Diurnal variation of cisplatin-induced renal toxicity in ICR mice.

Cisplatin (CDDP) is a platinum-based anticancer drug widely prescribed for its effectiveness in treating various forms of cancer. However, its major side effect is nephrotoxicity. Although several methods have been developed to mitigate CDDP-induced nephrotoxicity, an optimal approach has yet to be established. This study aimed to investigate the "chronotoxicity" of CDDP as a potential strategy to reduce its side effects. Male ICR mice were treated with CDDP (20 mg/kg, intraperitoneal injection, one shot) at zeitgeber time (ZT) 2 or ZT14 (light or dark phase). After 72 h, we collected plasma and kidney and evaluated several markers. We found that body weight change between ZT2 and ZT14 by CDDP was comparable. In contrast, many toxicological factors, such as plasma blood urine nitrogen, plasma creatinine, renal oxidative stress (malondialdehyde), DNA damage (γH2AX), acute kidney injury biomarker (KIM-1), and inflammation (Tnfα), were significantly induced at ZT14 compared to than that of ZT2. Our present data suggested that chronotoxicology might provide beneficial information on the importance of administration timings for toxic evaluations and unacceptable side effects.

Copper-induced diurnal hepatic toxicity is associated with Cry2 and Per1 in mice.

BACKGROUND: This study aimed to investigate diurnal variations in copper-induced hepatic toxicity and the molecular mechanisms underlying this chronotoxicity. METHODS: Male C57BL/6J mice were intraperitoneally injected with copper chloride (CuCl2) at zeitgeber time 2 (ZT2) or 14 (ZT14), twice per week for 5 or 8 weeks. Seventy-two hours after the final CuCl2 injection, the mice were euthanized, and plasma samples were collected. The livers and kidneys were collected and weighed. In vitro experiments were performed to assess cell viability and fluctuations in clock gene expression levels in Hepa1-6 cells after CuCl2 treatment. We examined copper homeostasis- and apoptosis-related genes under clock genes overexpression. RESULTS: Repeated CuCl2 administration for 8 weeks resulted in more severe toxicity at ZT14 compared to ZT2. CuCl2 administration at ZT14 elevated plasma aspartate aminotransferase, hepatic tumor necrosis factor-α, and interleukin-6 for 5 weeks, whereas the toxic effects of CuCl2 administration at ZT2 were weaker. Moreover, CuCl2 treatment inhibited Hepa1-6 cell viability in a dose-dependent manner. We observed increased expression of three clock genes (Ciart, Cry2, and Per1) after CuCl2 treatment. Among them, overexpression of Cry2 and Per1 accelerated CuCl2-induced inhibition of Hepa1-6 cell viability. Moreover, we found that the overexpression of Cry2 and Per1 regulates cleaved caspase-3 by modulating the copper transporter genes ATP7B and CTR1. CONCLUSION: These results suggest that CuCl2-induced diurnal toxicity is associated with Cry2 and Per1 expression through the regulation of copper transporter genes in mice.

Involvement of Bmal1 and Clock in Bromobenzene Metabolite-Induced Diurnal Renal Toxicity.

Circadian rhythms are endogenous oscillators that regulate 24 h behavioral and physiological processes. Our previous investigation demonstrated that bromobenzene metabolite (4-bromocatechol: 4-BrCA) exhibited chronotoxicity (i.e., the nephrotoxicity induced by 4-BrCA was observed during the dark phase, while not observed at light phase in mice). However, the molecular mechanism is still unknown. The aim of the present study is to investigate the cellular molecule(s) involved in the 4-BrCA-induced nephrotoxicity using mouse renal cortex tubular cell lines (MuRTE61 cells). We found that 4-BrCA showed dose dependent (0.01-1 mM) cell proliferation defect in MuRTE61 cells. By treating with 0.03 mM 4-BrCA, we demonstrated that major clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) were significantly downregulated. Interestingly, the expression levels of two genes, Bmal1 and Clock, continued to decrease after 3 h of treatment with 4-BrCA, while Cry1, Per1, and Per2 were unchanged until 24 h of treatment. Moreover, BMAL1 and CLOCK levels are higher at light phase. We speculated that BMAL1 and CLOCK might function defensively against 4-BrCA-induced nephrotoxicity since the expression levels of Bmal1 and Clock were rapidly decreased. Finally, overexpression of Bmal1 and Clock restored 4-BrCA-induced cell proliferation defect in MuRTE61 cells. Taken together, our results suggest that Bmal1 and Clock have protective roles against 4-BrCA-induced nephrotoxicity.

Involvement of Npas2 and Per2 modifications in zinc-induced acute diurnal toxicity in mice.

