Toru Kono

Hedgehog Promotes Neovascularization in Pancreatic Cancers by Regulating Ang-1 and IGF-1 Expression in Bone-Marrow Derived Pro-Angiogenic Cells

Background The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s) that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM)-derived cells. Methodology/Principal Findings Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs) were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity. Conclusions/Significance We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial tumorigenesis.

Exodus of Kampo, traditional Japanese medicine, from the complementary and alternative medicines: is it time yet?

ALTHOUGH HERBAL MEDICINE has been used throughout the world since time immemorial, its utilization in the Western World remains within the limited scope of practice largely of complementary and alternative medicine (CAM), the epicenter of so-called allopathic medicine. According to the data from 2007 National Health Interview Survey of 24,000 people conducted by the Center for Disease Control and Prevention’s National Center for Health Statistics, approximately 20% of U.S. adults reported use of herbal supplements. Results from the latest Annual Survey by the American Hospital Association revealed that 60% of the hospitals did not provide CAM therapies which include herbal medicine. Probable reasons for the low use of CAM in hospitals included lack of convincing, evidence-based information on herbal medicine for physicians, as well as the fears of contamination of Chinese herbal products with unexpected pharmaceutical ingredients (steroids, antibiotics, pain relievers, etc.) as reported by the Food and Drug Administration (FDA). Over the last few years, the FDA began shifting its focus to traditional Japanese medicines (called Kampo) whose exceptionally high quality and standardized ingredients were noteworthy of their attention.

Prognostic significance of overexpression of c-Met oncoprotein in cholangiocarcinoma

Background:Cholangiocarcinoma (CC) is a highly malignant carcinoma. We attempted to clarify the prognostic significance of c-Met overexpression and its association with clinicopathological factors in patients with CC.Patients and methods:One hundred and eleven patients with intrahepatic CC (IHCC) and 136 patients with extrahepatic CC (EHCC) who had undergone curative surgery were divided immunohistologically into c-Methigh and c-Metlow groups. Clinicopathological factors and outcomes were compared between the groups. c-Met and epidermal growth factor receptor (EGFR) expression was also examined in 10 CC cell lines.Results:The positivity of c-Met was 45.0% in IHCC and 68.4% in EHCC; c-Methigh expression was demonstrated in 11.7% of IHCC and 16.2% of EHCC. c-Methigh expression was significantly correlated with the 5-year survival rate for CC overall (P=0.0046) and for IHCC (P=0.0013), histopathological classification in EHCC, and for EGFR overexpression in both IHCC and EHCC. Coexpression and coactivation of c-Met and EGFR were also observed in CC cell lines. Multivariate analysis revealed that c-Methigh expression was an independent predictor of poor overall and disease-free survival in patients with IHCC.Conclusions:c-Met overexpression is associated with EGFR expression and is a poor prognostic factor in CC.

Colonic Vascular Conductance Increased by Daikenchuto via Calcitonin Gene-Related Peptide and Receptor-Activity Modifying Protein 1

