Clinical utility of serial analysis of circulating tumour cells for detection of minimal residual disease of metastatic nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an important cancer in Hong Kong. We aim to utilise liquid biopsies for serial monitoring of disseminated NPC in patients to compare with PET-CT imaging in detection of minimal residual disease. METHOD: Prospective serial monitoring of liquid biopsies was performed for 21 metastatic patients. Circulating tumour cell (CTC) enrichment and characterisation was performed using a sized-based microfluidics CTC chip, enumerating by immunofluorescence staining, and using target-capture sequencing to determine blood mutation load. PET-CT scans were used to monitor NPC patients throughout their treatment according to EORTC guidelines. RESULTS: The longitudinal molecular analysis of CTCs by enumeration or NGS mutational profiling findings provide supplementary information to the plasma EBV assay for disease progression for good responders. Strikingly, post-treatment CTC findings detected positive findings in 75% (6/8) of metastatic NPC patients showing complete response by imaging, thereby demonstrating more sensitive CTC detection of minimal residual disease. Positive baseline, post-treatment CTC, and longitudinal change of CTCs significantly associated with poorer progression-free survival by the Kaplan-Meier analysis. CONCLUSIONS: We show the potential usefulness of application of serial analysis in metastatic NPC of liquid biopsy CTCs, as a novel more sensitive biomarker for minimal residual disease, when compared with imaging.

Mechanical regulation of breast cancer migration and apoptosis via direct and indirect osteocyte signaling.

Bone metastases, the migration of cancers to bone, occur in 65-80% of patients with advanced breast cancer. Metastasized cancer cells interact with cells such as the bone-resorbing osteoclasts to alter bone remodeling. Exercise, often suggested as an intervention for cancer patients, regulates bone remodeling via osteocytes. Osteocytes also signal to endothelial cells, which may affect cancer cell extravasation. Therefore, we hypothesize that mechanically stimulated osteocytes can regulate processes in breast cancer bone metastasis. To test this, we exposed osteocytes to oscillatory fluid flow in vitro using parallel-plate flow chambers. We observed that conditioned medium from flow-stimulated osteocytes increased migration (by 45%) and reduced apoptosis (by 12%) of breast cancer cells. Conditioned medium from osteoclasts conditioned in flowed osteocytes' conditioned medium reduced migration (by 47%) and increased apoptosis (by 55%) of cancer cells. Cancer cell trans-endothelial migration was reduced by 34% toward flowed osteocytes' conditioned medium. This difference was abolished with ICAM-1 or IL-6 neutralizing antibodies. Conditioned medium from endothelial cells conditioned in flowed osteocytes' conditioned medium increased cancer cell apoptosis by 29%. To summarize, this study demonstrated mechanically stimulated osteocytes' potential to affect breast cancer cells not only through direct signaling, but also through osteoclasts and endothelial cells. The anti-metastatic potential of the indirect signalings is particularly exciting since osteocytes are further away from metastasizing cancer cells than osteoclasts and endothelial cells. Future studies into the effect of bone mechanical loading on metastases and its mechanism will assist in designing cancer intervention programs that lowers the risk for bone metastases.

Polysaccharide of Danggui Buxue Tang, an Ancient Chinese Herbal Decoction, Induces Expression of Pro-inflammatory Cytokines Possibly Via Activation of NFκB Signaling in Cultured RAW 264.7 Cells.

