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1.  Calreticulin is highly expressed in pancreatic cancer stem‐like cells 
Cancer Science  2016;107(11):1599-1609.
Cancer stem‐like cells (CSLCs) in solid tumors are thought to be resistant to conventional chemotherapy or molecular targeting therapy and to contribute to cancer recurrence and metastasis. In this study, we aimed to identify a biomarker of pancreatic CSLCs (P‐CSLCs). A P‐CSLC‐enriched population was generated from pancreatic cancer cell lines using our previously reported method and its protein expression profile was compared with that of parental cells by 2‐D electrophoresis and tandem mass spectrometry. The results indicated that a chaperone protein calreticulin (CRT) was significantly upregulated in P‐CSLCs compared to parental cells. Flow cytometry analysis indicated that CRT was mostly localized to the surface of P‐CSLCs and did not correlate with the levels of CD44v9, another P‐CSLC biomarker. Furthermore, the side population in the CRThigh/CD44v9low population was much higher than that in the CRTlow/CD44v9high population. Calreticulin expression was also assessed by immunohistochemistry in pancreatic cancer tissues (n = 80) obtained after radical resection and was found to be associated with patients' clinicopathological features and disease outcomes in the Cox proportional hazard regression model. Multivariate analysis identified CRT as an independent prognostic factor for pancreatic cancer patients, along with age and postoperative therapy. Our results suggest that CRT can serve as a biomarker of P‐CSLCs and a prognostic factor associated with poorer survival of pancreatic cancer patients. This novel biomarker can be considered as a therapeutic target for cancer immunotherapy.
PMCID: PMC5132278  PMID: 27561105
Biomarkers; calreticulin; cancer stem cells; pancreatic cancer; proteomics
2.  Integrin α2 Mediates Selective Metastasis to the Liver 
Cancer research  2009;69(18):7320-7328.
Cancers display distinct patterns of organ-specific metastasis. Comparative analysis of a broad array of cell membrane molecules on a liver-metastasizing subline of B16 melanoma versus the parental B16-F0 revealed unique up-regulation of integrin α2. The direct role of integrin α2 in hepatic metastasis was shown by comparison of high versus low-expressing populations, antibody blockade, and ectopic expression. Integrin α2–mediated binding to collagen type IV (highly exposed in the liver sinusoids) and collagen type IV–dependent activation of focal adhesion kinase are both known to be important in the metastatic process. Analysis of primary colorectal cancers as well as coexisting liver and lung metastases from individual patients suggests that integrin α2 expression contributes to liver metastasis in human colorectal cancer. These findings define integrin α2 as a molecule conferring selective potential for formation of hepatic metastasis, as well as a possible target to prevent their formation.
PMCID: PMC4857201  PMID: 19738067
3.  Involvement of angiotensin II type 2 receptor (AT2R) signaling in human pancreatic ductal adenocarcinoma (PDAC): a novel AT2R agonist effectively attenuates growth of PDAC grafts in mice 
Cancer Biology & Therapy  2015;16(2):307-316.
We have recently discovered the potential involvement of angiotensin II type 2 receptor (AT2R) signaling in pancreatic cancer using AT2R deficient mice. To examine the involvement of AT2R expression in human PDAC, expressions of AT2R as well as the major angiotensin II receptor (type 1 receptor, AT1R) in human PDAC and adjacent normal tissue was evaluated by immunohistochemistry and real time PCR using surgically dissected human PDAC specimens. In immunohistochemical analysis, relatively strong AT1R expression was detected consistently in both normal pancreas and PDAC areas, whereas moderate AT2R expression was detected in 78.5% of PDAC specimens and 100% of normal area of the pancreas. AT1R, but not AT2R, mRNA levels were significantly higher in the PDAC area than in the normal pancreas. AT2R mRNA levels showed a negative correlation trend with overall survival. In cell cultures, treatment with a novel AT2R agonist significantly attenuated both murine and human PDAC cell growth with negligible cytotoxicity in normal epithelial cells. In a mouse study, administrations of the AT2R agonist in tumor surrounding connective tissue markedly attenuated growth of only AT2R expressing PAN02 murine PDAC grafts in syngeneic mice. The AT2R agonist treatment induced apoptosis primarily in tumor cells but not in stromal cells. Taken together, our findings offer clinical and preclinical evidence for the involvement of AT2R signaling in PDAC development and pinpoint that the novel AT2R agonist could serve as an effective therapeutic for PDAC treatment.
PMCID: PMC4623015  PMID: 25756513
angiotensin II type 2 receptor (AT2R); apoptosis; pancreatic ductal adenocarcinoma; selective AT2R agonist
4.  Risk Factors and Managements of Bile Leakage After Hepatectomy 
World Journal of Surgery  2015;40:182-189.
The purpose of this study was to retrospectively determine the risk factors and evaluate the management of bile leakage.
Three hundred and thirty-four patients who underwent hepatectomy for Child classification grade A liver disease, without biliary reconstruction and laparoscopic procedures, between 2003 and 2013 were included. Risk factors were identified using multivariate analysis.
