Association between α-defensin 5 and the expression and function of P-glycoprotein in differentiated intestinal Caco-2 cells.

α-Defensin 5 is known to be secreted by Paneth cells in the small intestine and plays an important role in eliminating pathogenic microorganisms. It has been reported that a decrease in α-defensin 5 level in the human small intestine is a risk of inflammatory bowel disease (IBD). Furthermore, P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter superfamily, encoded by the ABCB1/MDR1 gene, plays an important role in the front line of host defense by protecting the gastrointestinal barrier from xenobiotic accumulation and may contribute to the development and persistence of IBD. Therefore, we examined the relationship between α-defensin 5 and the expression and function of P-gp using a human gastrointestinal model cell line (Caco-2). We found that MDR1 mRNA and P-gp protein level were increased in Caco-2 cells as well as α-defensin 5 secretion corresponded with the duration of cell culture. Exposure to α-defensin 5 peptide and recombinant tumor necrosis factor-α (TNF-α) significantly increased the expression and function P-gp. The mRNA levels of interleukin (IL)-8, IL-6, TNF-α, IL-1ß, and IL-2 were also increased following exposure to TNF-α, similar to α-defensin 5 treatment. These results suggest that α-defensin 5 regulates P-gp expression and function by increasing TNF-α expression in Caco-2 cells.

Requirement of CLIC4 Expression in Human Colorectal Cancer Cells for Sensitivity to Growth Inhibition by Fucoxanthinol.

BACKGROUND/AIM: Fucoxanthinol (FxOH), a marine carotenoid, induces apoptosis and anoikis in human colorectal cancer (CRC) DLD-1 cells via the down-regulation of chloride intracellular channel 4 (CLIC4) expression, a key molecule for apoptosis. However, whether FxOH is susceptible to CLIC4 expression and its regulatory mechanisms in human CRC cells remains unknown. We investigated the inhibitory effects of FxOH on six types of human CRC cells with CLIC4 regulation. MATERIALS AND METHODS: The association between FxOH and CLIC4 was investigated using gene knockdown, overexpression, and transcriptome analyses. RESULTS: CLIC4 expression in CRC cells was a significant factor associated with sensitivity to FxOH. CLIC4 regulates many cancer-related signals and participates in growth inhibition in FxOH-treated DLD-1 cells. Both CLIC4 knockdown and overexpression attenuated the inhibitory effects of FxOH on DLD-1 cells. CONCLUSION: Our findings suggest that the protein expression of CLIC4 and its regulating mechanisms play significant roles regarding cell death in human CRC cells by FxOH treatment. Further investigation by in vitro and in vivo models is needed to determine the effect of CLIC4.

A Biscuit Containing Fucoxanthin Prevents Colorectal Carcinogenesis in Mice.

Fucoxanthin (Fx) is a critical pigment required for photosynthesis in brown algae and microalgae. Fx is also a dietary marine carotenoid that with potent anticancer activity in vitro and in vivo. Some popular light meals for increased satiety, such as biscuits, cereals, and crackers, are frequently fortified with micronutrients for human health benefits. However, data on the anticancer potential of Fx-supplemented light meals in humans and animal models remain limited. In the present study, we investigated the anticancer effects of a Fx-supplemented biscuit using a carcinogenic murine azoxymethane/dextran sodium sulfate (AOM/DSS) model. We observed that periodic administration of biscuits containing 0.3% Fx (Fx-biscuit) at an interval of 3 days (each 15 h) per week for 15 weeks significantly inhibited colorectal carcinogenesis in AOM/DSS mice. Comprehensive gene analysis demonstrated that the Fx-biscuit significantly altered the expression of 138 genes in the colorectal mucosal tissue of the mice. In particular, the expression of heat shock protein 70 (HSP70) genes, Hspa1b (-35.7-fold) and Hspa1a (-34.9-fold), was markedly downregulated. HSP70 is a polyfunctional chaperone protein that is involved in cancer development. Compared to the control-biscuit group, the number of cells with markedly high fluorescence for HSP70 protein (HSP70high) in colorectal mucosal crypts and adenocarcinomas significantly reduced by 0.3- and 0.2-fold, respectively, in the Fx-biscuit group. Our results suggested that Fx-biscuit possesses chemopreventive potential in the colorectal cancer of AOM/DSS mice via the downregulation of HSP70.

