Proapoptotic activity of anti-CD52 monoclonal antibody, alemtuzumab (ALT) as well as ALT-affected apoptosis-regulatory mechanisms were assessed in tumor cells from 36 patients with chronic lymphocytic leukemia (CLL). Cells were treated in vitro for 24-48 h with ALT alone or in combination with rituximab (RTX), or purine nucleoside analogues (PNA), fludarabine and cladribine. Moreover, eight ALT-treated patients were examined in vivo. In 22/36 patients with the pre-treatment overexpression of Bax, Bak and Bid proteins, ALT induced a distinct (more than 50% from the baseline) increase in the incidence of apoptosis after 24 h of in vitro treatment. ALT-attributed CLL cell apoptosis was also detected after 24 h from in vivo ALT administration, with significantly downregulated Bcl-2 (P = 0.012) and Mcl-1 (P = 0.031). ALT combined with PNA or RTX exerted significantly higher proapoptotic effect in vitro than single agents, downregulating FLIP and Bcl-2 (ALT + PNA) or significantly increasing Bax expression (ALT + RTX; P = 0.007). In conclusion, the evidence of apoptotic CLL cells death in response to ALT, with deregulation of intrinsic apoptotic pathway, is presented. ALT and PNA or RTX trigger complementary changes in expression of proteins regulating cell propensity to undergo apoptosis, what provides molecular rationale for combining ALT with those agents.

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Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant, apoptosis-resistant B CD19(+)/CD5(+) cells. Populations of CLL cells are heterogeneous and consist primarily of quiescent cells with a minor subset of dividing cells. In this study the efficacy of a first-line in vivo therapy was compared with treatment by R-roscovitine (ROSC) alone or by purine analogues (cladribine and fludarabine) combined with maphosphamide for 14 CLL patients under ex vivo conditions. ROSC induced the highest reduction in numbers of living B-cells, coinciding with an increased rate of apoptosis. After 24 h the percentage of apoptotic cells in ROSC-treated cultures was markedly higher than in untreated controls. ROSC also induced strong activation of the apoptosome and effector caspases in CLL cells. During progression of apoptosis the plasma membrane became permeable, resulting in the release of activated caspases into the culture medium. Leukemic cells were more sensitive to ROSC than normal mononuclear cells. Treatment with ROSC did not affect the activating phosphorylation of CDK2 or CDK1. However, ROSC decreased phosphorylation of survivin, CDK7, and RNA-Pol II, resulting in inhibition of transcription elongation and subsequent down-regulation of levels of anti-apoptotic factors, thereby facilitating apoptosis. Unlike ROSC, two other purine analogues barely affected the cellular levels of anti-apoptotic proteins and more weakly activated effector caspases. In addition, the efficacies of in vivo and ex vivo therapies were found to be correlated. Marked between-patient differences in expression patterns of apoptosis-regulating factors in CLL cells were observed, explaining the variations in patients' sensitivity to therapy.

Cancer is the second leading cause of death in humans. Despite rapid developments in diagnostic methods and therapies, metastasis and resistance to administrated drugs are the main obstacles to successful treatment. Therefore, the main challenge should be the diagnosis and design of optimal therapeutic strategies for patients to increase their chances of responding positively to treatment and increase their life expectancy. In many types of cancer, a deregulation of multiple pathways has been found. This includes disturbances in cellular metabolism, cell cycle, apoptosis, angiogenesis, or epigenetic modifications. Additionally, signals received from the microenvironment may significantly contribute to cancer development. Chemical agents obtained from natural sources seem to be very attractive alternatives to synthetic compounds. They can exhibit similar anti-cancer potential, usually with reduced side effects. It was reported that natural compounds obtained from fruits and vegetables, e.g., polyphenols, flavonoids, stilbenes, carotenoids and acetogenins, might be effective against cancer cells in vitro and in vivo. Several published results indicate the activity of natural compounds on protein expression by its influence on transcription factors. They could also be involved in alterations in cellular response, cell signaling and epigenetic modifications. Such natural components could be used in our diet for anti-cancer protection. In this review, the activities of natural compounds, including anti-cancer properties, are described. The influence of natural agents on cancer cell metabolism, proliferation, signal transduction and epigenetic modifications is highlighted.

