Cytotoxic Activity of Melatonin Alone and in Combination with Doxorubicin and/or Dexamethasone on Diffuse Large B-Cell Lymphoma Cells in In Vitro Conditions.

Melatonin (MLT), a pineal gland hormone, not only regulates circadian and seasonal rhythms, but also plays an important role in many aspects of human physiology and pathophysiology. MLT is of great interest as a natural substance with anti-cancer activities. The aim of this study was to assess the cytotoxicity and apoptosis of MLT, used alone or in combination with one of the most active anti-cancer drugs, doxorubicin (DOX), and a well-known anti-inflammatory drug, dexamethasone (DEX), on a diffuse large B-cell lymphoma (DLBCL)-derived cell line. The cytotoxicity and cell cycle distribution were measured using propidium iodide staining, while apoptosis was assessed using the annexin-V binding method. Additionally, to elucidate the mechanisms of action, caspase-3, -8, and -9 and a decline in the mitochondrial potential were determined using flow cytometry. MLT inhibited cell viability as well as induced apoptosis and cell cycle arrest at the G0/G1 phase. The pro-apoptotic effect was exerted through both the mitochondrial and caspase-dependent pathways. Furthermore, we observed increased cytotoxic and pro-apoptotic activity as well as the modulation of the cell cycle after the combination of MLT with DOX, DEX, or a combination of DOX + DEX, compared with both drugs or MLT used alone. Our findings confirm that MLT is a promising in vitro anti-tumour agent that requires further evaluation when used with other drugs active against DLBCL.

Atypical Chronic Lymphocytic Leukemia-The Current Status.

A diagnosis of typical chronic lymphocytic leukemia (CLL) requires the presence of ≥5000 clonal B-lymphocytes/µL, the coexistence of CD19, CD20, CD5, and CD23, the restriction of light chain immunoglobulin, and the lack of expression of antigens CD22 and CD79b. Atypical CLL (aCLL) can be distinguished from typical CLL morphologically and immunophenotypically. Morphologically atypical CLL cells have been defined mainly as large, atypical forms, prolymphocytes, or cleaved cells. However, current aCLL diagnostics rely more on immunophenotypic characteristics rather than atypical morphology. Immunophenotypically, atypical CLL differs from classic CLL in the lack of expression of one or fewer surface antigens, most commonly CD5 and CD23, and the patient does not meet the criteria for a diagnosis of any other B-cell lymphoid malignancy. Morphologically atypical CLL has more aggressive clinical behavior and worse prognosis than classic CLL. Patients with aCLL are more likely to display markers associated with poor prognosis, including trisomy 12, unmutated IGVH, and CD38 expression, compared with classic CLL. However, no standard or commonly accepted criteria exist for differentiating aCLL from classic CLL and the clinical significance of aCLL is still under debate. This review summarizes the current state of knowledge on the morphological, immunophenotypic, and genetic abnormalities of aCLL.

Novel Clofarabine-Based Combinations with Polyphenols Epigenetically Reactivate Retinoic Acid Receptor Beta, Inhibit Cell Growth, and Induce Apoptosis of Breast Cancer Cells.

An epigenetic component, especially aberrant DNA methylation pattern, has been shown to be frequently involved in sporadic breast cancer development. A growing body of literature demonstrates that combination of agents, i.e. nucleoside analogues with dietary phytochemicals, may provide enhanced therapeutic effects in epigenetic reprogramming of cancer cells. Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF), a second-generation 2'-deoxyadenosine analogue, has numerous anti-cancer effects, including potential capacity to regulate epigenetic processes. Our present study is the first to investigate the combinatorial effects of ClF (used at IC50 concentration) with epigallocatechin-3-gallate (EGCG, tea catechin) or genistein (soy phytoestrogen), at physiological concentrations, on breast cancer cell growth, apoptosis, and epigenetic regulation of retinoic acid receptor beta (RARB) transcriptional activity. In MCF7 and MDA-MB-231 cells, RARB promoter methylation and expression of RARB, modifiers of DNA methylation reaction (DNMT1, CDKN1A, TP53), and potential regulator of RARB transcription, PTEN, were estimated using methylation-sensitive restriction analysis (MSRA) and quantitative real-time polymerase chain reaction (qPCR), respectively. The combinatorial exposures synergistically or additively inhibited the growth and induced apoptosis of breast cancer cells, followed by RARB hypomethylation with concomitant multiple increase in RARB, PTEN, and CDKN1A transcript levels. Taken together, our results demonstrate the ability of ClF-based combinations with polyphenols to promote cancer cell death and reactivate DNA methylation-silenced tumor suppressor genes in breast cancer cells with different invasive potential.

