Pentapeptide IIAEK ameliorates cholesterol metabolism via the suppression of intestinal cholesterol absorption in mice.

Dietary protein-derived peptides are effective in improving dyslipidemia and hypercholesterolemia. We previously identified a novel cholesterol-lowering pentapeptide IIAEK from milk beta-lactoglobulin. However, it remains unclear whether IIAEK affects the micellar solubility of cholesterol and the bile acid-binding ability to lower cholesterol. Moreover, there is no direct evidence that IIAEK inhibits intestinal cholesterol absorption and affects hepatic cholesterol and fecal steroid excretion in vivo. Herein, we showed that IIAEK did not affect the micellar solubility of cholesterol and the bile acid-binding ability. However, we found that IIAEK decreased serum and liver cholesterol levels and increased fecal steroid excretion in mice. Interestingly, IIAEK markedly suppressed the intestinal absorption of [3H]-cholesterol in mice. In conclusion, we found that IIAEK ameliorated cholesterol metabolism by suppressing intestinal cholesterol absorption without affecting in vitro micellar solubility of cholesterol and the bile acid-binding ability in mice.

Protamine-derived peptide RPR (Arg-Pro-Arg) ameliorates oleic acid-induced lipogenesis via the PepT1 pathway in HepG2 cells.

The protamine-derived peptide arginine-proline-arginine (RPR) can ameliorate lifestyle-related diseases such as obesity and hypercholesterolemia. Thus, we hypothesized that the hypolipidemic activity of RPR could attenuate events leading to non-alcoholic fatty liver disease. Addition of 2 m m oleic acid (OA) to the culture medium induced fatty liver conditions in HepG2 cells. The OA + RPR group showed significantly decreased cellular or medium triglyceride (TG) level compared with the OA group. Stearoyl-CoA desaturase-1 (SCD1) or sterol regulatory element-binding protein 1 (SREBP1) protein level was significantly lower in the OA + RPR group than in the OA group. In the R + P + R amino acid mixture-treated group, the TG level was not significantly different from that in the OA-treated group. The OA + RP- or OA + PR-treated groups showed significantly decreased cellular TG level compared with the OA group. Moreover, the effect of RPR disappeared when the peptide transporter 1 (PepT1) was knocked down with a siRNA. Collectively, our results demonstrated that RPR effectively ameliorated hepatic steatosis in HepG2 cells via the PepT1 pathway.

Eugeniin improves cholesterol metabolism in HepG2 cells and Caco-2 cells.

Considering the absence of prior studies on the cholesterol metabolism-improving effects of eugeniin, the present investigation aimed to explore the potential impact of eugeniin on cholesterol metabolism. This study sought to elucidate the molecular mechanisms involved in this process using HepG2 and Caco-2 cells treated with 5 µm eugeniin. The intracellular cholesterol levels in HepG2 and Caco-2 cells were significantly decreased in the 24-h eugeniin-treated group. The protein and messenger ribonucleic acid (mRNA) levels of the low-density lipoprotein receptor (LDLR) were increased, while 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase protein and mRNA levels were decreased in HepG2 cells 6 h of the eugeniin-treated group. Additionally, LDLR protein and mRNA levels were increased in HepG2 cells after 24 h of eugeniin treatment. In Caco-2, the protein and mRNA levels of ATP-binding cassette transporter 1 were increased after 24 h eugeniin treatment. This novel finding indicates that eugeniin improves cholesterol metabolism in human cell cultures.

[A Case of Achieving CR through Chemoradiotherapy for Postoperative Oligo Lymph Node Metastasis in Gastric Cancer].

We present a case of oligo lymph node metastasis in a 70s man who had previously undergone subtotal gastrectomy for advanced gastric cancer in the prepylorus. Postoperatively, adjuvant chemotherapy was administered for a duration of 1 year. During the third postoperative year, elevated tumor markers and lymph node enlargement prompted a diagnosis of lymph node metastasis. Subsequent chemoradiotherapy resulted in a complete response(CR), which has been sustained for 2 years without any recurrence. The outcomes of this case indicate that chemoradiotherapy stands as a viable treatment option for oligo lymphatic recurrence in gastric cancer.

The physiological blood concentration of phenylalanine-proline can ameliorate cholesterol metabolism in HepG2 cells.