Abstract. to improve the efficacy of therapeutic options in chronic lymphocytic leukemia (cll) an in vitro system to determine the response of mononuclear blood cells from blood of patients was elaborated. the study combines four approaches, i.e., cell viability, apoptosis rate, differential scanning calorimetry (DSc), and immunoblotting to develop personalized therapy protocols based on the cell sensitivity to drug exposure of individual cll patients. the complementary analyses were performed on 28 peripheral blood samples from previously untreated cll patients before therapy. the induction and progress of apoptosis in cll cells exposed in vitro to purine analogs combined with mafosfamide, i.e., cladribine + mafosfamide (CM) and fludarabine + mafosfamide (fM) were assessed using the above approaches. the changes in thermal profiles (decrease/loss of transition at 95±5˚C) coincided with an accumulation of apoptotic cells, a decrease in the number of viable cells, and differences in the expression of the apoptosis-related protein ParP-1. no significant changes were observed in the thermal profiles of nuclei isolated from cll cells resistant to the treatment. the complementary assays revealed a strong relationship between both the in vitro sensitivity of leukemia cells to drugs and the clinical response of the patients, determined usually after the sixth course of treatment (after ~6 months of therapy). as a summary of studies followed by complementary tests, our findings demonstrate the value of in vitro exposure of cll cell samples to drugs intended to treat cll patients, before their administration in order to recommend the most suitable and effective therapy for individual patients.

There is individual variation in the course of disease development and response to therapy of patients with chronic lymphocytic leukemia (CLL). Novel treatment options for CLL include a new generation of purine analogs, antibodies and inhibitors of specific cell signaling pathways, which typically induce apoptosis or necrosis. A prospective analysis of patient blood samples revealed that a combination of four tests allowed the most appropriate and effective type of treatment to be selected prior to drug administration, and for the analysis of leukemic cell sensitivity to anticancer drug(s) during disease development. The comparative analysis of blood from the stable and progressive form of CLL in an individual patient revealed diversity in the response to anticancer agents. CLL peripheral blood mononuclear cells were incubated with cladribine + mafosfamide (CM), fludarabine + mafosfamide, CM + rituximab, rituximab alone (Rit) or kinetin riboside (RK). A combination of cell viability, differential scanning calorimetry (DSC) profiles of nuclear preparations and poly(ADP-ribose) polymerase 1 (PARP-1) protein expression analysis of the leukemic cells was performed to evaluate the anticancer effects of the tested agents during CLL development. The results of the present study indicate that such studies are effective in determining the most appropriate anticancer drug and could monitor disease progression on an individual level. In addition, the results of the current study suggest that CLL progression leads to diversification of the cellular drug response. The most efficient apoptosis inducer for the patient was purine analog RK when the disease was stable, while the CM combination was the most effective agent for the progressive form of disease.

Using differential scanning calorimetry we analyzed the thermal profiles of nuclei from normal and B-cell chronic lymphocytic leukemia mononuclear cells. Intact nuclear fraction of normal mononuclear cells is characterized by four thermal transitions, i.e., at 60, 70, 83 and 103 degrees C. Leukemic nuclear samples revealed the transitions at 67 and 83 degrees C, however, in more aggressive stage of the disease additional thermal peaks at 76 and 93 degrees C were observed. Our very preliminary results revealed that mononuclear cell nuclear fraction from blood of patients responding to the used therapy, i.e., cladribine alone or its combination with mitoxantrone and cyclophosphamide indicates decrease (or even loss) of transition at 93 degrees C concomitant with increase of transition at 76 degrees C. A complementary study showed that in mononuclear cells of patients who appeared to be sensitive to chemotherapy the decrease of antiapoptotic Bcl-2 protein expression and signs of apoptotic morphology were observed.

Mitochondria play important roles as energetic centers. Mutations in mitochondrial DNA (mtDNA) were found in several diseases, including cancers. Studies on cytoplasmic hybrids (cybrids) confirm that directed mutation introduced into mtDNA could be a reason for cancer induction. Mitochondria could also be a factor linking cancer transformation and progression. The importance of mitochondria in cancer also confirms their involvement in the resistance to treatment. Resistance to treatment of cancer cells can frequently be a reason for glycolysis acceleration. It could be explained by cancer cells' high proliferation index and high energy request. The involvement of mitochondria in metabolic disturbances of several metabolic diseases, including cancers, was reported. These data confirm that cancer induction, as well as cancer progression, could have metabolic roots. The aberrant products observed in prostate cells involved in the Krebs cycle could promote cancer progression. These multiple relationships between alterations on a genetic level translated into disturbances in cellular metabolism and their potential relation with epigenetic control of gene expression make cancerogenesis more complicated and prognoses' success in studies on cancer etiology more distant in time.