Expression and Functional Characterization of Breast Cancer-Associated Cytochrome P450 4Z1 in Saccharomyces cerevisiae.

CYP4Z1 is an "orphan" cytochrome P450 (P450) enzyme that has provoked interest because of its hypothesized role in breast cancer through formation of the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). We expressed human CYP4Z1 in Saccharomyces cerevisiae and evaluated its catalytic capabilities toward arachidonic and lauric acids (AA and LA). Specific and sensitive mass spectrometry assays enabled discrimination of the regioselectivity of hydroxylation of these two fatty acids. CYP4Z1 generated 7-, 8-, 9-, 10-, and 11-hydroxy LA, whereas the 12-hydroxy metabolite was not detected. HET0016, the prototypic CYP4 inhibitor, only weakly inhibited laurate metabolite formation (IC50 ∼15 µM). CYP4Z1 preferentially oxidized AA to the 14(S),15(R)-epoxide with high regioselectivity and stereoselectivity, a reaction that was also insensitive to HET0016, but neither 20-HETE nor 20-carboxy-AA were detectable metabolites. Docking of LA and AA into a CYP4Z1 homology model was consistent with this preference for internal fatty acid oxidation. Thus, human CYP4Z1 has an inhibitor profile and product regioselectivity distinct from most other CYP4 enzymes, consistent with CYP4Z1's lack of a covalently linked heme. These data suggest that, if CYP4Z1 modulates breast cancer progression, it does so by a mechanism other than direct production of 20-HETE.

Phosphorylation within the cysteine-rich region of dystrophin enhances its association with ß-dystroglycan and identifies a potential novel therapeutic target for skeletal muscle wasting.

Mutations in dystrophin lead to Duchenne muscular dystrophy, which is among the most common human genetic disorders. Dystrophin nucleates assembly of the dystrophin-glycoprotein complex (DGC), and a defective DGC disrupts an essential link between the intracellular cytoskeleton and the basal lamina, leading to progressive muscle wasting. In vitro studies have suggested that dystrophin phosphorylation may affect interactions with actin or syntrophin, yet whether this occurs in vivo or affects protein function remains unknown. Utilizing nanoflow liquid chromatography mass spectrometry, we identified 18 phosphorylated residues within endogenous dystrophin. Mutagenesis revealed that phosphorylation at S3059 enhances the dystrophin-dystroglycan interaction and 3D modeling utilizing the Rosetta software program provided a structural model for how phosphorylation enhances this interaction. These findings demonstrate that phosphorylation is a key mechanism regulating the interaction between dystrophin and the DGC and reveal that posttranslational modification of a single amino acid directly modulates the function of dystrophin.

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.

Effects of transferrin conjugates of artemisinin and artemisinin dimer on breast cancer cell lines.

Transferrin (Tf) conjugates of monomeric artemisinin (ART) and artemisinin dimer were synthesized. The two conjugates, ART-Tf and dimer-Tf, retained the original protein structure, and formed stable aggregates in aqueous buffer. ART-Tf induced declines in proteins involved in apoptosis (survivin), cell cycling (cyclin D1), oncogenesis (c-myelocytomatosis oncogene product (c-MYC)), and dysregulated WNT signaling (beta-catenin) in both the human prostate (DU145) and breast (MCF7) cancer cell lines. Both ART-Tf and dimer-Tf induced down-regulation of survivin, c-MYC and mutated human epidermal growth factor receptor-2 (ERBB2 or HER2) in the BT474 breast cancer cell line. To our knowledge, this is the first demonstration that an ART derivative can cause a decline of ERBB2 in a human cancer cell line. Potential mechanisms for the observed effects are presented. Both transferrin conjugates strongly inhibited the growth of BT474 cells in the same concentration range that the conjugates caused declines in the levels of ERBB2, survivin, and c-MYC, while showing essentially no toxicity towards MCF10A normal breast cells.

Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells.

