pH-Responsive Artemisinin Derivatives and Lipid Nanoparticle Formulations Inhibit Growth of Breast Cancer Cells In Vitro and Induce Down-Regulation of HER Family Members

Artemisinin (ART) dimers show potent anti-proliferative activities against breast cancer cells. To facilitate their clinical development, novel pH-responsive artemisinin dimers were synthesized for liposomal nanoparticle formulations. A new ART dimer was designed to become increasingly water-soluble as pH declines. The new artemisinin dimer piperazine derivatives (ADPs) remained tightly associated with liposomal nanoparticles (NPs) at neutral pH but were efficiently released at acidic pH''s that are known to exist within solid tumors and organelles such as endosomes and lysosomes. ADPs incorporated into nanoparticles down regulated the anti-apoptotic protein, survivin, and cyclin D1 when incubated at low concentrations with breast cancer cell lines. We demonstrate for the first time, for any ART derivative, that ADP NPs can down regulate the oncogenic protein HER2, and its counterpart, HER3 in a HER2+ cell line. We also show that the wild type epidermal growth factor receptor (EGFR or HER1) declines in a triple negative breast cancer (TNBC) cell line in response to ADP NPs. The declines in these proteins are achieved at concentrations of NP109 at or below 1 µM. Furthermore, the new artemisinin derivatives showed improved cell-proliferation inhibition effects compared to known dimer derivatives.     

Feeding tricarboxylic acid cycle intermediates improves lactate consumption and antibody production in Chinese hamster ovary cell cultures

Media components play an important role in modulating cell metabolism and improving product titer in mammalian cell cultures. To sustain cell productivity, highly active oxidative metabolism is desired. Here we explored the effect of tricarboxylic acid (TCA) cycle intermediates supplementation on lactate metabolism and productivity in Chinese hamster ovary fed-batch cultures. Direct addition of 5 mM alpha-ketoglutarate (α-KG), malic acid, or succinic acid in the basal medium did not have any significant impact on culture performance. On the other hand, feeding α-KG, malic acid, and succinic acid in the stationary phase, either as a single solution or as a mixture, significantly improved lactate consumption, reduced ammonium accumulation, and led to higher cell specific productivity and antibody titer (~35% increase for the best condition). Delivering those intermediates as an acidic solution for pH control eliminated CO₂ sparging and accumulation. Feeding TCA cycle intermediates was also demonstrated to be superior to feeding lactic acid or pyruvic acid in titer improvement. Taken together, feeding TCA cycle intermediates was effective in improving lactate consumption and increasing product titer, which is likely due to enhanced oxidative metabolism in an extended duration.     

Redox regulation of calcium ion channels: chemical and physiological aspects

Reactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease.Changes in the intracellular free Ca²⁺ concentration are key triggers for diverse cellular functions. Ca²⁺ homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological- and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca²⁺ permeable ion channels such as voltage-gated (CaV) Ca²⁺ channels, transient receptor potential (TRP) channels and Orai channels.     

PS II model based analysis of transient fluorescence yield measured on whole leaves of Arabidopsis thaliana after excitation with light flashes of different energies

Our recently presented PS II model (Belyaeva et al., 2008) was improved in order to permit a consistent simulation of Single Flash Induced Transient Fluorescence Yield (SFITFY) traces that were earlier measured by Steffen et al. (2005) on whole leaves of Arabidopsis (A.) thaliana at four different energies of the actinic flash. As the essential modification, the shape of the actinic flash was explicitly taken into account assuming that an exponentially decaying rate simulates the time dependent excitation of PS II by the 10 ns actinic flash. The maximum amplitude of this excitation exceeds that of the measuring light by 9 orders of magnitude. A very good fit of the SFITFY data was achieved in the time domain from 100 ns to 10 s for all actinic flash energies (the maximum energy of 7.5 × 10¹⁶ photons/(cm² flash) is set to 100%, the relative energies of weaker actinic flashes were of ∼8%, 4%, ∼1%). Our model allows the calculation and visualization of the transient PS II redox state populations ranging from the dark adapted state, via excitation energy and electron transfer steps induced by pulse excitation, followed by final relaxation into the stationary state eventually attained under the measuring light. It turned out that the rate constants of electron transfer steps are invariant to intensity of the actinic laser flash. In marked contrast, an increase of the actinic flash energy by more than two orders of magnitude from 5.4 × 10¹⁴ photons/(cm² flash) to 7.5 × 10¹⁶ photons/(cm² flash), leads to an increase of the extent of fluorescence quenching due to carotenoid triplet (³Car) formation by a factor of 14 and of the recombination reaction between reduced primary pheophytin (Phe⁻) and P680⁺ by a factor of 3 while the heat dissipation in the antenna complex remains virtually constant.The modified PS II model offers new opportunities to compare electron transfer and dissipative parameters for different species (e.g. for the green algae and the higher plant) under varying illumination conditions.     