Danggui Buxue Tang (DBT) is an ancient Chinese herbal decoction containing two herbs, Astragali Radix (AR) and Angelicae Sinensis Radix (ASR): this herbal decoction serves as dietary supplement for women during menopause. DBT has been known to modulate immune responses, and its polysaccharide is proposed to be one of the active components. However, the polysaccharide-induced signaling in immune activation is not revealed. Here, we are identifying that the immune activation, triggered by DBT, could be mediated by polysaccharide. In cultured macrophages (RAW 264.7 cells), the application of polysaccharide-enriched extract of DBT significantly increased the expressions of mRNA and protein levels of interleukin-1ß, interleukin-6 and tumor necrosis factor. The induction was much stronger than the polysaccharide extract generated singly from AR, or from ASR, or from their simple mixture. The induced cytokine release in cultured macrophage was revealed to be triggered by activation of nuclear factor-kappa B (NF-κB) signaling, including (i) degradation of IkBα; (ii) translocation of NF-κB p65 from cytosol to nuclei; and (iii) activation of NF-κB transcriptional elements. These results verified the possible role of DBT polysaccharide in modulating immune responses. Copyright © 2016 John Wiley & Sons, Ltd.

Extract of Ziziphus jujuba Fruit (Jujube) Stimulates Expression of Enzymes Responsible for Heme Recycle via Anti-oxidant Response Element in Cultured Murine Macrophages.

Jujube, the fruit of Ziziphus jujuba Mill., is a functional food and commonly used as a health supplement worldwide. To study the beneficial role of jujube in heme iron recycling during erythrophagocytosis, the expression of heme oxygenase-1 (HO-1), biliverdin reductase A and B, and ferroportin were determined in jujube-treated cultured RAW 264.7 macrophages. Application of a chemically standardized jujube water extract in cultured RAW 264.7 cells for 24 h stimulated the expressions of HO-1, biliverdin reductase A, biliverdin reductase B, and ferroportin in a concentration-dependent manner, having the maximal responses from twofolds to threefolds. A plasmid containing anti-oxidant response element, a regulator for HO-1 transcription, was transfected into RAW 264.7 cells. Application of jujube water extract onto the transfected macrophages stimulated the anti-oxidant response element-mediated transcriptional activity by twofolds. These results supported the potential capacity of jujube by regulating expressions of heme iron recycling genes in cultured macrophages.

The extract of Ziziphus jujuba fruit (jujube) induces expression of erythropoietin via hypoxia-inducible factor-1α in cultured Hep3B cells.

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as health supplement or herbal medicine worldwide. To study the beneficial role of jujube in enhancing hematopoietic function, we investigated its roles on the expression of erythropoietin in cultured Hep3B human hepatocellular carcinoma cells. Application of chemically standardized jujube water extract stimulated erythropoietin expression in a dose-dependent manner, with the highest response by ~ 100 % of increase. A plasmid containing hypoxia response element, a critical regulator for erythropoietin transcription, was transfected into Hep3B cells. Application of jujube water extract onto the transfected cells induced the transcriptional activity of the hypoxia response element. To account for its transcriptional activation, the expression of hypoxia-inducible factor-1α was increased after treatment with jujube water extract: the increase was in both mRNA and protein levels. These results confirmed the hematopoietic function of jujube in the regulation of erythropoietin expression in liver cells.

The standardized extract of Ziziphus jujuba fruit (jujube) regulates pro-inflammatory cytokine expression in cultured murine macrophages: suppression of lipopolysaccharide-stimulated NF-κB activity.

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as a health supplement or herbal medicine worldwide. To study the beneficial role of jujube in regulating immune response, we investigated its roles on the expressions of pro-inflammatory cytokines in cultured macrophages. Application of chemically standardized jujube water extract for 24 h stimulated the transcriptional expression of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in cultured RAW 264.7 macrophages. In contrast, the pretreatment with jujube water extract suppressed the expression of IL-1ß and IL-6, but not for TNF-α in lipopolysaccharide (LPS)-stimulated macrophages. The IL-1ß and IL-6 cytokines in LPS-induced macrophages were suppressed by jujube water extract in both mRNA and protein levels. In parallel, the inhibition of jujube water extract on the transcriptional activity of nuclear factor-kappa B was revealed in LPS-induced macrophages. These results verified the bidirectional immune-modulatory roles of jujube by regulating the expressions of pro-inflammatory cytokines in macrophages.

