Monotherapy with a novel intervenolin derivative, AS-1934, is an effective treatment for Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori (H. pylori) infection causes various gastrointestinal diseases including gastric cancer. Hence, eradication of this infection could prevent these diseases. The most popular first-line treatment protocol to eradicate H. pylori is termed "triple therapy" and consists of a proton pump inhibitor (PPI), clarithromycin, and amoxicillin or metronidazole. However, the antibiotics used to treat H. pylori infection are hindered by the antibiotics-resistant bacteria and by their antimicrobial activity against intestinal bacteria, leading to side effects. Therefore, an alternative treatment with fewer adverse side effects is urgently required to improve the overall eradication rate of H. pylori. OBJECTIVE: The aim of this study was to assess the effectiveness and mechanism of action of an antitumor agent, intervenolin, and its derivatives as an agent for the treatment of H. pylori infection. RESULTS: We demonstrate that intervenolin, and its derivatives showed selective anti-H. pylori activity, including antibiotic-resistant strains, without any effect on intestinal bacteria. We showed that dihydroorotate dehydrogenase, a key enzyme for de novo pyrimidine biosynthesis, is a target and treatment with intervenolin or its derivatives decreased the protein and mRNA levels of H. pylori urease, which protects H. pylori against acidic conditions in the stomach. Using a mouse model of H. pylori infection, oral monotherapy with the intervenolin derivative AS-1934 had a stronger anti-H. pylori effect than the triple therapy commonly used worldwide to eradicate H. pylori. CONCLUSION: AS-1934 has potential advantages over current treatment options for H. pylori infection.

In vivo efficacy of ß-lactam/tripropeptin C in a mouse septicemia model and the mechanism of reverse ß-lactam resistance in methicillin-resistant Staphylococcus aureus mediated by tripropeptin C.

Natural lipopeptide antibiotic tripropeptin C (TPPC) revitalizes and synergistically potentiates the activities of the class of ß-lactam antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) but not against methicillin-sensitive S. aureus in vitro; however, the mode of action remains unclear. In the course of the study to reveal its mode of action, we found that TPPC inhibited the ß-lactamase production induced by cefotiam. This prompted us to focus on the ß-lactam-inducible ß-lactam-resistant genes blaZ (ß-lactamase) and mecA (foreign penicillin-binding protein), as they are mutually regulated by the blaZ/I/R1 and mecA/I/R1 systems. Quantitative reverse-transcription polymerase chain reaction analysis revealed that TPPC reversed ß-lactam resistance by reducing the expression of the genes blaZ and mecA, when treated alone or in combination with ß-lactam antibiotics. In a mouse/MRSA septicemia model, subcutaneous injection of a combination of TPPC and ceftizoxime demonstrated synergistic therapeutic efficacy compared with each drug alone. These observations strongly suggested that reverse ß-lactam resistance by TPPC may be a potentially effective new therapeutic strategy to overcome refractory MRSA infections.The Journal of Antibiotics advance online publication, 26 July 2017; doi:10.1038/ja.2017.88.

A novel monoclonal antibody targeting coxsackie virus and adenovirus receptor inhibits tumor growth in vivo.

To create a new anti-tumor antibody, we conducted signal sequence trap by retrovirus-meditated expression method and identified coxsackie virus and adenovirus receptor (CXADR) as an appropriate target. We developed monoclonal antibodies against human CXADR and found that one antibody (6G10A) significantly inhibited the growth of subcutaneous as well as orthotopic xenografts of human prostate cancer cells in vivo. Furthermore, 6G10A also inhibited other cancer xenografts expressing CXADR, such as pancreatic and colorectal cancer cells. Knockdown and overexpression of CXADR confirmed the dependence of its anti-tumor activity on CXADR expression. Our studies of its action demonstrated that 6G10A exerted its anti-tumor activity primarily through both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Moreover, 6G10A reacted with human tumor tissues, such as prostate, lung, and brain, each of which express CXADR. Although we need further evaluation of its reactivity and safety in human tissues, our results show that a novel anti-CXADR antibody may be a feasible candidate for cancer immunotherapy.

