Molecular subtypes of lung adenocarcinoma present distinct immune tumor microenvironments.

Overcoming resistance to immune checkpoint inhibitors is an important issue in patients with non-small-cell lung cancer (NSCLC). Transcriptome analysis shows that adenocarcinoma can be divided into three molecular subtypes: terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI), and squamous cell carcinoma (LUSQ) into four. However, the immunological characteristics of these subtypes are not fully understood. In this study, we investigated the immune landscape of NSCLC tissues in molecular subtypes using a multi-omics dataset, including tumor-infiltrating leukocytes (TILs) analyzed using flow cytometry, RNA sequences, whole exome sequences, metabolomic analysis, and clinicopathologic findings. In the PI subtype, the number of TILs increased and the immune response in the tumor microenvironment (TME) was activated, as indicated by high levels of tertiary lymphoid structures, and high cytotoxic marker levels. Patient prognosis was worse in the PP subtype than in other adenocarcinoma subtypes. Glucose transporter 1 (GLUT1) expression levels were upregulated and lactate accumulated in the TME of the PP subtype. This could lead to the formation of an immunosuppressive TME, including the inactivation of antigen-presenting cells. The TRU subtype had low biological malignancy and "cold" tumor-immune phenotypes. Squamous cell carcinoma (LUSQ) did not show distinct immunological characteristics in its respective subtypes. Elucidation of the immune characteristics of molecular subtypes could lead to the development of personalized immune therapy for lung cancer. Immune checkpoint inhibitors could be an effective treatment for the PI subtype. Glycolysis is a potential target for converting an immunosuppressive TME into an antitumorigenic TME in the PP subtype.

Tumor-infiltrating Leukocyte Profiling Defines Three Immune Subtypes of NSCLC with Distinct Signaling Pathways and Genetic Alterations.

Resistance to immune checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell-dominant, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC. Significance: The precise TIL profiling classified NSCLC into novel three immune subtypes that correlates with patient outcome, identifying subtype-specific molecular pathways and genomic alterations that should play important roles in constructing subtype-specific immune tumor microenvironments. These classifications of NSCLC based on TIL status are useful for developing personalized immune therapies for NSCLC.

Establishment of preanalytical conditions for microRNA profile analysis of clinical plasma samples.

The relationship between the expression of microRNAs (miRNAs) in blood and a variety of diseases has been investigated. MiRNA-based liquid biopsy has attracted much attention, and cancer-specific miRNAs have been reported. However, the results of analyses of the expression of these miRNAs vary among studies. The reproduction of results regarding miRNA expression levels could be difficult if there are differences in the data acquisition process. Previous studies have shown that the anticoagulant type used during plasma preparation and sample storage conditions could contribute to differences in measured miRNA levels. Thus, the impact of these preanalytical conditions on comprehensive miRNA expression profiles was examined. First, the miRNA expression profiles of samples obtained from healthy volunteers were analyzed using next-generation sequencing. Based on an analysis of the library concentration, human genome identification rate, ratio of unique sequences and expression profiles, the optimal preanalytical conditions for obtaining highly reproducible miRNA expression profiles were established. The optimal preanalytical conditions were as follows: ethylenediaminetetraacetic acid (EDTA) as the anticoagulant, whole-blood storage at room temperature within 6 hours, and plasma storage at 4°C or -20°C within 30 days. Next, plasma samples were collected from 60 cancer patients (3 facilities × 20 patients/facility), and miRNA expression profiles were analyzed. There were no significant differences in measurements except in the expression of erythrocyte-derived hsa-miR-451a. However, the variation in hsa-miR-451a levels was smaller among facilities than among individuals. This finding suggests that samples obtained from the same facility could show significantly different degrees of hemolysis across individuals. We found that the standardization of anticoagulant use and storage conditions contributed to reducing the variation in sample quality across facilities. The findings from this study could be useful in developing protocols for collecting samples from multiple facilities for cancer screening tests.

In vivo effects of mutant RHOA on tumor formation in an orthotopic inoculation model.

