Down-Regulation of DUSP6 Expression in Lung Cancer —Its Mechanism and Potential Role in Carcinogenesis

Our preliminary studies revealed that oncogenic KRAS (KRAS/V12) dramatically suppressed the growth of immortalized airway epithelial cells (NHBE-T, with viral antigen-inactivated p53 and RB proteins). This process appeared to be a novel event, different from the so-called premature senescence that is induced by either p53 or RB, suggesting the existence of a novel tumor suppressor that functions downstream of oncogenic KRAS. After a comprehensive search for genes whose expression levels were modulated by KRAS/V12, we focused on DUSP6, a pivotal negative feedback regulator of the RAS-ERK pathway. A dominant-negative DUSP6 mutant, however, failed to rescue KRAS/V12-induced growth suppression, but conferred a stronger anchorage-independent growth activity to the surviving subpopulation of cells generated from KRAS/V12-transduced NHBE-T. DUSP6 expression levels were found to be weaker in most lung cancer cell lines than in NHBE-T, and DUSP6 restoration suppressed cellular growth. In primary lung cancers, DUSP6 expression levels decreased as both growth activity and histological grade of the tumor increased. Loss of heterozygosity of the DUSP6 locus was found in 17.7% of cases and was associated with reduced expression levels. These results suggest that DUSP6 is a growth suppressor whose inactivation could promote the progression of lung cancer. We have here identified an important factor involved in carcinogenesis through a comprehensive search for downstream targets of oncogenic KRAS.Oncogenic mutations of KRAS occur very early in carcinogenesis of the lung and affect even premalignant lesions.^1,2 It is therefore likely that additional genetic and/or epigenetic alterations are necessary for lung neoplasms to develop into advanced form of cancers. The inactivation of putative tumor suppressors, p53 and p16INK4A/RB protein, and activation of telomerase, are generally accepted as crucial to the promotion carcinogenesis.^1,3 A recent study, however, reported that these alterations are not enough to provide primary bronchial epithelial cells with a fully malignant phenotype in vitro, ⁴ and suggested further alterations would be necessary for the progression of lung cancer.DUSP6, dual-specificity phosphatase 6, is a putative negative feedback regulator for the RAS-ERK pathway, playing physiologically important role(s) in the maintenance of cellular homeostasis in response to growth factors.^{5,6,7,8,9,10,11} Disruption of this feedback loop could therefore result in neoplastic, and even malignant, transformation by providing cells with enhanced growth activity. Actually, a series of previous studies reported the involvement of DUSP6 down-regulation caused by the hypermethylation of its promoter in the progression of pancreatic cancers.^{12,13,14,15,16} To our knowledge, however, there have been only a few reports investigating the significance of DUSP6 in other types of cancers including lung cancers.^4,17Our preliminary study revealed that oncogenic KRAS (KRAS/V12) dramatically suppressed growth of immortalized airway epithelial cells whose p53 and RB proteins were inactivated by viral antigens. This seemed a novel event different from so-called premature senescence that is induced by p53 or RB pathway,³ and suggested there might exist novel tumor suppressors downstream of oncogenic KRAS. Thus, we have been interested in identifying such suppressors. Also, our recent study demonstrated that insulin-like growth factor-binding proteins 2 and 4, downstream targets of oncogenic KRAS, suppress the growth of cancer cells in a negative-feedback manner and that disruption of this feedback loop, through their promoter’s hypermethylation, is possibly involved in the progression of lung cancers.¹⁸ It is worth noting that growth suppressors lie hidden downstream of the signal transduction pathway of an oncogene. Thus, we were prompted to search for downstream targets of oncogenic KRAS comprehensively, to further identify potential growth suppressors. Among genes whose expression was modulated by oncogenic KRAS, this study has focused on DUSP6, and elucidated its role in the KRAS-induced growth suppression and in carcinogenesis of the lung.     

