Phytochemical Profile, Antioxidant, Anti-Atopic, and Anti-Inflammatory Activities of Filipendula glaberrima Nakai at Different Growth Stages.

Since atopic dermatitis is an inflammatory skin disease, natural remedies, such as Filipendula glaberrima Nakai (FG), with anti-inflammatory properties are possible promising therapeutic options. This study aimed to investigate the therapeutic potential of FG extracts at different growth stages. Seven compounds were isolated from the FG leaf extracts using open-column chromatography, and they were analyzed using HPLC. The extracts were further evaluated for their total polyphenol and flavonoid content (TPC and TFC). The in vitro antioxidant properties of the FG extracts were evaluated using radical scavenging assays, whereas their anti-inflammatory activities were assessed by evaluating their ability to inhibit the production of inflammation-associated biomarkers using the Griess assay and ELISA, respectively. The MTT assay was used to evaluate the viability and cytotoxicity of the FG extracts in keratinocyte cell lines. The results showed that the full-flowering stage exhibited the highest TPC, TFC, and antioxidant activities, thus suggesting a positive correlation between these properties. All FG extracts showed significant anti-inflammatory activity by inhibiting the production of pro-inflammatory biomarkers in lipopolysaccharide-stimulated macrophages. Additionally, the FG extracts suppressed the production of cytokines and chemokines in keratinocytes, indicating their anti-atopic potential. HPLC analysis revealed that the full-flowering stage had the highest content of all the analyzed phytochemicals (gallic acid, (+)-catechin, hyperin, miquelianin, astragalin, afzelin, and quercetin). These results suggest that the full-flowering stage of FG is the most promising source for therapeutic applications owing to its superior phytochemical profile and biological activities. This study highlights the potential of FG extracts, particularly in its full-flowering stage, as a natural therapeutic agent for the management of inflammation-related diseases, and it can also serve as a reference for further research on FG.

hiPSC-derived macrophages improve drug sensitivity and selectivity in a macrophage-incorporating organoid culture model.

Accurate simulation of different cell type interactions is crucial for physiological and precisein vitrodrug testing. Human tissue-resident macrophages are critical for modulating disease conditions and drug-induced injuries in various tissues; however, their limited availability has hindered their use inin vitromodeling. Therefore, this study aimed to create macrophage-containing organoid co-culture models by directly incorporating human-induced pluripotent stem cell (hiPSC)-derived pre-macrophages into organoid and scaffold cell models. The fully differentiated cells in these organoids exhibited functional characteristics of tissue-resident macrophages with enriched pan-macrophage markers and the potential for M1/M2 subtype specialization upon cytokine stimulation. In a hepatic organoid model, the integrated macrophages replicated typical intrinsic properties, including cytokine release, polarization, and phagocytosis, and the co-culture model was more responsive to drug-induced liver injury than a macrophage-free model. Furthermore, alveolar organoid models containing these hiPSC-derived macrophages also showed increased drug and chemical sensitivity to pulmonary toxicants. Moreover, 3D adipocyte scaffold models incorporating macrophages effectively simulated in vivo insulin resistance observed in adipose tissue and showed improved insulin sensitivity on exposure to anti-diabetic drugs. Overall, the findings demonstrated that incorporating hiPSC-derived macrophages into organoid culture models resulted in more physiological and sensitivein vitrodrug evaluation and screening systems.

A Bee Trp-Arg Dense Peptide with Antiproliferation Efficacy against the Prostate Cancer Cell Line DU145.

Prostate cancer accounts for 14% of male cancer-related fatalities in the UK. Given the challenges associated with hormone-based therapies in the context of androgen-independent prostate cancer, there is an imperative need for research into anticancer drugs. N0821, a peptide belonging to the Trp-Arg dense region and derived from the homologous region of various bee species, shows substantial potential for an anticancer effect. Both MTT assays and 3D spheroid assays were conducted to substantiate its antiproliferation potential and strongly indicated the antiproliferation effect of N0820 (WWWWRWWRKI) and N0821 (YWWWWRWWRKI). Notably, the mechanism underlying this effect is related to the downregulation of CCNA2 and the upregulation of CCNE1. Cell cycle arrest results from the reduction of CCNA2 in the S/G2 phase, leading to the accumulation of CCNE1. Our peptides were predicted to make an α-helix structure. This can act as an ion channel in the cell membrane. Therefore, we analyzed genes implicated in the influx of calcium ions into the mitochondria. Trp-Arg dense-region peptides are known for their antibacterial properties in targeting cell membranes, making the development of resistance less likely. Hence, further research in this area is essential and promising.