Ras homolog family member A (RHOA) mutations are driver genes in diffuse­type gastric cancers (DGCs), and we previously revealed that RHOA mutations contribute to cancer cell survival and cell migration through their dominant negative effect on Rho­associated kinase (ROCK) signaling in vitro. However, how RHOA mutations contribute to DGC development in vivo is poorly understood. In the present study, the contribution of RHOA mutations to tumor morphology was investigated using an orthotopic xenograft model using the gastric cancer cell line MKN74, in which wild­type (WT) or mutated (Y42C and Y42S) RHOA had been introduced. When we conducted RNA sequencing to distinguish between the genes expressed in human tumor tissues from those in mouse stroma, the expression profiles of the tumors were clearly divided into a Y42C/Y42S group and a mock/WT group. Through gene set enrichment analysis, it was revealed that inflammation­ and hypoxia­related pathways were enriched in the mock/WT tumors; however, cell metabolism­ and cell cycle­related pathways such as Myc, E2F, oxidative phosphorylation and G2M checkpoint were enriched in the Y42C/Y42S tumors. In addition, the gene set related to ROCK signaling inhibition was enriched in the RHOA­mutated group, which indicated that a series of events are related to ROCK inhibition induced by RHOA mutations. Histopathological analysis revealed that small tumor nests were more frequent in RHOA mutants than in the mock or WT group. In addition, increased blood vessel formation and infiltration of macrophages within the tumor mass were observed in the RHOA mutants. Furthermore, unlike mock/WT, the RHOA­mutated tumor cells had little antitumor host reaction in the invasive front, which is similar to the pattern of mucosal invasion in clinical RHOA­mutated DGC. These transcriptome and pathological analyses revealed that mutated RHOA functionally contributes to the acquisition of DGC features, which will accelerate our understanding of the contribution of RHOA mutations in DGC biology and the development of further therapeutic strategies.

Pollen Allergy Suppression Effect by the Oral Administration of Acetic Acid Bacteria (Gluconacetobacter hansenii).

BACKGROUND/AIM: Gluconacetobacter hansenii (G. hansenii) is an acetic acid bacterium of vinegar production. Its anti-allergic effect on mice upon oral administration was examined. MATERIALS AND METHODS: The amount of LPS was measured by the Limulus reaction. Mice were sensitized by peritoneal and intranasal administration of cedar pollen and alum followed by oral administration of 30 or 150 mg/kg of heated G. hansenii cells. Pollen was administered intranasally to evaluate nasal symptoms, and at 8 weeks, IgE and IL-10 levels in blood were measured by ELISA. RESULTS: The amount of LPS in dried bacterial cells was 10.4±3.3 mg/g. In the cedar pollinosis model of mice, a significant reduction was observed in nose scratching of both groups administered with the bacterial cells (30, 150 mg/kg). CONCLUSION: G. hansenii contains LPS, and its oral administration showed an anti-allergic effect by a significant mitigation of the symptoms in a pollen allergy mouse model.

Difference in morphology and interactome profiles between orthotopic and subcutaneous gastric cancer xenograft models.

In xenograft models, orthotopic (ORT) engraftment is thought to provide a different tumor microenvironment compared with subcutaneous (SC) engraftment. We attempted to characterize the biological difference between OE19 (adenocarcinoma of the gastroesophageal junction) SC and ORT models by pathological analysis and CASTIN (CAncer-STromal INteractome) analysis, which is a novel method developed to analyze the tumor-stroma interactome framework. In SC models, SCID mice were inoculated subcutaneously with OE19 cells, and tumor tissues were sampled at 3 weeks. In ORT models, SCID mice were inoculated under the serosal membrane of the stomach wall, and tumor tissues were sampled at 3 and 6 weeks after engraftment. Results from the two models were then compared. Histopathologically, the SC tumors were well circumscribed from the adjacent tissue, with scant stroma and the formation of large ductal structures. In contrast, the ORT tumors were less circumscribed, with small ductal structures invading into abundant stroma. Then we compared the transcriptome profiles of human tumor cells with the mouse stromal cells of each model by species-specific RNA sequencing. With CASTIN analysis, we successfully identified several interactions that are known to affect the tumor microenvironment as being selectively enhanced in the ORT model. In conclusion, pathological analysis and CASTIN analysis revealed that ORT models of OE19 cells have a more invasive character and enhanced interaction with stromal cells compared with SC models.

DGC-specific RHOA mutations maintained cancer cell survival and promoted cell migration via ROCK inactivation.

