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With technology development, there is a growing need for an accurate simulation tools, allowing the best possible representation of the reality. Developed model found not only application in prototyping process but can provide significant knowledge into the artificial intelligent model extending their comprehension. Computational performance and accuracy of the numerical model, dedicated for granular flows are mostly a function of mathematical model that track the mutual interaction between particles. Currently the kinetic theory of granular flow or soft- and hard-sphere collision models are used for modeling particle interactions. Each of them suffers from some imperfection that often limit the problem sizes. The purpose of this work is building new mathematical approach, not only fast, but also accurate regarding predicting collisions and determining particle trajectories by application of machine learning technique.

The present paper describes the problem and effects of water scarcity and the possibility of rational use of this resource in the idea of a Circular Economy (CE) and sustainable development. Rational water management requires innovation, due to the growing demand for this raw material. It seems that water is widely available, e.g., in Poland, there is no problem with drought. Unfortunately, Polish water resources are shrinking and modern solutions, as well as the construction of new and modernisation of old infrastructure, are some of the few solutions that can protect against a shortage of potable water. Water is also an essential resource for economic development. It is used in every sector of the economy. Limited water resources lead to an inevitable energy transformation because, in its present state, the Polish energy industry consumes huge amounts of water. Due to the above statements, the authors propose a solution in the form of an interactive shower panel that contributes to more rational water management (e.g., in households or hotels) based on the latest technological achievements. This device enables the creation of water consumption statistics based on accurate liquid flow measurements and the transfer of data to the user’s mobile device. This innovation aims to make the user aware of the amount of water used, which in turn can contribute to lower water consumption.

Targeting of the TRAIL-DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD-O51.4, which is a TRAIL equipped with positively charged VEGFA-derived effector peptides. The study was performed in multiple cancer cell line- and patient-derived xenografts. A pharmacokinetic profile was established in monkeys. AD-O51.4 strongly inhibits tumor growth, even leading to complete long-term tumor remission. Neither mice nor monkeys treated with AD-O51.4 demonstrate symptoms of drug toxicity. AD-O51.4 exhibits a satisfactory half-life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD-O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD-O51.4-driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL-sensitive and TRAIL-resistant cancer cells, respectively. The FADD-dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD-O51.4 is capable of bypassing the refractoriness of TRAIL. AD-O51.4-driven cell death, which exceeds TRAIL activity, is achieved due to the N-terminally fused polypeptide, containing VEGFA-derived effector peptides. The high anticancer efficiency of AD-O51.4 combined with its safety has led to the entry of AD-O51.4 into toxicological studies.

The epidermal growth factor receptor (EGFR) is one of the most important and attractive targets for specific anticancer therapies. It is a robust regulator of pathways involved in cancer pathogenesis and progression. Thus far, clinical trials have demonstrated the benefits of monoclonal antibodies and synthetic tyrosine kinase inhibitors in targeting this receptor; however, novel strategies are still being developed. This article reviews the current state of efforts in targeting the EGFR in cancer therapy. Following a brief characterization of EGFR, we will present a complete list of anti-EGFR agents that are already approved, and available in clinical practice. Aside from the indications, we will present the sales forecasts and expiry dates of product patents for the selected agents. Finally, we discuss the novel anti-EGFR strategies that are currently in preclinical development.

The TNF-Related Apoptosis Inducing Ligand (TRAIL) cytokine triggers apoptosis specifically in cancer cells. Susceptibility of a given cell to TRAIL depends on the activity of regulatory proteins, one of the most important of which is BID. The aim of this study was to increase the cytotoxic potential of TRAIL against cancer cells. TRAIL was fused to the BH3 domain of BID. Hence, TRAIL acted not only as an anticancer agent, but also as a specific carrier for the BID fragment. Two fusion protein variants were obtained by genetic engineering, harboring two different linker sequences. The short linker allowed both parts of the fusion protein to fold into their native structures. The long linker influenced the structure of the fused proteins but nonetheless resulted in their highest cytotoxic activity. Optimal buffer formulation was determined for all the analyzed TRAIL variants. Fusing the BH3 domain of BID to TRAIL improved the cytotoxic potential of TRAIL. Further, these findings may be useful for the optimization of other anticancer drugs based on TRAIL, since the appropriate formulation would secure their native structures during prolonged storage.

