The Hippo-YAP signaling pathway drives CD24-mediated immune evasion in esophageal squamous cell carcinoma via macrophage phagocytosis.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies in the world with poor prognosis. Despite the promising applications of immunotherapy, the objective response rate is still unsatisfactory. We have previously shown that Hippo/YAP signaling acts as a powerful tumor promoter in ESCC. However, whether Hippo/YAP signaling is involved in tumor immune escape in ESCC remains largely unknown. Here, we show that YAP directly activates transcription of the "don't eat me" signal CD24, and plays a crucial role in driving tumor cells to avoid phagocytosis by macrophages. Mechanistically, YAP regulates CD24 expression by interacting with TEAD and binding the CD24 promoter to initiate transcription, which facilitates tumor cell escape from macrophage-mediated immune attack. Our animal model data and clinical data show that YAP combined with CD24 in tumor microenvironment redefines the impact of TAMs on the prognosis of ESCC patients which will provide a valuable basis for precision medicine. Moreover, treatment with YAP inhibitor altered the distribution of macrophages and suppressed tumorigenesis and progression of ESCC in vivo. Together, our study provides a novel link between Hippo/YAP signaling and macrophage-mediated immune escape, which suggests that the Hippo-YAP-CD24 axis may act as a promising target to improve the prognosis of ESCC patients. A proposed model for the regulatory mechanism of Hippo-YAP-CD24-signaling axis in the tumor-associated macrophages mediated immune escape.

Ferroptosis as a potential target for cancer therapy.

Ferroptosis is a recently discovered essential type of cell death that is mainly characterized by iron overload and lipid peroxidation. Emerging evidence suggests that ferroptosis is a double-edged sword in human cancer. However, the precise underlying molecular mechanisms and their differential roles in tumorigenesis are unclear. Therefore, in this review, we summarize and briefly present the key pathways of ferroptosis, paying special attention to the regulation of ferroptosis as well as its dual role as an oncogenic and as a tumor suppressor event in various human cancers. Moreover, multiple pharmacological ferroptosis activators are summarized, and the prospect of targeting ferroptosis in cancer therapy is further elucidated.

mRNA transcriptome profiling of human hepatocellular carcinoma cells HepG2 treated with Catharanthus roseus-silver nanoparticles.

BACKGROUND: The demand for the development of cancer nanomedicine has increased due to its great therapeutic value that can overcome the limitations of conventional cancer therapy. However, the presence of various bioactive compounds in crude plant extracts used for the synthesis of silver nanoparticles (AgNPs) makes its precise mechanisms of action unclear. AIM: To assessed the mRNA transcriptome profiling of human HepG2 cells exposed to Catharanthus roseus G. Don (C. roseus)-AgNPs. METHODS: The proliferative activity of hepatocellular carcinoma (HepG2) and normal human liver (THLE3) cells treated with C. roseusAgNPs were measured using MTT assay. The RNA samples were extracted and sequenced using BGIseq500 platform. This is followed by data filtering, mapping, gene expression analysis, differentially expression genes analysis, Gene Ontology analysis, and pathway analysis. RESULTS: The mean IC50 values of C. roseusAgNPs on HepG2 was 4.38 ± 1.59 µg/mL while on THLE3 cells was 800 ± 1.55 µg/mL. Transcriptome profiling revealed an alteration of 296 genes. C. roseusAgNPs induced the expression of stress-associated genes such as MT, HSP and HMOX-1. Cellular signalling pathways were potentially activated through MAPK, TNF and TGF pathways that are responsible for apoptosis and cell cycle arrest. The alteration of ARF6, EHD2, FGFR3, RhoA, EEA1, VPS28, VPS25, and TSG101 indicated the uptake of C. roseus-AgNPs via both clathrin-dependent and clathrin-independent endocytosis. CONCLUSION: This study provides new insights into gene expression study of biosynthesised AgNPs on cancer cells. The cytotoxicity effect is mediated by the aberrant gene alteration, and more interestingly the unique selective antiproliferative properties indicate the C. roseusAgNPs as an ideal anticancer candidate.

Combination of Goniothalamin and Sol-Gel-Derived Bioactive Glass 45S5 Enhances Growth Inhibitory Activity via Apoptosis Induction and Cell Cycle Arrest in Breast Cancer Cells MCF-7.

