Early committed polarization of intracellular tension in response to cell shape determines the osteogenic differentiation of mesenchymal stromal cells.

Within the heterogeneous tissue architecture, a comprehensive understanding of how cell shapes regulate cytoskeletal mechanics by adjusting focal adhesions (FAs) signals to correlate with the lineage commitment of mesenchymal stromal cells (MSCs) remains obscure. Here, via engineered extracellular matrices, we observed that the development of mature FAs, coupled with a symmetrical pattern of radial fiber bundles, appeared at the right-angle vertices in cells with square shape. While circular cells aligned the transverse fibers parallel to the cell edge, and moved them centripetally in a counter-clockwise direction, symmetrical bundles of radial fibers at the vertices of square cells disrupted the counter-clockwise swirling and bridged the transverse fibers to move centripetally. In square cells, the contractile force, generated by the myosin IIA-enriched transverse fibers, were concentrated and transmitted outwards along the symmetrical bundles of radial fibers, to the extracellular matrix through FAs, and thereby driving FA organization and maturation. The symmetrical radial fiber bundles concentrated the transverse fibers contractility inward to the linkage between the actin cytoskeleton and the nuclear envelope. The tauter cytoskeletal network adjusted the nuclear-actomyosin force balance to cause nuclear deformability and to increase nuclear translocation of the transcription co-activator YAP, which in turn modulated the switch in MSC commitment. Thus, FAs dynamically respond to geometric cues and remodel actin cytoskeletal network to re-distribute intracelluar tension towards the cell nucleus, and thereby controlling YAP mechanotransduction signaling in regulating MSC fate decision. STATEMENT OF SIGNIFICANCE: We decipher how cellular mechanics is self-organized depending on extracellular geometric features to correlate with mesenchymal stromal cell lineage commitment. In response to geometry constrains on cell morphology, symmetrical radial fiber bundles are assembled and clustered depending on the maturation state of focal adhesions and bridge with the transverse fibers, and thereby establishing the dynamic cytoskeletal network. Contractile force, generated by the myosin-IIA-enriched transverse fibers, is transmitted and dynamically drives the retrograde movement of the actin cytoskeletal network, which appropriately adjusts the nuclear-actomyosin force balance and deforms the cell nucleus for YAP mechano-transduction signaling in regulating mesenchymal stromal cell fate decision.

Drug Repurposing: The Mechanisms and Signaling Pathways of Anti-Cancer Effects of Anesthetics.

Cancer is one of the leading causes of death worldwide. There are only limited treatment strategies that can be applied to treat cancer, including surgical resection, chemotherapy, and radiotherapy, but these have only limited effectiveness. Developing a new drug for cancer therapy is protracted, costly, and inefficient. Recently, drug repurposing has become a rising research field to provide new meaning for an old drug. By searching a drug repurposing database ReDO_DB, a brief list of anesthetic/sedative drugs, such as haloperidol, ketamine, lidocaine, midazolam, propofol, and valproic acid, are shown to possess anti-cancer properties. Therefore, in the current review, we will provide a general overview of the anti-cancer mechanisms of these anesthetic/sedative drugs and explore the potential underlying signaling pathways and clinical application of these drugs applied individually or in combination with other anti-cancer agents.

Arsenic compounds induce apoptosis by activating the MAPK and caspase pathways in FaDu oral squamous carcinoma cells.

