Maya M. Zaharieva

Erufosine simultaneously induces apoptosis and autophagy by modulating the Akt-mTOR signaling pathway in oral squamous cell carcinoma.

We investigated the anticancer activity of erufosine in oral squamous carcinoma cell lines in terms of cell proliferation, colony formation, induction of autophagy/apoptosis, cell cycle and mTOR signaling pathway. Erufosine showed dose-dependent cytotoxicity in all cell lines, it induced autophagy as well as apoptosis, G2 cell cycle arrest and modulation of cyclin D1 expression. Further erufosine downregulated the phosphorylation of major components of mTOR pathway, like p-Akt at Ser473 and Thr308 residues, p-Raptor, p-mTOR, p-PRAS40 and its downstream substrates p-p70S6K and p-4EBP1 in a dose-dependent manner. The pre-treatment of tumor cells with p-mTOR siRNA increased cytotoxic effects of erufosine comparable to cisplatin but higher than rapamycin.

Triterpenoid saponins from the roots of Gypsophila trichotoma Wender.

Eleven triterpenoid saponins were isolated from the roots of Gypsophila trichotoma Wender. (G. trichotoma Wender. var. trichotoma) (Caryophyllaceae), together with one known compound. The structures were established on the basis of extensive NMR analysis ((1)H, (13)C NMR, COSY, TOCSY, ROESY, HSQC, and HMBC), completed by analysis of HR-ESI-MS and ESI-MS(n). The saponins have the commonly found gypsogenin as the aglycone substituted at C-3 with trisaccharide and at C-28 with oligosaccharide through a fucose residue, as saponins isolated from Gypsophila perfoliata L. originated from China. The oligosaccharide attached to C-28 is substituted with acetyl and (or) sulfate groups. Тhe cytotoxicity of the saponin extract from G. trichotoma was evaluated against a rat alveolar macrophage-like cell line NR8383 and human leukemia cell lines U937 and BV-173. The synergistic effect of the aminoacyl saponins, previously isolated from G. trichotoma, was tested for its ability to enhance the cytotoxicity of the targeted toxin in HER14 cells.

In vitro toxicological evaluation of a dinuclear platinum(II) complex with acetate ligands

In the present study the toxicological potential of a tumor-inhibiting dinuclear platinum(II) complex (bis(acetato)diammine-bis-μ-acetato diplatinum(II) dihydrate (BAP)) was evaluated, utilizing in vitro models of nephrotoxicity, myelosuppression and neurotoxicity. Regarding the discrepancies between the hallmark toxicity of the clinically utilized platinum drugs, we used three distinct referent compounds as follows cisplatin for the assessment of in vitro nephrotoxicity, carboplatin in case of cultured bone marrow cells and oxaliplatin for the determination of the in vitro neurotoxicty, respectively. The results obtained indicate that the investigated dinuclear complex is endowed by a lower potential to induce detrimental effects upon these typically susceptible platinum toxicity cellular populations as compared to the corresponding referent drugs. These findings, together with the previously encountered profound cytotoxic efficiency of this dinuclear platinum(II) complex against human tumor cell lines, recall for a further detailed evaluation of BAP as potential antineoplastic agent.

The expression level of the tumor suppressor retinoblastoma protein (Rb) influences the antileukemic efficacy of erucylphospho-N,N,N-trimethylpropylammonium (ErPC3)

The alkylphosphocholine erucylphospho-N,N,N-trimethylpropylammonium (ErPC3) is a promising new drug for treating various types of cancer. Its mechanism of action is not yet fully understood but is related to the Rb tumor suppressor protein. In the pre¬sent study, we investigated the role of decreased Rb expression levels for the an¬tileukemic efficacy of ErPC3 in BV-173 and K-562 CML-derived cell lines. We used antisense technique to knock down Rb levels in the two cell lines in addition to ErPC3 treatment. Cells with reduced Rb expression showed a diminished sensitivity to ErPC3 exposure, as determined by MTT (BV-173 and K-562) and clonogenicity as¬says (K-562 only), if concentrations below the IC50 were used. The feasibility of Rb knockdown varied between BV-173 and K-562 cells, with the former being distinctly more sensitive than the latter. We conclude that sufficient Rb levels are important for the cytotoxic and anticlonogenic effects of ErPC3 at levels below the IC50, but that higher concentrations of ErPC3 are less dependent on Rb status.