A chemically standardized extract of Ziziphus jujuba fruit (Jujube) stimulates expressions of neurotrophic factors and anti-oxidant enzymes in cultured astrocytes.

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as a health supplement worldwide. To study the role of jujube in brain benefits, the expression of neurotrophic factors and anti-oxidant enzymes in the jujube-treated cultured astrocytes was determined. Application of a chemical standardized water extract of jujube in cultured astrocytes for 24 h stimulated the expressions of nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in a concentration-dependent manner. The pre-treatment with H89, a protein kinase A inhibitor, attenuated the jujube-induced expression of neurotrophic factors. In parallel, the treatment of jujube water extract induced the transcriptional expressions of the enzymes responsible for anti-oxidation, i.e. NAD(P)H: quinine oxidoreductase 1, glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit and glutathione S-transferase, in a concentration-dependent manner. These results proposed the benefits of jujube in regulating expressions of neurotrophic factors and anti-oxidant enzymes in cultured astrocytes.

Using Machine Learning Algorithms to Pool Data from Meta-Analysis for the Prediction of Countermovement Jump Improvement.

To solve the research-practice gap and take one step forward toward using big data with real-world evidence, the present study aims to adopt a novel method using machine learning to pool findings from meta-analyses and predict the change of countermovement jump. The data were collected through a total of 124 individual studies included in 16 recent meta-analyses. The performance of four selected machine learning algorithms including support vector machine, random forest (RF) ensemble, light gradient boosted machine, and the neural network using multi-layer perceptron was compared. The RF yielded the highest accuracy (mean absolute error: 0.071 cm; R2: 0.985). Based on the feature importance calculated by the RF regressor, the baseline CMJ ("Pre-CMJ") was the most impactful predictor, followed by age ("Age"), the total number of training sessions received ("Total number of training_session"), controlled or non-controlled conditions ("Control (no training)"), whether the training program included squat, lunge, deadlift, or hip thrust exercises ("Squat_Lunge_Deadlift_Hipthrust_True", "Squat_Lunge_Deadlift_Hipthrust_False"), or "Plyometric (mixed fast/slow SSC)", and whether the athlete was from an Asian pacific region including Australia ("Race_Asian or Australian"). By using multiple simulated virtual cases, the successful predictions of the CMJ improvement are shown, whereas the perceived benefits and limitations of using machine learning in a meta-analysis are discussed.

Circulating Tumor Cell Enumeration for Serial Monitoring of Treatment Outcomes for Locally Advanced Esophageal Squamous Cell Carcinoma.

We aim to reveal the clinical significance and potential usefulness of dynamic monitoring of CTCs to track therapeutic responses and improve survival for advanced ESCC patients. Peripheral blood (PB) (n = 389) and azygos vein blood (AVB) (n = 13) samplings were recruited prospectively from 88 ESCC patients undergoing curative surgery from 2017 to 2022. Longitudinal CTC enumeration was performed with epithelial (EpCAM/pan-cytokeratins/MUC1) and mesenchymal (vimentin) markers at 12 serial timepoints at any of the pre-treatment, all of the post-treatments/pre-surgery, post-surgery follow-ups for 3-year, and relapse. Longitudinal real-time CTC analysis in PB and AVB suggests more CTCs are released early at pre-surgery and 3-month post-surgery into the circulation from the CTRT group compared to the up-front surgery group. High CTC levels at pre-treatments, 1-/3-month post-surgery, unfavorable changes of CTC levels between all post-treatment/pre-surgery and 1-month or 3-month post-surgery (Hazard Ratio (HR) = 6.662, p < 0.001), were independent prognosticators for curative treatment. The unfavorable pre-surgery CTC status was independent prognostic and predictive for neoadjuvant treatment efficacy (HR = 3.652, p = 0.035). The aggressive CTC clusters were more frequently observed in AVB compared to PB. Its role as an independent prognosticator with relapse was first reported in ESCC (HR = 2.539, p = 0.068). CTC clusters and longitudinal CTC monitoring provide useful prognostic information and potential predictive biomarkers to help guide clinicians in improving disease management.

Asarone from Acori Tatarinowii Rhizoma Potentiates the Nerve Growth Factor-Induced Neuronal Differentiation in Cultured PC12 Cells: A Signaling Mediated by Protein Kinase A.

Acori Tatarinowii Rhizoma (ATR), the rhizome of Acorus tatarinowii Schott, is being used clinically to treat neurological disorders. The volatile oil of ATR is being considered as an active ingredient. Here, α-asarone and ß-asarone, accounting about 95% of ATR oil, were evaluated for its function in stimulating neurogenesis. In cultured PC12 cells, application of ATR volatile oil, α-asarone or ß-asarone, stimulated the expression of neurofilaments, a bio-marker for neurite outgrowth, in a concentration-dependent manner. The co-treatment of ATR volatile oil, α-asarone or ß-asarone, with low concentration of nerve growth factor (NGF) potentiated the NGF-induced neuronal differentiation in cultured PC12 cells. In addition, application of protein kinase A inhibitors, H89 and KT5720, in cultures blocked the ATR-induced neurofilament expression, as well as the phosphorylation of cAMP-responsive element binding protein (CREB). In the potentiation of NGF-induced signaling in cultured PC12 cells, α-asarone and ß-asarone showed synergistic effects. These results proposed the neurite-promoting asarone, or ATR volatile oil, could be useful in finding potential drugs for treating various neurodegenerative diseases, in which neurotrophin deficiency is normally involved.

Jujube-containing herbal decoctions induce neuronal differentiation and the expression of anti-oxidant enzymes in cultured PC12 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Ziziphus jujuba (Mill.), known as Jujuba Fructus (JF) or jujube, is a well-known Traditional Chinese Medicine (TCM) for blood nourishment and sedation effect. Apart from prescribing as single herb alone, JF is very often being included in multi-herbal decoctions to prolong, enhance and harmonize pharmaceutical effects of decoctions while at the same time reducing toxicity. Here, we aimed to compare the protective and differentiating activities of three chemically standardized jujube-containing decoctions, including Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and ZaoTang (ZOT) in cultured PC12 cells. MATERIALS AND METHODS: The protein expressions of neurofilaments, including NF68, NF160 and NF200, under the herbal treatment were revealed by western blot. The determination of neurite outgrowth in cultured PC12 cells upon the treatment of herbal extracts was performed by light microscope equipped with a phase-contrast condenser and SPOT imaging software. The protective effect against tBHP-induced cytotoxicity under the herbal treatment was measured by MTT assay. A luciferase reporter construct carrying four repeats of anti-oxidant response element (ARE) and a downstream luciferase reporter gene luc2P was transfected into PC12 cells to study the transcriptional activation of ARE. The mRNA expression of antioxidant enzymes under the herbal treatment was analyzed by quantitative real-time PCR. RESULTS: These jujube-containing decoctions processed similar neuro-protective and brain beneficial properties. The herbal treatment induced the protein expression of neurofilaments. Neurite outgrowth was observed under the herbal treatment. In parallel, the pre-treatment of herbal extracts protected PC 12 cells against oxidative stress-induced apoptosis in a dose-dependent manner. Moreover, the herbal treatments triggered the mRNA expressions of relevant anti-oxidation genes, i.e. glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modulatory subunit (GCLM), glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase (NQO1) via the activation of anti-oxidant response element (ARE). CONCLUSION: The results therefore demonstrated neuro-protective and differentiating properties of the three closely related decoctions, and which subsequently illustrated the enhancement function of jujube within a multi-herbal decoction.

Chemical and biological assessment of Jujube (Ziziphus jujuba)-containing herbal decoctions: Induction of erythropoietin expression in cultures.

Jujubae Fructus, known as jujube or Chinese date, is the fruit of Ziziphus jujuba (Mill.), which not only serves as daily food, but acts as tonic medicine and health supplement for blood nourishment and sedation. According to Chinese medicine, jujube is commonly included in herbal mixtures, as to prolong, enhance and harmonize the efficiency of herbal decoction, as well as to minimize the toxicity. Here, we aim to compare the chemical and pharmacological properties of three commonly used jujube-containing decoctions, including Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and Zao Tang (ZOT). These decoctions share common herbs, i.e. Glycyrrhizae Radix et Rhizoma Praeparata cum Melle, Zingiberis Rhizoma Recens and Jujube, and they have the same proposed hematopoietic functions. The amount of twelve marker biomolecules deriving from different herbs in the decoctions were determined by LC-MS, and which served as parameters for chemical standardization. In general, three decoctions showed common chemical profiles but with variations in solubilities of known active ingredients. The chemical standardized decoctions were tested in cultured Hep3B cells. The herbal treatment stimulated the amount of mRNA and protein expressions of erythropoietin (EPO) via the activation of hypoxia response elements: the three herbal decoctions showed different activation. The results therefore demonstrated the hematopoietic function of decoctions and explained the enhancement of jujube function within a herbal mixture.

Transcriptional activity of acetylcholinesterase gene is regulated by DNA methylation during C2C12 myogenesis.

The expression of acetylcholinesterase (AChE), an enzyme hydrolyzes neurotransmitter acetylcholine at vertebrate neuromuscular junction, is regulated during myogenesis, indicating the significance of muscle intrinsic factors in controlling the enzyme expression. DNA methylation is essential for temporal control of myogenic gene expression during myogenesis; however, its role in AChE regulation is not known. The promoter of vertebrate ACHE gene carries highly conserved CG-rich regions, implying its likeliness to be methylated for epigenetic regulation. A DNA methyltransferase inhibitor, 5-azacytidine (5-Aza), was applied onto C2C12 cells throughout the myotube formation. When DNA methylation was inhibited, the promoter activity, transcript expression and enzymatic activity of AChE were markedly increased after day 3 of differentiation, which indicated the putative role of DNA methylation. By bisulfite pyrosequencing, the overall methylation rate was found to peak at day 3 during C2C12 cell differentiation; a SP1 site located at -1826bp upstream of mouse ACHE gene was revealed to be heavily methylated. The involvement of transcriptional factor SP1 in epigenetic regulation of AChE was illustrated here: (i) the SP1-driven transcriptional activity was increased in 5-Aza-treated C2C12 culture; (ii) the binding of SP1 onto the SP1 site of ACHE gene was fully blocked by the DNA methylation; and (iii) the sequence flanking SP1 sites of ACHE gene was precipitated by chromatin immuno-precipitation assay. The findings suggested the role of DNA methylation on AChE transcriptional regulation and provided insight in elucidating the DNA methylation-mediated regulatory mechanism on AChE expression during muscle differentiation.

Fruit of Ziziphus jujuba (Jujube) at two stages of maturity: distinction by metabolic profiling and biological assessment.

The fruit of Ziziphus jujuba, named as jujube or Chinese date, is used as a health supplement worldwide. Two kinds of jujubes are commonly found in the market: immature jujubes eaten as fruits, and mature jujubes employed as medicinal herbs. To study the variation of jujubes at two developmental stages, we investigated their chemical and biological properties by metabolic profiling and cellular assays. In NMR profiling, the levels of 11 metabolites were measured. Statistically differences in the levels of threonine, alanine, acetate, creatine, glucose, sucrose, and formate were found between mature and immature jujubes. In parallel, their neuro-protecting and erythropoietic activities were compared. The water extract of mature jujube possessed better effect in inducing neurofilament expression than that of the immature one, while immature jujube extract performed better in activating HRE-mediated transcriptional activity. These findings suggest the maturity of jujube has to be considered when it is being used for health food products.

