Towards understanding the cutting temperature in ultrasonic vibration-assisted drilling based on the dynamic contact characteristics between the cutting edge and workpiece.

Compared with conventional drilling (CD), ultrasonic vibration-assisted drilling(UVAD) is experimentally proven a promising method to reduce the cutting temperature. But sometimes cutting temperature also becomes higher in UVAD than in CD. To further make clear the cutting temperature mechanisms in UVAD, this study aims to study the effect of tool's ultrasonic vibration on the cutting heat generation and heat dissipation at a relatively micro level. Firstly, drilling experiments are designed to explore the variations of cutting heat under different ultrasonic vibrations. Then, to analyze the influence of ultrasonic vibration on the cutting heat theoretically, a kinematic model is developed to describe the dynamic contact between the cutting edge and workpiece in UVAD. Besides, a cutting heat analysis model based on the contact characteristics in UVAD is proposed to study and compare the variations of cutting heat generation. The effect of ultrasonic vibration on the cutting heat generation, heat dispassion, and the resultant cutting temperature under different machining in UVAD conditions are discussed. It is indicated from the theoretical analysis that more cutting heat tends to be produced due to the significantly increased sliding velocity on the cutting edge-workpiece interface when the ultrasonic vibration is applied. The analysis agrees with the experimental results that the cutting temperature in dry UVAD is higher than in dry CD. But on the other hand, ultrasonic vibration can also improve the lubrication and cooling effect under appropriate machining conditions, which is beneficial to the reduction in cutting temperature. The investigation shows the multifaceted influences of ultrasonic vibration on the cutting temperature in the drilling process in detail, which provides a reference for UVAD parameter optimization.

Upregulation of miR-345-5p suppresses cell growth of lung adenocarcinoma by regulating ras homolog family member A (RhoA) and Rho/Rho associated protein kinase (Rho/ROCK) pathway.

BACKGROUND: Microribose nucleic acids (miRNAs) are implicated in the progression of lung adenocarcinoma. MicroRNA-345-5p (miR-345-5p) is a recently identified anti-oncogene in some human cancers, but its functional role and possible molecular mechanism in lung adenocarcinoma remain unknown. This study aimed to identify the biological function and underlying mechanism of miR-345-5p in lung adenocarcinoma cells. METHODS: In this study, lung adenocarcinoma tissues and adjacent tissues were collected in the First Affiliated Hospital of Anhui Medical University between April 2016 and February 2017. The expression of miR-345-5p and ras homolog family member A (RhoA) in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines (A549, H1650, PC-9, and H441) was detected by reverse transcription quantitative polymerase chain reaction analysis. Functional assays including colony formation, flow cytometry analysis, wound healing, and transwell assays were performed to assess the proliferation, apoptosis, migration, and invasion of lung adenocarcinoma cells. In addition, RNA pulldown and luciferase reporter assays were conducted to evaluate the relationship between miR-345-5p and RhoA. Difference between the two groups was analyzed with Student's t test, while that among multiple groups was analyzed with one-way analysis of variance. RESULTS: MiR-345-5p expression displayed lower level in lung adenocarcinoma tissues (0.241 ± 0.095 vs.1.000 ± 0.233, t = 19.247, P < 0.001) and cell lines (F = 56.992, P < 0.001) than control tissues and cells. Functional experiments demonstrated that upregulation of miR-345-5p inhibited the malignant phenotypes of lung adenocarcinoma cells via suppressing cell proliferation, migration, invasion, and facilitating cell apoptosis. Additionally, RhoA was verified to be the downstream target of miR-345-5p. Expression of RhoA was downregulated by overexpression of miR-345-5p in PC-9 (0.321 ± 0.047 vs. 1.000 ± 0.127, t = 8.536, P < 0.001) and H1650 (0.398 ± 0.054 vs. 1.000 ± 0.156, t = 4.429, P = 0.011) cells. Rescue assays revealed that overexpression of RhoA rescued the suppressive effects of miR-345-5p upregulation on proliferation, migration, and invasion of lung adenocarcinoma cells. Further, miR-345-5p was found to regulate the Rho/Rho-associated protein kinase (ROCK) signaling pathway by downregulation of RhoA in lung adenocarcinoma cells. CONCLUSIONS: MiR-345-5p plays a tumor suppressor role in lung adenocarcinoma cells by downregulating RhoA to inactivate the Rho/ROCK pathway.