Suppression of C-C chemokine receptor 1 is a key regulation for colon cancer chemoprevention in AOM/DSS mice by fucoxanthin.

Fucoxanthin (Fx) has shown potential cancer chemopreventive functions in a carcinogenic murine azoxymethane/dextran sodium sulfate (AOM/DSS) model. However, the molecular mechanisms based on transcriptome profiles in vivo remain poorly understood. We investigated Fx-dependent alterations of the transcriptome with cancer-associated proteins in colorectal mucosal tissue obtained from AOM/DSS mice with or without Fx treatment. Fx administration (50 mg/kg body weight for 14 weeks) significantly prevented the onset of colorectal adenocarcinoma in AOM/DSS mice. A transcriptome analysis revealed that 11 signals, including adhesion, cell cycle, chemokine receptor, interleukin, MAPK, PI3K/AKT, p53, RAS, STAT, TGF-ß, and Wnt were remarkably altered by Fx administration. In particular, chemokine (C-C motif) receptor 1 (Ccr1), which is contained in a gene set related to cytokine-cytokine receptor interactions, was the only significantly down-regulated gene after Fx administration for both 7 and 14 weeks. CCR1, AKT, Cyclin D1, and Smad2 were found to play central roles in the 11 signals shown above. Fx administration significantly down-regulated CCR1 (0.3- and 0.5-fold in mucosal crypts and lamina propria, respectively), pAKT(Ser473) (0.2-fold in mucosal crypts), Cyclin D1 (0.4-fold in mucosal crypts), and pSmad2(Ser465/467) (0.7-fold in mucosal crypts) compared with proteins in these tissues of control mice after Fx administration for 14 weeks. Our findings suggested that Fx exerts a chemopreventive effect in AOM/DSS mice through attenuation of CCR1 expression along with 11 cancer-associated signals.

A Marine Carotenoid of Fucoxanthinol Accelerates the Growth of Human Pancreatic Cancer PANC-1 Cells.

Fucoxanthin and its metabolite fucoxanthinol (FxOH), highly polar xanthophylls, exert strong anticancer effects against many cancer cell types. However, the effects of Fx and FxOH on pancreatic cancer, a high mortality cancer, remain unclear. We herein investigated whether FxOH induces apoptosis in human pancreatic cancer cells. FxOH (5.0 µmol/L) significantly promoted the growth of human pancreatic cancer PANC-1 cells, but induced apoptosis in human colorectal cancer DLD-1 cells. A microarray-based gene analysis revealed that the gene sets of cell cycle, adhesion, PI3K/AKT, MAPK, NRF2, adipogenesis, TGF-ß, STAT, and Wnt signals in PANC-1 cells were markedly altered by FxOH. A western blot analysis showed that FxOH up-regulated the expression of integrin ß1 and PPARγ as well as the activation of pFAK(Tyr397), pPaxillin(Tyr31), and pAKT(Ser473) in PANC-1 cells, but exerted the opposite effects in DLD-1 cells. Moreover, the expression of FYN, a downstream target of integrin subunits, was up-regulated (7.4-fold by qPCR) in FxOH-treated PANC-1 cells. These results suggest that FxOH accelerates the growth of PANC-1 cells by up-regulating the expression of integrin ß1, FAK, Paxillin, FYN, AKT, and PPARγ.

Fucoxanthin Prevents Pancreatic Tumorigenesis in C57BL/6J Mice That Received Allogenic and Orthotopic Transplants of Cancer Cells.