Artemisinin is a plant-derived anti-malarial drug that has relatively low toxicity in humans and is activated by heme and/or intracellular iron leading to intracellular free radical formation. Interestingly, artemisinin has displayed anti-cancer activity, with artemisinin dimers being more potent than monomeric artemisinin. Intracellular iron uptake is regulated by the transferrin receptor (TfR), and the activity of artemisinin depends on the availability of iron. We examined the level of TfR in prostate cancer (PCa) tumor cells, synthesized two new artemisinin dimers, and evaluated the effect of dihydroartemisinin and artemisinin dimers, ON-2Py and 2Py, on proliferation and apoptosis in PCa cells. TfR was expressed in the majority of PCa bone and soft tissue metastases, all 24 LuCaP PCa xenografts, and PCa cell lines. After treatment with dihydroartemisinin, ON-2Py, or 2Py all PCa cell lines displayed dose-dependent decrease in cell number. 2Py was most effective in decreasing cell number. An increase in apoptotic events and growth arrest was observed in the C4-2 and LNCaP cell lines. Growth arrest was observed in PC-3 cells, but no significant change was observed in DU 145 cells. Treatment with 2Py resulted in a loss of the anti-apoptotic protein survivin in all four cell lines. 2Py treatment also decreased androgen receptor and prostate-specific antigen expression in C4-2 and LNCaP cells, with a concomitant loss of cell cycle regulatory proteins cyclin D1 and c-Myc. This study shows the potential use of artemisinin derivatives as therapeutic candidates for PCa and warrants the initiation of preclinical studies.

Transferrin receptor-dependent cytotoxicity of artemisinin-transferrin conjugates on prostate cancer cells and induction of apoptosis.

Artemisinin, a natural product isolated from Artemisia annua, contains an endoperoxide group that can be activated by intracellular iron to generate toxic radical species. Cancer cells over-express transferrin receptors (TfR) for iron uptake while most normal cells express nearly undetectable levels of TfR. We prepared a series of artemisinin-tagged transferrins (ART-Tf) where different numbers of artemisinin units are attached to the N-glycoside chains of transferrin (Tf). The Tf bearing approximately 16 artemisinins retains the functionality of both Tf and artemisinin. Reduction of TfRs by TfR siRNA transfection significantly impaired the ability of ART-Tf, but not dihydroartemisinin, to kill cells. We also demonstrate that the ART-Tf conjugate kills the prostate carcinoma cell line DU 145 by the mitochondrial pathway of apoptosis.

Determination and comparison of the Francisella tularensis subsp.novicida U112 proteome to other bacterial proteomes.

The proteins expressed by Francisella tularensis subsp. novicida U112 grown to midexponential phase were surveyed by nanoLC-tandem mass spectrometry (LC-MS/MS). To improve annotation of the genome and develop a technology to provide high-throughput analysis of the Francisella proteome in multiple conditions, we sought to establish a fast and simple analysis that would reduce as much as possible the false discovery rate. Our survey detected expression of 63.0% of the predicted proteome from the stable condition of growth in rich medium available at (www.francisella.org). On the basis of detection of essential proteins, we estimated coverage to be approximately 80% of the actual expressed proteome. This suggests that no less than 70% of the proteins could be expressed in this condition. This analysis revealed two previously unidentified protein coding open reading frames and validated 50% of the proteins annotated as hypothetical. On the basis of results of the screen to detect essential proteins, not all proteins expressed provide a measurable contribution to F.t. novicida growth in this condition. Comparison of this protein profile with other profiles previously published suggested that the genome size and number of genes involved in regulation have little effect on the number of proteins expressed in a given stable condition.

Comparison of a label-free quantitative proteomic method based on peptide ion current area to the isotope coded affinity tag method.

Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA) and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded affinity tag (ICAT) method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis.

Immunocytochemical detection of phosphatidylinositol 3-kinase activation by insulin and leptin.

Intracellular signaling mediated by phosphatidylinositol 3-kinase (PI3K) is important for a number of cellular processes and is stimulated by a variety of hormones, including insulin and leptin. A histochemical method for assessment of PI3K signaling would be an important advance in identifying specific cells in histologically complex organs that are regulated by growth factors and peptide hormones. However, current methods for detecting PI3K activity require either homogenization of the tissue or cells or the ability to transfect probes that bind to phosphatidylinositol 3,4,5 trisphosphate (PIP3), the reaction product of PI3K catalysis. Here we report the validation of an immunocytochemical method to detect changes in PI3K activity, using a recently developed monoclonal antibody to PIP3, in paraformaldehyde-fixed bovine aortic endothelial cells (BAECs) in culture and in hepatocytes of intact rat liver. Treatment with either insulin or leptin increased BAEC PIP3 immunoreactivity, and these effects were blocked by pretreatment with PI3K inhibitors. Furthermore, infusion of insulin into the hepatic portal vein of fasted rats caused an increase of PIP3 immunostaining in hepatocytes that was associated with increased serine phosphorylation of the downstream signaling molecule protein kinase B/Akt (PKB/Akt). We conclude that immunocytochemical PIP3 staining can detect changes in PI3K activation induced by insulin and leptin in cell culture and intact liver.