Bile leakage was observed in 30 (9.0 %) patients. Multivariate analysis demonstrated that type of hepatectomy (segmentectomy 1, medial sectionectomy, anterior sectionectomy, or central bisectionectomy) and operating time was independent risk factors for bile leakage. Among 30 patients with confirmed bile leakage, central type leakage that was in communication with the biliary tree occurred in 23 (76.7 %) patients and peripheral type, which was not in communication with the biliary tree, in 7 (23.3 %) patients. Ten patients were treated with only drainage. Endoscopic or percutaneous transhepatic procedures were performed in 15 cases with central type leakage. Ablation treatment using ethanol or minocycline was mainly performed for peripheral type leakage. Four cases with central type leakage had strictures of the right hepatic duct. Two of them were treated with ablation treatment, portal vein embolization, or fistulojejunostomy. Median duration from diagnosis to end of therapy was 77 days (11–323) in central type and 44 days (6–123) in peripheral type leakage, respectively.
Complex hepatectomy and operating time are independent risk factors for postoperative bile leakage. Biliary exploration should be performed as soon as possible after diagnosis, because most bile leakage is the central type. Central type of bile leakage is sometimes refractory to therapy, needing various treatments and requiring a long time for recovery.
PMCID: PMC4695499  PMID: 26159119
5.  Perioperative Microbiologic Monitoring of Sputum on Postoperative Day One as a Predictor of Pneumonia After Hepatectomy 
Journal of Gastrointestinal Surgery  2015;19(9):1662-1667.
The purpose of this study was to retrospectively evaluate microbial examination of sputum on postoperative day one (POD1) and to determine risk factors for postoperative pneumonia (POP) after hepatectomy.
Two hundred ninety-four patients who expectorated sputum on POD1 after hepatectomy between 2003 and 2014 were investigated. Sputum samples were submitted for microbial examination. Risk factors for POP were identified using multivariable analysis.
One hundred fifty-eight (53.7 %) of 294 patients had bacteria in their sputum on POD1. POP was observed in 24 (8.2 %) patients, with increased mortality in the patients with POP (0.74 vs 12.5 %, p < 0.01). Multivariate analysis demonstrated that a Brinkman index of >400 and bacteria in sputum on POD1 were independent risk factors for POP. Bacterial homology in sputum obtained on POD1 and onset day of POP was found in 13 of the 24 (54.2 %) patients with POP. In particular, in 13 patients with POP caused by methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa, homology was confirmed in 9 patients (69.2 %).
A Brinkman index ≥400 and bacteria in sputum on POD1 increased the risk of POP. Presence of bacteria in sputum on POD1 may be useful in determining early treatment against POP after hepatectomy.
PMCID: PMC4539353  PMID: 26105551
Postoperative pneumonia; Hepatectomy
6.  Influence of mesenchymal stem cells on stomach tissue engineering using small intestinal submucosa 
Small intestinal submucosa (SIS) is a biodegradable collagen-rich matrix containing functional growth factors. We have previously reported encouraging outcomes for regeneration of an artificial defect in the rodent stomach using SIS grafts, although the muscular layer was diminutive. In this study, we investigated the feasibility of SIS in conjunction with mesenchymal stem cells (MSCs) for regeneration of the gastrointestinal tract. MSCs from the bone marrow of green fluorescence protein (GFP)-transgenic Sprague–Dawley (SD) rats were isolated and expanded ex vivo. A 1 cm whole-layer stomach defect in SD rats was repaired using: a plain SIS graft without MSCs (group 1, control); a plain SIS graft followed by intravenous injection of MSCs (group 2); a SIS graft co-cultured with MSCs (group 3); or a SIS sandwich containing an MSC sheet (group 4). Pharmacological, electrophysiological and immunohistochemical examination was performed to evaluate the regenerated stomach tissue. Contractility in response to a muscarinic receptor agonist, a nitric oxide precursor or electrical field stimulation was observed in all groups. SIS grafts seeded with MSCs (groups 3 and 4) appeared to support improved regeneration compared with SIS grafts not seeded with MSCs (groups 1 and 2), by enabling the development of well-structured smooth muscle layers of significantly increased length. GFP expression was detected in the regenerated interstitial tissue, with fibroblast-like cells in the seeded-SIS groups. SIS potently induced pharmacological and electrophysiological regeneration of the digestive tract, and seeded MSCs provided an enriched environment that supported tissue regeneration by the SIS graft in the engineered stomach. © 2013 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.
PMCID: PMC4409104  PMID: 23913876
small intestinal submucosa; SIS; stomach tissue engineering; mesenchymal stem cell; extracellular matrix; GFP
7.  Prognostic factors in patients with septic shock in digestive surgery who have undergone direct hemoperfusion with polymyxin B-immobilized fibers: a retrospective observational study 
Direct hemoperfusion with polymyxin B-immobilized fiber (PMX-DHP) has been widely used for patients with septic shock around the world, but the prognostic factors have not been fully understood. We conducted a retrospective analysis to determine the prognostic factors in patients with septic shock who underwent PMX-DHP.
