An increasing trend of gastric cancer deaths and inadequate preventive measures in elderly adults.

BACKGROUND: Japan became the world's first country to cover Helicobacter pylori eradication for chronic gastritis under its National Health Insurance (NHI) system in February 2013. Thereafter, H. pylori eradication dramatically increased and gastric cancer deaths began to decrease in Japan. However, the details of gastric cancer deaths and its prevention in the very elderly have not been fully elucidated. METHODS: We analyzed the temporal trend of gastric cancer deaths referencing data from Ministry of Health, Labour and Welfare reports and "Cancer Statistics in Japan-2021" and assessed the numbers of H. pylori test and gastric cancer screening using a national database and a report of cancer screening in Shimane Prefecture, respectively. RESULTS: Although gastric cancer deaths in total population have clearly decreased since 2013, those in people aged 80 years and older are still increasing. People aged 80 years and older represent 9% of the total population and accounted for half of all gastric cancer deaths in 2020. The numbers of H. pylori eradication and gastric cancer screening in people aged 80 years and older were 25% and 25% of those in other generations, respectively. CONCLUSION: In spite of a dramatic increase in H. pylori eradication and a clear decrease in gastric cancer deaths in Japan, gastric cancer deaths in people aged 80 years and older are increasing. This might be due to fewer H. pylori eradication in the elderly than in other generations, indicating the difficulty of gastric cancer prevention in the very elderly.

Down-regulating Effect of a Standardized Extract of Cultured Lentinula edodes mycelia on Cortactin in Prostate Cancer Cells Is Dependent on Malignant Potential.

BACKGROUND/AIM: The incidence and mortality rates of prostate cancer have been increasing worldwide. Although prostate cancer cells grow slowly in the local original site, once the cancer cells spread to distant organs they grow rapidly and show very aggressive features. Cortactin is a protein that regulates the actin cytoskeleton and plays crucial roles in cancer metastasis. Up-regulated cortactin is correlated with the metastatic capacity of prostate cancer cells. AHCC®, a standardized extract of cultured Lentinula edodes mycelia, has been previously reported to have cortactin-down-regulating effects on human pancreatic cancer cells. In the present study, the effects of AHCC® treatment on cortactin levels in prostate cancer cells was evaluated. MATERIALS AND METHODS: LNCaP.FGC, DU145, and PC-3 are human prostate cancer cell lines. LNCaP.FGC is well differentiated, androgen-dependent, and poorly metastatic. DU145 is less differentiated, androgen-independent, and moderate metastatic. PC-3 is less differentiated, androgen-independent, and highly metastatic. The effects of AHCC® treatment on cortactin levels in prostate cancer cells was evaluated by western blot. RESULTS: In vitro AHCC® treatment decreased cortactin levels in LNCaP.FGC and DU145 cells but did not change those in PC-3 cells. CONCLUSION: AHCC® treatment down-regulated cortactin expression in poor and moderate metastatic LNCaP.FGC and DU145 cells but showed no effect on cortactin expression in the highly metastatic PC-3 cells. Further studies are required to elucidate the mechanism of the resistance to AHCC® treatment in highly metastatic PC-3 cells.

A Standardized Extract of Cultured Lentinula edodes mycelia Up-regulates COX-2 in Inflammation-related Malignant Progressive Fibrosarcoma Cell Clone QRsP-11.

BACKGROUND/AIM: Cyclooxygenase is an enzyme that transforms arachidonic acid to prostaglandins. Cyclooxygenase-2 (COX-2) is an isoform of cyclooxygenase. There exist many reports on the expression levels of COX-2 in cancer tissues, and prognosis of cancer patients has been reported to be related to COX-2 up-regulation. In the present study we assessed the suppressive effect of AHCC® on the expression of COX-2 in QRsP-11cells. MATERIALS AND METHODS: QR-32 is a clone which was derived from murine fibrosarcoma BMT-11 cells by treatment with quercetin. These clone cells regress spontaneously after injection into C57BL/6 mice. QRsP-11 is a clone derived from QR-32, showing very aggressive tumorigenicity. AHCC® is a standardized extract of cultured Lentinula edodes mycelia and has been reported to exert suppressive effects on various tumor-associated proteins including HSP27. The protein levels of COX-2 in QR-32 and QRsP-11 cells were compared by using western blotting. Furthermore, the expression levels of COX-2 were assessed in QRsP-11 cells after AHCC®-treatment. RESULTS: Western blot analysis showed a significant up-regulation of COX-2 in QRsP-11 cells compared to QR-32 cells. In vitro AHCC®-treatment increased COX-2 expression levels in QRsP-11 cells contrary to expectations. CONCLUSION: When using AHCC® in cancer treatment, it might be important to decrease COX-2 expression by means of non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin. Further studies are required to clarify the mechanism of up-regulation of COX-2 through AHCC®-treatment.

AT-hook DNA-binding motif-containing protein one knockdown downregulates EWS-FLI1 transcriptional activity in Ewing's sarcoma cells.

Ewing's sarcoma is the second most common bone malignancy in children or young adults and is caused by an oncogenic transcription factor by a chromosomal translocation between the EWSR1 gene and the ETS transcription factor family. However, the transcriptional mechanism of EWS-ETS fusion proteins is still unclear. To identify the transcriptional complexes of EWS-ETS fusion transcription factors, we applied a proximal labeling system called BioID in Ewing's sarcoma cells. We identified AHDC1 as a proximal protein of EWS-ETS fusion proteins. AHDC1 knockdown showed a reduced cell growth and transcriptional activity of EWS-FLI1. AHDC1 knockdown also reduced BRD4 and BRG1 protein levels, both known as interacting proteins of EWS-FLI1. Our results suggest that AHDC1 supports cell growth through EWS-FLI1.

A standardized extract of cultured Lentinula edodes mycelia downregulates cortactin in gemcitabine-resistant pancreatic cancer cells.

AHCC®, a standardized extract of cultured Lentinula edodes mycelia, enhances the therapeutic effects and reduces the adverse effects of chemotherapy. Our previous study reported that treatment with AHCC® downregulated the expression levels of tumor-associated proteins in the gemcitabine-resistant pancreatic cancer cell line, KLM1-R. However, to the best of our knowledge, the role of AHCC® in the inhibition of cell migration remains unexplored. Cortactin (CTTN), an actin nucleation-promoting factor, has been reported to be upregulated and correlated with migration, invasion and metastasis in pancreatic cancer cells. The present study aimed to investigate the effects of AHCC® on cell migration and the protein expression level of CTTN in KLM1-R cells. The Gene Expression Profiling Interactive Analysis (GEPIA2), an online bioinformatics platform, was used to analyze CTTN mRNA expression levels in pancreatic cancer tissues compared with normal pancreatic tissues. CTTN mRNA expression and its association with clinicopathological characteristics were assessed by using the GEPIA2 platform. Next, the effects of AHCC® on KLM1-R cell migration were investigated by in vitro wound-healing assay. The KLM1-R cells were treated with AHCC® at a concentration of 10 mg/ml for 48 h. Western blotting was performed on of cell lysates with anti-CTTN or anti-actin antibodies to assess the protein expression levels of CTTN. Bioinformatics analysis indicated that the mRNA expression level of CTTN increased in pancreatic cancer tissues. The increased mRNA expression levels of CTTN were inversely associated with clinicopathological characteristics, including disease stages and prolonged patient survival times. The administration of 10 mg/ml AHCC® significantly inhibited KLM1-R cells migration compared with controls. The protein expression levels of CTTN were significantly reduced in AHCC®-treated KLM1-R cells, whereas actin expression was not affected. The downregulation of CTTN indicated the anti-metastatic potential of AHCC® in pancreatic cancer cells. Overall, AHCC® may have the potential to be a complementary and alternative therapeutic approach in treating pancreatic cancer.

Proteomic Analysis of Hepatocellular Carcinoma Tissues With Encapsulation Shows Up-regulation of Leucine Aminopeptidase 3 and Phosphoenolpyruvate Carboxykinase 2.

BACKGROUND/AIM: Cancer is the most fatal disease worldwide whose most lethal characteristics are invasion and metastasis. Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. HCC often shows encapsulation, which is related to better prognosis. In this study, proteomic analysis of HCC tissues with and without encapsulation was performed, in order to elucidate the factors which play important roles in encapsulation. MATERIALS AND METHODS: Five HCC tissues surrounded by a capsule and five HCC tissues which broke the capsule were obtained from patients diagnosed with HCC who underwent surgical liver resection. Protein samples from these tissues were separated by two-dimensional gel electrophoresis (2-DE), and the protein spots whose expression was different between encapsulated and non-encapsulated HCC tissues were identified through gel imaging analysis software. The selected protein spots were analyzed and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Two-DE analysis showed 14 spots whose expression was different between encapsulated and non-encapsulated HCC tissues. Of these, 9 were up-regulated and 5 were down-regulated in HCC tissues without encapsulation. The validation by Western blot confirmed that leucine aminopeptidase 3 (LAP3) and phosphoenolpyruvate carboxykinase mitochondrial (PCK2) were up-regulated significantly in HCC tissues with a capsule, compared to HCC tissues that broke the capsule. CONCLUSION: These findings suggest that LAP3 and PCK2 could be factors responsible for the maintenance of encapsulation in HCC tissues.

