The Warburg effect: a score for many instruments in the concert of cancer and cancer niche cells.

Although Warburg's discovery of intensive glucose uptake by tumors, followed by lactate fermentation in oxygen presence of oxygen was made a century ago, it is still an area of intense research and development of new hypotheses that, layer by layer, unravel the complexities of neoplastic transformation. This seemingly simple metabolic reprogramming of cancer cells reveals an intriguing, multi-faceted nature that may link various phenomena including cell signaling, cell proliferation, ROS generation, energy supply, macromolecules synthesis/biosynthetic precursor supply, immunosuppression, or cooperation of cancerous cells with cancer-associated fibroblasts (CAFs), known as reversed Warburg effect. According to the current perception of the causes and consequences of the Warburg effect, PI3K/Akt/mTOR are the main signaling pathways that, in concert with the transcription factors HIF-1, p53, and c-Myc, modulate the activity/expression of key regulatory enzymes, including PKM2, and PDK1 to tune in the most optimal metabolic setting for the cancer cell. This in turn secures adequate levels of biosynthetic precursors, NADPH, NAD+, and rapid ATP production to meet the increased demands of intensively proliferating tumor cells. The end-product of "aerobic glycolysis", lactate, an oncometabolite, may provide fuel to neighboring cancer cells, and facilitate metastasis and immunosuppression together enabling cancer progression. The importance and possible applicability of the presented issue are best illustrated by numerous trials with various agents targeting the Warburg effect, constituting a promising strategy in future anti-cancer regimens. In this review, we present the key aspects of this multifactorial phenomenon, depicting the mechanisms and benefits behind the Warburg effect, and also pointing to selected aspects in the field of anticancer therapy.

Teaching "Biochemistry with Elements of Chemistry" in the COVID-19 pandemic.

The purpose of the presented article is to evaluate the students' perception of the online teaching educational model as a part of the "Biochemistry with Elements of Chemistry" course conducted during the COVID-19 pandemic at the Faculty of Medicine of the Jagiellonian University Medical College. The first part of the article reflects upon the pandemic impact on the transition from the in-person (standard format) to a complete remote learning format. The next part is based on the responses of the students to a questionnaire and presents an analysis of the students' preferences and perceptions regarding synchronous and asynchronous teaching methods. Students answered questions about the advantages and disadvantages of distance learning programs. They indicated the most suitable learning mode in terms of gaining knowledge and enhancing their motivation to learn. They listed factors that facilitated remote learning as well as those which made it difficult and posed a challenge to adjust to the new system, in all types of classes, that is, lectures, seminars, and laboratories. The last part of this paper presents the results of the students' performance in the pandemic-enforced system and compares them with the results from the previous class of students from the past year, when teaching was conducted mainly in the standard format. The conclusions from the given analysis may enable beneficial changes for the "Biochemistry with Elements of Chemistry" course for remote teaching in the future. It may also provide valuable insights for such types of courses conducted at other Universities and promote deliberations to continuously improve learning outcomes.

Antibacterial Therapy by Ag+ Ions Complexed with Titan Yellow/Congo Red and Albumin during Anticancer Therapy of Urinary Bladder Cancer.