Effect of arterial blood bicarbonate (HCO3-) concentration on the accuracy of STOP-Bang questionnaire screening for obstructive sleep apnea.

BACKGROUND: To evaluate the effect of arterial bicarbonate (HCO3-) concentration on the accuracy of STOP-Bang questionnaire (SBQ) screening for obstructive sleep apnea (OSA). METHODS: A total of 144 patients with suspected OSA were included. Polysomnograms (PSG) and blood gas analysis were performed, and the Epworth Sleepiness Scale (ESS), STOP-Bang questionnaire, and Berlin questionnaire were completed. The correlation between the arterial HCO3- concentration, apnea hypopnea index (AHI), and other related indicators was analyzed. The scoring results of the ESS, SBQ, and Berlin questionnaire were compared with the PSG results, and the sensitivity and specificity were calculated in the form of a four-cell table. The changes in the sensitivity and specificity of OSA screening after SBQ alone and combined with HCO3- concentration were compared, and ROC curves were drawn. RESULTS: Arterial HCO3- concentration was positively correlated with AHI (r = 0.537, P < 0.001). The ratio of HCO3- concentration ≥ 24.6 mmol/L in the non-OSA group was significantly lower than that in the OSA group (25.0% VS 80.8%, P < 0.001). The sensitivity of the SBQ was higher than that of the ESS (97.5% VS 81.7%, P < 0.001) and the Berlin questionnaire (97.5% VS 79.2%, P < 0.001). There was no statistical significance in the specificity of the three scales (25%, 37.5%, 37.5%). A combined SBQ score ≥ 3 and HCO3- concentration ≥ 24.6 mmol/L showed increased specificity and decreased sensitivity compared with an SBQ score ≥ 3 alone, with a corresponding AUC of 0.771 (P < 0.01) and 0.613 (P > 0.05), respectively. CONCLUSION: The sensitivity of the SBQ was better than that of the Berlin questionnaire and ESS. After combining arterial blood HCO3- concentration, the SBQ questionnaire increased the specificity of OSA prediction and decreased the sensitivity, which improved the accuracy of screening.

Multiplexed Nanomaterial-Based Sensor Array for Detection of COVID-19 in Exhaled Breath.

This article reports on a noninvasive approach in detecting and following-up individuals who are at-risk or have an existing COVID-19 infection, with a potential ability to serve as an epidemic control tool. The proposed method uses a developed breath device composed of a nanomaterial-based hybrid sensor array with multiplexed detection capabilities that can detect disease-specific biomarkers from exhaled breath, thus enabling rapid and accurate diagnosis. An exploratory clinical study with this approach was examined in Wuhan, China, during March 2020. The study cohort included 49 confirmed COVID-19 patients, 58 healthy controls, and 33 non-COVID lung infection controls. When applicable, positive COVID-19 patients were sampled twice: during the active disease and after recovery. Discriminant analysis of the obtained signals from the nanomaterial-based sensors achieved very good test discriminations between the different groups. The training and test set data exhibited respectively 94% and 76% accuracy in differentiating patients from controls as well as 90% and 95% accuracy in differentiating between patients with COVID-19 and patients with other lung infections. While further validation studies are needed, the results may serve as a base for technology that would lead to a reduction in the number of unneeded confirmatory tests and lower the burden on hospitals, while allowing individuals a screening solution that can be performed in PoC facilities. The proposed method can be considered as a platform that could be applied for any other disease infection with proper modifications to the artificial intelligence and would therefore be available to serve as a diagnostic tool in case of a new disease outbreak.

Myosin light chain kinase inhibitor ML7 improves vascular endothelial dysfunction and permeability via the mitogen-activated protein kinase pathway in a rabbit model of atherosclerosis.

Endothelial dysfunction (ED) and hyperpermeability are considered as the initiating steps in early atherosclerosis. Phosphorylation of myosin light chain (MLC) is key to cause vascular hyperpermeability via endothelial cell contraction. However, it is unclear whether MLC phosphorylation can also regulate the balance between contraction and relaxation of endothelial cells, thereby affecting endothelium-dependent diastolic function and leading to ED. The present study investigated relationships between ED and MLC phosphorylation and underlying mechanisms. Twenty-four male New Zealand white rabbits were randomly divided into three groups: control, AS, and ML7 (MLCK inhibitor) groups, and fed with normal diet, high-fat diet (HFD), and HFD plus oral ML7 (1 mg/kg daily) respectively. HFD-fed rabbits showed typical atheromatous lesions and endothelial hyperpermeability, and these lesions could be partly reversed following ML7 therapy. Western blotting revealed significant increased expression of myosin light chain kinase (MLCK) and phosphorylation of MLC, JNK, and ERK in the arterial wall of rabbits in the AS group compared with those of the control group (p < 0.05), whereas the ML7 group showed markedly decreased levels of these proteins compared with the AS group (p < 0.05). The endothelium-dependent relaxation rate was significantly reduced both in vitro and in vivo in AS group, and was improved using ML7 therapy. Taken together, these results indicate that MLCK expression and subsequent MLC phosphorylation increase vascular endothelial permeability and endothelium-dependent diastolic dysfunction by promoting endothelial cell contraction, which may be initiated by the activation of the MAP/ERK (MEK) and MAP/JNK (MEK) pathways.

Procyanidin B2 Suppresses Lipopolysaccharides-Induced Inflammation and Apoptosis in Human Type II Alveolar Epithelial Cells and Lung Fibroblasts.

Acute lung injury (ALI) is characterized by acute lung inflammation and apoptosis of alveolar epithelial cells (AECs) with a high morbidity and mortality. Procyanidin B2 (PCB2) is a naturally occurring flavonoid with anti-inflammatory activity. Our previous study demonstrated that PCB2 inhibited NLRP3 inflammasome signaling and ameliorated paraquat-induced ALI in rat, indicating the protective role of PCB2. As lipopolysaccharide (LPS) induced acute cell injury and dysfunction, we continued to evaluate the protective effects of PCB2 using LPS-treated human AECs and lung fibroblasts (LFs) model. We tested the effects of PCB2 on cell permeability, viability, apoptosis, nuclear factor-kappaB (NF-κB) activation, NLRP3 inflammasome activation, and proinflammatory cytokines production in LPS-treated human AECs and LFs. PCB2 prevented LPS-induced cell apoptosis, and increased the cell viability in LPS-treated human AECs and LFs. PCB2 inhibited LPS-induced Bax and active caspase-3 expression, and promoted Bcl-2 expression. PCB2 prevented LPS-induced tumor necrosis factor-α, interleukin-1ß expression, NF-κB activation, and NLRP3 inflammasome activation. PCB2 suppressed LPS-induced inflammation and apoptosis in human AECs and LFs by inhibiting NF-κB and NLRP3 inflammasome.

Multiple Copies in T-Cell Malignancy 1 (MCT-1) Promotes the Stemness of Non-Small Cell Lung Cancer Cells via Activating Interleukin-6 (IL-6) Signaling through Suppressing MiR-34a Expression.

BACKGROUND Although the oncogenic roles of multiple copies in T-cell malignancy 1 (MCT-1) have been revealed in multiple cancers, its effects on non-small cell lung cancer (NSCLC) progression are still uncertain. This study aimed to reveal the effects of MCT-1 on the stem cell-like traits of NSCLC cells. MATERIAL AND METHODS Western blot, real-time quantitative polymerase chain reaction (RT-qPCR), spheroid forming ability, and ALDH1 (aldehyde dehydrogenase 1) activity analysis were carried out to examine the effects of MCT-1/micrRNa-34 (miR-34a)/interleukin-6 (IL-6) on the stem cell-like characteristics of lung cancer cells. RESULTS MCT-1 knockdown reduced the spheroid forming ability, characterized as the decreased spheroid size and number. Additionally, MCT-1 knockdown decreased the expression of the NSCLC stemness markers and the activity of ALDH1. Moreover, MCT-1 knockdown decreased IL-6 secretion that promotes NSCLC cell stemness. Furthermore, MCT-1 knockdown increased the level of miR-34a, which attenuated the stemness of NSCLC cells through targeting IL-6R (IL-6 receptor) expression. CONCLUSIONS These results suggest MCT-1/miR-34a/IL-6/IL-6R axis is responsible for MCT-1-mediated effects on NSCLC cell stemness.