Twenty-nine patients with septic shock who underwent PMX-DHP were included in the study. The patients were divided into groups based on survival (n = 23) and non-survival (n = 6) 28 days after PMX-DHP, and the clinical data for the two groups before and after PMX-DHP were compared.
In non-survivors, the vasopressor dependency index before PMX-DHP was significantly higher (p = 0.046), and the leukocyte count before PMX-DHP was significantly lower (p = 0.024) than in survivors. Furthermore, base excess after PMX-DHP was significantly lower in non-survivors (p = 0.007) than in survivors. The optimal cutoff points of the vasopressor dependency index, leukocyte count, and base excess identified by receiver operating characteristic curves were 0.499/mmHg, 1360/μL, and −6.4 mmol/L, respectively. And the score using these three cutoffs, termed the prognostic score, was related to the prognosis of septic shock patients who underwent PMX-DHP (area under the curve = 0.946).
The prognostic score, using three parameters which are immediately and readily available in early phase after starting PMX-DHP, might be useful to predict the prognosis of these patients.
PMCID: PMC4908858  PMID: 27307992
Intra-abdominal infection; Hemodynamic dysfunction; Prognostic score
8.  An accurate prognostic staging system for hepatocellular carcinoma patients after curative hepatectomy 
International Journal of Oncology  2014;46(3):944-952.
The aim of this study was to develop an accurate predictive system for prognosis of hepatocellular carcinoma (HCC) patients after hepatectomy. We pooled data of clinicopathological features of 234 HCC patients who underwent curative hepatectomy. On the basis of the pooled data, we established a simple predictive staging system (PS score) scored by the mathematical product of tumor number and size, and degree of liver function. We compared the prognostic abilities of the PS score (score 0–3) with those of six well-known clinical staging systems. Then, we found that there were significant differences (P<0.05) in both disease-free survival (DFS) and overall survival (OS) between patients with different PS scores (PS score 0 vs. 1; PS score 1 vs. 2), and there was a significant difference in DFS, but not OS, between patients with PS score 2 and those with PS score 3. Moreover, the PS score had smaller values of the Akaike information criterion for both DFS and OS than any of the six well-known clinical staging systems. These results suggest that the PS score serves as a simple, accurate predictor for the prognosis of HCC patients after hepatectomy.
PMCID: PMC4324590  PMID: 25524574
hepatocellular carcinoma; recurrence; staging system; prognostic factor; Akaike information criterion
9.  Long-fragment DNA as a potential marker for stool-based detection of colorectal cancer 
Oncology Letters  2014;9(1):454-458.
Neoplastic cells that are exfoliated from the colorectal epithelium exhibit dysfunctional apoptotic mechanisms, and thus it is possible to identify high-molecular-weight DNA fragments (long DNA) in feces. In the present study, the sensitivity and specificity of fecal-based long DNA assays were evaluated for the detection of colorectal cancer (CRC). Feces were collected from 54 healthy volunteers and 130 patients with CRC prior to surgical treatment. The presence of long DNA of the adenomatosis polyposis coli, Kirsten rat sarcoma viral oncogene homolog (KRAS), B-raf proto-oncogene, serine/threonine kinase and p53 genes was assessed by polymerase chain reaction followed by electrophoresis. The identification of long DNA in feces was found to exhibit a sensitivity of 56.2% and specificity of 96.3% for CRC detection. In addition, long DNA was identified in the feces of 58/90 (64.4%) patients with distal CRC and 15/40 (37.5%) patients with proximal CRC. This study indicates the potential of the fecal long DNA assay as a non-invasive and easily performed method for the detection of individuals with CRC.
PMCID: PMC4247014  PMID: 25436008
colorectal cancer; stool DNA test
10.  Cancer stem-like sphere cells induced from de-differentiated hepatocellular carcinoma-derived cell lines possess the resistance to anti-cancer drugs 
BMC Cancer  2014;14:722.
Cancer stem cells (CSCs) are thought to play important roles in therapy-resistance. In this study, we induced cancer stem-like cells from hepatocellular carcinoma (HCC) cell lines using a unique medium, and examined their potential for resistance to anti-cancer drugs.
The human HCC cell lines SK-HEP-1 (SK), HLE, Hep 3B, and HuH-7 were used to induce cancer stem-like cells with our sphere induction medium supplemented with neural survival factor-1. NANOG and LIN28A were examined as stemness markers. Several surface markers for CSC such as CD24, CD44, CD44 variant, and CD90 were analyzed by flow-cytometry. To assess the resistance to anti-cancer drugs, the MTS assay, cell cycle analysis, and reactive oxygen species (ROS) activity assay were performed.
Poorly differentiated HCC derived SK and undifferentiated HCC derived HLE cell lines efficiently formed spheres of cells (SK-sphere and HLE-sphere), but well-differentiated HCC-derived HuH-7 and Hep 3B cells did not. SK-spheres showed increased NANOG, LIN28A, and ALDH1A1 mRNA levels compared to parental cells. We observed more CD44 variant-positive cells in SK-spheres than in parental cells. The cell viability of SK-spheres was significantly higher than that of SK cells in the presence of several anti-cancer drugs except sorafenib (1.7- to 7.3-fold, each P < 0.05). The cell cycle of SK-spheres was arrested at the G0/G1 phase compared to SK cells. SK-spheres showed higher ABCG2 and HIF1A mRNA expression and lower ROS production compared to parental cells.