For a number of years, oral cancer has remained in the top ten most common types of cancer, with an incidence rate that is steadily increasing. In total, ~75% oral cancer cases are associated with lifestyle factors, including uncontrolled alcohol consumption, betel and tobacco chewing, and the excessive use of tobacco. Notably, betel chewing is highly associated with oral cancer in Southeast Asia. Arsenic is a key environmental toxicant; however, arsenic trioxide has been used as a medicine for the treatment of acute promyelocytic leukemia, highlighting its anticancer properties. The present study aimed to investigate the role of arsenic compounds in the treatment of cancer, using FaDu oral squamous carcinoma cells treated with sodium arsenite (NaAsO2) and dimethyl arsenic acid (DMA). The results demonstrated that FaDu cells exhibited membrane blebbing phenomena and high levels of apoptosis following treatment with 10 µM NaAsO2 and 1 mM DMA for 24 h. The results of cell viability assay demonstrated that the rate of FaDu cell survival was markedly reduced as the concentration of arsenic compounds increased from 10 to 100 µM NaAsO2, and 1 to 100 mM DMA. Moreover, flow cytometry was carried out to further examine the effects of arsenic compounds on FaDu cell cycle regulation; the results revealed that treatment with NaAsO2 and DMA led to a significant increase in the percentage of FaDu cells in the sub­G1 and G2/M phases of the cell cycle. An Annexin V/PI double staining assay was subsequently performed to verify the levels of FaDu cell apoptosis following treatment with arsenic compounds. Furthermore, the results of the western blot analyses revealed that the expression levels of caspase­8, ­9 and ­3, and poly ADP­ribose polymerase, as well the levels of phosphorylated JNK and ERK1/2 were increased following treatment with NaAsO2 and DMA in the FaDu cells. On the whole, the results of the present study revealed that treatment with NaAsO2 and DMA promoted the apoptosis of FaDu oral cancer cells, by activating MAPK pathways, as well as the extrinsic and intrinsic apoptotic pathways.

Anticancer Effects of Midazolam on Lung and Breast Cancers by Inhibiting Cell Proliferation and Epithelial-Mesenchymal Transition.

Despite improvements in cancer treatments resulting in higher survival rates, the proliferation and metastasis of tumors still raise new questions in cancer therapy. Therefore, new drugs and strategies are still needed. Midazolam (MDZ) is a common sedative drug acting through the γ-aminobutyric acid receptor in the central nervous system and also binds to the peripheral benzodiazepine receptor (PBR) in peripheral tissues. Previous studies have shown that MDZ inhibits cancer cell proliferation but increases cancer cell apoptosis through different mechanisms. In this study, we investigated the possible anticancer mechanisms of MDZ on different cancer cell types. MDZ inhibited transforming growth factor ß (TGF-ß)-induced cancer cell proliferation of both A549 and MCF-7 cells. MDZ also inhibited TGF-ß-induced cell migration, invasion, epithelial-mesenchymal-transition, and Smad phosphorylation in both cancer cell lines. Inhibition of PBR by PK11195 rescued the MDZ-inhibited cell proliferation, suggesting that MDZ worked through PBR to inhibit TGF-ß pathway. Furthermore, MDZ inhibited proliferation, migration, invasion and levels of mesenchymal proteins in MDA-MD-231 triple-negative breast cancer cells. Together, MDZ inhibits cancer cell proliferation both in epithelial and mesenchymal types and EMT, indicating an important role for MDZ as a candidate to treat lung and breast cancers.

Aqueous extract of Arctium lappa L. root (burdock) enhances chondrogenesis in human bone marrow-derived mesenchymal stem cells.

