ABSTRACT
DNA repair processes play a role in the development of drug resistance which represents a huge obstacle to leukemia chemotherapy.Histone H2AX phosphorylation (ser139) (γH2AX) occurs rapidly at the onset of DNA double strand break (DSB) and is critical to the regulation of DSB repair.If DNA repair is successful,cells exposed to anti-neoplastic drugs will keep entering the cycle and develop resistance to the drugs.In this study,we investigated whether γH2AX can be used as an indicator of tumor chemosensitivity and a potential target for enhancing chemotherapy.K562 and multi-drug resistant cell line K562/A02 were exposed to adriamycin (ADR) and γH2AX formed.Flow cytometry revealed that percentage of cells expressing γH2AX was increased in a dose-dependent manner and the percentage of K562/A02 cells was lower than that of K562 cells when treated with the same concentration of ADR.In order to test the potential of γH2AX to reverse drug resistance,K562/A02 cells were treated with PI3K inhibitor LY294002.It was found that LY249002 decreased ADR-induced γH2AX expression and increased the sensitivity of K562/A02 cells to ADR.Additionally,the single-cell gel electrophoresis assay and the Western blotting showed that LY249002 enhanced DSBs and decreased the expression of repair factor BRCAl.These results illustrate chemosensitivity can partly be measured by detecting γH2AX and drug resistance can be reversed by inhibiting γH2AX.
ABSTRACT
The effects of betulinic acid (BA),a pentacyclic lupane-type triterpene,on the cell viability,cell cycle and apoptosis in human leukemia K562 cells were investigated.The effects of BA on the growth of K562 cells were studied by MTT assay.Apoptosis was assayed through Annexin V/propidium iodide (PI) double-labeled cytometry.The effects of BA on the cell cycle of K562 cells were studied by a PI method.The expression of Bax and capase-3 was detected by using Western blot.The results showed that BA was cytotoxic to K562 cells with an IC50 of 21.26 μg/mL at 24 h.After treating K562 cells with 10 μg/mL BA for 72 h,the number of cells was reduced by 58%.BA induced apoptosis of K562 cells in a time-and dose-dependent manner.The proportion of cells in G0/G1 and G2/M phases was decreased and that in S phase was increased after K562 cells were treated with BA for 24 h.BA treatment also increased the expression of the pro-apoptotic proteins Bax and caspase-3.It suggested that BA could inhibit the proliferation of K562 cells through the induction of cell cycle arrest and apoptosis.The antitumor effects of BA were related with up-regulation of the expression of Bax and caspase-3 proteins.BA may qualify for the development of new therapies for leukemia.
ABSTRACT
The anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 μmol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G0/G1 phase and decrease in S phase. After treatment with 0, 20, 60, 100 μmol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00±1.25)% to (58.84±0.32)% in G0/G1 phase, whereas it was decreased from (61.45±1.04)% to (35.82±1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3,bel-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is coneluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle,arrest cells at G0/G1 phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl.