Angiogenic protein profiling, phytochemical screening and in silico anti-cancer targets validation of stem, leaves, fruit, and seeds of Calotropis procera in human liver and breast cancer cell lines.

The main focus of anticancer drug discovery is on developing medications that are gentle on normal cells and should have the ability to target multiple anti-cancer pathways. Liver cancer is becoming a worldwide epidemic due to the highest occurring and reoccurring rate in some countries. Calotropis procera is a xerophytic herbal plant growing wildly in Saudi Arabia. Due to its anti-angiogenic and anticancer capabilities, "C. procera" is a viable option for developing innovative anticancer medicines. However, no study has been done previously, to discover angiogenic and anti-cancer targets which are regulated by C. procera in liver cancer. In this study, leaves, stems, flowers, and seeds of C. procera were used to prepare crude extracts and were fractionated into four solvents of diverse polarities. These bioactivity-guided solvent fractions helped to identify useful compounds with minimal side effects. The phytoconstituents present in the leaves and stem were identified by GC-MS. In silico studies were done to predict the anti-cancer targets by major bioactive constituents present in leaves and stem extracts. A human angiogenesis antibody array was performed to profile novel angiogenic targets. The results from this study showed that C. procera extracts are an ideal anti-cancer remedy with minimum toxicity to normal cells as revealed by zebrafish in vivo toxicity screening assays. The novel antiangiogenic and anticancer targets identified in this study could be explored to design medication against liver cancer.

Tolypothrix Dichloromethane Ethylacetate fraction (TDEF) inhibits cisplatin resistance H357 cell through PI3K/AKT/beta-catenin pathway.

Chemoresistance is one of the major factors for treatment failure in OSCC. Reprogramming chemoresistance cells to undergo drug induced apoptotic cell death is a feasible approach to overcome drug resistance. Cyanobacteria is considered important sources of lead compounds for the development of drugs for treating cancer chemoresistance. This study deals with the role of Tolypothrix Dichloromethane Ethyl acetate fraction (TDEF) inducing apoptosis in cisplatin resistance H357 cell (H357cisR) and the underlying mechanisms sensitizing the chemoresistance. TDEF showing effective activity against H357cisR with IC50-14.13±1.18 µg mL-1, inhibits proliferation and migration. Proteome apoptosis arrays were found to stimulate phosphorylation of p53, activation of proapoptotic proteins including BAX and cytochrome C (CYCS), caspase-3/9 (CASP3/9), suppression of anti-apoptotic proteins like Bcl2, survivin and increased expression of the cell cycle checkpoint protein p21, p27. TDEF induced apoptosis with cell death-transducing signals, that regulate the Matrix metalloproteinases (MMPs) by down-regulation of Bcl2 and up-regulation of Bax, triggering the cytochrome c release from mitochondria to cytosol thus triggered the activation of caspases-9 to activate downstream executioner caspase-3/7 required for apoptotic changes. The mechanistic pathway of apoptotic cell death in H357cisR was done through inhibiting ß-catenin through GSK3ß in turn activated by AKT. The phosphorylated ß-catenin leads to proteasome degradation and unable to translocation to nucleus thereby activating c-Myc, survivin, Cyclin D and upregulate p21 expression which lead to cell cycle arrest in G0/G1 phase.

Calotropis procera: A double edged sword against glioblastoma, inhibiting glioblastoma cell line growth by targeting histone deacetylases (HDAC) and angiogenesis.