The impact of agonists and antagonists of TLR3 and TLR9 on concentrations of IL-6, IL10 and sIL-2R in culture supernatants of peripheral blood mononuclear cells derived from patients with systemic lupus erythematosus.

Toll-like receptors (TLR), especially TLR3, 7 and 9, play an important role in the pathogenesis of systemic lupus erythematosus (SLE). In our study blood was collected from 16 patients with SLE and from 8 healthy volunteers. Concentrations of IL-6, IL-10 and sIL-2R were measured by ELISA in mononuclear cell culture supernatant after 24 hours of stimulation by agonists and antagonists of TLR3 and 9 (for TLR3-poli I/C, resveratrol and for TLR9-ODN2006, IRS 945). Stimulation of TLR9 by ODN2006 led to an increase of IL-6 concentration in cell culture supernatants from the cells of healthy volunteers compared with unstimulated cells from controls. Inhibition of TLR3 activation by resveratrol caused a significantly lower concentration of IL-10 in cell culture supernatants derived from both patients and healthy donors. Moreover, resveratrol significantly decreased the level of IL-10 and sIL-2R in culture supernatants of cells derived from patients with active disease compared to the inactive stage. A positive correlation was also found between IL-6 concentration following ODN2006 administration and disease activity. In conclusions, our results indicate that TLRs play a role in the modulation of the inflammatory response in SLE patients. This suppressive action on IL-10 synthesis demonstrated by resveratrol suggests that it may be useful in SLE therapy.

The distribution and potential prognostic value of SMAD protein expression in chronic lymphocytic leukemia.

The SMAD proteins are responsible for transducing signals from activated transforming growth factor-beta. This is the first study assessing the expression of SMAD-1/8, SMAD-2/3, SMAD-4, and SMAD-7 in chronic lymphocytic leukemia cells with regard to their clinical significance and potential prognostic value. Overexpression of SMAD-1/8 was observed in 160 chronic lymphocytic leukemia patients compared to 42 healthy volunteers (p = 0.023) and was associated with a more progressive course of the disease (p = 0.016). Moreover, the high expression of SMAD-1/8 correlated with other, well-established prognostic factors, including clinical stage (p = 0.010) and lymphocyte doubling time (p = 0.021). The expression of SMAD-4 was lower in chronic lymphocytic leukemia patients compared with the control group (p = 0.003). Importantly, lower SMAD-4 levels correlated with longer progression-free survival (p = 0.009), progressive course of the disease (p = 0.002), advanced clinical stage (p = 0.0004), elevated beta-2-microglobulin and lactate dehydrogenase levels (p < 0.05), shorter lymphocyte doubling time (p = 0.009), and CD38 antigen expression (p = 0.039). In addition, lower SMAD-4 expression correlated with lower apoptotic index (p = 0.0007) and lower expression of receptors for vascular endothelial growth factors VEGFR-1 and VEGFR-2. A significant association was found between the low expression of inhibitory protein SMAD-7 and both zeta-chain-associated protein kinase 70-negative cells (p = 0.04) and lower apoptotic index (p = 0.004). No differences were observed in SMAD-2/3 expression. In conclusion, our results demonstrate a significant correlation between greater SMAD-1/8 and lower SMAD-4 expression in chronic lymphocytic leukemia cells, as well as more progressive outcome and poor prognosis. These data provide supporting evidence that the expression of SMAD proteins plays an important role in disease development and may be considered as a novel, biologic prognostic factor in this disease.