RHOA missense mutations exist specifically in diffuse type gastric cancers (DGC) and are considered one of the DGC driver genes, but it is not fully understood how RHOA mutations contribute to DGC development. Here we examined how RHOA mutations affect cancer cell survival and cell motility. We revealed that cell survival was maintained by specific mutation sites, namely G17, Y42, and L57. Because these functional mutations suppressed MLC2 phosphorylation and actin stress fiber formation, we realized they act in a dominant-negative fashion against the ROCK pathway. Through the same inactivating mechanism that maintained cell survival, RHOA mutations also increased cell migration activity. Cell survival and migration studies on CLDN18-ARHGAP (CLG) fusions, which are known to be mutually exclusive to RHOA mutations, showed that CLG fusions complemented cell survival under RHOA knockdown condition and also induced cell migration. Site-directed mutagenesis analysis revealed the importance of the GAP domain and indicated that CLG fusions maintained RHOA in the inactive form. Taken together, these findings show that the inactivation of ROCK would be a key step in DGC development, so ROCK activation might provide novel therapeutic opportunities.

[Problems of Home-Visiting Speech-Language-Hearing Therapists in Dysphagia Management].

There are fewer reports by speech-language-hearing therapists than those by physical therapists or occupational therapists for visiting rehabilitation. Therefore, we examined the present situation with emphasis on professional roles of speech-language- hearing therapists working in visiting rehabilitation, patient tendency, and dysphagia rehabilitation. A questionnaire survey and interview survey were conducted on 6 speech-language-hearing therapists working in visiting rehabilitation. In the questionnaire, personal attributes, subject area, details of dysphagia rehabilitation, professional duties, and tendency of patient in charge were collected. In the interview survey, we asked about trends and request status, evaluation and training protocol for patients with dysphagia, activities related to pneumonia prevention, and future directions in the field. Results show that many linguistichearing experts worked with dysphagia patients, indicating that the needs for respiratory rehabilitation and dysphagia rehabilitation are high. In this survey, the environment surrounding visiting speech-language-hearing therapists and patients with dysphagia was clarified.

Immunopotentiator from Pantoea agglomerans 1 (IP-PA1) Promotes Murine Hair Growth and Human Dermal Papilla Cell Gene Expression.

BACKGROUND/AIM: The lipopolysaccharide (LPS)-like compound derived from Pantoea agglomerans (immunopotentiator from Pantoea agglomerans 1 (IP-PA1)) has been used not only as dietary supplement or cosmetic for humans, but also by Japanese veterinarians as an anti-tumor, anti-allergy, "keep a fine coat of fur" and hair growth-promoting functional food for dogs and cats. In the present study, we focused on the hair growth-promoting effects of IP-PA1 on a hair-shaved animal model and its mechanism of action. We also investigated its potential on gene expression after stimulating human dermal papilla cells with IP-PA1. MATERIALS AND METHODS: The hair on the back of a C3H/HeN mouse was shaved and IP-PA1 was orally administered or applied to the skin. The status of hair growth was observed and recorded for 14 days. Skin was collected and histological tissue examination was performed with respect to hair growth status using hematoxylin and eosin staining. After IP-PA1 administration (2 and 10 µg/ml) to human dermal papilla cell culture system for 24 h, fibroblast growth factor-7 (FGF-7) and vascular endothelial growth factor (VEGF) mRNA expression were measured using real-time polymerase chain reaction (PCR) analysis. RESULTS: IP-PA1, when given orally, showed a tendency to promote hair growth in mice. In addition, skin application also significantly promoted hair growth, while histopathological examinations further demonstrated hair elongation from dermal papilla cells. In the human dermal papilla cell culture system, significant FGF-7 and VEGF mRNA expressions were observed (p<0.05). CONCLUSION: An underlying mechanism of gene expression by which IP-PA1 promotes hair growth was suggested to be different from that of medicine and traditional hair tonics, such as minoxidil and adenosine.

Immunopotentiator from Pantoea agglomerans Prevents Atopic Dermatitis Induced by Dermatophagoides farinae Extract in NC/Nga Mouse.