BACKGROUND: Arctium lappa L. root (burdock root) has long been recommended for the treatment of different diseases in traditional Chinese medicine. Burdock root possesses anti-oxidative, anti-inflammatory, anti-cancer, and anti-microbial activities. The aim of the study was to elucidate whether aqueous extract of burdock root regulates mesenchymal stem cell proliferation and differentiation. METHODS: Human bone marrow-derived mesenchymal stem cells in 2D high density culture and in 3D micromass pellets were treated with chondrogenic induction medium and chondral basal medium in the absence or presence of aqueous extract of burdock root. The chondrogenic differentiation was accessed by staining glucosaminoglycans, immunostaining SOX9 and type II collagen and immuonblotting of SOX9, aggrecan and type II collagen. RESULTS: Treatment of aqueous extract of burdock root increased the cell proliferation of hMSCs. It did not have significant effect on osteogenic and adipogenic differentiation, but significantly enhanced chondrogenic induction medium-induced chondrogenesis. The increment was dose dependent, as examined by staining glucosaminoglycans, SOX9, and type II collagen and immunobloting of SOX9, aggrecan and type II collagen in 2D and 3D cultures. In the presence of supplemental materials, burdock root aqueous extract showed equivalent chondrogenic induction capability to that of TGF-ß. CONCLUSIONS: The results demonstrate that aqueous extract of Arctium lappa L. root promotes chondrogenic medium-induced chondrogenic differentiation. The aqueous extract of burdock root can even be used alone to stimulate chondrogenic differentiation. The study suggests that the aqueous extract of burdock root can be used as an alternative strategy for treatment purposes.

Simple In-House Fabrication of Microwells for Generating Uniform Hepatic Multicellular Cancer Aggregates and Discovering Novel Therapeutics.

Three-dimensional (3D) cell culture models have become powerful tools because they better simulate the in vivo pathophysiological microenvironment than traditional two-dimensional (2D) monolayer cultures. Tumor cells cultured in a 3D system as multicellular cancer aggregates (MCAs) recapitulate several critical in vivo characteristics that enable the study of biological functions and drug discovery. The microwell, in particular, has emerged as a revolutionary technology in the generation of MCAs as it provides geometrically defined microstructures for culturing size-controlled MCAs amenable for various downstream functional assays. This paper presents a simple and economical microwell fabrication methodology that can be conveniently incorporated into a conventional laboratory setting and used for the discovery of therapeutic interventions for liver cancer. The microwells were 400-700 µm in diameter, and hepatic MCAs (Huh-7 cells) were cultured in them for up to 5 days, over which time they grew to 250-520 µm with good viability and shape. The integrability of the microwell fabrication with a high-throughput workflow was demonstrated using a standard 96-well plate for proof-of-concept drug screening. The IC50 of doxorubicin was determined to be 9.3 µM under 2D conditions and 42.8 µM under 3D conditions. The application of photothermal treatment was demonstrated by optimizing concanavalin A-FITC conjugated silica-carbon hollow spheres (SCHSs) at a concentration of 500:200 µg/mL after a 2 h incubation to best bind with MCAs. Based on this concentration, which was appropriate for further photothermal treatment, the relative cell viability was assessed through exposure to a 3 W/cm2 near-infrared laser for 20 min. The relative fluorescence intensity showed an eight-fold reduction in cell viability, confirming the feasibility of using photothermal treatment as a potential therapeutic intervention. The proposed microwell integration is envisioned to serve as a simple in-house technique for the generation of MCAs useful for discovering therapeutic modalities for liver cancer treatment.

Coincidence Detection of Membrane Stretch and Extracellular pH by the Proton-Sensing Receptor OGR1 (GPR68).

The physical environment critically affects cell shape, proliferation, differentiation, and survival by exerting mechanical forces on cells. These forces are sensed and transduced into intracellular signals and responses by cells. A number of different membrane and cytoplasmic proteins have been implicated in sensing mechanical forces, but the picture is far from complete, and the exact transduction pathways remain largely elusive. Furthermore, mechanosensation takes place alongside chemosensation, and cells need to integrate physical and chemical signals to respond appropriately and ensure normal tissue and organ development and function. Here, we report that ovarian cancer G protein coupled receptor 1 (OGR1) (aka GPR68) acts as coincidence detector of membrane stretch and its physiological ligand, extracellular H+. Using fluorescence imaging, substrates of different stiffness, microcontact printing methods, and cell-stretching techniques, we show that OGR1 only responds to extracellular acidification under conditions of membrane stretch and vice versa. The level of OGR1 activity mirrors the extent of membrane stretch and degree of extracellular acidification. Furthermore, actin polymerization in response to membrane stretch is critical for OGR1 activity, and its depolymerization limits how long OGR1 remains responsive following a stretch event, thus providing a "memory" for past stretch. Cells experience changes in membrane stretch and extracellular pH throughout their lifetime. Because OGR1 is a widely expressed receptor, it represents a unique yet widespread mechanism that enables cells to respond dynamically to mechanical and pH changes in their microenvironment by integrating these chemical and physical stimuli at the receptor level.

