UNLABELLED: Aspirin exhibits potential as a repurposed agent for preventing and treating cancer, specifically PIK3CA-mutated (MT) colorectal cancer. However, the fundamental biological reasons for the particular antitumor activity of aspirin against PIK3CA-MT colorectal cancer are not yet fully elucidated. We have previously established that aspirin relates to glutamine metabolism in PIK3CA-MT colorectal cancer. In evaluating the antitumor effects of aspirin, it is essential to consider the tumor growth environment, such as hypoxia. The impact of the tumor microenvironment on aspirin efficacy remains unknown. Therefore, we aimed to investigate the mechanisms through which hypoxia influences aspirin- associated intracellular glutamine accumulation in PIK3CA-MT colorectal cancer cell lines. Connectivity Map analysis was used to explore the associations of aspirin with biological pathways in HT-29 (PIK3CA-MT) cells. RNA sequencing was performed on PIK3CA-wild type (WT)/mutant isogenic SW48 cell lines to assess aspirin’s effects under hypoxic conditions. Targeted metabolomics was employed to measure intracellular glutamine levels in PIK3CA-MT HCT116 cells following aspirin treatment and hypoxic stimulation. Bioinformatic analysis revealed associations of aspirin with amino acid metabolism and hypoxic pathways. Under hypoxic conditions, aspirin enhanced amino acid uptake in PIK3CA-MT cells. Targeted metabolomics showed that aspirin treatment and hypoxic stimulation additively upregulated intracellular glutamine levels in PIK3CA-MT cells. This increase was reversed by V-9302, an inhibitor of the glutamine transporter ASCT2, which demonstrated antitumor potential in combination with aspirin. Our findings suggest that the combination of aspirin and hypoxia significantly enhances intracellular glutamine accumulation in PIK3CA-MT colorectal cancer under hypoxic conditions, emphasizing glutamine metabolism as a potential therapeutic target to enhance aspirin’s efficacy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-42753-z.

Owing to their safety, plant-derived nanovesicles (PDNVs), particularly those derived from edible plants, are expected to be natural delivery carriers for microRNAs (miRNAs). Among PDNVs, Citrus limon-derived extracellular vesicle-like nanovesicles have been shown to exert antitumor effects. We previously reported that PDNVs derived from C. limon L. exert selective inhibitory effects on the proliferation of p53-inactivated colon cancer cells. However, miRNAs in C. limon L.-derived nanovesicles have not yet been identified, and it remains unclear which miRNAs are contained in these PDNVs. We herein extracted PDNVs from C. limon L., C. hassaku, and C. sudachi and identified and compared the miRNAs present within them via next-generation sequencing. A total of 158 miRNAs were identified, of which 109 were known miRNAs and 49 were novel miRNAs. Comparisons of miRNAs expressed by C. limon L., C. hassaku, and C. sudachi revealed 77 miRNAs that were commonly expressed. Among all the libraries, miR159a had the highest number of reads, and the same result was obtained when it was converted to TPM. The present results raise the possibility that miR159a could be involved in the biological properties of citrus‑derived nanovesicles, given that it was the most abundantly expressed miRNA.

Activation of BCR::ABL1 tyrosine kinase is the main pathogenic mechanism underlying chronic myeloid leukemia (CML) in 90% of affected patients. The prognosis for individuals with CML who receive treatment with BCR::ABL1 tyrosine kinase inhibitors (TKIs) such as imatinib, is promising, with a 5-year survival rate of >90%. However, unfortunately, 20-30% of patients who are treated with imatinib may become resistant to the BCR::ABL1 TKIs. The objective of the present study was to determine whether inhibitors of E3 ubiquitin-protein ligase Mdm2 (MDM2), a regulator of p53 that promotes apoptosis and is highly expressed in CML, could induce cell death in imatinib-resistant CML cells. Apoptosis and cell viability were evaluated using Annexin-V-positive cell count and caspase-3 activity, as well as trypan blue dye exclusion assay. Expression levels of MDM2, p53, Bax, Puma, Noxa, p21, and cleaved caspase-3 were determined via western blotting. MDM2 levels in both the cytoplasm and nucleus were found to be ~3-fold higher in K562/IR cells compared with K562 cells, while the levels of p53 in both cell structures were markedly lower. In addition, an examination of a publicly accessible database revealed that the levels of MDM2 were evidently greater in patients who did not respond to imatinib compared with those who did respond to the drug. NSC-66811 and Nutlin-3, MDM2 inhibitors, increased the percentage of Annexin-positive cells in K562/IR cells by 43 and 62% at concentrations of 10 and 25 µM, respectively. Furthermore, the MDM2 inhibitors increased the levels of Bax, Puma, Noxa, and p21 by increasing the expression of p53 and decreasing the expression of MDM2 in K562/IR cells. Additionally, pifithrin-α, a p53 inhibitor, suppressed MDM2 inhibitor-induced cell death in K562/IR cells. Overall, the findings of the present study highlight the therapeutic potential of MDM2 inhibitors for imatinib-resistant CML.