Despite substantial investments in anti-glioblastoma (GBM) drug discovery over the last decade, progress is limited to preclinical stages, with clinical studies frequently encountering obstacles. Angiogenic and histone deacetylase inhibitors (HDACi) have shown profound results in pre-clinical studies. Investigating a multicomponent anti-cancer remedy that disrupts the tumor angiogenic blood vessels and simultaneously disrupts HDACs, while inducing minimal side effects, is critically needed. The crude extracts derived from medicinal plants serve as a renewable reservoir of anti-tumor drugs, exhibiting reduced toxicity compared to chemically synthesized formulations. Calotropis procera is a traditional medicinal plant, and its anticancer potential against many cancer cell lines has been reported, however its antiangiogenic and HDAC inhibitory action is largely unknown. The anticancer activity of methanol leaf extract of C. procera was tested in three types of human glioblastoma cell lines. Wild-type and transgenic zebrafish embryos were used to evaluate developmental toxicity and angiogenic activity. A human angiogenic antibody array was used to profile angiogenic proteins in the U251 GM cell line. A real-time reverse transcriptase polymerase chain reaction (RT PCR) assay was used to detect the differential expression of eleven HDAC genes in U251 cells treated with C. procera extract. The extract significantly reduced the proliferation of all three types of GBM cell lines and the cytotoxicity was found to be more pronounced in U251 GM cells, with an IC50 value of 2.63 ± 0.23 µg/ml, possibly by arresting the cell cycle at the G2/M transition. The extract did not exhibit toxic effects in zebrafish embryos, even at concentrations as high as 1000 µg/ml. The extract also inhibited angiogenic blood vessel formation in the transgenic zebrafish model in a dose-dependent manner. The results from the angiogenic antibody array have suggested novel angiogenesis targets that can be utilized to treat GBM. Real-time RT PCR analysis has shown that C. procrea extract caused an upregulation of HDAC5, 7, and 10, while the mRNA of HDAC1, 2, 3 and 8 (Class I HDACs), and HDAC4, 6, and 9 (Class II) were downregulated in U251 GM cells. The cytotoxicity of the C. procera extract on GBM cell lines could be due to its dual action by regulation of both tumor angiogenesis and histone deacetylases enzymes. Through this study, the C. procera leaf extract has been suggested as an effective remedy to treat GBM with minimal toxicity. In addition, various novel angiogenic and HDAC targets has been identified which could be helpful in designing better therapeutic strategies to manage glioblastoma multiforme in human patients.

Expression of macrophage activation­specific factors in hyperplastic scar tissue during hyperplasia phase by antibody array blotting membrane assay and its clinical significance.

The expression of macrophage activation-specific factors in hyperplastic scar (HS) tissues during hyperplasia phase was detected by antibody array imprinted membrane method and the role of macrophage activation in the natural evolution of HS was explored. A total of 83 patients with HS admitted to the Affiliated Hospital of Beihua University (Jilin, China) between February 2021 and July 2021 were enrolled. The clinical data of the patients were retrospectively analyzed. These patients were divided into the hyperplasia HS group (n=26) and the decline HS group (the HS tissues ceased to grow and were in regression periods; n=57) according to the time of scar formation and clinical characteristics. The HS tissues were collected from patients in both groups. The contents of IL-12, IL-10, VEGF and basic fibroblast growth factor (bFGF) were detected by antibody array imprinted membrane method and the contents of IL-12, IL-10, VEGF and bFGF in tissues with various groups of tissues and clinical features were compared. The connection between macrophage activation-specific factors with VEGF and bFGF was analyzed using Pearson correlation analysis. The contents of IL-10 (9.48±1.06), VEGF (24.15±2.64) and bFGF (37.48±2.56) were much lower and IL-12 levels (16.45±0.85) were strongly higher in hyperplasia HS group compared with those in the decline HS group (14.56±1.26 for IL-10, 27.85±2.63 for VEGF, 43.15±3.16 for bFGF and 10.46±0.75 for IL-12, P<0.001). In the hyperplasia HS group, the contents of IL-10, VEGF and bFGF were obviously higher and the IL-12 levels were markedly lower in patients with age ≥30 years, protuberance height <2 mm, soft flexibility, low hyperemia degree and no concomitant symptoms than those in the patients with age <30 years, protuberance height ≥2 mm, hard flexibility, high hyperemia degree and concomitant symptoms (P<0.001). Pearson correlation analysis showed that IL-12 was negatively correlated with VEGF and bFGF (r=-0.328, 0.600, P<0.01). IL-10 was positively correlated with VEGF and bFGF (r=0.486, 0.684, respectively, P<0.001). In conclusion, macrophage activation-specific factors were abnormally expressed in hyperplasia HS, mainly M1 macrophages, accompanied by severe inflammatory reaction. The transformation of M1 macrophage into M2 macrophage usually occurred during the declining HS phase, which accelerated scar formation by promoting the formation of fibroblasts and angiogenesis. Detection of macrophage activation-specific factors may contribute to evaluate the clinical stage of HS.

Chicken muscle antibody array reveals the regulations of LDHA on myoblast differentiation through energy metabolism.

Myoblast proliferation and differentiation are highly dynamic and regulated processes in skeletal muscle development. Given that proteins serve as the executors for the majority of biological processes, exploring key regulatory factors and mechanisms at the protein level offers substantial opportunities for understanding the skeletal muscle development. In this study, a total of 607 differentially expressed proteins between proliferation and differentiation in myoblasts were screened out using our chicken muscle antibody array. Biological function analysis revealed the importance of energy production processes and compound metabolic processes in myogenesis. Our antibody array specifically identified an upregulation of LDHA during differentiation, which was associated with the energy metabolism. Subsequent investigation demonstrated that LDHA promoted the glycolysis and TCA cycle, thereby enhancing myoblasts differentiation. Mechanistically, LDHA promotes the glycolysis and TCA cycle but inhibits the ETC oxidative phosphorylation through enhancing the NADH cycle, providing the intermediate metabolites that improve the myoblasts differentiation. Additionally, increased glycolytic ATP by LDHA induces Akt phosphorylation and activate the PI3K-Akt pathway, which might also contribute to the promotion of myoblasts differentiation. Our studies not only present a powerful tool for exploring myogenic regulatory factors in chicken muscle, but also identify a novel role for LDHA in modulating myoblast differentiation through its regulation of cellular NAD+ levels and subsequent downstream effects on mitochondrial function.

Role of serum cytokines in the prediction of heart failure in patients with coronary artery disease.

AIMS: Coronary artery disease (CAD) is the most common cause of heart failure (HF). This study aimed to identify cytokine biomarkers for predicting HF in patients with CAD. METHODS AND RESULTS: Twelve patients with CAD without HF (CAD-non HF), 12 patients with CAD complicated with HF (CAD-HF), and 12 healthy controls were enrolled for Human Cytokine Antibody Array, which were used as the training dataset. Then, differentially expressed cytokines among the different groups were identified, and crucial characteristic proteins related to CAD-HF were screened using a combination of the least absolute shrinkage and selection operator, recursive feature elimination, and random forest methods. A support vector machine (SVM) diagnostic model was constructed based on crucial characteristic proteins, followed by receiver operating characteristic curve analysis. Finally, two validation datasets, GSE20681 and GSE59867, were downloaded to verify the diagnostic performance of the SVM model and expression of crucial proteins, as well as enzyme-linked immunosorbent assay was also used to verify the levels of crucial proteins in blood samples. In total, 12 differentially expressed proteins were overlapped in the three comparison groups, and then four optimal characteristic proteins were identified, including VEGFR2, FLRG, IL-23, and FGF-21. After that, the area under the receiver operating characteristic curve of the constructed SVM classification model for the training dataset was 0.944. The accuracy of the SVM classification model was validated using the GSE20681 and GSE59867 datasets, with area under the receiver operating characteristic curve values of 0.773 and 0.745, respectively. The expression trends of the four crucial proteins in the training dataset were consistent with those in the validation dataset and those determined by enzyme-linked immunosorbent assay. CONCLUSIONS: The combination of VEGFR2, FLRG, IL-23, and FGF-21 can be used as a candidate biomarker for the prediction and prevention of HF in patients with CAD.

ß-Lapachone Exerts Anticancer Effects by Downregulating p53, Lys-Acetylated Proteins, TrkA, p38 MAPK, SOD1, Caspase-2, CD44 and NPM in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells.

ß-lapachone (ß-Lap), a topoisomerase inhibitor, is a naturally occurring ortho-naphthoquinone phytochemical and is involved in drug resistance mechanisms. Oxaliplatin (OxPt) is a commonly used chemotherapeutic drug for metastatic colorectal cancer, and OxPt-induced drug resistance remains to be solved to increase chances of successful therapy. To reveal the novel role of ß-Lap associated with OxPt resistance, 5 µM OxPt-resistant HCT116 cells (HCT116-OxPt-R) were generated and characterized via hematoxylin staining, a CCK-8 assay and Western blot analysis. HCT116-OxPt-R cells were shown to have OxPt-specific resistance, increased aggresomes, upregulated p53 and downregulated caspase-9 and XIAP. Through signaling explorer antibody array, nucleophosmin (NPM), CD37, Nkx-2.5, SOD1, H2B, calreticulin, p38 MAPK, caspase-2, cadherin-9, MMP23B, ACOT2, Lys-acetylated proteins, COL3A1, TrkA, MPS-1, CD44, ITGA5, claudin-3, parkin and ACTG2 were identified as OxPt-R-related proteins due to a more than two-fold alteration in protein status. Gene ontology analysis suggested that TrkA, Nkx-2.5 and SOD1 were related to certain aggresomes produced in HCT116-OxPt-R cells. Moreover, ß-Lap exerted more cytotoxicity and morphological changes in HCT116-OxPt-R cells than in HCT116 cells through the downregulation of p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM. Our results indicate that ß-Lap could be used as an alternative drug to overcome the upregulated p53-containing OxPt-R caused by various OxPt-containing chemotherapies.

