PEG-appended beta-(1-->3)-D-glucan schizophyllan to deliver antisense-oligonucleotides with avoiding lysosomal degradation

Schizophyllan is a natural beta-(1-->3)-d-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO). As we already reported, when a homo-polynucleotide [e.g., poly(dA) or poly(C)] is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications for this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enabled us to introduce PEG only to the side chain of schizophyllan. This technique consisted of periodate oxidation of the glucose side chain and subsequent reaction between methoxypolyethylene glycol amine and the formyl terminate, followed by reduction with NaBH4. Subsequently, we made a complex from PEG-appended schizophyllan and an AS ODN sequence, and carried out an in vitro antisense assay, administrating the AS ODN complex to depress A375 c-myb mRNA of A375 melanoma cell lines. The PEG-SPG/AS ODN complex showed more enhanced antisnese effect than naked AS ODN dose, i.e., the same level as that of RGD-appended SPG. Here, the RGD system has been shown one on the most effective AS ODN carrier (Science 261 (1993) 1004-1012). When we added nigericin to the assay system, the antisense effect was not affected in the PEG-SPG system, on the other hand, it was almost eliminated in the RGD system. Nigericin is well known to interrupt transport from endosome to lysosome. Therefore, the difference between the PEG and RGD complexes indicates that, in the PEG system, AS ODN was able to escape from lysosomal degradation. The present work has thus proposed a new strategy to delivery AS ODN using schizophyllan as a new carrier.     

Mechanical properties and bond strength of silicone-based resilient denture liners

To evaluate the ease of manipulation and durability of 11 commercially available silicone-based resilient denture liners, extrusion force, hardness, weight change, and bond strength were determined. Extrusion force from the cartridge of each material ranged from 0.25 to 1.26 MPa at an extrusion rate of 1 cm/min. Durometer hardness, after set materials were stored in distilled water at 37 degrees C for one day, ranged from A5.9 to A47.7, and after four weeks their values increased by 4.0 to 275%. Bond strength ranged from 1.01 to 2.88 MPa after set materials were stored in distilled water at 37 degrees C for one day, but decreased to 0.59 to 1.99 MPa after 10,000 thermal cycles. These results suggested that except for one material, the rest of the evaluated materials exhibited good handling properties--for example, mixing and spreading of material can be done easily. However, some materials exhibited inadequate durability for clinical service, because hardness increased during storage and/or bond strength decreased after thermal cycling.     

Identification of DKC1 gene mutations in Japanese patients with X-linked dyskeratosis congenita

Dyskeratosis congenita (DC) is a rare inherited multisystem disorder characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leucoplakia. X-linked recessive inheritances are recognized in approximately 40% of the patients. DKC1 has been identified as the gene responsible for X-linked DC, and genetic analyses have been performed in a worldwide study. Here, we performed genetic analysis of five Japanese patients with presumed X-linked DC, and identified four mutations in the DKC1 gene, including two novel missense mutations (Q31K and T357A). Such genetic analysis is useful for the definite diagnosis and genetic counselling of patients.     

Usefulness of plasma brain-type natriuretic peptide level to differentiate left ventricular diastolic dysfunction from preserved diastolic function in patients with systolic dysfunction

The extent of left ventricular (LV) diastolic dysfunction is related to the finding that some patients with cardiomegaly due to LV systolic dysfunction have good exercise tolerance, although others have limited tolerance. A brain-type natriuretic peptide level of >/=104 pg/ml reliably enables the detection of relatively worse LV diastolic function in patients with systolic dysfunction, and this value may provide clinically useful information for the management of patients with cardiomegaly.     

Gefitinib ("Iressa", ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, reverses breast cancer resistance protein/ABCG2-mediated drug resistance

Gefitinib ("Iressa", ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K(i) value was 1.01 +/- 0.09 micromol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.     

Disruption of the inhibitor of apoptosis protein survivin sensitizes Bcr-abl-positive cells to STI571-induced apoptosis

The Bcr-abl oncogene induces hematopoietic cell transformation and protects cells from apoptosis; however, the mechanisms whereby Bcr-abl blocks apoptosis are poorly defined. We examined whether the inhibitor of apoptosis protein (IAP) family, in particular survivin, are regulated by Bcr-abl. Overexpression of Bcr-abl in Mo7e or BaF3 hematopoietic cells elevated survivin mRNA and protein concomitant with a 4-fold increase in survivin promoter activity. The region of the survivin promoter responding to Bcr-abl was narrowed down to a 116 bp fragment between nucleotides -1,194 and -1,078. The IAP family member IAP-like protein-2 was also up-regulated by Bcr-abl. Disruption of Bcr-abl in Bcr-abl-transduced BaF3 cells by small interfering RNA resulted in 3- to 4-fold reduction in survivin protein confirming the link between Bcr-abl and survivin. Survivin disruption in Bcr-abl-transduced Mo7e cells, or in K562 cells that endogenously express Bcr-abl, by transfection with dominant-negative or antisense survivin constructs promoted apoptosis induced by the Bcr-abl tyrosine kinase inhibitor STI571, which was accompanied by caspase-dependent cleavage of Bcr-abl, mitochondrial membrane potential disruption, and enhanced mitochondrial cytochrome c release. Although ectopic survivin protected K562 cells from apoptosis induced by STI571, it did not protect cells from apoptosis induced either by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of TRAIL plus Hemin. Our results identify a new signal pathway downstream of Bcr-abl, in addition to the Bcl-2 family involved in the antiapoptotic effects of Bcr-abl, and suggest that anti-survivin therapy may have utility in patients with chronic myelogenous leukemia.     