BACKGROUND Daikencyuto (DKT) is a traditional Japanese medicine (Kampo) and is a mixture of extract powders from dried Japanese pepper, processed ginger, ginseng radix, and maltose powder and has been used as the treatment of paralytic ileus. DKT may increase gastrointestinal motility by an up-regulation of the calcitonin gene-related peptide (CGRP). CGRP is also the most powerful vasoactive substance. In the present study, we investigated whether DKT has any effect on the colonic blood flow in rats. MATERIALS AND METHODS Experiments were performed on fasted anesthetized and artificially ventilated Wistar rats. Systemic mean arterial blood pressure and heart rate were recorded. Red blood cell flux in colonic blood flow was measured using noncontact laser tissue blood flowmetry, and colonic vascular conductance (CVC) was calculated as the ratio of flux to mean arterial blood pressure. We examined four key physiological mechanisms underlying the response using blocker drugs: CGRP1 receptor blocker (CGRP(8-37)), nitric oxide synthase inhibitor, vasoactive intestinal polypeptide (VIP) receptor blocker ([4-Cl-DPhe6, Leu17]-VIP), and substance P receptor blocker (spantide). Reverse transcription-polymerase chain reaction was used for the detection of mRNA of calcitonin receptor-like receptor, receptor-activity modifying protein 1, the component of CGRP 1 receptor and CGRP. After laparotomy, a cannula was inserted into the proximal colon to administer the DKT and to measure CVC at the distal colon. RESULTS Intracolonal administration of DKT (10, 100, and 300 mg/kg) increased CVC (basal CVC, 0.10 mL/mmHg) from the first 15-min observation period (0.14, 0.17, and 0.17 mL/mmHg, respectively) and with peak response at either 45 min (0.17 mL/mmHg by 10 mg/kg), or 75 and 60 min (0.23 and 0.21 mL/mmHg by 100 and 300 mg/kg, respectively). CGRP(8-37) completely abolished the DKT-induced hyperemia, whereas nitric oxide synthase inhibitor partially attenuated the DKT-induced hyperemia. [4-Cl-DPhe6, Leu17]-VIP and spantide did not affect the hyperemia. Japanese pepper significantly increased CVC at 45 min or later, whereas ginseng radix only showed a significant increase at 15 min. Reverse transcription-polymerase chain reaction showed that mRNA for calcitonin receptor-like receptor, receptor-activity modifying protein 1, and CGRP were expressed in rat colon and up-regulated by DKT. CONCLUSIONS The present study demonstrated that DKT increased CVC, which was mainly mediated by CGRP and its receptor components.

Sonic hedgehog derived from human pancreatic cancer cells augments angiogenic function of endothelial progenitor cells

Hedgehog signaling is important in the pathogenesis of pancreatic cancer. Several recent observations suggest the involvement of sonic hedgehog (SHH) in postnatal neovascularization. We identified a novel role for SHH in tumor‐associated angiogenesis in pancreatic cancer. Immunohistochemical analysis revealed that patched homolog 1 (PTCH1), both a receptor for and transcriptional target of hedgehog signaling, was expressed in a small fraction of endothelial cells within pancreatic cancer, but not in normal pancreatic tissue. When endothelial progenitor cells (EPC) isolated from human peripheral blood were cultured with supernatant from SHH‐transfected 293 cells or pancreatic cancer cells, mRNA levels of vascular endothelial growth factor (VEGF), stromal cell‐derived factor‐1 and angiopoietin‐1 were significantly increased, whereas no such induction was observed in human umbilical vein endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMVEC). HUVEC tube formation was stimulated when cocultured with EPC, and preconditioning EPC with supernatant from KP‐1 N pancreatic cancer cells highly expressing SHH significantly enhanced the effect. The effect was partially attenuated by specific inhibition of SHH with cyclopamine or a neutralizing antibody. These findings suggest that tumor‐derived SHH can induce angiogenesis, and this is mediated by its effects on EPC specifically. Targeting SHH would be a novel therapeutic approach that can inhibit not only proliferation of cancer cells but also EPC‐mediated angiogenesis. (Cancer Sci 2008; 99: 1131–1138)

Anti-colitis and -adhesion effects of daikenchuto via endogenous adrenomedullin enhancement in Crohn's disease mouse model