We explore the relation between correlation dimension, approximate entropy and sample entropy parameters, which are commonly used in nonlinear systems analysis. Using theoretical considerations we identify the points which are shared by all these complexity algorithms and show explicitly that the above parameters are intimately connected and mutually interdependent. A new geometrical interpretation of sample entropy and correlation dimension is provided and the consequences for the interpretation of sample entropy, its relative consistency and some of the algorithms for parameter selection for this quantity are discussed. To get an exact algorithmic relation between the three parameters we construct a very fast algorithm for simultaneous calculations of the above, which uses the full time series as the source of templates, rather than the usual 10%. This algorithm can be used in medical applications of complexity theory, as it can calculate all three parameters for a realistic recording of 10 4 points within minutes with the use of an average notebook computer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors became promising molecules for selective targeting of tumor cells without affecting normal tissue. Unfortunately, cancer cells have developed a number of mechanisms that confer resistance to TRAIL\Apo2L-induced apoptosis, which substantiates the need for development of alternative therapeutic strategies. Here we present a recombinant variant of TRAIL\Apo2L peptide, named AD-O53.2, fused to the peptide-derived from Smac/Diablo protein-the natural inhibitor of the apoptotic X-linked IAP (XIAP) protein considered as a pro-apoptotic agent. The proposed mechanism of action for this construct involves specific targeting of the tumor by TRAIL\Apo2L followed by activation and internalization of pro-apoptotic peptide into the cancer cells. While in the cytoplasm , the Smac\Diablo peptide inhibits activity of X-linked IAP (XIAP) proteins and promotes caspase-mediated apoptosis. AD-O53.2 construct was expressed in E.coli and purified by Ion Exchange Chromatography (IEC). Derived protein was initially characterized by circular dichroism spectroscopy (CD), HPLC-SEC chromatography, surface plasmon resonance, protease activation and cell proliferation assays. Our Smac/Diablo-TRAIL fusion variant was tested against a panel of cancer cells (including lung, colorectal, pancreatic, liver, kidney and uterine) and showed a potent cytotoxic effect with the IC50 values in femtomolar range for the most sensitive cell lines, while it remained ineffective against non-transformed HUVEC cells as well as isolated normal human and rat hepatocytes. Importantly, the construct was well tolerated by animals and significantly reduced the rate of the tumor growth in colon and lung adenocarcinoma animal models.

W artykule przedstawiono zastosowanie termografii aktywnej do badań nieniszczących połączeń zakładkowych blach spawanych laserowo bez pełnego przetopienia. Opisano opracowaną metodologię badań, sposób wytworzenia połączeń próbnych oraz zastosowane stanowisko badawcze. Uzyskane obrazy termograficzne zostały poddane analizie polegającej na wyznaczeniu maksymalnego kontrastu temperaturowego. Ocenę wykonanych połączeń przeprowadzono na podstawie analizy makrostruktury wybranych przekrojów poprzecznych próbek. Na podstawie wyników badań termograficznych stwierdzono, że zaproponowana metoda może być z powodzeniem zastosowana do nieniszczących badań połączeń zakładkowych spawanych laserowo. Active thermography as a new non-destructive testing method of laser welded lap joints Abs􏰤􏰁tract The application of active thermography for non-destructive testing of laser welded lap joints (with partial joint penetration) was presented in this article. The methodology, specimen preparation and applied apparatus were described. Thermal images of the specimens obtained during experiments were analyzed using procedure based on maximum thermal contrast determination. Prepared la- ser welded lap joints were examined using typical macro image analysis of selected cross sections of joints. The obtained experimental results showed that considered method has a great potential to be successfully applied for non-destructive testing of laser welded lap joints.

The development of novel therapeutic agents that activate the apoptotic pathways in tumor cells focuses laboratories around the world. Well characterized mechanisms that can result in cellular apoptosis are those induced by the death receptors of the tumor necrosis factor receptor superfamily (DRs) and their respective death ligands (e.g. TRAIL). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has no toxic effects on normal cells and induces apoptosis in various tumor cell types.

The investigation of etiopathology of arteriosclerosis shows close relationship between inflammatory processes and evolution of sclerotic lesions. In many patients acute coronary syndromes are caused by the rupture of hemodynamically insignificant sclerotic lesion and consequently thrombosis of coronary artery. The reason of instability of sclerotic plaque may be on going inflammation inside it. This paper reviews literature on the subject of relationship of atheromatosis, inflammation and infection.

Oxygen, carried mainly by erythrocytes, plays a crucial role in human organisms--as the terminal electron acceptor enables mitochondria functioning and energy production. Oxygen excess may be a cause of the damage of basic structural organism components--proteins, lipids and nucleinic acids. Thus, from the biological point of view, regulation of oxygen supply with its detection mechanisms is a critical process. Erythrocyte content optima-lization, in means of "benefit-loss" is a compromise between the necessity of anaerobic metabolism transit (in case of too low erythrocyte count), excessive increase in blood viscosity and non-productive, increased red blood cell turn-over with increased erythrocyte level. It is in fact a process of adaptation to changeable environmental conditions, life style and possible pathological processes. The study is a review of knowledge on the oxygen sensor and its connection to erythropoiesis regulation.

The risk of blood loss in open heart surgery procedures is related to a high level of homologous blood transfusion. Due to numerous possible complications connected with transfusion, as well as the increase in prices of blood preparations, methods of blood saving are of great interest. The aim of the study was to assess acute normovolemic hemo-dilution (ANH) efficacy and aprotinin administration in homologous blood usage limitations. The study was conducted in the group of 265 patients operated on during the period of 12 months. The control group consisted of patients operated on during the period 6 months. The scheme of ANH with the administration of aprotinin was applied in the second half-year period. A statistically significant reduction in the percentage of patients who required blood transfusions was observed (77.2%/53%). The scheme applied enabled to reduce procedure costs in regard to blood preparation purchase. The results have shown that ANH together with antifibrinolytic drug administration is not only an efficient but also profitable strategy of blood saving.

Quinolinic acid (QA) is a potent endogenous excitotoxin; elevation of its concentration in an organism has been implicated in the pathogenesis of various disorders. The purpose of this study was the assessment of QA impact on the process of erythropoiesis. Marked increase of QA concentration was observed in plasma and peripheral tissues of uremic rats. These changes were proportional to the amount of the removed renal tissue and positively correlated with the concentration of creatinine but negatively correlated with hematological parameters, i.e., hematocrit and Hb red blood cells count. The changes were accompanied by a slight decrease in the concentration of endogenic erythropoietin (EPO) in the plasma of animals with uremia. Chronic treatment with QA diminished the increase in EPO concentration after introduction of cobalt in rats. These changes were associated with the decrease in all hematological parameters after QA administration. The in vitro study in the conditions of hypoxia showed that QA inhibited the EPO release from HepG2 cells to the culture base. Additionally, in HepG2 cells QA had a dose-dependent inhibitory effect on hypoxia- and cobalt-induced EPO gene expression without any cell toxicity. In conclusion, the erythropoiesis in chronic renal failure could be attributed to the influence of QA on EPO synthesis. Thus we propose that QA can be a uremic toxin responsible for anemia in animals or patients with renal failure.

Background The tumor necrosis factor related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF superfamily that initiates apoptosis of tumor cells through the activation of their death receptors. The ability of TRAIL to selectively induce apoptosis of tumor cells but not normal cells makes it an attractive agent for cancer therapy. However, many cancer types have developed resistance mechanisms, such as dysfunctions of proapoptotic proteins. Hereby, we report a novel molecule - AD-O56.9, which is composed of the soluble fragment of TRAIL (acting as both carrier and also effector) fused with a cationic, alpha-helical (KLAKLAK)2 antimicrobial peptide (acting as effector). The (KLAKLAK)2 peptide fused to protein transduction domain can induce cancer cell death by triggering mitochondrial membrane permeabilization and swelling, resulting in the release of cytochrome c and induction of apoptosis. It creates also the capacity to cause aggregation of mitochondria that is also a mechanism of cytotoxic action. (KLAKLAK)2 peptide is equipped with protein transduction domain domain, to increase its internalization. To allow separation of TRAIL fragment from the effector peptide domain specifically in the tumor environment, we linked these two domains with a sequence motif recognized by MMP and uPa proteases, present in tumor cells membranes or their proximity. Methods AD-O56.9 protein was produced in E. coli and purified by IEC. The molecule was characterized biochemically and biophysically. MTT assay was used to estimate killing of carcinoma cells. Flow cytometric analysis was used to evaluate influence of the AD-O56.9 on plasma and mitochondrial membrane integrity, caspase 3 activation, PARP cleavage, as well as on the cell cycle of cancer cells. The tumoricidal activity of AD-O56.9 was evaluated in NOD/SCID mice bearing different types of tumor xenografts. Results AD-O56.9 exhibited cytotoxic effect on various cancer cell lines, both TRAIL-sensitive and TRAIL-resistant, but showed no toxic effect on normal cells. This protein was also highly cytotoxic against primary cancer cells. The component that overcomes resistance to TRAIL is RRRRRRRR(KLAKLAK)2 peptide, but only as a component of AD-O56.9 fusion protein. Analyzing cell cycle and plasma membrane integrity in relatively sensitive cell line (NCI-H460) and TRAIL-resistant cell line (A549) we showed that AD-O56.9 induced apoptosis in those cells. This protein led to activation of caspase 3, cleavage of PARP as well as caused strong depolarization of mitochondrial membrane. AD-O56.9 administration caused significant regression of TRAIL-sensitive MIA PaCa-2, OE19, Colo205 and TRAIL-resistant HepG2. Conclusions AD-O56.9 is able to induce cell death in many cancer cell lines, even TRAIL resistant and causes tumor regression in mice bearing human tumors. Obtained results make this molecule worth of further preclinical development. Citation Format: Bartlomiej Maciej Zerek, Michal Szymanik, Piotr Kamil Rozga, Anna Pieczykolan, Marlena Galazka, Katarzyna Bukato, Albert Jaworski, Katarzyna Poleszak, Sebastian Dominik Pawlak, Malgorzata Teska-Kaminska, Wojciech Strozek, Jerzy Szczepan Pieczykolan. AD-O56.9: A fusion of TRAIL/Apo2L with a membrane disrupting peptide as a novel anticancer therapeutic. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2773. doi:10.1158/1538-7445.AM2014-2773

Although standard chemotherapy is still one of the most effective treatment for many types of cancer - often causes side effects. Chemotherapeutic agents primarily damage cancer cells with a rapidly dividing and growing profile, thereby also destroy healthy cells with such characteristics, which leads to side effects. Scientists are continually working to identify new, safer drugs, methods of administering chemotherapy, and combinations of existing treatments that have fewer side effects. In recent years, drug delivery systems (DDS) based on a concept of conjugation anticancer agents to carrier protein have been developed, achieving a better clinical response and tolerability. Protein-drug conjugates represent a whole new concept for cancer treatment which, although highly effective, cause much fewer sides effects than traditional chemotherapy. The present work is aimed to create a new conjugate molecule which essentially demonstrates greater effect than the summarized effects of its components due to specific targeting to cancer cells. We generated AD-O64.4 complex molecule, consisting of fusion protein INF- γ - TRAIL/Apo2L conjugated with the anti-mitotic agent SN-38 via the thioether linker. Carrier protein is a fusion molecule which is composed of soluble fragment of TRAIL (acting as a targeting carrier and effector) fused with artificial dimer of IFN-γ (acting as effector). SN-38, a topoisomerase I inhibitor with low nanomolar potency, the active metabolite of irinotecan, cannot be given directly to patients because of its toxicity and poor solubility. Linking SN-38 to the carrier fusion protein results in selective drug delivery to tumors which consequently leads to increased amount of the drug reaching the tumors and minimized damage of healthy cells. Analysis of the conjugate was performed by LC-MS and resulted in determination of drug-protein ratio. Agregation studies of the molecule complex were conducted by DLS and fluorescent protein aggregation assay. Cytotoxicity was evaluated with MTT assay and efficacy was performed on female SCID mice xenograft model bearing Human Uterine Sarcoma (MES-SA/Dx5). Our results show an effective process of conjugation of anticancer compound SN-38 to the carrier protein which leads to formation of an active complex with enhanced efficacy. AD-O64.4 exhibited cytotoxic effect on various cancer cell lines, (IC50 about 10 ng/ml), but showed no toxic effect on normal cells. This conjugate was also highly cytotoxic against primary cancer cells. AD-O64.4 was highly active (up to 75% tumor remission) against subcutaneous multiple drug resistant, MES-SA/Dx5, tumor xenografts in severe combined immunodeficient mice with better activity compared to the carrier protein alone, as well as to combination of the carrier protein with free-drug. Overall, these results represent a novel and a valuable system for drug delivery to the tumor and its microenvironment. Citation Format: Wojciech Strozek, Anna Pieczykolan, Bartlomiej Zerek, Michal Szymanik, Albert Jaworski, Marlena Galazka, Katarzyna Bukato, Piotr Rozga, Sebastian Pawlak, Katarzyna Poleszak, Malgorzata Teska-Kaminska, Jerzy Pieczykolan. AD-O64.4 - a novel bioconjugate for tumor-targeted drug delivery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4471. doi:10.1158/1538-7445.AM2014-4471