Computer simulation of interaction of photosystem 1 with plastocyanin and ferredoxin

We designed 3D multiparticle computer models to simulate diffusion and interactions of spinach plastocyanin and ferredoxin with plant photosystem 1 in a solution. Using these models we studied kinetic characteristics of plastocyanin-photosystem 1 and ferredoxin-photosystem 1 complex formation at a variety of ionic strength values. The computer multiparticle models demonstrate non-monotonic dependences of complex formation rates on the ionic strength as the result of long-range electrostatic interactions. Our calculations show that the decrease in the association second-order rate constant at low values of the ionic strength is caused by the protein pairs spending more time in “wrong” orientations which do not satisfy the docking conditions and so do not form the final complex capable of the electron transfer.     

CHILD: a new tool for detecting low-abundance insertions and deletions in standard sequence traces

Several methods have been proposed for detecting insertion/deletions (indels) from chromatograms generated by Sanger sequencing. However, most such methods are unsuitable when the mutated and normal variants occur at unequal ratios, such as is expected to be the case in cancer, with organellar DNA or with alternatively spliced RNAs. In addition, the current methods do not provide robust estimates of the statistical confidence of their results, and the sensitivity of this approach has not been rigorously evaluated. Here, we present CHILD, a tool specifically designed for indel detection in mixtures where one variant is rare. CHILD makes use of standard sequence alignment statistics to evaluate the significance of the results. The sensitivity of CHILD was tested by sequencing controlled mixtures of deleted and undeleted plasmids at various ratios. Our results indicate that CHILD can identify deleted molecules present as just 5% of the mixture. Notably, the results were plasmid/primer-specific; for some primers and/or plasmids, the deleted molecule was only detected when it comprised 10% or more of the mixture. The false positive rate was estimated to be lower than 0.4%. CHILD was implemented as a user-oriented web site, providing a sensitive and experimentally validated method for the detection of rare indel-carrying molecules in common Sanger sequence reads.     

Dependence of Golgi apparatus integrity on nitric oxide in vascular cells: implications in pulmonary arterial hypertension

Although reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), its consequences on organellar structure and function within vascular cells is largely unexplored. We investigated the effect of reduced NO on the structure of the Golgi apparatus as assayed by giantin or GM130 immunofluorescence in human pulmonary arterial endothelial (HPAECs) and smooth muscle (HPASMCs) cells, bovine PAECs, and human EA.hy926 endothelial cells. Golgi structure was also investigated in cells in tissue sections of pulmonary vascular lesions in idiopathic PAH (IPAH) and in macaques infected with a chimeric simian immunodeficiency virus containing the human immunodeficiency virus (HIV)-nef gene (SHIV-nef) with subcellular three-dimensional (3D) immunoimaging. Compounds with NO scavenging activity including 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), methylene blue, N-acetylcysteine, and hemoglobin markedly fragmented the Golgi in all cell types evaluated as did monocrotaline pyrrole, while LY-83583, sildenafil, fasudil, Y-27632, Tiron, Tempol, or H(2)O(2) did not. Golgi fragmentation by NO scavengers was inhibited by diethylamine NONOate, was evident in HPAECs after selective knockdown of endothelial nitric oxide synthase using small interfering RNA (siRNA), was independent of microtubule organization, required the GTPase dynamin 2, and was accompanied by depletion of α-soluble N-ethylmaleimide-sensitive factor (NSF) acceptor protein (α-SNAP) from Golgi membranes and codispersal of the SNAP receptor (SNARE) Vti1a with giantin. Golgi fragmentation was confirmed in endothelial and smooth muscle cells in pulmonary arterial lesions in IPAH and the SHIV-nef-infected macaque with subcellular 3D immunoimaging. In SHIV-nef-infected macaques Golgi fragmentation was observed in cells containing HIV-nef-bearing endosomes. The observed Golgi fragmentation suggests that NO plays a significant role in modulating global protein trafficking patterns that contribute to changes in the cell surface landscape and functional signaling in vascular cells.     

Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects

Bisphenol A (BPA) is one of the highest volume chemicals produced worldwide. This compound is a building block of polycarbonate plastics often used for food and beverage storage, and BPA is also a component of epoxy resins that are used to line food and beverage containers. Studies have shown that BPA can leach from these and other products in contact with food and drink, and as a result, routine ingestion of BPA is presumed. This compound is also found in an enormous number of other products that we come into contact with daily, and therefore it is not surprising that it has been detected in the majority of individuals examined. BPA is a known endocrine disruptor. Although initially considered to be a weak environmental estrogen, more recent studies have demonstrated that BPA may be similar in potency to estradiol in stimulating some cellular responses. Moreover, emerging evidence suggests that BPA may influence multiple endocrine-related pathways. Studies in rodents have identified adverse effects of BPA at levels at or below the current acceptable daily intake level for this compound. The various reported adverse effects of BPA are reviewed, and potential mechanisms of BPA action are discussed. Much more investigation is needed to understand the potential adverse health effects of BPA exposure in humans and to understand the multiple pathways through which it may act. Although many questions remain to be answered, it is becoming increasingly apparent that exposure to BPA is ubiquitous and that the effects of this endocrine disruptor are complex and wide-ranging.