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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

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.

Two types of nanosized titanium dioxide, anatase (anTiO2) and rutile (rnTiO2), are widely used in industry, commercial products and biosystems. TiO2 has been evaluated as a Group 2B carcinogen. Previous reports indicated that anTiO2 is less toxic than rnTiO2, however, under ultraviolet irradiation anTiO2 is more toxic than rnTiO2 in vitro because of differences in their crystal structures. In the present study, we compared the in vivo and in vitro toxic effects induced by anTiO2 and rnTiO2. Female SD rats were treated with 500 ?g/ml of anTiO2 or rnTiO2 suspensions by intra-pulmonary spraying 8 times over a two week period. In the lung, treatment with anTiO2 or rnTiO2 increased alveolar macrophage numbers and levels of 8-hydroxydeoxyguanosine (8-OHdG); these increases tended to be lower in the anTiO2 treated group compared to the rnTiO2 treated group. Expression of MIP1??mRNA and protein in lung tissues treated with anTiO2 and rnTiO2 was also significantly up-regulated, with MIP1??mRNA and protein expression significantly lower in the anTiO2 group than in the rnTiO2 group. In cell culture of primary alveolar macrophages (PAM) treated with anTiO2 and rnTiO2, expression of MIP1??mRNA in the PAM and protein in the culture media was significantly higher than in control cultures. Similarly to the in vivo results, MIP1??mRNA and protein expression was significantly lower in the anTiO2 treated cultures compared to the rnTiO2 treated cultures. Furthermore, conditioned cell culture media from PAM cultures treated with anTiO2 had less effect on A549 cell proliferation compared to conditioned media from cultures treated with rnTiO2. However, no significant difference was found in the toxicological effects on cell viability of ultra violet irradiated anTiO2 and rnTiO2. In conclusion, our results indicate that anTiO2 is less potent in induction of alveolar macrophage infiltration, 8-OHdG and MIP1??expression in the lung, and growth stimulation of A549 cells in vitro than rnTiO2.

Zinc oxide (ZnO) is known to induce lung toxicity, including terminal bronchiolar epithelial hyperplasia, which gives rise to concerns that nanosized ZnO (nZnO) might lead to lung carcinogenesis. We studied the tumor promoting activity of nZnO by an initiation–promotion protocol using human c-Ha-ras proto-oncogene transgenic rats (Hras128 rats). The rats were given 0.2 % N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water for 2 weeks and then treated with 0.5 ml of 250 or 500 μg/ml nZnO suspension by intra-pulmonary spraying once every 2 weeks for a total of 7 times. Treatment with nZnO particles did not promote DHPN-induced lung carcinogenesis. However, nZnO dose-dependently caused epithelial hyperplasia of terminal bronchioles (EHTB) and fibrosis-associated interstitial pneumonitis (FAIP) that were independent of DHPN treatment. Tracing the fate of EHTB lesions in wild-type rats indicated that the hyperplastic lesions almost completely disappeared within 12 weeks after the last nZnO treatment. Since nZnO particles were not found in the lung and ZnCl2 solution induced similar lung lesions and gene expression profiles, the observed lesions were most likely caused by dissolved Zn2+. In summary, nZnO did not promote carcinogenesis in the lung and induced EHTB and FAIP lesions that regressed rapidly, probably due to clearance of surplus Zn2+ from the lung. Electronic supplementary material The online version of this article (doi:10.1007/s00204-013-1086-5) contains supplementary material, which is available to authorized users.

Tribbles-related protein 3 (TRB3) has been shown to be a crucial modulator of tumorigenesis. However, the precise role and the functional morphology of TRB3 are not clearly understood. To elucidate these enigmas we established the cell line, M2TRB3, by introducing the human TRB3 gene and protein in Cl66M2 (M2) mouse mammary tumor cells. This cell line stably expressed the TRB3 gene and protein. After 72 h of cell culture, there was a 34% increase in the growth of M2TRB3 cells compared to the control M2 mock cells. The mean volume of the tumors originating from the M2TRB3 cells was significantly increased by 38% when compared to the mean volume of the M2 mock tumors, and the proliferating cell nuclear antigen (PCNA) labeling index in the M2TRB3 tumors was higher when compared to that of the M2 and M2 mock cells. In the tumor tissue samples, the mean diameter of nuclei in the M2TRB3 tumor cells (9.4±0.3 µm) showed a significant increase compared to that of the M2 mock tumor cells (7.0±0.2 µm). M2TRB3 cells also showed a marked increase in the population of tetraploid or octaploid nuclei compared to M2 mock cells bearing mainly either diploid or tetraploid nuclei. Western blot analysis revealed the overexpression of cyclin B1 and cyclin D1 in M2TRB3 cells when compared to that in the M2 mock cells. These novel findings provide further evidence that TRB3 promotes cell proliferation and chromosomal instability by causing polyploidization during development.