Gastric cancer deaths by age group in Japan: Outlook on preventive measures for elderly adults.

In February 2013, Japan became the first country in the world to cover Helicobacter pylori eradication for chronic gastritis under its National Health Insurance (NHI) system. Now that eradication therapy is covered by NHI, its usage has increased dramatically, and gastric cancer deaths have begun to decrease. We undertook a detailed epidemiological analysis to investigate effects of expanded NHI coverage for H. pylori eradication therapy on gastric cancer deaths in specific age groups. Numbers of gastric cancer deaths were determined by referencing data from Ministry of Health, Labour and Welfare reports and "Cancer Statistics in Japan - 2018" published by the Foundation for Promotion of Cancer Research. Gastric cancer deaths across all age groups have been clearly decreasing since 2013, but deaths of people aged 80 years and older are still increasing. The number of gastric cancer deaths in people aged in their 80s was 2 times higher than in people aged in their 70s and 4 times higher than in people aged in their 60s. The number of people in their 80s who had an endoscopy was less than half that of people in their 60s and 70s. The eradication therapy has increased dramatically, and gastric cancer deaths are clearly decreasing in Japan. However, this decrease in deaths has not extended to elderly adults aged in their 80s, which suggests that measures to prevent gastric cancer in people aged 80 years and older will be critical to achieving the mission of eliminating gastric cancer in Japan.

Metformin Regulates the Expression of CD133 Through the AMPK-CEBPß Pathway in Hepatocellular Carcinoma Cell Lines.

CD133 is a cellular surface protein, which has been reported to be a cancer stem cell marker, and thus is considered a potential target for cancer treatment. Metformin, one of the biguanides used for the treatment of diabetes, is also known to reduce the risk of cancer development and cancer stem-like cells (CSCs), including the expression of CD133. However, the mechanism underlying the reduction of the expression of CD133 by metformin is not yet understood. This study shows that metformin suppressed CD133 expression mainly by affecting the CD133 P1 promoter via adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling but not the mammalian target of rapamycin (mTOR). AMPK inhibition rescued the reduction of CD133 by metformin. Further experiments demonstrated that CCAAT/enhancer-binding protein beta (CEBPß) was upregulated by metformin and that two isoforms of CEBPß reciprocally regulated the expression of CD133. Specifically, the liver-enriched activator protein (LAP) isoform increased the expression of CD133 by directly binding to the P1 promoter region, whereas the liver-enriched inhibitory protein (LIP) isoform suppressed the expression of CD133. Consistent with these findings, a three dimensional (3D) culture assay and drug sensitivity assay demonstrated that LAP-overexpressing cells formed large spheroids and were more resistant to 5-fluorouracil (5-FU) treatment, whereas LIP-overexpressing cells were more sensitive to 5-FU and showed combined effects with metformin. Our results indicated that metformin-AMPK-CEBPß signaling plays a crucial role in regulating the gene expression of CD133. Additionally, regulating the ratio of LAP/LIP may be a novel strategy for targeting CSCs for the treatment of cancer.

CUB Domain-containing Protein 1 (CDCP1) Is Down-regulated by Active Hexose-correlated Compound in Human Pancreatic Cancer Cells.