Stromal cells positively and negatively modulate the growth of cancer cells: stimulation via the PGE2-TNFα-IL-6 pathway and inhibition via secreted GAPDH-E-cadherin interaction.

Fibroblast-like stromal cells modulate cancer cells through secreted factors and adhesion, but those factors are not fully understood. Here, we have identified critical stromal factors that modulate cancer growth positively and negatively. Using a cell co-culture system, we found that gastric stromal cells secreted IL-6 as a growth and survival factor for gastric cancer cells. Moreover, gastric cancer cells secreted PGE2 and TNFα that stimulated IL-6 secretion by the stromal cells. Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems. GAPDH bound to E-cadherin and downregulated the mTOR-p70S6 kinase pathway. These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors. We propose that negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy.

New metastatic model of human small-cell lung cancer by orthotopic transplantation in mice.

Small-cell lung cancer (SCLC) is an aggressive cancer with high metastatic ability and novel strategies against the metastasis are urgently needed to improve SCLC treatment. However, the mechanism of metastasis of SCLC remains largely to be elucidated. For further studies of SCLC metastasis, we developed a new orthotopic transplantation model in mice. We established a GFP-labeled subline from the human SCLC cell line DMS273 and transplanted them orthotopically into the lung of nude mice with Matrigel. The GFP-labeled cells showed significant metastatic activity and formed metastatic foci in distant tissues such as bone, kidney, and brain, as observed in SCLC patients. From a bone metastasis focus of the mouse, we isolated another subline, termed G3H, with enhanced metastatic potential and higher hepatocyte growth factor (HGF) expression than the parental line. Further studies indicated that the HGF/MET signaling pathway was involved in in vitro motility and invasion activities of the G3H cells and treatments with MET inhibitors decreased formation of distant metastases in our orthotopic model using G3H cells. These data indicated that our model mimics the clinical aspect of SCLC such as metastatic tropism and autocrine of HGF/MET signaling. Compared with other orthotopic SCLC models, our model has a superior ability to form distant metastases. Therefore, our model will provide a valuable tool for the study of SCLC metastasis.

Antitumor effects of tyropeptin-boronic acid derivatives: New proteasome inhibitors.

The proteasome degrades numerous regulatory proteins that are critical for tumor growth. Thus, proteasome inhibitors are promising antitumor agents. New proteasome inhibitors, such as tyropeptins and tyropeptin-boronic acid derivatives, have a potent inhibitory activity. Here we report the antitumor effects of two new tyropeptin-boronic acid derivatives, AS-06 and AS-29. AS-06 and AS-29 significantly suppress the degradation of the proteasome-sensitive fluorescent proteins in HEK293PS cells, and induce the accumulation of ubiquitinated proteins in human multiple myeloma cells. We show that these derivatives also suppress the degradation of the NF-κB inhibitor IκB-α and the nuclear translocation of NF-κB p65 in multiple myeloma cells, resulting in the inhibition of NF-κB activation. Furthermore, we demonstrate that AS-06 and AS-29 induce apoptosis through the caspase-8 and caspase-9 cascades. In a xenograft mouse model, i.v. administration of tyropeptin-boronic acid derivatives inhibits proteasome in tumors and clearly suppresses tumor growth in mice bearing human multiple myeloma. Our results indicate that tyropeptin-boronic acid derivatives could be lead therapeutic agents against human multiple myeloma.

Intervenolin, a new antitumor compound with anti-Helicobacter pylori activity, from Nocardia sp. ML96-86F2.

Because stromal cells can regulate the growth and metastasis of tumor cells, a compound that modulates the interaction between the stromal cells and the tumor cells can control the tumor progression. In the course of our screening for such a compound, we have isolated a new compound, intervenolin, from the culture broth of Nocardia sp. ML96-86F2. Intervenolin inhibits the growth of human gastric and colorectal cancer cell lines in the coculture with the respective organ-derived stromal cells more strongly than that of the cancer cells cultured alone. Intervenolin shows antitumor effect against a xenograft model of human colorectal cancer cells in vivo. Furthermore, intervenolin exerts selective anti-Helicobacter pylori effect.