BACKGROUND/AIM: Pantoea agglomerans LPS (immunopotentiator from Pantoea agglomerans 1: IP-PA1) has been reported to have anti-inflammatory effects in in vitro and in vivo models. The aim of the present study was to investigate the effects of orally-administered IP-PA1 on atopic dermatitis (AD) symptoms induced by Dermatophagoides farinae body extract (DFE) in NC/Nga mice. MATERIALS AND METHODS: Using the NC/Nga AD murine model, mice were orally administered 0.1% (High) or 0.01% (Low) water-containing IP-PA1. Skin lesion assessment and blood collection from the caudal vein was performed on days 0, 7, 21 and 31. On day 31, all mice were sacrificed and blood, skin, spleen, as well as intestine samples, were obtained. RESULTS: Assessment score of the skin lesion and serum immunoglobulin E (IgE) level of both IP-PA1 groups were significantly lower than that of the DFE group on days 14 and 21. The serum periostin and thymus and activation-regulated chemokine (TARC) level of IP-PA1-Low group was significantly lower than that of the DFE group on day 31. On histological examination of the skin, hyperplasia of epidermal and dermal layers and infiltration of inflammatory cells were suppressed by IP-PA1 administration. Deposition of periostin was observed in the DFE group skin tissue. Moreover, the CD4(+)/CD8(+) ratio of splenic T-cells increased by IP-PA1 administration. CONCLUSION: IP-PA1 administration may have an inhibitory effect on AD skin lesions.

Recurrent gain-of-function mutations of RHOA in diffuse-type gastric carcinoma.

Diffuse-type gastric carcinoma (DGC) is characterized by a highly malignant phenotype with prominent infiltration and stromal induction. We performed whole-exome sequencing on 30 DGC cases and found recurrent RHOA nonsynonymous mutations. With validation sequencing of an additional 57 cases, RHOA mutation was observed in 25.3% (22/87) of DGCs, with mutational hotspots affecting the Tyr42, Arg5 and Gly17 residues in RHOA protein. These positions are highly conserved among RHO family members, and Tyr42 and Arg5 are located outside the guanine nucleotide-binding pocket. Several lines of functional evidence indicated that mutant RHOA works in a gain-of-function manner. Comparison of mutational profiles for the major gastric cancer subtypes showed that RHOA mutations occur specifically in DGCs, the majority of which were histopathologically characterized by the presence of poorly differentiated adenocarcinomas together with more differentiated components in the gastric mucosa. Our findings identify a potential therapeutic target for this poor-prognosis subtype of gastric cancer with no available molecularly targeted drugs.

The relationship between clinicopathological factors and the reduction of pyrimidine nucleoside phosphorylase activity after preoperative administration of 5'-deoxy-5-fluorouridine.

AIM: The response to fluoropyrimidine chemotherapeutic drugs is different in individual tumors. Predictive biomarkers of antitumor effects by these drugs are unknown. 5'-Deoxy-5-fluorouridine (5'-DFUR), a fluoro-pyrimidine chemotherapeutic drug, is converted to 5-fluorouracil (5-FU) by pyrimidine nucleoside phosphorylase (PyNPase). It is suggested that 5'-DFUR will efficiently exert antitumor effects via PyNPase in tumor tissues. The change of PyNPase activity in tumor tissues following 5'-DFUR administration may reflect antitumor effects, and may be useful for detecting predictive factors of antitumor effects. The aim of this study was to search for predictive factors of antitumor effects by analyzing the relationship between clinicopathological factors and the change of PyNPase activity in colorectal tumor tissues after preoperative 5'-DFUR administration. PATIENTS AND METHODS: PyNPase activity in colorectal tissues from 45 patients with colorectal tumors was measured using an ELISA method. RESULTS: The reduction rate of PyNPase activity in colorectal tumor tissues after preoperative 5'-DFUR administration was correlated with significant differences in lymphatic invasion, stage, and histologic classification. It is suggested that lymphatic invasion, stage (distant metastasis), and histologic classification may be predictive factors for evaluating antitumor effects and selecting 5-FU-based chemotherapeutic drugs for patients with colorectal tumors.

Mechanism for maintaining homeostasis in the immune system of the intestine.