Angiogenesis, the process of new blood vessels formation, is essential for tumor growth and metastasis. Since tumors are unable to grow beyond a millimeter diameter in the absence of angiogenic factors, it was postulated that blocking of angiogenic factors could provide a new strategy to inhibit tumor leading to a potential novel anticancer therapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) was considered as a promising anticancer agent due its remarkable ability to induce apoptosis in cancer cells without harming normal cells. However, in clinical practice TRAIL efficacy was too low to become the effective single therapy agent. Here we present a novel fusion protein based on TRAIL/Apo2L and equipped with an antiangiogenic effector peptide originating from VEGF exon 6 that binds to natural VEGF receptors and competes with their natural ligand. The proposed fusion protein, AD-O51.4, consists of extracellular soluble portion of TRAIL linked to two tandemly arranged copies of the effector peptide, separated by a sequence that contains the motif recognized by tumor-specific proteases (MMP's, uPa). The AD-O51.4 fusion molecule exerts its dual cytotoxic and antiangiogenic activity upon interaction with cellular death and VEGF receptors on cancer endothelial cells. In consequence, the peptide blocks the binding of VEGF to its receptors, preventing new vessels formation. In addition, due to the presence of VEGF receptors on cancer cells, they can be also targeted by VEGF receptors-binding domain of AD-O51.4 making them susceptible for TRAIL-induced apoptosis. Indeed, an extensive panel of discovery-stage studies of AD-O51.4 showed broad cytotoxic activity on primary cancer cell lines but no toxic effects on normal cells and strong antitumor activity on subcutaneous and orthotropic xenograft models with complete tumor remission. Based on these data, early preclinical part of the program was initiated. An LC\MS-based method, specific for the intact molecule, was set up as the detection and quantification tool, enabling detection of AD-O51.4 in different biological matrices. Safety of the molecule was evaluated using healthy hepatocytes form humans and cynomolgus monkeys. To verify tumor targeting capacity of the molecule, tumor-specific accumulation in subcutaneous xenograft model of human colorectal cancer as a function of time was assessed using imaging techniques. Tumor specific accumulation was also confirmed using LC\MS method even after single administration. To analyse the pharmacological properties of the AD-O51.4, PK profiles were evaluated in Wistar rats and cynomoglus monkeys. Finally, strong antitumor activity was confirmed using PDX models of human lung, colon, rectal and pancreatic cancer. Currently the molecule is approaching formal ADME\Tox panel. Citation Format: Jerzy S. Pieczykolan, Anna Pieczykolan, Piotr Rozga, Sebastian Pawlak, Bartlomiej Zerek, Malgorzata Teska - Kaminska, Szymanik Michal, Albert Jaworski, Karl R. Erlemann, Jacek Capala. Preclinical evaluation of the novel fusion molecule with high dual antiangiogenic and anticancer potential. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2600. doi:10.1158/1538-7445.AM2014-2600