Zinc (Zn) is one of the most essential trace elements in the body and an integral part of many enzyme systems. Zn deficiency is characterized by growth retardation, loss of appetite, and impaired immune function. In contrast, Zn overdoses can be associated with liver, kidney, and stomach damage. We focused on the "chronotoxicity," or the relationship between injection time and severity of chemical toxicity. The aim of this study was to investigate the chronotoxicity of Zn and the in vivo factors involved. Seven-week-old male ICR mice were administered Zn at six different time points per day (zeitgeber time [ZT]: ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22). Mortality was monitored for 7-days after administration. The mice were tolerant to Zn administered at ZT2 and ZT6, and were highly sensitive at ZT14 and ZT18. Furthermore, when mice were administered a non-lethal dose of Zn, the levels of hepatic injury indicators (AST and ALT) were much higher at ZT14 than at ZT2. To explore the mechanism of Zn-induced diurnal hepatotoxicity, we performed an in vitro experiment, focusing on the clock genes. We found that Zn downregulated the expression levels of several clock genes, neuronal PAS domain protein 2 (Npas2) and Peroid2 (Per2), in Hepa1-6 cells. Interestingly, overexpression of both Npas2 and Per2 restored Zn-induced toxicity in Hepa1-6 cells. Since NPAS2 and PER2 are known to modulate the hepatic injury induced by carbon tetrachrolide or acetaminophen, our results suggest that Zn-induced diurnal toxicity may be associated with modulation of Npas2 and Per2 gene expression.

Extracts of Musa basjoo induce growth inhibition and changes in the protein expression of cell cycle control molecules in human colorectal cancer cell lines.

Musa basjoo (MB) is a species of the banana plant belonging to the genus Musa that has been used as a folk medicine. However, evidence-based biological activities and the molecular mechanism of action of MB are unknown. Thus, the aim of the present study was to examine whether the crude dried leaf extracts of MB inhibit the growth of colorectal (HT29 and HCT116) and other types (HepG2, MCF-7 and PC-3) of human cancer cell lines. Crude extracts of MB inhibited the growth of cells with IC50 values of 136 µg/ml (acetone extract, HT29), 51 µg/ml (acetone extract, HCT116), 45 µg/ml (acetone extract, HepG2), 40 µg/ml (acetone extract, MCF-7), 29 µg/ml (acetone extract, PC-3), 175 µg/ml (methanol extract, HT29), 137 µg/ml (methanol extract, HCT116), 102 µg/ml (methanol extract, HepG2), 85 µg/ml (methanol extract, MCF-7), and 85 µg/ml (methanol extract, PC-3) in colony formation assays, and 126 µg/ml (acetone extract, HT29), 68 µg/ml (acetone extract, HCT116), 260 µg/ml (methanol extract, HT29), and 216 µg/ml (methanol extract, HCT116) in MTT assays. Thin layer chromatography analysis revealed the potential existence of aromatic compounds in the acetone extract of MB. Flow cytometric analysis indicated that the percentage of cells in G1 increased, and this was associated with a concomitant decrease of cells in the S and/or G2-M phases of the cell cycle. When colorectal cancer cells were treated with acetone extract of MB, there was a marked decrease in the levels of expression of the cyclin D1, cyclin E, cdk2 and cdk4 proteins and a marked increase in the levels of the expression of the p21CIP1, p27KIP1, and p53 proteins, but those of apoptosis-associated protein PARP did not change. There was a tendency for acetone extract of MB to inhibit xenograft tumor growth in mice. Collectively, the crude extracts of MB contain active components that exert growth inhibition of human cancer cells. This is the first systematic study of the anticancer activity of MB and may broaden insights into the possible clinical approach of specific herbal medicines.

Palmitoyl piperidinopiperidine, a novel derivative of 10­hydroxy­2­decenoic acid, as a potent and selective anticancer agent against human colon carcinoma cell lines.

