Microminipig, a non-rodent experimental animal optimized for life science research: preface

Fuji Micra Inc. has recently achieved success in a challenging and prospective project that produces the smallest pig in the world, the "Microminipig", at a breeding farm at the foothills of Mt. Fuji in Japan. Microminipigs weigh approximately 7.0 kg at 6 months of age when they are mature. Microminipigs have been provided to several research organizations in Japan as a non-rodent experimental animal optimized for life science research.     

Painless acute pancreatitis associated with sorafenib treatment: a case report

Sorafenib is a multikinase inhibitor that is used for the treatment of metastatic renal-cell carcinoma. We report the case of a patient with painless acute pancreatitis associated with sorafenib treatment. The patient was a 71-year-old man who had undergone surgery for left renal carcinoma and tumor thrombus in the inferior vena cava and right atrium (IVC-RA). After a follow-up period of 3 years, he developed right adrenal metastasis and received interferon (IFN)-alpha treatment. One year later, progression of the adrenal metastasis was observed, and he was admitted to a hospital for treatment with sorafenib, which was administered at a dose of 800 mg/day. Two weeks later, he developed painless acute pancreatitis associated with sorafenib treatment. Thereafter, sorafenib treatment was discontinued, and he was treated with conservative therapy. Three weeks later, he was discharged. Even though painless acute pancreatitis associated with sorafenib treatment is rare, the possible development of painless acute pancreatitis in patients undergoing sorafenib treatment must be kept in mind.     

Laparoscopy-assisted subtotal gastrectomy with very small remnant stomach: a novel surgical procedure for selected early gastric cancer in the upper stomach

Total gastrectomy or proximal gastrectomy is usually performed either as an open procedure or laparoscopically for the treatment of early gastric cancer (EGC) in the upper stomach. However, quality of life after either total or proximal gastrectomy is not so satisfactory. The authors report a novel surgical procedure, laparoscopy-assisted subtotal gastrectomy (LAsTG), by which a very small remnant stomach is preserved, for the surgery of selected EGCs in the upper stomach. Twenty-three patients with EGC in the upper stomach underwent LAsTG. After lymph node dissection and mobilization of the stomach, the stomach was transected about 2 cm proximal to the tumor and a very small remnant stomach was preserved. An anvil was inserted transorally into the remnant stomach by using the OrVil™ system. The reconstruction method was Roux-en-Y, and hemidouble-stapling gastrojejunostomy with a circular stapler was performed intracorporeally. There were no intraoperative complications or conversions to open surgery. Mean operation time and blood loss were 266.7 min and 54.6 ml, respectively. The overall incidence of early postoperative complications was 17.4%, and two patients underwent reoperation because of duodenal stump leakage and stenosis of the Y-anastomosis, respectively. During the follow-up period, two patients experienced gastrojejunostomy stenosis and both were treated successfully by endoscopic balloon dilation. LAsTG may be performed in selected patients with EGC in the upper stomach. With the described method, a very small remnant stomach can be preserved.     

Mesenchymal Stem Cells Display Tumor-Specific Tropism in an RCAS/Ntv-a Glioma Model

Bone marrow-derived mesenchymal stem cells (MSCs) have been shown to localize to gliomas and deliver therapeutic agents. However, the clinical translation of MSCs remains poorly defined because previous studies relied on glioma models with uncertain relevance to human disease, typically xenograft models in immunocompromised mice. To address this shortcoming, we used the RCAS/Ntv-a system, in which endogenous gliomas that recapitulate the tumor and stromal features of human gliomas develop in immunocompetent mice. MSCs were harvested from bonemarrowof Ntv-a mice and injected into the carotid artery of Ntv-a mice previously inoculated with RCAS-PDGF-B and RCAS-IGFBP2 to induce malignant gliomas (n = 9). MSCs were labeled with luciferase for in vivo bioluminescence imaging (BLI). After intra-arterial injection, BLI revealed MSCs in the right frontal lobe in seven of nine mice. At necropsy, gliomas were detected within the right frontal lobe in all these mice, correlating with the location of the MSCs. In the twomice without MSCs based on BLI, no tumor was found, indicating thatMSC localization was tumor specific. In another cohort of mice (n = 9), MSCs were labeled with SP-DiI, a fluorescent vital dye. After intra-arterial injection, fluorescence microscopy revealed SP-DiI-labeled MSCs throughout tumors 1 to 7 days after injection but not in nontumoral areas of the brain. MSCs injected intravenously did not localize to tumors (n = 12). We conclude that syngeneic MSCs are capable of homing to endogenous gliomas in immunocompetent mice. These findings support the use of MSCs as tumor-specific delivery vehicles for treating gliomas.     