Midazolam inhibits chondrogenesis via peripheral benzodiazepine receptor in human mesenchymal stem cells.

Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high-density culture performed with TGF-ß-driven chondrogenic induction medium. Treatment of the Midazolam dose-dependently inhibited chondrogenesis, examined using Alcian blue-stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor-ß-induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam-induced congenital malformations of the musculoskeletal system through PBR.

Lis1 dysfunction leads to traction force reduction and cytoskeletal disorganization during cell migration.

Cell migration is a critical process during development, tissue repair, and cancer metastasis. It requires complex processes of cell adhesion, cytoskeletal dynamics, and force generation. Lis1 plays an important role in the migration of neurons, fibroblasts and other cell types, and is essential for normal development of the cerebral cortex. Mutations in human LIS1 gene cause classical lissencephaly (smooth brain), resulting from defects in neuronal migration. However, how Lis1 may affect force generation in migrating cells is still not fully understood. Using traction force microscopy (TFM) with live cell imaging to measure cellular traction force in migrating NIH3T3 cells, we showed that Lis1 knockdown (KD) by RNA interference (RNAi) caused reductions in cell migration and traction force against the extracellular matrix (ECM). Immunostaining of cytoskeletal components in Lis1 KD cells showed disorganization of microtubules and actin filaments. Interestingly, focal adhesions at the cell periphery were significantly reduced. These results suggest that Lis1 is important for cellular traction force generation through the regulation of cytoskeleton organization and focal adhesion formation in migrating cells.

Caveolin-1 Controls Hyperresponsiveness to Mechanical Stimuli and Fibrogenesis-Associated RUNX2 Activation in Keloid Fibroblasts.

Keloids are pathological scars characterized by excessive extracellular matrix production that are prone to form in body sites with increased skin tension. CAV1, the principal coat protein of caveolae, has been associated with the regulation of cell mechanics, including cell softening and loss of stiffness sensing ability in NIH3T3 fibroblasts. Although CAV1 is present in low amounts in keloid fibroblasts (KFs), the causal association between CAV1 down-regulation and its aberrant responses to mechanical stimuli remain unclear. In this study, atomic force microscopy showed that KFs were softer than normal fibroblasts with a loss of stiffness sensing. The decrease of CAV1 contributed to the hyperactivation of fibrogenesis-associated RUNX2, a transcription factor germane to osteogenesis/chondrogenesis, and increased migratory ability in KFs. Treatment of KFs with trichostatin A, which increased the acetylation level of histone H3, increased CAV1 and decreased RUNX2 and fibronectin. Trichostatin A treatment also resulted in cell stiffening and decreased migratory ability in KFs. Collectively, these results suggest a role for CAV1 down-regulation in linking the aberrant responsiveness to mechanical stimulation and extracellular matrix accumulation with the progression of keloids, findings that may lead to new developments in the prevention and treatment of keloid scarring.

DDR1 promotes E-cadherin stability via inhibition of integrin-ß1-Src activation-mediated E-cadherin endocytosis.

Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase of collagen, is primarily expressed in epithelial cells. Activation of DDR1 stabilises E-cadherin located on the cell membrane; however, the detailed mechanism of DDR1-stabilised E-cadherin remains unclear. We performed DDR1 knockdown (Sh-DDR1) on Mardin-Darby canine kidney cells to investigate the mechanism of DDR1-stabilised E-cadherin. Sh-DDR1 decreased junctional localisation, increased endocytosis of E-cadherin, and increased physical interactions between E-cadherin and clathrin. Treatment of the dynamin inhibitor Dyngo 4a suppressed Sh-DDR1-induced E-cadherin endocytosis. In addition, the phosphorylation level of Src tyrosine 418 was increased in Sh-DDR1 cell junctions, and inhibition of Src activity decreased Sh-DDR1-induced E-cadherin endocytosis. To characterise the molecular mechanisms, blocking integrin ß1 decreased Src activity and E-cadherin junctional localisation in Sh-DDR1 cells. Photoconversion results showed that inhibition of Src activity rescued E-cadherin membrane stability and that inhibition of integrin ß1-Src signalling decreased stress fibres and rescued E-cadherin membrane stability in Sh-DDR1 cells. Taken together, DDR1 stabilised membrane localisation of E-cadherin by inhibiting the integrin ß1-Src-mediated clathrin-dependent endocytosis pathway.

Spatial distribution of filament elasticity determines the migratory behaviors of a cell.

Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.

Cordycepin induced MA-10 mouse Leydig tumor cell apoptosis by regulating p38 MAPKs and PI3K/AKT signaling pathways.

The p38 MAPKs play important roles in the regulation of balance between cell survival and cell death on the development of various cancers. However, the roles of p38 MAPKs regulating apoptotic effects on Leydig tumor cells remain unclear. In the present study, we showed that cordycepin (3'-deoxyadenosine) selectively induced apoptosis in MA-10 mouse Leydig tumor cells through regulating the p38 MAPK and PI3K/AKT signaling pathways. Cordycepin reduced viability in MA-10, TM4, and NT2/D1 cells, but not cause cell death of primary mouse Leydig cells on moderate concentration. Cordycepin increased reactive oxygen species (ROS) levels, which is associated with the induction of apoptosis as characterized by positive Annexin V binding, activation of caspase-3, and cleavage of PARP. Inhibition of p38 MAPKs activity by SB203580 significantly prevented cordycepin-induced apoptosis in MA-10 cells. Co-treatment with wortmannin or the autophagy inhibitor 3-methyladenine (3-MA) elevated levels of apoptosis in cordycepin-treated MA-10 cells. Moreover, cordycepin activated p53, p21 and TGFß; and downregulated CDK2. The antitumour activity of cordycepin-treated MA-10 cells was significantly distinct in severe combined immunodeficiency (SCID) mice in vivo. These results suggested that cordycein is a highly selective treatment to induce MA-10 cells apoptosis via p38 MAPKs signaling.

Mechanical phenotype of cancer cells: cell softening and loss of stiffness sensing.

The stiffness sensing ability is required to respond to the stiffness of the matrix. Here we determined whether normal cells and cancer cells display distinct mechanical phenotypes. Cancer cells were softer than their normal counterparts, regardless of the type of cancer (breast, bladder, cervix, pancreas, or Ha-RasV12-transformed cells). When cultured on matrices of varying stiffness, low stiffness decreased proliferation in normal cells, while cancer cells and transformed cells lost this response. Thus, cancer cells undergo a change in their mechanical phenotype that includes cell softening and loss of stiffness sensing. Caveolin-1, which is suppressed in many tumor cells and in oncogene-transformed cells, regulates the mechanical phenotype. Caveolin-1-upregulated RhoA activity and Y397FAK phosphorylation directed actin cap formation, which was positively correlated with cell elasticity and stiffness sensing in fibroblasts. Ha-RasV12-induced transformation and changes in the mechanical phenotypes were reversed by re-expression of caveolin-1 and mimicked by the suppression of caveolin-1 in normal fibroblasts. This is the first study to describe this novel role for caveolin-1, linking mechanical phenotype to cell transformation. Furthermore, mechanical characteristics may serve as biomarkers for cell transformation.

Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation.