Summary The recent widespread application of Cre/loxP technology has resulted in a new generation of conditional animal models that can better recapitulate many salient features of human disease. These models benefit from the ability to monitor the expression and functionality of Cre protein. We have generated a conditional (Cre/loxP dependent) LacZ reporter rat (termed the LacZ541 rat) to monitor Cre in transgenic rats. When LacZ541 rats were bred with another transgenic rat line expressing Cre recombinase under the control of the CAG promoter, LacZ/Cre double transgenic embryos displayed ubiquitous expression of LacZ, and when LacZ541 rats were bred with transgenic rats expressing Cre/loxP-dependent oncogenic H- or K-ras, LacZ was expressed in the lesions resulting from the activation of the oncogene. The LacZ541 rat enables evaluation of the performance of Cre-expressing systems which are based upon transgenic rats or somatic gene transfer vectors and provides efficient and simple lineage marking. genesis 51:268–274. © 2013 Wiley Periodicals, Inc.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most debilitating malignancies in humans, and one of the reasons for this is the inability to diagnose this disease early in its development. To search for biomarkers that can be used for early diagnosis of PDAC, we established a rat model of human PDAC in which expression of a human K-ras(G12V) oncogene and induction of PDAC are regulated by the Cre/lox system. In the present study, transgenic rats bearing PDAC and control transgenic rats with normal pancreatic tissues were used for metabolomic analysis of serum and pancreatic tissue by non-targeted and targeted gas chromatography-mass spectrometry and transcriptomic analysis of pancreatic tissue by microarray. Comparison of the metabolic profiles of the serum and pancreatic tissue of PDAC-bearing and control rats identified palmitoleic acid as a metabolite, which was significantly decreased in the serum of PDAC-bearing animals. Transcriptomic analysis indicated that several transcripts involved in anaerobic glycolysis and nucleotide degradation were increased and transcripts involved in the trichloroacetic acid cycle were decreased. Other transcripts that were changed in PDAC-bearing rats were adenosine triphosphate citrate lyase (decreased: fatty acid biosynthesis), fatty acid synthase (increased: fatty acid biosynthesis) and arachidonate 5-lipoxygenase activating protein (increased: arachidonic acid metabolism). Overall, our results suggest that the decreased serum levels of palmitoleic acid in rats with PDAC was likely due to its decrease in pancreatic tissue and that palmitoleic acid should be investigated in human samples to assess its diagnostic significance as a serum biomarker for human PDAC.

The animal model is a powerful and fundamental tool in the field of biochemical research including toxicology, carcinogenesis, cancer therapeutics and prevention. In the carcinogenesis animal model system, numerous examples of preneoplastic lesions have been isolated and investigated from various perspectives. This may indicate that several options of endpoints to evaluate carcinogenesis effect or therapeutic outcome are presently available; however, classification of preneoplastic lesions has become complicated. For instance, these lesions include aberrant crypt foci (ACF), dysplastic ACF, flat ACF, β-catenin accumulated crypts, and mucin-depleted foci. These lesions have been induced by commonly used chemical carcinogens such as azoxymethane (AOM), 1,2-dimethylhydrazine (DMH), methylnitrosourea (MUN), or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Investigators can choose any procedures or methods to examine colonic preneoplastic lesions according to their interests and the objectives of their experiments. Based on topographical, histopathological, and biological features of colon cancer preneoplastic lesions in the animal model, we summarize and discuss the character and implications of these lesions.

Despite recent advancements in multidisciplinary treatments, the overall survival and quality of life of patients with advanced head and neck squamous cell carcinoma (HNSCC) have not improved significantly over the past decade. Molecular targeted therapies, which have been addressed and advanced by the concept of “oncogene addiction”, have demonstrated only limited successes so far. To explore a novel clue for clinically effective targeted therapies, we analyzed the molecular circuitry of HNSCC through the lens that HNSCC is an evolving system. In the trajectory of this somatic evolution, HNSCC acquires biological robustness under a variety of selective pressures including genetic, epigenetic, micro‐environmental and metabolic stressors, which well explains the major mechanism of “escaping from oncogene addiction”. On the other hand, this systemic view appears to instruct us approaches to target latent vulnerability of HNSCC that is masked behind the plasticity and evolvability of this complex adaptive system., Highlights ► There is an urgent need to develop a novel conceptual framework for the treatment of HNSCC.► The biological robustness of HNSCC was analyzed through a somatic evolution model.► This model well explains the mechanism of “escaping from oncogene addiction”.► We discuss about the possible approaches to target vulnerability of evolving HNSCC.

OBJECTIVES Novel biomarkers for pancreatic ductal adenocarcinoma (PDAC) are urgently needed because of its poor prognosis. We have previously established an animal model for human PDAC using transgenic rats in which expression of a human K-ras(G12V) oncogene is regulated by the Cre/lox system. Using this model, we searched for candidate circulating microRNAs (miRNAs) for use as novel clinical diagnostic biomarkers for PDAC. METHODS Rats bearing PDACs were generated using our model. MicroRNA expression in serum and pancreatic tissues of PDAC and control rats was compared by microarray analysis. Rat serum levels of 28 miRNAs identified by microarray analysis and 4 miRNAs previously reported to be high in plasma of PDAC patients were quantified by real-time quantitative reverse transcription polymerase chain reaction. RESULTS Quantification by real-time quantitative polymerase chain reaction revealed that miR-155, miR-21, and miR-210 were higher in serum of PDAC rats, similar to plasma of patients with PDAC. In addition, miR-18a, miR-203, miR-30b-5p, miR-31, miR-369-5p, miR-376a, and miR-541 were higher and miR-375 was lower in the serum of PDAC rats. CONCLUSION We identified 4 previously unreported miRNAs (miRNA-203, miRNA-369-5p, miRNA-376a, and miRNA-375) whose expression is significantly different in PDAC rats compared to control rats. These miRNAs need to be quantitated in humans as potential novel clinical diagnostic biomarkers for PDAC.

Lactoferrin is a major component of biologically important mucosal fluids and of the specific granules of neutrophils. Understanding its biological function is essential for understanding neutrophil- and mucosal-mediated immunity. In this review, we reevaluate the in vivo functions of human lactoferrin (hLF) emphasizing in vivo studies and in vitro studies performed in biologically relevant fluids. We discuss the evidence in the literature that supports (or does not support) proposed roles for hLF in mucosal immunity and in neutrophil function. We argue that the current literature supports a microbiostatic role, but not a microbicidal role, for hLF in vivo. The literature also supports a role for hLF in inhibiting colonization and infection of epithelial surfaces by microorganisms and in protecting tissues from neutrophil-mediated damage. Using this information, we briefly discuss hLF in the context of the complex biological fluids in which it is found.

Titanium dioxide (TiO(2)) is used in sunscreens and cosmetics as an ultraviolet light screen. TiO(2) has carcinogenic activity in the rat lung, but its effect on the skin has not been reported. We examined the promoting/carcinogenic effect of nano-size TiO(2) particles using a two-stage skin model. c-Ha-ras proto-oncogene transgenic (Hras128) rats, which are sensitive to skin carcinogenesis, and their wild-type siblings were exposed to ultraviolet B radiation on shaved back skin twice weekly for 10 weeks; then the shaved area was painted with a 100 mg/ml TiO(2) suspension twice weekly until sacrifice. All rats were killed at week 52 except for female Hras128 rats which were sacrificed at week 16 because of early mammary tumor development. Skin tumors developed in male Hras128 rats and mammary tumors developed in both sexes of Hras128 rats and in wild-type female rats, but tumor incidence was not different from controls. TiO(2) particles were detected in the upper stratum corneum but not in the underlying skin tissue layers. TiO(2) particles also did not penetrate a human epidermis model in vitro. Our data suggest that TiO(2) does not cause skin carcinogenesis, probably due to its inability to penetrate through the epidermis and reach underlying skin structures.