In-Depth Serum Proteomics Reveals the Trajectory of Hallmarks of Cancer in Hepatitis B Virus-Related Liver Diseases.

Hepatocellular carcinoma (HCC) is a prevalent cancer in China, with chronic hepatitis B (CHB) and liver cirrhosis (LC) being high-risk factors for developing HCC. Here, we determined the serum proteomes (762 proteins) of 125 healthy controls and Hepatitis B virus-infected CHB, LC, and HCC patients and constructed the first cancerous trajectory of liver diseases. The results not only reveal that the majority of altered biological processes were involved in the hallmarks of cancer (inflammation, metastasis, metabolism, vasculature, and coagulation) but also identify potential therapeutic targets in cancerous pathways (i.e., IL17 signaling pathway). Notably, the biomarker panels for detecting HCC in CHB and LC high-risk populations were further developed using machine learning in two cohorts comprised of 200 samples (discovery cohort = 125 and validation cohort = 75). The protein signatures significantly improved the area under the receiver operating characteristic curve of HCC (CHB discovery and validation cohort = 0.953 and 0.891, respectively; LC discovery and validation cohort = 0.966 and 0.818, respectively) compared to using the traditional biomarker, alpha-fetoprotein, alone. Finally, selected biomarkers were validated with parallel reaction monitoring mass spectrometry in an additional cohort (n = 120). Altogether, our results provide fundamental insights into the continuous changes of cancer biology processes in liver diseases and identify candidate protein targets for early detection and intervention.

The effects of encapsulation on NK cell differentiation potency of C-kit+ hematopoietic stem cells via identifying cytokine profiles.

Natural killer cells (NK cells) can kill cancerous cells without prior sensitization. This feature makes them appealing candidates for cellular therapy. Due to the degradation rate and controlled release of these matrices, hydrogels hold great promise in cell differentiation. The study aims to investigate the effect of encapsulated alginate-gelatin on the differentiation potential of C-kit+ cells toward NK cells which are mediated by cytokines detection. Under both encapsulated and unencapsulated conditions, C-kit+ cells can differentiate into NK cells. In the following, real-time PCR and western blotting were done to investigate the mRNA and protein expression, respectively. Determine cytokine profiles from the collected culture medium conducted a Cytokine antibody array. The differentiated cells were then co-cultured with Molt-4 cells to examine the expression levels of INF-γ, TNF-α, and IL-10 using real-time-PCR. There was a substantial change in protein expression of the Notch pathway. Also, the encapsulation increased the mRNA expression of INF-γ and TNF-α in Molt-4 cells. Based on these findings, the encapsulation effects on the differentiation of C-kit+ cells toward NK cells could be related to the secreted cytokines such as interleukin-10 and INF-γ and the Notch protein expression.

Anti-drug resistance, anti-inflammation, and anti-proliferation activities mediated by melatonin in doxorubicin-resistant hepatocellular carcinoma: in vitro investigations.

Hepatocellular carcinoma (HCC) is the major life-threatening primary liver malignancy in both sexes all over the world. Unfortunately, the majority of patients are diagnosed at later stages because HCC does not elicit obvious symptoms during its early incidence. Consequently, most individuals escape the first-line HCC treatments and are treated with chemotherapy. Regrettably, the therapeutic outcomes for those patients are usually poor because of the development of multidrug resistance phenomena. Furthermore, most anti-HCC therapies cause severe undesired side effects that notably interfere with the life quality of such patients. Accordingly, there is an important need to search for an alternative therapeutic drug or adjuvant which is more efficient with safe or even minimal side effects for HCC treatment. Melatonin was recently reported to exert intrinsic antitumor activity in different cancers. However, the regulatory pathways underlying the antitumor activity of melatonin are poorly understood in resistant liver cells. Furthermore, a limited number of studies have addressed the therapeutic role of melatonin in HCC cells resistant to doxorubicin chemotherapy. In this study, we investigated the antitumor effects of melatonin in doxorubicin-resistant HepG2 cells and explored the regulatory pivotal targets underlying these effects. To achieve our aim, an MTT assay was used to calculate the 50% inhibitory concentration of melatonin and evaluate its antiproliferative effect on resistant cells. Additionally, qRT-PCR was used to quantify genes having a role in drug resistance phenotype (ABCB1, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, and ABCG2); apoptosis (caspases-3, and -7, Bcl2, Bax, and p53); anti-oxidation (NRF2); expression of melatonin receptors (MT1, MT2, and MT3); besides, programmed death receptor PD-1 gene. The active form of the caspase-3 enzyme was estimated by ELISA. A human inflammatory antibody membrane array was employed to quantify forty inflammatory factors expressed in treated cells. We observed that melatonin inhibited the proliferation of doxorubicin-resistant HepG2 cells in a dose-dependent manner after 24-h incubation time with a calculated IC50 greater than 10 mM (13.4 mM), the expression levels of genes involved in drug resistance response (ABCB1, ABCC1, ABCC5, and ABCG2) were downregulated. Also, the expression of caspase-3, Caspase-7, NRF2, and p53 genes were expressed at higher levels as compared to control (DMSO-treated cells). An active form of caspase-3 was confirmed by ELISA. Moreover, the anti-inflammatory effect of melatonin was detected through the calculated fold change to control which was reduced for various mediators that have a role in the inflammation pathway. The current findings introduce melatonin as a promising anti-cancer treatment for human-resistant HCC which could be used in combination with current chemotherapeutic regimens to improve the outcome and reduce the developed multidrug resistance.