Background and Objectives: In previous studies, we reported that the assessment of the cumulative thermal dose in the crystalline lens, conducted through computational modeling utilizing a supercomputer and the biothermal transport equation, exhibited a significant association with the incidence of nuclear cataracts. In this study, we have investigated the types of proteins that expressed underlying 35.0 °C (normal-temp) and 37.5 °C (warming-temp) by using the shotgun liquid chromatography (LC) with tandem mass spectrometry (MS/MS)-based global proteomic approach. Materials and Methods: We have discussed the changes in protein expression in warmed iHLEC-NY2 cells using Gene Ontology analysis and a label-free semiquantitative method based on spectral counting. Results: In iHLEC-NY2, 615 proteins were detected, including 307 (49.9%) present in both lenses cultured at normal-temp and warming-temp, 130 (21.1%) unique to the lens cultured at normal-temp, and 178 (29.0%) unique to the lens cultured at warming-temp. Furthermore, LC-MS/MS analysis showed that warming decreased the expression of actin, alpha cardiac muscle 1, actin-related protein 2, putative tubulin-like protein alpha-4B, ubiquitin carboxyl-terminal hydrolase 17-like protein 1, ubiquitin-ribosomal protein eL40 fusion protein, ribosome biogenesis protein BMS1 homolog, histone H2B type 1-M, and histone H2A.J. in iHLEC-NY2. Conclusions: The decreases in the specific protein levels of actin, tubulin, ubiquitin, ribosomes, and histones may be related to cataract development under warming conditions. This investigation could provide a critical framework for understanding the correlation between temperature dynamics and the development of nuclear cataracts.

Previous studies have reported that a strong correlation between the estimated cumulative thermal exposure in the crystalline lens and the incidence of nuclear cataracts; however, the precise relationship between temperature and cataracts remains to be fully elucidated. In the present study, the shotgun liquid chromatography/mass spectroscopy‑based global proteomic approach was applied to investigate cataract‑inducing factors in lens cultured at normal (35.0˚C) and slightly warmer (37.5˚C) conditions. In the rat lens, 190 proteins (total) were identified. Of these, 48 proteins (25.3%) were found in lenses cultured at both 35.0˚C and 37.5˚C. Moreover, 85 proteins (44.7%) were unique to lenses cultured at 35.0˚C, while 57 proteins (30.0%) were unique to lenses cultured at 37.5˚C. Protein expression changes in rat lenses cultured at 37.5˚C were examined using a label‑free semiquantitative approach that uses spectral counting and Gene Ontology analysis. Filensin and vimentin protein expression, key factors in maintaining lens structure, were decreased. These findings may serve as a valuable indicator for elucidating the relationship between temperature and the onset of nuclear cataracts.