【目的】肺がん患者が持つ麻薬性鎮痛薬に対する負のイメージの払拭と, 正しい知識の啓蒙を目的として集団勉強会を実施し, 麻薬性鎮痛薬に対する認識と, 勉強会の実施効果を調査した. 【方法】勉強会への参加同意が得られた肺がん入院患者を対象に薬剤師による麻薬性鎮痛薬に関する勉強会を実施した. 勉強会前後にアンケート調査を行った. 【結果】60名の参加患者から回答が得られた. 負のイメージに関連した設問, 麻薬性鎮痛薬の知識に関連した設問のすべてにおいて勉強会後には認識の改善, 知識の向上が認められた. 【考察】勉強会により負のイメージを完全に払拭することはできなかったが, 勉強会は患者教育を行ううえで1つの有用な手法であると考えられた. 患者が麻薬性鎮痛薬導入に抵抗を感じることは, 疼痛緩和医療の遅れをきたしQOLの低下を招くおそれがあるため, 事前に正しい知識を啓蒙しておくことが必要であると考えられる. Palliat Care Res 2011; 6(1): 109-118

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

Bisphosphonates are expected to be effective at preventing tumor metastasis to bone tissue. Since protein kinase C (PKC) plays a crucial role in cancer progression, we examined the effect of bisphosphonates on PKC expression to clarify the mechanism behind the inhibition of the bone metastasis of prostate cancer by bisphosphonates. We found that pamidronate inhibits PKC protein expression and PKC activity in prostate cancer PC-3 cells. PKC protein expression was markedly reduced by treatment with 100 microM of pamidronate. The inhibitory effect of PKC expression by pamidronate was specific for PKCalpha and PKCzeta. Nitrogen-containing bisphosphonates are known to inhibit the mevalonate pathway, but the effect of pamidronate on PKC expression was not due to the inhibition of this pathway. Urokinase-type plasminogen activator (uPA) is one of the critical proteins in tumor metastasis and decreased in bisphosphonate-treated PC-3 cells. We also showed that uPA expression was suppressed by PKC inhibitors (calphostin C and staurosporine) and induced by a PKC activator (PMA) in PC-3 cells, suggesting that the inhibition of uPA by bisphosphonates is involved in PKC inhibition. This is the first finding that bisphosphonates suppress PKC expression in cancer cells. These results strongly suggest that one of the mechanisms behind the inhibitory effect of bisphosphonates on tumor bone metastasis is mediated by PKC inhibition.

Opioid analgesics possess a number of side effects, among which constipation and nausea/vomiting occur most frequently. Although pretreatment with laxatives and antiemetics for the prophylaxis of opioid-induced constipation and nausea/vomiting, respectively, is recommended, such side effects are still a matter of concern in clinical setting.We first surveyed the prevalence of premedication in 83 cancer patients who took opioid analgesics and the incidence of such side effects. Subsequently, intervention was carried out to promote premedication, and the effectiveness of the intervention was evaluated in 107 patients.Prophylactic treatment with laxatives and antiemetics were conducted in 57% and 52%, respectively. The most frequently prescribed laxatives and antiemetics were magnesium oxide in combination with pantethine, a mild stimulant laxative, and prochlorperazine, respectively. The lack of premedication increased the risk of constipation (odds ratio, 5.25; 95% confidence intervals, 1.93-14.31; p = 0.001) and vomiting (4.67, 1.04-21.04; p = 0.045). Intervention such as provision of drug information to physicians, verification of prescription orders, and instructions to patients increased the rates of prophylactic medications to 93% (p0.001) for laxatives and 81% (p0.001) for antiemetics. The incidence of side effects was lowered from 36% to 9% (p0.001) for constipation, from 28% to 17% for nausea (p = 0.077), and from 16% to 4% for vomiting (p = 0.0085).Intervention to promote prophylactic medication was highly effective in reducing the risk of opioid-induced constipation and nausea/vomiting.

There is growing interest in the use of natural compounds for the treatment and prevention of a wide variety of diseases, including cancer. Several herb-derived components are currently evaluated in preclinical studies as potential cancer chemopreventive agents. We have recently found that several herbal plants in the Ryukyu Islands, or any other natural compound, have a potential chemopreventive effect on biomarkers of colon carcinogenesis and a growth inhibitory effect on human cancer cells. These studies together with related in vivo and in vitro investigations are summarized and discussed in this review.