Peptides and Anti-peptide Antibodies for Small- and Medium-Scale Peptide and Anti-peptide Affinity Microarrays.

This chapter describes the principles for selection of antigenic peptides for the development of anti-peptide antibodies suitable for microarray-based multiplex affinity assays and optional mass spectrometry detection. The methods described here are mostly applicable to small- and medium-scale multiplex affinity assay and microarrays. Although the same principles of peptide selection may also be applied to larger-scale arrays (with 100+ features), informatics software and printing methods may well differ. Due to the sheer number of proteins/peptides to be processed and analyzed, dedicated software with high processing capacity and enterprise-level array robotics may be required for larger-scale efforts. This report aims to provide practical advice to those seeking to develop or use arrays with up to ~100 different peptide or protein features.

Met/HGFR triggers detrimental reactive microglia in TBI.

The complexity of signaling events and cellular responses unfolding in neuronal, glial, and immune cells upon traumatic brain injury (TBI) constitutes an obstacle in elucidating pathophysiological links and targets for intervention. We use array phosphoproteomics in a murine mild blunt TBI to reconstruct the temporal dynamics of tyrosine-kinase signaling in TBI and then scrutinize the large-scale effects of perturbation of Met/HGFR, VEGFR1, and Btk signaling by small molecules. We show Met/HGFR as a selective modifier of early microglial response and that Met/HGFR blockade prevents the induction of microglial inflammatory mediators, of reactive microglia morphology, and TBI-associated responses in neurons and vasculature. Both acute and prolonged Met/HGFR inhibition ameliorate neuronal survival and motor recovery. Early elevation of HGF itself in the cerebrospinal fluid of TBI patients suggests that this mechanism has translational value in human subjects. Our findings identify Met/HGFR as a modulator of early neuroinflammation in TBI with promising translational potential.

Anti-leukemic principle(s) from Momordica charantia seeds induce differentiation of HL-60 cells through ERK/MAPK signalling pathway.

Myeloid leukemia is one of the major causes of deaths among elderly with very poor prognosis. Due to the adverse effects of existing chemotherapeutic agents, plant-derived components are being screened for their anti-leukemic potential. Momordica charantia (bitter gourd) possesses a variety of therapeutic activities. We have earlier demonstrated anti-leukemic activity of acetone extract of M. charantia seeds. The present study reports purification of differentiation inducing principle(s) from further fractionated seed extract (hexane fraction of the acetone extract, Mc2-Ac-hex) using HL-60 cells. Out of the 5 peak fractions (P1-P5) obtained from normal phase HPLC of the Mc2-Ac-hex, only peak fraction 3 (P3) induced differentiation of HL-60 cells as evident from an increase in NBT-positive cells and increased expression of cell surface marker CD11b. The P3 differentiated the HL-60 cells to granulocytic lineage, established by increased CD15 (granulocytic cell surface marker) expression in the treated cells. Further, possible molecular mechanism and the signalling pathway involved in the differentiation of HL-60 cells were also investigated. Use of specific signalling pathway inhibitors in the differentiation study, and proteome array analysis of the treated cells collectively revealed the involvement the of ERK/MAPK mediated pathway. Partial characterization of the P3 by GC-MS analysis revealed the presence of dibutyl phthalate, and derivatives of 2,5-dihydrofuran to be the highest among the 5 identified compounds. This study thus demonstrated that purified differentiation-inducing principle(s) from M. charantia seed extract induce HL-60 cells to granulocytic lineage through ERK/MAPK signalling pathway. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00547-x.