The present study, to the best of our knowledge, is the first systematic study of the inhibitory effects of palmitoyl piperidinopiperidine (PPI; Japan Patent no. 5597427), on colon carcinogenesis. PPI exhibited marked growth inhibitory activity in several human colon carcinoma cell lines, with IC50 values of approximately 0.5­2.2 µM. In silico docking analysis indicated that PPI could bind to the SH2 domain of signal transducer and activator of transcription 3 (STAT3). PPI markedly inhibited the transcriptional activity of the SW837 cell line. Flowcytometric analysis demonstrated that PPI induced an increase in the number of cells in the G1 phase of the cell cycle, and induced sub­G1 fractions of cells at a higher concentration level of PPI. In the HT29 and SW837 cells, western blot analyses exhibited that in whole cell lysates, PPI induced a marked decrease in the expression levels of p­STAT3, but not in the levels of STAT3 in these cells. PPI also induced a marked decrease in the expression levels of both STAT3 and p­STAT3 in the chromatin fraction. In addition, PPI affected the protein expression levels of cyclin D1, p53, Bcl­2, Bcl­xL and vascular endothelial growth factor (VEGF). In the HT29 cells, PPI induced a marked and dose­dependent increase in the expression levels of Bax, cleaved caspase­3, cleaved caspase­7, cleaved caspase­8, cleaved caspase­9 and cleaved poly (ADP­ribose) polymerase (PARP). In animal model systems, PPI inhibited the growth of implanted carcinoma cells, and also induced a significant decrease in the multiplicity of colonic aberrant crypt foci. In addition, a marked and dose­dependent inhibition of angiogenesis of the chick chorioallantoic membrane was observed. As regards the possible molecular mechanisms, it is suggested that the inhibition of STAT3 by PPI may affect the function of molecules that are related to apoptosis, angiogenesis and cell cycle progression, eventually contributing to the PPI­induced growth inhibitory effects.

Sasa veitchii extract induces anticancer effects via inhibition of cyclin D1 expression in MCF-7 cells.

Sasa veitchii and other Sasa species are traditional medicinal herbs belonging to a group of Japanese bamboos collectively called Kumazasa, and these species possess the potential for a wide variety of uses. The present study aimed to elucidate the anticancer mechanisms exerted by S. veitchii extract (SE) against a human breast cancer cell line, MCF-7 cells. Freeze-dried Sunchlon® was used as the SE, and cell proliferation activity was measured using the [3H]-thymidine incorporation assay. Induction of apoptosis was assessed via Annexin V and caspase-3 fluorescent staining, the induction of necrosis was measured via propidium iodide staining, and cell cycle-related protein expression was determined using western blotting. The IC50 value of the SE was 7.7 µg/mL in MCF-7 cells. Although the primary active ingredient in Sunchlon® is sodium copper chlorophyllin (0.25%), the present results indicated that ingredients other than SCC exert anti-cancer activities (the IC50 value of SCC was 715 µg/mL), and late apoptosis or necrosis was induced in an SE dose-dependent manner. The expression levels of cyclin D1 and Cdk6 were decreased after SE treatment, and there was no change in the Cdk1/2 expression levels. Additionally, the expression of the necrosis-related cell death indicators RIP1 and RIP3 was increased in response to high-dose SE treatments, and this was indicative of cells preparing for programmed cell death. SE induces cell death in MCF-7 cells via the inhibition of cyclin D1 expression at low concentrations, and this extract induces programmed necrosis (necroptosis) by potentiating RIP1/RIP3 expression.

Evaluation of a biomarker for the diagnosis of pancreas cancer using an animal model.

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.

Persistent Pleural Lesions and Inflammation by Pulmonary Exposure of Multiwalled Carbon Nanotubes.

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.

Therapeutic and Preventive Effects of Osteoclastogenesis Inhibitory Factor on Osteolysis, Proliferation of Mammary Tumor Cell and Induction of Cancer Stem Cells in the Bone Microenvironment.

BACKGROUND: 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.

Lethal chronotoxicity induced by seven metal compounds in mice.

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.

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial.

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 intratracheally instilled into the rat lung induce development of pleural malignant mesothelioma and lung tumors.

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.

C5a inhibitor protects against ischemia/reperfusion injury in rat small intestine.

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.

Heterogeneity of tumor cells in the bone microenvironment: Mechanisms and therapeutic targets for bone metastasis of prostate or breast cancer.

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 TGFß 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.

In vivo18F-fluorodeoxyglucose-positron emission tomography/computed tomography imaging of pancreatic tumors in a transgenic rat model carrying the human KRASG12V oncogene.

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 <2 mm in diameter, none of which were detected by 18F-FDG PET/CT or CECT. At eight weeks post-treatment, the pancreatic tumors were heterogeneously visualized by 18F-FDG-PET/CT and CECT in two of the three rats. Furthermore, the autopsies confirmed that all three rats had developed pancreatic tumors. These novel findings provide evidence that the FDG-PET/CT imaging system is a valuable tool for the evaluation of the carcinogenic process, and one which may aid in treatment and preventive methods for pancreatic tumors in mammalian models. A limitation associated with the early detection of PDACs warrants further investigation.

Chemokine (C-C motif) ligand 3 detection in the serum of persons exposed to asbestos: A patient-based study.