In vivo imaging of proteasome inhibition using a proteasome-sensitive fluorescent reporter.

A proteasome degrades numerous regulatory proteins that are critical for tumor growth and is therefore recognized as a promising anticancer target. Determining proteasome activity in the tumors of mice bearing xenografts is essential for the development of novel proteasome inhibitors. We developed a system for in vivo imaging of proteasome inhibition in the tumors of living mice, using a proteasome-sensitive fluorescent reporter, ZsProSensor-1. This reporter consists of a green fluorescent protein, ZsGreen, fused to mouse ornithine decarboxylase, which is degraded by the proteasome without being ubiquitinated. In stably transfected cells expressing ZsProSensor-1, the fluorescent reporter was rapidly degraded under steady-state conditions, whereas it was stabilized in the presence of proteasome inhibitors. Subcutaneous inoculation of the transfected cells into nude mice resulted in tumor formation. When the proteasome inhibitor bortezomib was intravenously administered to mice bearing these tumors, the ZsProSensor-1 protein accumulated in the tumors and emitted a fluorescent signal in a dose-dependent manner. Robust fluorescence was sustained for 3 days and then gradually decreased to baseline levels within 15 days. Intravenous administration of bortezomib also showed potent antitumor activity. In contrast, oral administration of bortezomib did not result in fluorescent protein accumulation in tumors or exhibit any antitumor activity. These results indicate that in vivo imaging using the ZsProSensor-1 fluorescent protein can be used as an indicator of antitumor activity and will be a powerful tool for the development of novel proteasome inhibitors.

NBRI16716A, a new antitumor compound against human prostate cancer cells, produced by Perisporiopsis melioloides Mer-f16716.

Prostate stroma can regulate the growth and metastasis of prostate cancer through the tumor-stromal cell interactions. Thus, small molecules that modulate the tumor-stromal cell interactions will have a chance to become new antitumor drugs. In the course of our screening of the modulators, we isolated three new natural compounds, NBRI16716A (1), NBRI16716B (2) and NBRI16716C (3), from the fermentation broth of Perisporiopsis melioloides Mer-f16716, although compound 2 was already reported as a chemical degradation product of isotriornicin. Compounds 1 and 2 inhibited the growth of human prostate cancer DU-145 cells in the coculture with human prostate stromal cells (PrSCs) more strongly than that of DU-145 cells alone. Furthermore, both compounds showed antitumor effect against xenograft models of DU-145 cells and PrSCs in vivo.

NBRI17671, a new antitumor compound, produced by Acremonium sp. CR17671.

The interaction between the receptor for advanced glycation end-product (RAGE) and amphoterin has an important role in tumor growth and metastasis. Because the abrogation of the interaction results in the inhibition of the tumor growth and metastasis, we designed a screening system for an inhibitor of the interaction between RAGE and amphoterin. In the course of our screening of the inhibitor, we isolated a novel natural compound NBRI17671 (1) from the fermentation broth of Acremonium sp. CR17671. We also modified 1 into a more active NBRI17671al (2). Although 1 at 50 g ml(-1) weakly inhibited binding of various cells to amphoterin, 2 at 50 g ml(-1) inhibited it by >50% of control. Compound 2 effectively inhibited the tumor growth of glioma and lung tumor xenografts in mice at 25 mg kg(-1). Furthermore, 2 was found to downregulate mitogen-activated protein kinase (MAPK) activity in the tumor cells.

Mitochondrial inhibitors show preferential cytotoxicity to human pancreatic cancer PANC-1 cells under glucose-deprived conditions.