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

In previous studies, we found that sulindac sulfide and exisulind (sulindac sulfone, Aptosyn) cause growth inhibition, arrest cells in the G1 phase of the cell cycle, and induce apoptosis in human breast cancer cell lines. These effects were associated with decreased expression of cyclin D1. The present study focuses on the effects of sulindac sulfide and exisulind on hormone signaling components in breast cancer cells. We found that estrogen receptor (ER)–positive and progesterone receptor (PR)–positive T47D breast cancer cells were somewhat more sensitive to growth inhibition by sulindac sulfide or exisulind than ER-negative PR-negative MB-MDA-468 breast cancer cells. Further studies indicated that sulindac sulfide and exisulind caused marked down-regulation of expression of the ER and PR-A and PR-B in T47D cells. However, neither compound caused a major change in expression of the retinoic acid receptor α (RARα), RARβ, or RARα in T47D cells. Sulindac sulfide and exisulind also caused a decrease in expression of the ER in estrogen-responsive MCF-7 breast cancer cells. Both compounds also markedly inhibited estrogen-stimulated activation of an estrogen-responsive promoter in transient transfection reporter assays. Treatment of T47D cells with specific protein kinase G (PKG) activators did not cause a decrease in ER or PR expression. Therefore, although sulindac sulfide and exisulind can cause activation of PKG, the inhibitory effects of these two compounds on ER and PR expression does not seem to be mediated by PKG. Our findings suggest that the growth inhibition by sulindac sulfide and exisulind in ER-positive and PR-positive human breast cancer cells may be mediated, in part, by inhibition of ER and PR signaling. Thus, these and related compounds may provide a novel approach to the prevention and treatment of human breast cancers, especially those that are ER positive.

Sphingolipids display a wide spectrum of biological activities, including cell growth, differentiation and apoptosis. However, precise mechanisms by which these compounds exert anticancer or cancer-preventive effects are not known. In the present study, we evaluated the preventive efficacy of enriched dietary monoglucosylceramide 1-O-beta-glucosyl-N-2'-hydroxyarachidoyl-4,8-sphingadienine (G(1)CM) on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) and beta-catenin-accumulated crypt (BCAC) formation in F344 rats during initiation stage. We also examined whether G(1)CM affects cell proliferation and apoptosis in these lesions. Pure G(1)CM was isolated from rice bran. Forty-two rats were divided randomly into five experimental groups. Rats in groups 1-3 were given subcutaneous injections of DMH (40 mg/kg body weight) once a week for 2 weeks. One week before the first injection of DMH, rats in groups 2 and 3 were fed a diet containing 200 and 1,000 p.p.m. G(1)CM, respectively, for 5 weeks. Rats in group 4 were fed a diet containing 1,000 p.p.m. G(1)CM. Rats in group 5 were given the basal diet alone and served as untreated controls. The experiment was terminated 5 weeks after the start. Dietary G(1)CM at both doses (groups 2 and 3) significantly inhibited the induction of ACF and BCAC (P

Oral squamous cell carcinoma is one of the most common human neoplasms, and prevention of this malignancy requires a better understanding of its carcinogenesis process. To this end, we tried to establish an animal model using the human c-Ha-ras proto-oncogene-carrying transgenic (Tg) rats and the carcinogen 4-nitroquinoline 1-oxide (4-NQO). 4-NQO (20 p.p.m.) was administered to Tg and non-Tg rats for 8 weeks in their drinking water, and then the occurrence of tongue carcinogenesis was compared during the experimental period of 22 weeks. In addition, we determined the DNA ploidy in tongue lesions and examined the immunohistochemical expression of five biomarkers such as cyclin D1, glutathione S-transferase placental form, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and beta-catenin. Next, the cancer chemopreventive effects of nimesulide, pioglitazone and a synthetic geranylated derivative, which have been reported to be inhibitors of tongue carcinogenesis, were examined in Tg rats treated with 4-NQO. Either during or after treatment with 4-NQO in the drinking water, tongue dysplasia and tumors were observed on the tongues of both Tg and non-Tg rats, with a greater incidence and multiplicity in Tg rats. Histopathologically, squamous cell dysplasia, papilloma and carcinoma with or without invasion were present in the tongue. Immunohistochemistry revealed that expression levels against five biomarkers increase with disease progression, and the changes correlated with those of the DNA ploidy pattern. Interestingly, a strong expression of COX-2, iNOS and beta-catenin was observed on the invasive front of squamous cell carcinomas. A subsequent chemoprevention study using Tg rats showed that the chemicals tested suppressed the occurrence of tongue carcinomas when they were administered after 4-NQO-exposure. These results may thus indicate that our 4-NQO-induced Tg rat tongue carcinogenesis model simulates many aspects of human oral carcinogenesis and it can be applied for an analysis of oral cancer development while also helping to identify potentially effective cancer chemopreventive agents against oral cancer.

