Implications of glutathione-S transferase P1 in MAPK signaling as a CRAF chaperone: In memory of Dr. Irving Listowsky.

Glutathione-S transferase P1 (GSTP1) is one of the glutathione-S transferase isozymes that belong to a family of phase II metabolic isozymes. The unique feature of GSTP1 compared with other GST isozymes is its relatively high expression in malignant tissues. Thus, clinically, GSTP1 serves as a tumor marker and as a refractory factor against certain types of anticancer drugs through its primary function as a detoxifying enzyme. Additionally, recent studies have identified a chaperone activity of GSTP1 involved in the regulation the function of various intracellular proteins, including factors of the growth signaling pathway. In this review, we will first describe the function of GSTP1 and then extend the details onto its role in the mitogen-activated protein kinase signal pathway, referring to the results of our recent study that proposed a novel autocrine signal loop formed by the CRAF/GSTP1 complex in mutated KRAS and BRAF cancers. Finally, the possibilities of new therapeutic approaches for these cancers by targeting this complex will be discussed.

Resolution of fibrosis by siRNA HSP47 in vitamin A-coupled liposomes induces regeneration of chronically injured livers.

BACKGROUND AND AIM: In chronic hepatic diseases where treatment strategies are not available, deposited fibrotic tissues deteriorate the intrinsic regeneration capacity of the liver by creating special restrictions. Thus, if the anti-fibrosis modality is efficiently applied, the regeneration capacity of the liver should be reactivated even in such refractory hepatic diseases. METHODS: Rat liver fibrosis was induced by dimethyl-nitrosamine (DMN). Another liver fibrosis model was established in CCl4 treated Sox9CreERT2ROSA26: YFP mice. To resolve hepatic fibrosis, vitamin A-coupled liposomes containing siRNA HSP47 (VA-liposome siHSP47) were employed. EpCAM + hepatic progenitor cells from GFP rats were transplanted to DMN rat liver to examine their trans-differentiation into hepatic cells after resolution of liver fibrosis. RESULTS: Even under continuous exposure to such strong hepatotoxin as DMN, rats undergoing VA-liposome siHSP47 treatment showed an increment of DNA synthesis of hepatocytes with the concomitant restoration of impaired liver weight and normalization of albumin levels. These results were consistent with the observation that GFP + EpCAM hepatic progenitor cells transplanted to DMN rat liver, trans-differentiated into GFP + mature hepatic cells after VA-liposome siHSP47 treatment. Another rodent model also proved regeneration potential of the fibrotic liver in CCl4 administered Sox9CreERT2ROSA26: YFP mice, VA-liposome siHSP47 treatment-induced restoration of liver weight and trans-differentiation of YEP + Sox9 + cells into YFP + hepatic cells, although because of relatively mild hepatotoxicity of CCl4, undamaged hepatocytes also proliferated. CONCLUSIONS: These results demonstrated that regeneration of chronically damaged liver indeed occurs after anti-fibrosis treatment even under continuous exposure to hepatotoxin, which promises a significant benefit of the anti-fibrosis therapy for refractory liver diseases.

Chemoprevention of pancreatic cancer by inhibition of glutathione-S transferase P1.

Pancreatic cancer is among the most refractory malignancies with poor prognosis. Thus, preventive approaches, in addition to the development of novel therapeutic strategies are essential for this type of cancer. KRAS mutations occur very early in the development of pancreatic cancers and could be targeted for its prevention, yet specific inhibitors for mutated KRAS are lacking. Accordingly, Glutathione-S Transferase p1 (GSTP1), which we recently found to be an autocrine stimulator of mutated KRAS signaling, is predicted to be an alternative target for chemoprevention of pancreatic cancer. In this study, chemopreventive effects of O-Hexadecyl-γ-glutamyl-S-benzyl-cysteinyl-D-phenyl glycine-Ethylester (HGBPE), which we previously synthesized to inhibit GSTP1 activity, was analyzed for its effect on the prevention of a rat pancreatic carcinogenesis model induced by 7,12-dimethyl-benzanthracene (DMBA). Rats administered with DMBA were grouped into five cohorts. In the treated group I, which was treated neither with HGBPE nor vehicle, sequential appearance of precancerous lesions, ductal complexes, and adenocarcinoma was confirmed as previously reported. We also confirmed in this group that mutations of KRAS and expression of GSTP1 simultaneously occurred in the ductal complex. To rats of groups II and IV, HGBPE was administered, and vehicle to those of group III and V. In groups of II and IV, the incidence of both ductal complex and adenocarcinoma were significantly lower than those in groups III and V. These data clearly suggest the efficacy of HGBP as a potential chemopreventive agent for pancreatic cancer.

