Bufalin Induces Reactive Oxygen Species Dependent Bax Translocation and Apoptosis in ASTC-a-1 Cells

Bufalin has been shown to induce cancer cell death through apoptotic pathways. However, the molecular mechanisms are not well understood. In this study, we used the confocal fluorescence microscopy (CFM) to monitor the spatio-temporal dynamics of reactive oxygen species (ROS) production, Bax translocation and caspase-3 activation during bufalin-induced apoptosis in living human lung adenocarcinoma (ASTC-a-1) cells. Bufalin induced ROS production and apoptotic cell death, demonstrated by Hoechst 33258 staining as well as flow cytometry analysis. Bax redistributed from cytosol to mitochondria from 12 to 48 h after bufalin treatment in living cells expressed with green fluorescent protein Bax. Treatment with the antioxidant N-acetyl-cysteine (NAC), a ROS scavenger, inhibited ROS generation and Bax translocation and led to a significant protection against bufalin-induced apoptosis. Our results also revealed that bufalin induced a prominent increase of caspase-3 activation blocked potently by NAC. Taken together, bufalin induced ROS-mediated Bax translocation, mitochondrial permeability transition and caspase-3 activation, implying that bufalin induced apoptosis via ROS-dependent mitochondrial death pathway in ASTC-a-1 cells

Image informatics for studying signal transduction in cells interacting with 3D matrices

Cells sense and respond to chemical stimuli on their environment via signal transduction pathways, complex networks of proteins whose interactions transmit chemical information. This work describes an implementation of image informatics, imaging-based methodologies for studying signal transduction networks. The methodology developed focuses on studying signal transduction networks in cells that interact with 3D matrices. It utilizes shRNA-based knock down of network components, 3D high-content imaging of cells inside the matrix by spectral multi-photon microscopy, and single-cell quantification using features that describe both cell morphology and cell-matrix adhesion pattern. The methodology is applied in a pilot study of TGFβ signaling via the SMAD pathway in fibroblasts cultured inside porous collagen-GAG scaffolds, biomaterials similar to the ones used clinically to induce skin regeneration. Preliminary results suggest that knocking down all rSMAD components affects fibroblast response to TGFβ1 and TGFβ3 isoforms in different ways, and suggest a potential role for SMAD1 and SMAD5 in regulating TGFβ isoform response. These preliminary results need to be verified with proteomic results that can provide solid evidence about the particular role of individual components of the SMAD pathway.National Institutes of Health (U.S.) (RO1 NS051320)Singapore-MIT Alliance for Research and Technolog

Photoacoustic tomography with novel optical contrast agents based on gold nanocages or nanoparticles containing near-infrared dyes

Poly(ethylene glycol)-coated Au nanocages have been evaluated as a potential near-infrared (NIR) contrast agent for photoacoustic tomography (PAT). Previously, Au nanoshells were found to be an effective NIR contrast agent for PAT; however, Au nanocages, with their more compact sizes (100 nm for Au nanoshells) and larger optical absorption cross-sections, should be better suited for in vivo applications. In this study, we tested Au nanocages as a contrast agent for PAT. The result suggests that Au nanocages are promising contrast agents for our applications. We also present PAT results when novel, dye-containing nanoparticles are used as contrast agents

Mesenchymal stem cells alleviate bacteria‐induced liver injury in mice by inducing regulatory dendritic cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102718/1/hep26670.pd

Rapid direct aperture optimization via dose influence matrix based piecewise aperture dose model.