Procyanidin B2 protects rats from paraquat-induced acute lung injury by inhibiting NLRP3 inflammasome activation.

Paraquat is a commonly used heterocyclic herbicide and has high toxicity by causing acute lung injury. There is no effective treatment for paraquat poisoning. We evaluated the effects of procyanidin B2, a natural dietary phytochemical, on paraquat-induced lung injury in rats. Paraquat was used to induce acute lung injury of rats, which were administered with procyanidin B2. The lung injury was evaluated by measuring the lung/body weight ratio, the histology and PMNs count. The oxidative stress was assessed by detecting ROS-mediated indices in the BALF. The expression of IL-1ß and IL-18 were detected by RT-PCR and ELISA. The levels of NLRP3 inflammasome components including NLRP3, ASC and caspase-1 were detected by western blot. The lung injury in the paraquat-induced models in NLRP3 gene silenced animals was compared with the same lung injury model treated with procyanidin B2. Administration of procyanidin B2 significantly reduced paraquat-induced lung injury with lower BALF PMNs count, MPO activity, MDA level and elevated SOD activity. Procyanidin B2 suppressed expression of IL-1ß and IL-18 at both RNA and protein levels, similar to the NLRP3 gene silenced rats. Compared to paraquat-induced group, procyanidin B2 showed remarkably decreased NLRP3, ASC and caspase-1 signals in the lung tissues in a dose-dependent manner. Procyanidin B2 significantly suppressed the activation of NLRP3 inflammasome in the lung tissue induced by paraquat in the rat model. This finding revealed a novel mechanism by which procyanidin B2 exerts anti-inflammatory effects and their clinical benefits in health.

Fenretinide inhibits the proliferation and migration of human liver cancer HepG2 cells by downregulating the activation of myosin light chain kinase through the p38­MAPK signaling pathway.

N-(4-hydroxyphenyl)retinamide (4-HPR or fenretinide), which is a synthetic analog of all­trans retinoic acid (ATRA), effectively inhibits the growth of several types of tumor cells; however, its molecular mechanism remains unclear. We found that 4­HPR altered the morphology of human liver cancer HepG2 cells and also inhibited their proliferation and suppressed the colony formation in a dose­ and time­dependent manner. A wound healing assay revealed that 4­HPR significantly hindered HepG2 cell migration, and that this was accompanied by the phosphorylation of p38­MAPK (mitogen­activated protein kinase). Mechanistically, the MAPK­specific inhibitor SB203580 attenuated the inhibitory effects of 4­HPR on the migration of HepG2 cells. Moreover, we also observed that 4­HPR inhibited the activation and expression of myosin light chain kinase (MLCK) in HepG2 cells. Simultaneously, 4­HPR lowered the expression of F­actin and promoted the expression of E­cadherin. ML­7, a selective inhibitor of MLCK, significantly inhibited the migration of HepG2 cells while increasing the phosphorylation of p38­MAPK and the expression of E­cadherin, and decreasing the activation of MLCK and the expression of F­actin. In conclusion, 4­HPR inhibited the proliferation and migration of HepG2 cells, and p38­MAPK plays an important role in regulating these 4­HPR effects by reducing the activation of MLCK. The present study suggests that 4­HPR may be a potent antimetastatic agent.

In vivo effects of the NLRP1/NLRP3 inflammasome pathway on latent respiratory virus infection.