A CRAF/glutathione-S-transferase P1 complex sustains autocrine growth of cancers with KRAS and BRAF mutations.

The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (mKRAS) and BRAF (mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRAS and mBRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRAS and mBRAF cells in vitro and suppressed tumorigenesis of the xenografted mKRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers.

Vitamin A-coupled liposomes containing siRNA against HSP47 ameliorate skin fibrosis in chronic graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (SCT) is characterized by multiorgan fibrosis and profoundly affects the quality of life of transplant survivors. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in collagen synthesis in myofibroblasts. We explored the role of HSP47 in the fibrotic process of cutaneous chronic GVHD in mice. Immunohistochemical analysis showed massive fibrosis with elevated amounts of collagen deposits and accumulation of F4/80+ macrophages, as well as myofibroblasts expressing HSP47 and retinol-binding protein 1 in the skin after allogeneic SCT. Repeated injection of anti-colony-stimulating factor (CSF-1) receptor-blocking antibodies significantly reduced HSP47+ myofibroblasts in the skin, indicating a macrophage-dependent accumulation of myofibroblasts. Vitamin A-coupled liposomes carrying HSP47 small interfering RNA (siRNA) (VA-lip HSP47) delivered HSP47 siRNA to cells expressing vitamin A receptors and knocked down their HSP47 in vitro. Intravenously injected VA-lip HSP47 were specifically distributed to skin fibrotic lesions and did not affect collagen synthesis in healthy skin. VA-lip HSP47 knocked down HSP47 expression in myofibroblasts and significantly reduced collagen deposition without inducing systemic immunosuppression. It also abrogated fibrosis in the salivary glands. These results highlight a cascade of fibrosis in chronic GVHD; macrophage production of transforming growth factor ß mediates fibroblast differentiation to HSP47+ myofibroblasts that produce collagen. VA-lip HSP47 represent a novel strategy to modulate fibrosis in chronic GVHD by targeting HSP47+ myofibroblasts without inducing immunosuppression.

Treatment of pulmonary fibrosis with siRNA against a collagen-specific chaperone HSP47 in vitamin A-coupled liposomes.

BACKGROUND: Pulmonary fibrosis is a life-threatening pathological state of progressive interstitial lung diseases, such as idiopathic pulmonary fibrosis. Myofibroblasts are known to play a critical role in the pathogenesis of pulmonary fibrosis. This study aimed to evaluate the inhibitory effect of a small interfering RNA (siRNA) on a collagen-specific chaperone heat shock protein 47 (HSP47). The siRNA was preferentially delivered to myofibroblasts in a bleomycin (BLM)-induced pulmonary fibrosis rat model using siRNA against HSP47, encapsulated in a vitamin A-coupled liposome (VA-lip-siRNA HSP47). METHODS AND RESULTS: Male Sprague-Dawley rats were treated with an intratracheal injection of BLM or phosphate buffered saline followed by an intravenous injection of VA-lip-siRNA HSP47 three times per week under preventive administration schedules from day 1 to day 21 and therapeutic administration schedules from day 15 to day 35. The expression of HSP47 after the treatment was assessed by immunoblotting. The specific delivery of VA-lip-siRNA HSP47 conjugated with 6'-carboxyfluoresce into myofibroblasts was examined by immunofluorescence staining. The effect of VA-lip-siRNA HSP47 on fibrosis was analyzed by morphological and biochemical methods. Preferential delivery of VA-lip-siRNA HSP47 to myofibroblasts in fibrotic areas in BLM-treated rats was verified by immunofluorescence staining. Treatment of VA-lip-siRNA HSP47 clearly suppressed HSP47 expression and induced apoptosis of myofibroblasts in the lung of BLM-treated rats. Hydroxyproline levels and inflammatory cytokines in the lungs, and the number of inflammatory cells in the bronchial alveolar lavage of BLM-treated rats were significantly suppressed by the treatment. Morphological assessment showed that VA-lip-siRNA HSP47 also significantly improved the morphological pulmonary fibrosis of BLM-treated rats in both preventive and therapeutic schedules. CONCLUSIONS: These results suggest that VA-lip-siRNA HSP47 improves pulmonary fibrosis in not only preventive, but also therapeutic schedules, and thus, this drug delivery system should provide a novel therapy for refractory pulmonary fibrosis.