BACKGROUND/AIM: We have previously reported that treatment of pancreatic cancer cells with active hexose-correlated compound (AHCC), an extract of a basidiomycete mushroom, decreases the levels of tumor-associated proteins including heat-shock protein 27 (HSP27), heat shock factor 1 (HSF1) and sex-determining region Y-box 2 (SOX2). The transmembrane glycoprotein, CUB domain-containing protein 1 (CDCP1) has been reported to be up-regulated in various cancers, and be associated with invasion and metastasis. The aim of this study was to examine the effect of AHCC on the expression of CDCP1 in KLM1-R cells. MATERIALS AND METHODS: Gemcitabine-resistant pancreatic cancer cells (KLM1-R) were treated with AHCC (10 mg/ml) for 48 h. Western blot analysis of cell extracts with anti-CDCP1 or anti-actin antibodies was performed to assess the expression of CDCP1. RESULTS: Expression of CDCP1 was reduced by AHCC treatment of KLM1-R cells, whereas expression of actin was not affected. The ratio of intensities of CDCP1/actin in AHCC-treated KLM1-R cells was significantly suppressed (p<0.05) compared to untreated cells. CONCLUSION: AHCC down-regulated CDCP1 expression and inhibited the malignant progression of pancreatic cancer cells.

Amigo2-upregulation in Tumour Cells Facilitates Their Attachment to Liver Endothelial Cells Resulting in Liver Metastases.

Since liver metastasis is the main cause of death in cancer patients, we attempted to identify the driver gene involved. QRsP-11 fibrosarcoma cells were injected into the spleens of syngeneic mice to isolate tumour sub-populations that colonize the liver. Cells from liver metastatic nodules were established and subsequently injected intrasplenically for selection. After 12 cycles, the cell subline LV12 was obtained. Intravenous injection of LV12 cells produced more liver metastases than QRsP-11 cells, whereas the incidence of lung metastases was similar to that of QRsP-11 cells. LV12 cells adhered to liver-derived but not to lung-derived endothelial cells. DNA chip analysis showed that amphoterin-induced gene and open reading frame 2 (Amigo2) was overexpressed in LV12 cells. siRNA-mediated knockdown of Amigo2 expression in LV12 cells attenuated liver endothelial cell adhesion. Ex vivo imaging showed that suppression of Amigo2 in luciferase-expressing LV12 cells reduced attachment/metastasis to liver to the same level as that observed with QRsP-11 cells. Forced expression of Amigo2 in QRsP-11 cells increased liver endothelial cell adhesion and liver metastasis. Additionally, Amigo2 expression in human cancers was higher in liver metastatic lesions than in primary lesions. Thus, Amigo2 regulated tumour cell adhesion to liver endothelial cells and formation of liver metastases.

Fermented Brown Rice and Rice Bran with Aspergillus oryzae (FBRA) Prevents Inflammation-Related Carcinogenesis in Mice, through Inhibition of Inflammatory Cell Infiltration.

We have established an inflammation-related carcinogenesis model in mouse, in which regressive QR-32 cells subcutaneously co-implanted with a foreign body-gelatin sponge-convert themselves into lethal tumors due to massive infiltration of inflammatory cells into the sponge. Animals were fed with a diet containing 5% or 10% fermented brown rice and rice bran with Aspergillus oryzae (FBRA). In 5% and 10% FBRA diet groups, tumor incidences were lower (35% and 20%, respectively) than in the non-treated group (70%). We found that FBRA reduced the number of inflammatory cells infiltrating into the sponge. FBRA administration did not cause myelosuppression, which indicated that the anti-inflammatory effects of FBRA took place at the inflammatory lesion. FBRA did not have antitumor effects on the implanted QRsP-11 tumor cells, which is a tumorigenic cell line established from a tumor arisen after co-implantation of QR-32 cells with sponge. FBRA did not reduce formation of 8-hydroxy-2'-deoxyguanine adducts, a marker of oxidative DNA damage in the inflammatory lesion; however, it reduced expression of inflammation-related genes such as TNF-α, Mac-1, CCL3 and CXCL2. These results suggest that FBRA will be an effective chemopreventive agent against inflammation-related carcinogenesis that acts by inhibiting inflammatory cell infiltration into inflammatory lesions.

Fascin regulates chronic inflammation-related human colon carcinogenesis by inhibiting cell anoikis.

