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Many approaches have been taken to identify new biomarkers of pancreatic ductal carcinoma (PDC). Since animal models can be sampled under controlled conditions, better standardization is possible compared with heterogeneous human studies. Transgenic rats with conditional activation of oncogenic RAS in pancreatic tissue develop PDC that closely resembles the biological and histopathological features of human PDC. Using this model, we evaluated the usefulness of leucine-rich α2-glycoprotein-1 (LRG-1) as a serum marker. In this study, we found that LRG-1 was overexpressed in rat PDC compared with normal pancreas tissue of the control rats. Serum levels of LRG-1 were also significantly higher in rats bearing PDC than in controls. Importantly, chronic pancreatitis in male Wistar Bonn/Kobori rats, which is a widely accepted as a model of chronic pancreatitis, did not cause serum levels of LRG-1 to become elevated. These results strongly support serum LRG-1 as a candidate biomarker for noninvasive diagnosis of PDC. Our models of pancreas cancer provide a useful strategy for evaluation of candidate markers applicable to human cancer.

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.

Multiwalled carbon nanotubes (MWCNT) have a fibrous structure and physical properties similar to asbestos and have been shown to induce malignant mesothelioma of the peritoneum after injection into the scrotum or peritoneal cavity in rats and mice. For human cancer risk assessment, however, data after administration of MWCNT via the airway, the exposure route that is most relevant to humans, is required. The present study was undertaken to investigate the carcinogenicity of MWCNT‐N (NIKKISO) after administration to the rat lung. MWCNT‐N was fractionated by passing it through a sieve with a pore size of 25 μm. The average lengths of the MWCNT were 4.2 μm before filtration and 2.6 μm in the flow‐through fraction; the length of the retained MWCNT could not be determined. For the present study, 10‐week‐old F344/Crj male rats were divided into five groups: no treatment, vehicle control, MWCNT‐N before filtration, MWCNT‐N flow‐through and MWCNT‐N retained groups. Administration was by the trans‐tracheal intrapulmonary spraying (TIPS) method. Rats were administered a total of 1 mg/rat during the initial 2 weeks of the experiment and then observed up to 109 weeks. The incidences of malignant mesothelioma and lung tumors (bronchiolo‐alveolar adenomas and carcinomas) were 6/38 and 14/38, respectively, in the three groups administered MWCNT and 0/28 and 0/28, respectively, in the control groups. All malignant mesotheliomas were localized in the pericardial pleural cavity. The sieve fractions did not have a significant effect on tumor incidence. In conclusion, administration of MWCNT to the lung in the rat induces malignant mesothelioma and lung tumors.

Translocation of multiwalled carbon nanotubes (MWCNTs) from the lung to the pleural cavity, deposition of the fibers in the pleural tissue, induction of pleural fibrosis, and mesothelial proliferation have been found in rodents administered MWCNTs by different pulmonary exposure methods. However, whether the translocation and deposition and the subsequent pleural inflammation are associated with the pleural lesions is unclear. In the present study, male F344 rats were given 250 μg of two types of MWCNTs, with crocidolite as a positive control, 2 times/week for 4 weeks by intratracheal spraying. At 24 h and at 3 months after the last spraying, the rats were sacrificed for histological examination of the lung and chest wall; pleural cavity lavage was also collected at sacrifice for observation of pleural inflammatory reactions. The results indicated that intratracheally sprayed MWCNTs, like crocidolite fibers, translocated into the pleural cavity, deposited in the pleura, and induced persistent infiltration of immune cells into the pleural cavity, persistent pleural fibrosis, and mesothelial proliferation. The number of MWCNT fibers detected in the pleural cavity lavage was parallel to the number of infiltrating immune cells, which were mainly composed of macrophages. Analysis of cytokines in the fluids of the pleural cavity lavages by suspension array indicated that levels of IL-2, IL-18, and IP-10 were significantly increased both at 24 h and at 3 months after the last spraying. In vitro proliferation assays revealed that a mixture of IL-2, IL-18, and IP-10, but not any of these cytokines alone, promoted cell proliferation of human fibroblasts and mesothelial cells. These results suggest that translocated and deposited MWCNTs induce subsequent pleural inflammation and that increased IL-2, IL-18, and IP-10 synergistically promote the development of pleural fibrosis and mesothelial proliferation.

