Inhibition of branched-chain alpha-keto acid dehydrogenase kinase augments the sensitivity of ovarian and breast cancer cells to paclitaxel.

CONTEXT: Many cancer patients who initially respond to chemotherapy eventually develop chemoresistance, and to address this, we previously conducted a RNAi screen to identify genes contributing to resistance. One of the hits from the screen was branched-chain α-keto acid dehydrogenase kinase (BCKDK). BCKDK controls the metabolism of branched-chain amino acids (BCAAs) through phosphorylation and inactivation of the branched-chain α-keto acid dehydrogenase complex (BCKDH), thereby inhibiting catabolism of BCAAs. METHODS: We measured the impact on paclitaxel sensitivity of inhibiting BCKDK in ovarian and breast cancer cell lines. RESULTS: Inhibition of BCKDK using siRNA or two chemical inhibitors (BCKDKi) was synergistic with paclitaxel in both breast and ovarian cancer cells. BCKDKi reduced levels of BCAA and the addition of exogenous BCAA suppressed this synergy. BCKDKi inactivated the mTORC1-Aurora pathway, allowing cells to overcame M-phase arrest induced by paclitaxel. In some cases, cells almost completed cytokinesis, then reverted to a single cell, resulting in multinucleate cells. CONCLUSION: BCKDK is an attractive target to augment the sensitivity of cancer cells to paclitaxel.

Predicting Bone Formation in Mesenchymal Stromal Cell-Seeded Hydrogels Using Experiment-Based Mathematical Modeling.

In vitro bone formation by mesenchymal stromal cells encapsulated in type-1 collagen hydrogels is demonstrated after a 28-day in vitro culture period. Analysis of the hydrogels is carried out by X-ray microcomputed tomography, histology, and immunohistochemistry, which collectively demonstrates that bone formation in the hydrogels was quantifiably proportional to the initial collagen concentration, and subsequently the population density of seeded cells. This was established by varying the initial collagen concentration at a constant cell seeding density (3 × 105 cells/0.3 mL hydrogel), and separately varying cell seeding density at a constant collagen concentration (1 mg/mL). Using these data, a mathematical model is presented for the total hydrogel volume and mineralization volume based on the observed linear contraction dynamics of cell-seeded collagen gels. The model parameters are fitted by comparing the predictions of the mathematical model for the hydrogel and mineralized volumes on day 28 with the experimental data. The model is then used to predict the hydrogel and mineralization volumes for a range of hydrogel collagen concentrations and cell seeding densities, providing comprehensive input/output descriptors for generating mineralized hydrogels for bone tissue engineering. It is proposed that this quantitative approach will be a useful tool for generating in vitro manufactured bone tissue, defining input parameters that yield predictable output measures of tissue maturation. Impact statement This article describes a simple yet powerful quantitative description of in vitro tissue-engineered bone by combining experimental data with mathematical modeling. The overall aim of the article is to examine what is currently known about cell-mediated collagen contraction, and demonstrate that this phenomenon can be exploited to tailor bone formation by choosing a specific set of input parameters in the form of cell seeding density and collagen hydrogel concentration. Our study utilizes a clinically relevant cell source (human mesenchymal stem cells) with a biomaterial that has received regulatory approval for use in humans (collagen type 1), and hence could be useful for clinical applications, as well as furthering our understanding of cell/extracellular matrix interactions in determining in vitro bone tissue formation.

Quantum Sensing in a Physiological-Like Cell Niche Using Fluorescent Nanodiamonds Embedded in Electrospun Polymer Nanofibers.

Fluorescent nanodiamonds (fNDs) containing nitrogen vacancy (NV) centers are promising candidates for quantum sensing in biological environments. This work describes the fabrication and implementation of electrospun poly lactic-co-glycolic acid (PLGA) nanofibers embedded with fNDs for optical quantum sensing in an environment, which recapitulates the nanoscale architecture and topography of the cell niche. A protocol that produces uniformly dispersed fNDs within electrospun nanofibers is demonstrated and the resulting fibers are characterized using fluorescent microscopy and scanning electron microscopy (SEM). Optically detected magnetic resonance (ODMR) and longitudinal spin relaxometry results for fNDs and embedded fNDs are compared. A new approach for fast detection of time varying magnetic fields external to the fND embedded nanofibers is demonstrated. ODMR spectra are successfully acquired from a culture of live differentiated neural stem cells functioning as a connected neural network grown on fND embedded nanofibers. This work advances the current state of the art in quantum sensing by providing a versatile sensing platform that can be tailored to produce physiological-like cell niches to replicate biologically relevant growth environments and fast measurement protocols for the detection of co-ordinated endogenous signals from clinically relevant populations of electrically active neuronal circuits.

Immobilized WNT Proteins Act as a Stem Cell Niche for Tissue Engineering.

The timing, location, and level of WNT signaling are highly regulated during embryonic development and for the maintenance of adult tissues. Consequently the ability to provide a defined and directed source of WNT proteins is crucial to fully understand its role in tissue development and to mimic its activity in vitro. Here we describe a one-step immobilization technique to covalently bind WNT3A proteins as a basal surface with easy storage and long-lasting activity. We show that this platform is able to maintain adult and embryonic stem cells while also being adaptable for 3D systems. Therefore, this platform could be used for recapitulating specific stem cell niches with the goal of improving tissue engineering.

The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkalinization.

Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal alkalinization contributes to the cytotoxic activity of obatoclax.

Liquid Crystalline Ordered Collagen Substrates for Applications in Tissue Engineering.

This report describes methods for fabricating substrates with anisotropic order from a single solution of high concentration collagen. By exploiting the intrinsic property of collagen to behave as a cholesteric liquid crystal, we demonstrate first the production of dense collagen films containing anisotropic fibers by simple dialysis and polymerization in ammonia vapor. We then utilized shear driven alignment of collagen using viscous extrusion to produce aligned collagen fibers. Next we describe an evaporation technique to observe crystalline growth into the collagen, which serves to template the substrate prior to fibrillogenesis. The ordered substrates supported osteogenic differentiation of hMSCs and also oriented growth of hMSCs. We also demonstrate using Raman spectroscopy that the local protein concentration in the substrates influenced the molecular orientation of collagen, Finally, we compare the resultant textures in the substrates with section of native cornea and tendon using polarized light microscopy, which showed remarkable similarities in terms of both anisotropy and second order chiral structure. These rapid, cost-effective methods could potentially serve a range of different applications in tissue engineering.