We have previously reported that the dipeptide Phe-Pro affects lipid metabolism in vivo and in vitro, but very little is known regarding the mechanism of action of Phe-Pro after it is absorbed by the intestines via PepT1. In this study, we administered a single oral dose of Phe-Pro to rats and quantified its concentration in the portal plasma using LC-TOF/MS analysis. Additionally, the physiological blood concentration of Phe-Pro was added to the lipid accumulation model of HepG2 cells to decrease intracellular cholesterol and increase the expression of CYP7A1 and PPARα mRNA levels. Moreover, we analyzed the binding of PPARα and Phe-Pro using AlphaFold2. We found that Phe-Pro is a ligand for PPARα. To the best of our knowledge, this is the first study that shows Phe-Pro to be present in the portal plasma. We found for the first time that Phe-Pro ameliorated cholesterol metabolism in HepG2 cells.

The blockade of interleukin-33 released by hepatectomy would be a promising treatment option for cholangiocarcinoma.

Interleukin-33 (IL-33), an alarmin released during tissue injury, facilitates the development of cholangiocarcinoma (CCA) in a murine model. However, it is unclear whether IL-33 is associated with human CCA. The aim of this study was to support the following hypothesis: IL-33 is released during hepatectomy for CCA, subsequently facilitating the development of subclinical CCA and eventually leading to recurrent disease. IL-33 expression was assessed in various samples from both humans and mice including resected liver and paired plasma samples collected at hepatectomy and after surgery, and its influences on recurrent disease and patient prognosis were determined. Homogenized human liver samples with high or low IL-33 expression were added to the culture medium of human CCA cells, and the changes in proliferation and migration were evaluated. To examine the effects of inhibiting the IL-33 release induced by hepatectomy, syngraft transplantation of murine CCA cells was performed in C57BL/6J mice with or without IL-33 blockade. The amount of IL-33 released into the plasma during hepatectomy correlated with the background liver expression. High expression of IL-33 in the liver was an independent risk factor for recurrence. Homogenized liver tissue strongly expressing IL-33 increased both the proliferation and migration of tumor cells. Mice who underwent hepatectomy exhibited CCA progression in the remnant liver, whereas blockade of IL-33 during hepatectomy inhibited tumor progression. Thus, we concluded that surgery for CCA with curative intent paradoxically induced IL-33 release, which facilitated CCA recurrence, and anti-IL-33 therapy during hepatectomy might reduce the risk of CCA recurrence.

Ellagic acid affects mRNA expression levels of genes that regulate cholesterol metabolism in HepG2 cells.

Ellagic acid has been shown to improve cholesterol metabolism in animal studies, but the molecular mechanisms underlying this function have not been fully understood. We performed DNA microarray analysis to elucidate the effects of ellagic acid on cholesterol metabolism in HepG2 hepatocytes. This revealed that the expression levels of several genes related to cholesterol metabolism, including the low-density lipoprotein receptor (LDLR), were changed by ellagic acid treatment. Using a real-time PCR and immunoblot we confirmed that ellagic acid treatment up-regulated mRNA and protein expression level of the LDLR. Moreover, In the presence of 25 µM ellagic acid, extracellular apoB protein and MTP mRNA levels were significantly decreased. These findings indicate that ellagic acid improves cholesterol metabolism through the up-regulation of LDLR, down-regulation of MTP mRNA and reduces extracellular apoB levels. The ellagic acid-induced up-regulation of LDLR occurred via the extracellular signal-regulated kinase (ERK) signaling pathway in HepG2 hepatocytes. Abbreviations: LDLR: low-density lipoprotein receptor; apoB: apolipoprotein B; PKC: diacylglycerol-protein kinase C; MAPK: mitogen-activated protein kinase; ERK: p42/44 extracellular signal-regulated kinase; JNK: c-Jun N-terminal kinase; VLDLR: very low density lipoprotein receptor; PPARδ: peroxisome proliferator-activated receptor δ; SREBPs: sterol regulatory element-binding proteins; MTP: microsomal triacylglycerol transfer protein; LPDS: lipoprotein-deficient serum.

[A Case of Disseminated Carcinomatosis of the Bone Marrow Developing 15 Months after Gastrectomy].

The patient was a 60-year-old man who underwent distal gastrectomy for gastric cancer. The pathological diagnosis was Stage ⅡB. He received adjuvant chemotherapy(capecitabine plus oxaliplatin: CapeOX)for 6months and the postoperative course was uneventful. One year and 3 months after surgery, he visited the outpatient department for acute lower back pain. Blood tests showed elevated ALP(3,752 U/L), LDH(308 U/L), and CA19-9(69.4 U/mL)levels. Bone scintigraphy showed multiple bone metastases to the femora, ischium, iliac bone, vertebrae, sternum, costae, and scapulae in a super bone scan. The onset of disseminated intravascular coagulation(DIC)was observed later. The patient was diagnosed with disseminated carcinomatosis of the bone marrow. Radiation therapy was performed and anti-RANKL monoclonal antibody was administered for the bone metastases. Recombinant human soluble thrombomodulin was administered for DIC. He received chemotherapy( TS-1 plus cisplatin: SP)but died 4 months after the diagnosis. The prognosis of disseminated carcinomatosis of the bone marrow is extremely poor. We report this case along with a literature review.

Possible involvement of Enterococcus infection in the pathogenesis of chronic pancreatitis and cancer.

(Aim) Bacterial infection underlies the pathogenesis of many human diseases, including acute and chronic inflammation. Here, we investigated a possible role for bacterial infection in the progression of chronic pancreatitis. (Materials and Methods) Pancreatic juice was obtained from patients with pancreatic cancer (n = 20) or duodenal cancer/bile duct cancer (n = 16) and subjected to PCR using universal primers for the bacterial 16S ribosomal RNA gene. Bacterial species were identified by PCR using bile samples from four pancreatic cancer patients. PCR products were subcloned into T-vectors, and the sequences were then analyzed. Immunohistochemical and serologic analyses for Enterococcus faecalis infection were performed on a large cohort of healthy volunteers and patients with chronic pancreatitis or pancreatic cancer and on mice with caerulein-induced chronic pancreatitis. The effect of E. faecalis antigens on cytokine secretion by pancreatic cancer cells was also investigated. (Results) We found that 29 of 36 pancreatic juice samples were positive for bacterial DNA. Enterococcus and Enterobacter species were detected primarily in bile, which is thought to be a pathway for bacterial infection of the pancreas. Enterococcus faecalis was also detected in pancreatic tissue from chronic pancreatitis and pancreatic cancer patients; antibodies to E. faecalis capsular polysaccharide were elevated in serum from chronic pancreatitis patients. Enterococcus-specific antibodies and pancreatic tissue-associated E. faecalis were detected in mice with caerulein-induced chronic pancreatitis. Addition of Enterococcus lipoteichoic acid and heat-killed bacteria induced expression of pro-fibrotic cytokines by pancreatic cancer cells in vitro. (Conclusion) Infection with E. faecalis may be involved in chronic pancreatitis progression, ultimately leading to development of pancreatic cancer.

Identification of the active protein in rice bran protein having an inhibitory activity of cholesterol micellar solubility.

In our previous study, rice bran protein (RBP) inhibited cholesterol micellar solubility in vitro and decreased serum cholesterol level in rats. In the present study, RBP was separated and purified by size-exclusion chromatography and reversed-phase chromatography. The active protein of RBP related to cholesterol micellar solubility was identified as lectin and non-specific lipid-transfer protein 1 using MALDI-TOF mass spectrometry analysis.

Calreticulin is highly expressed in pancreatic cancer stem-like cells.

Cancer stem-like cells (CSLCs) in solid tumors are thought to be resistant to conventional chemotherapy or molecular targeting therapy and to contribute to cancer recurrence and metastasis. In this study, we aimed to identify a biomarker of pancreatic CSLCs (P-CSLCs). A P-CSLC-enriched population was generated from pancreatic cancer cell lines using our previously reported method and its protein expression profile was compared with that of parental cells by 2-D electrophoresis and tandem mass spectrometry. The results indicated that a chaperone protein calreticulin (CRT) was significantly upregulated in P-CSLCs compared to parental cells. Flow cytometry analysis indicated that CRT was mostly localized to the surface of P-CSLCs and did not correlate with the levels of CD44v9, another P-CSLC biomarker. Furthermore, the side population in the CRThigh /CD44v9low population was much higher than that in the CRTlow /CD44v9high population. Calreticulin expression was also assessed by immunohistochemistry in pancreatic cancer tissues (n = 80) obtained after radical resection and was found to be associated with patients' clinicopathological features and disease outcomes in the Cox proportional hazard regression model. Multivariate analysis identified CRT as an independent prognostic factor for pancreatic cancer patients, along with age and postoperative therapy. Our results suggest that CRT can serve as a biomarker of P-CSLCs and a prognostic factor associated with poorer survival of pancreatic cancer patients. This novel biomarker can be considered as a therapeutic target for cancer immunotherapy.

[A Case of Surgical Resection for Gallbladder Cancer with Para-Aortic Lymph Node Metastasis Following Neoadjuvant Chemotherapy with Gemcitabine, Cisplatin, and TS-1].