Every organism possesses a mechanism for maintaining homeostasis. We have focused on the immune system as a system that helps maintain homeostasis of the body, and particularly on the intestine as the largest organ of immunity in the body. We have also focused our research on the mechanism that responds to foreign substances in the intestine, especially the toll-like receptors (TLR). The activation of myeloid differentiation primary response gene 88 (MyD88) signal transduction as a response to TLR in the intestine is believed to contribute to the maintenance of homeostasis of the body through the homeostasis of the intestine. Furthermore, significant findings were reported in which signal transduction from TLR4 was essential for the maintenance and regulation of the intestine. These results strongly suggest the possibility that homeostasis in the intestine is maintained by TLR4, and signaling by TLR4 after exposure to lipopolysaccharide (LPS) probably has a role in regulating homeostasis. It is expected that the prevention and treatment of various diseases using TLR4 will continue to develop. As LPS is a substance that enhances the activity of TLR4, it will also attract attention as a valuable substance in its own right.

Intestinal macrophages involved in the homeostasis of the intestine have the potential for responding to LPS.

Recently, there has been interest in the tertiary functions of food, those that maintain human health. Moreover, lipopolysaccharides (LPS), which are components of Gram-negative bacteria, have been found to be highly effective in activating innate immunity and have been rediscovered as new functional food materials. In this review, we discuss the significance of LPS as a food component with reference to these tertiary functions based on recent findings. There is special emphasis on the plasticity of responses to LPS by intestinal macrophages. According to the macrophage-network theory, local macrophages cooperate with other tissue macrophages. For this reason, this review also discusses the possibility that information is transferred throughout the body from intestinal macrophages.

Improvement of allergic dermatitis via regulation of the Th1/Th2 immune system balance by macrophages activated with lipopolysaccharide derived from Pantoea agglomerans (IP-PA1).

Recently, the incidence of allergies has been increasing, especially in advanced countries. The cause of these allergies is believed to be a failure in the immune system balance that has been caused by changes in the living environment. The incidence of allergy shows a negative correlation with the decrease of infectious diseases in childhood. It has been suggested that the key to alleviating allergies is to activate innate immunity by exposure to microbial components such as lipopolysaccharides (LPS). The activation of innate immunity is expected to normalize the T-helper type 1 and 2 (Th1/Th2) immune system balance and to suppress the excessive reaction of Th2 type responses that cause immunoglobulin (Ig) E-dependent allergies. This study introduces information on how the activation of macrophages, which are important in innate immunity, by LPS derived from Pantoea agglomerans (IP-PA1) caused suppressive effects on type I allergic reactions and improved allergic dermatitis. We also summarize our hypothesis that regulating the immune system balance using LPS to stimulate macrophages may be an important procedure for preventing and improving allergic dermatitis.

ROS and innate immunity.

Oxygen is converted into reactive oxygen (RO) by radiation, light, the electron transport system in mitochondria, or by other enzymes and is regulated by the action of antioxidative enzymes which convert RO into an inactive state. Reactive oxygen species (ROS) have a biocidal effect on invading bacteria and they can also injure the cells of the host. For this reason, RO is considered as a general cause of aging and contributes to lifestyle-related diseases and cancer. However, for any organism that uses oxygen as an energy source, RO is inevitably produced and has important biological significance. Apart from the direct activity of RO, recent studies have shown that it functions as a second messenger of signal transduction. In this review, the recent findings related to ROS/nitric oxide (NO) and especially of its relationship to innate immunity are summarized.

Utility and safety of LPS-based fermented flour extract as a macrophage activator.

The immune system is part of the homeostasis system. Our research is focused on ways to maintain health, with an emphasis on the role of macrophages. We have hypothesized that tissue macrophages form a systemic network which we believe contributes to the homeostasis system, and have named it the 'macrophage network.' This network creates a dynamic equilibrium situation where macrophages control homeostasis. Our research is based on this macrophage network theory as we believe that the innate immune system provides the foundation for the homeostasis system. As part of our research, we have demonstrated that macrophage activation can provide protection and therapeutic effects for various diseases. Therefore, we have also focused on lipopolysaccharide (LPS). We proved that the LPS of Pantoea agglomerans (which we have named IP-PA1) was useful in preventing various health disorders and in restoring health when administered via the oral or transdermal route. We also developed a 'fermented flour extract', which consists largely of IP-PA1. For LPS to become a valuable commodity, it is very important to provide assurance about safety (when administered orally or transdermally) to build confidence. For this reason, we tested fermented flour extract (in which the major component was IP-PA1) to confirm that it was safe. The results of these safety trials confirmed that oral and transdermal administration of fermented flour extract was very safe. Thus, we believe that fermented flour extract is a new substance that will have applications in health food, cosmetics, animal feeds, fisheries feeds and drugs industries.