Involvement of Pancreatic Stellate Cells in Regeneration of Remnant Pancreas after Partial Pancreatectomy.

BACKGROUND AND OBJECTIVES: Mechanism of regeneration of remnant pancreas after partial pancreatectomy (PX) is still unknown. In this study, effect of siRNA against the collagen specific chaperone, HSP47, which inhibits collagen secretion from activated pancreas stellate cells (aPSCs), and induces their apoptosis, on regeneration of remnant pancreas was determined. METHODS: Pancreatectomy was performed according to established methods. Proliferation of cells was assessed by BrdU incorporation. Immunostaining of HSP47 was employed to identify PSCs. Progenitor cells were identified by SOX9 staining. Acinar cells were immunostained for amylase. Co-culture of acinar cells with aPSCs were carried out in a double chamber with a cell culture insert. siRNA HSP47 encapsulated in vitamin A-coupled liposome (VA-lip siRNA HSP47) was delivered to aPSCs by iv injection. RESULTS: In remnant pancreas of 90% PX rat, new areas of foci were located separately from duodenal areas with normal pancreatic features. After PX, BrdU uptake of acinar cells and islet cells significantly increased, but was suppressed by treatment with VA-lip siRNA HSP47. BrdU uptake by acinar cells was augmented by co-culturing with aPSCs and the augmentation was nullified by siRNA HSP47. BrdU uptake by progenitor cells in foci area was slightly enhanced by the same treatment. New area which exhibited intermediate features between those of duodenal and area of foci, emerged after the treatment. CONCLUSION: aPSCs play a crucial role in regeneration of remnant pancreas, proliferation of acinar and islet cells after PX through the activity of secreted collagen. Characterization of new area emerged by siRNA HSP47 treatment as to its origin is a future task.

Panitumumab in combination with irinotecan plus S-1 (IRIS) as second-line therapy for metastatic colorectal cancer.

BACKGROUND: Irinotecan plus S-1 (IRIS) is the only oral fluoropyrimidine-based regimen reported to be non-inferior to FOLFIRI and widely used in clinical practice for metastatic colorectal cancer (mCRC) patients. However, the combination of IRIS plus an anti-EGFR agent has not been evaluated previously. This study aimed to investigate the feasibility and efficacy of IRIS with panitumumab as second-line therapy for wild-type KRAS mCRC. METHODS: Main inclusion criteria were patients with wild-type KRAS mCRC refractory to one prior chemotherapy regimen for mCRC, ECOG PS 0-2, and age ≥20 years. Patients received panitumumab (6 mg/kg) and irinotecan (100 mg/m(2)) on days 1 and 15 and S-1 (40-60 mg according to body surface area) twice daily for 2 weeks, repeated every 4 weeks. The primary endpoint was the feasibility of the therapy. The secondary endpoints were response rate (RR), progression-free survival (PFS), and overall survival (OS). RESULTS: A total of 36 patients received protocol treatment in eight centers. Of these, 23 patients (63.9 %) completed protocol treatment, demonstrating achievement of the primary endpoint. The most frequent grade 3/4 toxicities were diarrhea (16.7 %), acne-like rash (13.9 %), and neutropenia (11.1 %). The overall RR was 33.3 % (12/36). Of these patients, five underwent conversion surgery. Median PFS and OS were 9.5 months (95 % CI 3.5-15.4 months) and 20.1 months (95 % CI 16.7-23.2 months), respectively. CONCLUSION: IRIS plus panitumumab has an acceptable toxicity profile and a promising efficacy in patients with previously treated wild-type KRAS mCRC. Accordingly, this regimen can be an additional treatment option for second-line chemotherapy in wild-type KRAS mCRC.

Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence.

