Organic cation transporter OCT6 mediates cisplatin uptake and resistance to cisplatin in lung cancer.

PURPOSE: The purposes of this study were to determine whether organic cation transporters (OCTs) can mediate platinum uptake, and whether OCT down-regulation confers resistance against cisplatin (CDDP) in cancer cells. METHODS: Two lung cancer cell lines, PC-6 and PC-14, and their CDDP-resistant derivatives, PC-6/CDDP and PC-14/CDDP, were analyzed. OCT expression levels were assayed using quantitative RT-PCR and Western blotting. Additionally, the effect of OCT6 overexpression, induced by transfection of the OCT6 gene SLC22A16 using a forced expression vector, on cellular sensitivity to CDDP and on intracellular platinum accumulation was measured using PC-14/CDDP cells. RESULTS: Both gene and protein expression of OCT6 were decreased in both CDDP-resistant cell lines compared with their expression in their respective parental cells. Intracellular accumulation of platinum was decreased in PC-14/CDDP cells compared with the parental cells after CDDP treatment. Furthermore, OCT6 overexpression induced by transfection of the OCT6 gene (SLC22A16) forced expression vector-sensitized PC-14/CDDP cells to CDDP and oxaliplatin (L-OHP) concomitant with increased intracellular concentration of platinum. CONCLUSION: OCT6 is a mediator of platinum uptake in cancer cells, and down-regulation of OCT6 is possibly one of the mechanisms of resistance against cisplatin in lung cancer.

Significance of c-MET overexpression in cytotoxic anticancer drug-resistant small-cell lung cancer cells.

The c-MET receptor tyrosine kinase is the receptor for hepatocyte growth factor. Recently, activation of the c-MET/hepatocyte growth factor signaling pathway was associated with poor prognosis in various solid tumors and was one of the mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib. But the link between c-MET activation and the cytotoxic anticancer drug has not been fully examined. Here, we found that the enhanced expression and activation of c-MET in cytotoxic anticancer agent-resistant small-cell lung cancer cells. Downregulation of c-MET expression by siRNA against the c-MET gene or inhibition of c-MET activation by SU11274, a c-MET inhibitor, in the resistant cells altered resistance to the cytotoxic anticancer agent. These results indicated that c-MET overexpression might play an important role in acquired resistance to cytotoxic anticancer drugs. Furthermore, the number of c-MET gene loci was increased in the resistant cells compared to the parental cells. In conclusion, increased c-Met expression through an increase in the number of c-MET gene loci is one of the mechanisms of acquired resistance to cytotoxic anticancer drugs. Our results add a new strategy, the targeting of c-MET, for overcoming resistance to cytotoxic agents in small-cell lung cancer.

Thymidylate synthase gene copy number as a predictive marker for response to pemetrexed treatment of lung adenocarcinoma.

BACKGROUND: Pemetrexed is a key drug for therapy of non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: In a search for biomarkers for study of the efficacy of pemetrexed treatment, we examined the thymidylate synthase (TYMS) copy number in NSCLC cell lines and in clinical NSCLC samples treated with pemetrexed, combined with platinum drugs. RESULTS: TYMS copy numbers in lung adenocarcinoma cell lines were significantly lower than in squamous cell carcinoma (p=0.0105), and a significant correlation was found between the TYMS copy number and the 50% inhibitory concentration value for pemetrexed in all 17 lung cancer cell lines tested (r=0.6814, p=0.0026). Moreover, TYMS copy number was significantly lower in clinical NSCLC samples responsive to treatment with pemetrexed combined with platinum drugs (p=0.0067). Furthermore, the decrease in the baseline CT size measurement of pemetrexed combined with platinum drug treatment correlated significantly with TYMS copy number (r=0.7967, p=0.0011). CONCLUSION: To our knowledge, this is the first report of a significant association between TYMS copy number and response to pemetrexed treatment in tumor biopsy specimens. Our results suggest that TYMS copy number could be a predictive biomarker for pemetrexed based chemotherapy.

Involvement of intermediate filament nestin in cell growth of small-cell lung cancer.