Evaluation of the Drug-Induced Liver Injury Potential of Saxagliptin through Reactive Metabolite Identification in Rats.

A liver injury was recently reported for saxagliptin, which is a dipeptidyl peptidase-4 (DPP-4) inhibitor. However, the underlying mechanisms of saxagliptin-induced liver injury remain unknown. This study aimed to evaluate whether saxagliptin, a potent and selective DPP-4 inhibitor that is globally used for treating type 2 diabetes mellitus, binds to the nucleophiles in vitro. Four DPP-4 inhibitors, including vildagliptin, were evaluated for comparison. Only saxagliptin and vildagliptin, which both contain a cyanopyrrolidine group, quickly reacted with L-cysteine to enzyme-independently produce thiazolinic acid metabolites. This saxagliptin-cysteine adduct was also found in saxagliptin-administered male Sprague-Dawley rats. In addition, this study newly identified cysteinyl glycine conjugates of saxagliptin and 5-hydroxysaxagliptin. The observed metabolic pathways were hydroxylation and conjugation with cysteine, glutathione, sulfate, and glucuronide. In summary, we determined four new thiazoline-containing thiol metabolites (cysteine and cysteinylglycine conjugates of saxagliptin and 5-hydroxysaxagliptin) in saxagliptin-administered male rats. Our results reveal that saxagliptin can covalently bind to the thiol groups of cysteine residues of endogenous proteins in vivo, indicating the potential for saxagliptin to cause drug-induced liver injury.

Identification of a Novel ERK5 (MAPK7) Inhibitor, MHJ-627, and Verification of Its Potent Anticancer Efficacy in Cervical Cancer HeLa Cells.

Extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase (MAPK) family, is involved in key cellular processes. However, overexpression and upregulation of ERK5 have been reported in various cancers, and ERK5 is associated with almost every biological characteristic of cancer cells. Accordingly, ERK5 has become a novel target for the development of anticancer drugs as inhibition of ERK5 shows suppressive effects of the deleterious properties of cancer cells. Herein, we report the synthesis and identification of a novel ERK5 inhibitor, MHJ-627, and verify its potent anticancer efficacy in a yeast model and the cervical cancer HeLa cell line. MHJ-627 successfully inhibited the kinase activity of ERK5 (IC50: 0.91 µM) and promoted the mRNA expression of tumor suppressors and anti-metastatic genes. Moreover, we observed significant cancer cell death, accompanied by a reduction in mRNA levels of the cell proliferation marker, proliferating cell nuclear antigen (PCNA), following ERK5 inhibition due to MHJ-627 treatment. We expect this finding to serve as a lead compound for further identification of inhibitors for ERK5-directed novel approaches for oncotherapy with increased specificity.

Wound healing effects of Asparagus lucidus Lindl extract through the phosphorylation of ERK1/2.