Reduced expression of the retinoblastoma protein shows that the related signaling pathway is essential for mediating the antineoplastic activity of erufosine.

Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G2 phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by eru-fosine in the expression of these proteins and abrogated the induction of G2 arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status.

Modeling and Technoeconomic Analysis of Algae for Bioenergy and Coproducts

Abstract The complex use of the algae biomass highlights the trends in green and innovative technologies of high priorities in EU program Horizon 2020. This part of the chapter will combine the obtained key knowledge about the modeling of different photobioreactor (PBR) designs. Innovative approaches and achievements in modeling of microalgal kinetics connected with light intensity, hydrodynamics, and mass transfer phenomena in PBRs will be underlined. Furthermore, we will discuss applicability of the complex models to optimize overall microalgae process for bioenergy production. In the second part of the chapter technoeconomic analysis will be performed to determine cost viability of algae process for bioenergy. Such study varies depending upon algae-processing techniques, involved equipment, and downstream steps of desired products. The most industrially viable algae metabolite products for bioenergy and some high-value coproducts will be discussed through the life cycle assessment approach.

Complex mathematical analysis of photobioreactor system

Modeling as a tool solves extremely difficult tasks in life sciences. Recently, schemes of culturing of microalgae have received special attention because of its unique features and possible uses in many industrial applications for renewable energy production and high value products isolation. The goal of this review is to present the use of system analysis theory applied to microalgae culturing modeling and process development. The review mainly focuses on the modeling of the key steps of autotrophic growth under the integral biorefinery concept of the microalgae biomass. The system approach follows systematically a procedure showing the difficulties by modeling of sub‐systems. The development of microalgae kinetics and computational fluid dynamics (CFD) studies were analyzed in details as sub‐systems in advanced design of photobioreactor (PBR). This review logically follows the trends of the modeling procedure and clarifies how this approach may save time and money during the research efforts. The result of this work is a successful development of a complex PBR mathematical analysis in the frame of the integral biorefinery concept.

Migratory birds along the Mediterranean/Black Sea Flyway as carriers of zoonotic pathogens

At the crossroad between Europe, Asia, and Africa, Bulgaria is part of the Mediterranean - Black Sea Flyway (MBSF) used by millions of migratory birds. In this study, bird species migrating through Bulgaria were investigated as carriers of zoonotic pathogens. In total, 706 birds belonging to 46 species were checked for the presence of various bacterial pathogens (Campylobacter, Yersinia, Salmonella, Listeria, Escherichia coli, Staphylococcus aureus, Francisella tularensis, Coxiella burnetii, Borrelia burgdorferi, and Brucella spp.). From 673 birds we investigated fecal samples, from the remaining 33, blood samples. We detected Campylobacter 16S rDNA gene in 1.3% of birds, but none were of pathogenic Campylobacter jejuni and Campylobacter coli species. Escherichia coli 16S rDNA gene was found in 8.8% of the birds. Out of 34 birds that transported Yersinia enterocolitica strains (5.05%), only 1 carried a pathogenic isolate. Three birds (0.4%) were carriers of nonpathogenic Salmonella strains. Four avian samples (0.6%) were positive for Listeria monocytogenes and 1 (0.15%) was positive for Brucella spp. None of the birds tested carried the tick-borne pathogens C. burnetii or B. burgdorferi sensu lato. Antibiotic-resistant strains were detected, suggesting that migratory birds could be reservoirs and spreaders of bacterial pathogens as well as antibiotic resistance genes.

