Study on the mechanism of apoptosis of myelocytic leukemia cell lines induced by curcumin

Objective: To investigate the mechanism of apoptosis of myelocytic leukemia lines NB4, K562 and THP-1 cells induced by curcumin. Methods: After the cells were treated with curcumin at different concentration (25, 12.5, 6.25, 3.125, 0 μmol/L) for various times (0, 12, 24, 48 h), flow cytometry (FCM) was used to determine the rate of apoptosis of cells. After the cells were treated with curcumin at 25 μmol/L for 24 h, flow cytometry was used to determine the expression level of Fas, and Western blot was performed to determine the expression of Caspase-8 and Caspase-9. Results: (1) Curcumin could induced the apoptosis of NB4, K562 and THP-1 cells in a time- and dose-dependent manner. After the cells were treated with curcumin at 25 μmol/L for 48 h, the rate of apoptosis of cells was over fifty percent. (2) Fas level showed remarkable increase (P < 0.01) after above cells were treated with 25 μmol/L curcumin for 24 h. (3) The apoptosis proteins of Caspase-8 and Cas- pase-9 were also increased obviously (P < 0.01) after the cells were treated with 25 μmol/L curcumin for 24 h. Conclusion: The molecular pathway of apoptosis of myelocytic leukemia lines induced by curcumin are concerned with death receptor and mitochondria.

ANALYSIS OF APOPTOSIS BY DNA END LABELING METHOD (TDT) IN LEUKEMIA CELL LINES HL-60 AND U937

Antitumor-drug-indnced apoptosis in leukemia cell lines HL-60 and U937 was quantitatively analyzed with TDT (terminal deoxynuleotidyl transferase-mediated nick-end labeling) approach, which allows to labal the ends of DNA broken strands in apoptotic cells by biotinlated dUTP which to avidin-FITC. In this way the apoptotic cells show nuorescent when the labeling cells were emitted by UV light microscope or laser-activated now cell sorting at r=480. In our study, HL-6o and U937 cell lines were cocultured respectively with cisdiaminodicaicroplatinum (CDDP), hydroxycamptothecinum (HCT) and vindesini sulfa (VCR) for 18 hours.By calculating percentages of apoptotic cells with TDT mothod, we were able to show that the two cell lines gave different sensitivity to the drugs. HL-60 showed high sensitivity to CDDP but U937 cells were more sensitive to other two drugs, HCT and VCR. Meanwhile we compared the results of obtained by DNA gel electrophoresis with that by TDT. We found that gel electrophoresis is not sensitive enough to reveal apoptosis since there was no ladder structure, a typical electrophoresis pattern for apoptosis, appeared until the apoptotic cells reached or over 13%. And we report in this paper as first time that three forms of apoptotic cells could be detected under fluorescent microscope, which we called as spot form and crescent form and assembling form in terms of distribution of light spots within cell nuclei. It seemed that the spot form was at an early stage of apoptosis and the crescent form rcprtscntcd a later stage of apoptosis.

ADVANCES IN THE STUDY ON LEUKEMIA STRAINS AND LEUKEMIA CELL LINES IN CHINA

The study and establishment of leukemia strains and leukemia cell lines have made great achievement in China.It has been extended from mice to rat model and related to explore human leukemia cell line and heterotransplantable tumor strains. The cellular types of leukemia strain have included Iymphocytic,tmyelocytic,ecythroleukmia and megakaryoblastic cell strain.

ESTABLISHMENT OF A CELL LINE INFECTED WITH SRS MOUSE LEUKEMIA VIRUS AND ITS BIOLOGICAL CHARACTERISTICS

NIH/3T3 cells were infected with a cell-free extract from the mouse SRS ascltic tumor and propagated at vitro. Experimental results showed that type A and type C virus particles were observed in the cytoplasm of infected NIH/ 3T3 cells by electron-microscopy, X-C assay and reverse transcriptase were both positive. Free proviral-DN A of the leukemia vims was found in the infected NIH/3T3 cell by Southern blot hybridization.Morphologically, the NIH/3T3 cells infected in vitro appeared spherical and formed monolayer cell colonies of various sizes after 24 - 48 hours. When the cultured cells were Inoculated into nude mice (BALB/ c, nu nu ) subcutaneously, flbrosarcoma was Induced, 100%. Inoculation of the cell-free extract of the same cultured cells into Inbred SW-1 newborn mice, resulted in the production of lymphocytic leukemia and lymphoma in 52. 3% within 191 days.

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.