Large areas of tumor are nutrient-starved and hypoxic due to a disorganized vascular system. Therefore, we screened small molecules to identify cytotoxic agents that function preferentially in nutrient-starved conditions. We found that efrapeptin F had preferential cytotoxicity to nutrient-deprived cells compared with nutrient-sufficient cells. Because efrapeptin F acts as a mitochondrial complex V inhibitor, we examined whether inhibitors of complex I, II, III, and V function as cytotoxic agents preferentially in nutrient-deprived cells. Interestingly, these inhibitors showed preferential cytotoxicity to nutrient-deprived cells and caused cell death under glucose-limiting conditions, irrespective of the presence or absence of amino acids and/or serum. In addition, these inhibitors were preferentially cytotoxic to nutrient-deprived cells even under hypoxic conditions. Further, efrapeptin F showed antitumor activity in vivo. These data indicate that mitochondrial inhibitors show preferential cytotoxicity to cancer cells under glucose-limiting conditions, and these inhibitors offer a promising strategy for anticancer therapeutic.

Leucinostatin A inhibits prostate cancer growth through reduction of insulin-like growth factor-I expression in prostate stromal cells.

Targeting stroma in tumor tissues is an attractive new strategy for cancer treatment. We developed in vitro coculture system, in which the growth of human prostate cancer DU-145 cells is stimulated by prostate stromal cells (PrSC) through insulin-like growth factor I (IGF-I). Using this system, we have been searching for small molecules that inhibit tumor growth through modulation of tumor-stromal cell interactions. As a result, we have found that leucinostatins and atpenins, natural antifungal antibiotics, inhibit the growth of DU-145 cells cocultured with PrSC more strongly than that of DU-145 cells alone. In this study we examined the antitumor effects of these small molecules in vitro and in vivo. When DU-145 cells were coinoculated with PrSC subcutaneously in nude mice, leucinostatin A was found to significantly suppress the tumor growth more than atpenin B. The antitumor effect of leucinostatin A in vivo was not obtained against the tumors of DU-145 cells alone. RT-PCR experiments revealed that leucinostatin A specifically inhibited IGF-I expression in PrSC without effect on expressions of other IGF axis molecules. Leucinostatins and atpenins are known to abrogate mitochondrial functions. However, when we used mitochondrial DNA-depleted, pseudo-rho(0) cells, we found that one of leucinostain A actions certainly depended on mitochondrial function, but it actually inhibited the growth of DU-145 cells more strongly in coculture with pseudo-rho(0) PrSC and reduced IGF-I expression in pseudo-rho(0) PrSC. Taken together, our results suggested that leucinostatin A inhibited prostate cancer cell growth through reduction of IGF-I expression in PrSC.

Highly tumorigenic human androgen receptor-positive prostate cancer cells overexpress angiogenin.

We have recently established a highly tumorigenic cell line, LNCaP-CR, derived from human androgen-dependent prostate cancer LNCaP cells. In the present study, we examined the genes responsible for the high tumorigenicity of LNCaP-CR cells. The cDNA microarray analysis and protein array of secreted factors indicated angiogenin (ANG), an angiogenic factor, as a candidate gene. Reverse transcription-polymerase chain reaction and immunoassay confirmed that LNCaP-CR cells expressed high levels of ANG but not vascular endothelial growth factor (VEGF), compared with the parental LNCaP cells. We also proved that another tumorigenic androgen receptor-positive prostate cancer cell line, 22Rv1, secretes higher levels of ANG than VEGF. To assess the contribution of ANG to the highly tumorigenic phenotype, we transfected the ANG gene into LNCaP cells in order to overexpress ANG, and also transfected ANG small interfering RNA-expressing constructs into LNCaP-CR cells to downregulate ANG. Overexpression of ANG in LNCaP cells did not affect their growth in vitro, but it significantly enhanced tumorigenicity and angiogenesis in vivo. In contrast, knockdown of ANG expression in LNCaP-CR cells also did not affect the growth in vitro, but it led to a significant decrease in tumorigenicity and angiogenesis. Taken together, ANG is one of the genes responsible for the high tumorigenicity of LNCaP-CR cells. Thus, our results support the idea that ANG is an attractive target for cancer therapy and show that LNCaP-CR cells are useful for studying ANG action and experimental therapeutic approaches targeting ANG.