By a proteomics-based approach, we identified an overexpression of fascin in colon adenocarcinoma cells (FPCKpP-3) that developed from nontumorigenic human colonic adenoma cells (FPCK-1-1) and were converted to tumorigenic by foreign-body-induced chronic inflammation in nude mice. Fascin overexpression was also observed in the tumors arising from rat intestinal epithelial cells (IEC 6) converted to tumorigenic in chronic inflammation which was induced in the same manner. Upregulation of fascin expression in FPCK-1-1 cells by transfection with sense fascin cDNA converted the cells tumorigenic, whereas antisense fascin-cDNA-transfected FPCKpP-3 cells reduced fascin expression and lost their tumor-forming ability in vivo. The tumorigenic potential by fascin expression was consistent with their ability to survive and grow in the three-dimensional multicellular spheroids. We found that resistance to anoikis (apoptotic cell death as a consequence of insufficient cell-to-substrate interactions), which is represented by the three-dimensional growth of solid tumors in vivo, was regulated by fascin expression through caspase-dependent apoptotic signals. From these, we demonstrate that fascin is a potent suppressor to caspase-associated anoikis and accelerator of the conversion of colonic adenoma cells into adenocarcinoma cells by chronic inflammation.

Chronic inflammation-derived nitric oxide causes conversion of human colonic adenoma cells into adenocarcinoma cells.

It has been suggested that nitric oxide (NO) derived from chronically inflamed tissues is a cause of carcinogenesis. We herein demonstrated that administration of an inducible NO synthase inhibitor, aminoguanidine, significantly suppressed the tumorigenic conversion of human colonic adenoma (FPCK-1-1) cells into adenocarcinoma (FPCK/Inflam) cells accelerated by foreign body-induced chronic inflammation in nude mice. To determine whether NO directly promotes carcinogenesis, we exposed FPCK-1-1 cells continuously to chemically generated NO (FPCK/NO), and periodically examined their tumorigenicity. FPCK/NO cells formed tumors, whereas vehicle-treated cells (FPCK/NaOH) did not. We selected a tumorigenic population from FPCK/NO cells kept it in three-dimensional (3D) culture where in vivo-like multicellular spheroidal growth was expected. FPCK/Inflam cells developed large spheroids whereas FPCK/NO cells formed tiny but growing compact aggregates in 3D culture. Meanwhile, FPCK-1-1 and FPCK/NaOH cells underwent anoikis (apoptotic cell death consequential on insufficient cell-to-substrate interactions) through activation of caspase 3. The survived cells in the 3D culture (FPCK/NO/3D), which were derived from FPCK/NO cells, showed a similar tumor incidence to that of FPCK/Inflam cells. These results showed that NO was one of the causative factors for the acceleration of colon carcinogenesis, especially in the conversion from adenoma to adenocarcinoma in the chronic inflammatory environment.

hypoxia-inducible factors activate CD133 promoter through ETS family transcription factors.

CD133 is a cellular surface protein that has been reported to be a cancer stem cell marker, and thus it is considered to be a potential target for cancer treatment. However, the mechanism regulating CD133 expression is not yet understood. In this study, we analyzed the activity of five putative promoters (P1-P5) of CD133 in human embryonic kidney (HEK) 293 cells and colon cancer cell line WiDr, and found that the activity of promoters, particularly of P5, is elevated by overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α). Deletion and mutation analysis identified one of the two E-twenty six (ETS) binding sites (EBSs) in the P5 region as being essential for its promoter activity induced by HIF-1α and HIF-2α. In addition, a chromatin imunoprecipitation assay demonstrated that HIF-1α and HIF-2α bind to the proximal P5 promoter at the EBSs. The immunoprecipitation assay showed that HIF-1α physically interacts with Elk1; however, HIF-2α did not bind to Elk1 or ETS1. Furthermore, knockdown of both HIF-1α and HIF-2α resulted in a reduction of CD133 expression in WiDr. Taken together, our results revealed that HIF-1α and HIF-2α activate CD133 promoter through ETS proteins.

Enhancement of in vitro cell motility and invasiveness of human malignant pleural mesothelioma cells through the HIF-1α-MUC1 pathway.

In this study, we examined the effects of hypoxia on the malignancy of human malignant pleural mesothelioma (MPM) cell lines, and found (1) hypoxia enhanced motility and invasiveness of human malignant pleural mesothelioma (MPM) cells; (2) this phenomenon resulted from increased expression of sialylated MUC1 through the activation of HIF-1 pathway; (3) two HIF-binding sites located in the promoter region of MUC1 were important for MUC1 transactivation under hypoxia. These findings are useful for better understanding molecular mechanisms of aggressive behavior of MPM cells and for targeting them in the clinical therapies for MPM patients.

The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1-AMPK complex.

AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic α subunit and regulatory ß and γ subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the α-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPKα2-binding protein. Artemis was found to co-immunoprecipitate with AMPKα2, and the co-localization of Artemis with AMPKα2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPKα2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPKα2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex.