The usefulness of mucin-depleted foci (MDF), which have recently been proposed as a new preneoplastic biomarker in rat colon carcinogenesis, was histologically investigated in rat colonic tissues treated with 1,2-dimethylhydrazine dihydrochloride (DMH). The relationship among aberrant crypt foci (ACF), MDF and beta-catenin accumulated crypts (BCAC) was examined by comparing the corresponding computer-captured images. Twelve male F344 rats were given DMH s.c. at a dose of 40 mg/kg body weight, once a week for 2 weeks, and randomly divided into two groups. Rats in group 1 were given normal drinking water, while those in group 2 were given drinking water containing indomethacin (IND) at 16 ppm for 6 weeks. All animals were sacrificed 8 weeks after the first DMH treatment. The resected colons were fixed in 10% formalin, and stained with Alcian blue for observation of ACF and MDF. Histological and immunohistochemical analysis revealed that the numbers of ACF, MDF and overlapping lesions in group 2 (treated with IND) were significantly decreased, compared with those in group 1. The number of BCAC in group 2 was also significantly lower than that in group 1. The reduction (61.5%) of MDF by IND was much greater than that (29.3%) of ACF. Analyses of the computer-captured images indicated that MDF had more frequent dysplastic changes and overexpression of beta-catenin than did ACF. MDF having over 4 crypts or MDF with the appearance of ACF corresponded well to BCAC. These results suggest that MDF may be useful as an early biomarker in colon carcinogenesis.

Hepatoma is one of the most frequently occurring cancers worldwide. However, effective chemotherapeutic agents for this disease have not been developed. Acyclic retinoid, a novel synthetic retinoid, can reduce the incidence of postsurgical recurrence of hepatoma and improve the survival rate. OSI-461, a potent derivative of exisulind, can increase intracellular levels of cyclic GMP, which leads to activation of protein kinase G and induction of apoptosis in cancer cells. In the present study, we examined the combined effects of acyclic retinoid plus OSI-461 in the HepG2 human hepatoma cell line. We found that the combination of as little as 1.0 μmol/L acyclic retinoid and 0.01 μmol/L OSI-461 exerted synergistic inhibition of the growth of HepG2 cells. Combined treatment with low concentrations of these two agents also acted synergistically to induce apoptosis in HepG2 cells through induction of Bax and Apaf-1, reduction of Bcl-2 and Bcl-xL, and activation of caspase-3, -8, and -9. OSI-461 enhanced the G0-G1 arrest caused by acyclic retinoid, and the combination of these agents caused a synergistic decrease in the levels of expression of cyclin D1 protein and mRNA, inhibited cyclin D1 promoter activity, decreased the level of hyperphosphorylated forms of the Rb protein, induced increased cellular levels of the p21CIP1 protein and mRNA, and stimulated p21CIP1 promoter activity. Moreover, OSI-461 enhanced the ability of acyclic retinoid to induce increased cellular levels of retinoic acid receptor β and to stimulate retinoic acid response element-chloramphenicol acetyltransferase activity. A hypothetical model involving concerted effects on p21CIP1 and retinoic acid receptor β expression is proposed to explain these synergistic effects. Our results suggest that the combination of acyclic retinoid plus OSI-461 might be an effective regimen for the chemoprevention and chemotherapy of human hepatoma and possibly other malignancies.

The modifying effect of dietary Peucedanum japonicum (PJ), which is a traditional herb in the Ryukyu Islands and is an anti-oxidant, on azoxymethane (AOM)-induced rat colon carcinogenesis was examined. Male F344 rats were divided into six groups: rats in groups 1-4 were given subcutaneous injection of AOM (20 mg/kg body weight) once a week for 2 weeks. Rats in groups 2, 3 and 4 were fed the diets containing 0.2 and 1% PJ and 0.025% chlorogenic acid, respectively. We observed modification of the preneoplastic lesions of both aberrant crypt foci (ACF) and beta-catenin accumulated crypts (BCAC) in colon carcinogenesis, microscopically and immunohistochemically. The numbers of ACF consisting of more than four aberrant crypts per rat in groups 2 (3.2+/-1.7) and 3 (3.0+/-3.2) were significantly lower than that of group 1 (10.8+/-4.9; P0.05, respectively). The mean number of BCAC in both groups 2 (0.88+/-0.48/cm2/rat) and 3 (0.81+/-0.34/cm2/rat) was significantly lower than that in group 1 (2.13+/-0.54/cm2/rat; P0.0001, respectively). In addition, proliferating cell nuclear antigen labeling indices in group 2 (10.98+/-2.03) and group 3 (9.85+/-2.62) were significantly lower than that in group 1 (14.87+/-3.93; P0.001 and P0.0001, respectively). These findings indicate that PJ inhibits both ACF formation and accumulation of beta-catenin, and that PJ also reduces the cell proliferation activity, suggesting that PJ may have chemopreventive potential for colon carcinogenesis.