A 60-year-old woman who visited a doctor during postoperative follow-up for breast cancer was found to have elevated CEA levels. Investigation with FDG-PET CT following enhanced CT suggested gallbladder cancer(GBC)accompanied with para-aortic lymph node(PAL)metastasis. Although we could not obtain any histological evidence, we recommended she be treated as a patient with advanced GBC, and she received 8 courses of chemotherapy consisting of gemcitabine, cisplatin, and TS-1, following the protocol of a clinical trial. After the chemotherapy, imaging exhibited down-staging of the GBC, indicating tumor shrinkage and disappearance of the accumulation of FDG at the tumor sites. We first performed cholecystectomy to obtain histological evidence, and subsequently performed pancreaticoduodenectomy and partial hepatectomy at the gallbladder bed. The pathological diagnosis was pT2N1M0, Stage III B, Evans Grade II b; curative grade A surgery was recommended because the PAL did not contain a tumor any more. The prognosis of advanced GBC with PAL metastasis is extremely poor, but conversion therapy with combined modality therapy including neoadjuvant chemotherapy is a useful strategy to achieve a curative resection.

Synthesis of oolongtheanins and their inhibitory activity on micellar cholesterol solubility in vitro.

The synthesis of oolongtheanins (1a-d) was accomplished from EGC and/or EGCg in three steps. Oolongtheanin-3'-O-gallate (1b) showed more potent inhibitory activity on micellar cholesterol solubility than did EGCg.

Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins.

Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins.

L-Cysteine-induced up-regulation of the low-density lipoprotein receptor is mediated via a transforming growth factor-alpha signalling pathway.

Sulphur-containing amino acids regulate plasma cholesterol levels in animals and humans. However, their mechanism of action remains unclear. Low-density lipoprotein receptor (LDLR) plays an important role in cholesterol metabolism. We therefore investigated the effects of sulphur-containing amino acids on the expression of LDLR in hepatocytes. HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium with or without sulphur-containing amino acids and cysteine-containing compounds. We found that L-cysteine increased LDLR mRNA and enhanced LDLR gene promoter activity through the extracellular-signal-related kinase and p38 mitogen-activated protein kinase signalling pathways in HepG2 cells. Moreover, we observed that L-cysteine stimulated the release of transforming growth factor-alpha (TGF-α) and that TGF-α increased the LDLR mRNA levels. This study provides a report of the L-cysteine mediated up-regulation of the LDLR expression via TGF-α signalling pathway. Our findings provide insights into cholesterol homeostasis and amino acid signalling.

Development of a novel transgenic rice with hypocholesterolemic activity via high-level accumulation of the α' subunit of soybean ß-conglycinin.

Soybean 7S globulin, known as ß-conglycinin, has been shown to regulate human plasma cholesterol and triglyceride levels. Furthermore, the α' subunit of ß-conglycinin has specifically been shown to possess low-density lipoprotein (LDL)-cholesterol-lowering activity. Therefore, accumulation of the α' subunit of ß-conglycinin in rice seeds could lead to the production of new functional rice that could promote human health. Herein, we used the low-glutelin rice mutant 'Koshihikari' (var. a123) and suppressed its glutelins and prolamins, the major seed storage proteins of rice, by RNA interference. The accumulation levels of the α' subunit in the lines with suppressed glutelin and prolamin levels were >20 mg in 1 g of rice seeds, which is considerably higher than those in previous studies. Oral administration of the transgenic rice containing the α' subunit exhibited a hypocholesterolemic activity in rats; the serum total cholesterol and LDL cholesterol levels were significantly reduced when compared to those of the control rice (var. a123). The cholesterol-lowering action by transgenic rice accumulating the α' subunit induces a significant increase in fecal bile acid excretion and a tendency to increase in fecal cholesterol excretion. This is the first report that transgenic rice exhibits a hypocholesterolemic activity in rats in vivo by using the ß-conglycinin α' subunit.

High-level production of lactostatin, a hypocholesterolemic peptide, in transgenic rice using soybean A1aB1b as carrier.

Hypercholesterolemia, a form of cardiovascular disease, is one of the leading causes of deaths worldwide. Lactostatin (Ile-Ile-Ala-Glu-Lys), derived from ß-lactoglobulin in cow's milk, is a bioactive peptide with hypocholesterolemic activity higher than sitosterol, a known anti-hypercholesterolemic drug. Here, we successfully developed a transgenic rice accumulating a much higher level of lactostatin by inserting 29 IIAEK sequences into the structurally flexible (nonconserved) regions of soybean seed storage protein, A1aB1b, and introducing it into LGC-1 (low glutelin content mutant 1) as host variety. A1aB1b containing 29 lactostatins was expressed in the endosperm of rice seed cells by using seed specific promoters and sorted into novel compartments distinct from normal PB-I (ER-derived protein body) and PB-II (protein storage vacuoles). Transgenic rice seeds accumulated approximately 2 mg of lactostatins/g of dry seeds, which is relatively high compared with previous reports. Our findings suggest that the introduction of a high copy number of bioactive peptide into seed storage proteins as carrier is one of the effective means in producing higher amounts of bioactive peptides in rice.