Background: Combination of natural products with chemically synthesised biomaterials as cancer therapy has attracted great interest lately. Hence, this study is aimed at investigating the combined effects of goniothalamin and bioactive glass 45S5 (GTN-BG) and evaluating their anticancer properties on human breast cancer cells MCF-7. Methods: The BG 45S5 was prepared using the sol-gel process followed by characterisation using PSA, BET, SEM/EDS, XRD, and FTIR. The effects of GTN-BG on the proliferation of MCF-7 were assessed by MTT, PrestoBlue, and scratch wound assays. The cell cycle analysis, Annexin-FITC assay, and activation of caspase-3/7, caspase-8, and caspase-9 assays were determined to further explore its mechanism of action. Results: The synthesised BG 45S5 was classified as a fine powder, having a rough surface, and contains mesopores of 12.6 nm. EDS analysis revealed that silica and calcium elements are the primary components of BG powders. Both crystalline and amorphous structures were detected with 73% and 27% similarity to Na2Ca2(Si2O7) and hydroxyapatite, respectively. The combination of GTN-BG was more potent than GTN in inhibiting the proliferation of MCF-7 cells. G0/G1 and G2/M phases of the cell cycle were arrested by GTN and GTN-BG. The percentage of viable cells in GTN-BG treatment was significantly lower than that in GTN. In terms of activation of initiator caspases for both extrinsic and intrinsic apoptosis pathways, caspase-8 and caspase-9 were found more effective in response to GTN-BG than GTN. Conclusion: The anticancer effect of GTN in MCF-7 cells was improved when combined with BG. The findings provide significant insight into the mechanism of GTN-BG against MCF-7 cells, which can potentially be used as a novel anticancer therapeutic approach.

Potential Antioxidant and Anti-Inflammatory Effects of Spilanthes acmella and Its Health Beneficial Effects: A Review.

Oxidative stress and inflammation are two common risk factors of various life-threatening disease pathogenesis. In recent years, medicinal plants that possess antioxidant and anti-inflammatory activities were extensively studied for their potential role in treating and preventing diseases. Spilanthes acmella (S. acmella), which has been traditionally used to treat toothache in Malaysia, contains various active metabolites responsible for its anti-inflammatory, antiseptic, and anesthetic bioactivities. These bioactivities were attributed to bioactive compounds, such as phenolic, flavonoids, and alkamides. The review focused on the summarization of in vitro and in vivo experimental reports on the antioxidant and anti-inflammatory actions of S. acmella, as well as how they contributed to potential health benefits in lowering the risk of diseases that were related to oxidative stress. The molecular mechanism of S. acmella in reducing oxidative stress and inflammatory targets, such as inducible nitric oxide synthase (iNOS), transcription factors of the nuclear factor-κB family (NF-κB), cyclooxygenase-2 (COX-2), and mitogen-activated protein kinase (MAPK) signaling pathways were discussed. Besides, the antioxidant potential of S. acmella was measured by total phenolic content (TPC), total flavonid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and superoxide anion radical scavenging (SOD) and thiobarbituric acid reactive substance (TBARS) assays. This review revealed that S. acmella might have a potential role as a reservoir of bioactive agents contributing to the observed antioxidant, anti-inflammatory, and health beneficial effects.

Phytochemical Analysis, Antioxidant and Bone Anabolic Effects of Blainvillea acmella (L.) Philipson.

Blainvillea acmella (L.) Philipson [Asteraceae] (B. acmella) is an important medicinal plant native to Brazil, and it is widely known as a toothache plant. A plethora of studies have demonstrated the antioxidant activities of B. acmella and few studies on the stimulatory effects on alkaline phosphatase (ALP) secretion from bone cells; however, there is no study on its antioxidant and anabolic activity on bone cells. The study aimed to evaluate the phytochemical contents of aqueous and ethanol extracts of B. acmella using gas chromatography mass spectrometry (GCMS) and liquid chromatography time of flight mass spectrometry (LCTOFMS) along with the total phenolic (TPC) and flavonoid (TFC) contents using Folin-Ciocalteu and aluminum colorimetric methods. The extracts of B. acmella leaves were used to scavenge synthetic-free radicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. The bone anabolic effects of B. acmella extracts on MC3T3-E1 cells were measured with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoium bromide (MTT) at 1, 3, 5, and 7 days, Sirius-red and ALP at 7 and 14 days, and Alizarin Red S at 14 and 21 days. Comparatively, ethanol extract of B. acmella (BaE) contributed higher antioxidant activities (IC50 of 476.71 µg/ml and 56.01 ± 6.46 mg L-ascorbic acid/g against DPPH and FRAP, respectively). Anabolic activities in bone proliferation, differentiation, and mineralization were also higher in B. acmella of ethanol (BaE) than aqueous (BaA) extracts. Positive correlations were observed between phenolic content (TPC and TFC) to antioxidant (ABTS and FRAP) and anabolic activities. Conversely, negative correlations were present between phenolic content to antioxidant (DPPH) activity. These potential antioxidant and bone anabolic activities in BaE might be due to the phytochemicals confirmed through GCMS and LCTOFMS, revealed that terpenoids of α-cubebene, cryophyllene, cryophyllene oxide, phytol and flavonoids of pinostrobin and apigenin were the compounds contributing to both antioxidant and anabolic effects in BaE. Thus, B. acmella may be a valuable antioxidant and anti-osteoporosis agent. Further study is needed to isolate, characterize and elucidate the underlying mechanisms responsible for the antioxidant and bone anabolic effects.