Neokestose is considered to have a prebiotic function. However, the physiological activity of neokestose remains unknown. Neokestose has a blastose, a sucrose analog, in its structure. We previously demonstrated that oral administration of blastose to diabetic rats suppressed the increase in plasma glucose (PG) concentration after sucrose administration. Therefore, neokestose might have a similar effect. In this study, we investigated the effects of neokestose on PG concentrations and the mechanism of its action. We first administered neokestose orally to streptozotocin-induced diabetic rats and observed that the expected consequent increase in PG concentration was significantly suppressed. Next, we examined the inhibitory effect of neokestose on glycosidase activity, but observed only a slight inhibitory effect. Therefore, we hypothesized that neokestose might be hydrolyzed by gastric acid to produce blastose. We performed an acid hydrolysis of neokestose using artificial gastric juice. After acid hydrolysis, peaks corresponding to neokestose and its decomposition products including blastose were observed. Therefore, we suggest that neokestose and blastose, a decomposition product, synergistically inhibit glycosidase activity. These findings support the potential use of neokestose as a useful functional oligosaccharide that can help manage plasma glucose concentrations in patients with diabetes mellitus.

Maple syrup is a natural sweetener consumed worldwide. Active ingredients of maple syrup possess antitumor effects; however, these ingredients are phenolic compounds. The present study aimed to investigate components other than phenolic compounds that may have antitumor effects against colorectal cancer (CRC). Cell proliferation assays demonstrated that treatment with the more than 10,000 molecular weight fraction significantly inhibited viability in DLD‑1 cells. Therefore, we hypothesized that the protein components of maple syrup may be the active ingredients in maple syrup. We obtained protein components from maple syrup by ammonium sulfate precipitation, and treatment with the protein fraction of maple syrup (MSpf) was found to exhibit a potential antitumor effect. MSpf‑treated DLD‑1 colon adenocarcinoma cells exhibited significantly decreased proliferation, migration and invasion. In addition, upregulation of LC3A and E‑cadherin and downregulation of MMP‑9 expression levels were observed following MSpf treatment. Investigation of the components of MSpf suggested that it was primarily formed of advanced glycation end products (AGEs). Therefore, whether AGEs in MSpf affected the STAT3 pathway through the binding to its receptor, receptor of AGE (RAGE), was assessed. MSpf treatment was associated with decreased RAGE expression and STAT3 phosphorylation. Finally, to determine whether autophagy contributed to the inhibitory effect of cell proliferation following MSpf treatment, the effect of MSpf treatment on autophagy induction following bafilomycin A1 treatment, a specific autophagy inhibitor, was assessed. The inhibitory effect of MSpf treatment on cell proliferation was enhanced through the inhibition of autophagy by bafilomycin A1 treatment. These results suggested that AGEs in MSpf suppressed cell proliferation and epithelial‑mesenchymal transition through inhibition of the STAT3 signaling pathway through decreased RAGE expression. Therefore, AGEs in MSpf may be potential compounds for the development of antitumor drugs for the treatment of CRC with fewer adverse effects compared with existing antitumor drugs.

In the original publication [...].

Edible plant-derived nanovesicles have been explored as effective materials for preventing colorectal cancer (CRC) incidence, dependent on gene status, as a K-Ras-activating mutation via the macropinocytosis pathway. Approximately 70% of CRC harbors the p53 mutation, which is strongly associated with a poor prognosis for CRC. However, it has not been revealed whether p53 inactivation activates the macropinocytosis pathway or not. In this study, we investigated parental cells, wild-type or null for p53 treated with Citrus limon L.-derived nanovesicles, as potential materials for CRC prevention. Using ultracentrifugation, we obtained C. limon L.-derived nanovesicles, the diameters of which were approximately 100 nm, similar to that of the exosomes derived from mammalian cells. C. limon L.-derived nanovesicles showed inhibitory effects on cell growth in not p53-wild, but also in p53-inactivated CRC cells. Furthermore, we revealed that the macropinocytosis pathway is activated by p53 inactivation and C. limon L.-derived nanovesicles were up taken via the macropinocytosis pathway. Notably, although C. limon L.-derived nanovesicles contained citrate, the inhibitory effects of citrate were not dependent on the p53 status. We thus provide a novel mechanism for the growth inhibition of C. limon L.-derived nanovesicles via macropinocytosis and expect to develop a functional food product containing them for preventing p53-inactivation CRC incidence.

