Pure total flavonoids from Citrus paradisi Macfadyen act synergistically with arsenic trioxide in inducing apoptosis of Kasumi-1 leukemia cells in vitro

To investigate the potential effects of pure total flavonoid compounds（PTFCs） from Citrus paradisi Macfadyen separately or combined with arsenic trioxide on the proliferation of human myeloid leukemia cells and the mechanisms underlying the action of PTFCs. The effects of PTFCs separately or combined with arsenic trioxide on the proliferation and apoptosis of leukemia cells were determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT）, fluorescence microscopy, and flow cytometry. Their effects on the expression levels of apoptosisrelated regulators were determined by Western blot assay. PTFCs combined with arsenic trioxide significantly inhibited the growth of Kasumi-1 cells, and apoptosis was confirmed by flow cytometry analysis. Hoechst 33258 staining showed more significant morphological changes and more apoptosis following the combined treatment. Western blots showed changes in the expression of genes for poly ADP-ribose polymerase（PARP）, caspase 3/9, and P65. The results indicated that PTFCs separately or combined with arsenic trioxide inhibited proliferation of leukemia cells in vitro and induced their apoptosis by modulating the expression of apoptosis-related regulator genes.

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.

Pure Total Flavonoids from Citrus paradisi Macfad InduceLeukemia Cell Apoptosis In Vitro

Objective： To investigate the potential effect of pure total flavonoids from Citrus paradisi Macfad peel（PTFC） on the proliferation and apoptosis of human myeloid leukemia cells Kasumi-1, HL-60 and K562, and the underlying mechanisms. Methods： PTFC was extracted from Citrus paradisi Macfad peel and was identified by high performance liquid chromatography. The effect of PTFC on the proliferation and apoptosis of leukemia cells were determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide, fluorescent microscopy and flow cytometry, respectively. The effect of PTFC on the expression levels of apoptosis-related regulators was determined by Western blot assay. Results： Treatment with PTFC inhibited leukemia cell proliferation in a dose-and time-dependent manner and triggered Kasumi-1 cell apoptosis. Treatment with PTFC significantly increased the levels of activated poly adenosine diphosphate-ribosepolymerase and caspase-3/-9, but reduced the levels of Mcl-1 expression in Kasumi-1 cells. However, PTFC did not obviously induce HL-60 cell apoptosis. Conclusion： PTFC inhibited leukemia cell proliferation and induced their apoptosis by modulating apoptosisrelated regulator expression in leukemia cells in vitro.

New insight into the oncogenic mechanism of the retroviral oncoprotein Tax

Human T cell leukemia virus type 1(HTLV-1),an etio-logical factor that causes adult T cell leukemia and lym-phoma(ATL),infects over 20 million people worldwide.About 1 million of HTLV-1-infected patients develop ATL,a highly aggressive non-Hodgkin's lymphoma without an effective therapy.The pX region of the HTLV-1 viral genome encodes an oncogenic protein,Tax,which plays a central role in transforming CD4+ T lymphocytes by deregulating oncogenic signaling pathways and promoting cell cycle progression.Expression of Tax following viral entry is critical for promoting survival and proliferation of human T cells and is required for initiation of oncogenesis.Tax exhibits diverse functions in host cells,and this oncoprotein primarily targets IκB kinase complex in the cytoplasm,resulting in persistent activation of NF-κB and upregulation of its responsive gene expressions that are crucial for T cell survival and cell cycle progression.We here review recent advances for the pathological roles of Tax in modulating IκB kinase activity.We also discuss our recent observation that Tax connects the IκB kinase complex to autophagy pathways.Understanding Tax-mediated pathogenesis will provide insights into development of new therapeutics in controlling HTLV-1-associated diseases.