The present study aimed to investigate the effects of nucleotide-binding domain leucine-rich repeat protein (NLRP)1/NLRP3 inflammasome pathways on latent viral infection of the respiratory tract. A total of 55 BALB/c mice were assigned to the control, bleomycin (BLM)­treated, murine cytomegalovirus (MCMV), MCMV+BLM and MCMV+BLM+CD4+ T­cell groups. The viral loads were detected in the salivary glands, kidney, liver and lung tissues via polymerase chain reaction (PCR). The weight, lung coefficient and hydroxyproline (HYP) were detected. HE and Masson staining were performed to score for alveolitis and degree of pulmonary fibrosis. Reverse transcription­quantitative PCR and western blot were applied to assess the expression levels of the NLRP inflammasome components caspase­1, interleukin (IL)­1ß and IL­18. ELISA was used to evaluate the expression levels of caspase­1, tumor necrosis factor (TNF)­α, IL­1ß and IL­18. The weight of the mice decreased, and the lung coefficient and HYP content increased in the BLM, MCMV, MCMV+BLM and MCMV+BLM+CD4+ T­cell groups compared with those in the control group. Compared with the control group, mice in the BLM, MCMV+BLM and MCMV+BLM+CD4+ T­cell groups had obviously increased alveolitis and degrees of pulmonary fibrosis, increased mRNA expression levels of caspase­1, IL­1ß and IL­18, and increased protein expression levels of caspase­1(p20), mature IL­1ß and mature IL­18. The values in the MCMV+BLM group were also higher than those in the BLM group and those in the MCMV+BLM+CD4+ T­cell group. The serum levels of caspase­1, TNF­α, IL­1ß and IL­18 in the serum of mice in the MCMV+BLM group were significantly higher than those in the BLM group. Compared with the MCMV+BLM group, the MCMV+BLM+CD4+ T­cell group had decreased levels of caspase­1, TNF­α, IL­1ß and IL­18 (all P<0.05). These results demonstrated that the activation of the NLRP1 and NLRP3 inflammasome pathways may contribute to pulmonary fibrosis caused by latent MCMV infection in mice.

Hyperbaric oxygen rescues lung cancer cells from chemical hypoxia-induced low differentiation and apoptosis resistance.

Hypoxia induces vigorous growth and a higher malignant phenotype in solid tumors. Hyperoxic treatment using hyperbaric oxygen (HBO) has previously been shown as a highly effective method to attenuate hypoxia. We aimed to investigate the effect of HBO on hypoxia-induced malignancy of lung cancer cells. Cobalt chloride (CoCl2) was used to induce chemical hypoxia in lung cancer cell line A549. Hypoxic inducible factor-1α (HIF-1α) expression, lactate dehydrogenase (LDH) activity, migration and invasion capacity, expression profiles of epithelial-mesenchymal transition (EMT) markers and apoptotic markers were assessed in CoCl2-treated A549 cells, with or without HBO treatment. Chemical hypoxia caused by CoCl2 resulted in high LDH activity, increased migration and invasion, decreased E-cadherin/N-cadherin ratio, enhanced EMT phenotype, higher Bcl-2/Bax ratio and elevated GRP78 expression. HBO treatment could significantly attenuate hypoxia-induced LDH activity, migration and invasion, restore hypoxia-reduced E-cadherin/N-cadherin ratio and EMT phenotype, as well as hypoxia-induced Bcl-2/Bax ratio, and repress GRP78 expression. HBO could serve as a reliable adjuvant treatment targeting the hypoxia microenvironment in solid tumors.

Effects of hyperbaric oxygen treatment on gastric cancer cell line SGC7901.

Hyperbaric oxygen (HBO) has been previously identified as an effective adjunct treatment option for the management of brain injury, diabetic ulcers and chronic wounds. However, the roles of HBO as an adjunctive therapy for tumors remain controversial. The present research project was performed to explore the effects of HBO treatment on proliferation, autophagy and endoplasmic reticulum stress response of the gastric cancer cell line, SGC7901. The present study demonstrated that, after subjecting SGC7901 cells to HBO treatment, the increase in cell proliferation was significant, compared with that of the control group. In addition, there was a significant increase in LC3-phosphatidylethanolamine conjugate (LC3-II) level, as well as binding immunoglobulin protein level, and a significant decrease in CCAAT-enhancer-binding protein homologous protein level. These suggested that hyperbaric oxygen treatment alone may promote proliferation and cell survival of gastric cancer cell SGC7901, and inhibit apoptosis through regulating cell autophagy and oxidative stress.

All-Trans Retinoic Acid Inhibits Human Colorectal Cancer Cells RKO Migration via Downregulating Myosin Light Chain Kinase Expression through MAPK Signaling Pathway.