Modulations of cytoskeletal organization and focal adhesion turnover correlate to tumorigenesis and epithelial-mesenchymal transition (EMT), the latter process accompanied by the loss of epithelial markers and the gain of mesenchymal markers (e.g., vimentin). Clinical microarray results demonstrated that increased levels of vimentin mRNA after chemotherapy correlated to a poor prognosis of breast cancer patients. We hypothesized that vimentin mediated the reorganization of cytoskeletons to maintain the mechanical integrity in EMT cancer cells. By using knockdown strategy, the results showed reduced cell proliferation, impaired wound healing, loss of directional migration, and increased large membrane extension in MDA-MB 231 cells. Vimentin depletion also induced reorganization of cytoskeletons and reduced focal adhesions, which resulted in impaired mechanical strength because of reduced cell stiffness and contractile force. In addition, overexpressing vimentin in MCF7 cells increased cell stiffness, elevated cell motility and directional migration, reoriented microtubule polarity, and increased EMT phenotypes due to the increased ß1-integrin and the loss of junction protein E-cadherin. The EMT-related transcription factor slug was also mediated by vimentin. The current study demonstrated that vimentin serves as a regulator to maintain intracellular mechanical homeostasis by mediating cytoskeleton architecture and the balance of cell force generation in EMT cancer cells.

ß-PIX controls intracellular viscoelasticity to regulate lung cancer cell migration.

Cancer metastasis occurs via a progress involving abnormal cell migration. Cell migration, a dynamic physical process, is controlled by the cytoskeletal system, which includes the dynamics of actin organization and cellular adhesive organelles, focal adhesions (FAs). However, it is not known whether the organization of actin cytoskeletal system has a regulatory role in the physiologically relevant aspects of cancer metastasis. In the present studies, it was found that lung adenocarcinoma cells isolated from the secondary lung cancer of the lymph nodes, H1299 cells, show specific dynamics in terms of the actin cytoskeleton and FAs. This results in a higher level of mobility and this is regulated by an immature FA component, ß-PIX (PAK-interacting exchange factor-ß). In H1299 cells, ß-PIX's activity was found not to be down-regulated by sequestration onto stress fibres, as the cells did not bundle actin filaments into stress fibres. Thus, ß-PIX mainly remained localized at FAs, which allowed maturation of nascent adhesions into focal complexes; this resulted in actin polymerization, increased actin network integrity, changes in the intracellular microrheology at the peripheral of the cell, and cell polarity, which in turn regulated cell migration. Perturbation of ß-PIX caused an inhibition of cell migration, including migration velocity, accumulated distance and directional persistence. Our results demonstrate the importance of ß-PIX to the regulation of high mobility of lung adenocarcinoma cell line H1299 and that this occurs via regulation of FA dynamics, changes in actin cytoskeleton organization and cell polarity.

GEF-H1 controls focal adhesion signaling that regulates mesenchymal stem cell lineage commitment.

Focal adhesions (FAs) undergo maturation that culminates in size and composition changes that modulate adhesion, cytoskeleton remodeling and differentiation. Although it is well recognized that stimuli for osteogenesis of mesenchymal stem cells (MSCs) drive FA maturation, actin organization and stress fiber polarization, the extent to which FA-mediated signals regulated by the FA protein composition specifies MSC commitment remains largely unknown. Here, we demonstrate that, upon dexamethasone (osteogenic induction) treatment, guanine nucleotide exchange factor H1 (GEF-H1, also known as Rho guanine nucleotide exchange factor 2, encoded by ARHGEF2) is significantly enriched in FAs. Perturbation of GEF-H1 inhibits FA formation, anisotropic stress fiber orientation and MSC osteogenesis in an actomyosin-contractility-independent manner. To determine the role of GEF-H1 in MSC osteogenesis, we explore the GEF-H1-modulated FA proteome that reveals non-muscle myosin-II heavy chain-B (NMIIB, also known as myosin-10, encoded by MYH10) as a target of GEF-H1 in FAs. Inhibition of targeting NMIIB into FAs suppresses FA formation, stress fiber polarization, cell stiffness and osteogenic commitments in MSCs. Our data demonstrate a role for FA signaling in specifying MSC commitment.