In the traditional two-step procedure used in intensity-modulated radiation therapy, fluence map optimization (FMO) is performed first, followed by use of a leaf sequencing algorithm (LSA). By contrast, direct aperture optimization (DAO) directly optimizes aperture leaf positions and weights. However, dose calculation using the Monte Carlo (MC) method for DAO is often time-consuming. Therefore, a rapid DAO (RDAO) algorithm is proposed that uses a dose influence matrix based piecewise aperture dose model (DIM-PADM). In the proposed RDAO algorithm, dose calculation is based on the dose influence matrix instead of MC. The dose dependence of aperture leafs is modeled as a piecewise function using the DIM. The corresponding DIM-PADM-based DAO problem is solved using a simulated annealing algorithm.The proposed algorithm was validated through application to TG119, prostate, liver, and head and neck (H&N) cases from the common optimization for radiation therapy dataset. Compared with the two-step FMO-LSA procedure, the proposed algorithm resulted in more precise dose conformality in all four cases. Specifically, for the H&N dataset, the cost value for the planned target volume (PTV) was decreased by 32%, whereas the cost value for the two organs at risk (OARs) was decreased by 60% and 92%. Our study of the proposed novel DIM-PADM-based RDAO algorithm makes two main contributions: First, we validate the use of the proposed algorithm, in contrast to the FMO-LSA framework, for direct optimization of aperture leaf positions and show that this method results in more precise dose conformality. Second, we demonstrate that compared to MC, the DIM-PADM-based method significantly reduces the computational time required for DAO

Selective cell targeting with light-absorbing microparticles and nanoparticles

We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manne

Taxol induces paraptosis independent of both protein synthesis and MAPK pathway

Our recent studies have shown that high concentration of taxol induced a caspase-independent paraptosis-like cell death and cytoplasmic vacuolization derived predominantly from endoplasmic reticulum (ER) swelling in human lung carcinoma cell lines (ASTC-a-1). In this report, we further explored the relationship between taxol-induced cell death and vacuolization, and the roles of protein synthesis, mitogen-activated protein kinase kinases (MEK), c-jun N-terminal kinase (JNK) and P38 in taxol-induced paraptosis. Enhanced green fluorescent protein (EGFP) was used to probe the cell morphological change, while ER-targeted red fluorescent protein (er-RFP) was used to probe ER spatial distribution. Real-time monitoring of the ER swelling dynamics during the formation of vacuolization inside single living cells co-expressing EGFP and er-RFP further demonstrated that taxol-induced cytoplasmic vacuolization was from the ER restructuring due to fusion and swelling. PI staining showed that taxol-induced vacuolization was not necrosis. These results further demonstrated that the taxol-induced cell death was neither apoptosis nor necrosis, and fitted the criteria of paraptosis characterized by cytoplasmic vacuolization, caspase-independence, lack of apoptotic morphology and insensitivity to broad caspase inhibitor. Our data further indicated that taxol-induced paraptosis required neither protein synthesis nor the participation of MEK, JNK, and P38, which was different from the insulin-like growth factor I receptor (IGFIR)-induced paraptosis. These results suggest that high concentration of taxol activates an alternative paraptotic cell death pathway

Photobleaching-based quantitative analysis of fluorescence resonance energy transfer inside single living cell

The current advances of fluorescence microscopy and new fluorescent probes make fluorescence resonance energy transfer (FRET) a powerful technique for studying protein-protein interactions inside living cells. It is very hard to quantitatively analyze FRET efficiency using intensitybased FRET imaging microscopy due to the presence of autofluorescence and spectral crosstalks. In this study, we for the first time developed a novel photobleaching-based method to quantitatively detect FRET efficiency (Pb-FRET) by selectively photobleaching acceptor. The Pb-FRET method requires two fluorescence detection channels: a donor channel (CH1) to selectively detect the fluorescence from donor, and a FRET channel (CH2) which normally includes the fluorescence from both acceptor and donor due to emission spectral crosstalk. We used the Pb-FRET method to quantitatively measure the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside single living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by Pb-FRET inside living cells was verified by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM). The temporal resolution of Pb-FRET method is in second time-scale for ROI photobleaching, even in microsecond time-scale for spot photobleaching. Our results demonstrate that the Pb- FRET method is independent of photobleaching degree, and is very useful for quantitatively monitoring proteinprotein interactions inside single living cell