HER2 overexpression increases sensitivity to gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, through inhibition of HER2/HER3 heterodimer formation in lung cancer cells

Gefitinib (Iressa), an epidermal growth factor receptor targeting drug, has been clinically useful for the treatment of patients with non-small cell lung cancer (NSCLC). Gefitinib is currently being applied in clinical studies as either a monotherapy, or as part of a combination therapy against prostate, head and neck, gastric, breast, and colorectal tumors. However, success rates vary between different tumor types, and thus it is important to understand which molecular target(s) are responsible for limiting the therapeutic efficacy of the drug. In this study, we ask whether expression of HER2 affects sensitivity to gefitinib in human lung cancer cells. We established two clones, LK2/HER2-32 and LK2/HER2-57, by transfecting HER2 cDNA into LK2, a NSCLC line with a low expression level of HER2. We observed no mutations in exons 18, 19, and 21 of EGFR gene in LK2, LK2/mock- and two HER2-trasfectants when we observed in-frame deletion mutations (E746-A750) adjacent to K745 in a gefitinib-sensitive NSCLC cell line, PC9. These LK2/HER2-32 and LK2/HER2-57 were much more sensitive to the cytotoxic effects of gefitinib than the parental LK2 lines. Treatment with 0.5 to 1 micromol/L gefitinib specifically blocked Akt activation in both HER2-transfectant lines, but not in the parental LK2 cells. Extracellular signal-regulated kinase-1/2 activation, however, was not blocked by gefitinib up to 10 micromol/L in either the parent or transfectant lines. Gefitinib was also shown to induce cell cycle arrest in the G1-S phase, and an accompanying increase of p27Kip1 was observed. LK2/HER2 transfectants showed constitutive formation of HER2/HER3 heterodimer, which were seen to associate with a regulatory subunit of phosphoinositide-3-kinase, p85alpha, when active. Treatment of LK2/HER2 cells with gefitinib markedly decreased the formation of HER2/HER3 heterodimers, HER3 basal phosphorylation, and the association of p85alpha with HER3. This study is the first to show that under basal growth conditions, HER2 sensitizes low-EGFR NSCLC cell lines to growth inhibition by gefitinib.     

RM2 antigen (beta1,4-GalNAc-disialyl-Lc4) as a new marker for prostate cancer

Although prostate-specific antigen (PSA) has been widely used for early detection of prostate cancer, PSA has problems with specificity and prediction of pathological stage. Therefore, a new marker for prostate cancer is urgently required. We examined expression of a novel carbohydrate antigen, beta1,4-GalNAc-disialyl-Lc(4), defined by the monoclonal antibody RM2, in prostate cancer using 75 cases of radical prostatectomy specimens. RM2 immunoreactivity was negative to weak in all benign glands, and weak to moderate in high-grade prostatic intraepithelial neoplasia. In prostatic adenocarcinoma, RM2 immunoreactivity was negative to weak (lower expression) in 20 cases, and moderate to strong (higher expression) in 55 cases. A clear difference of RM2 expression level was observed between Gleason patterns 3 and >/=4. Higher expression of RM2 antigen was significantly associated with primary Gleason pattern >/=4, high Gleason score (>/=8), larger tumor volume and advanced tumor stage. Furthermore, 5-year PSA failure-free survival was significantly lower in the higher expression group. However, no significant relationship was observed between RM2 expression level and preoperative serum PSA. Western blot analysis in prostate cancer cell lines PC3 and LNCap revealed that major 49-kDa and minor 39-kDa glycoproteins were common to both cells, but there was an increase of 59- and 125-kDa glycoproteins unique to LNCap and an increase of 88- and 98-kDa glycoproteins unique to PC3. RM2 antigen is a new histological marker for prostate cancer that may reflect the Gleason grading system. Identification of the glycoproteins carrying the RM2 antigen will provide new insights into the properties of prostate cancer.     

Multidetector row computed tomography for diagnosing intraductal extension of breast carcinoma

OBJECTIVES: Several reports supported the association of higher ipsilateral breast tumor recurrence rates with positive or intermediate margins compared with negative pathologic margins. Precise evaluation of intraductal component and adequate surgical margin are important factors affecting the tumor recurrence after breast conserving surgery. Numerous studies have reported the utility of magnetic resonance imaging for diagnosing developing intraductal extension of breast cancer, but few have investigated multidetector-row computed tomography (MD-CT). The present study evaluated the clinical utility of MD-CT for detecting intraductal extension of breast carcinoma, and analyzed clinical parameters affecting the detection of intraductal extension under MD-CT. METHODS: Subjects comprised 44 patients grouped into three categories according to degree of intraductal extension of the main tumor under MD-CT (Intraductal spread grade 1 approximately 3: IDS 1 approximately 3). Tumors were also categorized histopathologically (p-IDS 0 approximately 3), and CT-pathological correlations were examined retrospectively. Clinical parameters were evaluated to determine the affect on detection of intraductal components. RESULTS: MD-CT detected 44 breast lesions (100%). Sensitivity for detection of intraductal component was 81.2%, specificity was 67.8%, and accuracy was 72.7%. Regarding extent of intraductal components, significant correlations were found between histopathological and MD-CT findings. A strong correlation was found in postmenopausal women between T2 tumor and high histological grade. CONCLUSIONS: MD-CT findings of intraductal extension from breast carcinoma correlate with histological degree of intraductal extension, and MD-CT may be useful for preoperative assessment of breast-conserving surgery, particularly for postmenopausal women with histological high nuclear grade and T2 tumor.