Proteomic analysis of nuclear matrix proteins during arsenic trioxide induced apoptosis in leukemia K562 cells

Background Arsenic trioxide (As2O3) has been identified as a very potent antiacute leukemic agent However its role in apoptosis needs to be elucidated As2O3 interferes with the proliferation and survival of tumor cells via a variety of mechanisms Drugtarget interactions at the level of nuclear matrix (NM) may be critical events in the induction of cell death by As2O3 This study dealt with As2O3-target interactions at the level of NM in chronic myelogenous leukemia cell line K562 by proteomics Methods K562 cells were cultured in MEM and treated with different concentrations of As2O3 The nuclear matrix proteins were analyzed by highresolution twodimensional gel electrophoresis and computerassisted image analysis Results As2O3 significantly inhibited the growth of chronic myelogenous leukemia cell line K562 at low concentrations While more than 200 protein spots were shared among the nuclear matrices, about 18 distinct spots in the nuclear matrices were found characteristic for As2O3 treated cells Conclusions: As2O3 induces apoptosis in K562 cells in a dose and timedependent manner Our results demonstrated that for the detection of the onset of apoptosis, the alteration in the composition of nuclear matrix proteins was a more sensitive indicator than nucleosomal DNA fragmentation test These results indicated that As2O3 might be clinically useful in the treatment of chronic myelogenous leukemia. The changes of nuclear matrix proteins in the treated cells can be used as a useful indicator for this treatment

Reversal effect of bufalin on multidrug resistance in K562/VCR vincristine-resistant leukemia cell line

OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin(ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry(FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein(P-gp), multidrug-associated protein-1(MRP1), Bcl-x L and Bax protein were measured by immunocytochemistry.RESULTS: The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002,0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77,respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07%(P<0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-x L and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.CONCLUSION: Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-x L/Bax ratio.

Construction of antisense Bmi-1 expression plasmid and its inhibitory effect on K562 cells proliferation

Background Bmi-1 gene determines the proliferative capacity of normal and leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid leukemia cells in both humans and mice. This study aimed at assessing the effect of antisense Bmi-1 expression on K562 cells proliferation and p16 protein （p16） expression. Methods A transcriptional repressor, Bmi-1 cDNA was cloned by reverse transcriptase polymerase chain reaction （RT-PCR） of its mRNA from K562 cells. A plasmid expressing antisense Bmi-1 mRNA was then constructed by reverse design of PCR primers and cloned to the plasmid pLNCX2; G418 was added to the medium after the plasmid was successfully introduced in K562 cells by lipofectin-mediated DNA transfection. The effects of the antisense expression on the proliferation of K562 cells were analyzed by using microcuhure tetrazolium and colony forming. Cell cycle was analyzed by using flow cytometry. The p16 expression of K562 cells was observed by immunoflorescence histochemical stain. Results K562 cells transfected with antisense Bmi-1 plasmid grew significantly slower than that of controls （the parental K562 and cells transfected with empty plasmid）. The colony forming ability of antisense Bmi-1 plasmid transfected ceils decreased significantly （P 〈0. O1 ） compared with controls. The p16 expression of cells transfected with antisense Bmi-1 was upgraded more apparently than that of controls. Conclusion The antisense Bmi-1 gene can inhibit the growth of K562 cell and upgrade expression of p16 in K562 cells.

N-（Substituted benzyl）-3,5-bis（benzylidene）-4-piperidones： Synthesis and Preliminary Anti-leukemia Activity （I）

A series of novel N-（substituted benzyl）-3,5-bis（benzylidene）-4-piperidones 5a--50 were synthesized with substituted benzylamines as raw materials via a series of Michael addition, Dieckmann condensation, hydrolysis decarboxylation and aldol condensation. The structures were confirmed by 1↑H NMR, IR, MS techniques and elemental analysis. Assay-based antiproliferative activity study using leukemic cell lines K562 revealed that most of the title compounds have high effectiveness in inhibiting leukemia K562 cells proliferation, among which the compounds 5g （IC50=7.81 μg·mL^-1）, 5k （IC50=6.35μg·mL^-1）, 51 （IC50=7.20 μg·mL^-1）, and 50 （IC50=5.79 μg·mL^-1） have better inhibition activities than standard 5-fluorouracil （IC50=8.56 μg·mL^-1）.

Synthesis and Anti-leukemia Evaluation of Tetrahydro-4H-pyrano[3,2-c]pyridines and Corresponding Anti-CD14 Monoclonal Antibody Conjugates

A series of novel tetrahydro-4H-pyrano[3,2-c]pyridines（3a-3p） were synthesized and found to possess potent antiproliferative activity against leukemia K562 cells in vitro. Preliminary bioassay indicates that compounds 3a and 3e afford the best activity, the IC50 values of them were 6.93 and 7.51μg/mL, respectively, which were lower than that of the anticancer drug 5-FU（1C50=8.56μg/mL）. To reduce the toxicity of compounds 3a-3p to the prolife- ration of normal bematopoietic cells, a tumor targeted CD14 monoclonal antibody（McAb） was used in conjugation with compounds 3a--3p to get conjugates 4a--4p, respectively. The inhibitory activities of conjugates 4a--4p toward K562 cells were discovered to approach those of compounds 3a--3p. In the presence of CD14 McAb, tumor cells were found to be much more susceptible to conjugates 3a--3p than normal hematopoietic cells. Therefore, the toxicity of conjugates 4a--4p to normal hematopoietic cells declined obviously. For example, as for the toxicity of compound 3a compared with that of compound 4a, the value of ICs0 increased from 35.90 μmol/L to 39.52 μmol/L.