Transdifferentiation of vitamin A-storing hepatic stellate cells (HSCs) to vitamin A-depleted myofibroblastic cells leads to liver fibrosis. Vitamin A regulates lipid accumulation and gene transcription, suggesting that vitamin A is involved in the maintenance of HSC quiescence under a physiological condition. However, the precise mechanism remains elusive because there is no appropriate in vitro culture system for quiescent HSCs. Here, we show that treatment of quiescent HSCs with vitamin A partially maintained the accumulation of lipid droplets and expression of quiescent HSC markers (glial fibrillary acidic protein, peroxisome proliferator-activator receptor-γ and CCAAT/enhancer-binding protein-α) and also the expression of myofibroblastic markers (α-smooth muscle actin, heat shock protein 47 and collagen type I). On the other hand, combined treatment with vitamin A and insulin sustained the characteristic of HSC quiescence and completely suppressed the expression of myofibroblastic markers through activation of the JAK2/STAT5 signaling pathway and increased expression of sterol regulatory element binding protein-1. These treated HSCs transdifferentiated to myofibroblastic cells under a culture condition with fetal bovine serum. The results suggest an important role of vitamin A and insulin in the maintenance of HSC quiescence under a physiological condition.

Activated hepatic stellate cells are dependent on self-collagen, cleaved by membrane type 1 matrix metalloproteinase for their growth.

Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVß1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVß1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies.

Treatment of pancreatic fibrosis with siRNA against a collagen-specific chaperone in vitamin A-coupled liposomes.

BACKGROUND AND OBJECTIVE: Fibrosis associated with chronic pancreatitis is an irreversible lesion that can disrupt pancreatic exocrine and endocrine function. Currently, there are no approved treatments for this disease. We previously showed that siRNA against collagen-specific chaperone protein gp46, encapsulated in vitamin A-coupled liposomes (VA-lip-siRNAgp46), resolved fibrosis in a model of liver cirrhosis. This treatment was investigated for pancreatic fibrosis induced by dibutyltin dichloride (DBTC) and cerulein in rats. METHODS: Specific uptake of VA-lip-siRNAgp46, conjugated with 6'-carboxyfluorescein (FAM) by activated pancreatic stellate cells (aPSCs), was analysed by fluorescence activated cell sorting (FACS). Intracellular distribution of VA-lip-siRNAgp46-FAM was examined by fluorescent microscopy. Suppression of gp46 expression by VA-lip-siRNAgp46 was assessed by immunoblotting. Collagen synthesis in aPSCs was assayed by dye-binding. Specific delivery of VA-lip-siRNAgp46 to aPSCs in DBTC rats was verified following intravenous VA-lip-siRNA-FAM and (3)H-VA-lip-siRNAgp46. The effect of VA-lip-siRNA on pancreatic histology in DBTC- and cerulein-treated rats was determined by Azan-Mallory staining and hydroxyproline content. RESULTS: FACS analysis revealed specific uptake of VA-lip-siRNAgp46-FAM through the retinol binding protein receptor by aPSCs in vitro. Immunoblotting and collagen assay verified knockdown of gp46 and suppression of collagen secretion, respectively, by aPSCs after transduction of VA-lip-siRNAgp46. Specific delivery of VA-lip-siRNAgp46 to aPSCs in fibrotic areas in DBTC rats was confirmed by fluorescence and radioactivity 24 h after the final injection. 10 systemic VA-lip-siRNAgp46 treatments resolved pancreatic fibrosis, and suppressed tissue hydroxyproline levels in DBTC- and cerulein-treated rats. CONCLUSION: These data suggest the therapeutic potential of the present approach for reversing pancreatic fibrosis.

Efficacy and safety of micafungin as an empirical antifungal therapy for suspected fungal infection in neutropenic patients with hematological disorders.

This prospective multicenter study was performed to clarify the efficacy and safety of micafungin (MCFG) as an empirical antifungal therapy for suspected fungal infection in patients with hematological disorders and neutropenia. Three hundred and eighty-eight patients were enrolled; 151 patients with possible fungal infection diagnosed by radiological imaging or serological testing and 237 patients with refractory fever were included in this study. The mean dose and duration of treatment with MCFG were 154.6 mg/day and 14.0 days, respectively. The clinical response rate for patients with possible fungal infection and refractory fever was 60.1% and 65.3%, respectively. Even in persistent neutropenic patients with a neutrophil count of <500/µL throughout the MCFG treatment, the clinical response rate was 46.9%. Ninety-one drug-related adverse events (DAEs) were observed in 56 patients (14.4%) and 9 serious DAEs were observed in 6 patients (1.5%). Neither daily dose nor duration of MCFG treatment affected the incidence of DAEs. It was confirmed that MCFG has adequate clinical efficacy and is safe for the treatment of suspected fungal infections in patients with hematological disorders and neutropenia.