BACKGROUND: Nestin is a class VI intermediate filament protein expressed in stem/progenitor cells during the development of the central nervous system. Nestin is detected in various types of tumors and is involved in malignant processes. This study investigated the expression and function of nestin in small-cell lung cancer (SCLC). METHODS: Expression of nestin and achaete-scute homolog 1 (ASH1) was studied in 21 lung cancer cell lines. To assess the function of nestin, a short hairpin RNA (shRNA) targeting nestin was transfected into two SCLC cell lines (DMS53 and SBC3), and cloned cells that showed apparent down-regulation of nestin were obtained. Nestin expression was also studied immunohistochemically in surgically resected SCLC primary tumors and metastatic SCLC tumors obtained from autopsy cases. RESULT: Nestin was expressed in nine of 10 SCLC cell lines. The nestin expression level was significantly higher in SCLC cell lines than in NSCLC cell lines (P < 0.01). There was a statistically significant positive correlation between the expression levels of nestin and ASH1 in SCLC cell lines. Nestin knock-down cells created by transfection with shRNA exhibited decreased invasion and cell proliferation capabilities. Furthermore, nestin was detected in SCLC tumor cells and tumor vessels in all clinical tumor specimens. CONCLUSION: Nestin is expressed in SCLC in association with neuroendocrine features and participates in malignant phenotypes, including cell growth. Therefore, nestin may be a novel therapeutic target for SCLC.

Preclinical rationale for synergistic interaction of pemetrexed and cytotoxic nucleoside analogues.

Cytotoxic nucleoside analogues are widely used in cancer chemotherapy. We used the cytosine arabinoside (Ara-C)-resistant erythroleukaemia cell line K562 and the Ara-C-sensitive myeloid leukaemia cell line HL60 to examine the differential expression of molecular markers. We found increased expression levels of deoxycytidine kinase (dCK) and human equilibrative nucleoside transporter 1 (hENT1) and decreased levels of multidrug resistance protein 5 (ABCC5) and ribonucleoside reductase subunit M1 (RRM1) expression in Ara-C-sensitive HL60 cells. We previously established the pemetrexed (MTA)-resistant small cell lung cancer cell lines PC6/MTA-0.4 and PC6/MTA-1.6 and found that MTA-resistant cells are more sensitive to gemcitabine (GEM) and Ara-C compared with parental PC-6 cells. We examined the molecular markers for GEM and Ara-C sensitivity in MTA-resistant cells and found increased gene expression of dCK and hENT1. Furthermore, treatment with MTA resulted in increased expression of dCK and hENT1 and decreased expression of ABCC5 and RRM1, concomitant with the alteration of the resistance to Ara-C in Ara-C-resistant K562 cells. These results provide evidence that the chemotherapeutic activity of the combination of MTA and cytotoxic nucleoside analogues is synergistic with regard to the alteration of metabolic molecules.

C609T polymorphism of NAD(P)H quinone oxidoreductase 1 as a predictive biomarker for response to amrubicin.

INTRODUCTION: Amrubicin is a promising agent in the treatment of lung cancer, but predictive biomarkers have not yet been described. NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme known to metabolize amrubicinol, the active metabolite of amrubicin, to an inactive compound. We examined the relationship between NQO1 and amrubicinol cytotoxicity. METHODS: Gene and protein expression of NQO1, amrubicinol cytotoxicity, and C609T single-nucleotide polymorphism of NQO1 were evaluated in 29 lung cancer cell lines: 14 small cell lung cancer (SCLC) and 15 non-SCLC (NSCLC). The involvement of NQO1 in amrubicinol cytotoxicity was evaluated by small interfering RNA against NQO1. RESULTS: A significant inverse relationship between both gene and protein expression of NQO1 and amrubicinol cytotoxicity was found in all cell lines. Treatment with NQO1 small interfering RNA increased amrubicinol cytotoxicity and decreased NQO1 expression in both NSCLC and SCLC cells. Furthermore, cell lines genotyped homozygous for the 609T allele showed significantly lower NQO1 protein expression and higher sensitivity for amrubicinol than those with the other genotypes in both NSCLC and SCLC cells. CONCLUSIONS: NQO1 expression is one of the major determinants for amrubicinol cytotoxicity, and C609T single-nucleotide polymorphism of NQO1 could be a predictive biomarker for response to amrubicin treatment.

Over-expression of MDR1 in amrubicinol-resistant lung cancer cells.