Glycodendrimer PPI as a Potential Drug in Chronic Lymphocytic Leukaemia. The Influence of Glycodendrimer on Apoptosis in In Vitro B-CLL Cells Defined by Microarrays.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) cells are characterized by failures in the apoptosis pathway and increased proliferation, resulting in the progressive accumulation of B-lymphocytes in blood. Despite the wide range of antileukaemic drugs, CLL remains an incurable disease. However, a breakthrough is expected which will allow more effective treatment. OBJECTIVE: The study investigates the influence of poly(propyleneimine) (PPI) dendrimer with peripheral amino groups, 30% of which were coated with maltotriose (PPI-G4-OS-Mal-III), on CLL cells, and demonstrates that it acts through the induction of the apoptotic mechanism. It is important to note that the dendrimer was used as a drug itself and not as a drug carrier. METHOD: CLL and normal lymphocytes were treated in vitro with the dendrimer, either alone or in combination with fludarabine (FA). The percentages of apoptotic and necrotic cells, and the protein expression, were checked using a flow cytometer. Gene expression was screened using a two-colour microarray with 60-mer probes. RESULTS: The results confirm that PPI-G4-OS-Mal-III influences the viability of CLL cells in vitro and does not exert any significant harmful effect on normal lymphocytes. The dendrimer appears to significantly influence gene and protein expression in CLL cells. CONCLUSION: Since dendrimers can be specifically targeted, they may be very effective in CLL therapy, especially since in vitro PPI-G4-OS-Mal-III has been found to have stronger effect than fludarabine.

Pro-Apoptotic Activity of New Honokiol/Triphenylmethane Analogues in B-Cell Lymphoid Malignancies.

Honokiol and triphenylmethanes are small molecules with anti-tumor properties. Recently, we synthesized new honokiol analogues (HAs) that possess common features of both groups. We assessed the anti-tumor effectiveness of HAs in B-cell leukemia/lymphoma cells, namely in chronic lymphocytic leukemia (CLL) cells ex vivo and in pre-B-cell acute lymphoblastic leukemia (Nalm-6), Burkitt lymphoma (BL; Raji), diffuse large B-cell lymphoma (DLBCL; Toledo) and multiple myeloma (MM; RPMI 8226) cell lines. Four of these compounds appeared to be significantly active against the majority of cells examined, with no significant impact on healthy lymphocytes. These active HAs induced caspase-dependent apoptosis, causing significant deregulation of several apoptosis-regulating proteins. Overall, these compounds downregulated Bcl-2 and XIAP and upregulated Bax, Bak and survivin proteins. In conclusion, some of the HAs are potent tumor-selective inducers of apoptosis in ex vivo CLL and in BL, DLBCL and MM cells in vitro. Further preclinical studies of these agents are recommended.

Immune checkpoints: Cytotoxic T-lymphocyte antigen 4 and programmed cell death protein 1 in breast cancer surgery.

Immune checkpoints refer to a plethora of inhibitory pathways built into the immune system, and recent studies have emphasized the role of these checkpoints in carcinogenesis. The aim of the present study was to evaluate two major immune checkpoints, the cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), in the serum of 35 patients with stage I and II breast cancer. Serum concentrations of CTLA-4 and PD-1 were measured at three time points: i) Preoperatively; ii) during anesthesia following the harvesting of sentinel nodes (SNs); and iii) 24 h postoperatively. Control samples were obtained from 25 healthy, age-matched females. Assessment of CTLA-4 and PD-1 expression levels was conducted using flow cytometry. A statistically significant difference in PD-1 expression was identified between breast cancer patients preoperatively and healthy controls (26.31±11.87 vs. 12.72±8.15; P<0.0001). In addition, a statistically significant association was found between CTLA-4 and PD-1 levels prior to surgery (P=0.0084). In addition, CTLA-4 expression was associated with age (P=0.0453), with elevated levels of CTLA-4 detected in older breast cancer patients. Higher PD-1 expression levels were observed in T2 tumors compared with T1 tumors prior to surgery and intraoperatively; however, the differences were not statistically significant. Furthermore, a decrease in PD-1 levels was observed subsequent to harvesting SNs with metastasis, but not in SN-negative patients (P=0.05). A negative correlation was also observed between PD-1 expression and progesterone receptor (PR) status following surgery (P=0.024). These results provided a basis for further investigation of immune checkpoints in breast cancer. Breast cancer patients exhibit an altered profile of immune checkpoint markers, with higher concentrations of PD-1 observed in larger, PR-negative tumors.

Sulforaphane Alone and in Combination with Clofarabine Epigenetically Regulates the Expression of DNA Methylation-Silenced Tumour Suppressor Genes in Human Breast Cancer Cells.