According to the World Health Organization report, the increasing antibiotic resistance of microorganisms is one of the biggest global health problems. The percentage of bacterial strains showing multidrug resistance (MDR) to commonly used antibiotics is growing rapidly. Therefore, the search for alternative solutions to antibiotic therapy has become critical to combat this phenomenon. It is especially important as frequent and recurring infections can cause cancer. One example of this phenomenon is urinary tract infections that can contribute to the development of human urinary bladder carcinoma. This tumor is one of the most common malignant neoplasms in humans. It occurs almost three times more often in men than in women, and in terms of the number of cases, it is the fifth malignant neoplasm after prostate, lung, colon, and stomach cancer. The risk of developing the disease increases with age. Despite the improvement of its treatment methods, the current outcome in the advanced stages of this tumor is not satisfactory. Hence, there is an urgent need to introduce innovative solutions that will prove effective even in the advanced stage of the disease. In our study, a nanosystem based on ionic silver (Ag+) bound to a carrier-Titan yellow (TY) was analyzed. The possibility of binding the thus formed TY-Ag system to Congo red (CR) and albumin (BSA) was determined. TY-Ag binding to CR provides for better nanosystem solubility and enables its targeted intracellular transport and binding to immune complexes. The binding of TY-Ag or CR-TY-Ag to albumin also protects the system against the uncontrolled release of silver ions. It will also allow the delivery of silver in a targeted manner directly to the desired site in the case of intravenous administration of such a system. In this study, the MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values of the TY-Ag or BSA-TY-Ag systems were determined in two reference strains (Escherichia coli and Staphylococcus aureus). The paper presents nanosystems with a size of about 40-50 nm, with an intense antibacterial effect obtained at concentrations of 0.019 mM. We have also discovered that TY-Ag free or complexed with BSA (with a minimal Ag+ dose of 15-20 µM) inhibited cancer cells proliferation. TY-Ag complex diminished migration and effectively inhibited the T24 cell viability and induced apoptosis. On the basis of the obtained results, it has been shown that the presented systems may have anti-inflammatory and antitumor properties at the same time. TY-Ag or BSA-TY-Ag are new potential drugs and may become in future important therapeutic compounds in human urinary bladder carcinoma treatment and/or potent antimicrobial factors as an alternative to antibiotics.

Influence of metformin on HIF-1 pathway in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is defined as plasma cells malignancy, developing in the bone marrow. At the beginning of the disease, the malignant plasma cells are dependent on bone marrow microenvironment, providing growth and survival factors. Importantly, the recent studies pointed hypoxia as an important factor promoting progression of MM. In particular, hypoxia-triggered HIF-1 signaling was shown to promote chemoresistance, angiogenesis, invasiveness and induction of immature phenotype, suggesting that strategies targeting HIF-1 may contribute to improvement of anti-myeloma therapies. METHODS: The Western Blot and RT-PCR techniques were applied to analyze the influence of metformin on HIF-1 pathway in MM cells. To evaluate the effect of metformin on the growth of MM cell lines in normoxic and hypoxic conditions the MTT assay was used. The apoptosis induction in metformin treated hypoxic and normoxic cells was verified by Annexin V/PI staining followed by FACS analysis. RESULTS: Our results showed, for the first time, that metformin inhibits HIF-1 signaling in MM cells. Moreover, we demonstrated the effect of metformin to be mainly oxygen dependent, since the HIF-1 pathway was not significantly affected by metformin in anoxic conditions as well as after application of hypoxic mimicking compound, CoCl2. Our data also revealed that metformin triggers the growth arrest without inducing apoptosis in either normoxic or hypoxic conditions. CONCLUSIONS: Taken together, our study indicates metformin as a promising candidate for developing new treatment strategies exploiting HIF-1 signaling inhibition to enhance the overall anti-MM effect of currently used therapies, that may considerably benefit MM patients.

Scaffold vascularization method using an adipose-derived stem cell (ASC)-seeded scaffold prefabricated with a flow-through pedicle.