Background Cancer growth and development is tightly related to both new vessels formation for tissue remodeling and inhibition of anti-apoptotic signals. Vascular endothelial growth factor (VEGF) is important for vascular development in physiological and pathological processes. Blockade of VEGF pathway has been shown to inhibit both pathological angiogenesis and tumor growth. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been under intense scientific evaluation because of its ability to induce apoptosis in cancer cells while omitting normal cells. However, in most cases it wasn't potent enough to become the effective single therapy agent. The AD-O51.4 is a novel anticancer fusion protein. It consists of a recombinant variant of TRAIL/Apo2L fragment, which is linked to the repeated antiangiogenic effector peptide sequence derived from the 6th exon of VEGF ligand. The peptide sequences are separated by a motif recognized by tumor-specific proteases (MMP's, uPa). The structure and biophysical properties of AD-O51.4 should be mostly derived from TRAIL/Apo2L - the carrier component of the fusion molecule. The AD-O51.4 should be targeted to both types: VEGF and TRAIL receptors and lead to sequestration of the VEGF receptors on malignant and endothelial cells making them susceptible to apoptosis induced through TRAIL/Apo2L dead receptors. Methods The AD-O51.4 has been produced in E. coli cells and purified using IEX chromatography. Its structure and biophysical properties were verified using circular dichroism (CD), size-exclusion chromatography (SEC), proteolytic digestion of activation sequence and finally direct N-terminus sequencing. The VEGF and TRAIL/Apo2L receptors specificity was confirmed using surface plasmon resonance (SPR). Antitumor activity was analyzed on a panel of established cancer cell lines and xenograft model of human cancer. Results The analysis of AD-O51.4 revealed secondary structure rich with beta-sheets and a trimeric form of the fusion molecule what is typical for TRAIL/Apo2L ligand. The molecule also displays strong specific binding for both classes of receptors: VEGF and TRAIL/Apo2L what confirms potential antiangiogenic and proapoptotic properties. Sequencing and specific digestion endorsed molecule identity. Finally specific cytotoxic and antitumor activities were confirmed for AD-O51.4. Conclusion We demonstrated that AD-O51.4 fusion protein has well established structure corresponding to its main component TRAIL/Apo2L. The structure of VEGF-derived peptides determines its specific interaction with therapeutic targets and as a consequence its antitumor properties. The obtained results confirm that a combination of two effectors in one protein molecule may be an effective way of anticancer compounds development. Citation Format: Sebastian D. Pawlak, Jerzy S. Pieczykolan, Bartlomiej Zerek, Katarzyna Poleszak, Malgorzata Teska-Kaminska, Marlena Galaska, Michal Szymanik, Albert Jaworski, Anna Pieczykolan, Katarzyna Bukato, Wojciech Strozek, Piotr K. Rozga. Biochemical and biophysical characterization of AD-O51.4 a novel anticancer biological therapeutic agent with dual mechanism of action. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3796. doi:10.1158/1538-7445.AM2014-3796

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising antitumor agent, but its clinical utility in human malignancies is limited by the resistance mechanism evolved in many cancers types, including overexpresion of antiapoptotic Bcl-2 like proteins and lack or disfunction of proapoptotic agents. Bid is well known proapoptotic factor able to inhibit Bcl-2 proteins after activation by caspases 8 or 10. Bid may also promote apoptosis by forming selective voltage dependent anion-channels in mitochondrial membrane through interaction with Bax protein. AD-O57.4 is a fusion protein consisting of a short peptide derived from Bid linked to an amino terminus of soluble TRAIL domain. The separating sequence contains additional motif recognized by MMPs and uPa proteases that enables cutting-off and releasing the proapoptotic peptide in tumor environment. Activity of AD-O57.4 was examined using a MTT assay on NCI panel and primal, patient derived cancer cell lines. Apoptosis was analyzed by caspase 3 activation and mitochondrial membrane potential change. The expression of Bid was determined by Western Blot analysis. Various of tested cell lines were very sensitive to AD-O57.4 protein with IC50 value between 0.06-173 ng/ml, whereas 5 cell lines showed weak sensitivity or remained resistant. Cytotoxic activity of TRAIL was significantly lower in comparison with AD-O57.4 protein. AD-O57.4 induced relatively strong mitochondrial depolarization both in moderate sensitive cell line (NCI-H460) and TRAIL-resistant cell line (A549). Analysis of caspase 3 activation and expression of Bid protein showed very fast induction of apoptosis by AD-O57.4 in comparison to TRAIL protein. Strong antitumor activity of AD-O57.4 fusion protein was analyzed on the xenograft models of series of human cancer cell lines regarding TRAIL sensitive human colorectal carcinoma Colo205, TRAIL resistant, hepatocellular carcinoma HepG2 and human uterine sarcoma (MES-SA/Dx5) - a multidrug resistant cell line where it caused almost complete tumor regression and showed much higher efficacy than TRAIL alone or standard chemotherapeutic agent. We demonstrated that AD-O57.4 protein has broad in vitro cytotoxic activity against a panel of cancer cell lines and in vivo antitumor activity on xenograft model. Sensitization by AD-O57.4 was dependent on short peptide derived from Bid protein indicating its role for mitochondrial signal amplification in the proapoptotic TRAIL activity. The use of a peptide derived from Bid protein causes a significant increase in potency of TRAIL activity and therefore TRAIL resistant cancer cells can be sensitized by the fusion peptide which can connect extrinsic and intrinsic pathways of apoptosis. Citation Format: Jerzy S. Pieczykolan, Sebastian D. Pawlak, Bartlomiej M. Zerek, Piotr K. Rozga, Anna Pieczykolan, Michal Szymanik, Albert Jaworski, Marlena Galazka, Wojciech Strozek, Katarzyna Wiciejowska, Malgorzata Teska-Kaminska, Lukasz Kutner. Fusion protein capable to initiate two apoptotic pathways in cancer cells resistant to classical PARA's. [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 5488. doi:10.1158/1538-7445.AM2013-5488