BACKGROUND/AIM: Sporadic breast cancer is frequently associated with aberrant DNA methylation patterns that are reversible and responsive to environmental factors, including diet. In the present study, we investigated the effects of sulforaphane (SFN), a phytochemical from cruciferous vegetables, on the methylation and expression of PTEN and RARbeta2 tumour suppressor genes as well as on the expression of regulators of DNA methylation reaction, DNMT1 , p53 , and p21 , in MCF-7 and MDA-MB-231 human breast cancer cells with different invasive potential. We also evaluate the role of SFN epigenetic effects in support of therapy with clofarabine (ClF) that was recently shown to modulate the epigenome as well. METHODS: Promoter methylation and gene expression were estimated using methylation-sensitive restriction analysis and real-time PCR, respectively. RESULTS: In both MCF-7 and MDA-MB-231 cells, SFN at IC 50 (22 and 46 µ M , respectively) and a physiologically relevant 10 µ M concentration lead to hypomethylation of PTEN and RARbeta2 promoters with concomitant gene upregulation. The combination of SFN and ClF enhances these effects, resulting in an increase in cell growth arrest and apoptosis at a non-invasive breast cancer stage. CONCLUSIONS: Our findings provide evidence that SFN activates DNA methylation-silenced tumour suppressor genes in breast cancer cells and may contribute to SFN-mediated support of therapy with an anti-cancer drug, ClF, increasing its applications in solid tumours.

Pro-Apoptotic Activity of Ruxolitinib Alone and in Combination with Hydroxyurea, Busulphan, and PI3K/mTOR Inhibitors in JAK2-Positive Human Cell Lines.

BACKGROUND: The JAK2V617F mutation plays a crucial role in the pathogenesis of myeloproliferative neoplasms (MPNs). Inhibition of JAK2 activity by ruxolitinib (RX) results in growth inhibition and apoptosis of cells carrying the JAK2V617F mutation however the exact mechanisms regulating apoptosis have not been fully elucidated. OBJECTIVES: This study assessed the potential cytotoxicity of RX against JAK2-positive human cell lines (SET-2 and HEL), either alone or in combination with hydroxyurea, busulphan, rapamycin or LY294002. MATERIAL AND METHODS: Cell viability, the apoptosis rate (annexin-V staining), drop of mitochondrial transmembrane potential (Δψm) and caspase activation, were measured using flow cytometry. Additionally, the expression of several apoptosis-regulating proteins was evaluated. RESULTS: RX showed cytotoxicity against both SET-2 and HEL cell lines. The main mechanism of this action was apoptosis, with significant drop of Δψm, caspase-3 and -9 activation, and moderate activation of caspase-8 (only for SET-2 cells). Corresponding to enhanced apoptosis, the expression levels of some apoptosis-regulating proteins were changed, the most pronounced in both cell lines being up-regulation of Bax and down-regulation of Bcl-2 proteins. Additionally, up-regulation of Bak and Bad (SET-2) and down-regulation of Mcl-1 (HEL) were observed. Of the studied compounds, a combination of RX + LY294002 induced the greatest cytotoxicity in both SET-2 and HEL cell lines, and rapamycin the least. CONCLUSIONS: This study shows that the combination of RX and a PI3K kinase inhibitor provokes a significant pro-apoptotic effect in JAK2V617F mutated cells, which may justify the beginning of clinical trials based on the combination of these drugs.

Prognostic value of inhibitor of apoptosis protein family expression in patients with acute myeloid leukemia.

The inhibitor of apoptosis protein (IAP) family acts as an inhibitor of apoptosis pathways. The potential prognostic value of the expression of selected IAP family members, XIAP, cIAP-1, cIAP-2 and survivin protein, was evaluated with regard to treatment response and survival of 56 newly diagnosed adult patients with acute myeloid leukemia (AML). The presence of these IAP members influenced the achievement of a complete response (CR). In addition, overall survival (OS) was influenced by low survivin expression in univariate and multivariate analysis (p = 0.014 and p = 0.013, respectively). A strong correlation was observed between members of the IAP family (XIAP and cIAP-1, XIAP and cIAP-2, cIAP-1 and cIAP-2, p < 0.001 for all comparisons), while Smac/DIABLO demonstrated an inverse correlation with XIAP, cIAP-1 and cIAP-2 (p < 0.001 for all comparisons). Further studies should be undertaken to better demonstrate the mode of action of IAP members, as well as their prognostic and therapeutic potentials.