BACKGROUND: Vascularization is important for the clinical application of tissue engineered products. Both adipose-derived stem cells (ASCs) and surgical prefabrication can be used to induce angiogenesis in scaffolds. Our aim was to compare the angiogenic potential of ASC-seeded scaffolds combined with scaffold prefabrication with that of non-seeded, non-prefabricated scaffolds. METHODS: For prefabrication, functional blood vessels were introduced into the scaffold using a flow-through pedicle system. ASCs were isolated from rat fat deposits. Three-dimensional-printed cylindrical poly-ε-caprolactone scaffolds were fabricated by fused deposition modelling. Three groups, each containing six rats, were investigated by using non-seeded, ASC-seeded, and osteogenic induced ASC-seeded scaffolds. In each group, one rat was implanted with two scaffolds in the inguinal region. On the right side, a scaffold was implanted subcutaneously around the inferior epigastric vessels (classic prefabrication group). On the left side, the inferior epigastric vessels were placed inside the prefabricated scaffold in the flow-through pedicle system (flow-through prefabrication group). The vessel density and vascular architecture were examined histopathologically and by µCT imaging, respectively, at 2 months after implantation. RESULTS: The mean vessel densities were 10- and 5-fold higher in the ASC-seeded and osteogenic induced ASC-seeded scaffolds with flow-through prefabrication, respectively, than in the non-seeded classic prefabricated group (p < 0.001). µCT imaging revealed functional vessels within the scaffold. CONCLUSION: ASC-seeded scaffolds with prefabrication showed significantly improved scaffold vasculogenesis and could be useful for application to tissue engineering products in the clinical settings.

Aberrant promoter methylation may be responsible for the control of CD146 (MCAM) gene expression during breast cancer progression.

The CD146 (also known as MCAM, MUC-18, Mel-CAM) was initially reported in 1987, as a protein crucial for the invasiveness of malignant melanoma. Recently, it has been confirmed that CD146 has been involved in progression and poor overall survival of many cancers including breast cancer. Importantly, in independent studies, CD146 was reported to be a trigger of epithelial to mesenchymal transition in breast cancer cells. The goal of our current study was to verify the potential involvement of epigenetic mechanism behind the regulation of CD146 expression in breast cancer cells, as it has been previously reported in prostate cancer. First, we analysed the response of breast cancer cell lines, differing in the initial CD146 mRNA and protein content, to epigenetic modifier, 5-aza-2-deoxycytidine, and subsequently the methylation status of CD146 gene promoter was investigated, using direct bisulfite sequencing. We observed that treatment with demethylating agent led to induction of CD146 expression in all analysed breast cancer cell lines, both at mRNA and protein level, what was accompanied by increased expression of selected mesenchymal markers. Importantly, CD146 gene promoter analysis showed aberrant CpG island methylation in 2 out of 3 studied breast cancer cells lines, indicating epigenetic regulation of CD146 gene expression. In conclusion, our study revealed, for the first time, that aberrant methylation maybe involved in expression control of CD146, a very potent EMT inducer in breast cancer cells. Altogether, the data obtained may provide the basis for novel therapies as well as diagnostic approaches enabling sensitive and very accurate detection of breast cancer cells.

The Epigenetic Modifier 5-Aza-2-deoxycytidine Triggers the Expression of CD146 Gene in Prostate Cancer Cells.

BACKGROUND/AIM: During cancer progression cells undergo epithelial-to-mesenchymal transition (EMT). Although EMT is a complex process, recently, it has been reported that CD146 overexpression in prostate cancer cells is sufficient to induce mesenchymal phenotype. The following study aimed to investigate whether the expression of CD146 is altered by an epigenetic modifier in prostate cancer cells, in vitro. MATERIALS AND METHODS: Three human prostate cancer cell lines were treated with 5-aza-2-deoxycytidine; the expression of CD146 and EMT-related factors was analyzed by RT-PCR and western Blot. The methylation status of the CD146 promoter area was assessed using bisulfite sequencing. RESULTS: Our data showed that, the expression of CD146 was evidently increased in all three studied cell lines in response to a demethylating agent, both at the mRNA and protein level, suggesting epigenetic regulation of the analyzed gene. However, there was no methylation in the studied CpG island in CD146 gene promoter. Moreover, the demethylating agent induced the expression of EMT-related transcription factors (SNAI1, SNAI2, TWIST1 and ZEB1), the pattern of which differed among the cell lines, as well as alterations in cell morphology; altogether accounting for the mesenchymal phenotype. CONCLUSION: The demethylating agent 5-aza-2-deoxycytidine triggers the expression of CD146 in prostate cancer cells independently on the methylation status of the analyzed CpG island fragment in CD146 gene promoter. Moreover, demethylation treatment induces a mesenchymal profile in prostate cancer cells.

