[Show abstract][Hide abstract] ABSTRACT: Improvement of transduction and augmentation of cytotoxicity are crucial for adenoviruses (Ad)-mediated gene therapy for cancer. Down-regulated expression of type 5 Ad (Ad5) receptors on human tumors hampered Ad-mediated transduction. Furthermore, a role of the p53 pathways in cytotoxicity mediated by replication-competent Ad remained uncharacterized.
We constructed replication-competent Ad5 of which the E1 region genes were activated by a transcriptional regulatory region of the midkine or the survivin gene, which is expressed preferentially in human tumors. We also prepared replication-competent Ad5 which were regulated by the same region but had a fiber-knob region derived from serotype 35 (AdF35). We examined the cytotoxicity of these Ad and a possible combinatory use of the replication-competent AdF35 and Ad5 expressing the wild-type p53 gene (Ad5/p53) in esophageal carcinoma cells. Expression levels of molecules involved in cell death, anti-tumor effects in vivo and production of viral progenies were also investigated.
Replication-competent AdF35 in general achieved greater cytotoxic effects to esophageal carcinoma cells than the corresponding replication-competent Ad5. Infection with the AdF35 induced cleavages of caspases and increased sub-G1 fractions, but did not activate the autophagy pathway. Transduction with Ad5/p53 in combination with the replication-competent AdF35 further enhanced the cytotoxicity in a synergistic manner. We also demonstrated the combinatory effects in an animal model. Transduction with Ad5/p53 however suppressed production of replication-competent AdF35 progenies, but the combination augmented Ad5/p53-mediated p53 expression levels and the downstream pathways.
Combination of replication-competent AdF35 and Ad5/p53 achieved synergistic cytotoxicity due to enhanced p53-mediated apoptotic pathways.
[Show abstract][Hide abstract] ABSTRACT: Previous studies investigating cancer cells cultured at acidic pH have shown that the expression level of ~700 genes were more than two-fold higher than those of the cells cultured in alkaline medium at pH 7.5. The aim of the present study was to confirm whether these acidosis-induced genes are expressed in human cancer tissues. Therefore, 7 genes were selected from our previous study, which encoded interleukin 32 (IL-32), lysosomal H(+) transporting ATPase, V0 subunit d2 (ATP6V0D2), tumor necrosis factor receptor superfamily, member 9 (TNFRSF9), amphiregulin, schwannoma-derived growth factor (AREG), v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3), PRR5-ARHGAP8 (LOC553158) and dimethylglycine dehydrogenase (DMGDH), and their expression was examined in human clinical specimens from patients with cancer. In addition, the expression of the gene encoding manganese superoxide dismutase (MnSOD) was examined. The specimens from patients with colon, stomach and renal cancer showed increased MnSOD, IL-32, and TNFRSF9 transcripts compared to those from non-tumorous regions of the same patients. Notably, an elevated expression of ATP6V0D2 was found in the specimens from patients with stomach cancer, whereas the expression was decreased in those from patients with colon and renal cancer. The expression of LOC553158 was upregulated in colon and stomach cancer specimens. These results indicate that the investigation of gene expression under acidic conditions is useful for the development of novel cancer markers and/or chemotherapeutic targets.
Full-text · Article · Nov 2014 · Molecular and Clinical Oncology
[Show abstract][Hide abstract] ABSTRACT: Interferons (IFNs) have been tested for the therapeutic effects in various types of malignancy, but mechanisms of the anti-tumors effects and the differential biological activities among IFN members are dependent on respective cell types. In this study, we examined growth inhibitory activities of type I and III IFNs on 5 kinds of human mesothelioma cells bearing wild-type p53 gene, and showed that type I IFNs but not type III IFNs decreased the cell viabilities. Moreover, growth inhibitory activities and up-regulated expression levels of the major histocompatibility complexes class I antigens were greater with IFN-β than with IFN-α treatments. Cell cycle analyses demonstrated that type I IFNs increased S- and G2/M-phase populations, and subsequently sub-G1-phase fractions. The cell cycle changes were also greater with IFN-β than IFN-α treatments, and these data collectively showed that IFN-β had stronger biological activities than IFN-α in mesothelioma. Type I IFNs-treated cells increased p53 expression and the phosphorylation levels, and activated apoptotic pathways. A combinatory use of IFN-β and cisplatin or pemetrexed, both of which are the current first-line chemotherapeutic agents for mesothelioma, produced synergistic anti-tumor effects, which were also evidenced by increased sub-G1-phase fractions. These data demonstrated firstly to our knowledge that IFN-β produced synergistic anti-tumor effects with cisplatin or pemetrexed on mesothelioma through up-regulated p53 expression.