TNF-α, IL-6 and hsCRP in patients with melancholic, atypical and anxious depression: an antibody array analysis related to somatic symptoms.

Background: The association between inflammation and major depressive disorder (MDD) remains poorly understood, given the heterogeneity of patients with MDD. Aims: We investigated inflammatory markers, such as interleukin (IL)-6, high-sensitivity C reactive protein (hsCRP) and tumour necrosis factor-α (TNF-α) in melancholic, atypical and anxious depression and explored whether baseline inflammatory protein levels could indicate prognosis. Methods: The sample consisted of participants (aged 18-55 years) from a previously reported multicentre randomised controlled trial with a parallel-group design registered with ClinicalTrials.gov, including melancholic (n=44), atypical (n=37) and anxious (n=44) patients with depression and healthy controls (HCs) (n=33). Subtypes of MDD were classified according to the 30-item Inventory of Depressive Symptomatology, Self-Rated Version and the 17-item Hamilton Depression Rating Scale. Blood levels of TNF-α, IL-6 and hsCRP were assessed using antibody array analysis. Results: Patients with MDD, classified according to melancholic, atypical and anxious depression subtypes, and HCs did not differ significantly in baseline TNF-α, IL-6 and hsCRP levels after adjustment. In patients with anxious depression, hsCRP levels increased significantly if they experienced no pain (adjusted (adj.) p=0.010) or mild to moderate pain (adj. p=0.038) compared with those with severe pain. However, the patients with anxious depression and severe pain showed a lower trend in hsCRP levels than patients with atypical depression who experienced severe pain (p=0.022; adj. p=0.155). Baseline TNF-α (adj. p=0.038) and IL-6 (adj. p=0.006) levels in patients in remission were significantly lower than those in patients with no remission among the participants with the atypical depression subtype at the eighth-week follow-up. Conclusions: This study provides evidence of differences in inflammatory proteins in patients with varied symptoms among melancholic, atypical and anxious depression subtypes. Further studies on the immunoinflammatory mechanism underlying different subtypes of depression are expected for improved individualised therapy. Trial registration number: NCT03219008.

Human amniotic mesenchymal stem cells-derived IGFBP-3, DKK-3, and DKK-1 attenuate liver fibrosis through inhibiting hepatic stellate cell activation by blocking Wnt/ß-catenin signaling pathway in mice.

BACKGROUND: Liver fibrosis is an outcome of restoring process in chronic liver injury. Human amniotic mesenchymal stem cells (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammatory potential which makes them suitable for treating liver fibrosis. This study aimed to explore the effect and mechanism of hAMSCs on liver fibrosis. METHODS: hAMSCs were transplanted into carbon tetrachloride (CCl4)-induced liver fibrosis mice via tail vein, and the effects of hAMSCs on hepatic fibrosis were assessed. The effects of hAMSCs and hAMSCs conditional medium (CM) on the activation of hepatic stellate cells (HSCs) were investigated in vivo and in vitro. Antibody array assay was used to identify the cytokines secreted by hAMSCs that may inhibit the activation of HSCs. Finally, the underlying mechanisms were explored by assessing IGF-1R/PI3K/AKT and GSK3ß/ß-catenin signaling pathways in the activated HSCs (LX-2) with hAMSCs and hAMSCs transfected with corresponding siRNAs. RESULTS: Our results showed that hAMSCs possessed the characterizations of mesenchymal stem cells. hAMSCs significantly reduced liver fibrosis and improved liver function in mice by inhibiting HSCs activation in vivo. Both hAMSCs and hAMSC-CM remarkably inhibited the collagen deposition and activation of LX-2 cells in vitro. Antibody array assay showed that insulin-like growth factor binding protein-3 (IGFBP-3), Dickkopf-3 (DKK-3), and Dickkopf-1 (DKK-1) were highly expressed in the co-culture group and hAMSC-CM group compared with LX-2 group. Western blot assay demonstrated that IGFBP-3, DKK-3, and DKK-1 derived from hAMSCs inhibit LX-2 cell activation through blocking canonical Wnt signaling pathway. CONCLUSIONS: Our results demonstrated that IGFBP-3, Dkk3, and DKK-1 secreted by hAMSCs attenuated liver fibrosis in mice through inhibiting HSCs activation via depression of Wnt/ß-catenin signaling pathway, suggesting that hAMSCs or hAMSC-CM provides an alternative therapeutic approach for the treatment of liver fibrosis.

Mechanisms and Minimization of False Discovery of Metabolic Bioorthogonal Noncanonical Amino Acid Proteomics.

Metabolic proteomics has been widely used to characterize dynamic protein networks in many areas of biomedicine, including in the arena of tissue aging and rejuvenation. Bioorthogonal noncanonical amino acid tagging (BONCAT) is based on mutant methionine-tRNA synthases (MetRS) that incorporates metabolic tags, for example, azidonorleucine [ANL], into newly synthesized proteins. BONCAT revolutionizes metabolic proteomics, because mutant MetRS transgene allows one to identify cell type-specific proteomes in mixed biological environments. This is not possible with other methods, such as stable isotope labeling with amino acids in cell culture, isobaric tags for relative and absolute quantitation and tandem mass tags. At the same time, an inherent weakness of BONCAT is that after click chemistry-based enrichment, all identified proteins are assumed to have been metabolically tagged, but there is no confirmation in mass spectrometry data that only tagged proteins are detected. As we show here, such assumption is incorrect and accurate negative controls uncover a surprisingly high degree of false positives in BONCAT proteomics. We show not only how to reveal the false discovery and thus improve the accuracy of the analyses and conclusions but also approaches for avoiding it through minimizing nonspecific detection of biotin, biotin-independent direct detection of metabolic tags, and improvement of signal to noise ratio through machine learning algorithms.

Inhibitory effect of membrane­free stem cell components derived from adipose tissues on skin inflammation in keratinocytes.

Inflammatory disorders of the skin are major public health concerns due to constant exposure to external stimuli. Skin cells are associated with prominent immune mechanisms to defend against adverse reactions. In the present study, the anti­inflammatory properties of membrane­free stem cell components (MFSCC) from adipose tissue­derived stem cells (ADSCs) and their basic preventive effects on skin wrinkle formation using human keratinocytes (HaCaT) and fibroblast (Detroit 551) cells, were investigated. Initially, a human inflammation antibody array was used on tumor necrosis factor­α (TNF­α)/interferon­Î³ (IFN­Î³)­induced and MFSCC­treated HaCaT cells. Array spots revealed three differential proteins, interleukin (IL)­1 F1 (IL­1α), IL­6, and TIMP2. Of these three proteins, IL­6 was significantly downregulated by MFSCC treatment. Western blot analysis revealed that IL­6 and its key downstream proteins JAK2 and STAT3 were suppressed in MFSCC­treated HaCaT cells. Further analysis revealed that MFSCC decreased the expression of TNF­α/IFN­Î³­induced phosphorylated (p)­IκB­α, p­p65, p­JNK, p­ERK, and p­p38 by inhibiting the activation of MAPK and NF­κB pathways. Treatment of Detroit 551 cells with MFSCC increased COL1A1 and elastin but suppressed matrix metalloproteinase (MMP)­1 and MMP­8 protein expression levels. Collectively, these data indicated that MFSCC exhibited a primary inhibitory effect on inflammation and wrinkle formation in skin. These results provide a basis for further extensive studies and application of MFSCC in treating skin inflammatory disorders.

Identification of Growth Factors, Cytokines and Mediators Regulated by Artemisia annua L. Polyphenols (pKAL) in HCT116 Colorectal Cancer Cells: TGF-ß1 and NGF-ß Attenuate pKAL-Induced Anticancer Effects via NF-κB p65 Upregulation.

The anticancer effects of natural phytochemicals are relevant to the modulation of cytokine signaling pathways in various cancer cells with stem-like properties as well as immune cells. The aim of this study was to elucidate a novel anticancer mechanism of Artemisia annua L. polyphenols (pKAL) involved in the regulation of growth factors, cytokines and mediators in stem-like HCT116 colorectal cancer cells. Through RayBiotech human L-1000 antibody array and bioinformatics analysis, we show here that pKAL-induced anticancer effects are associated with downregulation of growth factor and cytokine signaling proteins including TGFA, FGF16, PDGFC, CCL28, CXCR3, IRF6 and SMAD1. Notably, we found that TGF-ß signaling proteins such as GDF10, ENG and TGFBR2 and well-known survival proteins such as NGF-ß, VEGFD and insulin were significantly upregulated by pKAL. Moreover, the results of hematoxylin staining, cell viability assay and Western blot analysis demonstrated that TGF-ß1 and NGF-ß attenuated pKAL-induced anticancer effects by inhibiting pKAL-induced downregulation of caspase-8, NF-κB p65 and cyclin D1. These results suggest that certain survival mediators may be activated by pKAL through the TGF-ß1 and NGF-ß signaling pathways during pKAL-induced cell death and thus, strategies to inhibit the survival signaling are inevitably required for more effective anticancer effects of pKAL.