Calycosin orchestrates the functions of Danggui Buxue Tang, a Chinese herbal decoction composing of Astragali Radix and Angelica Sinensis Radix: An evaluation by using calycosin-knock out herbal extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Danggui Buxue Tang (DBT) is a classical Chinese herbal decoction containing two herbs, Astragali Radix (AR) and Angelicae Sinensis Radix (ASR), which serves as dietary supplement for treating women menopausal syndromes. Pharmacological studies indicate that DBT has estrogenic, erythropoietic and osteogenic properties; however, the action mechanism for this complex herbal decoction is not known. Calycosin, a major flavonoid in AR, shares similar structure with ß-estradiol, and thus which is hypothesized to be the critical compound of DBT. Here, we aim to investigate the role of calycosin in DBT in terms of its biological functions by using a calycosin-depleted DBT decoction (DBT(Δcal)). The biological functions of DBT(Δcal) and parental DBT were systematically compared. MATERIALS AND METHODS: In order to standardize DBT decoction, four chemical markers were determined and quantified by HPLC. A semi-preparative HPLC method was utilized to prepare DBT(Δcal). The authenticity of DBT(Δcal) was evaluated by LC-QQQ-MS/MS. To reveal the effect of calycosin on DBT functions, several cell assays related to the known properties of DBT were revealed, including estrogenic, erythropoietic and osteogenic functions. RESULTS: As compared to parental DBT, the estrogenic, erythropoietic and osteogenic abilities were markedly reduced in DBT(Δcal). However, calycosin alone did not show significant responses. CONCLUSIONS: Our results suggest that calycosin is a bioactive chemical in DBT decoction, and which could play a key linker in orchestrating multi-components of DBT as to achieve maximal functions. These discoveries should be invaluable in drug development and in investigating the modernization of traditional Chinese medicine from a new perspective.

A standardized extract of the fruit of Ziziphus jujuba (Jujube) induces neuronal differentiation of cultured PC12 cells: a signaling mediated by protein kinase A.

The fruit of Ziziphus jujuba Mill., known as Chinese date or jujube, is consumed as a health supplement worldwide. To study the role of jujube in brain benefits, its effects on neuronal differentiation of PC12 cells were studied. Application of jujube water extract induced neurite outgrowth of PC12 cells, >25% of which were differentiated; this effect was similar to that of nerve growth factor. In parallel, the expressions of neurofilaments (NFs) in jujube-treated cultures showed a dose-dependent increase, with the highest inductions by ∼150% for NF68 and NF160 and by ∼100% for NF200. Application of H89, a protein kinase A inhibitor, attenuated jujube-induced neurite outgrowth of the cultures. Besides, using jujube extract induced the phosphorylation of cAMP responsive element binding protein on PC12 cells, which was blocked by H89. These results support the use of jujube as a food supplement for the prevention of neurodegenerative diseases in which neurotrophin deficiency is involved.

Chemical and biological assessment of Ziziphus jujuba fruits from China: different geographical sources and developmental stages.

Chinese date, the fruit of Ziziphus jujuba Mill., has thousands of years cultivation history, and about 700 cultivars of dates in China. Two types of dates are commonly found in the market: (i) fresh immature dates consumed as fruits, and (ii) dried mature dates used as Chinese medicines. Here, chemical and biological properties of these dates were revealed. Different sources of dates showed similar chemical profiles; however, the amounts of identified chemicals showed a great variation. The amount of nucleotides, flavonoids and polysaccharides in dates could be affected by its maturity and drying process. In parallel, the antioxidative functions of their extracts were compared. The date extracts protected PC12 cells against tBHP-induced cytotoxicity, and which also stimulated the transcriptional activity of antioxidant response element. The antioxidative effects were varied among different dates. The current results suggested the optimization of sources and specific usage of different maturity dates.