[Show abstract][Hide abstract] ABSTRACT: Many previous studies in animal models and clinical investigations have suggested that statins are useful chemotherapeutics against rheumatoid arthritis, whereas in vitro experiments using synovial cell lines showed no significant effect of statins on cell proliferation until now. Since synovial fluid in rheumatoid joint knee was found to be acidic, we examined the effect of statins on human synovial sarcoma cell line SW982 cells in acidic medium. Statins suppressed the proliferation of SW982 cells at pH6.7, while the suppression was very weak in pH7.5 medium. It was shown that the suppression was caused by the decrease in geranylgeranyl diphosphate, suggesting that a geranylgeranylated protein(s) has an essential role in cell proliferation of SW982 cells under acidic conditions. Our present data clearly implied that statins had high efficacy against SW982 cells in acidic medium whose pH is close to that of rheumatoid arthritis loci in patients. These results lead us to anticipate that screening of chemicals having high therapeutic efficacy in acidic medium promotes the development of new microenvironment-dependent medicines for chemotherapies against rheumatoid arthritis.
No preview · Article · Jun 2013 · International immunopharmacology
[Show abstract][Hide abstract] ABSTRACT: We examined anti-tumor effects of zoledronic acid (ZOL), one of the bisphosphonates agents clinically used for preventing loss of bone mass, on human mesothelioma cells bearing the wild-type p53 gene. ZOL-treated cells showed activation of caspase-3/7, -8 and -9, and increased sub-G1 phase fractions. A combinatory use of ZOL and cisplatin (CDDP), one of the first-line anti-cancer agents for mesothelioma, synergistically or additively produced the cytotoxicity on mesothelioma cells. Moreover, the combination achieved greater anti-tumor effects on mesothelioma developed in the pleural cavity than administration of either ZOL or CDDP alone. ZOL-treated cells as well as CDDP-treated cells induced p53 phosphorylation at Ser 15, a marker of p53 activation, and up-regulated p53 protein expression levels. Down-regulation of p53 levels with siRNA however did not influence the ZOL-mediated cytotoxicity but negated the combinatory effects by ZOL and CDDP. In addition, ZOL treatments augmented cytotoxicity of adenoviruses expressing the p53 gene on mesothelioma. These data demonstrated that ZOL-mediated augmentation of p53, which was not linked with ZOL-induced cytotoxicity, played a role in the combinatory effects with a p53 up-regulating agent, and suggests a possible clinical use of ZOL to mesothelioma with anti-cancer agents.
[Show abstract][Hide abstract] ABSTRACT: Although it is now well known that some diseased areas, such as cancer nests, inflammation loci, and infarction areas, are acidified, little is known about cellular signal transduction, gene expression, and cellular functions under acidic conditions. Our group showed that different signal proteins were activated under acidic conditions compared with those observed in a typical medium of around pH 7.4 that has been used until now. Investigations of gene expression under acidic conditions may be crucial to our understanding of signal transduction in acidic diseased areas. In this study, we investigated gene expression in mesothelioma cells cultured at an acidic pH using a DNA microarray technique. After 24 h culture at pH 6.7, expressions of 379 genes were increased more than twofold compared with those in cells cultured at pH 7.5. Genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors numbered 35, 32, and 17 among the 379 genes, respectively. Since the functions of 78 genes are unknown, it can be argued that cells may have other genes for signaling under acidic conditions. The expressions of 37 of the 379 genes were observed to increase after as little as 2 h. After 24 h culture at pH 6.7, expressions of 412 genes were repressed more than twofold compared with those in cells cultured at pH 7.5, and the 412 genes contained 35, 76, and 7 genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors, respectively. These results suggest that the signal pathways in acidic diseased areas are different, at least in part, from those examined with cells cultured at a pH of around 7.4.