Optimization of biogenic synthesis of silver nanoparticles from flavonoid-rich Clinacanthus nutans leaf and stem aqueous extracts.

Background: Silver nanoparticles (AgNPs) are widely used in food industries, biomedical, dentistry, catalysis, diagnostic biological probes and sensors. The use of plant extract for AgNPs synthesis eliminates the process of maintaining cell culture and the process could be scaled up under a non-aseptic environment. The purpose of this study is to determine the classes of phytochemicals, to biosynthesize and characterize the AgNPs using Clinacanthus nutans leaf and stem extracts. In this study, AgNPs were synthesized from the aqueous extracts of C. nutans leaves and stems through a non-toxic, cost-effective and eco-friendly method. Results: The formation of AgNPs was confirmed by UV-Vis spectroscopy, and the size of AgNP-L (leaf) and AgNP-S (stem) were 114.7 and 129.9 nm, respectively. Transmission electron microscopy (TEM) analysis showed spherical nanoparticles with AgNP-L and AgNP-S ranging from 10 to 300 nm and 10 to 180 nm, with average of 101.18 and 75.38 nm, respectively. The zeta potentials of AgNP-L and AgNP-S were recorded at -42.8 and -43.9 mV. X-ray diffraction analysis matched the face-centred cubic structure of silver and was capped with bioactive compounds. Fourier transform infrared spectrophotometer analysis revealed the presence of few functional groups of phenolic and flavonoid compounds. These functional groups act as reducing agents in AgNPs synthesis. Conclusion: These results showed that the biogenically synthesized nanoparticles reduced silver ions to silver nanoparticles in aqueous condition and the AgNPs formed were stable and less toxic.

Regulation of Hippo/YAP signaling and Esophageal Squamous Carcinoma progression by an E3 ubiquitin ligase PARK2.

Objective: Esophageal squamous cell carcinoma (ESCC) is one of the most commonly diagnosed cancer types in China. Recent genomic sequencing analysis indicated the over-activation of Hippo/YAP signaling might play important roles for the carcinogenic process and progression for ESCC patients. However, little is known about the molecular mechanisms that controls Hippo signaling activity in ESCC. Our previous studies indicated that PLCE1-an important risk factor for ESCC-linked to ESCC progression through snail signaling, during this period, we found PARK2 was an important downstream target of PLCE1-snail axis. PARK2 was decreased in ESCC human samples, and correlated with good prognosis in ESCC patients. Further research showed that PARK2 could inhibit YAP, which functions as key downstream effectors of the Hippo pathway. Here, we aim to reveal the molecular mechanisms of PARK2 modulated Hippo pathway in ESCC. Methods: To evaluate the function of PARK2 in ESCC, we used a tissue microarray (TMA) of 223 human ESCC patients and immunohistochemistry to analyze the correlation between PARK2 expression and clinicopathologic variables. Depletion of endogenous PARK2 and YAP from ESCC cells using CRISPR/Cas9 technologies. Flow cytometry and EdU cell proliferation assay were used to detect proliferation of ESCC cells. Nude mice subcutaneous injection and Ki-67 staining were used to evaluate tumor growth in vivo. Migration and invasion assays were performed. In addition, lung metastasis models in mice were used to validate the function of PARK2 in vivo. Identification of PARK2 involved in hippo pathway was achieved by expression microarray screening, double immunofluorescence staining and co-immunoprecipitation assays. The RNA-seq analysis results were validated through quantitative real-time PCR (qRT-PCR) analysis. The protein half-life of YAP was analyzed by Cycloheximide assay, and the TEAD activity was detected by Luciferase reporter assays. Results: Clinical sample of ESCC revealed that low PARK2 expression correlated with late tumor stage (P < 0.001), poor differentiation (P < 0.04), lymph node (P < 0.001) and distant metastasis (P = 0.0087). Multivariate Cox proportional regression analysis further revealed that PARK2 expression (P = 0.032) is an independent prognostic factor for the overall survival of ESCC patients. Besides, the immunohistochemistry results showed that PARK2 negatively correlated with YAP protein level (P < 0.001). PARK2 depletion promotes ESCC progression both through Hippo/YAP axis, while PARK2 overexpression suppresses ESCC tumor progression by Hippo signaling. Co-IP and ubiquitination assays revealed that PARK2 could interact with YAP in the cytosol and promotes YAP K48-linked ubiquitination at K90 sites. Conclusion: Clinical sample analysis and mechanistic study have validated PARK2 as a tumor suppressor for ESCC. Multivariate Cox proportional regression analysis further revealed that PARK2 is an independent prognostic factor for the overall survival of ESCC patients. Cellular and molecular mechanisms in this study showed that PARK2 associated with YAP protein in the cytosol, promoted YAP ubiquitination and proteasome-dependent degradation in ESCC cells. Therefore, as a novel modulator for Hippo signaling, modulation of PARK2 activity or gene expression level could be an appealing strategy to treat esophageal.