Toll-like receptor 7-mediated enhancement of contextual fear memory in mice.

Toll-like receptor (TLR) 7 recognizes viral single-stranded RNA and triggers production of the type I interferons (IFNs) IFN-α and IFN-ß. Imiquimod, a synthetic TLR7 ligand, induces production of type I IFNs and is used clinically as an antiviral and antitumor drug. In the present study, we examined the effect of imiquimod on conditioned and innate fear behaviors in mice. Imiquimod was administered 2, 4, or 15 h before contextual fear conditioning. Imiquimod treatment 4 or 15 h before fear conditioning significantly enhanced context-dependent freezing behavior. This imiquimod-induced enhancement of fear-related behaviors was observed 120 h after fear conditioning. In contrast, imiquimod failed to enhance context-dependent freezing behavior in TLR7 knockout mice. Imiquimod had no significant effect on pain threshold or on innate fear-related behavior, as measured by the elevated plus-maze. The levels of type I IFN mRNA in the brain were significantly increased at 2 h after imiquimod treatment. Imiquimod also increased interleukin (IL)-1ß mRNA expression in the brain at 4 h following administration, while mRNA expression of F4/80, a macrophage marker, was unaffected by imiquimod treatment. Our findings suggest that TLR7-mediated signaling enhances contextual fear memory in mice, possibly by inducing the expression of type I IFNs and IL-1ß in the brain.

Development of a quantitative bioassay to assess preventive compounds against inflammation-based carcinogenesis.

Reducing cancer incidence and mortality by use of cancer-chemopreventive agents is an important goal. We have established an in vitro bioassay that is able to screen large numbers of candidate chemicals that are positive for prevention of inflammation-related carcinogenesis. To accomplish this we have added candidate chemicals or vehicles and freshly isolated, fluorescent dye-labeled inflammatory cells that were overlaid on TNF-alpha-stimulated mouse endothelial cells in a 96-well plate. Inhibition of inflammatory cell attachment to the endothelial cells by the chemicals was quantified by the intensity of fluorescence from the adherent inflammatory cells after removing unattached cells. Using this assay, we selected two chemicals, auraptene and turmerones, for further study. As an in vivo test, diets containing these test chemicals were administered to mice with a piece of foreign body, gelatin sponge, that had been implanted to cause inflammation, and we found that the number of inflammatory cells that infiltrated into the subcutaneously implanted gelatin sponge was reduced compared to that found in the mice fed with a control diet. Moreover, diets containing either of the two chemicals prevented inflammation-based carcinogenesis in a mouse model. We found that the compounds reduced not only the number of infiltrating cells but also the expression of inducible nitric oxide synthase (iNOS) or formation of 8-hydroxy-2'-deoxyguanine (8-OHdG) in the infiltrated cells. Moreover, both compounds but not controls sustained the reducing activity in the inflammatory lesion, and this finding was confirmed by using non-invasive in vivo electron spin resonance. The newly established in vitro screening assay will be useful for finding biologically effective chemopreventive agents against inflammation-related carcinogenesis.

p53 transactivation is involved in the antiproliferative activity of the putative tumor suppressor RBM5.

RBM5 (RNA-binding motif protein 5) is a nuclear RNA binding protein containing 2 RNA recognition motifs. The RBM5 gene is located at the tumor suppressor locus 3p21.3. Deletion of this locus is the most frequent genetic alteration in lung cancer, but is also found in other human cancers. RBM5 is known to induce apoptosis and cell cycle arrest but the molecular mechanisms of RBM5 function are poorly understood. Here, we show that RBM5 is important for the activity of the tumor suppressor protein p53. Overexpression of RBM5 enhanced p53-mediated inhibition of cell growth and colony formation. Expression of RBM5 augmented p53 transcriptional activity in reporter gene assays and resulted in increased mRNA and protein levels for endogenous p53 target genes. In contrast, shRNA-mediated knockdown of endogenous RBM5 led to decreased p53 transcriptional activity and reduced levels of mRNA and protein for endogenous p53 target genes. RBM5 affected protein, but not mRNA, levels of endogenous p53 after DNA damage suggest that RBM5 contributes to p53 activity through post-transcriptional mechanisms. Our results show that RBM5 contributes to p53 transcriptional activity after DNA damage and that growth suppression and apoptosis mediated by RBM5 are linked to activity of the tumor suppressor protein p53.