The kinetics of hematopoietic niche cytokines and their influence on mobilization efficacy and timing in patients with hematological malignancies.

The bone marrow niche functions are modulated by complicated cytokines network. The aim of our study was to evaluate the levels of VCAM-1, VEGF, MMP-9 and SDF during mobilization of CD34+ cells in patients with hematological malignancies. Thirty four patients were enrolled to the study (19F, 15 M) at median age of 57 years. The group consisted of patients with multiple myeloma (26) and lymphoma (8). The mobilization procedures comprised chemotherapy and then G-CSF. Blood samples were collected before chemotherapy (N = 34) and on the day of the first apheresis (N = 26). Cytokines were evaluated with ELISA assay. We observed significant increase in VCAM-1 levels during mobilization. On contrary, VEGF and SDF levels decreased during mobilization procedure. The levels of MMP-9 were stable during mobilization. We divided patients according to baseline cytokines levels below and above median into "low" and "high" expressors. The group of VEGF "low" expressors had longer median time of G-CSF treatment before first apheresis than 'high' expressors. Baseline VEGF levels correlated adversely with duration of G-CSF treatment before first apheresis. Patients were also divided according to median cytokines levels at apheresis into "low" and "high" expressors. "High" VCAM-1 expressors had higher CD34+in peripheral blood as well as higher CD34+numbers collected during first apheresis than "low" expressors. In conclusion, the levels of niche cytokines change significantly during mobilization in patients with hematopoietic malignancies. Baseline VEGF can influence timing of mobilization. Higher VCAM-1 corresponds with higher mobilization efficacy.

Angiopoietins in haematopoietic stem cell mobilisation in patients with haematological malignancies.

BACKGROUND: The bone marrow niche contains different types of cells including osteoblasts and endothelial progenitors, all of which interact and take part in the process of mobilisation. The aim of our study was to evaluate the levels of cytokines (osteopontin and angiopoietins 1 and 2) active in the bone marrow niche during the mobilisation of haematopoietic stem cells for autologous transplantation. MATERIALS AND METHODS: Forty-eight patients (24 females, 24 males), median age 56.5 years, entered the study. The group consisted of patients with multiple myeloma (n=34), lymphoma (n=13) and acute myeloid leukaemia (n=1). Blood samples were collected before chemotherapy and on the day of the first apheresis. Cytokines were evaluated by enzyme-linked immunosorbent assays. Additionally, circulating endothelial cells were assessed by flow cytometry. RESULTS: The median concentration of angiopoietin 1 at the time of apheresis was lower than that at baseline (2.7 vs 7.8 ng/mL, p<0.001). In contrast, the median level of angiopoietin 2 increased during the mobilisation procedure (3.6 vs 2.8 ng/mL, p=0.001). The patients were divided according to the number of days of granulocyte colony-stimulating factor treatment before the first apheresis into "early" (median) mobilisers. The group of "early mobilisers" had higher baseline angiopoietin 1 levels (median=11.6 ng/mL) than those of the "late mobilisers" (median=6.0 ng/mL, p=0.05). An adverse correlation was observed between duration of granulocyte colony-stimulating factor treatment and baseline angiopoietin 1 level. Baseline angiopoietin 1 levels correlated with numbers of circulating endothelial cells. Low angiopoietin 2 level increased the chance of poor mobilisation. CONCLUSIONS: The angiogenic processes can influence the timing of mobilisation. Angiopoietins 1 and 2 need further evaluation in the context of mobilisation.

Jagged-1: a new promising factor associated with favorable prognosis in patients with acute myeloid leukemia.

In this study Jagged-1 and Dll-1 surface expression as well as Notch-1 receptor intracellular domain (Notch-1-IC) expression were assessed by multi-color flow cytometry in leukemic blasts obtained from 88 patients with acute myeloid leukemia (AML). CD34+peripheral blood stem cells (PBSCs) were used as a control. The median expression of Jagged-1 and Dll-1 was significantly higher in AML blasts than in PBSCs (p=0.001 and p=0.002, respectively). Higher expression of Notch-1-IC was detected in patients with poor-risk karyotype as compared to good- and intermediate-risk groups (p=0.035). In our study, poor-risk cytogenetics and low (

trans-Platinum(II) complex of 3-aminoflavone - synthesis, X-ray crystal structure and biological activities in vitro.