Characterization and Optimization of the Seeding Process of Adipose Stem Cells on the Polycaprolactone Scaffolds.

The purpose of the current study was to evaluate the usefulness of adipose-derived stem cells (ASCs) for bone injury therapy. Lipoaspirates were collected from the abdomen regions of 17 healthy female donors (mean age 49 ± 6 years) using Coleman technique or Body-jet liposuction. In the present study, the primary objective was the in vitro characteristics of human ASCs. The secondary objective was the optimization of the cell seeding process on 3D-printed scaffolds using polycaprolactone (PCL) or polycaprolactone covered with tricalcium phosphate (PCL + 5% TCP). Biological evaluation of human ASC showed high efficiency of isolation obtaining a satisfying amount of homogeneous cell populations. Results suggest that ASCs can be cultured in vitro for a long time without impairing their proliferative capacity. Growth kinetics shows that the highest number of cells can be achieved in passage 5 and after the 16th passage; there is a significant decrease of cell numbers and their proliferative potential. The percentage of colony forming units from the adipose stem cells is 8% ± 0.63% (p < 0.05). It was observed that the accumulation of calcium phosphate in the cells in vitro, marked with Alizarin Red S, was increased along with the next passage. Analysis of key parameters critically related to the cell seeding process shows that volume of cell suspension and propagation time greatly improve the efficiency of seeding both in PCL and PCL + 5% TCP scaffolds. The cell seeding efficiency did differ significantly between scaffold materials and cell seeding methods (p < 0.001). Increased seeding efficiency was observed when using the saturation of cell suspension into scaffolds with additional incubation. Alkaline phosphatase level production in PCL + 5% TCP scaffold was better than in PCL-only scaffold. The study results can be used for the optimization of the seeding process and quantification methods determining the successful implementation of the preclinical model study in the future tissue engineering strategies.

Application of case study to introduce medical students to molecular biology techniques used in HIV diagnostics.

Diagnostic molecular biology is a fast developing discipline of laboratory medicine widely used in numerous medical branches such as oncology, hematology, immunology, internal medicine, or infectious diseases, which will certainly have a major impact on clinical medicine in the near future. Nowadays, educational process is forced to face the quickly growing overflow of easily accessible data and properly guide the students not to be lead astray in the information chaos. Hence, in view of the foregoing, it appears obvious that modern medical education should put particular stress on selective acquiring, interpreting, and applying integrated multidisciplinary knowledge rather than on just absorbing and memorizing huge amount of scattered information. The presented case study aims at familiarizing the students with basic molecular biology techniques such as enzyme-linked immunosorbent assay, Western blot, and quantitative reverse transcription-polymerase chain reaction. Importantly, it is not limited only to discussing and learning the principles of the assays mentioned earlier, but it also shows their practical application in a particular diagnostic process and give the guidelines on how to explain and interpret exemplary results. In parallel, the way the case study is constructed allows a tutor to lead students into discussion on clinical aspects related to HIV infection what should eventually create complete picture of a HIV diagnostic process, thereby integrating basic knowledge of molecular biology laboratory techniques, HIV biology, and immunological response. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):355-360, 2019.

Analysis of Malignant Melanoma Cell Lines Exposed to Hypoxia Reveals the Importance of PFKFB4 Overexpression for Disease Progression.

BACKGROUND/AIM: Most melanomas develop in hypoxic conditions. Since hypoxia via HIF-1 induces glycolysis, a process essential for malignant melanoma growth/survival, the goal of this study was to analyze the influence of hypoxia on the expression of HIF-1 target genes involved in glucose metabolism. MATERIALS AND METHODS: The response of melanoma cell lines to hypoxic conditions was analyzed by RT-PCR and western blotting. A Kaplan-Meier survival analysis for patients with high and low expression level of PFKFB4 was performed. Further analysis of patients' data was performed using the R/Bioconductor environment. RESULTS: Induction of PFKFB4 gene expression can be considered a crucial mechanism behind glycolysis enhancement in hypoxic melanoma cells. Analysis of a publicly available database revealed that high PFKFB4 expression contributes to poor prognosis of melanoma patients. CONCLUSION: Currently available anti-melanoma therapeutic strategies may significantly benefit from agents targeting PFKFB4 activity.