Fucoxanthin (Fx) is a marine carotenoid with anti-inflammatory and anti-cancer properties in various animal models of carcinogenesis. However, there is currently no information on the effects of Fx in animal models of pancreatic cancer. We investigated the chemopreventive effects of Fx in C57BL/6J mice that received allogenic and orthotopic transplantations of cancer cells (KMPC44) derived from a pancreatic cancer murine model (Ptf1aCre/+; LSL-krasG12D/+). Using microarray, immunofluorescence, western blot, and siRNA analyses, alterations in cancer-related genes and protein expression were evaluated in pancreatic tumors of Fx-administered mice. Fx administration prevented the adenocarcinoma (ADC) development of pancreatic and parietal peritoneum tissues in a pancreatic cancer murine model, but not the incidence of ADC. Gene and protein expressions showed that the suppression of chemokine (C-C motif) ligand 21 (CCL21)/chemokine receptor 7 (CCR7) axis, its downstream of Rho A, B- and T-lymphocyte attenuator (BTLA), N-cadherin, αSMA, pFAK(Tyr397), and pPaxillin(Tyr31) were significantly suppressed in the pancreatic tumors of mice treated with Fx. In addition, Ccr7 knockdown significantly attenuated the growth of KMPC44 cells. These results suggest that Fx is a promising candidate for pancreatic cancer chemoprevention that mediates the suppression of the CCL21/CCR7 axis, BTLA, tumor microenvironment, epithelial mesenchymal transition, and adhesion.

Detection of Cells Displaying High Expression of CLIC4 in Tumor Tissue of Patients With Colorectal Cancer.

BACKGROUND/AIM: Chloride intracellular channel 4 (CLIC4) is associated with the progression of colorectal cancer (CRC). However, quantitative differences in CLIC4 expression in epithelial and stromal cells of normal mucosal tissue (NT), cancer adjacent to normal colorectal mucosal tissue (NAT), and CRC tissue remain unclear. MATERIALS AND METHODS: We investigated the number of CLIC4 high-expressing (CLIC4high) cells in colorectal tissue of CRC patients and healthy individuals. RESULTS: The number of CLIC4high cells in malignant epithelial cells at early cancerous lesions was significantly higher than that in NAT, but was significantly lower or tended to become low corresponding to the progression of colorectal carcinogenesis. Meanwhile, the number of CLIC4high cells in the stromal tissue remained low in NAT compared to late lesions. CONCLUSION: The number of CLIC4high cells is a useful predictor in determining the pathological condition in both malignant epithelial and stromal tissues of CRC patients.

An analytical survey of benzotriazole UV stabilizers in plastic products and their endocrine-disrupting potential via human estrogen and androgen receptors.

Benzotriazole UV stabilizers (BUVSs) are added to various materials to prevent damage from UV-irradiation. Recently, there has been great concern regarding the endocrine-disrupting effects of exposure to microplastic-derivative BUVSs in particular. In this study, we measured the concentrations of nine representative BUVSs in the plastic bottle caps of 10 beverages, 4 food packages, and 4 plastic shopping bags purchased from Japanese grocery stores by GC-MS analysis, and found that eight BUVSs, except for 2-(3,5-di-tert-butyl-2-hydroxyphenyl)-2H-benzotriazole (UV-320), were detected from these plastic products. In particular, 2-(2-hydroxy-5-methylphenyl) benzotriazole (UV-P) and 2-(2-hydroxy-3-tert-butyl-5-methylphenyl)-5-chlorobenzotriazole (UV-326) were detected from all the bottle caps at concentrations in the order of ng/g. In addition, we characterized the agonistic and/or antagonistic activities against human estrogen receptors (ERα/ß) and androgen receptor (AR) of 13 BUVSs. Results revealed that, among the 13 BUVSs, UV-P, 2-(5-tert-butyl-2-hydroxyphenyl) benzotriazole (UV-PS), 2-[2-hydroxy-5-[2-(methacryloyloxy)ethyl]phenyl]-2H-benzotriazole (UV-090) and 2-(2-hydroxy-5-tert-octylphenyl)-benzotriazole (UV-329) showed ERα and/or ERß agonistic activity, with UV-P being the most potent based on these assays. On the other hand, UV-320 and 2-(3-s-butyl-5-tert-butyl-2-hydroxyphenyl) benzotriazole (UV-350) showed both ERα and ERß antagonistic activities, and 2-(3,5-di-tert-amyl-2-hydroxylphenyl) benzotriazole (UV-328) and UV-329 acted as ERß antagonists. In the AR assay, UV-P and 2-(3-allyl-2-hydroxy-5-methylphenyl)-2H-benzotriazole (UV-9) showed AR antagonistic activity although none of the test compounds showed AR agonistic activity. Taken together, our findings suggest that a series of BUVSs are present in our environments via plastic materials and several of these compounds possess endocrine-disrupting potential, such as ERα/ß agonistic and/or antagonistic activity and AR antagonistic activity. UV-P and its structurally similar compounds, in particular, appear to be a cause for concern.