The aim of the present study is to investigate the "chronotoxicity" of seven metal compounds (Hg, Pb, Ni, Cr, Cu, Zn, or Fe) by assessing how their toxicity varies with circadian periodicity. Male ICR mice were injected with each metal compound intraperitoneally at 6 different time points over the course of a day (zeitgeber time [ZT]: ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22). Mortality was then monitored until 14 days after the injection. Our investigation demonstrated that mice were tolerant against Ni toxicity during dark phase, on the other hand, they were tolerant against Cr toxicity during light phase. The chronotoxicity of Hg and Pb seemed to be biphasic. Further, mice were susceptible to toxicities against Cu and Zn in the time zone during which light and dark were reversed. Interestingly, no significant differences were observed for Fe exposure at any time of the day. Our results propose that the chronotoxicology may provide valuable information regarding the importance of injection timing for not only toxicity evaluation tests but also the reproducibility of animal experiments. Furthermore, our data suggests that chronotoxicology may be an important consideration when evaluating the quality of risk assessments for night shift workers who may be exposed to toxic substances at various times of the day.

Acute mesenteric ischemia (AMI) is caused by considerable intestinal injury, which is associated with intestinal ischemia followed by reperfusion. To elucidate the mechanisms of ischemia/reperfusion injuries, a C5a inhibitory peptide termed AcPepA was used to examine the role of C5a anaphylatoxin, induction of inflammatory cells, and cell proliferation of the intestinal epithelial cells in an experimental AMI model. In this rat model, the superior mesenteric artery was occluded and subsequently reperfused (Induce-I/R). Other groups were treated with AcPepA before ischemia or reperfusion. Induce-I/R induced injuries in the intestine and AcPepA significantly decreased the proportion of severely injured villi. Induce-I/R induced secondary receptor for C5a-positive polymorphonuclear leukocytes in the vessels and CD204-positive macrophages near the injured site; this was correlated with hypoxia-induced factor 1-alpha-positive cells. Induction of these inflammatory cells was attenuated by AcPepA. In addition, AcPepA increased proliferation of epithelial cells in the villi, possibly preventing further damage. Therefore, Induce-I/R activates C5a followed by the accumulation of polymorphonuclear leukocyte and hypoxia-induced factor 1-alpha-producing macrophages, leading to villus injury. AcPepA, a C5a inhibitory peptide, blocks the deleterious effects of C5a, indicating it has a therapeutic effect on the inflammatory consequences of experimental AMI.

Exposure to asbestos results in serious risk of developing lung and mesothelial diseases. Currently, there are no biomarkers that can be used to diagnose asbestos exposure. The purpose of the present study was to determine whether the levels or detection rate of chemokine (C-C motif) ligand 3 (CCL3) in the serum are elevated in persons exposed to asbestos. The primary study group consisted of 76 healthy subjects not exposed to asbestos and 172 healthy subjects possibly exposed to asbestos. The secondary study group consisted of 535 subjects possibly exposed to asbestos and diagnosed with pleural plaque (412), benign hydrothorax (10), asbestosis (86), lung cancer (17), and malignant mesothelioma (10). All study subjects who were possibly exposed to asbestos had a certificate of asbestos exposure issued by the Japanese Ministry of Health, Labour and Welfare. For the primary study group, levels of serum CCL3 did not differ between the two groups. However, the detection rate of CCL3 in the serum of healthy subjects possibly exposed to asbestos (30.2%) was significantly higher (P

