Daidzein Protects Caco-2 Cells against Lipopolysaccharide-Induced Intestinal Epithelial Barrier Injury by Suppressing PI3K/AKT and P38 Pathways

The intestinal epithelium provides an important barrier against bacterial endotoxin translocation, which can regulate the absorption of water and ions. The disruption of epithelial barrier function can result in water transport and tight junction damage, or further cause diarrhea. Therefore, reducing intestinal epithelial barrier injury plays an important role in diarrhea. Inflammatory response is an important cause of intestinal barrier defects. Daidzein improving the barrier integrity has been reported, but the effect on tight junction proteins and aquaporins is not well-described yet, and the underlying mechanism remains indistinct in the human intestinal epithelium. This study aimed to investigate the effects and mechanisms of daidzein on intestinal epithelial barrier injury induced by LPS, and a barrier injury model induced by LPS was established with human colorectal epithelial adenocarcinoma cell line Caco-2 cells. We found that daidzein protected the integrity of Caco-2 cell monolayers, reversed LPS-induced downregulation of ZO-1, occludin, claudin-1, and AQP3 expression, maintained intercellular junction of ZO-1, and suppressed NF-κB and the expression of inflammatory factors (TNF-α, IL-6). Furthermore, we found that daidzein suppressed the phosphorylation of the PI3K/AKT and P38 pathway-related proteins and the level of the related genes, and the PI3K/AKT and P38 pathway inhibitors increased ZO-1, occludin, claudin-1, and AQP3 expression. The study showed that daidzein could resist LPS-induced intestinal epithelial barrier injury, and the mechanism is related to suppressing the PI3K/AKT and P38 pathways. Therefore, daidzein could be a candidate as a dietary supplementation or drug to prevent or cure diarrhea.     

Induction of G2/M Cell Cycle Arrest via p38/p21Waf1/Cip1-Dependent Signaling Pathway Activation by Bavachinin in Non-Small-Cell Lung Cancer Cells

Lung cancer is the most commonly diagnosed malignant cancer in the world. Non-small-cell lung cancer (NSCLC) is the major category of lung cancer. Although effective therapies have been administered, for improving the NSCLC patient’s survival, the incident rate is still high. Therefore, searching for a good strategy for preventing NSCLC is urgent. Traditional Chinese medicine (TCM) are brilliant materials for cancer chemoprevention, because of their high biological safety and low cost. Bavachinin, which is an active flavanone of Proralea corylifolia L., possesses anti-inflammation, anti-angiogenesis, and anti-cancer activities. The present study’s aim was to evaluate the anti-cancer activity of bavachinin on NSCLC, and its regulating molecular mechanisms. The results exhibited that a dose-dependent decrease in the cell viability and colony formation capacity of three NSCLC cell lines, by bavachinin, were through G2/M cell cycle arrest induction. Meanwhile, the expression of the G2/M cell cycle regulators, such as cyclin B, p-cdc2^Y15, p-cdc2^T161, and p-wee1, was suppressed. With the dramatic up-regulation of the cyclin-dependent kinase inhibitor, p21^Waf1/Cip1, the expression and association of p21^Waf1/Cip1 with the cyclin B/cdc2 complex was observed. Silencing the p21^Waf1/Cip1 expression significantly rescued bavachinin-induced G2/M cell accumulation. Furthermore, the expression of p21^Waf1/Cip1 mRNA was up-regulated in bavachinin-treated NSCLC cells. In addition, MAPK and AKT signaling were activated in bavachinin-added NSCLC cells. Interestingly, bavachinin-induced p21^Waf1/Cip1 expression was repressed after restraint p38 MAPK activation. The inhibition of p38 MAPK activation reversed bavachinin-induced p21^Waf1/Cip1 mRNA expression and G2/M cell cycle arrest. Collectively, bavachinin-induced G2/M cell cycle arrest was through the p38 MAPK-mediated p21^Waf1/Cip1-dependent signaling pathway in the NSCLC cells.     