Downregulated expression of organic anion transporting polypeptide (Oatp) 2b1 in the small intestine of rats with acute kidney injury.

The expression of transporters on the apical and basal membranes of renal tubular cells is modulated under acute kidney injury (AKI). However, little is known about alterations in non-renal transporters in the tissues other than the kidney under AKI situation. This study aimed to assess the modulation of organic anion transporting polypeptide (Oatp) 1a2 and Oatp2b1 expression/function in the small intestine of rats with drug-induced AKI. AKI was induced by intraperitoneal administration of cisplatin at a dose of 5 mg/kg. On day 3 after cisplatin administration, morphological changes in the small intestine, Oatp1a2 and Oatp2b1 expression, and absorption of pravastatin and theophylline were evaluated. Non-negligible atrophy was observed in the jejunum and ileum of the AKI rats. However, the absorption of theophylline was not affected. While intestinal Oatp2b1 expression was markedly decreased in the AKI rats, no alteration was observed in Oatp1a2 expression. The plasma levels of pravastatin after intraluminal administration declined significantly in the AKI rats. However, no such decline was observed after intravenous administration. This study suggested that the responses of intestinal Oatps to experimentally induced AKI was not unidirectional and that pravastatin absorption was governed more potently by Oatp2b1 than by Oatp1a2 in the rat intestine.

Fucoxanthin and Colorectal Cancer Prevention.

Colorectal cancer (CRC), which ranks among the top 10 most prevalent cancers, can obtain a good outcome with appropriate surgery and/or chemotherapy. However, the global numbers of both new cancer cases and death from CRC are expected to increase up to 2030. Diet-induced lifestyle modification is suggested to be effective in reducing the risk of human CRC; therefore, interventional studies using diets or diet-derived compounds have been conducted to explore the prevention of CRC. Fucoxanthin (Fx), a dietary carotenoid, is predominantly contained in edible brown algae, such as Undaria pinnatifida (wakame) and Himanthalia elongata (Sea spaghetti), which are consumed particularly frequently in Asian countries but also in some Western countries. Fx is responsible for a majority of the anticancer effects exerted by the lipophilic bioactive compounds in those algae. Interventional human trials have shown that Fx and brown algae mitigate certain risk factors for CRC; however, the direct mechanisms underlying the anti-CRC properties of Fx remain elusive. Fx and its deacetylated type "fucoxanthinol" (FxOH) have been reported to exert potential anticancer effects in preclinical cancer models through the suppression of many cancer-related signal pathways and the tumor microenvironment or alteration of the gut microbiota. We herein review the most recent studies on Fx as a potential candidate drug for CRC prevention.

Effect of Fucoxanthinol on Pancreatic Ductal Adenocarcinoma Cells from an N-Nitrosobis(2-oxopropyl)amine-initiated Syrian Golden Hamster Pancreatic Carcinogenesis Model.