Acyclic retinoid (ACR), a novel synthetic retinoid, has recently been demonstrated by us to inhibit the in vitro growth of human hepatoma cells, and this effect was associated with decreased expression of cell cycle-related molecules. These results, taken together with previous in vitro and clinical studies with ACR, suggest that this agent may be useful in the chemoprevention and therapy of hepatoma and possibly other human malignancies. In the present study, we further examined the molecular effects of ACR on the HepG2 human hepatoma cell line, focusing on the expression of nuclear retinoid receptors and the cell cycle inhibitor protein p21CIP1. Reverse transcription-PCR assays and Western blot analyses indicated that these cells express retinoic acid receptors (RARs) α, β, and γ, retinoid X receptors (RXRs) α and β, and peroxisome proliferator-activated receptors (PPAR) γ mRNA. Treatment with ACR caused a rapid induction within 3 h of RARβ mRNA and the related protein, but there was no significant change in the levels of the mRNA or proteins for RARs α and γ, RXRs α and β, and PPARγ. There was also a rapid increase in p21CIP1 mRNA and protein in HepG2 cells treated with ACR, and this induction occurred via a p53-independent mechanism. In transient transfection reporter assays, we cotransfected the retinoic acid response element-chloramphenicol acetyltransferase (CAT) reporter gene into HepG2 cells together with a RARβ expression vector. RARβ expression markedly stimulated CAT activity (up to about 4-fold) after the addition of ACR. However, CAT activity in the presence of ACR was only about 2-fold higher than that in the absence of ACR, when cells were cotransfected with RARs α and γ or RXRα. These findings suggest that the growth inhibitory effects of ACR are mediated at least in part through RARβ and that both RARβ and p21CIP1 play critical roles in the molecular mechanisms of growth inhibition induced by ACR.

The purpose of this study was to determine whether black cohosh contains constituents that inhibit the growth of human breast cancer cells, and therefore might eventually be useful in the prevention or treatment of breast cancer. Black cohosh rhizomes were extracted with methanol/water and fractionated by solvent-solvent partitioning to yield three fractions: hexane, ethyl acetate and water. The ethyl acetate fraction displayed the highest potency in two cell-based assays, growth inhibition and cell cycle analysis. This fraction inhibited growth of both the ER+ MCF7 and ER-MDA-MB-453 human breast cancer cell lines with IC50 values of about 20 and 10 micro g/ml, respectively. It also induced cell cycle arrest at G1 when tested at 30 micro g/ml and at G2/M at 60 micro g/ml in MCF7 cells. This suggests that the extract contains a mixture of components with the more active (or more abundant) causing G1 arrest and the less active causing G2/M arrest. We then examined specific components in this extract. The triterpene glycoside fraction obtained by polyamide column chromatography, and the specific triterpene glycosides actein, 23-epi-26-deoxyactein and cimiracemoside A, inhibited growth of the MCF7 human breast cancer cells and induced cell cycle arrest at G1. The most potent compound, actein, decreased the level of cyclin D1, cdk4 and the hyperphosphorylated form of the pRb protein and increased the level of p21cip1 in MCF7 cells, changes that may contribute to the arrest in G1. Further studies are in progress to identify the mechanisms by which actein and related compounds present in black cohosh inhibit growth of human breast cancer cells.

背景:明細胞髄膜腫は, 通常の髄膜腫と好発年齢および部位が異なるまれな亜型である. 脊髄に好発し, 髄膜との癒着が乏しい例では神経鞘腫などとの鑑別が困難である.症例:患者は17歳, 女性. 7ヵ月前より左腰部痛出現し, 画像上, 腰部硬膜内に腫瘍を指摘され, 切除術を施行された. 腫瘍はグリコーゲンを有する淡明な細胞質と異型性の乏しい核をもつ細胞からなり, 小塊状の膠原線維が散在し, 不明瞭なwhorl形成が認められることから明細胞髄膜腫と診断された.結論:術中所見でも馬尾神経との関係が強く, 神経鞘腫との鑑別が困難であったが, 術中細胞診により髄膜腫と診断することができ, 有用であった.