BACKGROUND AND AIMS Adrenomedullin (ADM) is a member of the calcitonin family of regulatory peptides, and is reported to have anti-inflammatory effects in animal models of Crohns disease (CD). We investigated the therapeutic effects of daikenchuto (DKT), an extracted Japanese herbal medicine, on the regulation of endogenous ADM in the gastrointestinal tract in a CD mouse model. METHODS Colitis was induced in mice by intrarectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS); afterwards, DKT was given orally. Colonic damage was assessed on day 3 by macroscopic and microscopic observation, enzyme immunoassays of proinflammatory cytokines in the colonic mucosa, and serum amyloid A (SAA), a hepatic acute-phase protein. To determine the involvement of ADM, an ADM antagonist was instilled intrarectally before DKT administration. The effect of DKT on ADM production by intestinal epithelial cells was evaluated by enzyme immunoassay and real-time PCR. RESULTS DKT significantly attenuated mucosal damage and colonic inflammatory adhesions, and inhibited elevations of SAA in plasma and the proinflammatory cytokines TNFα and IFNγ in the colon. Small and large intestinal epithelial cells produced higher levels of ADM after DKT stimulation. A DKT-treated IEC-6 cell line also showed enhanced ADM production at protein and mRNA levels. Abolition of this effect by pretreatment with an ADM antagonist shows that DKT appears to exert its anti-colitis effect via up-regulation of endogenous ADM in the intestinal tract. CONCLUSION DKT exerts beneficial effects in a CD mouse model through endogenous release and production of ADM. Endogenous ADM may be a therapeutic target for CD.

Epithelial transient receptor potential ankyrin 1 (TRPA1)-dependent adrenomedullin upregulates blood flow in rat small intestine

The functional roles of transient receptor potential (TRP) channels in the gastrointestinal tract have garnered considerable attention in recent years. We previously reported that daikenchuto (TU-100), a traditional Japanese herbal medicine, increased intestinal blood flow (IBF) via adrenomedullin (ADM) release from intestinal epithelial (IE) cells (Kono T et al. J Crohns Colitis 4: 161-170, 2010). TU-100 contains multiple TRP activators. In the present study, therefore, we examined the involvement of TRP channels in the ADM-mediated vasodilatatory effect of TU-100. Rats were treated intraduodenally with the TRP vanilloid type 1 (TRPV1) agonist capsaicin (CAP), the TRP ankyrin 1 (TRPA1) agonist allyl-isothiocyanate (AITC), or TU-100, and jejunum IBF was evaluated using laser-Doppler blood flowmetry. All three compounds resulted in vasodilatation, and the vasodilatory effect of TU-100 was abolished by a TRPA1 antagonist but not by a TRPV1 antagonist. Vasodilatation induced by AITC and TU-100 was abrogated by anti-ADM antibody treatment. RT-PCR and flow cytometry revealed that an IEC-6 cell line originated from the small intestine and purified IE cells expressed ADM and TRPA1 but not TRPV1. AITC increased ADM release in IEC cells remarkably, while CAP had no effect. TU-100 and its ingredient 6-shogaol (6SG) increased ADM release dose-dependently, and the effects were abrogated by a TRPA1 antagonist. 6SG showed similar TRPA1-dependent vasodilatation in vivo. These results indicate that TRPA1 in IE cells may play an important role in controlling bowel microcirculation via ADM release. Epithelial TRPA1 appears to be a promising target for the development of novel strategies for the treatment of various gastrointestinal disorders.

Daikenchuto (TU-100) ameliorates colon microvascular dysfunction via endogenous adrenomedullin in Crohn’s disease rat model

BackgroundDaikenchuto (TU-100), a traditional Japanese medicine, has been reported to up-regulate the adrenomedullin (ADM)/calcitonin gene-related peptide (CGRP) system, which is involved in intestinal vasodilatation. The microvascular dysfunction of the intestine in Crohn’s disease (CD), due to down-regulation of the ADM/CGRP system, is etiologically related to the recurrence of CD. Therefore, we investigated the vasodilatory effect of TU-100 in a CD rat model.MethodsColitis was induced by the rectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in rats. Laser Doppler blood flowmetry was used to measure colonic blood flow. ADM, CGRP, and their receptors in the ischemic colon were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme immunoassays. Additionally, we determined whether the intestinal epithelial cell line IEC-6 released ADM in response to TU-100.ResultsTU-100 increased blood flow in ischemic segments of the colon but not in hyperemic segments. Pretreatment with an antibody to ADM abolished the vasodilatory effect of TU-100. CGRP levels and βCGRP mRNA expression were decreased in the ischemic colon, while protein and mRNA levels of ADM were unchanged. Hydroxy α-sanshool, the main constituent of TU-100, was the most active component in improving blood flow. Additionally, both TU-100 and hydroxy α-sanshool enhanced the release of ADM from IEC-6 cells.ConclusionsIn the ischemic colon, endogenous βCGRP, but not ADM, was decreased. Thus, it was concluded that TU-100 ameliorated microvascular dysfunction by the up-regulation of endogenous ADM in the CD rat model. TU-100 may be a possible therapeutic agent for gastrointestinal ischemia-related diseases including CD.