BACKGROUND/AIM: Fucoxanthinol (FxOH) is a marine carotenoid metabolite with potent anti-cancer activity. However, little is known about the efficacy of FxOH in pancreatic cancer. In the present study, we investigated the inhibitory effect of FxOH on six types of cells cloned from N-nitrosobis(2-oxopropyl)amine (BOP)-induced hamster pancreatic cancer (HaPC) cells. MATERIALS AND METHODS: FxOH action and its molecular mechanisms were investigated in HaPC cells using flow-cytometry, comprehensive gene array, and western blotting analyses. RESULTS: FxOH (5.0 µM) significantly suppressed the growth of four out of six types of HaPC cells. Moreover, FxOH significantly suppressed cell cycle, chemokine, integrin, actin polymerization, microtubule organization and PI3K/AKT and TGF-ß signals, and activated caspase-3 followed by apoptosis and anoikis induction in HaPC-5 cells. CONCLUSION: FxOH may have a high potential as a cancer chemopreventive agent in a hamster pancreatic carcinogenesis model.

Fucoxanthin Prevents Colorectal Cancer Development in Dextran Sodium Sulfate-treated Apc Min/+ Mice.

BACKGROUND/AIM: A xanthophyll of fucoxanthin (Fx) is a potential chemopreventive agent. Familial adenomatous polyposis (FAP) is an inherited disease that is associated with a high risk of developing colorectal cancer. However, it remains unclear whether Fx can modify colorectal tumorigenesis in ApcMin/+ mice, a model mouse for human FAP. MATERIALS AND METHODS: We investigated the chemopreventive effect of Fx in dextran sodium sulfate (DSS)-treated ApcMin/+ mice. RESULTS: Administration of Fx in the diet for 5 weeks significantly suppressed the number of colorectal adenocarcinomas in DSS-treated male ApcMin/+ mice, although the treatment did not affect the occurrence of colorectal dysplastic crypts and adenoma in the mice. In addition, Fx down-regulated cyclin D1 expression (0.6-fold) in colorectal mucosa of ApcMin/+ mice when compared with that of the control mice. CONCLUSION: Fx possesses chemopreventive potential against progression of colorectal carcinogenesis in ApcMin/+ mice that receive inflammatory stimuli.

Analysis of α-Defensin 5 Secretion in Differentiated Caco-2 Cells: Comparison of Cell Bank Origin.

α-Defensin 5 has a particularly broad antibacterial spectrum; it eliminates pathogenic microorganisms and regulates intestinal flora. Although Caco-2 cells are similar to small intestinal cells, it is unclear whether they secrete α-defensin 5. Therefore, we investigated whether Caco-2 cells secrete α-defensin 5 and determined the secretion mechanism using cells from three cell banks (ATCC, DSMZ, and RIKEN). The Caco-2 cell proliferation rate increased with the number of culture days, irrespective of cell bank origin. On the other hand, the alkaline phosphatase activity, which affects cell differentiation and the mRNA levels of several cytokines, such as interleukin 8 (IL-8), IL-6, IL-1ß, tumor necrosis factor-α (TNF-α), and IL-2, in the Caco-2 cells fluctuated with the number of culture days, and differed for each cell bank. α-Defensin 5 secretion was detected in all three cell bank Caco-2 cells; particularly, the ATCC Caco-2 cells grew linearly depending on the cell culture day as well as the levels of IL-8 and TNF-α mRNA. This suggested that α-defensin 5 secretion in the ATCC Caco-2 cells was associated with fluctuations in the mRNA levels of various cytokines, such as IL-8 and TNF-α. In conclusion, Caco-2 cells may be a simple model for screening health food components and drugs that affect α-defensin 5 secretion.

A Fucoxanthinol Induces Apoptosis in a Pancreatic Intraepithelial Neoplasia Cell.