Fructosyl oligosaccharides, including fructo-oligosaccharide (FOS), are gaining popularity as functional oligosaccharides and have been found in various natural products. Our previous study suggested that maple syrup contains an unidentified fructosyl oligosaccharide. Because these saccharides cannot be detected with high sensitivity using derivatization methods, they must be detected directly. As a result, an analytical method based on charged aerosol detection (CAD) that can detect saccharides directly was optimized in order to avoid relying on these structures and physical properties to clarify the profile of fructosyl oligosaccharides in maple syrup. This analytical method is simple and can analyze up to hepta-saccharides in 30 min. This analytical method was also reliable and reproducible with high validation values. It was used to determine the content of saccharides in maple syrup, which revealed that it contained not only fructose, glucose, and sucrose but also FOS such as 1-kestose and nystose. Furthermore, we discovered a fructosyl oligosaccharide called neokestose in maple syrup, which has only been found in a few natural foods. These findings help to shed light on the saccharides profile of maple syrup.

The tricarboxylic acid (TCA) cycle is one of the most important pathways of energy metabolism, and the profiles of its components are influenced by factors such as diseases and diets. Therefore, the differences in metabolic profile of TCA cycle between healthy and cancer cells have been the focus of studies to understand pathological conditions. In this study, we developed a quantitative method to measure TCA cycle metabolites using LC-MS/MS to obtain useful metabolic profiles for development of diagnostic and therapeutic methods for cancer. We successfully analyzed 11 TCA cycle metabolites by LC MS/MS with high reproducibility by using a PFP column with 0.5% formic acid as a mobile phase. Next, we analyzed the concentration of TCA cycle metabolites in human cell lines (HaCaT: normal skin keratinocytes; A431: skin squamous carcinoma cells; SW480: colorectal cancer cells). We observed reduced concentration of succinate and increased concentration of citrate, 2-hydroxyglutarate, and glutamine in A431 cells as compared with HaCaT cells. On the other hand, decreased concentration of isocitrate, fumarate, and α-ketoglutarate and increased concentration of malate, glutamine, and glutamate in A431 cells were observed in comparison with SW480 cells. These findings suggested the possibility of identifying disease-specific metabolites and/or organ-specific metabolites by using this targeted metabolomic analysis.

The authors wish to make the following corrections to this paper [...].

Circulating tumor cells (CTCs) are involved in metastasis; thus, one of the most important approaches for identifying metastatic cancer is to detect CTCs in blood. In the present study, we examined whether directly analyzing cells with capillary electrophoresis (CE) could distinguish cancer cells from normal cells, based on differences in cell surface glycosylation. We compared human colorectal cancer (CRC) cell lines to a normal colon epithelium cell line. Our results demonstrated that direct CE analysis could successfully distinguish between CRC and normal cells with high reproducibility, based on migration times. We found that the weighted-average migration time was significantly shorter for CRC cells than for normal cells. Next, we observed changes in the electrophoretic behaviors of CRC cells by adding five different types of lectins. When Aleuria aurantia lectin was added, migration delays were observed in CRC cells, but not in normal colon cells. Therefore, by focusing on shifts in migration time after adding specific lectins, we could distinguish cancer cells from normal cells. These findings suggested that this diagnostic method of directly analyzing cells with CE after adding specific lectin(s) could be useful for detecting the difference in the sugar moieties on a surface of normal and cancer cells.

Enhanced expression of cyclophilin A (CypA) in colorectal cancer (CRC) was reported; however, how CypA influences CRC progression is not clear. Therefore, we examine the effects of CypA on CRC cell progression. Knockdown of CypA in SW480 cells significantly inhibited cell migration and invasion but had no effect on cell proliferation. In addition, upregulation of E-cadherin and downregulation of N-cadherin and Snail expression were observed by CypA knockdown. These results suggested that CypA knockdown inhibited cell migration and invasion by suppressing epithelial-mesenchymal transition. CypA knockdown was also associated with increased p38 phosphorylation, and the p38 inhibitor treatment led to increase in the number of invasive CypA-knockdown SW480 cells. Therefore, CypA may be a potential therapeutic target in preventing CRC metastasis.