All-trans-retinoic acid (ATRA) inhibits the invasive and metastatic potentials of various cancer cells. However, the underlying mechanism is unclear. Here, we demonstrate that ATRA inhibited colorectal cancer cells RKO (human colon adenocarcinoma cell) migration by downregulating cell movement and increasing cell adhesion. ATRA inhibited the expression and activation of myosin light chain kinase (MLCK) in RKO cells, while the expression level of MLC phosphatase (MLCP) had no change in RKO cells treated with or without ATRA. The expression and activity of MLC was also inhibited in RKO cells exposed to ATRA. Intriguingly, ATRA increased the expression of occludin messenger RNA (mRNA) and protein and its localization on cell membrane. However, ATRA did not change the expression of zonula occludens 1 (ZO-1), but increased the accumulation of ZO-1 on RKO cells membrane. ML-7, an inhibitor of MLCK, significantly inhibited RKO cell migration. Furthermore, knockdown of endogenous MLCK expression inhibited RKO migration. Mechanistically, we showed that MAPK-specific inhibitor PD98059 enhanced the inhibitory effect of ATRA on RKO migration. In contrast, phorbol 12-myristate 13-acetate (PMA) attenuated the effects of ATRA in RKO cells. Moreover, knocking down endogenous extracellular signal-regulated kinase (ERK) expression inhibited MLCK expression in the RKO cells. In conclusion, ATRA inhibits RKO migration by reducing MLCK expression via extracellular signal-regulated kinase 1/Mitogen-activated protein kinase (ERK1/MAPK) signaling pathway.

Protein tyrosine phosphatase PTPRB regulates Src phosphorylation and tumour progression in NSCLC.

Protein tyrosine-phosphatases (PTPs) play important roles in various biological processes. Deregulation in PTP function has been implicated in carcinogenesis and tumour progression in many cancer types. However, the role of protein tyrosine phosphatase receptor type B (PTPRB) in non-small-cell lung cancer (NSCLC) tumorigenesis has not been investigated. Lentiviral vector expressing PTPRB cDNA or shRNA was infected into A549 and H1299 cell lines, followed by cell proliferation, colony formation, soft agar and invasion assays. A549 xenograft mouse model was used to evaluate in vivo function of PTPRB. Quantitative polymerase chain reaction (PCR) was used to measure PTPRB expression in NSCLC patient samples. Kaplan Meier analysis was performed to assess association between PTPRB expression and patient overall survival (OS). Multivariate analysis was performed to evaluate prognostic significance of PTPRB. Overexpression of PTPRB reduced cell proliferation rate, colony formation efficiency, soft agar growth and cell invasion in A549 and H1299 cells, as well as tumour growth rate in A549 xenograft. Knockdown of PTPRB increased Src phosphorylation and cell invasion, which was reversed by Src inhibitor PP2. Additionally, PTPRB was down-regulated in NSCLC patient and was associated with patient OS. PTPRB regulates Src phosphorylation and tumorigenesis in NSCLC. PTPRB may serve as an independent prognostic biomarker for NSCLC patients.

A novel all-trans retinoic acid derivative 4-amino­2­trifluoromethyl-phenyl retinate inhibits the proliferation of human hepatocellular carcinoma HepG2 cells by inducing G0/G1 cell cycle arrest and apoptosis via upregulation of p53 and ASPP1 and downregulation of iASPP.

4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, was reported to function as a tumor inhibitor in various types of cancer cells in vitro. However, little is known concerning its antitumor effect on human hepatocellular carcinoma (HCC) HepG2 cells. The aims of the present study were to investigate the effects of ATPR on the proliferation of HepG2 cells and to explore the probable mechanisms. A series of experiments were performed following the treatment of HepG2 cells with ATRA and ATPR. MTT and plate colony formation assays were used to measure the cell viability. To confirm the influence on proliferation, flow cytometry was used to detect the distribution of the cell cycle. Apoptosis was observed by Hoechst staining and flow cytometry. In addition, to characterize the underlying molecular mechanisms, immunofluorescence was applied to observe the distribution of p53. The transcription and translation levels of p53 were analyzed by real-time quantitative RT-PCR (qRT-PCR) and western blotting. The expression levels of murine double minute 2 (MDM2), apoptosis stimulating proteins of p53 (ASPP), cell cycle- and apoptosis-associated proteins were detected by western blotting. After HepG2 cells were incubated with ATRA and ATPR, the viability of the HepG2 cells was inhibited in a dose- and time-dependent manner. As well, ATPR significantly suppressed HepG2 cell colony formation and arrested cells at the G0/G1 phase, while ATRA had no obvious effects. Both Hoechst staining and flow cytometry unveiled the apoptosis of HepG2 cells. Moreover, the fluorescent density of p53 was higher in the nuclei after exposure to ATPR than that in the ATRA group. HepG2 cells treated with ATPR showed elevated mRNA and protein levels of p53 when compared with these levels in the ATRA-treated cells. Western blotting showed that ATPR increased ASPP1, p21 and Bax expression and decreased MDM2, iASPP, cyclin D and E, cyclin-dependent kinase 6 (CDK6) and Bcl-2 expression, while CDK4 and ASPP2 expression were scarcely altered. Consequently, ATPR exerted a better inhibitory effect on the proliferation of HepG2 cells than ATRA through increased expression of p53 and ASPP1 and downregulation of iASPP, thereby resulting in G0/G1 cell cycle arrest and apoptosis.