[Show abstract][Hide abstract] ABSTRACT: Besides amino acid decarboxylation, the ADP biosynthetic pathway was reported to enhance survival under extremely acidic conditions in Escherichia coli (Sun et al., J. Bacteriol. 193∶ 3072-3077, 2011). E. coli has two pathways for ATP synthesis from ADP: glycolysis and oxidative phosphorylation. We found in this study that the deletion of the F(1)Fo-ATPase, which catalyzes the synthesis of ATP from ADP and inorganic phosphate using the electro-chemical gradient of protons generated by respiration in E. coli, decreased the survival at pH 2.5. A mutant deficient in hemA encoding the glutamyl tRNA reductase, which synthesizes glutamate 1-semialdehyde also showed the decreased survival of E. coli at pH 2.5. Glutamate 1-semialdehyde is a precursor of heme synthesis that is an essential component of the respiratory chain. The ATP content decreased rapidly at pH 2.5 in these mutants as compared with that of their parent strain. The internal pH was lowered by the deletion of these genes at pH 2.5. These results suggest that respiration and the F(1)Fo-ATPase are still working at pH 2.5 to enhance the survival under such extremely acidic conditions.
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Genetic characterization of malignant mesothelioma shows a homozygous deletion of the INK4A/ARF locus, which results in inactivation of the p53 pathways.
We examined possible antitumor effects of adenoviruses with a deletion of the E1B-55kD gene (Ad-delE1B55) on mesothelioma and investigated combinatory actions with the first-line chemotherapeutic agents.
Ad-delE1B55 produced cytotoxicity on mesothelioma cells, which was associated with p53 phosphorylation, pRb dephosphorylation, and cleavage of caspases. Ad-delE1B55-infected cells displayed hyperploidy at the cell-cycle analysis and showed enlarged nuclear configurations. Combination of Ad-delE1B55 plus cisplatin or pemetrexed produced antitumor effects in vitro. Furthermore, Ad-delE1B55 and cisplatin showed combinatory effects in an orthotopic animal model.
Cell death caused by Ad-delE1B55 is attributable to cell-cycle arrest at M-phase checkpoint followed by activated apoptotic pathways, and combination of the first-line chemotherapeutic agents and the oncolytic adenovirus is a potential therapeutic for mesothelioma.
Full-text · Article · Dec 2012 · Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer
[Show abstract][Hide abstract] ABSTRACT: We examined whether zoledronic acid (ZOL), the third generation of bisphosphonates, produced cytotoxic effects on human mesothelioma cells in vitro and in vivo, and investigated a possible involvement of p53, Ras, and extracellular signal-regulated kinase1/2 (ERK1/2) pathways.
Cytotoxicity and cell cycles were assessed with a colorimetric assay and flow cytometry, respectively. Expression levels of apoptosis-linked proteins and prenylation of small guanine-nucleotide-binding regulatory proteins were tested with p53-small interfering RNA, an ERK kinase1/2-inhibitor, and prenyl alcohols. The antitumor activity was examined in an orthotopic animal model.
ZOL treatments suppressed growth of mesothelioma cells bearing the wild-type p53 gene through apoptosis induction accompanied by activation of caspases, or S-phase arrest by up-regulated cyclin A and B1. ZOL induced p53 phosphorylation and subsequent activation of the downstream pathways. Down-regulated p53 expression with the small interfering RNA, however, showed that both apoptosis and S-phase arrest were irrelevant to the p53 activation. Geranylgeranyl but not farnesyl pyrophosphate inhibited ZOL-induced apoptosis and S-phase arrest, and the geranylgeraniol supplement decreased ZOL-mediated Rap1A but not Ras unprenylation. Inhibition of ERK1/2 pathways suppressed ZOL-induced apoptosis but not S-phase arrest. We further demonstrated that ZOL, administrated intrapleurally, inhibited the tumor growth in the pleural cavity.
These data indicate that ZOL induces apoptosis or S-phase arrest, both of which are independent of p53 activation and Ras unprenylation, and suggest that ZOL is a possible therapeutic agent to mesothelioma partly through non-Ras- and ERK1/2-mediated pathways.
Full-text · Article · Apr 2012 · Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer
[Show abstract][Hide abstract] ABSTRACT: The inside of the human body is maintained within a narrow pH range around 7.4, but cancer nests are acidified, mainly due to insufficient blood circulation and impaired respiration. The activities of all enzymes are strictly dependent on pH, and hence many metabolic processes catalyzed by the pH-dependent enzymes decline as pH decreases in such acidified diseased areas, leading us to argue that the efficacy of inhibitors varies under different pH conditions. However, there have been few reports to examine inhibitor efficacy in acidic conditions. The main reason for this insufficient investigation may be that no reliable method for the screening of such medicines has been developed. Our group has invented a useful method for this purpose, and has found that several medicines have high efficacy as anti-cancer reagents under acidic conditions. In this article, we introduce this novel method applied for a patent and recent findings that encourage us to use this simple method for developing new anti-cancer drugs.