Nigrosporins B,a Potential Anti-Cervical Cancer Agent,Induces Apoptosis and Protective Autophagy in Human Cervical Cancer Ca Ski Cells Mediated by PI3K/AKT/mTOR Signaling Pathway

Nigrosporins B, an anthraquinone derivative obtained from the secondary metabolites of marine fungus Nigrospora oryzae. In this study, we characterized the distinctive anti-cancer potential of Nigrosporins B in vitro and underlying molecular mechanisms in human cervical cancer Ca Ski cells for the first time. The results of MTT assay showed that Nigrosporins B significantly inhibited the proliferation of multiple tumor cells in a dose-dependent manner, especially for the Ca Ski cells with an IC₅₀ of 1.24 µM. Nigrosporins B exerted an apoptosis induction effect on Ca Ski cells as confirmed by flow cytometry, AO/EB dual fluorescence staining, mitochondrial membrane potential analysis and western blot assay. In addition, Nigrosporins B induced obvious autophagy accompanied with the increase of autophagic vacuoles and the acceleration of autophagic flux as indicated by Cyto-ID staining, mRFP-GFP-LC3 adenovirus transfection and western blot analysis. Interestingly, the combination of Nigrosporins B with the three autophagy inhibitors all significantly enhanced the cytotoxicity of Nigrosporins B on Ca Ski cells, indicating that the autophagy induced by Nigrosporins B might protect Ca Ski cells from death. Furthermore, we found that Nigrosporins B inhibited the phosphorylation of PI3K, AKT, mTOR molecules and increased the protein expression levels of PTEN and p-AMPKα in a dose-dependent manner, suggesting that Nigrosporins B induced apoptosis and protective autophagy through the suppression of the PI3K/AKT/mTOR signaling pathway. Together, these findings revealed the anti-cervical cancer effect of Nigrosporins B and the underlying mechanism of action in Ca Ski cells, it might be as a promising alternative therapeutic agent for human cervical cancer.     

A Natural CHI3L1—Targeting Compound,Ebractenoid F,Inhibits Lung Cancer Cell Growth and Migration and Induces Apoptosis by Blocking CHI3L1/AKT Signals

Our previous big data analyses reported a strong association between CHI3L1 expression and lung tumor development. In this present study, we investigated whether a CHI3L1-inhibiting natural compound, ebractenoid F, inhibits lung cancer cell growth and migration and induces apoptosis. Ebractenoid F concentration-dependently (0, 17, 35, 70 µM) and significantly inhibited the proliferation and migration of A549 and H460 lung cancer cells and induced apoptosis. In the mechanism study, we found that ebractenoid F bound to CHI3L1 and suppressed CHI3L1-associated AKT signaling. Combined treatment with an AKT inhibitor, LY294002, and ebractenoid F synergistically decreased the expression of CHI3L1. Moreover, the combination treatment further inhibited the growth and migration of lung cancer cells and further induced apoptosis, as well as the expression levels of apoptosis-related proteins. Thus, our data demonstrate that ebractenoid F may serve as a potential anti-lung cancer compound targeting CHI3L1-associated AKT signaling.     

Andrographolide Exhibits Anticancer Activity against Breast Cancer Cells (MCF-7 and MDA-MB-231 Cells) through Suppressing Cell Proliferation and Inducing Cell Apoptosis via Inactivation of ER-α Receptor and PI3K/AKT/mTOR Signaling

Breast cancer is the most common cancer among women worldwide. Chemotherapy followed by endocrine therapy is the standard treatment strategy after surgery or radiotherapy. However, breast cancer is highly resistant to the treatments leading to the recurrence of breast cancer. As a result, the development of alternative medicines derived from natural plants with fewer side effects is being emphasized. Andrographolide isolated from Andrographis paniculata is one of the potential substances with anti-cancer properties in a variety of cell types, including breast cancer cells. This study aims to investigate the anti-cancer effects of andrographolide in breast cancer cells by evaluating cell viability and apoptosis as well as its underlying mechanisms through estrogen receptor (ER)-dependent and PI3K/AKT/mTOR signaling pathways. Cell viability, cell apoptosis, mRNA or miRNA, and protein expression were examined by MTT assay, Annexin V-FITC, qRT-PCR, and Western blot analysis, respectively. MCF-7 and MDA-MB-231 cell viability was reduced in a concentration- and time-dependent manner after andrographolide treatment. Moreover, andrographolide induced cell apoptosis in both MCF-7 and MDA-MB-231 cells by inhibiting Bcl-2 and enhancing Bax expression at both mRNA and protein levels. In MCF-7 cells, the ER-positive breast cancer, andrographolide showed an inhibitory effect on cell proliferation through downregulation of ERα, PI3K, and mTOR expression levels. Andrographolide also inhibited MDA-MB-231 breast cancer cell proliferation via induction of cell apoptosis. However, the inhibition of MCF-7 and MDA-MB-231 cell proliferation of andrographolide treatment did not disrupt miR-21. Our findings showed that andrographolide possesses an anti-estrogenic effect by suppressing cell proliferation in MCF-7 cells. The effects were comparable to those of the anticancer drug fulvestrant in MCF-7 cells. This study provides new insights into the anti-cancer effect of andrographolide on breast cancer and suggests andrographolide as a potential alternative from the natural plant for treating breast cancer types that are resistant to tamoxifen and fulvestrant.     

Detection of non‐small cell lung cancer cells based on microfluidic polarization microscopic image analysis

In early diagnosis of lung cancer, a polarization microscopy is a powerful tool to obtain the optical information of biological tissues. In this paper, a new microfluidic polarization imaging and analysis method was proposed for the detection and classification of cancer‐associated fibroblasts and the two kinds of non‐small cell lung cancer cells, A549 and H322. A polarizing microscopy system was constructed based on a commercial microscope to obtain 3*3 Mueller matrix of cells. Based on the Muller matrix decomposition algorithm and analysis in spatial domain and frequency domain, appropriate classification parameters were selected for the characterization of different polarization characteristics of cells. Finally, the logistic regression models based on machine learning were applied to determine optimal feature parameters and classify cells. This method integrated the morphological information of the cells, and the polarization characteristics of the cells in different polarization states. It is for the first time that the polarization microscopic image analysis method has been applied to the detection and classification of non‐small cell lung cancer cells. The results show that the presented microfluidic polarization microscopic image analysis method could classify cells effectively. Compared with the Muller matrix measurement and calculation methods, the method proposed in this paper was greatly simplified in both the acquisition of polarized images and the analysis and processing of polarized images.     

Simultaneous determination of gemcitabine and its main metabolite, dFdU, in plasma of patients with advanced non-small-cell lung cancer by high-performance liquid chromatography-tandem mass spectrometry

Gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC) is a pyrimidine antimetabolite employed against several human malignancies. It undergoes intracellular activation to the pharmacologically active triphosphate form (dFdCTP) and metabolic inactivation to the metabolite 2',2'-difluorodeoxyuridine (dFdU). In order to investigate the human plasma pharmacokinetics of dFdC and dFdU, we developed and validated an HPLC-MS/MS method, adding 2'-deoxycytidine as internal standard and simply precipitating the protein with acetonitrile. The method requires a small sample (125 microl), and it is rapid and selective, allowing good resolution of peaks from the plasma matrix in only 7 min. It is sensitive, precise and accurate, with overall precision, expressed as CV%, always less than 10.0% for both analytes and high recovery: > or = 80%. The limits of detection for dFdC and dFdU were 0.1 and 1.1 ng/ml, but considering the high concentrations in the plasma of patients investigated, we set the limit of quantitation at 20 ng/ml (0.08 microM) for dFdC and 250 ng/ml for dFdU, and validated the assay up to the dFdC concentration of 6.0 microg/ml (22.8 microM). The method was successfully used to study the drug pharmacokinetics in patients with advanced non-small-cell lung cancer in a phase II trial with gemcitabine administered as a fixed dose-rate infusion.     

Inhibition of cell proliferation,invasion and migration by the cardenolides digitoxigenin monodigitoxoside and convallatoxin in human lung cancer cell line

Cardiac glycosides consist of a large family of naturally derived compounds that are clinically used to treat congestive heart failure, and also present anticancer properties. In this study, the cytotoxic effects of two cardenolides, digitoxigenin monodigitoxoside (DGX) and convallatoxin (CON) were screened in four human tumour cell lines. Both compounds showed anti-proliferative effects in all tumour cells, at nanomolar concentrations. Since the human lung cancer cell line A549 was the most sensitive, we investigated the anti-proliferative, anti-migratory and anti-invasive effects of these cardenolides. DGX and CON reduced A549 cell migration, being able to reduce more than 90% of cell invasion. Their effects on the expression of key regulators of metastatic mechanism showed decreased levels of MMP-2, MMP-9 and p-FAK. Both compounds also presented low toxicity for healthy cells. Finally, this work provides the first insights into the effects of these cardenolides on key steps of lung cancer metastasis.     

Implication of leucyl-tRNA synthetase 1 (LARS1) over-expression in growth and migration of lung cancer cells detected by siRNA targeted knock-down analysis

Molecular mechanism of lung carcinogenesis and its aggressive nature is still largely elusive. To uncover the biomarkers related with tumorigenesis and behavior of lung cancer, we screened novel differentially expressed genes (DEG) in A549 lung cancer cell line by comparison with CCD-25Lu, normal pulmonary epithelial cell line, using annealing control primer(ACP)-based GeneFishing system. Of the DEGs, over-expression of leucyl-tRNA synthetase 1 (LARS1) was prominent and this up-regulation was confirmed by immunoblotting and real-time quantitative RT-PCR analysis. In addition to A549 cell line, primary lung cancer tissues also expressed higher level of LARS1 mRNA than their normal counter tissues. To explore the oncogenic potential of LARS1 over-expression in lung cancer, we knocked-down LARS1 by treating siRNA and observed the tumor behavior. LARS1 knock-down cells showed reduced ability to migrate through transwell membrane and to form colonies in both soft agar and culture plate. Taken together, these findings suggest that LARS1 may play roles in migration and growth of lung cancer cells, which suggest its potential implication in lung tumorigenesis.     

Preparative isolation and purification of steroidal glycoalkaloid from the ripe berries of Solanum nigrum L. by preparative HPLC–MS and UHPLC–TOF‐MS/MS and its anti‐non‐small cell lung tumors effects in vitro and in vivo

Overcoming epidermal growth factor receptor resistance is a critical problem that needs to be solved in clinical practice. Drugs that downregulate the fatty acid synthase‐epidermal growth factor receptor will become novel treatments for non‐small cell lung cancer. Solanum nigrum, extracted with water at 4°C, shows strong cytotoxic activity and inhibits tumor growth in Lewis tumor bearing‐mice in a dose‐dependent manner. A novel active compound in S. nigrum, solaoiacid, was successfully separated and purified from S. nigrum by preparative high‐performance liquid chromatography with mass spectrometry and ultra high performance liquid chromatography with time‐of‐flight tandem mass spectrometry. The IC₅₀ of solaoiacid on lung cancer cells was 2.3 µmol/L, which was significantly lower than that of the known steroidal glycoalkaloid. Label‐free proteomics and STRING Network analysis were used to identify significantly deregulated proteins in lung cancer cells that were treated with the fresh ripe fruit extracts of S. nigrum. S. nigrum regulates multiple signal pathways, including the epidermal growth factor receptor pathway. S. nigrum downregulated 24 main proteins with direct roles in fatty acid biosynthesis. Both S. nigrum and solaoiacid showed strong downregulation of the fatty acid synthase‐epidermal growth factor receptor and anti‐non‐small cell lung cancer effects, and thus will become a novel drug for the treatment of non‐small cell lung cancer.     

Enhanced skin anti-inflammatory and moisturizing action of gold nanoparticles produced utilizing Diospyros kaki fruit extracts

Inflammatory skin diseases (ISD) cause very severe itchy skin and dryness which is now a days an important issue which has to be taken care. Nanotechnology plays a main role in manufacturing cosmetic ingredients at a nanoscale size. Among different nanoparticles, gold (Au) is one of the non-toxic materials synthesized organically or inorganically. For synthesizing nanoparticles (NPs), using inorganic methods may cause some toxicity to cells, but using organic synthesis like plant extract is less toxic and environmentally friendly. Therefore, we synthesized DK-AuNPs using Diospyros kaki fruit extract. UPLC-MS/MS was used to evaluate phytochemicals responsible for converting salt into nanoparticles. The DK-AuNPs were characterized to confirm the formation of NPs. Furthermore, we analyzed the activity of DK-AuNPs on human keratinocytes (HaCaT cells). The DK-AuNPs showed 98.2 % cell survival upto 200 µg/mL against HaCaT cells. Additionally, compared to DK treatment, DK-AuNPs therapy decreased ROS production in TNF-α/IFN-γ (T + I) stimulated HaCaT cells by 68.7 %, whereas DK treatment reduced ROS generation by 27.8 %. Moreover, the skin anti-inflammatory potential and moisturizing effect of DK-AuNPs were analyzed using HaCaT cells. Furthermore, skin inflammatory activity biomarkers were downregulated through the MAPK/NFκB signaling pathway and showed significant inhibition by DK-AuNPs. Also, the skin moisturizing biomarkers such as HAS (1–3) were upregulated and HYAL (1–2) were downregulated by PI3K/AKT/NFκB through HAS2 regulation. Therefore, skin anti-inflammatory and moisturizing activity were enhanced by treatment with DK-AuNPs. In summary, we conclude that the DK-AuNPs could be a new alternative for skin disease.