Midazolam induces apoptosis in MA-10 mouse Leydig tumor cells through caspase activation and the involvement of MAPK signaling pathway.

PURPOSE: The present study aims to investigate how midazolam, a sedative drug for clinical use with cytotoxicity on neuronal and peripheral tissues, induced apoptosis in MA-10 mouse Leydig tumor cells. METHODS: The apoptotic effect and underlying mechanism of midazolam to MA-10 cells were investigated by flow cytometry assay and Western blotting methods. RESULTS: Data showed that midazolam induced the accumulation of the MA-10 cell population in the sub-G1 phase and a reduction in the G2/M phase in a time- and dose-dependent manner, suggesting an apoptotic phenomenon. Midazolam could also induce the activation of caspase-8, -9, and -3 and poly (ADP-ribose) polymerase proteins. There were no changes in the levels of Bax and cytochrome-c, whereas Bid was significantly decreased after midazolam treatment. Moreover, midazolam decreased both pAkt and Akt expression. In addition, midazolam stimulated the phosphorylation of p38 and c-Jun NH2-terminal kinase but not extracellular signal-regulated kinase. CONCLUSION: Midazolam could induce MA-10 cell apoptosis through the activation of caspase cascade, the inhibition of pAkt pathway, and the induction of p38 and c-Jun NH2-terminal kinase pathways.

Ionizing radiation induces autophagy in human oral squamous cell carcinoma.

PURPOSE: Irradiation-induced autophagy has been reported in several types of cancers, however, the relationship between irradiation and autophagy in human oral squamous cell carcinoma (OSCC) has not yet been described. In this study we investigated the induction of autophagy in cell lines by exposing them to ionizing irradiation. METHODS: Human OSCC OC3 and SAS cell lines were used in this study. Cell viability and induction of autophagy were determined under irradiation treatment. The GFP-LC3 puncta formation and the levels of LC3-II as indicators of autophagy were detected by fluorescence microscopy and Western blot method. The signaling pathways involved in irradiation-mediated autophagy were also determined by Western blot method. RESULTS: Irradiation decreased cell viability only in OC3 cells, while autophagic machinery and related signaling pathways were found to be elevated after irradiation in OC3 and SAS cells. However, autophagic degradation determined by the reduction of p62 levels was only found in OC3 cells, suggesting autophagosome accumulation took place in SAS cells. In addition, irradiation accompanied with rapamycin treatment elevated autophagy formation and induced death of OC3 cells. CONCLUSIONS: These results suggested that induction of autophagy might provide an advantageous strategy to increase the anticancer effects of radiotherapy in patients with OSCCs.

Cell adhesion and mechanical stimulation in the regulation of mesenchymal stem cell differentiation.

Stem cells have been shown to have the potential to provide a source of cells for applications to tissue engineering and organ repair. The mechanisms that regulate stem cell fate, however, mostly remain unclear. Mesenchymal stem cells (MSCs) are multipotent progenitor cells that are isolated from bone marrow and other adult tissues, and can be differentiated into multiple cell lineages, such as bone, cartilage, fat, muscles and neurons. Although previous studies have focused intensively on the effects of chemical signals that regulate MSC commitment, the effects of physical/mechanical cues of the microenvironment on MSC fate determination have long been neglected. However, several studies provided evidence that mechanical signals, both direct and indirect, played important roles in regulating a stem cell fate. In this review, we summarize a number of recent studies on how cell adhesion and mechanical cues influence the differentiation of MSCs into specific lineages. Understanding how chemical and mechanical cues in the microenvironment orchestrate stem cell differentiation may provide new insights into ways to improve our techniques in cell therapy and organ repair.