Manabu Murakami

Correlation of Glut-1 glucose transporter expression with [18F]FDG uptake in non-small cell lung cancer

Abstract. Positron emission tomography (PET) with [18F]2-fluoro-2-deoxy-D-glucose (FDG) may show negative results for bronchioloalveolar lung carcinoma. We investigated the correlation of Glut-1 glucose transporter expression with [18F]FDG uptake in non-small cell lung cancer. Thirty-two patients with 34 non-small cell lung cancers (7 bronchioloalveolar carcinomas, 23 non-bronchioloalveolar adenocarcinomas, 3 squamous cell carcinomas, and 1 adenosquamous cell carcinoma) were studied. Final diagnoses were established by histology (via thoracotomy) in all patients. [18F]FDG PET was performed 40xa0min after i.v. injection of 185xa0MBq [18F]FDG. For semi-quantitative analysis of [18F]FDG uptake, standardized uptake values (SUVs) were calculated. Glut-1 expression was studied in terms of the immunohistochemistry of paraffin sections using anti-Glut-1 antibody to determine the intensity (0–3) of Glut-1 immunoreactivity and percentage of the Glut-1-positive area. Of seven bronchioloalveolar carcinomas, six (85.7%) were negative for the expression of Glut-1, while only one (4.3%) of 23 non-bronchioloalveolar adenocarcinomas was negative (P<0.0001). The percentages of Glut-1-positive area, as well as the SUVs, were significantly lower in bronchioloalveolar carcinomas (n=7) (2.86%±7.56% and 1.25±0.75, respectively) than in non-bronchioloalveolar adenocarcinomas (n=23) (54.83%±25.64%, P<0.0001, and 3.94±1.93, P=0.001, respectively). The degree of cell differentiation correlated with the percentage of Glut-1-positive area and SUVs in adenocarcinoma of the lung. Correlations between SUVs and the intensity of Glut-1 immunoreactivity were also significant (intensities 0 and 1, n=11, SUV 1.47±0.63; intensities 2 and 3, n=23, SUV 4.78±2.13; P<0.0001). The percentage of Glut-1-positive area correlated significantly with SUVs (n=34, r=0.658, P<0.01). Overexpression of Glut-1 correlated with high [18F]FDG uptake. These findings suggest that Glut-1 expression is related to [18F]FDG uptake in non-small cell lung cancer. Glut-1 expression, as well as [18F]FDG uptake, correlated with the degree of cell differentiation in adenocarcinomas, and both Glut-1 expression and [18F]FDG uptake were significantly lower in bronchioloalveolar carcinomas than in non-bronchioloalveolar carcinomas.

Suppression of azoxymethane‐induced colon carcinogenesis in male F344 rats by mandarin juices rich in β‐cryptoxanthin and hesperidin

We have reported protective effects of dietary administration of a powder “CHRP” containing high amounts of β‐cryptoxanthin and hesperidin prepared from a Satsuma mandarin (Citrus unshiu Marc.) juice on azoxymethane (AOM)‐induced rat aberrant crypt foci through suppression of crypt cell proliferation and/or induction of detoxifying enzymes. In the present study, we investigated the modifying effects of a commercial Satsuma mandarin (Citrus unshiu Marc.) juice (MJ) and those of MJ2 and MJ5, which were prepared from MJ and are richer in β‐cryptoxanthin and hesperidin than MJ, on the occurrence of colonic tumors induced by AOM in male F344 rats. Rats were given 2 weekly s.c. injections of AOM (20 mg/kg body weight) to induce colonic neoplasms. They also received MJ, MJ2, or MJ5 as a drinking water at night for 36 weeks, starting 1 week after the last dosing of AOM. AOM exposure produced colonic adenocarcinoma with an incidence of 69% and a multiplicity of 0.76 ± 0.57/rat at week 38. MJ, MJ2, and MJ5 administration significantly reduced the frequency of colonic carcinoma [MJ: 35% (49% reduction), p < 0.02; MJ2: 20% (64% reduction), p = 0.0028; and MJ5: 15% (78% reduction), p < 0.00021] and multiplicity [MJ: 0.40 ± 0.58 (47% reduction), p < 0.05; MJ2: 0.25 ± 0.43 (67% reduction), p < 0.005; and MJ5: 0.15 ± 0.36 (80% reduction), p < 0.001]. Also, the numbers of cancer cells positive for proliferative cell nuclear antigen (PCNA) and cyclin D1 in colonic tumors were lowered by these treatments. In addition, treatment with MJ, MJ2, or MJ5 significantly increased apoptotic index in colonic adenocarcinoma. These findings might suggest effective chemopreventive ability of MJs, especially MJ5, in colon tumorigenesis. Int. J. Cancer 88:146–150, 2000.