One of the most important mechanisms of tumorgenesis is formation of new blood vessels and tissue remodeling. Vascular endothelial growth factor (VEGF) is one of the most important and widely studied proangiogenic factors, that has an important role in many physiological and pathological processes. Blockade of VEGF pathway has been shown to inhibit both pathological angiogenesis and tumor growth. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) was considered as a promising anticancer agent due its remarkable ability to induce apoptosis in cancer cells without harming normal cells. However, in clinical practice TRAIL efficacy was too low to made it an effective single therapy agent. Here we present a novel fusion protein based on TRAIL/Apo2L, equipped with an antiangiogenic, effector peptide, fused to its N- terminus. The effector role plays a 7-amino acid-long peptide that binds to natural VEGF receptors and competes with their natural ligand. The proposed fusion protein AD-O51.4 consists of extracellular soluble portion of TRAIL linked to two tandemly arranged copies of the effector peptide separated by a sequence containing the motif recognized by tumor-specific proteases (MMP's, uPa). The TRAIL-targeted peptide is able to bind and sequester the VEGF receptors on malignant (cancer) and endothelial cells. As a consequence, the peptide blocks the binding of VEGF ligand, preventing formation of new vessels. In addition, a lot of literature data confirm expression of the VEGF receptors on the surfaces of various cancer cell lines. Due to the presence of VEGF receptors, those cell lines can be also targeted by our VEGF receptor-blocking peptide making them susceptible for TRAIL-induced apoptosis. In vitro, AD-O51.4 showed cytotoxic effect on various cancer cell lines at IC50 below 0.1 ng/mL and no toxic effects on normal cells. Cytotoxic activity of TRAIL alone was significantly lower in comparison to AD-O51.4. We demonstrated that TRAIL/Apo2L-VEGF-antagonist is a strong activator of caspase 3 and Bid processing. Additionally, its direct antiangiogenic activity was confirmed with the ring aortic assay and HUVEC spheroid assay. Strong antitumor activity of novel fusion molecule was also confirmed in xenograft model of multidrug resistant human uterine sarcoma MES-SA/Dx5 where our protein caused complete tumor remission and showed much higher efficacy than TRAIL alone. The similar effect was observed using human colorectal adenocarcinoma Colo205 and human pancreatic carcinoma MIA PaCa-2 xenografts, as well as an orthotropic model of human lung carcinoma NCI-H460-luc2. Citation Format: Jerzy S. Pieczykolan, Piotr Rozga, Anna Pieczykolan, Bartlomiej Zerek, Sebastian D. Pawlak, Michal Szymanik, Albert Jaworski, Marlena Galazka, Katarzyna Wiciejowska, Wojciech Strozek, Malgorzata Teska - Kaminska, Lukasz Kutner, Anna Grochot - Przeczek, Neli Kahmakowa - Trojanowska. Novel fusion molecule with high dual antiangiogenic and anticancer potential. [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 5073. doi:10.1158/1538-7445.AM2013-5073