Effect of Ecabet Sodium Enema on mildly to moderately active ulcerative proctosigmoiditis: an open-label study

OBJECTIVES:Ecabet sodium (ES), a nonabsorbable antigastric ulcer agent, has been shown to adhere to the region of an ulcer. It topically enhances gastric mucosal defensive factors such as the endogenous prostaglandins, capsaicin-sensitive sensory nerves, nitric oxide, and mucin. All of these mucosal defensive factors play an important role in maintaining the mucosal integrity of the colon and rectum. Therefore, we investigated the effect of ES in patients with mildly to moderately active ulcerative proctosigmoiditis.METHODS:In an open-label study, seven patients with mildly to moderately active ulcerative colitis (UC) who had an inflamed mucosa in the rectum and/or sigmoid and were resistant to 4-wk topical and systemic standard treatment were treated with an ES enema b.i.d. for 14 days. The enema consisted of ES (1 g) and tepid water (20 or 50 ml). These patients were assessed by the Clinical Activity Index, colonoscopically, and histologically before and after the ES therapy. The ES therapy was started after obtaining informed consent from the patients.RESULTS:Six of the seven patients responded to therapy and achieved clinical, endoscopic, and histological remissions. One patient was withdrawn because of increased stool frequency. All six patients who completed the study showed a significant change in the mean Clinical Activity Index score from 5.3 ± 1.4 (mean ± SD) to 0.5 ± 0.8 (p < 0.05), in the colonoscopic score from 3.0 ± 0.9 to 0.8 ± 0.4 (p < 0.05), and in the histological score from 2.7 ± 0.5 to 0.5 ± 0.6 (p < 0.05), and achieved remission at the end of the study. There were no side effects attributable to the ES therapy. Five of the six patients are still in clinical remission after a median follow-up period of 5 months.CONCLUSIONS:The ES enemas proved to be a safe and potentially useful adjuvant therapy currently available for treating patients with mildly to moderately active ulcerative proctosigmoiditis. A controlled study is necessary to confirm our results.

Efficacy of goshajinkigan for peripheral neurotoxicity of oxaliplatin in patients with advanced or recurrent colorectal cancer.

Peripheral neurotoxicity is the major limiting factor for oxaliplatin therapy. Goshajinkigan (GJG), a traditional Japanese herbal medicine, was recently shown to be effective in protecting against the neurotoxicity of taxanes in Japan. We retrospectively investigated the effect of GJG on peripheral neurotoxicity associated with oxaliplatin therapy. Ninety patients with metastatic colorectal cancer that received FOLFOX4 or modified FOLFOX6 therapy were assigned to receive one of the following adjuncts: oral GJG at 7.5 g day−1 (Group A, n = 11), intravenous supplementation of calcium gluconate and magnesium sulfate (1 g each before and after FOLFOX) (Group B, n = 14), combined GJG and calcium gluconate and magnesium sulfate therapies (Group C, n = 21), or no concomitant therapy (Group D, n = 44). The incidence of peripheral neurotoxicity was investigated when the cumulative dose of oxaliplatin exceeded 500 mg m−2. When the cumulative dose of oxaliplatin exceeded 500 mg m−2, the incidence of neuropathy (all grades) in Groups A–D was 50.0%, 100%, 78.9%, and 91.7%, respectively. It was lowest in the group that received GJG alone. Concomitant administration of GJG reduced the neurotoxicity of oxaliplatin in patients that received chemotherapy for colorectal cancer.