No preview · Article · Dec 2011 · Recent Patents on Biomedical Engineering
[Show abstract][Hide abstract] ABSTRACT: ATP participates in many cellular metabolic processes as a major substrate to supply energy. Many systems for acidic resistance
(AR) under extremely acidic conditions have been reported, but the role of ATP has not been examined. To clarify whether or
not ATP is necessary for the AR in Escherichia coli, the AR of mutants deficient in genes for ATP biosynthesis was investigated in this study. The deletion of purA or purB, each of which encodes enzymes to produce AMP from inosinate (IMP), markedly decreased the AR. The content of ATP in these
mutants decreased rapidly at pH 2.5 compared to that of the wild type. The AR was again decreased significantly by the mutation
of adk, which encoded an enzyme to produce ADP from AMP. The DNA damage in the purA and purB mutants was higher than that in the wild type. These results demonstrated that metabolic processes that require ATP participate
in survival under extremely acidic conditions, and that one such system is the ATP-dependent DNA repair system.
Full-text · Article · Jun 2011 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: The roles of OmpC and OmpF in acidic resistance (AR) were examined. When ompC and ompF were deleted, AR was decreased. The decreased level of AR seen in the mutant that was deficient in ompC and ompF was elevated by the addition of glutamate, but not by the addition of arginine or lysine. The expression levels of adiA and cadB were diminished by the deletion of ompC and ompF, and the conversion of arginine to agmatine and lysine to cadaverine by intact cells were reduced in the mutant. The expression of gadA/gadB was not affected by the deletion of ompC and ompF. These results suggest that the transport of arginine, lysine, and their decarboxylated products through OmpC and/or OmpF is essential for the survival of Escherichia coli cells under extremely acidic conditions.
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli has three major K(+) uptake systems, Trk, Kup, and Kdp, which have been studied extensively at near neutral pH. However, the function of these transporters under acidic conditions is not well understood, although growth and survival under acidic conditions are important for bacterial pathogenesis. In this study, we examined the expression and activity of Kdp under acidic conditions and found that the transport activity of Kdp is decreased at low pH and that the expression of kdp is regulated by the internal K(+) concentration in a pH-independent manner. Consequently, the low activity of Kdp was compensated for by the induction of its elevated expression by low K(+) accumulation via Kdp at acidic pH.
[Show abstract][Hide abstract] ABSTRACT: In tumor cell masses, the extracellular pH decreases below 6.5. The effect of external acidic pH on the efficacy of 24 chemical compounds including molecular-targeted inhibitors and anti-tumor reagents was investigated in human cancer cells. Lovastatin showed no cytotoxicity in mesothelioma or pancreatic carcinoma cells at concentrations up to 10 μM and pH around 7.4, but 10 μM lovastatin decreased the survival of these cells below 40% at acidic pH. Lovastatin inhibits HMG-CoA reductase, resulting in a decrease in the levels of cholesterol and prenylated proteins. An inhibitor of the former pathway showed pH-independent cytotoxic activity, whereas an inhibitor of the latter pathway had stronger activity at acidic pH. The inhibitory efficacy of cantharidin also increased at acidic pH. On the other hands, no pH dependency or slightly impaired efficacy at low pH conditions was observed in other 20 reagents, and especially, the activity of aphidicolin was suppressed under acidic conditions. These results suggested that screening under acidic conditions would be useful for developing new chemotherapeutic reagents.
[Show abstract][Hide abstract] ABSTRACT: The first enantioselective total synthesis of tangutorine has been achieved, wherein a Pd-catalyzed asymmetric allylic amination using a chiral diaminophosphine oxide (DIAPHOX) preligand was the key step.
[Show abstract][Hide abstract] ABSTRACT: Cellular signal transduction is initiated by the binding of extracellular ligands to membrane receptors. Receptors are often expressed in excess, and cells are activated when a small number of receptors bind ligands. Intracellular signal proteins are activated at a high level soon after ligand binding, and the activation level decreases in a negative feedback manner without ligand clearance. Why are excess receptors required? What is the physiological significance of the negative feedback regulation?
To answer these questions, we developed a Monte Carlo simulation program to kinetically analyze signal pathways using the model in which ligands are bound to receptors and then membrane complexes with other membrane proteins are formed. Our simulation results showed that excess receptors are not required for cell activation when the dissociation constant (Kd) of the ligand-receptor complex is 10-10 M or less. However, such low Kd values cause delayed signal shutdown after ligand clearance from the extracellular space. In contrast, when the Kd was 10-8 M and the ligand level was less than 1 muM, excess receptors were required for prompt signal propagation and rapid signal cessation after ligand clearance. An initial increase in active cytosolic signal proteins to a high level is required for rapid activation of cellular signal pathways, and a low level of active signal proteins is essential for the rapid shutdown of signal pathways after ligand clearance.