Our novel method successfully induced cancer stem-like cells, which possessed chemoresistance that was related to the cell cycle, drug efflux, and ROS.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-722) contains supplementary material, which is available to authorized users.
PMCID: PMC4190290  PMID: 25260650
Cancer stem cell; HCC; Sphere; Chemoresistance
11.  A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population 
International Journal of Oncology  2014;45(5):1857-1866.
Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLCenriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy.
PMCID: PMC4203336  PMID: 25118635
cancer stem cells; cell culture method; pancreatic cancer
12.  Adoptive immunotherapy with MUC1-mRNA transfected dendritic cells and cytotoxic lymphocytes plus gemcitabine for unresectable pancreatic cancer 
We previously reported the clinical efficacy of adoptive immunotherapy (AIT) with dendritic cells (DCs) pulsed with mucin 1 (MUC1) peptide and cytotoxic T lymphocytes (CTLs). We also reported that gemcitabine (GEM) enhances anti-tumor immunity by suppressing regulatory T cells. Therefore, in the present study, we performed combination therapy with AIT and GEM for patients with unresectable or recurrent pancreatic cancer.
Patients and methods
Forty-two patients with unresectable or recurrent pancreatic cancer were treated. DCs were generated by culture with granulocyte macrophage colony-stimulating factor and interleukin-4 and then exposed to tumor necrosis factor-α. Mature DCs were transfected with MUC1-mRNA by electroporation (MUC1-DCs). MUC1-CTLs were induced by co-culture with YPK-1, a human pancreatic cancer cell line, and then with interleukin-2. Patients were treated with GEM, while MUC1-DCs were intradermally injected, and MUC1-CTLs were intravenously administered.
Median survival time (MST) was 13.9 months, and the 1-year survival rate was 51.1%. Of 42 patients, one patient had complete response (2.4%), three patients had partial response (7.1%) and 22 patients had stable disease (52.4%). The disease control ratio was 61.9%. The MST and 1-year survival rate of 35 patients who received more than 1 × 107 MUC1-DCs per injection was 16.1 months and 60.3%, respectively. Liver metastasis occurred in only 5 patients among 35 patients without liver metastasis before treatment. There were no severe toxicities associated with AIT.
AIT with MUC1-DCs and MUC1-CTLs plus GEM may be a feasible and effective treatment for pancreatic cancer.
PMCID: PMC4074851  PMID: 24947606
Pancreatic cancer; MUC1; Dendritic cell; Cytotoxic lymphocyte; Gemcitabine; Immunotherapy
13.  Relationship Between Cytokine Gene Polymorphisms and Risk of Postoperative Pneumonia with Esophageal Cancer 
Journal of Gastrointestinal Surgery  2014;18(7):1247-1253.
We retrospectively evaluated the relationship between cytokine gene polymorphisms and development of postoperative pneumonia after esophagectomy.
In 120 patients who underwent esophagectomy, serum samples were obtained to measure levels of serum interleukin (IL)-6 and IL-10 at four time points (preoperatively, postoperative day (POD)0, POD1, and POD3). DNA extracted from peripheral blood in all patients was analyzed to determine polymorphisms of cytokines such as tumor necrosis factor-α -1031 T/C, IL-1β -511C/T, IL-6 -634C/G, and IL-10 -819 T/C.
Postoperative pneumonia arose in 34 patients (28.3 %). Perioperative serum IL-10 levels were significantly higher for IL-10 -819 C/T + C/C genotypes than for T/T genotypes (POD0 16.7 ± 2.84 vs. 8.54 ± 0.87 pg/ml, p = 0.0002; POD1 14.0 ± 2.64 vs. 8.8 ± 0.87 pg/ml, p = 0.0143; POD3 8.9 ± 2.67 vs. 4.4 ± 0.52 pg/ml, p = 0.0076). The frequency of the IL-10 -819 T/T genotype was significantly higher in patients with postoperative pneumonia than in patients without pneumonia (p = 0.0323). Multivariate analysis of factors such as sex, smoking, length of operation, field of lymph node dissection, and IL-10 polymorphism identified IL-10 polymorphism as independent predictor of postoperative pneumonia.
Patients with IL-10 -819 T/T genotype may be at high risk for postoperative pneumonia after esophagectomy.
PMCID: PMC4057631  PMID: 24804995
IL-10 polymorphism; Postoperative pneumonia; Esophagectomy
14.  A phase ΙI study of five peptides combination with oxaliplatin-based chemotherapy as a first-line therapy for advanced colorectal cancer (FXV study) 
We previously conducted a phase I trial for advanced colorectal cancer (CRC) using five HLA-A*2402-restricted peptides, three derived from oncoantigens and two from vascular endothelial growth factor (VEGF) receptors, and confirmed safety and immunological responses. To evaluate clinical benefits of cancer vaccination treatment, we conducted a phase II trial using the same peptides in combination with oxaliplatin-based chemotherapy as a first-line therapy.