BACKGROUND: Skin is the outermost part of the human body and is essential in maintaining body homeostasis. In the event of skin injury, rapid wound repair is crucial to protect the body. In this study, we investigated the wound-healing properties of Asparagus lucidus Lindl extract by promoting keratinocyte proliferation. METHODS: To evaluate the effect of Asparagus lucidus Lindl extract on skin regeneration, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to measure keratinocyte proliferation, while an in vitro wound-healing assay was performed to evaluate wound closure through keratinocyte re-epithelialization. The intracellular mechanisms of the extract were studied using Western blot analysis to measure the phosphorylated forms of mitogen-activated protein kinases and protein kinase B. The mRNA expression of cell cycle-related genes was analyzed using quantitative real time-PCR analysis. A murine in vivo wound-healing assay was also conducted to observe the effect of the extract on wound closure. RESULTS: Asparagus lucidus Lindl extract induced 131.15% keratinocyte proliferation compared to the control in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The in vitro wound-healing assay showed that the extract improved wound closure by 216.94% through keratinocyte re-epithelialization. Western blot analysis revealed that the phosphorylated form of extracellular signal-regulated kinase 1/2 was increased by extract treatment. Quantitative real time-PCR analysis showed a dose-dependent increase in the mRNA expression of c-fos, c-jun, and VEGF. The in vivo wound-healing assay showed a significant increase (22.13%) of wound closure compared to the control on day 5. CONCLUSION: Asparagus lucidus Lindl extract promotes keratinocyte proliferation by activating the extracellular signal-regulated kinase 1/2 pathway and up-regulating the mRNA expression of c-fos, c-jun, and vascular endothelial growth factor.

Alpinia japonica extract induces apoptosis of hepatocellular carcinoma cells through G0/G1 cell cycle arrest and activation of JNK.

Hepatic cancer was the third most prevalent cause of cancer-related death worldwide in 2018, and its incidence is increasing. While therapeutic agents for hepatic cancer have improved, these agents can cause serious side effects, including damage to healthy tissues. To overcome this limitation, more than 3,000 plants have been used globally as common alternatives for cancer treatment. The anti-cancer activity of Alpinia japonica, one of the traditional herbal medicines (Korean name: Kkot-yang-ha), was investigated. Water extract of A. japonica (AJ) decreased the cell viability of hepatic cancer cells. AJ extract showed greater than 70% loss of mitochondrial potential in HepG2 cells as demonstrated by JC-1 staining. Apoptosis was induced by treatment with AJ extract as shown through FACS analysis, and G0/G1 phase arrest of 76.66% HepG2 cells was confirmed through cell cycle analysis and quantitative RT-PCR. Improper regulation of ERK1/2 might contribute to cell death, and JNK activation is necessary for apoptosis induced by stress stimuli. AJ extract stimulated the phosphorylation of JNK and ERK1/2, mitogen-activated protein kinases (MAPKs), in HepG2 cells. AJ extract has anticancer activity by inhibiting cell cycle progression, leading to apoptosis of hepatic cancer cells. This extract could potentially be used as a therapeutic agent for hepatic cancer.

Potent and Selective Inhibition of CYP1A2 Enzyme by Obtusifolin and Its Chemopreventive Effects.

Obtusifolin, a major anthraquinone component present in the seeds of Cassia tora, exhibits several biological activities, including the amelioration of memory impairment, prevention of breast cancer metastasis, and reduction of cartilage damage in osteoarthritis. We aimed to evaluate the inhibitory effects of obtusifolin and its analogs on CYP1A enzymes, which are responsible for activating procarcinogens, and investigate its inhibitory mechanism and chemopreventive effects. P450-selective substrates were incubated with human liver microsomes (HLMs) or recombinant CYP1A1 and CYP1A2 in the presence of obtusifolin and its four analogs. After incubation, the samples were analyzed using liquid chromatography-tandem mass spectrometry. Molecular docking simulations were performed using the crystal structure of CYP1A2 to identify the critical interactions between anthraquinones and human CYP1A2. Obtusifolin potently and selectively inhibited CYP1A2-mediated phenacetin O-deethylation (POD) with a Ki value of 0.031 µM in a competitive inhibitory manner in HLMs, whereas it exhibited negligible inhibitory effect against other P450s (IC50 > 28.6 µM). Obtusifolin also inhibited CYP1A1- and CYP1A2-mediated POD and ethoxyresorufin O-deethylation with IC50 values of <0.57 µM when using recombinant enzymes. Our molecular docking models suggested that the high CYP1A2 inhibitory activity of obtusifolin may be attributed to the combination of hydrophobic interactions and hydrogen bonding. This is the first report of selective and potent inhibitory effects of obtusifolin against CYP1A, indicating their potential chemopreventive effects.