The effect of short-term intensive insulin therapy on inflammatory cytokines in patients with newly diagnosed type 2 diabetes.

BACKGROUND: Diabetes mellitus was a chronic low-grade inflammatory disease and had increased circulating inflammatory cytokines and acute phase proteins. We aimed to identify the changes of inflammatory cytokines in newly diagnosed type 2 diabetic patients after short-term intensive insulin therapy using continuous subcutaneous insulin infusion (CSII). METHODS: Thirty-three newly diagnosed type 2 diabetic patients were enrolled between September 2020 to December 2020. Expression of 40 inflammatory cytokines of the patients were tested with RayBiotech antibody array before and after 1 week of intensive insulin therapy of CSII. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out to explore the signaling pathway involved in the therapy. RESULTS: Five inflammatory cytokines were downregulated significantly after 1 week of CSII therapy. They were interleukin-6 receptor (IL-6R), regulated upon activation normal T-cell expressed and secreted (RANTES), intercellular adhesion molecule-1 (ICAM-1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and platelet-derived growth factor type BB (PDGF-BB) (p < 0.05 and foldchange <0.83). Among patients with baseline glycated hemoglobin (HbA1c) < 10%, three proinflammatory cytokines were decreased significantly after therapy: IL-6R, RANTES, and ICAM-1. As for the patients with baseline HbA1c ≥ 10%, eight inflammatory cytokines were inhibited significantly after the treatment, including ICAM-1, IL-6R, RANTES, TIMP-1, TIMP-2, macrophage inflammatory protein-1 beta (MIP-1ß), PDGF-BB, and tumor necrosis factor receptor type II (TNF RII). No matter which subgroup of baseline HbA1c level was considered, the decreased cytokines after CSII therapy were significantly involved in TNF signaling pathway. Nuclear factor-kappa B (NF-κB) signaling pathway was mainly enriched in patients with baseline HbA1c ≥ 10%. CONCLUSIONS: A panel of 40 inflammatory cytokines, measured by protein microarray, were evaluated for 1 week of CSII treatment in newly diagnosed type 2 diabetic patients. After treatment, many proinflammatory cytokines decreased. In the higher baseline HbA1c subgroup, more proinflammatory cytokines improved. No matter which subgroup of HbA1c level was considered, IL-6R, RANTES, and ICAM-1, which were involved in TNF signaling pathway, decreased significantly after CSII therapy. This was the first report showing that the cytokines of IL-6R, TIMP-2, PDGF-BB, and TNF RII decreased after the CSII therapy.

Monoclonal antibodies capable of binding SARS-CoV-2 spike protein receptor-binding motif specifically prevent GM-CSF induction.

A severe acute respiratory syndrome (SARS)-like coronavirus 2 (SARS-CoV-2) has recently caused a pandemic COVID-19 disease that infected approximately 94 million and killed more than 2,000,000 people worldwide. Like the SARS-CoV, SARS-CoV-2 also employs a receptor-binding motif (RBM) of its envelope spike protein for binding the host angiotensin-converting enzyme 2 (ACE2) to gain viral entry. Currently, extensive efforts are being made to produce vaccines against a surface fragment of a SARS-CoV-2, such as the spike protein, in order to boost protective antibodies that can inhibit virus-ACE2 interaction to prevent viral entry. It was previously unknown how spike protein-targeting antibodies would affect innate inflammatory responses to SARS-CoV-2 infections. Here we generated a highly purified recombinant protein corresponding to the RBM of SARS-CoV-2, and used it to screen for cross-reactive monoclonal antibodies (mAbs). We found two RBM-binding mAbs that competitively inhibited its interaction with human ACE2, and specifically blocked the RBM-induced GM-CSF secretion in both human peripheral blood mononuclear cells and murine macrophage cultures. Our findings have suggested a possible strategy to prevent SARS-CoV-2-elicited "cytokine storm," and revealed a potentially anti-inflammatory and protective mechanism for SARS-CoV-2 spike-based vaccines.