YM155, a selective survivin suppressant, inhibits tumor spread and prolongs survival in a spontaneous metastatic model of human triple negative breast cancer

Metastatic triple negative breast cancer [TNBC, with negative expression of estrogen and progesterone receptors and no overexpression of HER2/neu (ErbB-2)] remains a major therapeutic challenge because of its poor overall prognosis and lack of optimal targeted therapies. Survivin has been implicated as an important mediator of breast cancer cell growth and dysfunctions in apoptosis, and its expression correlates with a higher incidence of metastases and patient mortality; thus, survivin is an attractive target for novel anti-cancer agents. In previous studies, we identified YM155 as a small molecule that selectively suppresses survivin expression. YM155 inhibits the growth of a wide range of human cancer cell lines. Tumor regression induced by YM155 is associated with decreased intratumoral survivin expression, increased apoptosis and a decreased mitotic index. In the present study, we evaluated the antitumor efficacy of YM155 both in vitro and in vivo using preclinical TNBC models. We found that YM155 suppressed survivin expression, including that of its splice variants (survivin 2B, δEx3 and 3B), resulting in decreased cellular proliferation and spontaneous apoptosis of human TNBC cells. In a mouse xenograft model, continuous infusion of YM155 led to the complete regression of subcutaneously established tumors. Furthermore, YM155 reduced spontaneous metastases and significantly prolonged the survival of animals bearing established metastatic tumors in the MDA-MB-231-Luc-D3H2-LN orthotopic model. These results suggest that the survivin-suppressing activity of YM155 may offer a novel therapeutic option for patients with metastatic TNBC.     

(-)-Epigallocatechin-3-gallate induces apoptosis in gastric cancer cell lines by down-regulating survivin expression

The polyphenol (-)-epigallocatechin-3-gallate (EGCG) is a green tea constituent, which has been shown to inhibit cancer cell growth in vitro, in vivo and in epidemiological studies. In this study, we investigated its effects in gastric cancer cell lines. Five gastric cancer cell lines, the MKN-1, MKN-28, MKN-45, NUGC-3 and TMK-1, were found to be sensitive to EGCG treatment. Of all the cell lines tested, NUGC-3 was the most sensitive. EGCG treatment of NUGC-3 cells induced apoptosis, which was confirmed by sub-G1 analysis, caspase-Glo assay and Western blotting against cleaved PARP and cleaved caspase-3. EGCG treatment lowered survivin and increased Bax and TRAIL expression. Furthermore, EGCG induced p73 activation in NUGC-3 cells. Small interfering RNA against p73 diminished EGCG effects on survivin expression and cell viability. These results show that EGCG induces cell death in gastric cancer cells by apoptosis via inhibition of survivin expression downstream of p73. This study provides a novel mechanism whereby EGCG potentially inhibits cancer cell growth, concluding that EGCG may be a potential candidate in anti-survivin cancer therapy.     

Anti-adult T-cell leukemia effects of Bidens pilosa

We evaluated the effects of Bidens pilosa, a plant found in tropical and subtropical regions, and investigated the molecular pathways responsible for the anti-adult T-cell leukemia (ATL) effect. Water extracts of B. pilosa had growth suppressive effects on human T-cell leukemia virus type 1 (HTLV-1)-infected T-cell lines and ATL cells. B.?pilosa extracts arrested cells in G1 cell cycle and induced apoptosis of HTLV-1-infected T-cell lines. B. pilosa extracts inhibited also the phosphorylation of IκB kinase β and IκBα, and NF-κB-DNA binding, in conjunction with reduction of expression of proteins involved in G1/S cell cycle transition and suppression of apoptosis. Reactive oxygen species played a role in B.?pilosa-mediated suppression of NF-κB activity. B.?pilosa extracts also inhibited the expression of JunB and JunD, resulting in suppression of AP-1-DNA binding. In animals harboring tumors of HTLV-1-infected T-cell origin, treatment with B. pilosa extracts suppressed tumor growth. Our results suggest that B. pilosa is a potentially useful medicinal plant for treatment of ATL.     