Spontaneous large-scale lymphoid neogenesis and balanced autoimmunity versus tolerance in the stomach of H+/K+-ATPase-reactive TCR transgenic mouse.

Autoimmunity is often accompanied by the development of ectopic lymphoid tissues in the target organ, and these tissues have been believed to have close relevance to the severity of the disease. However, the true relationship between the extent of such lymphoid structures and the intensity or type of immune responses mediated by self-reactive T cells has remained unclear. In the present study, we generated transgenic mice expressing TCR from an autoimmune gastritis (AIG)-inducing Th1 cell clone specific for one of the major stomach self-Ags, H(+)/K(+)-ATPase alpha subunit. The transgenic mice spontaneously develop massive lymphoid neogenesis with a highly organized tissue structure in the gastric mucosa, demonstrating Ag-specific, T cell-mediated induction of the lymphoid tissues. Nevertheless, the damage of surrounding tissue and autoantibody production were considerably limited compared with those in typical AIG induced by neonatal thymectomy. Such a moderate pathology is likely due to the locally restricted activation and Th2 skewing of self-reactive T cells, as well as the accumulation of naturally occurring regulatory T cells in the target organ. Altogether, the findings suggest that lymphoid neogenesis in chronic autoimmunity does not simply correlate with the destructive response; rather, the overall activation status of the T cell network, i.e., the balance of self-reactivity and tolerance, in the local environment has an impact.

Insulin-like growth factor I secreted from prostate stromal cells mediates tumor-stromal cell interactions of prostate cancer.

Prostate cancer shows high expression of type I insulin-like growth factor (IGF-I) receptor (IGF-IR) and prostate stromal cells (PrSC) produce IGF-I. Although high plasma level of IGF-I is a risk factor of prostate cancer, the significance of the prostate stromal IGF-I in the regulation of prostate cancer remains elusive. Here we show that the stromal IGF-I certainly regulates the development of prostate cancer. Coinoculation of PrSC increased the growth of human prostate cancer LNCaP and DU-145 tumors in severe combined immunodeficient mice. The conditioned medium of PrSC, as well as IGF-I, induced phosphorylation of IGF-IR and increased the growth of LNCaP and DU-145 cells. PrSC, but not LNCaP and DU-145 cells, secreted significant amounts of IGF-I. Coculture with PrSC increased the growth of DU-145 cells in vitro but the pretreatment of PrSC with small interfering RNA of IGF-I did not enhance it. Furthermore, various chemical inhibitors consisting of 79 compounds with approximately 60 different targets led to the finding that only IGF-IR inhibitor suppressed the PrSC-induced growth enhancement of DU-145 cells. Thus, these results show that the prostate stromal IGF-I mediates tumor-stromal cell interactions of prostate cancer to accelerate tumor growth, supporting the idea that the IGF-I signaling is a valuable target for the treatment of prostate cancer.

A new teleocidin analog from Streptomyces sp. MM216-87F4 induces substance P release from rat dorsal root ganglion neurons.

A new teleocidin analog was isolated from the fermentation medium of Streptomyces sp. MM216-87F4 and its structure was elucidated as 14-O-(N-acetylglucosaminyl) teleocidin A (GlcNAc-TA). GlcNAc-TA induces the translocation of protein kinases Calpha and theta fused with enhanced green fluorescent protein (PKCalpha-EGFP and PKCtheta-EGFP) to the plasma membrane in stable transfectants, and reduces intracellular calcium mobilization induced by agonists of G-protein coupled receptors in various cell lines without causing irritation of the mouse ear. Further, GlcNAc-TA sensitizes the release of excitatory neuropeptides substance P induced by capsaicin from primary-cultured dorsal root ganglion (DRG) neurons of the rat and GlcNAc-TA alone also triggers substance P release in a dose-dependent manner. This study provides the first observation that a teleocidin analog without a free hydroxyl group at C-14 acts as a PKC activator and directly induces the release of excitatory neuropeptide.