Targeting the hypoxia pathway in malignant plasma cells by using 17-allylamino-17-demethoxygeldanamycin.

Multiple myeloma (MM) is characterized as a clonal expansion of malignant plasma cells in the bone marrow, which is often associated with pancytopenia and osteolytic bone disease. Interestingly, myeloma-infiltrated bone marrow is considered to be hypoxic, providing selection pressure for a developing tumour. Since HSP90 was shown to participate in stabilization of the subunit of the key transcription factor HIF-1, which controls the hypoxic response, the aim of this study was to investigate the influence of a HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), on MM cells cultured under low oxygenation conditions. We confirmed that 17-AAG inhibits hypoxic induction of the HIF-1 target genes in malignant plasma cells and demonstrate the concentration range of severe hypoxia-specific cytotoxicity. Next, we selected the malignant plasma cells under severe hypoxia/re-oxygenation culture conditions in the presence or absence of 17-AAG and subsequently, the cells which survived were further expanded and analyzed. Interestingly, we have noticed significant changes in the survival and the response to anti-MM drugs between the parental cell lines and those selected in cyclic severe hypoxia in the presence and absence of 17-AAG. Importantly, we also observed that the lack of oxygen itself, irrespectively of HIF-1 inhibition, is the main/pivotal factor driving the selection process in the experiments presented here.

Three-dimensional printed polycaprolactone-based scaffolds provide an advantageous environment for osteogenic differentiation of human adipose-derived stem cells.

The capacity of bone grafts to repair critical size defects can be greatly enhanced by the delivery of mesenchymal stem cells (MSCs). Adipose tissue is considered the most effective source of MSCs (ADSCs); however, the efficiency of bone regeneration using undifferentiated ADSCs is low. Therefore, this study proposes scaffolds based on polycaprolactone (PCL), which is widely considered a suitable MSC delivery system, were used as a three-dimensional (3D) culture environment promoting osteogenic differentiation of ADSCs. PCL scaffolds enriched with 5% tricalcium phosphate (TCP) were used. Human ADSCs were cultured in osteogenic medium both on the scaffolds and in 2D culture. Cell viability and osteogenic differentiation were tested at various time points for 42 days. The expression of RUNX2, collagen I, alkaline phosphatase, osteonectin and osteocalcin, measured by real-time polymerase chain reaction was significantly upregulated in 3D culture. Production of osteocalcin, a specific marker of terminally differentiated osteoblasts, was significantly higher in 3D cultures than in 2D cultures, as confirmed by western blot and immunostaining, and accompanied by earlier and enhanced mineralization. Subcutaneous implantation into immunodeficient mice was used for in vivo observations. Immunohistological and micro-computed tomography analysis revealed ADSC survival and activity toward extracellular production after 4 and 12 weeks, although heterotopic osteogenesis was not confirmed - probably resulting from insufficient availability of Ca/P ions. Additionally, TCP did not contribute to the upregulation of differentiation on the scaffolds in culture, and we postulate that the 3D architecture is a critical factor and provides a useful environment for prior-to-implantation osteogenic differentiation of ADSCs. Copyright © 2016 John Wiley & Sons, Ltd.

Gradients in pore size enhance the osteogenic differentiation of human mesenchymal stromal cells in three-dimensional scaffolds.