Effect of acyl chains of phosphatidylcholines on the pharmacokinetics of menatetrenone incorporated in O/W lipid emulsions prepared with phosphatidylcholines and soybean oil in rats

Oil-in-water (O/W) lipid emulsions were prepared with phosphatidylcholines (PCs) of various acyl chains and soybean oil (SO) using a microfluidizer system, and the pharmacokinetics of menatetrenone incorporated in these oil particles were examined at the clinical injection volume (0.1 mL kg(-1)) in rats. The plasma half-life of menatetrenone incorporated in the oil particles prepared with SO and dipalmitoylphosphatidylcholine (DPPC) (SO/DPPC) was longer than that prepared with SO and eggyolk phosphatides (EYP) (SO/EYP) by 3 fold, while those of menatetrenone as oil particles prepared with SO and either dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC) or dilinoleoyl phosphatidylcholine (DLoPC) (SO/DLPC, SO/DMPC, SO/DSPC, SO/DOPC and SO/DLoPC, respectively) were similar to that of menatetrenone as SO/EYP. The menatetrenone uptake by the liver was not significantly different from that as SO/EYP in all SO/PCs examined, but the menatetrenone uptake by the spleen as SO/DPPC and SO/DSPC was higher than that as SO/EYP. The menatetrenone uptake by the lungs as SO/DPPC was also higher than that as SO/EYP. These findings suggest that SO/DPPC is a good candidate drug carrier for the prolonged plasma circulation of lipophilic drugs.     

Genotype-dependent down-regulation of gene expression and function of MDR1 in human peripheral blood mononuclear cells under acute inflammation

Recent advances in pharmacogenomics have suggested the association of clinical outcome of glucocorticoid-based anti-inflammatory therapy with a single nucleotide polymorphism at position 3435 in exon 26 (C3435T) of the MDR1 gene. In the present study, the effects of the MDR1 C3435T genotype on the time-dependent profiles of gene expression and function of MDR1/P-glycoprotein were evaluated in peripheral blood mononuclear cells (PBMCs) under lipopolysaccharide (LPS)-induced experimental acute inflammation. LPS treatment resulted in the rapid elevation of IL-1beta and TNF-alpha mRNA levels relative to beta-actin mRNA at 1 h, with a subsequent slight decrease at 3 h after the treatment, while the down-regulation of the relative concentration of MDR1 mRNA was found at 3 h, not at 1 h, after LPS treatment. Here, the C3435T genotype-dependent down-regulations of MDR1 mRNA level were found for CC(3435) and CT(3435), but not for TT(3435), and were 64.1+/-10.1%, 71.4+/-5.9% and 100.0+/-22.5% (+/-S.D.), respectively, of their respective baseline levels, which were independent of C3435T (0.010+/-0.005, 0.011+/-0.013 and 0.009+/-0.006 (+/-S.D.), respectively). The C3435T genotype-dependent down-regulation was supported by the increase of the intracellular accumulation of calcein in PBMCs treated with LPS for 72 h, and the increase was more predominant for CC(3435) than TT(3435). These data suggested that glucocorticoid-based anti-inflammatory therapy might be more effective for C(3435)-allele carriers than non-carriers.     

Haloperidol is an inhibitor but not substrate for MDR1/P-glycoprotein

The involvement of the multidrug resistant transporter MDR1/P-glycoprotein in the penetration of haloperidol into the brain and absorption in the intestine was investigated to examine its role in inter/intra-individual variability, using the porcine kidney epithelial cell line LLC-PK(1) and its MDR1-overexpressing transfectant, LLC-GA5-COL150. The inhibitory effect of haloperidol on other MDR1 substrates was also investigated in terms of the optimization of haloperidol-based pharmacotherapy. The transepithelial transport of [(3)H]haloperidol did not differ between the two cell lines, and vinblastine, a typical MDR1 substrate, had no effect on the transport, suggesting that haloperidol is not a substrate for MDR1, and it is unlikely that MDR function affects haloperidol absorption and brain distribution, and thereby the response to haloperidol. However, haloperidol was found to have an inhibitory effect on the MDR1-mediated transport of [(3)H]digoxin and [(3)H]vinblastine with an IC50 value of 7.84+/-0.76 and 3.60+/-0.64 microM, respectively, suggesting that the intestinal absorption, not distribution into the brain, of MDR1 substrate drugs could be altered by the co-administration of haloperidol in the clinical setting, although further clinical studies are needed.     