We examined the effects of recombinant human osteoclastogenesis inhibitory factor (hOCIF) on osteolysis, proliferation of mammary tumor cells, and induction of cancer stem cells (CSCs) in the tumor-bone and tumor-subcutaneous microenvironments (TB- and TS-microE). Methods: Mouse mammary tumor cells were transplanted onto the calvaria or into a subcutaneous lesion of female mice, creating a TB-microE and a TS-microE, and the mice were then treated with hOCIF. To investigate the preventive effects of hOCIF, mice were treated with hOCIF before tumor cell implantation onto the calvaria (Pre), after (Post), and both before and after (Whole). The number of CSCs and cytokine levels were evaluated by IHC and ELISA assay, respectively. Results: hOCIF suppressed osteolysis, and growth of mammary tumors in the TB-microE, but not in the TS-microE. In the Pre, Post, and Whole groups, hOCIF suppressed osteolysis, and cell proliferation. hOCIF increased mouse osteoprotegrin (mOPG) levels in vivo, which suppressed mammary tumor cell proliferation in vitro. These preventive effects were observed in the dose-dependent. hOCIF did not affect the induction of CSCs in either microenvironment. Conclusion: While receptor activator of NF-κB ligand (RANKL) targeting therapy may not affect the induction of CSCs, RANKL is a potential target for prevention as well as treatment of breast cancer bone metastasis., International Journal of Molecular Sciences, 19(3), 888; 2018

Acute mesenteric ischemia (AMI) is caused by considerable intestinal injury, which is associated with intestinal ischemia followed by reperfusion. To elucidate the mechanisms of ischemia/reperfusion injuries, a C5a inhibitory peptide termed AcPepA was used to examine the role of C5a anaphylatoxin, induction of inflammatory cells, and cell proliferation of the intestinal epithelial cells in an experimental AMI model. In this rat model, the superior mesenteric artery was occluded and subsequently reperfused (Induce‐I/R). Other groups were treated with AcPepA before ischemia or reperfusion. Induce‐I/R induced injuries in the intestine and AcPepA significantly decreased the proportion of severely injured villi. Induce‐I/R induced secondary receptor for C5a‐positive polymorphonuclear leukocytes in the vessels and CD204‐positive macrophages near the injured site; this was correlated with hypoxia‐induced factor 1‐alpha‐positive cells. Induction of these inflammatory cells was attenuated by AcPepA. In addition, AcPepA increased proliferation of epithelial cells in the villi, possibly preventing further damage. Therefore, Induce‐I/R activates C5a followed by the accumulation of polymorphonuclear leukocyte and hypoxia‐induced factor 1‐alpha‐producing macrophages, leading to villus injury. AcPepA, a C5a inhibitory peptide, blocks the deleterious effects of C5a, indicating it has a therapeutic effect on the inflammatory consequences of experimental AMI.

Bone is the most common target organ of metastasis of prostate and breast cancers. This produces considerable morbidity due to skeletal-related events, SREs, including bone pain, hypercalcemia, pathologic fracture, and compression of the spinal cord. The mechanism of bone metastasis is complex and involves cooperative reciprocal interaction among tumor cells, osteoblasts, osteoclasts, and the mineralized bone matrix. The interaction between the metastatic tumor and bone stromal cells has been commonly referred to as the "vicious cycle". Tumor cells stimulate osteoblasts, which in turn stimulate osteoclasts through the secretion of cytokines such as the TNF family member receptor activator of nuclear κB ligand (RANKL). Activated osteoclasts degrade the bone matrix by producing strong acid and proteinases. Bone degradation by osteoclasts releases TGFβ and other growth factors stored in the bone matrix, that further stimulate tumor cells. Bone modifying agents, targeting osteoclast activity, such as bisphosphonate and RANKL antibodies are considered as the standard of care for reducing SREs of patients with bone metastatic diseases. These agents decrease osteoclast activity and delay worsening of skeletal pain and aggravation of bone metastatic diseases. While the management of SREs by these agents may improve patients' lives, this treatment does not address the specific issues of the patients with bone metastasis such as tumor dormancy, drug resistance, or improvement of survival. Here, we review the mechanisms of bone metastasis formation, tumor heterogeneity in the bone microenvironment, and conventional therapy for bone metastatic diseases and discuss the potential development of new therapies targeting tumor heterogeneity in the bone microenvironment.