The incidence of diabetes mellitus (DM) is increasing rapidly and is associated with changes in dietary habits. Although restrictions in the use of sweeteners may prevent the development of DM, this might reduce the quality of life of patients with DM. Therefore, there has been a great deal of research into alternative sweeteners. In the search for such sweeteners, we analyzed the carbohydrate content of maple syrup and identified a novel oligosaccharide composed of fructose and glucose, linked at the C-4 of glucose and the C-6 of fructose. This oligosaccharide inhibited the release of fructose from sucrose by invertase (IC50: 1.17 mmol/L) and the decomposition of maltose by α-(1-4) glucosidase (IC50: 1.72 mmol/L). In addition, when orally administered together with sucrose to rats with DM, the subsequent plasma glucose concentrations were significantly lower than if the rats had been administered sucrose alone, without having any effect on the insulin concentration. These findings suggest that this novel oligosaccharide might represent a useful alternative sweetener for inclusion in the diet of patients with DM and may also have therapeutic benefits.

Maple syrup is a natural sweetener that is consumed worldwide. It has been previously reported that dark-colored maple syrup exerts an inhibitory effect on colorectal cancer (CRC) proliferation and invasion. In the present study, the underlying mechanism of CRC cell growth inhibition was examined with dark-colored maple syrup treatment using a shotgun liquid chromatography-tandem mass spectrometry-based global proteomic approach. Applying a semi-quantitative method based on spectral counting, 388 proteins were identified with expression changes of >1.5-fold following dark-colored maple syrup treatment. Gene Ontology analysis revealed that these proteins possessed cell cycle-associated functions. It was also indicated that CRC cells treated with dark-colored maple syrup exhibited decreased proliferating cell nuclear antigen (PCNA) expression and S-phase cell cycle arrest. Dark-colored maple syrup treatment also resulted in altered expression of cell cycle-associated genes, including cyclin-dependent kinase (CDK)4 and CDK6. In conclusion, these data suggested that dark-colored maple syrup induced S-phase cell cycle arrest in CRC cells by reducing the expression of PCNA and regulating cell cycle-associated genes. These findings suggest that dark-colored maple syrup may be a source of compounds for the development of novel drugs for colorectal cancer treatment.

Collagen peptides (CPs), derived by hydrolyzing collagen with chemicals or enzymes, are often used as functional materials, due to their various bioactivities and high bioavailability. A previous study by our group reported that collagen from soft‑shelled turtle, Pelodiscus sinensis, induces keratinocytes to undergo epithelial‑mesenchymal transition and facilitates wound healing. Therefore, CPs derived from soft‑shelled turtle collagen may have useful effects on the skin. In the present study, the functional effects of CPs on human skin were examined by analyzing CP‑treated human keratinocytes with a shotgun liquid chromatography/mass spectrometry‑based global proteomic approach. A semi‑quantitative method based on spectral counting was applied and 211 proteins that exhibited >2‑fold changes in expression after CP treatment were successfully identified. Based on a Gene Ontology analysis, the functions of these proteins were indicated to be closely linked with protein processing. In addition, CP treatment significantly increased the expression of calpain‑1, a calcium‑dependent intracellular cysteine protease. Furthermore, CP‑treated keratinocytes exhibited elevated interleukin (IL)‑1α and IL‑8 expression and reduced IL‑6 expression. CPs also induced the expression of proteins implicated in cell‑cell adhesion and the skin barrier. Therefore, CPs from soft‑shelled turtle may provide significant benefits for maintaining the biological environment of the skin, and may be useful as components of pharmaceuticals and medical products.

Soft-shelled turtles (Pelodiscus sinensis) are widely distributed in some Asian countries, and we previously reported that soft-shelled turtle tissue could be a useful material for collagen. In the present study, we performed shotgun liquid chromatography (LC)/mass spectrometry (MS)-based global proteomic analysis of collagen-administered human keratinocytes to examine the functional effects of collagen from soft-shelled turtle on human skin. Using a semiquantitative method based on spectral counting, we were able to successfully identify 187 proteins with expression levels that were changed more than twofold by the administration of collagen from soft-shelled turtle. Based on Gene Ontology analysis, the functions of these proteins closely correlated with cell-cell adhesion. In addition, epithelial-mesenchymal transition was induced by the administration of collagen from soft-shelled turtle through the down-regulation of E-cadherin expression. Moreover, collagen-administered keratinocytes significantly facilitated wound healing compared with nontreated cells in an in vitro scratch wound healing assay. These findings suggest that collagen from soft-shelled turtle provides significant benefits for skin wound healing and may be a useful material for pharmaceuticals and medical care products. © 2017 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2403-2413, 2018.