Simultaneous determination of single nucleotide polymorphisms of MDR1 genes by electrochemical DNA chip

The on-chip genotyping system ("the electrochemical DNA chip") has been developed as a more cost-effective genotyping system and was applied to MDR1 genotyping in the present study, which is required for wide use in clinical application and for personalized medication based on genotype. The electrochemical DNA chip was optimized and applied to simultaneous genotyping of four MDR1 polymorphisms (T-129C, C1236T, G2677(A,T) and C3435T) using synthetic model oligonucleotide DNA and human genomic DNA. The electrochemical DNA chip successfully gave the T-129C, C1236T, G2677(A,T) and C3435T genotypes, which were completely consistent with those determined by direct sequencing. In conclusion, the electrochemical DNA chip is useful for simultaneous determination of some genotypes and haplotypes, and efficient genotyping using this system can support future genotype-phenotype studies at a large scale.     

S-2474, a novel nonsteroidal anti-inflammatory drug, rescues cortical neurons from human group IIA secretory phospholipase A(2)-induced apoptosis

The elevated level of group IIA secretory phospholipase A(2) (sPLA(2)-IIA) activity contributes to neurodegeneration in the cerebral cortex after ischemia. The up-regulation of cyclooxygenase-2 (COX-2) is also relevant to cerebral ischemia in humans. Studies of ischemia with COX-2 inhibitors suggest a clinical benefit. In the present study, we investigated effects of S-2474 on sPLA(2)-IIA-induced cell death in primary cultures of rat cortical neurons, which was established as an in vitro model of brain ischemia. S-2474 is a novel nonsteroidal anti-inflammatory drug (NSAID), which inhibits COX-2 and contains the di-tert-butylphenol antioxidant moiety. S-2474 significantly prevented neurons from undergoing sPLA(2)-IIA-induced cell death. S-2474 completely ameliorated sPLA(2)-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. sPLA(2) also generated neurotoxic prostaglandin D(2) (PGD(2)) and free radicals from neurons before cell death. S-2474 significantly inhibited the sPLA(2)-IIA-induced generation of PGD(2). The present cortical cultures contained few non-neuronal cells, indicating that S-2474 affected neuronal survival directly, but not indirectly via non-neuronal cells. The inhibitory effect of S-2474 on COX-2 might contribute to its neuroprotective effect. In conclusion, S-2474 exhibits neuroprotective effects against sPLA(2)-IIA. Furthermore, the present study suggests that S-2474 may possess therapeutic potential for stroke via ameliorating neurodegeneration.     

N-Acetyltransferase2 genotype correlated with isoniazid acetylation in Japanese tuberculous patients

Isoniazid (INH) is metabolized by polymorphic N-acetyltransferase2 (NAT2). In the present study, the relationship between the NAT2 genotype and the INH acetylator phenotype was examined in Japanese tuberculous patients and compared with healthy subjects. Subjects were classified according to the genotyping into NAT2*5B (allele4), NAT2*6A (allele3) and NAT2*7B (allele2), using the PCR-RFLP method. Twelve healthy subjects and 7 tuberculous patients participated in the INH acetylator phenotyping study, in which each subject was administered an oral dose of INH, followed by urine sampling for 24 h. Urinary concentrations of INH and N-acetylisoniazid (AcINH) were measured by the HPLC method. The urinary recoveries of INH (% of dose) in healthy subjects in relation to NAT2 genotyping were as follows: 6.4+/-2.2 in the homozygotes for the wild-type allele, 10.7+/-2.2 in the compound heterozygotes for the mutant allele, and 38.6+/-6.4 in the homozygotes for the mutant allele. In the patients study, the findings in the corresponding three groups were 4.0+/-1.7, 8.8 and 18.3+/-9.3. Although no significant difference was found because of the lower systemic exposure of INH in patients compared with healthy subjects, there were differences in the disposition kinetics of INH between subjects with and without mutations in the NAT2 gene, and these findings were observed not only in healthy subjects but also in patients who had comedicated drugs and hepatic dysfunctions. The findings indicated that the metabolism of INH by NAT2 is clearly impaired in subjects with mutations in the NAT2 gene, and thus genotyping for three NAT2 point mutations was adequate to predict the metabolism of INH in Japanese tuberculous patients as well as healthy subjects. This NAT2 genotyping could become a useful alternative to TDM for INH.     