Establishment of a highly tumorigenic LNCaP cell line having inflammatory cytokine resistance.

Human androgen-dependent prostate cancer LNCaP cells are low tumorigenic even in immunodeficient mice and were killed by the synergistic effect of inflammatory cytokines, IL-beta and IL-6. To establish a highly tumorigenic LNCaP cell line, we isolated the cytokine-resistant LNCaP-CR cell line and examined the phenotypes. The parental LNCaP cells were induced to commit apoptosis by the addition of IL-1beta and IL-6, but LNCaP-CR cells showed strong resistance against the cytokine action. However, LNCaP-CR cells did not exhibit any resistance to various antitumor drugs investigated. While LNCaP cells formed only palpable tumors in SCID mice, LNCaP-CR cells readily made tumors and their growth was significantly higher than that of LNCaP cells. Moreover, LNCaP tumor-bearing mice gained the weight gradually, but LNCaP-CR tumor-bearing mice significantly lost their body weight. LNCaP-CR cells still responded to androgen action and expressed AR, erbB2, IL-1R, IL-6R, gp130, STAT3, p21, Bcl-2 and caspase-3 as well as LNCaP cells. These results indicate that LNCaP-CR cell line is a new type of tumorigenic LNCaP cell lines and should be useful for identifying responsible genes of tumorigenicity, cytokine resistance, and also cachexia.

Some biochemical and histochemical properties of human liver serine dehydratase.

In rat, serine dehydratase (SDH) is abundant in the liver and known to be a gluconeogenic enzyme, while there is little information about the biochemical property of human liver serine dehydratase because of its low content and difficulty in obtaining fresh materials. To circumvent these problems, we purified recombinant enzyme from Escherichia coli, and compared some properties between human and rat liver serine dehydratases. Edman degradation showed that the N-terminal sequence of about 75% of human serine dehydratase starts from MetSTART-Met2-Ser3- and the rest from Ser3-, whereas the N-terminus of rat enzyme begins from the second codon of MetSTART-Ala2-. The heterogeneity of the purified preparation was totally confirmed by mass spectrometry. Accordingly, this observation in part fails to follow the general rule that the first Met is not removed when the side chain of the penultimate amino acid is bulky such as Met, Arg, Lys, etc. There existed the obvious differences in the local structures between the two enzymes as revealed by limited-proteolysis experiments using trypsin and Staphylococcus aureus V8 protease. The most prominent difference was found histochemically: expression of rat liver serine dehydratase is confined to the periportal region in which many enzymes involved in gluconeogenesis and urea cycle are known to coexist, whereas human liver serine dehydratase resides predominantly in the perivenous region. These findings provide an additional support to the previous notion suggested by physiological experiments that contribution of serine dehydratase to gluconeogenesis is negligible or little in human liver.

A microplate assay for selective measurement of growth of epithelial tumor cells in direct coculture with stromal cells.

Stromal cells play an important role in regulating epithelial malignancies through diffusible factors and adhesion. Modulation of the tumor-stromal cell interaction is an attractive target for new antitumor strategies. To screen for a modulator of the interaction, we have now developed a quantitative colorimetric assay for measurement of tumor cell growth in coculture with stromal cells using rhodanile blue dye. Rhodanile blue specifically stained cytokeratin-positive tumor cells in the coculture. When human prostate carcinoma cells LNCaP, PC-3 and DU-145 were cocultured with normal prostate stromal cells (PrSC) in a microplate, growth of the prostate cancer cells in the coculture was selectively measured by the rhodanile blue staining method. Using this system, we searched for a modulator of the tumor-stromal cell interaction among clinically used drugs and natural products. As a result, we found that 5-fluorouracil, bleomycin and phthoxazolin A inhibit prostate cancer cell growth more strongly in coculture with PrSC than that in monoculture. Without need to pre-label cells and transfect a marker gene, our new method is simple, rapid and thus useful for screening for modulators of the tumor-stromal cell interaction. Furthermore, our results suggest that low molecular weight compounds modulate the tumor-stromal cell interaction.