Chemopreventive Effect of Bovine Lactoferrin on 4‐Nitroquinoline 1‐Oxide‐induced Tongue Carcinogenesis in Male F344 Rats

The modifying effects of dietary feeding of bovine lactoferrin (bLF) on tongue carcinogenesis initiated with 4‐nitroquinoline 1‐oxide (4‐NQO) were investigated in male F344 rats. The activities of phase II detoxifying enzymes, glutathione S‐transferase (GST) and quinone reductase (QR), polyamine content and ornithine decarboxylase (ODC) activity in the tongue were also examined for mechanistic analysis of possible modifying effects of bLF on carcinogenesis. At 7 weeks of age, all animals except those treated with bLF alone and untreated rats were given 20 ppm 4‐NQO in drinking water for 8 weeks to induce tongue neoplasms. Starting 7 days before 4‐NQO exposure, experimental groups were fed experimental diets containing bLF (0.2% and 2%) for 10 weeks (“initiation feeding”). Starting 1 week after the cessation of exposure to 4‐NQO, the other experimental groups given 4‐NQO and a basal diet were fed the experimental diets for 22 weeks (“postinitiation feeding”). At week 32, the incidence and multiplicity of tongue neoplasms in the “initiation feeding’ groups of 0.2% and 2% bLF and the “post‐initiation feeding” group of 0.2% bLF were lower than those of the 4‐NQO alone group, but without statistical significance. However, “post‐initiation feeding” of 2% bLF caused a significant reduction in the incidence (20% vs. 55%, P=0.02418) and multiplicity (0.25±0.54 vs. 0.70±0.71, P< 0.05) of tongue squamous cell carcinoma (by 64%, P=0.02418). bLF treatment elevated liver and tongue GST activities and liver QR activity. The “post‐initiation feeding with 2% bLF significantly decreased QR activity, proliferating cell nulcear antigen‐positive index and ODC activity in the tongue. In addition, feeding with bLF decreased tongue polyamine content. These results suggest that bLF, when given at the 2% dose level during the post‐initiation phase, exerts chemopreventive action against tongue tumorigenesis through modification of cell proliferation activity and/or the activities of detoxifying enzymes.

Inhibitory effect of RNAi on Japanese encephalitis virus replication in vitro and in vivo.

Flaviviruses include many insect‐mediated small viruses and still cause serious problems in the world. In humans, JEV can cause acute meningioencephalomyelitis, resulting in fatality rates of 5 to 40%. RNA‐interference (RNAi) as an antiviral mechanism was originally discovered in plants and then found in the specific suppression of gene expression of other organisms such as Caenorhabditis elegans, Drosophila and vertebrates. As JEV is an RNA virus, RNAi could be a reasonable approach for therapeutic purposes to use against Japanese encephalitis. In this study, we examined the effect of RNAi on JEV replication. Viral reproduction in Vero cells was decreased to 7.2% and 39.0% of control by the transfection of small interference RNAs, JCR and JN3R at 250 NM, respectively. Under the transfection of 5 μg/ml pJRi which produces stem‐loop RNAi, viral reproduction was decreased to about 10% of control. Western blot analysis indicated that RNAi inhibited the translation level. We used pJRi in the animal experiment. After the inoculation of viruses at 5× 103 PFU, pJRi at 1.0 and 5.0 μg/g was injected into mice i.p. JEV‐infected control mice (n=5) died within 15 days. pJRi (1.0 or 5.0 μg/g)‐medicated mice survived 40 or 80% at 15 days. The data clearly indicate that pJRi has highly potent inhibitory activity against JEV replication in vivo. The results in vivo and in vitro provide evidence that JEV replication was efficiently inhibited by RNAi and RNAis could be used as an antiviral drug against JEV infection.