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 < 0.001) than for the control group (6.6%). The pleural plaque, benign hydrothorax, asbestosis, and lung cancer groups had serum CCL3 levels and detection rates similar to that of healthy subjects possibly exposed to asbestos. The CCL3 chemokine was detected in the serum of 9 of the 10 patients diagnosed with malignant mesothelioma. Three of the patients with malignant mesothelioma had exceptionally high CCL3 levels. Malignant mesothelioma cells from four biopsy cases and an autopsy case were positive for CCL3, possibly identifying the source of the CCL3 in the three malignant mesothelioma patients with exceptionally high serum CCL3 levels. In conclusion, a significantly higher percentage of healthy persons possibly exposed to asbestos had detectable levels of serum CCL3 compared to healthy unexposed control subjects.

Rat N-ERC/mesothelin as a marker for in vivo screening of drugs against pancreas cancer.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease, which is usually diagnosed in an advanced stage. We have established transgenic rats carrying a mutated K-ras gene controlled by Cre/loxP activation. The animals develop PDA which is histopathologically similar to that in humans. Previously, we reported that serum levels of N-ERC/mesothelin were significantly higher in rats bearing PDA than in controls. In the present study, to determine whether serum levels of N-ERC/mesothelin correlated with tumor size, we measured N-ERC/mesothelin levels in rats bearing PDA. Increased serum levels of N-ERC/mesothelin correlated with increased tumor size. This result indicates an interrelationship between the serum level of N-ERC/mesothelin and tumor size. We next investigated the effect of chemotherapy on serum N-ERC/mesothelin levels. Rat pancreatic cancer cells were implanted subcutaneously into the flank of NOD-SCID mice. In the mice treated with 200 mg/kg gemcitabine, tumor weight and the serum level of N-ERC/mesothelin were significantly decreased compared to controls. These results suggest that serum N-ERC/mesothelin measurements might be useful for monitoring response to therapy.

A novel transgenic mouse model carrying human Tribbles related protein 3 (TRB3) gene and its site specific phenotype.

Tribbles related protein 3 (TRB3) pseudokinase plays a crucial role in cell proliferation, migration and morphogenesis during development. In our recent study, an introduction of human TRB3 gene into mouse mammary tumor cells caused an increase of proliferation of tumor cells and their nuclear size. In the current study, to examine whether this gene causes de novo morphological changes in a specific organ site we have developed a novel variation of the transgenic mouse model that conditionally expresses human TRB3 (hTRB3) gene using Cre-recombinase (Cre)/loxP recombination system. By injecting hTRB3 transgene construct into pronuclei of mouse embryo, we eventually obtained four hTRB3 mice. The gene expression was controlled by infection of adenovirus-expressing Cre via the tail vein of hTRB3 mouse. In Cre-mediated hTRB3 mouse, expression of the hTRB3 protein was detected in the cytoplasm of hepatocytes in the liver. Expression of this protein was also seen in lymphocytes in the spleen, glomerular endothelial cells, and epithelial cells of collecting duct of the kidney. In hepatocytes of the hTRB3 mouse, nuclear size was significantly greater than that of the wild type mouse, indicating that hTRB3 can play a role at least in part in hepatic morphogenesis. The present animal model may provide a system for evaluation of de novo morphological changes induced by a specific transgene in a specific organ site.

Inhibition of intestinal polyp growth by oral ingestion of bovine lactoferrin and immune cells in the large intestine.

Studies using animal models have demonstrated that ingestion of bovine lactoferrin (bLF) inhibits carcinogenesis in the colon and other organs of experimental animals. As a result of these studies, a blinded, randomized, controlled clinical trial was conducted in the National Cancer Center Hospital, Tokyo, Japan to determine whether ingestion of bLF had an effect on the growth of colorectal polyps in humans. Patients with colorectal polyps ≤5 mm diameter and likely to be adenomas ingested 0, 1.5, or 3.0 g bLF daily for 1 year. Ingestion of 3.0 g bLF suppressed the growth of colorectal polyps and increased the level of serum human lactoferrin in trial participants 63 years old or younger. The purpose of the present study was to investigate correlations between immune parameters and changes in polyp size. Trial participants with regressing polyps had increased NK cell activity, increased serum hLF levels (indicating increased neutrophil activity), and increased numbers of CD4+ cells in the polyps. These findings are consistent with a correlation between higher immune activity and suppression of colorectal polyps. In addition, participants with regressing polyps had lower numbers of PMNs and increased numbers of S100A8+ cells in the polyps, consistent with a correlation between lower inflammatory potential in the colon and suppression of colorectal polyps. Trial participants ingesting bLF had increased serum hLF levels, a possible increase in systemic NK cell activity, and increased numbers of CD4+ and CD161+ cells in the polyps. Taken together, our findings suggest that bLF suppressed colorectal polyps by enhancing immune responsiveness.