Miniature Dielectric Barrier Discharge Nonthermal Plasma Induces Apoptosis in Lung Cancer Cells and Inhibits Cell Migration

Traditional cancer treatments like radiotherapy and chemotherapy have drawbacks and are not selective for killing only cancer cells. Nonthermal atmospheric pressure plasmas with dielectric barrier discharge (DBD) can be applied to living cells and tissues and have emerged as novel tools for localized cancer therapy. The purpose of this study was to investigate the different effects caused by miniature DBD (mDBD) plasma to A549 lung cancer cells. In this study, A549 lung cancer cells cultured in 12 well plates were treated with mDBD plasma for specified treatment times to assess the changes in the size of the area of cell detachment, the viability of attached or detached cells, and cell migration. Furthermore, we investigated an innovative mDBD plasma-based therapy for localized treatment of lung cancer cells through apoptotic induction. Our results indicate that plasma treatment for 120 sec causes apoptotic cell death in 35.8% of cells, while mDBD plasma treatment for 60 sec, 30 sec, or 15 sec causes apoptotic cell death in 20.5%, 14.1%, and 6.3% of the cell population, respectively. Additionally, we observed reduced A549 cell migration in response to mDBD plasma treatment. Thus, mDBD plasma system can be a viable platform for localized lung cancer therapy

The Cytoskeleton Effectors Rho-Kinase (ROCK) and Mammalian Diaphanous-Related (mDia) Formin Have Dynamic Roles in Tumor Microtube Formation in Invasive Glioblastoma Cells

Glioblastoma (GBM) is a progressive and lethal brain cancer. Malignant control of actin and microtubule cytoskeletal mechanics facilitates two major GBM therapeutic resistance strategies—diffuse invasion and tumor microtube network formation. Actin and microtubule reorganization is controlled by Rho-GTPases, which exert their effects through downstream effector protein activation, including Rho-associated kinases (ROCK) 1 and 2 and mammalian diaphanous-related (mDia) formins (mDia1, 2, and 3). Precise spatial and temporal balancing of the activity between these effectors dictates cell shape, adhesion turnover, and motility. Using small molecules targeting mDia, we demonstrated that global agonism (IMM02) was superior to antagonism (SMIFH2) as anti-invasion strategies in GBM spheroids. Here, we use IDH-wild-type GBM patient-derived cell models and a novel semi-adherent in vitro system to investigate the relationship between ROCK and mDia in invasion and tumor microtube networks. IMM02-mediated mDia agonism disrupts invasion in GBM patient-derived spheroid models, in part by inducing mDia expression loss and tumor microtube network collapse. Pharmacological disruption of ROCK prevented invasive cell-body movement away from GBM spheres, yet induced ultralong, phenotypically abnormal tumor microtube formation. Simultaneously targeting mDia and ROCK did not enhance the anti-invasive/-tumor microtube effects of IMM02. Our data reveal that targeting mDia is a viable GBM anti-invasion/-tumor microtube networking strategy, while ROCK inhibition is contraindicated

Targeting the mDia Formin-Assembled Cytoskeleton Is an Effective Anti-Invasion Strategy in Adult High-Grade Glioma Patient-Derived Neurospheres

High-grade glioma (HGG, WHO Grade III–IV) accounts for the majority of adult primary malignant brain tumors. Failure of current therapies to target invasive glioma cells partly explains the minimal survival advantages: invasive tumors lack easily-defined surgical margins, and are inherently more chemo- and radioresistant. Much work centers upon Rho GTPase-mediated glioma invasion, yet downstream Rho effector roles are poorly understood and represent potential therapeutic targets. The roles for the mammalian Diaphanous (mDia)-related formin family of Rho effectors have emerged in invasive/metastatic disease. mDias assemble linear F-actin to promote protrusive cytoskeletal structures underlying tumor cell invasion. Small molecule mDia intramimic (IMM) agonists induced mDia functional activities including F-actin polymerization. mDia agonism inhibited polarized migration in Glioblastoma (WHO Grade IV) cells in three-dimensional (3D) in vitro and rat brain slice models. Here, we evaluate whether clinically-relevant high-grade glioma patient-derived neuro-sphere invasion is sensitive to formin agonism. Surgical HGG samples were dissociated, briefly grown as monolayers, and spontaneously formed non-adherent neuro-spheres. IMM treatment dramatically inhibited HGG patient neuro-sphere invasion, both at neuro-sphere embedding and mid-invasion assay, inducing an amoeboid morphology in neuro-sphere edge cells, while inhibiting actin- and tubulin-enriched tumor microtube formation. Thus, mDia agonism effectively disrupts multiple aspects of patient-derived HGG neuro-sphere invasion

A microwave-assisted solution combustion synthesis to produce europium-doped calcium phosphate nanowhiskers for bioimaging applications