Lumicanは糖鎖修飾されることが知られているが、産生される臓器や細胞により異なる糖鎖修飾を受けることが示唆されている。今回、膵臓癌細胞でのみ産生される特異糖鎖修飾を受けたlumicanの存在について検討するため、消化器癌細胞より産生されるlumicanの精製を試みた。その結果、食道癌と膵臓癌細胞から産生・分泌されるlumicanを精製することができた。また、そのlumicanの発現パターンの違いを検討した所、膵臓癌細胞特異的と思われるlumicanが発現していた。このことから、この膵臓特異的に発現するlumicanを標的とした新たな診断法の開発につながる可能性が考えられた。

ルミカンの発現量と細胞増殖能には正の相関性があり、細胞浸潤能とは負の相関性があることが報告されている。今回、ルミカンが膵癌細胞の増殖や浸潤に関わる機構について検討するため、ルミカン発現調製細胞を用い、それらの細胞の発現タンパク質の変動を網羅的に解析した。その結果、24種類のタンパク質がルミカンの発現と相関性があることが明らかとなった。この中にはアポトーシスのマーカーとして知られているタンパク質やMMP-9の活性化と関連があるタンパク質が含まれていたことから、ルミカンはアポトーシスやこれらのタンパク質の発現に関わることで細胞増殖や浸潤を制御していると考えられた。

線維芽細胞増殖因子受容体2IIIc(FGFR-2IIIc)は、ヒト膵臓癌症例の約70%に発現しており、FGFR-2IIIc陽性症例は術後の肝転移再発までの期間の短縮を認めた。FGFR-2IIIc遺伝子発現ベクターを遺伝子導入した膵臓癌培養細胞は、細胞増殖能の亢進と、皮下移植および同所移植腫瘍の体積の増大が見られた。FGFR-2IIIcに対するポリクローナル抗体を作成し、膵臓癌培養細胞に投与したところ、細胞増殖能と遊走能が抑制された(Ishiwata,Am J Pathol,2012)。FGFR-2の別のアイソフォームであるFGFR-2IIIbへのスプライシングを促進させるepithelial splicing regulatory protein(ESRP)1を遺伝子導入すると、膵臓癌の転移が抑制された。以上より、膵臓癌のFGFR-2IIIcの発現制御は、膵臓癌の治療標的となると考えられた。

浸潤性膵管癌の間質におけるルミカンの発現は、癌の浸潤や予後の悪化と関連があることが報告されている。今回、遺伝子導入法を用いて、培養膵臓癌細胞によって分泌されるルミカンの機能について検討した。ルミカン過剰発現膵癌細胞は、分子量70kDaのルミカンを過剰に分泌し、それに伴いERKが活性化した。また、遺伝子導入したことにより細胞増殖能が亢進するとともにラミニンに対する接着能が増加した。さらに細胞浸潤能が抑制され、それに関連してMMP-9の活性が抑制された。一方、siRNAを用いてルミカンの発現を抑制すると逆の結果が得られた。このことから、膵臓癌細胞によって分泌される70kDaのルミカンは、膵臓癌細胞の増殖や浸潤に重要な役割を果たしていると考えられた。

ケラチノサイト増殖因子(KGF)とその受容体(KGFR)は膵臓癌の増殖・浸潤に重要な役割を果たしている。培養膵臓癌細胞にリコンビナントKGFを投与すると、濃度依存的に膵臓癌細胞の増殖が進行し、それに関連してERKやp38といったMAPKが活性化された。一方、プロテオグリカンの一つであるルミカンの膵臓癌における発現量を調節したところ、ルミカンの発現量と細胞増殖及びERKの活性化に正の相関関係が認められた。KGFとKGFRの結合の安定化にはプロテオグリカンが必要なことから、ルミカンはKGF/KGFR系の活性化を制御することで膵臓癌の増殖を制御している可能性が考えられた。