Small fractures in bone tissue can heal by themselves, but in case of larger defects current therapies are not completely successful due to several drawbacks. A possible strategy relies on the combination of additive manufactured polymeric scaffolds and human mesenchymal stromal cells (hMSCs). The architecture of bone tissue is characterized by a structural gradient. Long bones display a structural gradient in the radial direction, while flat bones in the axial direction. Such gradient presents a variation in bone density from the cancellous bone to the cortical bone. Therefore, scaffolds presenting a gradient in porosity could be ideal candidates to improve bone tissue regeneration. In this study, we present a construct with a discrete gradient in pore size and characterize its ability to further support the osteogenic differentiation of hMSCs. Furthermore, we studied the behaviour of hMSCs within the different compartments of the gradient scaffolds, showing a correlation between osteogenic differentiation and ECM mineralization, and pore dimensions. Alkaline phosphatase activity and calcium content increased with increasing pore dimensions. Our results indicate that designing structural porosity gradients may be an appealing strategy to support gradual osteogenic differentiation of adult stem cells.

Influence of internal pore architecture on biological and mechanical properties of three-dimensional fiber deposited scaffolds for bone regeneration.

Fused deposition modeling has been used to fabricate three-dimensional (3D) scaffolds for tissue engineering applications, because it allows to tailor their pore network. Despite the proven flexibility in doing so, a limited amount of studies have been performed to evaluate whether specific pore shapes have an influence on cell activity and tissue formation. Our study aimed at investigating the influence of internal pore architecture on the biological and mechanical properties of 3D scaffolds seeded with mesenchymal stromal cells. Polycaprolactone scaffolds with six different geometries were fabricated. The 3D samples were manufactured with different lay-down pattern of the fibers by varying the layer deposition angle from 0°/15°/30°, to 0°/30°/60°, 0°/45°/90°, 0°/60°/120°, 0°/75°/150°, and 0°/90°/180°. The scaffolds were investigated by scanning electron microscopy and micro computed tomographical analysis and displayed a fully interconnected pore network. Cell proliferation and differentiation toward the osteogenic lineage were evaluated by DNA, alkaline phosphatase activity, and polymerase chain reaction. The obtained scaffolds had structures with open porosity (50%-60%) and interconnected pores ranging from 380 to 400 µm. Changing the angle deposition affected significantly the mechanical properties of the scaffolds. With increasing the angle deposition between successive layers, the elastic modulus increased as well. Cellular studies also showed influence of the internal architecture on cell adhesion and proliferation within the 3D construct, yet limited influence on cell differentiation was observed.

A bioactive hybrid three-dimensional tissue-engineering construct for cartilage repair.

The aim was to develop a hybrid three-dimensional-tissue engineering construct for chondrogenesis. The hypothesis was that they support chondrogenesis. A biodegradable, highly porous polycaprolactone-grate was produced by solid freeform fabrication. The polycaprolactone support was coated with a chitosan/polyethylene oxide nanofibre sheet produced by electrospinning. Transforming growth factor-ß3-induced chondrogenesis was followed using the following markers: sex determining region Y/-box 9, runt-related transcription factor 2 and collagen II and X in quantitative real-time polymerase chain reaction, histology and immunostaining. A polycaprolactone-grate and an optimized chitosan/polyethylene oxide nanofibre sheet supported cellular aggregation, chondrogenesis and matrix formation. In tissue engineering constructs, the sheets were seeded first with mesenchymal stem cells and then piled up according to the lasagne principle. The advantages of such a construct are (1) the cells do not need to migrate to the tissue engineering construct and therefore pore size and interconnectivity problems are omitted and (2) the cell-tight nanofibre sheet and collagen-fibre network mimic a cell culture platform for mesenchymal stem cells/chondrocytes (preventing escape) and hinders in-growth of fibroblasts and fibrous scarring (preventing capture). This allows time for the slowly progressing, multiphase true cartilage regeneration.

Tanespimycin and tipifarnib exhibit synergism in inducing apoptosis in melanoma cell lines from later stages of tumor progression.