A novel KRAS-mediated transgenic rat model has previously been demonstrated, in which animals develop multiple pancreatic ductal adenocarcinoma (PDAC) that is histologically similar to human PDAC within two weeks. Positron emission tomography (PET)/computed tomography (CT) is commonly used for the diagnosis and staging of PDAC in humans, and can be adopted for optimal use in animal experiments. The aim of the present study was to evaluate the carcinogenic process in a rat pancreatic carcinoma model using small-animal multimodality imaging systems. The utility of fluorodeoxyglucose (FDG)-PET/CT in detecting the location and size of PDAC during tumor development in the present transgenic rat model was assessed. A small animal multimodality PET/CT system and contrast-enhanced CT (CECT) system were used for the imaging analysis of KRASG12V male transgenic rats (n=6), which developed pancreatic tumors following the administration of an injection of Cre recombinase (Cre)-carrying adenovirus. Laparotomies performed at six weeks post-treatment revealed that all three (100%) Cre-expressing rats developed pancreatic tumors that were

Musa basjoo (MB) is a tropical evergreen tree growing mainly in subtropical or tropical countries. MB has been used globally as a folk medicine such as antipyretic, diuretic, and hemostatic drugs for centuries but evidence-based biological activities and molecular mechanism of action of MB are unknown. Therefore, in the current study we examined whether the crude extracts of MB exert anticancer activity in HT29 and HCT116 human colon carcinoma cell lines. Dried leaves of MB samples were extracted with acetone or methanol. Crude extracts of MB were then dissolved in dimethylsulfoxide (DMSO) and used for the following experiments. Growth inhibition was determined by colony or MTT assays in these cell lines. Cells were treated with increasing concentrations (12.5 to 200 μg/mL in colony assays, 25 to 400 μg/mL in MTT assays) of acetone/methanol extracts of MB in DMEM/5%FBS. Untreated control cells were treated with DMSO alone. Crude extracts of MB inhibited the growth of cells with IC50 values of 118 µg/mL (acetone extract, HT29), 75 µg/mL (acetone extract, HCT116), >200 µg/mL (methanol extract, HT29), 141 µg/mL (methanol extract, HCT116) in colony assays, and with IC50 values of 137 µg/mL (acetone extract, HT29), 73 µg/mL (acetone extract, HCT116), 240 µg/mL (methanol extract, HT29), 248 µg/mL (methanol extract, HCT116) in MTT assays. Acetone extract was used in the flowcytometry and western blot analyses because it showed stronger growth inhibition than methanol extract in both cell lines. Flowcytometry analysis indicated that when HT29 and HCT116 cells were treated with 100 µg/mL acetone extract of MB for 96h, the percentage of cells in G1 increased by 5.4% and this was associated with a concomitant decrease of cells in the S and G2-M phases of the cell cycle. Acetone extract of MB did not cause subG1 fraction in either HT29 or HCT116 cell lines. The results indicate that acetone extract of MB causes carcinoma cells to arrest in the G1 phase. We then performed western blot analysis to determine whether treatment of carcinoma cells with acetone extract of MB alters cellular levels of the G1 cell cycle control proteins cyclin D1, cdk4 and the cell cycle inhibitor protein p21CIP1. When HT29 and HCT116 cells were treated with 50 and 100 µg/mL acetone extract of MB for 96h, there was a marked decrease in the levels of expression of the cyclin D1 and cdk4 proteins and a marked increase in the levels of expression of the p21CIP1 protein. Thus, a decrease in cyclin D1 may cooperate with the induction of p21CIP1 to arrest cells in G1 and thereby further contribute to MB-induced growth inhibition. Taken together, the crude extracts of MB contain active component(s) that exert growth inhibition of human colon carcinoma cells. The current study is the first systematic examination of the anticancer activity of MB and may provide a novel approach to the chemoprevention and/or chemotherapy of human colon carcinoma. Citation Format: Harutoshi Matsumoto, Saeko Ando, Katsumi Fukamachi, Mitsuru Futakuchi, Kazunori Kimura, Naoki Yoshimi, Masumi Suzui. Growth inhibition of the crude extracts of Musa basjoo in human colon carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2178. doi:10.1158/1538-7445.AM2017-2178