The present kinetic analysis revealed that excess receptors and negative feedback regulation promote activation and cessation of signal transduction with a low amount of extracellular ligand.
Full-text · Article · Feb 2009 · Cell Communication and Signaling
[Show abstract][Hide abstract] ABSTRACT: The major histone-like Escherichia coli protein, HU, is composed of alpha and beta subunits respectively encoded by hupA and hupB in Escherichia coli. A mutant deficient in both hupA and hupB grew at a slightly slower rate than the wild type at pH 7.5. Growth of the mutant diminished with a decrease in pH, and no growth was observed at pH 4.6. Mutants of either hupA or hupB grew at all pH levels tested. The arginine-dependent survival at pH 2.5 was diminished approximately 60-fold by the deletion of both hupA and hupB, whereas the survival was slightly affected by the deletion of either hupA or hupB. The mRNA levels of adiA and adiC, which respectively encode arginine decarboxylase and arginine/agmatine antiporter, were low in the mutant deficient in both hupA and hupB. The deletion of both hupA and hupB had little effect on survival at pH 2.5 in the presence of glutamate or lysine, and expression of the genes for glutamate and lysine decarboxylases was not impaired by the deletion of the HU genes. These results suggest that HU regulates expression of the specific set of genes required for growth and survival in acidic environments.
No preview · Article · Feb 2009 · Current Microbiology
[Show abstract][Hide abstract] ABSTRACT: In solid tumor and inflammation loci, low pH conditions have been observed as a consequence of either a lack of sufficient vascularization or excess activity of tumor cells, and T cells have been reported to infiltrate tumors and inflammation sites. However, it remains unclear how extracellular acidic environments affect immune cell function. A previous report proposed that a different signal transduction cascade might occur under low pH conditions in Jurkat T cells (Fukamachi T, Saito H, Kakegawa T, Kobayashi H. Different proteins are phosphorylated under acidic environments in Jurkat cells. Immunol Lett 2002;82:155-8). In this study, we investigated the protein phosphotyrosine level in Jurkat and Jurkat mutant cells under different pH conditions. The ZAP-70 phosphorylation level increased under acidic environments. P38 MAPK was more activated at acidic pH. The level of active p38 was low in mutant P116 deficient in ZAP-70, and interestingly the level remained consistently low at all pH values tested. The activation of ERK was not stimulated at low pH. These results suggest that extracellular low pH stimulates or enhances TCR signaling via ZAP-70 and p38.
No preview · Article · Feb 2008 · Immunology Letters
[Show abstract][Hide abstract] ABSTRACT: In vertebrates, mRNAs containing a 5'-terminal oligopyrimidine (TOP) motif are coordinately post-transcriptionally regulated. Binding of specific proteins to this element has been proposed to downregulate expression of TOP mRNAs at the level of translational initiation. We previously reported that rapamycin induces binding activity to the TOP element of ribosomal protein (r-protein) L32 mRNA. In this study, we adapt DEAE-cellulose/oligo dT-cellulose tandem column chromatography to purify TOP element-binding proteins from bovine submaxillary lymph nodes (SLN). We also show by northwestern blot analysis that two proteins of molecular weight 47kDa (47BP) and 43kDa (43BP) specifically bind to a (32)P-labeled riboprobe containing TOP regulatory element of the r-protein L32. Microsequencing of the purified 47BP revealed an internal sequence of 15 amino acids identical to the consensus sequence of the 2x RBD-Gly family. Western blot analysis of the cytoplasm fractions using an AUF1 antibody revealed that these two proteins are p45 AUF1 and p42 AUF1. Increases of the four isoforms of AUF1 protein were observed in 100,000g supernatant fractions of rapamycin-administered rat SLN. Furthermore, decreases of p45 AUF1 and p42 and/or p40 AUF1 were observed in the polysomal fractions of BJAB cells in which translation of TOP mRNAs was selectively suppressed by rapamycin treatment. Taken together, these results suggest that AUF1 is a TOP mRNA-binding protein that may participate in the translational suppression of TOP mRNAs resulting from rapamycin treatment.
Full-text · Article · Oct 2007 · Archives of Biochemistry and Biophysics