Biocompatible nanoparticles possessing fluorescent properties offer attractive possibilities for multifunctional bioimaging and/or drug and gene delivery applications. Many of the limitations with current imaging systems center on the properties of the optical probes in relation to equipment technical capabilities. Here we introduce a novel high aspect ratio and highly crystalline europium-doped calcium phosphate nanowhisker produced using a simple microwave-assisted solution combustion synthesis method for use as a multifunctional bioimaging probe. X-ray diffraction confirmed the material phase as europium-doped hydroxyapatite. Fluorescence emission and excitation spectra and their corresponding peaks were identified using spectrofluorimetry and validated with fluorescence, confocal and multiphoton microscopy. The nanowhiskers were found to exhibit red and far red wavelength fluorescence under ultraviolet excitation with an optimal peak emission of 696 nm achieved with a 350 nm excitation. Relatively narrow emission bands were observed, which may permit their use in multicolor imaging applications. Confocal and multiphoton microscopy confirmed that the nanoparticles provide sufficient intensity to be utilized in imaging applications

Dia-Interacting Protein (DIP) Imposes Migratory Plasticity in mDia2-Dependent Tumor Cells in Three-Dimensional Matrices

<div><p>Tumor cells rely upon membrane pliancy to escape primary lesions and invade secondary metastatic sites. This process relies upon localized assembly and disassembly cycles of F-actin that support and underlie the plasma membrane. Dynamic actin generates both spear-like and bleb structures respectively characterizing mesenchymal and amoeboid motility programs utilized by metastatic cells in three-dimensional matrices. The molecular mechanism and physiological trigger(s) driving membrane plasticity are poorly understood. mDia formins are F-actin assembly factors directing membrane pliancy in motile cells. mDia2 is functionally coupled with its binding partner DIP, regulating cortical actin and inducing membrane blebbing in amoeboid cells. Here we show that mDia2 and DIP co-tether to nascent blebs and this linkage is required for bleb formation. DIP controls mesenchymal/amoeboid cell interconvertability, while CXCL12 induces assembly of mDia2:DIP complexes to bleb cortices in 3D matrices. These results demonstrate how DIP-directed mDia2-dependent F-actin dynamics regulate morphological plasticity in motile cancer cells.</p> </div

An mDia2/ROCK Signaling Axis Regulates Invasive Egress from Epithelial Ovarian Cancer Spheroids

<div><p>Multi-cellular spheroids are enriched in ascites of epithelial ovarian cancer (OvCa) patients. They represent an invasive and chemoresistant cellular population fundamental to metastatic dissemination. The molecular mechanisms triggering single cell invasive egress from spheroids remain enigmatic. mDia formins are Rho GTPase effectors that are key regulators of F-actin cytoskeletal dynamics. We hypothesized that mDia2-driven F-actin dynamics promote single cell invasive transitions in clinically relevant three-dimensional (3D) OvCa spheroids. The current study is a dissection of the contribution of the F-actin assembly factor mDia2 formin in invasive transitions and using a clinically relevant ovarian cancer spheroid model. We show that RhoA-directed mDia2 activity is required for tight spheroid organization, and enrichment of mDia2 in the invasive cellular protrusions of collagen-embedded OVCA429 spheroids. Depleting mDia2 in ES-2 spheroids enhanced invasive dissemination of single amoeboid-shaped cells. This contrasts with spheroids treated with control siRNA, where a mesenchymal invasion program predominated. Inhibition of another RhoA effector, ROCK, had no impact on ES-2 spheroid formation but dramatically inhibited spheroid invasion through induction of a highly elongated morphology. Concurrent inhibition of ROCK and mDia2 blocked single cell invasion from ES-2 spheroids more effectively than inhibition of either protein alone, indicating that invasive egress of amoeboid cells from mDia2-depleted spheroids is ROCK-dependent. Our findings indicate that multiple GTPase effectors must be suppressed in order to fully block invasive egress from ovarian cancer spheroids. Furthermore, tightly regulated interplay between ROCK and mDia2 signaling pathways dictates the invasive capacities and the type of invasion program utilized by motile spheroid-derived ovarian cancer cells. As loss of the gene encoding mDia2, <i>DRF3,</i> has been linked to cancer progression and metastasis, our results set the stage for understanding molecular mechanisms involved in mDia2-dependent egress of invasive cells from primary epithelial tumors.</p></div

DIP LRR induces amoeboid motility in invading mesenchymal breast cancer cells.

<p>(A, B) MDA-MB-231 cells were treated with DMSO vehicle, 20 µm MMP-inhibitor GM6001 or were transfected with the indicated CFP-fusion proteins and were embedded in type-I collagen gels and stained as above. Insets validate the CFP-transfection. (C) Elongation indices were calculated as in 3C. (D) MDA-MB-231 cells transfected with either CFP-DIP LRR or CFP-DIP LRR N555A for 48 hrs were embedded in collagen gels overnight, stained with phalloidin and anti-pMLC2 antibodies. (E) MDA-MB-231 cells transfected for 48 hrs with CFP, CFP-DIP LRR or CFP-DIP LRR treated with 90 µM Y27632 reverse-invaded for 24 hrs through transwells coated with collagen I gels. Upon fixation, transwells were imaged by confocal microscopy. CFP pixels were calculated for 10 µm optical slices.</p

Melanoma chondroitin sulphate proteoglycan regulates cell spreading through Cdc42, Ack-1 and p130\u3csup\u3ecas\u3c/sup\u3e

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumors. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-α4β1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine- phosphorylated p130cas, an adaptor protein important in tumor cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130cas. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130cas. Our findings indicate that MCSP may modify tumor growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization