| Objects Thioredoxin reductase (TrxR) incites pro-survival effects,enhances tumorproliferation,increases transcription factor activity,and inhibits apoptosis.TrxR is considered animportant indicator of prognosis because of its promoting tumor cells growth andprogress.BBSKE is a novel TrxR inhibitor that can significantly inhibit TrxR activitiesspecifically to induce apoptosis in cancer cells.The research aim is to explore the activity ofTrxR in chronic myelogenous leukemia (CML) cells. Furthermore,it would be found of theanti-leukemia effect of BBSKE in vitro,in order to provide experimental basis for the treatmentof CML in clincal.Methods K562 cell lineage and the mononuclear cells from the fresh bone marrow ofCML patients or healthy adults were cultured, and the activity of TrxR was analyzed by insulinreduction assay. The K562 cells were divided into control group one and experimental groupone.The mononuclear cells from the CML patients were divided into control group two andexperimental group two.The control groups were added nothing,while the experimental groupswere added different concentrations of BBSKE.The activity of TrxR on the two groups cellswere measured by insulin redction assay.The inhibitions of proliferation were measured byCCK-8 assay.The anti-leukemia effects of BBSKE were observed by laser scanning confocalmicroscope,agarose gel electrophoresis and flow cytometry with Annexin V-FITC/PI staining.Results 1.TrxR activities of K562 cell lineage and mononuclear cells from the fresh bonemarrow of CML patients were significantly higher than normal bone marrowmononuclearcells(P<0.05).2.The insulin reduction assay showed in experimental group one,10μmol/L BBSKE for 24or 48 hours could inhibit TrxR activity.The inhibition rates were (21.8±0.51)% or(23.7±0.43)%,respecctively,and were statistically significant compared with those of theuntreated K562 cells.The inhibition of BBSKE on K562 cell lineage was time and dosedependent. In experimental group two,10μmol/L BBSKE for 24 or 48 hours could also inhibitTrxR activity.The inhibition rates were (16.5±0.69)% or (18.7±0.58)%,respecctively,and werestatistically significant compared with the control group two.3.The CCK-8 assay showed the proliferation of K562 were inhibited by BBSKE atconcentration of 1μmol/L,2μmol/L,4μmol/L,8μmol/L and 16μmol/L for 24 hours.The inhibition rates were (5.0±0.72) %,(9.6±0.69) %,(20.8±0.41) %,(48.9±0.19) % and (68.3±0.54)%,respectively.There were statistically significant of these five groups(P<0.05). The inhibitionrates of the experimental group two at concentration of 1μmol/L,2μmol/L,4μmol/L, 8μmol/Land 16μmol/L for 24 hours were (3.8±0.26) %,(6.1±0.93) %,(17.4±0.61) %,(46.8±0.65) % and(66.8±0.28) %.There were statistically significant of these five groups(P<0.05),respectively.There were statistically significant of these five groups (P<0.05).And theinhibitions of BBSKE on CML cells were dose dependent. The apoptosis of CML cells couldbe induced after CML cells being treated with BBSKE, and the role of BBSKE was time anddose dependent. The IC50 values at 24 hours,48 hours and 72 hours of BBSKE in experimentalgroup one were 9.23μmol/L,5.14μmol/L and 3.81μmol/L,respectively. And in experimentalgroup two were 10.01μmol/L,5.64μmol/L and 4.10μmol/L,respectively.4. After being treated with BBSKE,the cells showed the typical apoptosis morphologicalchanges.Under Laser confocal microscopic evacuation of FITC-Annexin V/PI,positive cellsrevealed the typically early and final phase with morphologic characteristic of apoptosis.In theearly phase of cells apoptosis,the membranes are green and the nucleus are not red,and in themiddle and final phrases of cells apoptosis,the membranes are green and the nucleus are red,thenucleus broken into pieces and cells split into apoptosis bodies in different sizes,which are thetypical apoptosis morphological changes. The typical DNA"ladder"bans appeared by agarosegel electrophoresis.By the flow cytometry,the apoptotic rate of K562 cells at concentration of10μmol/L BBSKE after treated for 24 hours was (10.28±2.74) %,and the apoptotic rate ofK562 at concentration of 10μmol/L platinum after treated for 24 hours was (7.80±1.22) %,andthe apoptotic rate of control groups was (0.93±0.22) %.There were statistically significant ofthese three groups.10μmol/L BBSKE for 48 hours could also induce apoptosis of fresh bonemarrow cells from the patients of CML, and the apoptotic rate was (5.7±0.48)%.Conclusions TrxR activity in chronic myeloid leukemia cells was significantly higherthan that of normal cells. BBSKE inhibits the TrxR activity and the proliferation of CML cellsby inducing apoptosis.It might be a potential medication for chronic myeloid leukemia.
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