The primary objective of the study was the response rates (RR). Progression free survival (PFS), overall survival (OS), and immunological parameters were evaluated as secondary objective. The planned sample size was more than 40 patients for both HLA2402-matched and -unmatched groups. All patients received a cocktail of five peptides (3 mg each) mixed with 1.5 ml of IFA which was subcutaneously administered weekly for the first 12 weeks followed by biweekly administration. Presence or absence of the HLA-A*2402 genotype were used for classification of patients into two groups.
Between February 2009 and November 2012, ninety-six chemotherapy naïve CRC patients were enrolled under the masking of their HLA-A status. Ninety-three patients received mFOLFOX6 and three received XELOX. Bevacizumab was added in five patients. RR was 62.0% and 60.9% in the HLA-A*2402-matched and -unmatched groups, respectively (p = 0.910). The median OS was 20.7 months in the HLA-A*2402-matched group and 24.0 months in the unmatched group (log-rank, p = 0.489). In subgroup with a neutrophil/lymphocyte ratio (NLR) of < 3.0, patients in the HLA-matched group did not survive significantly longer than those in the unmatched group (log-rank, p = 0.289) but showed a delayed response.
Although no significance was observed for planned statistical efficacy endpoints, a delayed response was observed in subgroup with a NLR of < 3.0. Biomarkers such as NLR might be useful for selecting patients with a better treatment outcome by the vaccination.
Trial registration
Trial registration: UMIN000001791.
PMCID: PMC4021539  PMID: 24884643
Peptide vaccine; Peptide cocktail; Colorectal cancer; Phase II study; FOLFOX; Chemotherapy
15.  A phase I study of combination vaccine treatment of five therapeutic epitope-peptides for metastatic colorectal cancer; safety, immunological response, and clinical outcome 
To evaluate the safety of combination vaccine treatment of multiple peptides, phase I clinical trial was conducted for patients with advanced colorectal cancer using five novel HLA-A*2402-restricted peptides, three peptides derived from oncoantigens, ring finger protein 43 (RNF43), 34 kDa-translocase of the outer mitochondrial membrane (TOMM34), and insulin-like growth factor–II mRNA binding protein 3 (KOC1), and the remaining two from angiogenesis factors, vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2.
Eighteen HLA- A*2402-positive colorectal cancer patients who had failed to standard therapy were enrolled in this study. 0.5 mg, 1.0 mg or 3.0 mg each of the peptides was mixed with incomplete Freund’s adjuvant and then subcutaneously injected at five separated sites once a week. We also examined possible effect of a single site injection of “the cocktail of 5 peptides” on the immunological responses. ELISPOT assay was performed before and after vaccinations in the schedule of every 4 weeks.
The vaccine treatment using multiple peptides was well tolerated without any severe treatment-associated systemic adverse events. Dose-dependent induction of peptide-specific cytotoxic T lymphocytes was observed. The single injection of “peptides cocktail” did not diminish the immunological responses. Regarding the clinical outcome, one patient achieved complete response and 6 patients revealed stable disease for 4 to 7 months. The median overall survival time (MST) was 13.5 months. Patients, in which we detected induction of cytotoxic T lymphocytes specific to 3 or more peptides, revealed significantly better prognosis (MST; 27.8 months) than those with poorer immune responses (MST; 3.7 months) (p = 0.032).
Our cancer vaccine treatment using multiple peptides is a promising approach for advanced colorectal cancer with the minimum risk of systemic adverse reactions.
Clinical trial registration
UMIN-CTR number UMIN000004948.
PMCID: PMC4007571  PMID: 24612787
Peptide vaccine; Peptide cocktail; Colorectal cancer; Phase I study
16.  A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients 
Proteome Science  2013;11:33.
We report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals.
Five cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera.
This protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals.
PMCID: PMC3750938  PMID: 23866785
Antigen chip; Antibody profiling; Cysteine-tag; Maleimide; Hepatitis C virus; Hepatocellular carcinoma; Tumor-associated antigen; Autoantibody
17.  Evaluation of host quality of life and immune function in breast cancer patients treated with combination of adjuvant chemotherapy and oral administration of Lentinula edodes mycelia extract 
OncoTargets and therapy  2013;6:853-859.
Anthracycline-based chemotherapies for breast cancer are well known to have adverse effects and can also negatively affect host immune function. There is therefore a necessity for an adjuvant that maintains the quality of life (QOL) and immune function of cancer patients receiving anthracycline-based chemotherapies.
Patients and methods
The present study investigated the effectiveness of the concomitant use of Lentinula edodes mycelia extract (LEM), an oral immunomodulator, with FEC75 (5-fluorouracil + epirubicin + cyclophosphamide) therapy on host QOL and immune function in breast cancer patients with nodal metastases. Ten breast cancer patients with nodal metastases receiving surgery were enrolled in this study. Treatment with 5-fluorouracil (500 mg/m2), epirubicin (75 mg/m2), and cyclophosphamide (500 mg/m2) was performed every 21 days for two courses, and LEM (1800 mg/day by mouth) was administered during the second course.