Complications arising from clinical application of composite polycaprolactone/bioactive glass ceramic implants for craniofacial reconstruction: A prospective study.

This study aimed to demonstrate the in vitro performance of a novel polymer-ceramic composite incorporating polycaprolactone (PCL) and bioactive glass (BGS-7), and investigate its clinical outcomes in craniofacial reconstruction. After preparation of the material, the biochemical properties of the composite PCL/BGS-7 implant were tested to evaluate apatite formation in simulated body fluid (SBF). Changes in the implant surface after soaking in the SBF were determined using field-emission scanning electron microscopy. For clinical application of the implant, patients with craniofacial defects were prospectively enrolled to receive three-dimensional (3D)-printed PCL/BGS-7 implants. Clinical outcomes were investigated by reviewing postoperative complications, including wound problems, allergic responses, hematoma, seroma, implant displacement, and bone union. The accuracy of reconstruction was assessed by measuring the surface error between the reconstructed and mirrored models. Upon exposure of the PCL/BGS-7 implant to SBF, apatite particles were actively developed on the surface of the PCL/BGS-7 sample, showing favorable bone-binding capacity. Regarding the clinical application, seven patients with craniofacial defects were included. The clinical outcome was favorable in terms of complications, except in one patient, who presented with delayed wound healing due to previous irradiation. The patients showed improvements in symmetry, with a significant change in mean ± SD surface error between preoperative (5 ± 3 mm) and postoperative (1.5 ± 0.65 mm) status (p = 0.018). Wthin the limitations of the study it seems that the PCL/BGS-7 implants might be a relevant option for repairing craniofacial bone defects, owing to its favorable bone-binding property and clinical safety, with few complications.

Suppression of DYRK1A/B Drives Endoplasmic Reticulum Stress-mediated Autophagic Cell Death Through Metabolic Reprogramming in Colorectal Cancer Cells.

BACKGROUND/AIM: We previously identified KS40008 (4-(3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzene-1,2-diol), a novel inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase family (DYRK) 1A/B, which exhibited high enzymatic activity and cell proliferation-inhibitory effects in colorectal cancer (CRC) cell lines. In the present study, we aimed to elucidate the antitumor mechanisms of KS40008. MATERIALS AND METHODS: To assess the cytotoxicity of KS40008, we utilized a human cell line and organoid model and performed a CCK-8 assay and real-time cell analysis. Mitochondrial function was determined through mitochondrial staining, mito-stress test, and glycolysis test. In addition, we investigated the mechanisms of cancer cell death induced by KS40008 through immunoblotting, real-time quantitative polymerase chain reaction, reactive oxygen species staining, and immunofluorescence staining. RESULTS: KS40008 exhibited significant cytotoxicity in CRC and non-CRC cell lines, and organoid models compared to 5-fluorouracil, a conventional chemotherapeutic drug. Moreover, KS40008-induced inhibition of DYRK1A/B led to mitochondrial dysfunction and endoplasmic reticulum stress, promoting autophagic cancer cell death. CONCLUSION: KS40008 exerts antitumor activity through the inhibition of DYRK1A/B. Here, we demonstrated a mechanism by which KS40008 affects endoplasmic reticulum stress-mediated autophagy through the induction of mitochondrial stress, leading to cytotoxicity in CRC.

Adenophora triphylla var. japonica Inhibits Candida Biofilm Formation, Increases Susceptibility to Antifungal Agents and Reduces Infection.