BACKGROUND/AIM: Fucoxanthinol (FxOH), a predominant metabolite from fucoxanthin (Fx), can exert potential anti-cancer effects in various cancers. However, limited data are available on the effect of FxOH or Fx on pancreatic cancer. The present study investigated the effect of FxOH on a cell line derived from pancreatic cancer tissue developed in Ptf1aCre/+; LSL-k-rasG12D/+ mice. MATERIALS AND METHODS: Using flow-cytometric, microarrays, and western blotting analyses, alterations in FxOH-induced apoptosis-related gene expression and protein levels were evaluated in a mice pancreatic cancer cell line, KMPC44. RESULTS: FxOH significantly arrested the cells at S phase along with suppression of many gene sets, such as cytokine- cytokine receptor interaction and cell adhesion molecule CAMS. Moreover, attenuated protein levels for cytokine receptors, adhesion, phosphatidylinositol-3 kinase/protein kinase B, and mitogen-activated protein kinase were observed. CONCLUSION: FxOH may prevent pancreatic cancer development in a murine cancer model.

Alteration of fecal microbiota by fucoxanthin results in prevention of colorectal cancer in AOM/DSS mice.

Fucoxanthin (Fx), a marine carotenoid found in edible brown algae, is well known for having anticancer properties. The gut microbiota has been demonstrated as a hallmark for colorectal cancer progression in both humans and rodents. However, it remains unclear whether the gut microbiota is associated with the anticancer effect of Fx. We investigated the chemopreventive potency of Fx and its effect on gut microbiota in a mouse model of inflammation-associated colorectal cancer (by azoxymethane/dextran sulfate sodium treatment). Fx administration (30 mg/kg bw) during a 14 week period significantly inhibited the multiplicity of colorectal adenocarcinoma in mice. The number of apoptosis-like cleaved caspase-3high cells increased significantly in both colonic adenocarcinoma and mucosal crypts. Fx administration significantly suppressed Bacteroidlales (f_uc; g_uc) (0.3-fold) and Rikenellaceae (g_uc) (0.6-fold) and increased Lachnospiraceae (g_uc) (2.2-fold), compared with those of control mice. Oral administration of a fecal suspension obtained from Fx-treated mice, aimed to enhance Lachnospiraceae, suppress the number of colorectal adenocarcinomas in azoxymethane/dextran sulfate sodium-treated mice with a successful increase in Lachnospiraceae in the gut. Our findings suggested that an alteration in gut microbiota by dietary Fx might be an essential factor in the cancer chemopreventive effect of Fx in azoxymethane/dextran sulfate sodium-treated mice.

Continuity of Tumor Microenvironmental Suppression in AOM/DSS Mice by Fucoxanthin May Be Able to Track With Salivary Glycine.

BACKGROUND/AIM: Fucoxanthin (Fx) is a potent anticancer carotenoid, demonstrated by mouse cancer models. We recently showed the decrease of salivary glycine could represent an attenuation of tumor microenvironment (TME) formation in an azoxymethane/dextran sodium sulfate (AOM/DSS) colon cancer mouse model. However, it remains unclear whether the salivary glycine is an indicator for continuous TME suppression of Fx in the mice. MATERIALS AND METHODS: In the present study, we time-dependently analyzed salivary metabolites in AOM/DSS mice, and investigated candidate markers to evaluate the continuous inhibition of colonic TME formation and carcinogenesis in the mice with and without Fx. RESULTS: Fx attenuated the incidence and/or multiplicity of colonic lesions developed in AOM/DSS mice. The number of apoptosis-like cleaved caspase-3high cells was significantly increased, and colonic cancer stem cell-like CD44high/EpCAMhigh cells and cancer-associated fibroblast-like αSMAhigh cells were significantly decreased in colon mucosal tissue by Fx administration. Salivary glycine at 4, 11 and 14 weeks after the final DSS exposure in the Fx-treated mice showed successful and consecutive decreases of 0.5-, 0.4- and 0.7-fold respectively compared to that of control mice. CONCLUSION: Salivary glycine is a valuable indicator that could evaluate sustained efficacy of cancer chemopreventive effects of Fx in AOM/DSS mice.