PKC-interacting protein (PKCI), also designated histidine triad nucleotide-binding protein 1, belongs to the histidine triad (HIT) family of proteins. Its structure is highly conserved from bacteria to humans and shares homology with the tumor-suppressor gene fragile histidine triad (FHIT). Although it was originally thought to inhibit PKC, its actual physiologic function is not known. Therefore, we used the technique of homologous recombination to generate homozygous deleted PKCI -/- mice. These mice display normal fetal and adult development. However, when mouse embryo fibroblasts were established from 13.5-day embryos and serially passaged the PKCI -/- cells displayed an increase in growth rate and underwent spontaneous immortalization, whereas the PKCI +/+ cells senesced and ceased growing. Furthermore, the PKCI -/- mouse embryo fibroblasts displayed increased resistance to cytotoxicity by ionizing radiation. In view of these findings we examined possible effects of PKCI on susceptibility to carcinogenicity. Both PKCI +/+ and PKCI -/- mice were treated with the chemical carcinogen N -nitrosomethylbenzylamine (NMBA) by intragastric administration and killed 12 weeks later. As expected with this protocol, NMBA induced squamous tumors (both papillomas and carcinomas) of the forestomach. The incidence, multiplicity per mouse, volume, and degree of malignancy of these tumors were significantly greater in the PKCI -/- than in the PKCI +/+ mice. Furthermore, four adenomas and one adenocarcinoma of the glandular stomach were found in the NMBA-treated PKCI -/- mice but no tumors of the glandular stomach were found in the NMBA-treated PKCI +/+ mice or in any of the untreated mice. Taken together, these findings suggest that, like FHIT, PKCI may normally play a tumor-suppressor role. The possible role of PKCI as a tumor suppressor in humans remains to be determined.

The agent N-methyl-N-nitrosourea (MNU) is a direct acting carcinogen and induces well-differentiated adenocarcinoma on the rat gastric mucosa. In this study, 27 histopathologically verified gastric carcinomas induced in male F344 rats were analyzed for mutations in the N-terminal phosphorylation sites (codons 1-51) of the beta-catenin gene by using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) assays. In parallel studies, the specific localization of the beta-catenin protein was also examined by immunohistochemical analysis. No mutations in the beta-catenin gene were found in any of 27 gastric carcinomas induced by MNU. Immunohistochemical analysis resulted in the beta-catenin protein to be localized in the plasma membrane but cytoplasmic and/or nuclear accumulation of beta-catenin was not identified in any of these carcinomas. These results suggest that mutations in the beta-catenin gene are less contributory to the development of rat gastric carcinomas induced by MNU. This animal model may provide a system for evaluating the mechanism of human gastric carcinogenesis that is not associated with beta-catenin gene mutations.

In a recent study on head and neck squamous cell carcinoma (HNSCC) cells we found that epigallocatechin-3-gallate (EGCG), a major biologically active component of green tea, inhibited activation of the epidermal growth factor receptor (EGFR) and related signaling pathways. Since activation of EGFR signaling pathways is associated with angiogenesis, we examined the effects of EGCG on vascular endothelial growth factor (VEGF) production by YCU-H891 HNSCC and MDA-MB-231 breast carcinoma cell lines, because we found that both of these cell lines display autocrine activation of transforming growth factor-alpha (TGF-alpha)/EGFR signaling and produce high levels of VEGF. Treatment with EGCG inhibited the constitutive activation of the EGFR, Stat3, and Akt in both cell lines. These changes were associated with inhibition of VEGF promoter activity and cellular production of VEGF. Mechanistic studies indicated that inhibition of Stat3, but not mitogen-activated protein kinase kinase (MEK)1 or phosphatidylinositol 3'-kinase (PI3K), significantly decreased VEGF promoter activity. However, the inhibitory effects of a dominant negative Stat3 on VEGF expression was not as strong as that produced by EGCG. An analysis of alternative pathways indicated that EGCG strongly inhibited the constitutive activation of NF-kappa B in both cell lines, and an NF-kappa B inhibitor strongly inhibited VEGF production. These results suggest that EGCG inhibits VEGF production by inhibiting both the constitutive activation of Stat3 and NF-kappa B, but not extracellular-signal-regulated kinase (ERK) or Akt, in these cells. Therefore, EGCG may be useful in treating HNSCC and breast carcinoma because it can exert both antiproliferative and antiangiogenic activities.