Intravenous administration of auto serum-expanded autologous mesenchymal stem cells in stroke.

Transplantation of human mesenchymal stem cells has been shown to reduce infarct size and improve functional outcome in animal models of stroke. Here, we report a study designed to assess feasibility and safety of transplantation of autologous human mesenchymal stem cells expanded in autologous human serum in stroke patients. We report an unblinded study on 12 patients with ischaemic grey matter, white matter and mixed lesions, in contrast to a prior study on autologous mesenchymal stem cells expanded in foetal calf serum that focused on grey matter lesions. Cells cultured in human serum expanded more rapidly than in foetal calf serum, reducing cell preparation time and risk of transmissible disorders such as bovine spongiform encephalomyelitis. Autologous mesenchymal stem cells were delivered intravenously 36-133 days post-stroke. All patients had magnetic resonance angiography to identify vascular lesions, and magnetic resonance imaging prior to cell infusion and at intervals up to 1 year after. Magnetic resonance perfusion-imaging and 3D-tractography were carried out in some patients. Neurological status was scored using the National Institutes of Health Stroke Scale and modified Rankin scores. We did not observe any central nervous system tumours, abnormal cell growths or neurological deterioration, and there was no evidence for venous thromboembolism, systemic malignancy or systemic infection in any of the patients following stem cell infusion. The median daily rate of National Institutes of Health Stroke Scale change was 0.36 during the first week post-infusion, compared with a median daily rate of change of 0.04 from the first day of testing to immediately before infusion. Daily rates of change in National Institutes of Health Stroke Scale scores during longer post-infusion intervals that more closely matched the interval between initial scoring and cell infusion also showed an increase following cell infusion. Mean lesion volume as assessed by magnetic resonance imaging was reduced by >20% at 1 week post-cell infusion. While we would emphasize that the current study was unblinded, did not assess overall function or relative functional importance of different types of deficits, and does not exclude placebo effects or a contribution of recovery as a result of the natural history of stroke, our observations provide evidence supporting the feasibility and safety of delivery of a relatively large dose of autologous mesenchymal human stem cells, cultured in autologous human serum, into human subjects with stroke and support the need for additional blinded, placebo-controlled studies on autologous mesenchymal human stem cell infusion in stroke.

Randomized double-blind trial of sulindac and etodolac to eradicate aberrant crypt foci and to prevent sporadic colorectal polyps.

PURPOSE: On the basis of the results of our preliminary trial suggesting that aberrant crypt foci (ACF) could be eradicated by short-term administration of sulindac, in the present study, we explored the feasibility of using ACF as surrogate markers for chemoprevention of colorectal cancer. EXPERIMENTAL DESIGN: Randomly assigned to sulindac (300 mg daily), etodolac (400 mg daily), and placebo groups were 189 subjects without polyps or who had undergone polypectomy. Drugs were administered for 2 months. ACF in the rectal region were counted by magnifying endoscopy. Occurrence of polyps was evaluated at 12 months. A planned interim analysis was conducted. RESULTS: ACF number at 2 months was significantly suppressed in the sulindac group (P = 0.0075), but not in the etodolac group (P = 0.73). In the sulindac group, the numbers of adenomas plus hyperplastic polyps (total polyps) and adenomas at 12 months were significantly (P = 0.02) and marginally (P = 0.064) lower, respectively, in comparison with the placebo group; no such difference was observed in the etodolac group. In analysis of only polypectomized subjects, the numbers of total polyps and adenomas in the sulindac group were even more markedly lower, with P values of 0.014 and 0.034, respectively. A similar tendency was confirmed by analyses of the incidence of polyps at 12 months. Suppression rates of total polyps and adenomas in ACF responders to sulindac were significantly greater than in nonresponders. In all groups, compliance was more than 90% and no intolerable adverse effects were observed. CONCLUSIONS: ACF may be useful as surrogate lesions for chemoprevention of colorectal cancer.

Pure red cell aplasia associated with Good's syndrome accompanied by decreased stem cell factor production in the bone marrow.