This paper describes the synthesis of trans-bis-(3-aminoflavone)dichloridoplatinum(ii) (trans-Pt(3-af)2Cl2; TCAP) for use as a potential anticancer compound, and the evaluation of its structure by elemental and spectral analyses, and X-ray crystallography. The complex demonstrated a significant cytotoxic effect against human and murine cancer cell lines, as well as weaker toxicity towards healthy cells (human peripheral blood lymphocytes) in comparison with cisplatin. Various biochemical and morphological methods confirm that the proapoptotic activity of trans-Pt(3-af)2Cl2 is markedly higher than the reference cisplatin. Our results suggest that trans-Pt(3-af)2Cl2 may have a different antitumour specificity from that of cisplatin.

Cytotoxic and apoptosis-inducing effects of bendamustine used alone and in combination with rituximab on chronic lymphocytic leukemia cells in vitro.

AIM: The aim of our study was to compare the cytotoxic effects of bendamustine (BENDA) and rituximab (RIT) used either alone or in combination and to evaluate the influence of the above mentioned drugs on apoptosis measured as changes in mitochondrial transmembrane potential (Δψm), expression of caspases and selected apoptosis-regulating proteins in freshly isolated peripheral blood mononuclear cells of chronic lymphocytic leukemia (CLL) patients. MATERIALS/METHODS: Cytotoxic effect of tested drugs, as well as induction of apoptosis, drop in Δψm and expression of selected proteins involved in regulation of apoptosis were assessed in 48 hour cultures containing autologous serum (AS) using flow cytometry. BENDA was used at the concentration of 40 µg/ml and RIT at the concentration of 10 µg/ml. Control cultures were incubated without drugs. RESULTS: BENDA used either alone or in combination with RIT strongly induced apoptosis as well as enhanced expression of selected apoptotic proteins, especially those involved in the intrinsic apoptotic pathway: P53, PUMA and BAX, which cause mitochondrial transmembrane potential changes leading to activation of caspase-9 and -3. CONCLUSIONS: Our results indicate that both BENDA and RIT participate in the induction of apoptosis of CLL lymphocytes in vitro in the presence of AS in the culture medium. The drug-induced apoptosis occurs mainly via intrinsic pathway and activation of P53 and PUMA proteins, however the extrinsic pathway is likely to be involved as well. We also found that the combination of these drugs induces the expression of P53, caspase-8 and -9 more potently than either of them used separately.

Pro-apoptotic effect of an anti-CD37 scFv-Fc fusion protein, in combination with the anti-CD20 antibody, ofatumumab, on tumour cells from B-cell malignancies.

SMIP-016, a new anti-tumour agent, is a mouse/human chimeric fusion protein built on the ADAPTIR™ (modular protein therapeutic) platform targeting human CD37. In this study, for the first time, we examined pro-apoptotic activity of SMIP-016 in combination with monoclonal anti-CD20 antibody, ofatumumab (HuMax-CD20) in de novo chronic lymphocytic leukaemia (CLL) cells and in different B-cell neoplasm-derived lines. In CLL cells SMIP-016 exerted significant cytotoxicity (versus control - p=0.01). In the in vitro models, SMIP-016 was also distinctly active against Raji line (Burkitt lymphoma; BL) (versus control - p=0.007), Riva-1 line (diffuse large B-cell lymphoma; DLBCL) (versus control - p=0.002) and RPMI 8226 line (multiple myeloma cells; MM) (versus control - p=0.03). In studies combining SMIP-016 and ofatumumab, the cytotoxicity against CLL cells was significantly higher than the agents used alone (p<0.03). Remarkably enhanced cytotoxic activity of SMIP-016 and ofatumumab in combination was also observed in Raji and Riva-1 cell lines (p<0.01 and p<0.003, respectively). Importantly, both agents induced cytotoxicity at very low concentrations which suggests that potential side-effects may be decreased in clinical practice. The mechanism responsible for cytotoxicity of SMIP-016 in all the examined models was connected with caspase-dependent apoptosis. In majority of cell types SMIP-016 induced overexpression of Bax protein, as well as downregulation of Bcl-2, cIAP1 (p<0.03) and Smac/DIABLO (p<0.003) apoptosis-regulating proteins. In conclusion, our study demonstrated high pro-apoptotic activity of SMIP-016, especially in combination with ofatumumab, against ex vivo CLL cells, and BL or DLBCL in vitro cell lines. Thus, further preclinical studies in in vivo models are warranted, as this combination may be a promising therapeutic concept for treatment of those malignancies.