The final goal of the current study is to create a promising lead compound that targets human colon carcinoma. After discovering in vitro antiproliferative activity of a naturally occurring compound, 10-hydroxy-2-decenoic acid, we designed and synthesized over 150 derivatives. The initial screening resulted in the discovery of three primary lead compounds, we then proceeded with modifications in side chain and eventually obtained a second-generation lead compound, named PPI (patented in 2014), whose IC50 was 0.3 μM. We focused on the transcription factor STAT3 that is able to drive carcinogenesis, apoptosis, and angiogenesis-related genes. In silico docking simulation exhibited that PPI can bind to SH2 domain of STAT3, suggesting that this drug blocks dimerization of STAT3, thereby PPI inactivates function of STAT3. PPI inhibited transcriptional activity of STAT3 when SW480, SW837, and HT29 human colon carcinoma cells were treated with 3.7 to 5 μM PPI for 24h. Flowcytometry analysis indicated that when HT29 cells were treated with 1 μM PPI, the percentage of cells in G1 increased by 5% and this was associated with a concomitant decrease of cells in the S and G2-M phases of the cell cycle. The results indicate that PPI causes carcinoma cells to arrest in the G1 phase. When HT29 cells were treated with 10 μM PPI, subG1 fraction and DNA fragmentation were seen, indicating induction of apoptosis. We then performed western blot analysis to determine whether treatment of carcinoma cells with PPI alters cellular levels of the G1 cell cycle control protein cyclin D1 and apoptosis/angiogenesis-related molecules. When HT29 cells were treated with PPI for 24 to 48h, there was a marked decrease in the levels of expression of the Bcl-2, Bcl-xL, and VEGF proteins and a marked increase in the levels of expression of the cleaved caspase 3, 7, 8, 9, and PARP proteins. PPI also inhibited expression levels and nuclear tanslocation of pSTAT3. To see if PPI exerts preventive effect on tumor promotion, F344 rats received sc injections of a carcinogen azoxymethane and then treated with soybean oil or PPI. There was a dose dependent decrease in multiplicity of aberrant crypt foci (ACF) in the colon. PPI inhibited the occurrence of larger ACF consisting of more than 4 aberrant crypts, indicating inhibitory effect of PPI on tumor promotion. In a mouse xenograft model, PPI decreased the size and number of blood vessels in the tumor. All mice survived without causing significant body weight loss during experiment. No histopathological abnormality was found in any of organ site. To confirm the effects of PPI on angiogenesis, we performed chorioallantoic membrane assays and found a dose-dependent decrease in average number of blood vessels. Inhibition of STAT3 by PPI may affect the function of molecules that are related to apoptosis, angiogenesis, and cell cycle progression and eventually contributes to PPI-induced growth inhibition. Citation Format: Masumi Suzui, Saeko Ando, Harutoshi Matsumoto, Katsumi Fukamachi, Mitsuru Futakuchi, Naoki Yoshimi. A new anticancer agent derived from decenoic acid for the treatment of colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 182. doi:10.1158/1538-7445.AM2017-182