N-Acetyltransferase 2 genotype correlates with sulfasalazine pharmacokinetics after multiple dosing in healthy Japanese subjects

Sulfapyridine (SP) is metabolized by polymorphic N-acetyltransferase 2 (NAT2) [EC 2.3.1.5]. In this study, the correlation between the NAT2 genotype and the pharmacokinetics of SP after multiple oral dosing of sulfasalazine (SASP) was examined to elucidate the effect of multiple dosing on the predictability of the phenotype by NAT2 genotyping. Seven healthy subjects were classified into two groups; the homozygotes for the wild-type allele, NAT2*4/*4 (Group I) and the compound heterozygotes for the mutant allele (NAT2*4/*6A or NAT2*4/*7B) (Group II). All received once-daily 1 g of SASP (Salazopyrin) orally for 8 d. Plasma concentrations and urinary recoveries of SASP, SP and N-acetylsulfapyridine (AcSP) were monitored for 8 d. At 24 h on Day 1, the plasma concentration of SASP was lower and those of SP and AcSP were higher in Group II compared with Group I, but there was no significant difference. The plasma concentration ratio of AcSP to SP (AcSP/SP) tended to be lower in Group II. Urinary recoveries of SP and AcSP were increased in Group II, and their ratio was slightly reduced in Group II. Multiple dosing for 8 d resulted in an increase in the plasma concentrations of SASP, SP and AcSP. The difference between Group I and II was marked compared with single dosing, resulting in a significant difference in the plasma concentration of SP and the ratio of AcSP/SP. The simple input-output pharmacokinetic model applied for the analysis of plasma concentrations and urinary recoveries of SP and AcSP suggested the acetylation of SP into AcSP was 2.7-fold reduced in Group II (p=0.064).     

Conjugation with L-Glutamate for in vivo brain drug delivery

In vitro studies have shown that conjugation of a model compound [p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD)] with L-Glu can improve D-MOD permeation through the bovine brain microvessel endothelial cell monolayers (Sakaeda et al., 2000). The transport of this D-MOD-L-Glu conjugate is facilitated by the L-Glu transport system. In this paper, we evaluate the in vivo brain delivery of model compounds (i.e. D-MOD, p-nitro-D-phenylalanine (p-nitro-D-Phe), 5,7-dichlorokynurenic acid (DCKA) and D-kyotorphin) and their L-Glu conjugates. DCKA was also conjugated with L-Asp and L-Gln amino acids. The analgesic activities of D-kyotorphin and its L-Glu conjugate were also evaluated. The results showed that the brain-to-plasma concentration ratio of D-MOD-L-Glu was higher than the D-MOD alone; however, the plasma concentration of both compounds were the same. The plasma concentration of p-nitro-D-Phe-L-Glu conjugate was higher than the parent p-nitro-D-Phe; however, the brain-to-plasma concentration ratio of p-nitro-D-Phe was higher than its conjugate. On the other hand, both DCKA and DCKA conjugates have a low brain-to-plasma concentration ratio due to their inability to cross the blood-brain barrier (BBB). The L-Asp and L-Glu conjugates of DCKA have elevated plasma concentrations relative to DCKA; however, the DCKA-L-Gln conjugate has the same plasma concentration as DCKA. For D-kyotorphin, both the parent and the L-Glu conjugate showed similar analgesic activity. In conclusion, conjugation of a non-permeable drug with L-Glu may improve the drug's brain delivery; however, this improvement may depend on the physicochemical and receptor binding properties of the conjugate.