Suppressing growth, migration, and invasion of human hepatocellular carcinoma HepG2 cells by Catharanthus roseus­silver nanoparticles.

Application of silver nanoparticles serves as a new approach in cancer treatment due to its unique features. Biosynthesis of silver nanoparticles using plant is advantageous since they are easily accessible, nontoxic and produce quicker reaction compared to other methods. To evaluate the cytotoxicity, mechanism of cell death and DNA damage of biosynthesized Catharanthus roseus-silver nanoparticles on human liver cancer (HepG2) cells. The antiproliferative activity of Catharanthus roseus­silver nanoparticles was measured using MTT assay. The cytotoxic effects were further evaluated by measuring nitric oxide and reactive oxygen species (ROS). The mechanism of cell death was determined by annexin-FITC/propidium iodide, mitochondrial membrane potential (MMP) and cell cycle assays. The assessment of DNA damage was evaluated using Comet assay method. The uptake of the nanoparticles were evaluated by Transmission Electron Microscopy (TEM). Catharanthus roseus­silver nanoparticles has inhibited the proliferation of HepG2 cells in a time-dependent manner with a median IC50 value of 3.871 ± 0.18 µg/mL. The concentration of nitrite and ROS were significantly higher than control. The cell death was due to apoptosis associated with MMP loss, cell cycle arrest, and extensive DNA damage. TEM analysis indicated the presence of free nanoparticles and endosomes containing the nanoparticles. The findings show that Catharanthus roseus­silver nanoparticles have produced cytotoxic effects on HepG2 cells and thus may have a potential to be used as an anticancer treatment, particularly for hepatocellular carcinoma.

Determination and Quantification of the Vinblastine Content in Purple, Red, and White Catharanthus Roseus Leaves Using RP-HPLC Method.

Purpose: To determine and quantify vinblastine in different varieties of Catharanthus roseus using reversed-phase HPLC method. Methods: The liquid chromatographic separation was performed using a reversed phase C18, Microsorb - MV column (250 mm x 4.6 mm, 5 µm) at room temperature and eluted with a mobile phase containing methanol - phosphate buffer (5 mM, pH 6.0) - acetonitrile with different proportion gradient elution at a flow rate of 2.0 mL min-1 and detection at 254 nm. Results: The HPLC method was utilized for the quantification of vinblastine in purple, red and white varieties of Catharanthus roseus leaves. The separation was achieved in less than 8 min. The peak confirmation was done based on the retention times and UV spectra of the reference substance. The method was validated with respect to linearity, precision, recovery, limit of detection and quantification. Results showed that the purple variety gives 1.2 and 1.5 times more vinblastine concentration compared to the white and pink varieties, respectively. Conclusion: The obtained results from different varieties are thus useful for the purpose of vinblastine production from Catharanthus roseus plant.

Catharanthus roseus Aqueous Extract is Cytotoxic to Jurkat Leukaemic T-cells but Induces the Proliferation of Normal Peripheral Blood Mononuclear Cells.

Research on natural products has been widely used as a strategy to discover new drugs with potential for applications in complementary medicines because they have fewer side effects than conventional drugs. The aim of the present study was to evaluate the in vitro cytotoxic effects of crude aqueous Catharanthus roseus extract on Jurkat cells and normal peripheral blood mononuclear cells (PBMCs). The aqueous extract was standardised to vinblastine by high performance liquid chromatography (HPLC) and was used to determine cytotoxicity by the MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. DNA fragmentation assay was employed to determine if cell death was due to apoptosis. The results showed that the aqueous extract induced cell death of Jurkat cells at 24, 48 and 72 hours post-treatment in a time- and dose-dependent manner. However, cells treated at 48 and 72 hours produced higher cytotoxic effects with half maximal inhibitory concentration (IC50) values of 2.55 µg/ml and 2.38 µg/ml, respectively. In contrast, the extract induced normal PBMC proliferation, especially after 24 hours treatment with 1000 µg/ml. This result indicates that the C. roseus crude aqueous extract showed differential effects of inhibiting the proliferation of the Jurkat cell line and promoting the growth of PBMCs. These data suggest that the extract may be applicable for modulating the normal and transformed immune cells in leukaemia patients.