Rose polyphenols exert antiobesity effect in high-fat-induced obese mice by regulating lipogenic gene expression.

Obesity presents a major risk factor in the development of cardiovascular diseases. Recent reports indicate that many kinds of polyphenols have the potential to prevent metabolic diseases. We hypothesized that rose polyphenols (ROSE) have the effect of improvement in lipid metabolism. In this study, we investigated whether rose polyphenols affected lipid metabolism and exerted antiobesity. To clarify the mechanism, C57BL/6J mice were fed a high-fat diet containing 0.25% ROSE for 35 days. Compared with the control group, body weight gain and adipose tissue weight in the 0.25% ROSE group were significantly decreased. Serum cholesterol and hepatic triglyceride concentrations significantly decreased, whereas fecal triglyceride was significantly increased in the 0.25% ROSE group. Liver stearoyl-CoA desaturase 1 (Scd1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), and acyl-CoA:cholesterol acyltransferase 1 (Acat1) mRNA as well as protein stearoyl-CoA desaturase 1 concentrations were significantly lower in the 0.25% ROSE group than that in the control group. The mRNA and the protein concentrations of adipose triglyceride lipase, hormone-sensitive lipase, and peroxisomal acylcoenzyme A oxidase 1 in white adipose tissue were significantly higher in the 0.25% ROSE group than that in the control group. The components in rose polyphenols were quantified by liquid chromatography-tandem mass spectrometry, and we consider that ellagic acid plays an important role in an antiobesity effect because the ellagic acid content is the highest among polyphenols in rose polyphenols. In summary, rose polyphenols exhibit antiobesity effects by influencing lipid metabolism-related genes and proteins to promote lipolysis and suppress lipid synthesis.

Thymic atypical carcinoid tumors with elevated mitotic counts in a patient with multiple endocrine neoplasia: A case report.

Thymic neuroendocrine tumors associated with multiple endocrine neoplasia are only defined as carcinoid and are not associated with large-cell neuroendocrine carcinoma (LCNEC). We report the case of a multiple endocrine neoplasia type 1 patient with atypical carcinoid tumors with elevated mitotic counts (AC-h), an intermediate condition between carcinoid and LCNEC. A 27-year-old man underwent surgery for an anterior mediastinal mass and was diagnosed with thymic LCNEC. Fifteen years later, a mass appeared at the same site, which was determined to be a postoperative recurrence based on the pathological results of a needle biopsy and the clinical course. The patient's disease remained stable for 10 months on anti-programmed death-ligand 1 antibody and platinum-containing chemotherapy. The needle biopsy specimen was submitted for next-generation sequencing, which revealed a MEN1 gene mutation, and after further examination, a diagnosis of multiple endocrine neoplasia type 1 was made. A re-examination of the surgical specimen from 15 years prior showed that it corresponded to AC-h. Although thymic AC-h is classified as thymic LCNEC according to the current definition, our data suggests that a search for multiple endocrine neoplasia is warranted in such patients.

Primary tracheal small-cell carcinoma detected 11 months after surgery for pulmonary large-cell neuroendocrine carcinoma: A case report.

Primary tracheal small-cell carcinoma is rare, and is often treated using small-cell lung cancer guidelines given that no standard treatment has been established for it. We report a patient in whom nodules appeared in the trachea and left main bronchus 11 months after surgery for pulmonary large-cell neuroendocrine carcinoma; a biopsy revealed small-cell carcinoma. Given the absence of malignant lesions elsewhere in the body, the lesions were diagnosed as primary tracheal small-cell carcinoma. Respiratory failure progressed rapidly owing to airway stenosis caused by the growing lesion, and the patient required nasal high-flow therapy. However, the lesions shrank a few days after commencing first-line chemotherapy, and his respiratory failure resolved. Accelerated hyperfractionated radiotherapy was administered in conjunction with the third course of chemotherapy, and the patient ultimately achieved a complete response. Although the lesions were initially suspected of being postoperative recurrence of pulmonary large-cell neuroendocrine carcinoma, the fact that the biopsy revealed them to be primary tracheal small-cell carcinoma indicates that intra-airway nodules that appear after lung cancer surgery may possibly be primary tracheal tumors.