(1) Background: Candida is the most common cause of fungal infections worldwide, but due to the limited option of antifungal therapies, alternative strategies are required. (2) Methods: Adenophora triphylla var. japonica extract was used for the biofilm formation assay using RPMI1640. The combinatorial antifungal assay, the dimorphic transition assay, and the adherence assay were done to see the influence of inhibition of biofilm formation. qRT-PCR analysis were performed to check the gene expression. (3) Results: Adenophora triphylla var. japonica extract inhibited the Candida biofilm formation. Treatment of extract increased the antifungal susceptibility of miconazole from a 37% reduction in fungal growth to 99.05%, and also dose-dependently reduced the dimorphic transition of Candida and the attachment of Candida to HaCaT cells. The extract blocked the expression of hyphal-related genes, extracellular matrix genes, Ras1-cAMP-PKA pathway genes, Cph2-Tec1 pathway gene, and MAP kinase pathway gene. (4) Conclusions: In this study, the treatment of Adenophora triphylla var. japonica extract showed inhibition of fungal biofilm formation, activation of antifungal susceptibility, and reduction of infection. These results suggest that fungal biofilm formation is a good target for the development of antifungal adjuvants, and Adenophora triphylla var. japonica extract should be a good candidate for biofilm-associated fungal infections.

Synthesis of novel 1H-Pyrazolo[3,4-b]pyridine derivatives as DYRK 1A/1B inhibitors.

As DYRK1A and 1B inhibitors, 1H-pyrazolo[3,4-b]pyridine derivatives were synthesized. Mostly, 3-aryl-5-arylamino compounds (6) and 3,5-diaryl compounds (8 and 9) were prepared and especially, 3,5-diaryl compound 8 and 9 showed excellent DYRK1B inhibitory enzymatic activities with IC50 Values of 3-287 nM. Among them, 3-(4-hydroxyphenyl), 5-(3,4-dihydroxyphenyl)-1H-pyrazolo[3,4-b]pyridine (8h) exhibited the highest inhibitory enzymatic activity (IC50 = 3 nM) and cell proliferation inhibitory activity (IC50 = 1.6 µM) towards HCT116 colon cancer cells. Also compound 8h has excellent inhibitory activities in patient-derived colon cancer organoids model as well as in 3D spheroid assay model of SW480 and SW620. The docking study supported that we confirmed that compound 8h binds to DYRK1B through various hydrogen bonding interactions and hydrophobic interactions.

Reporting Apparent Biclonal Immunoglobulin-A Monoclonal Proteins with Identical Light Chains.

It is generally not well appreciated that Immunoglobulin A (IgA) can present as apparent biclonal monoclonal protein (M-protein) comprising monomers and polymers, despite the fact that Kyle pointed this out in 1999. In this clinical communication, we report on 3 patients with Ig-A multiple myeloma (MM) who displayed this phenomenon. Of these 3 patients, 2 had identical kappa light chains suggesting biclonality, while the other had 2 lambda light chains. Because the Ig-A M-proteins are clustered in the beta-area, accurate densitometric quantification is made difficult. Hence, we suggest assaying Ig-A levels and free light chains in concert with the M-protein levels to monitor these patients on therapy. In conclusion, when encountering biclonal Ig-A M-proteins with identical light chains, the laboratorian should add the 2 values and present one composite number to the clinician.

Robustaflavone induces G0/G1 cell cycle arrest and apoptosis in human umbilical vein endothelial cells and exhibits anti-angiogenic effects in vivo.

We investigated the anti-angiogenic and pro-apoptotic effects of robustaflavone (RF), a naturally occurring biflavonoid, on human umbilical vein endothelial cells (HUVECs). RF inhibited HUVEC proliferation and showed cytotoxicity that inhibited HUVEC viability. RF-induced apoptosis was characterized by flow cytometry and caspase 3 analysis. We found that RF increased the number of sub-G1 cells and terminal deoxynucleotidyl transferase dUTP nick end-labeled cells. Additionally, RF induced caspase 3 and poly (ADP-ribose) polymerase activation. Potential molecular targets were identified using a human apoptosis antibody array. RF upregulated Bax, Bad, cleaved caspase 3, p21, and phosphorylated p53 levels. RF induced mitochondrial membrane potential loss and the release of cytochrome c and apoptosis-inducing factor. Cell cycle arrest at G0/G1 phase and the downregulation of Cdk4, Cdk6, and cyclin D1 expression were induced by RF. In vivo anti-angiogenic effects were investigated using a tumor allograft animal model and a Matrigel plug assay. RF reduced the volumes and weights of CT-26 cell-derived tumors. The blood vessel density was significantly decreased in RF-treated tumors. RF also inhibited VEGF-A-stimulated blood vessel formation in vivo in Matrigel plugs. These results suggest that RF can potentially inhibit angiogenesis-dependent tumor growth and metastasis.