Many anticancer strategies rely on efficient induction of apoptosis. The need for development of drug combinations with a strong pro-apoptotic activity is of particular interest in melanoma resistant to currently available chemotherapeutic regimes. We studied the pro-apoptotic properties of combination of tanespimycin+tipifarnib in five melanoma cell lines representing various stages of tumor progression. Our results show that in cells derived from vertical- and metastatic-phase the combination of tested drugs is strongly cytotoxic and efficient in inducing apoptosis, as evidenced by activation of caspase-9 and caspase-3 and enhanced fragmentation of DNA.

Consistent quasistatic and acoustic elasticity determination of poly-L-lactide-based rapid-prototyped tissue engineering scaffolds.

This paper is concerned with reliable and physically sound elasticity determination of rapid-prototyped tissue engineering scaffolds made of poly-L-lactide (PLLA), with and without small portions of tricalcium phosphate (TCP) inclusions. At the level of overall scaffolds, that is, that of several millimeters, multiple uniaxial loading-unloading (quasistatic) tests were performed, giving access to the scaffolds' Young's moduli, through stress-strain characteristics during unloading. In addition, acoustic tests with 0.05 MHz frequency delivered an independent access to elastic properties, in terms of the normal components of the scaffolds' stiffness tensors. The latter strongly correlate, in a linear fashion, with the Young's moduli from the unloading tests, revealing porosity independence of Poisson's ratio. The magnitude of the latter is in full agreement with literature data on polymers. Both of these facts underline that both ultrasound tests and quasistatic unloading tests reliably provide the elastic properties of tissue engineering scaffolds.

Tipifarnib and tanespimycin show synergic proapoptotic activity in U937 cells.

BACKGROUND: Farnesyltransferase inhibitor tipifarnib (R115777) has been used for treatment of hematological malignancies; however, its observed anticancer effect was limited. This prompted us to search for inhibitors that would show synergic, proapoptotic effect when combined with R115777. We decided to study LY294002, which inhibits PI-3 kinase, and tanespimycin (17AAG), which inhibits Hsp90--a chaperone for a number of proteins, including Akt kinase. METHODS: The effect of drugs, used alone or in combination, was tested in U937 cells (human leukemic monocyte lymphoma), which are often used as a model for liquid tumor. The number of viable cells was evaluated with trypan blue staining, while apoptosis was assessed by presence of active caspase-3 and terminal dUTP nick-end labeling of DNA (TUNEL). RESULTS: At concentrations in which R115777, LY294002 and 17AAG were only slowing down the proliferation rate, when used separately, the combination of R115777 + LY294002 and R115777 + 17AAG significantly reduced the number of cells and induced cellular apoptosis. CONCLUSIONS: Our results suggest that the combination of R115777 + 17AAG could be useful in treating some of the hematological malignancies.

Cytochalasin D, LY294002 and olomoucine synergize in promoting death of melanoma cells through activation of caspase-3 and apoptosis.

Many of the current anticancer therapies rely on the induction of apoptosis, and several mechanisms that protect cells against apoptosis may be upregulated in tumors. A growing body of evidence suggests that single drugs with a clearly defined intracellular target may be less efficient in arresting tumor growth and induction of apoptosis than multitargeted strategies. To prove that this is also the case for melanoma, we studied five cell lines, which represent different stages of tumor progression. We tested cell viability, terminal dUTP nick-end labeling and activation of caspase-3 upon exposure to cytochalasin D, LY294002 and olomoucine, added either alone or in various combinations. The obtained data were compared with effects caused by staurosporine. The results show that whereas staurosporine efficiently induced apoptosis in all tested melanoma cell lines, the other drugs had only moderate effects when administered alone. In contrast, the combinations of drugs were more effective in inducing caspase-3 activity and reducing cell viability. In particular, the triple combination of cytochalasin D+LY294002+olomoucine was almost as effective as staurosporine in inducing caspase-3 activity and apoptosis. These results prove that it is possible to design new pharmacological strategies that will effectively induce caspase-3 activity and apoptosis in melanoma. The possible explanations of the observed synergy between the tested drugs are also discussed.