NRAS and BRAF mutation frequency in primary oral mucosal melanoma

Oral mucosal melanoma (OMM) is a fatal sarcoma of unknown etiology. Histological morphology and genetic events are distinct from those of its cutaneous counterpart. Mutation and up-regulation of c-kit has been identified in OMM which may activate downstream molecules such as RAS and RAF. These molecules are involved in the mitogen-activated protein kinase (MAPK) pathway leading to tremendous cell proliferation and survival. NRAS and BRAF mutation and protein expression have been studied in other melanoma subtypes. The purpose of this study was to determine RAS protein expression and NRAS and BRAF mutation in 18 primary OMM cases using immunohistochemistry and mutation analysis. Results showed that RAS is intensely expressed in both in situ and invasive OMMs. However, NRAS mutation was only observed in 2/15 polymerase chain reaction (PCR) amplified cases both of which were silent mutations. On the other hand, BRAF missense mutations were observed only in 1/15 cases with PCR amplification. NRAS and BRAF mutations were independent from previously reported c-kit mutations. The classical V600E BRAF mutation was not found; instead a novel V600L was observed suggesting that the oncogenic event in OMM is different from that in skin melanoma. The low frequency of NRAS and BRAF mutations indicate that these genes are not common, but probable events in OMM pathogenesis, most likely independent of c-kit mutation.     

A 4-week versus a 3-week schedule of gemcitabine monotherapy for advanced pancreatic cancer: a randomized phase II study to evaluate toxicity and dose intensity

Background

This randomized phase II study compared the efficacy and toxicity between 4-week and 3-week schedules of gemcitabine monotherapy in advanced pancreatic cancer.

Methods

Patients with advanced pancreatic cancer were randomly assigned to either a 4-week schedule (gemcitabine at 1000?mg/m2 as a 30-min infusion weekly for 3 consecutive weeks every 4?weeks) or a 3-week schedule (gemcitabine at 1000?mg/m2 as a 30-min infusion weekly for 2 consecutive weeks every 3?weeks). The primary endpoint was the compliance rate during the first 8?weeks between the two groups.

Results

A total of 90 patients were enrolled. The compliance rate during the first 8?weeks was the same (53.3%). For the 4- and 3-week schedules, the tumor response rates were 14.2 and 17.1% (p?=?0.92), median progression free survival was 112 and 114?days (p?=?0.82), and median overall survival was 206 and 250?days (p?=?0.84), respectively. Grade 3?? neutropenia was the major adverse event in both schedules: 37.7 and 35.5% (p?=?0.82). In contrast, thrombocytopenia (platelet count <70000/mm3) was significantly higher for the 4-week schedule: 26.6 and 4.4% (p?=?0.008). The mean received dose intensity was equal: 588 and 550?mg/m2/week (p?=?0.14).

Conclusions

The 3-week schedule of gemcitabine did not improve the compliance rate during 8?weeks compared with the 4-week schedule, but it attained a comparable efficacy with lower toxicity. Further investigation will be needed to introduce it into daily practice. Clinical trial registration number: UMIN ID 974.     

Use of quantitative PCR to evaluate methods of bacteria sampling in periodontal patients

Periodontal disease is associated with specific periodontal pathogens and may persist as gingivitis or progress to more severe disease. The bacteria involved in disease initiation and progression have not been identified. We used quantitative polymerase chain reaction (PCR) to compare the levels of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, and total bacteria detected by different sampling methods. On the basis of the results of clinical examinations, 57 patients were divided into 3 groups: healthy group (group A), gingivitis group (group B), and periodontitis group (group C). Bacterial samples were collected from saliva, mouthwash, and by paper-point sampling of gingival crevicular fluid (GCF), and the samples were analyzed with quantitative PCR targeting 16S rRNA. The numbers of total bacteria in samples of GCF, saliva, and mouthwash were 10? to 10?, 10?, and 10?, respectively, per milliliter. The number of P. gingivalis in GCF samples was lower than 10 in group A; however, in groups B and C, the values were 103 and 10?, respectively, indicating that the number of P. gingivalis increased with worsening clinical status. Findings were similar in the samples of saliva and mouthwash. The numbers of T. forsythia showed a pattern similar to that of P. gingivalis in all 3 samples. These results suggest that saliva and mouthwash samples are clinically useful for bacterial testing of periodontal diseases by quantitative PCR. In addition, mouthwash sampling is more feasible and straightforward than saliva sampling.