PURPOSE: Amrubicin, a totally synthetic 9-aminoanthracycline anticancer drug, has shown promising activity for lung cancer, but little is known about the mechanism of resistance for this agent. This study was aimed to clarify the role of P-glycoprotein (P-gp) in amrubicinol, an active metabolite of amrubicin, resistance in lung cancer cells. METHODS: Amrubicinol-resistant cell line PC-6/AMR-OH was developed by continuously exposing the small-cell lung cancer cell line PC-6 to amrubicinol. Gene expression level of MDR1, which encodes P-gp, and intracellular accumulation of amrubicinol were evaluated by PC-6 and PC-6/AMR-OH cells. The involvement of MDR1 in amrubicinol resistance was evaluated by treatment with P-gp inhibitor verapamil and small interfering RNA (siRNA) against MDR1. Also, expression levels and single-nucleotide polymorphisms (SNPs) of MDR1 in 22 lung cancer cell lines were examined, and the relationships between these factors and sensitivity to amrubicinol were evaluated. RESULTS: The MDR1 gene was increased approximately 4,500-fold in PC-6/AMR-OH cells compared with PC-6 cells, and intracellular accumulation of amrubicinol in PC-6/AMR-OH cells was decreased to about 15 percent of that in PC-6 cells. Treatment with verapamil and siRNA against MDR1 significantly increased the sensitivity to amrubicinol in PC-6/AMR-OH cells with increased cellular accumulation of amrubicinol. Meanwhile, neither MDR1 gene expression levels nor SNPs of the gene were associated with amrubicinol sensitivity. CONCLUSIONS: Results of this study indicate that increased MDR1 expression and P-gp activity confer acquired resistance to amrubicinol. In contrast, neither expression level nor SNPs of MDR1 are likely to be predictive markers for amrubicin activity.

[A case of spinal osteomyelitis due to Mycobacterium intracellulare with pulmonary Mycobacterium avium complex].

A 76-year-old woman was admitted to our hospital in October 2007 because of fever and anorexia. Chest computed tomography demonstrated consolidation and reticular shadows in the right middle lobe and left lingula. Pulmonary nontuberculous mycobacteriosis was diagnosed based on isolation of Mycobacterium intracellulare from her sputum. Her symptoms, which included fever, were improved by chemotherapy with clarithromycin and ethambutol, but the treatment was halted due to adverse effects. Subsequently, she complained of lumbago from April 2008, and spinal osteomyelitis from the T-4 through T-5 vertebral bodies was suspected based on the findings of magnetic resonance imaging. Spinal osteomyelitis due to Mycobacterium intracellulare was diagnosed based on isolation of the bacteria from a tissue culture taken from the vertebral body. Surgery was performed after chemotherapy with clarithromycin, rifampicin and moxifloxacin. Spinal osteomyelitis caused by nontuberculous mycobacterium infection should be considered due to a worldwide increase in reported cases of pulmonary nontuberculous mycobacteriosis.

[A case of Legionnaires' disease with abnormal neurological findings showing the corpus callosum abnormality on brain MRI].

A 47-year-old man was admitted to our hospital because of dysarthric speech and high fever. His brain magnetic resonance imaging (MRI) showed high intensity in the splenium of the corpus callosum (SCC) on diffusion-weighted and FLAIR imaging. His chest CT showed ground-glass attenuation in bilateral lung upper lobes, and consolidation in the right lower lobe. Because of his neurological findings and radiographic imaging, we started antibiotic therapy using quinolone, which is effective for Legionellosis. Later, Legionella species were identified by sputum culture. Pneumonia was relieved after antibiotic therapy and in parallel with clinical improvement, and the MRI findings also resolved. We report a patient who showed abnormal neurological findings and abnormality on brain MRI, both of which were reversible, in the setting of acute Legionnaires' disease.

[Management of difficult-to-treat asthma using a flow sheet for systematic evaluation and therapeutic intervention].

BACKGROUND: Although most patients of asthma can be controlled by inhaled corticosteroid (ICS), some patients remain uncontrolled even after the introduction of ICS treatment. In management of such difficult-to-treat asthma, systematic review including additional differential diagnosis and avoidance of exacerbating factors is very important. METHODS: Here we postulate a flow sheet presenting an algorithm which intends to achieve better asthma control following ATS refractory asthma guidance. For patients with poor controlled asthma even after using ICS more than moderate dose, we used the sheet in our daily outpatient management and investigated whether we could improve the control in such patients. The sheet was constructed by an algorithm which included (1) reevaluation of inhalation technique of ICS; (2) additional differential diagnosis of COPD and other similar diseases; and (3) reevaluation of presence of exacerbating factors. RESULTS: In our outpatient department, seven clinicians managed 22 difficult-to-treat asthma patients using this sheet. Additional factors which might worsen asthma control could be detected in 21 patients (95.5%). Firstly, smoking was disclosed in 8 patients (36.4%). Secondly, keeping pets was identified in 7 patients (31.8%). 5 patients (22.7%) were diagnosed as COPD rather than asthma and 4 patients (18.2%) were diagnosed as having rhinosinusitis. Some improvement of asthma control was achieved in 9 patients (40.9%). CONCLUSIONS: Reevaluation of refractory asthma patients using our newly developed flow sheet is essential and it may facilitate understanding of management of difficult-to-treat asthma.