In the first course, hematological toxicity was observed and host QOL and immune function were exacerbated. In the second course, however, the number of white blood cells and lymphocytes did not decrease and host QOL was maintained. Furthermore, the cytotoxic activities of natural killer (NK) and lymphokine-activated killer cells and the proportion of activated NK and NK T-cells in lymphocytes were maintained in the second course.
It has been suggested that the concomitant use of LEM with FEC75 therapy can maintain host QOL and immune function, and offer important implications for an application of LEM as a useful oral adjuvant to anthracycline-based chemotherapies.
PMCID: PMC3711972  PMID: 23874107
clinical study; breast cancer; anthracycline; Lentinula edodes; immunity; quality of life
18.  ABCB6 mRNA and DNA methylation levels serve as useful biomarkers for prediction of early intrahepatic recurrence of hepatitis C virus-related hepatocellular carcinoma 
International Journal of Oncology  2013;42(5):1551-1559.
The poor prognosis of hepatocellular carcinoma (HCC) can be explained largely by the high rate of intrahepatic recurrence (IHR). Identification of genes related to IHR is needed to improve the poor prognosis and important for personalized medicine. Eighty-one HCC specimens were used in this study. We screened for IHR-related genes by DNA microarray analysis. The validation of screening was performed by using real-time PCR. The methylation levels in genomic DNAs were measured by quantitative methylation-specific PCR. Six hepatoma cell lines were used for examination of ABCB6 expressional regulation. Time-to-event analyses for recurrence after surgery were analyzed by Kaplan-Meier analysis and Cox regression analysis with cutoff values obtained from receiver operating characteristic (ROC) analysis. We confirmed that ABCB6 mRNA levels were significantly higher in hepatitis C virus (HCV)-related HCCs with early IHR compared to HCV-related HCCs without early IHR (2.5-fold, P=0.01) and the corresponding non-cancerous livers (3.1-fold, P=0.05). Experiments with cell lines showed correlation between DNA methylation and mRNA levels of ABCB6. ROC analysis revealed that mRNA levels (0.81 area under the curve, 88% sensitivity and 72% specificity) and DNA methylation levels (0.81 area under the curve, 80% sensitivity and 80% specificity) of ABCB6 in HCV-related HCCs allowed for the accurate discrimination of the development of early IHR. Cox regression analysis revealed that ABCB6 mRNA levels was an independent risk factor for IHR of HCV-related HCC. Aberrant mRNA and DNA methylation levels of ABCB6 may serve as useful predictive biomarkers for early IHR of HCV-related HCC.
PMCID: PMC3661224  PMID: 23483087
ABCB6; hepatocellular carcinoma; prognosis
19.  Susceptibility of hepatoma-derived cells to histone deacetylase inhibitors is associated with ID2 expression 
International Journal of Oncology  2013;42(4):1159-1166.
Downregulation of inhibitor of DNA binding 2 (ID2) is associated with poor prognosis in cases of hepatocellular carcinoma (HCC). Therefore, to search for effective antitumor drugs for the treatment of HCC exhibiting poor prognostic indicators, we used two HCC-derived cell lines (HuH-7 and HLE) to alter ID2 levels. Specifically, ID2 expression was knocked down in HuH-7 cells via transfection with ID2-specific small interfering RNAs and separately ID2 was overexpressed in HLE cells via an ID2 expression plasmid vector. To assess the effect of antitumor drugs, MTS assay was performed. Annexin V staining was used to evaluate apoptosis and real-time RT-PCR was used to measure mRNA levels. ID2 knockdown cells were more susceptible to histone deacethylase (HDAC) inhibitors including sodium butyrate (NaB), sodium 4-phenyl-butyrate, tricostatin A, suberoylanilide hydroxamic acid, MS-275, apicidin and HC-toxin. Conversely, cells that overexpressed ID2 were less susceptible than control cells to HDAC inhibitors. NaB-induced apoptosis was inversely correlated with ID2 expression. Expression of the anti-apoptotic mRNA BCL2 was induced by NaB in control cells, but this induction of BCL2 was inhibited by ID2 knockdown and strengthened by ID2 overexpression. Expression of another anti-apoptotic mRNA, BCL2L1, was decreased by NaB administration and then partially recovered. However, in ID2 knockdown cells, BCL2L1 levels did not recover from NaB-induced suppression. ID2 affected the susceptibility of two HCC-derived cell lines to an HDAC inhibitor by regulating the expression of anti-apoptotic genes. Therefore, HDAC inhibitors may be effective for the treatment of HCC for which the prognosis is poor based on ID2 downregulation and ID2 could serve as a marker that is predictive of the clinical response to HDAC inhibitors.
PMCID: PMC3622658  PMID: 23403953
inhibitor of DNA binding 2; hepatocellular carcinoma; histone deacethylase inhibitor; apoptosis
20.  Germline copy number variations associated with breast cancer susceptibility in a Japanese population 
Tumour Biology  2012;34(2):947-952.
Although copy number variations (CNVs) are expected to affect various diseases, little is known about the association between CNVs and breast cancer susceptibility. Therefore, we investigated this relation. Array comparative genomic hybridization was performed to search for candidate CNVs related to breast cancer susceptibility. Subsequent quantitative real-time polymerase chain reaction was carried out for confirmation. We found seven CNV markers associated with breast cancer risk. The means of the relative copy numbers of patients with a history of breast cancer and women in the control group were 0.8 and 1.8 for Hs06535529_cn on 1p36.12 (P < 0.0001), 2.9 and 2.2 for Hs03103056_cn on 3q26.1 (P < 0.0001), 1.2 and 1.8 for Hs03899300_cn on 15q26.3 (P < 0.0001), 1.0 and 1.5 for Hs03908783_cn on 15q26.3 (P < 0.0001), and 1.1 and 1.7 for Hs03898338_cn on 15q26.3 (P < 0.0001), respectively. Interestingly, nine or more copies of Hs04093415_cn on 22q12.3 were found only in 8/193 (4.1 %) patients with a history of breast cancer and in none of the controls (P = 0.0081). Similarly, 12 or more copies of Hs040908898_cn on 22q12.3 were found only in 7/193 (3.6 %) patients with a history of breast cancer and in none of the controls (P = 0.016). A combination of two CNVs resulted in 80.3 % sensitivity, 80.6 % specificity, 82.4 % positive predictive value, and 78.3 % negative predictive value for the prediction of breast cancer susceptibility. These findings may lead to a new means of risk assessment for breast cancer. Confirmatory studies using independent data sets are needed to support our findings.
PMCID: PMC3597278  PMID: 23275252
CNV; Breast cancer susceptibility; CGH; Real-time PCR; Digital PCR
21.  Luminal A and luminal B (HER2 negative) subtypes of breast cancer consist of a mixture of tumors with different genotype 
BMC Research Notes  2012;5:376.
The St Gallen International Expert Consensus 2011 has proposed a new classification system for breast cancer. The purpose of this study was to elucidate the relationship between the breast cancer subtypes determined by the new classification system and genomic characteristics.
Invasive breast cancers (n = 363) were immunohistochemically classified as follows: 111 (30.6%) as luminal A, 95 (26.2%) as luminal B (HER2 negative), 69 (19.0%) as luminal B (HER2 positive), 41 (11.3%) as HER2, and 47 (12.9%) as basal-like subtypes.
The high expression of Ki-67 antigen was detected in 236 tumors; no cases of luminal A subtype showed high expression of the Ki-67 antigen, but more than 85% of tumors of the other subtypes showed high expression. In addition, DNA ploidy and chromosomal instability (CIN) were assessed using imaging cytometry and FISH, respectively. In this series, 336 (92.6%) tumors consisted of 129 diploid/CIN- and 207 aneuploid/CIN + tumors. Diploid/CIN- and aneuploid/CIN+ features were detected in 64.9% and 27.9% of luminal A, 41.1% and 49.5% of luminal B (HER2-), 11.6% and 81.2% of luminal B (HER2+), 4.9% and 90.2% of HER2, and 17.0% and 76.6% of basal-like subtypes, respectively. Unlike the luminal B (HER2+), HER2 and basal-like subtypes, the luminal A and luminal B (HER2-) subtypes were heterogeneous in terms of DNA ploidy and CIN.
It is reasonable to propose that the luminal A and luminal B (HER2-) subtypes should be further divided into two subgroups, diploid/CIN- and aneuploid/CIN+, based on their underlying genomic status.
PMCID: PMC3413599  PMID: 22830453
22.  Is there diversity among UGT1A1 polymorphism in Japan? 
AIM: To investigate into the diversity of UGT1A1 polymorphism across three different districts in Japan and highlight genetic differences among the population in Japan.
METHODS: We enrolled 50 healthy volunteers from each of the Yamaguchi (western part of Japan), Kochi (southern part of Japan) and Akita (northern part of Japan) prefectures. Blood samples (7 mL) were collected from each participant and stored in EDTA for subsequent genotyping by fragment size analysis, direct sequencing and TaqMan assay of UGT1A1*28, UGT1A7*3/UGT1A9*22 and UGT1A1*93/UGT1A1*6/UGT1A1*27/UGT1A1*60/UGT1A7 (-57), respectively.
RESULTS: The only statistically significant differences in allele polymorphisms among the group examined were for UGT1A1*6. The Akita population showed more UGT1A1*6 heterozygosity (P = 0.0496).
CONCLUSION: Our study revealed no regional diversity among UGT1A1, UGT1A7 or UGT1A9 polymorphisms in Japan.
PMCID: PMC3406281  PMID: 22848786
UGT1A1 gene; Polymorphism; Diversity
23.  Breast cancer cell lines carry cell line-specific genomic alterations that are distinct from aberrations in breast cancer tissues: Comparison of the CGH profiles between cancer cell lines and primary cancer tissues 
BMC Cancer  2010;10:15.
Cell lines are commonly used in various kinds of biomedical research in the world. However, it remains uncertain whether genomic alterations existing in primary tumor tissues are represented in cell lines and whether cell lines carry cell line-specific genomic alterations. This study was performed to answer these questions.