Alkylphospholipids are signal transduction modulators with potential for anticancer therapy

BACKGROUND Alkylphospholipids (APLs) are synthetically derived from cell membrane components, which they target and thus modify cellular signalling and cause diverse effects. This study reviews the mechanism of action of anticancer, antiprotozoal, antibacterial and antiviral activities of ALPs, as well as their clinical use. METHODS A literature search was used as the basis of this review. RESULTS ALPs target lipid rafts and alter phospholipase D and C signalling cascades, which in turn will modulate the PI3K/Akt/mTOR and RAS/RAF/MEK/ERK pathways. By feedback coupling, the SAPK/JNK signalling chain is also affected. These changes lead to a G2/M phase cell cycle arrest and subsequently induce programmed cell death. The available knowledge on inhibition of AKT phosphorylation, mTOR phosphorylation and Raf down-regulation renders ALPs as attractive candidates for modern medical treatment, which is based on individualized diagnosis and therapy. Corresponding to their unusual profile of activities, their side effects result from cholinomimetic activity mainly and focus on the gastrointestinal tract. These aspects together with their bone marrow sparing features render APCs well suited for modern combination therapy. Although the clinical success has been limited in cancer diseases so far, the use of miltefosine against leishmaniosis is leading the way to better understanding their optimized use. CONCLUSION Recent synthetic programs generate congeners with the increased therapeutic ratio, liposomal formulations, as well as diapeutic (or theranostic) derivatives with optimized properties. It is anticipated that these innovative modifications will pave the way for the further successful development of ALPs.

Abstract 4352: Down regulation of retinoblastoma protein expressionimpedes the antileukemic activity of erufosine.

Erufosine is a new antineoplastic agent from the class of alkylphosphocholines, which interferes with AKT phosphorylation, the related signal transduction and confers apoptosis. An improved understanding of its mode of action is important for its potential clinical application. The present study demonstrates that erufosine9s antileukemic efficacy is related to the level of cellular retinoblastoma (Rb) protein expression. Stable Rb-knockdown in SKW-3 leukemia T-cells was induced by lentiviral delivery of constructs expressing short-hairpin-RNA species with 21 or 27 bp lengths. The cytotoxicity of erufosine was tested via MTT proliferation test on thus engineered cell populations with reduced Rb expression levels. Likewise, the clonogenicity of these engineered cell populations was determined by CFU assay before and after treatment with erufosine. The effects on cell cycle were investigated by FACS analysis. The expression of signal transduction factors was examined by RT-PCR and immunoblot. The Rb protein expression levels in the shRNA-transduced cells ranged from 1%- 36% of wild type or nonsense control. Low Rb expression correlated with significantly diminished antiproliferative and anticlonogenic activities of erufosine (p 50 . Erufosine induced a G2 arrest, and complete Rb-deficiency aggravated this condition. With regard to proteins involved in cell cycle and apoptosis induction, Rb-deficiency reduced the expression of cyclin D3, that of the subsequently regulated kinase Cdk 4 and activated caspases 3 and 9, followed by PARP-cleavage. In partial variance, erufosine reduced cyclin D3 at RNA and protein levels, but affected not or only slightly the expression of its partner kinase Cdk 4. Rb-knockdown impaired the cytotoxic effect of erufosine significantly and contributed to the formation of a resistant cell fraction. It is concluded that Rb is a main mediator of erufosine9s antileukemic activity and this suggests that the effect of the drug in patients might be predicted by determining the Rb expression status. Citation Format: Maya M. Zaharieva, Milen Kirilov, Minquang Chai, Stefan M. Berger, Spiro M. Konstantinov, Martin R. Berger. Down regulation of retinoblastoma protein expressionimpedes the antileukemic activity of erufosine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4352. doi:10.1158/1538-7445.AM2013-4352