Intracellular localization of CD14 protein in intestinal macrophages.

BACKGROUND: Our research is focused on intestinal macrophages, which are believed to contribute to the maintenance of intestinal homeostasis. In addition, intestinal macrophages are unique in that there is an impairment of expression of tumor necrosis factor (TNF) from lipopolysaccharide (LPS). This characteristic can be attributed to the lack or poor level of expression of toll-like receptor 4 (TLR4) or CD14 on the membrane of intestinal macrophages. We therefore decided to identify where CD14 was localized in intestinal macrophages. MATERIALS AND METHODS: The endoplasmic reticulum and Golgi apparatus were double stained and the intracellular localization in the intestinal macrophages was observed using a confocal laser microscope. RESULTS: CD14 of peritoneal macrophages was expressed both in the endoplasmic reticulum and Golgi apparatus. By contrast, intestinal macrophages expressed very little CD14 on the cellular membrane. CD14 was present in the endoplasmic reticulum of intestinal macrophages, but was rare in the Golgi apparatus. CONCLUSION: The lack of expression of CD14 on the cell membrane of intestinal macrophages may be caused by transport interference from the endoplasmic reticulum to the Golgi apparatus.

The role of toll-like receptor 2 in survival strategies of Mycobacterium tuberculosis in macrophage phagosomes.

Mycobacterium tuberculosis (Mtb), an intracellular pathogen, is phagocytosed by alveolar macrophage but it is not digested; it survives, proliferates and establishes Mtb infections. The long-term survival mechanism of Mtb is not yet clear. The host's immune response to Mtb is mainly mediated by a Toll-like receptor 2 (TLR2) in macrophages. In the early stage of the immune response by macrophage activation through TLR2, the proliferation of Mtb is suppressed and there is a direct bactericidal effect or induction of apoptosis in infected macrophages. This indicates that TLR2 signaling functions as a defense system against Mtb infection. However, TLR2 signaling from Mtb also appears to be part of the Mtb strategy to escape immune responses by macrophages, such as has been observed when there has been a decrease in MHC-II expression or antigen-processing activity. TLR signaling is reported both to be and not be involved in the maturation of phagosomes, indicating the possibility of contrary influences. In this review, we summarize immune responses of macrophages through TLR2 in Mtb infection, its involvement in phagosome maturation and we describe survival strategies of Mtb through TLR2 signaling.

Linoleic acid metabolite suppresses skin inflammation and tumor promotion in mice: possible roles of programmed cell death 4 induction.

(+/-)-13-Hydroxy-10-oxo-trans-11-octadecenoic acid (13-HOA) is one of the lipoxygenase metabolites of linoleic acid (LA) from corn germ. Recently, we reported that this metabolite suppressed the expression of lipopolysaccharide-induced proinflammatory genes in murine macrophages by disrupting mitogen-activated protein kinases and Akt pathways. In this study, we investigated the inhibitory effects of 13-HOA on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in ears and skin, as well as tumor promotion in female ICR mice. Pretreatment with 13-HOA (1600 nmol) inhibited ear edema formation by 95% (P < 0.05) in an inflammation test and reduced tumor incidence and the number of tumors per mouse by 40 and 64% (P < 0.05 each), respectively, in a two-stage skin carcinogenesis model. Histological examinations revealed that it decreased epidermal thickness, the number of infiltrated leukocytes and cell proliferation index. Furthermore, 13-HOA (8-40 muM) suppressed TPA-induced anchorage-independent growth of JB6 mouse epidermal cells by 70-100%, whereas LA was virtually inactive. 13-HOA (40 muM) inhibited TPA-induced activator protein-1 transactivation but not extracellular signal-regulated kinase1/2 activation. Interestingly, 13-HOA (40 muM and 1600 nmol in JB6 cells and mouse skin, respectively) induced expression of programmed cell death 4 (Pdcd4), a novel tumor suppressor protein. To our knowledge, this is the first report of a food factor that is able to induce Pdcd4 expression. Collectively, our results indicate that 13-HOA may be a novel anti-inflammatory and antitumor chemopreventive agent with a unique mode of action.