Promotion or suppression of experimental metastasis of B16 melanoma cells after oral administration of lapachol.

Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] is a vitamin K antagonist with antitumor activity. The effect of lapachol on the experimental metastasis of murine B16BL6 melanoma cells was examined. A single oral administration of a high toxic dose of lapachol (80-100 mg/kg) 6 h before iv injection of tumor cells drastically promoted metastasis. This promotion of metastasis was also observed in T-cell-deficient mice and NK-suppressed mice. In vitro treatment of B16BL6 cells with lapachol promoted metastasis only slightly, indicating that lapachol promotes metastasis primarily by affecting host factors other than T cells and NK cells. A single oral administration of warfarin, the most commonly used vitamin K antagonist, 6 h before iv injection of tumor cells also drastically promoted the metastasis of B16BL6 cells. The promotion of metastasis by lapachol and warfarin was almost completely suppressed by preadministration of vitamin K3, indicating that the promotion of metastasis by lapachol was derived from vitamin K antagonism. Six hours after oral administration of lapachol or warfarin, the protein C level was reduced maximally, without elongation of prothrombin time. These observations suggest that a high toxic dose of lapachol promotes metastasis by inducing a hypercoagulable state as a result of vitamin K-dependent pathway inhibition. On the other hand, serial oral administration of low non-toxic doses of lapachol (5-20 mg/kg) weakly but significantly suppressed metastasis by an unknown mechanism, suggesting the possible use of lapachol as an anti-metastatic agent.

Involvement of protein kinase C in taxol-induced polyploidization in a cultured sarcoma cell line

Taxol was found to inhibit the proliferation and to induce the polyploidization of cultured methylcholanthrene-induced sarcoma cells (Meth-A cells). To investigate whether protein kinase C is involved in taxol-induced polyploidization, phorbol 12-myristate 13-acetate (PMA), which regulates the activity of protein kinase C, was used along with taxol to treat the cells. We found that PMA did not interfere with the proliferation and did not induce polyploidization by itself. However, at low concentration, taxol, which by itself did not induce polyploidization, clearly induced polyploidization in the presence of PMA. To explore the mechanism by which PMA potentiates polyploidization, the levels of the G1 checkpoint-related proteins cyclin E and cdk2, and those of the G2 checkpoint-related proteins cyclin B and cdc2 were determined by flow cytometry. We found that both G1 and G2 checkpoint-related proteins increased during the induction of polyploidization. To verify the relationship between protein kinase C and tubulin polymerization, flow cytometry was used to determine the total content of tubulin protein, and morphological observation was used to examine spindle organization. PMA did not affect the taxol-induced increase in tubulin protein, but markedly potentiated taxol-induced spindle disorganization. These findings suggest that protein kinase C plays an important role in regulating the induction of polyploidization in Meth-A cells.

RhoGDIβ lacking the N-terminal regulatory domain suppresses metastasis by promoting anoikis in v-src-transformed cells

Rho guanine nucleotide dissociation inhibitors (RhoGDIs) regulate the activity of Rho family GTPases. RhoGDIβ (LyGDI/GDID4/RhoGDI2) has two caspase cleavage sites after Asp19 and Asp55. The resulting cleavage products, ΔN(1–19)RhoGDIβ and ΔN(1–55)RhoGDIβ, are expressed in cells under conditions that activate caspases. ΔN(1–19)RhoGDIβ, which can inhibit GDP dissociation, is implicated in the process of apoptosis, whereas the physiological roles for ΔN(1–55)RhoGDIβ, which lacks the ability to inhibit GDP dissociation, are largely unknown. To explore the roles of ΔN(1–55)RhoGDIβ, we examined the phenotypes of v-src-transformed metastatic fibroblasts transfected with plasmids for expressing ΔN(1–55)RhoGDIβ. Although the expression of ΔN(1–55)RhoGDIβ had no effect on the rate of growth in vitro, it suppressed experimental metastasis and decreased the rate of growth in vivo. In addition, ΔN(1–55)RhoGDIβ-expressing cells had enhanced adhesion to fibronectin, laminin, and collagens but reduced retention in the lung after intravenous injection. Also, the expression of ΔN(1–55)RhoGDIβ promoted anoikis without affecting the levels of activated Rac1 or Cdc42. Furthermore, ΔN(1–55)RhoGDIβ did not affect the expression or phosphorylation of focal adhesion kinase, p44/p42 mitogen-activated protein kinases, or Akt1 before or after induction of anoikis. Thus, ΔN(1–55)RhoGDIβ appears to promote anoikis by undefined mechanisms, thereby suppressing metastasis in v-src-transformed fibroblasts.

Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells polyploidized by demecolcine

Abstract. The nuclear morphology of polyploidized cells was examined in V79 Chinese hamster cells polyploidized by demecolcine or K‐252a, inhibitors of spindle fibre formation and protein kinases, respectively. A variety of nuclear morphologies, including multinuclei, were observed in V79 cells polyploidized by demecolcine but not by K‐252a, which produced mononuclear cells. A lack of synchrony in the nuclear cycle was observed among nuclei in multinuclear polyploidized cells. Partial DNA fragmentation, defined as DNA fragmentation of a nucleus in a multinuclear cell, was detected using the TUNEL method in V79 cells polyploidized by demecolcine but not by K‐252a. Apoptosis occurred earlier in cell populations treated with demecolcine than in these treated with K‐252a once the drugs were removed from the medium, suggesting that polyploidized cells with separate nuclei tend to apoptose earlier than those with mononuclei.

Activation of Rac1 by Rho-guanine nucleotide dissociation inhibitor-β with defective isoprenyl-binding pocket

Rho‐guanine nucleotide dissociation inhibitor‐β (RhoGDIβ), a regulator for Rho GTPases, is implicated in cancer cell progression. We reported that C‐terminal truncated RhoGDIβ (ΔC(166–201)‐RhoGDIβ) promoted metastasis through activating Rac1 signaling pathway in ras‐transformed fibroblast cells. To better understand the mechanism of Rac1 activation by ΔC(166–201)‐RhoGDIβ during metastasis, the amount of GTP‐bound Rac1 was measured as the activation level of Rac1 in cells expressing various mutant RhoGDIβ with sequential C‐terminal deletions. Three C‐terminal hydrophobic amino acid residues (Trp191, Leu193, and Ile195) supposed to interact with isoprenyl groups of Rac1, was indispensable for a proper regulation of Rac1 activation/inhibition. Deletion of this region led RhoGDIβ to continuously associate with GTP‐bound Rac1, provoking constitutive activation of Rac1. Thus, impaired interaction of RhoGDIβ with Rac1 isoprenyl groups possibly makes RhoGDIβ function as a positive regulator for Rac1 during metastasis.

Morphology and Gene Analysis of Hybrids Between Two Congeneric Sea Stars With Different Modes of Development

The sea star Astropecten scoparius has feeding bipinnarian larvae, whereas its congener Astropecten latespinosus has nonfeeding barrel-shaped larvae. To investigate evolutionary changes in the development of asteroids, we performed reciprocal crosses between these two species with different larval forms. In the cross between A. scoparius eggs and A. latespinosus sperm, embryos developed into bipinnaria-like larvae. The larvae exhibited either a functional digestive system (a maternal feature) or a nonfunctional digestive system with the tip of the archenteron not connected to the stomodeum (a paternal characteristic). However, in the reciprocal cross between A. latespinosus eggs and A. scoparius sperm, barrel-shaped larvae resembling those of A. latespinosus were produced, in addition to bipinnaria-like larvae, some with functional digestive systems and some with nonfunctional ones. Juveniles were produced from all types of crosses. 18S rDNA was used as a gene marker in cycle sequencing analysis to investigate the genetic features of these juveniles. The sequences of juveniles from bipinnaria-like larvae showed double-peak nucleotide signals, indicating a biparental genome. On the other hand, juveniles from barrel-shaped larvae from A. latespinosus eggs and A. scoparius sperm showed the same sequence as A. latespinosus juveniles. This suggests that bipinnaria-like larvae of both crosses are always hybrids, whereas barrel-shaped larvae develop parthenogenetically.