Rhein inhibits the growth of Propionibacterium acnes by blocking NADH dehydrogenase-2 activity.

Introduction. Rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid) has various properties, including anti-inflammatory, antioxidant and anticancer activities. However, the mechanism underlying the role of rhein in antimicrobial activity remains largely unknown.Aim. This study aims to identify potential natural compounds of rhein that are capable of inhibiting Cutibacterium acnes and elucidate the effects of rhein on NADH dehydrogenase-2 activity in C. acnes.Methodology. The anti-C. acnes activity of compounds was analysed using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), the paper disc diffusion test and the checkerboard dilution test. To check whether rhein was inhibitory, putative type II NADH dehydrogenase (NDH-2) of C. acnes was analysed, cloned and expressed in Escherichia coli, and then NDH-2 purification was assessed with Ni-NTA before rhein inhibition of NADH dehydrogenase-2 activity was checked with ferricyanide [K3Fe(CN)6] as a substrate.Results. The results showed that the MIC of rhein against C. acnes was 6.25 µg ml-1, while the MBC was 12.5 µg ml-1, and there was a 38 mm inhibition zone in the paper disc diffusion test. Rhein showed an additive two- to fourfold reduction of the MIC value with four antibiotics on the checkerboard dilution test. The purified NADH dehydrogenase gene product showed a size of approximately 51 kDa and had a V max of 23 µmol and a K m of 280 µm. The inhibitory effect of rhein against NADH dehydrogenase-2 activity was non-competitive with ferricyanide [K3Fe(CN)6] with a K i value of 3.5-4.5 µm.Conclusion. This study provided evidence of the inhibitory effects of rhein on the growth of C. acnes by blocking of NADH dehydrogenase-2 activity. This mechanism of inhibitory activity in the reduction of ROS formation and ATP productivity should be further tested in C. acnes and the question of whether rhein inhibits the natural growth of C. acnes should be investigated.

A Novel Selective 11ß-HSD1 Inhibitor, (E)-4-(2-(6-(2,6-Dichloro-4-(Trifluoromethyl)Phenyl)-4-Methyl-1,1-Dioxido-1,2,6-Thiadiazinan-2-yl)Acetamido)Adamantan-1-Carboxamide (KR-67607), Prevents BAC-Induced Dry Eye Syndrome.

Dry eye syndrome is the most common eye disease and it is caused by various reasons. As the balance of the tear film that protects the eyes is broken due to various causes, it becomes impossible to properly protect the eyes. In this study, the protective effects and underlying mechanisms of topical (E)-4-(2-(6-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-methyl-1,1-dioxido-1,2,6-thiadiazinan-2-yl)acetamido)adamantan-1-carboxamide (KR-67607), a novel selective 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) inhibitor, were investigated in benzalkonium chloride (BAC)-induced dry eye syndrome. BAC-treated rat eyes induced significant increases in ocular surface damage, decreased corneal thickness, corneal basement membrane destruction in the conjunctival epithelium, and expression of pro-inflammatory cytokines tumor necrosis factor-α and 11ß-HSD1. These effects of BAC were reversed by topical KR-67607 treatment. Furthermore, KR-67607 decreased 4-hydroxynonenal expression and increased antioxidant and mucus secretion in BAC-treated rat eyes. Taken together, a novel selective 11ß-HSD1 inhibitor can prevent BAC-induced dry eye syndrome by inhibiting pro-inflammatory cytokine and reactive oxygen species expression via the inhibition of both 11ß-HSD1 activity and expression.

iTRAQ-Based Quantitative Proteomic Comparison of 2D and 3D Adipocyte Cell Models Co-cultured with Macrophages Using Online 2D-nanoLC-ESI-MS/MS.

The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDHα, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.