Ameliorative effect of melatonin against increased intestinal permeability in diabetic rats: possible involvement of MLCK-dependent MLC phosphorylation.

The increased intestinal permeability and functional impairment play an important role in type 2 diabetes (T2D), and melatonin may possess enteroprotection properties. Therefore, we used streptozotocin-induced diabetic rat model to investigate the regulation of intestinal permeability by melatonin. Rats were randomly divided into three groups, including control, diabetes mellitus (DM), and DM rats treated with melatonin. Melatonin was administered (10 mg/kg/day) by gavage for 24 weeks. The DM rats significantly increased the serum fasting blood glucose and lipid levels, which were alleviated by melatonin treatment. Importantly, the intestinal epithelial permeability was significantly increased in DM rats but was ameliorated following treatment with melatonin. These findings also indicated the expression of myosin light chain kinase (MLCK) and phosphorylation of MLC targeting subunit (MYPT) induced myosin light chain (MLC) phosphorylation level was markedly elevated in hyperglycemic and hyperlipidemic status. They were partly associated with down-regulated membrane type 1 and 2 (MT1 and MT2) expression, and up-regulated Rho-associated protein kinase (ROCK) expression and increased extracellular signal-regulated kinase (ERK) phosphorylation. However, the changes in target protein expression were reversed by melatonin. In conclusion, our results show melatonin beneficial effects on impaired intestinal epithelial permeability in T2D by suppressing ERK/MLCK- and ROCK/MCLP-dependent MLC phosphorylation.

Melatonin inhibits the Migration of Colon Cancer RKO cells by Down-regulating Myosin Light Chain Kinase Expression through Cross-talk with p38 MAPK.

BACKGROUND: Melatonin, which is mainly produced by the pineal gland, has a good inhibitory effect on cell growth of multiple cancer types. However, the underlying molecular mechanisms of anti-tumor activity for colon cancer have not been fully elucidated. In this study, we investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms. MATERIALS AND METHODS: The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. The relative mRNA levels of MLCK was assessed by real-time quantitative RT-PCR. Western blotting analysis was performed to examine the expression of MLCK, phosphorylation of myosin light chain (pMLC) and p38 (pp38). RESULTS: The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Furthermore, the phosphorylation level of p38, which showed the same trend, was also reduced when cells were treated by melatonin. In addition, ML-7 (25umol/l) could down-regulate the phosphorylation of p38. CONCLUSIONS: Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK.

Apoptotic events induced by high glucose in human hepatoma HepG2 cells involve endoplasmic reticulum stress and MAPK's activation.