Cytotoxic activity of the amphibian ribonucleases onconase and r-amphinase on tumor cells from B cell lymphoproliferative disorders.

Although major advancements in antitumor treatment have been observed, several B cell-derived malignancies still remain incurable. A promising approach that involves targeting RNA either by the use of specific antisense oligonucleotides or cytostatic/cytotoxic ribonucleases (RNases) is being promoted. Two amphibian RNases, onconase (ONC; ranpirnase) and, more recently, r-amphinase (r-Amph), have already been tested, but thus far, mostly on solid tumors. In this study, for the first time we provide comprehensive data on ex vivo and in vivo cytotoxic activity of ONC or r-Amph against cancer cells from different B cell lymphoid malignancies, together with their detailed mode of antitumor action. Our data revealed strong pro-apoptotic activity of ONC and r-Amph in both chronic lymphocytic leukemia and aggressive B cell lymphomas, with less impact on acute lymphoblastic leukemia or multiple myeloma cells. Moreover, the antitumor action of ONC and r-Amph was markedly selective against neoplastic cells sparing normal, healthy control­derived lymphocytes.

Expression of toll-like receptors 3, 7, and 9 in peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology. The results of experimental studies point to the involvement of innate immunity receptors-toll-like receptors (TLR)-in the pathogenesis of the disease. The aim of the study was to assess the expression of TLR3, 7, and 9 in the population of peripheral blood mononuclear cells (PBMC) and in B lymphocytes (CD19(+)), T lymphocytes (CD4(+) and CD8(+)) using flow cytometry. The study group included 35 patients with SLE and 15 healthy controls. The patient group presented a significantly higher percentage of TLR3- and TLR9-positive cells among all PBMCs and their subpopulations (CD3(+), CD4(+), CD8(+), and CD19(+) lymphocytes) as well as TLR7 in CD19(+) B-lymphocytes, compared to the control group. There was no correlation between the expression of all studied TLRs and the disease activity according to the SLAM scale, and the degree of organ damage according to the SLICC/ACR Damage Index. However, a correlation was observed between the percentage of various TLR-positive cells and some clinical (joint lesions) and laboratory (lymphopenia, hypogammaglobulinemia, anaemia, and higher ESR) features and menopause in women. The results of the study suggest that TLR3, 7, and 9 play a role in the pathogenesis of SLE and have an impact on organ involvement in SLE.

Spontaneous in vitro apoptosis of de novo chronic lymphocytic leukemia cells correlates with risk of the disease progression.

Despite significant progress in treatment, chronic lymphocytic leukemia (CLL) still remains an incurable disease. Major advances have been recently made to understand the molecular pathogenesis underlying CLL, but defects in apoptosis are considered to be the most important factors. Although neoplastic cells are resistant to apoptosis in vivo, they show decreased level of spontaneous in vitro apoptosis, with significant differences among CLL patients. This work compares the level of spontaneous CLL cell apoptosis with prognostic factors and clinical course of the disease. In vitro spontaneous apoptosis of peripheral blood lymphocytes was analyzed using Annexin-V assay (confirmed by TUNEL method) in 135 treatment naïve patients with CLL. Levels of apoptosis after 48 h culture in patients with stable disease were found to be significantly higher than in the group with progressive course of the disease (P = 0.015). Moreover, the level of spontaneous apoptosis after 24 and 48 h of incubation correlated inversely with the progression free survival (P = 0.026 and 0.009, respectively). These results suggest that in vitro spontaneous apoptosis of CLL cells may be a simple and cheap prognostic test which is relatively quick to use, and can predict the course of the disease and response to treatment.