to gemcitabine. These results indicate that modulation of ABCC5 activity could be used to increase the gemcitabine sensitivity in NSCLC. Previously, we found a decreased expression of deoxycytidine kinase in H23/GEM-R cells, and further investigation in this study showed an increased expression of ribonucleotide reductase subunit 1 in H23/GEM-R cells. We therefore also examined the effect of modifying the expression of both genes on gemcitabine resistance. We found that using small interfering RNA to decrease the expression of ribonucleotide reductase subunit 1 resulted in a decreased resistance to gemcitabine in H23/GEM-R cells. Furthermore, pretreatment with pemetrexed resulted in an increased deoxycytidine kinase expression concomitant with the alteration of the resistance to gemcitabine in H23/GEM-R cells. The determinants for sensitivity and the acquired resistance in gemcitabine are quite different; nonetheless, modification of these factors may increase the efficacy of gemcitabine in the treatment of NSCLC. [Mol Cancer Ther 2006;5(7):1800–6]

Differential Expression of Facilitative Glucose Transporter (GLUT) Genes in Primary Lung Cancers and Their Liver Metastases

Glucose uptake is mediated by members of the facilitative glucose transporter (GLUT) family. Malignant cells take up more glucose than their normal counterparts. The aim of this study was to investigate the gene expression levels of the GLUT family, especially GLUT1, GLUT3, and GLUT5 in primary lung cancer, metastatic liver tumors, and normal lung tissues, and to compare the expression levels of primary and metastatic tumors with those of normal tissues. We analyzed 105 autopsy samples (35 primary lung tumors, 35 corresponding normal lung tissues, 25 normal liver tissues, and 10 metastatic liver tumors) from 35 patients using the quantitative reverse transcription polymerase chain reaction. The GLUT1 gene expression levels in primary lung tumors were significantly higher than those in normal lung tissues. In liver metastatic lesions, the GLUT3 and GLUT5 gene expression levels were significantly higher than those in primary lung tumors, but there were no differences in GLUT1 expression levels between primary and metastatic liver tumors. Our results show that the gene expression pattern of the GLUT family is different between primary and metastatic liver tumors and suggest that the energy transporters in metastatic tumors may be different from those in primary tumors.

Expression of γ-glutamylcysteine synthetase (γ-GCS) and multidrug resistance-associated protein (MRP), but not human canalicular multispecific organic anion transporter (cMOAT), genes correlates with exposure of human lung cancers to platinum drugs

We examined the steady-state levels of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS), multidrug resistance-associated protein (MRP) and human canalicular multispecific organic anion transporter (cMOAT) in human lung cancer specimens to elucidate their roles in relation to platinum drug resistance in vivo. Seventy-six autopsy samples (38 primary tumours and their corresponding normal lung tissues) obtained from 38 patients were analysed using the quantitative reverse transcription polymerase chain reaction (RT-PCR) method. Both subunits (heavy and light subunits) of gamma-GCS expression levels of normal lung and tumour tissues exposed to platinum drugs during life were significantly higher than those of non-exposed tissues, whereas only the MRP expression levels of tumours were elevated in association with ante-mortem platinum drug exposure. The gamma-GCS and MRP expression levels correlated significantly. The cMOAT expression levels did not correlate with ante-mortem platinum drug exposure. Next, we monitored gamma-GCS heavy subunit expression levels in peripheral mononuclear cells of eight previously untreated lung cancer patients after platinum drug administration, which revealed that these drugs induced gamma-GCS expression in vivo. These results suggest that gamma-GCS expression is induced by platinum drugs in vivo and/or the physiological stress response to xenobiotics.

MRP7/ABCC10 expression is a predictive biomarker for the resistance to paclitaxel in non-small cell lung cancer.

We used the paclitaxel-resistant human small cell lung cancer subline PC-6/TAX1-1, selected from PC-6 cells by paclitaxel, to test whether MRP7/ABCC10 (ABCC10) confers paclitaxel resistance. We found that gene expression of both ABCB1/MDR1 (ABCB1) and ABCC10 was higher in PC-6/TAX1-1 cells than in PC-6 cells. The expression levels of ABCC10 showed a significant inverse correlation with paclitaxel sensitivity (r = 0.574; P < 0.05) in 17 non-small cell lung cancer (NSCLC) cells unlike the expression levels of ABCB1. Pretreatment with the ABCC10 inhibitor sulfinpyrazone altered the sensitivity to paclitaxel in ABCC10-expressing NSCLC cells, concomitant with increased intracellular paclitaxel accumulation. These findings suggest that expression of the ABCC10 gene is induced by paclitaxel and that ABCC10 confers paclitaxel resistance by enhancing the efflux for paclitaxel. To confirm this hypothesis, we tested the effect on paclitaxel cytotoxicity of decreasing the expression of ABCC10 by small interfering RNA and found that this enhanced paclitaxel cytotoxicity in NCI-H23 cells concomitant with increased intracellular paclitaxel accumulation. These data indicate that ABCC10 may be one of the biomarkers for paclitaxel resistance in NSCLC. [Mol Cancer Ther 2008;7(5):1150–5]

Increased expression of the MRP5 gene is associated with exposure to platinum drugs in lung cancer.

To investigate the role of the multidrug resistance‐associated protein (MRP1) homologue MRP5 in relation to platinum drug resistance, we examined the steady‐state levels of the mRNAs for MRP5 in both lung cancer cell lines and peripheral mononuclear cells (PMN) after exposure to platinum drug and in normal lung and lung cancer tissue specimens. Firstly, we examined MRP5 gene expression levels in 80 autopsy samples (40 primary tumors and 40 corresponding normal lung tissues) from 40 patients who had died from lung cancer. Next, we monitored MRP5 gene expression levels within 24 hr in both lung cancer cell lines incubated with cisplatin and in PMN from 10 previously untreated lung cancer patients after carboplatin administration alone. The MRP5 gene expression levels were assessed by quantitative reverse transcription polymerase chain reaction or RNase protection assay. The MRP5 expression levels in normal lung tissues and in tumors from patients exposed to platinum drugs during their lifetime were significantly higher than those in tissues from non‐exposed patients. On the other hand, the MRP5 expression levels were not rapidly induced by platinum drugs either in lung cancer cell lines or in PMN within 24 hr. Our results suggest that increased expression levels of the MRP5 gene are associated with exposure to platinum drugs in lung cancer in vivo and/or the chronic stress response to xenobiotics. Int. J. Cancer 86:95–100, 2000.