To investigate whether endoplasmic reticulum (ER) stress participates in the induction of apoptosis in HepG2 cells exposed to high glucose and explore its probable mechanism. A series of experiments were performed following HepG2 cells treated with different concentrations of glucose for 48 h. The apoptosis was detected by means of Hoechst staining and flow cytometry. Caspase-3 activity assay was performed by measuring the pNA (p-nitroaniline) to indirectly reveal the catalytic activity of caspase-3. The expression levels of apoptosis-, ER stress-associated proteins and MAPKs were analyzed by western blot. To further characterize the molecular mechanisms, the effects of antioxidant alpha-lipoic acid (ALA) and specific inhibitors for JNK and p38 (SP600125 and SB203580, respectively) were examined by Hoechst staining, immunofluorescence, and western blot. After HepG2 cells were incubated with high glucose for 48 h, both Hoechst staining and flow cytometry analyses unveiled the apoptosis of HepG2 cells. Caspase-3 activity assay revealed that the activity of caspase-3 was enhanced. Western blot showed an enhancement of pro-caspase-9 degradation, a reduction of Bcl-2/Bax ratio, a decrease in GRP78 expression, and increases in CHOP and p47/phox levels. In addition, western blot analysis presented that phosphorylation of p38 and JNK was triggered and that the expression of ASK1 was elevated. In the case of the contributions of oxidative stress and the MAPK signaling pathways, all ALA, SP600125 and SB203580 were able to largely rescue high glucose-induced apoptosis. High glucose induced the apoptosis in HepG2 cells through the activation of ASK1-p38/JNK pathway mediated by ER stress and oxidative stress.

A novel all-trans retinoid acid derivative N-(3-trifluoromethyl- phenyl)- retinamide inhibits lung adenocarcinoma A549 cell migration through down-regulating expression of myosin light chain kinase.

AIM: To observe the effects of a novel all-trans retinoid acid (ATRA) derivative, N-(3-trifluoromethyl-phenyl)- retinamide (ATPR), on lung adenocarcinoma A549 cells and to explore the potential mechanism of ATPR inhibiting of A549 cell migration. MATERIALS AND METHODS: The cytotoxicity of ATRA and ATPR on A549 cells was assessed using MTT assay. Wound healing assays were used to analyze the influences of ATRA, ATPR, ML-7 (a highly selective inhibitor of myosin light chain kinase (MLCK)), PMA (an activator of MAPKs) and PD98059 (a selective inhibitor of ERK1/2) on the migration of A549 cells. Expression of MLCK and phosphorylation of myosin light chain (MLC) were assessed by Western blotting. RESULTS: ATRA and ATPR inhibited the proliferation of A549 cells in a dose- and time-dependent manner, and the effect of ATPR was much more remarkable compared with ATRA. Relative migration rate and migration distance of A549 cells both decreased significantly after treatment with ATPR or ML-7. The effect on cell migration of PD98059 combining ATPR treatment was more notable than that of ATPR alone. Moreover, compared with control groups, the expression levels of MLCK and phosphorylated MLC in A549 cells were both clearly reduced in ATRA and ATPR groups. CONCLUSIONS: ATPR could suppress the migration and invasion of A549 cells, and the mechanism might be concerned with down- regulating the expression of MLCK in the ERK-MAPK signaling pathway, pointing to therapeutic prospects in lung cancer.

Melatonin inhibits the migration of human lung adenocarcinoma A549 cell lines involving JNK/MAPK pathway.

OBJECTIVE: Melatonin, an indolamine produced and secreted predominately by the pineal gland, exhibits a variety of physiological functions, possesses antioxidant and antitumor properties. But, the mechanisms for the anti-cancer effects are unknown. The present study explored the effects of melatonin on the migration of human lung adenocarcinoma A549 cells and its mechanism. METHODS: MTT assay was employed to measure the viability of A549 cells treated with different concentrations of melatonin. The effect of melatonin on the migration of A549 cells was analyzed by wound healing assay. Occludin location was observed by immunofluorescence. The expression of occludin, osteopontin (OPN), myosin light chain kinase (MLCK) and phosphorylation of myosin light chain (MLC), JNK were detected by western blots. RESULTS: After A549 cells were treated with melatonin, the viability and migration of the cells were inhibited significantly. The relative migration rate of A549 cells treated with melatonin was only about 20% at 24 h. The expression level of OPN, MLCK and phosphorylation of MLC of A549 cells were reduced, while the expression of occludin was conversely elevated, and occludin located on the cell surface was obviously increased. The phosphorylation status of JNK in A549 cells was also reduced when cells were treated by melatonin. CONCLUSIONS: Melatonin significantly inhibits the migration of A549 cells, and this may be associated with the down-regulation of the expression of OPN, MLCK, phosphorylation of MLC, and up-regulation of the expression of occludin involving JNK/MAPK pathway.