Array-based comparative genomic hybridization (CGH) was employed with 4030 bacterial artificial chromosomes (BACs) that cover the genome at 1.0 megabase resolution to analyze DNA copy number aberrations (DCNAs) in 35 primary breast tumors and 24 breast cancer cell lines. DCNAs were compared between these two groups. A tissue microdissection technique was applied to primary tumor tissues to reduce the contamination of samples by normal tissue components.
The average number of BAC clones with DCNAs was 1832 (45.3% of spotted clones) and 971 (24.9%) for cell lines and primary tumor tissues, respectively. Gains of 1q and 8q and losses of 8p, 11q, 16q and 17p were detected in >50% of primary cancer tissues. These aberrations were also frequently detected in cell lines. In addition to these alterations, the cell lines showed recurrent genomic alterations including gains of 5p14-15, 20q11 and 20q13 and losses of 4p13-p16, 18q12, 18q21, Xq21.1 and Xq26-q28 that were barely detected in tumor tissue specimens. These are considered to be cell line-specific DCNAs. The frequency of the HER2 amplification was high in both cell lines and tumor tissues, but it was statistically different between cell lines and primary tumors (P = 0.012); 41.3 ± 29.9% for the cell lines and 15.9 ± 18.6% for the tissue specimens.
Established cell lines carry cell lines-specific DCNAs together with recurrent aberrations detected in primary tumor tissues. It must therefore be emphasized that cell lines do not always represent the genotypes of parental tumor tissues.
PMCID: PMC2836299  PMID: 20070913
24.  Increased serum cell-free DNA levels in relation to inflammation are predictive of distant metastasis of esophageal squamous cell carcinoma 
Distant metastasis hinders a favorable outcome for patients with esophageal squamous cell carcinoma (ESCC) by limiting the surgical cure. The levels of cell-free DNA (cfDNA) in the blood have served as a predictor for metastasis and recurrence in distant organs in liver cancer. Thus, this study tested the clinical efficacy of serum cfDNA levels as a predictive marker for distant metastasis of ESCC. We investigated cfDNA levels in a cohort of 101 ESCC patients and 46 age- and gender-matched control patients with benign disease. We found that serum cfDNA levels were significantly higher in the ESCC patients than in the control patients (P=0.034). In the ESCC patients, serum cfDNA levels were positively associated with tumor size and cytokeratin 19 fragment (CYFRA 21-1) expression (r=0.416 and r=0.573, respectively). An increase in cfDNA levels was also associated with host inflammation status including C-reactive protein levels and neutrophil and monocyte numbers in the peripheral blood. Serum cfDNA levels tended to be higher in advanced tumors when compared to early stage tumors. We found that serum cfDNA levels were significantly higher in ESCC patients with distant metastasis than in those without (P=0.011). Logistic regression analysis showed that serum cfDNA levels represented only one independent risk factor for distant metastasis among the five factors tested including gender, age, cfDNA levels, CYFRA 21-1 and squamous cell carcinoma antigen levels (P=0.0414). These results suggest that increased serum cfDNA levels may serve as a useful predictor for distant metastasis of ESCC.
PMCID: PMC3490399  PMID: 23136599
cell-free DNA; esophageal squamous cell carcinoma; real-time PCR; prognosis; serum
25.  Frizzled-7 as a Potential Therapeutic Target in Colorectal Cancer1 
Neoplasia (New York, N.Y.)  2008;10(7):697-705.
We investigated whether one of the Wnt receptors, frizzled-7 (FZD7), functions in the canonical Wnt signaling pathway of colorectal cancer (CRC) cells harboring an APC or CTNNB1 mutation and may be a potential therapeutic target for sporadic CRCs. The expression level of FZD gene family members in colon cancer cells and primary CRC tissues were determined by real-time PCR. Activation of the Wnt signaling pathway was evaluated by TOPflash assay. The expression level of Wnt target genes was determined by real-time polymerase chain reaction and/or Western blot analysis. Cell growth and cell invasion were assessed by MTS and matrigel assays, respectively. Among 10 FZD gene family members, FZD7 mRNA was predominantly expressed in six colon cancer cell lines with APC or CTNNB1 mutation. These six cell lines were transfected with FZD7 cDNA together with a TOPflash reporter plasmid, resulting in a 1.5- to 24.3-fold increase of Tcf transcriptional activity. The mRNA expression levels of seven known Wnt target genes were also increased by 1.5- to 3.4-fold after transfection of FZD7 cDNA into HCT-116 cells. The six cell lines were then cotransfected with FZD7-siRNA and a TOPflash reporter plasmid, which reduced Tcf transcriptional activity to 20% to 80%. FZD7-siRNA was shown to significantly decrease cell viability and in vitro invasion activity after transfection into HCT-116 cells. Our present data demonstrated that FZD7 activates the canonical Wnt pathway in colon cancer cells despite the presence of APC or CTNNB1 mutation and that FZD7-siRNA may be used as a therapeutic reagent for CRCs.
PMCID: PMC2435005  PMID: 18592008

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