Anticancer effect of XAV939 is observed by inhibiting lactose dehydrogenase A in a 3-dimensional culture of colorectal cancer cells.

XAV939, a tankyrase inhibitor, exerts an anticancer effect in 3-dimensional (3D) cultured SW480 cells, however this is not exhibited in 2-dimensional (2D) cultured SW480 cells. In the current study, XAV939 induced a 3.7-fold increase in cellular apoptosis in 3D culture but not in the 2D culture. However, no significant changes were indicated in cell cycle distribution in the 2D or 3D culture. Based on the observation that protein expression, which was associated with the glycolytic pathway, was increased in the 3D culture, the effect of XAV939 on the patterns of glycolytic protein expression was assessed. XAV939 was revealed to decrease lactose dehydrogenase A (LDHA) expression in 3D cultured SW480 cells, but only exerted a small effect in the 2D culture. The coadministration of XAV939 with the LDHA inhibitor FX11 decreased proliferation in 3D cultured SW480 cells compared with the single administration of FX11, while there was no additive effect in the 2D culture. The lactate assay also indicated that XAV939 decreased lactate secretion in the 3D cell culture but not in the 2D culture. These results suggest that XAV939 exerts an anticancer effect through inhibition of LDHA in the 3D culture.

Development of a parallel microbore hollow fiber enzyme reactor platform for online 18O-labeling: Application to lectin-specific lung cancer N-glycoproteome.

We introduce a simple online 18O-labeling protocol for protein samples that uses a parallelizing microbore hollow fiber enzyme reactor (mHFER) as an alternative tool for online proteolytic digestion. Online 18O-labeling is performed by separately attaching two mHFERs in parallel to a 10-port switching valve with a high-pressure syringe pump and two syringes containing 16O- or 18O-water. 16O-/18O-labeled peptides are formed in this manner and simultaneously analyzed online using nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) without any residual trypsin activity. The usefulness of a parallel mHFER platform (P-mHFER) in 18O-labeling was tested using both cytochrome C and alpha-1-acid-glycoprotein to verify the incorporation level of two 18O atoms into tryptic peptides and to provide a quantitative assessment with varied mixing ratios. Additionally, our 18O-labeling approach was used to study the serum N-glycoproteome from lung cancer patients and controls to evaluate the applicability of lectin-based quantitative N-glycoproteomics. We successfully quantified 76 peptides (from 62 N-glycoproteins). Nineteen of these peptides from lung cancer serum were up-/down-regulated at least 2.5-fold compared to controls. As a result, the P-mHFER-based online 18O-labeling platform presented here can be a simple and reproducible way to allow quantitative proteomic analysis of diverse proteome samples.

Quantitative Proteomic Analysis of 2D and 3D Cultured Colorectal Cancer Cells: Profiling of Tankyrase Inhibitor XAV939-Induced Proteome.

Recently there has been a growing interest in three-dimensional (3D) cell culture systems for drug discovery and development. These 3D culture systems better represent the in vivo cellular environment compared to two-dimensional (2D) cell culture, thereby providing more physiologically reliable information on drug screening and testing. Here we present the quantitative profiling of a drug-induced proteome in 2D- and 3D-cultured colorectal cancer SW480 cells using 2D nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS) integrated with isobaric tags for relative and absolute quantitation (iTRAQ). We identified a total of 4854 shared proteins between 2D- and 3D-cultured SW480 cells and 136/247 differentially expressed proteins (up/down-regulated in 3D compared to 2D). These up/down-regulated proteins were mainly involved in energy metabolism, cell growth, and cell-cell interactions. We also investigated the XAV939 (tankyrase inhibitor)-induced proteome to reveal factors involved in the 3D culture-selective growth inhibitory effect of XAV939 on SW480 cells. We identified novel XAV939-induced proteins, including gelsolin (a possible tumor suppressor) and lactate dehydrogenase A (a key enzyme of glycolysis), which were differentially expressed between 2D- and 3D-cultured SW480 cells. These results provide a promising informative protein dataset to determine the effect of XAV939 on the expression levels of proteins involved in SW480 cell growth.