A 79-year-old Japanese woman diagnosed with pure red cell aplasia (PRCA) associated with thymoma and hypogammaglobulinemia (Good's syndrome) was successfully treated with cyclosporine-A after a thymectomy. We further studied the etiology of this case. A burst-forming unit erythroid (BFU-E) assay with SCF restored erythropoiesis in vitro. SCF production was reduced in bone marrow stromal cells; however, it was restored in vitro and in vivo after cyclosporine-A treatment.

Phase II study of S-1, docetaxel and cisplatin combination chemotherapy in patients with unresectable metastatic gastric cancer.

PURPOSE: We evaluated the activity and toxicity of docetaxel, cisplatin, and S-1 (DCS) combination chemotherapy in patients with unresectable metastatic gastric cancer. METHODS: Patients with histologically proven, unresectable metastatic gastric adenocarcinoma, performance status (PS) 0-2, and no prior chemotherapy were eligible. Patients received oral S-1 (40 mg/m(2) b.i.d.) on days 1-14 and intravenous cisplatin (60 mg/m(2)) and docetaxel (60 mg/m(2)) on day 8 every 3 weeks. RESULTS: Thirty-four patients were enrolled between March 2005 and April 2007. Three patients were considered ineligible and did not receive the DSC therapy. Clinical characteristics were as follows: median age, 63 years (range, 44-77); PS, 0/1/2: 23/8/0; women/men, 8/23; and well-differentiated/undifferentiated adenocarcinoma, 10/21. The objective response rate was 87.1% with 1 complete response (3.2%) and 26 partial responses (83.9%) in 31 assessable patients. Four had stable disease (12.9%) but none had progressive disease. Of these 27 responders, 8 (25.8%) achieved downstaging and 7 (22.6%) underwent curative surgery. The median survival time and progression-free survival were 687 days [confidence interval (95% CI), 600.0-1,138.1] and 226 days (95% CI, 182.5-379.3), respectively. Most common grade 3/4 hematologic toxicity was neutropenia (77.4%). Most common grade 3 nonhematologic toxicities included anorexia (35.5%) and nausea (32.3%). All treatment-related toxicities resolved, and no toxic deaths were observed. CONCLUSIONS: DCS combination chemotherapy is highly active against unresectable metastatic gastric cancer and can be given safely with proper management of adverse events. Further studies of this combination are warranted.

Interleukin-2 gene transfer potentiates the alpha-galactosylceramide-stimulated antitumor effect by the induction of TRAIL in NKT and NK cells in mouse models of subcutaneous and metastatic carcinoma.

Alpha-Galactosylceramide (alpha-GalCer) is a potent CD1d ligand that activates natural killer like T-cells (NKT), leading to the production of helper T (Th) 1 and Th2 cytokines that mediate various immunemodulatory and antitumor effects. Here, we determined whether the administration of adenovirus-vector-encoding mouse interleukin-2 (AdmIL-2) can augment the antitumor effect of alpha-GalCer on subcutaneous and metastatic tumors in mice. Mice were intraperitoneally injected with alpha-GalCer on days 7, 10 and 13 after tumor inoculation, with or without intratumoral injection of AdmIL-2 on day 7. alpha-GalCer treatment increased the serum levels of interferon-gamma, while intratumoral injection of AdmIL-2 elevated serum IL-2 levels. A combination of alpha-GalCer and AdmIL-2 (alpha-GalCer/AdmIL-2) inhibited the in vivo tumor growth and improved the survival of tumor-bearing mice, as compared to the use of a single agent. Experiments on spontaneous metastasis models revealed that alpha-GalCer/AdmIL-2 reduced lung metastasis and prolonged survival, as compared to control groups. In addition, the splenic and liver mononuclear cells from mice treated with alpha-GalCer/AdmIL-2 showed enhanced cytolytic activity against NK-sensitive YAC-1 and NK-resistant 3LL tumors. Moreover, alpha-GalCer/AdmIL-2 treatment expanded the absolute numbers of lung and liver NK, NKT and T-cells as well as the TNF-related apoptosis-inducing ligand (TRAIL) expression of these cells. This study shows the efficacy of alpha-GalCer/AdmIL-2 immunomodulatory therapy, and provides a cellular mechanism on how it exerts the antitumor effects.

Suppressive effect of sulindac on branch duct-intraductal papillary mucinous neoplasms.

BACKGROUND: When considering surgery for branch duct-intraductal papillary mucinous neoplasms (BD-IPMNs) with suspected malignancy, it should be recognized that these lesions are frequently multifocal and are usually found in elderly patients with potential comorbidities that could affect the outcome of surgery. Clinical trials of chemoprevention have been conducted for a wide variety of malignancies. METHODS: Twenty-two BD-IPMN patients participated in the trial at our institution from June 2004 to January 2007. Ten of the 22 patients who rejected surgical therapy although their lesions or clinical symptoms met the criteria for surgical resection of the International Association of Pancreatology guidelines were assigned to the treatment group. Sulindac (150 mg twice daily) and omeprazole (20 mg once daily) were administered to these patients for 18 months. The remaining 12 patients comprised the control group. Branch duct diameter and mural nodule heights were monitored by either magnetic resonance cholangiopancreatography (MRCP) or computed tomography (CT) and by endoscopic ultrasonography (EUS). RESULTS: Both branch duct diameter and mural nodule height of BD-IPMNs in the treatment group were significantly reduced, while those in the control group were unchanged. Immunohistochemical staining for cyclooxygenase-1 and -2 was negative in hyperplasia, adenoma and carcinoma portions of resected pancreatic specimens but was clearly positive for glutathione-S-transferase pi (GST-pi), suggesting that GST-pi is a putative target molecule for sulindac. CONCLUSIONS: Although a larger scale randomized controlled study is needed in future, the present results suggest the promise of chemoprevention of carcinoma derived from BD-IPMNs by sulindac.

Drug resistance is dramatically restored by hedgehog inhibitors in CD34+ leukemic cells.

Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers and in cancer stem cells. However, the contribution of Hh signaling to leukemic cell regulation has remained unclear. In this study, we assessed the possibility that Hh pathway activation contributes to the survival and drug resistance of cluster of differentiation (CD)34+ leukemia cells. Hh signaling in leukemic cell lines and primary leukemic cells was screened by reverse transcription - polymerase chain reaction (RT-PCR) and a Hh signaling reporter assay. We found that Hh signaling is active in several human acute myeloid leukemia (AML) cells, especially primary CD34+ leukemic cells and cytokine-responsive CD34+ cell lines such as Kasumi-1, Kasumi-3 and TF-1. These CD34+ cells express the downstream effectors glioma-associated oncogene homolog (GLI)1 or GLI2, indicative of active Hh signaling. Moreover, inhibition of Hh signaling with the naturally derived Smoothened antagonist cyclopamine, endogenous Hh inhibitor hedgehog-interacting protein or anti-hedgehog neutralizing antibody induced apoptosis after 48 h of exposure, although these CD34+ cell lines exhibited resistance to cytarabine (Ara-C). In contrast, cyclopamine failed to affect growth or survival in U937 and HL-60 cell lines that lack expression of Hh receptor components, confirming that the effect of Hh inhibition is specific. Furthermore, combination with 10 microM cyclopamine significantly reduced drug resistance of CD34+ cell lines and primary CD34+ leukemic cells to Ara-C. These results suggest that aberrant Hh pathway activation is a feature of some CD34+ myeloid leukemic cells and Hh inhibitors may have a therapeutic role in the treatment of AML.

[Effective CDDP arterial infusion with DSM for liver metastasis of malignant melanoma complicating DIC due to intratumoral hemorrhage--a case report].

A 65-year-old male, who had been diagnosed with melanoma of stage IIB and treated by chemotherapy since 2003 at the Dermatology Department, was referred to our department for liver metastasis of melanoma that had become resistant to chemotherapeutic agents. In 2006, he started receiving hepatic arterial infusion of CDDP. He was admitted to the hospital on an emergency basis for general fatigue the next May. Blood tests revealed anemia and thrombocytopenia. Contrast CT showed aggravation of liver metastasis. Contrast ultrasonography revealed nodular contrast enhancement at the margin of the tumor. On the basis of image findings and blood test results, DIC due to intratumoral hemorrhage was diagnosed. CDDP arterial infusion with DSM resulted in improved DIC, and he was able to be discharged. Taken together, attention has to be paid to the potential for emergency complications of DIC due to liver metastasis of melanoma with intratumoral hemorrhage. Moreover, it was shown that arterial infusion with DSM was effective for liver metastasis of melanoma.