| Homoharringt()nine(HHT) is a cephalotaxine ester derived from an evergreentree found widely thro11ghout Southern China,which has anticancer activity.In China,HHT was firstly use(1 to dea[ with acute nonlymphocytic leukemia (ANLL) andattained better therapeutic effect. In this study, we tried to elucidate the relationshipbetween the apoptosis of acute leukemic celIs induced by HHT in vitro and clinicaleffect. Additionally, it was uncertain whether HHT-induced apoptosis was reIated tohuman telomerase reverse transcriptase (hTERT), telomerase activity and theexpression of survivin gene, a new apoptosis-related gene.Bone marrow cells from 37 cases (26 cases with de novo ANLL, l1 cases withde novo ALL) with acute leukemia (AL) were cultured with HHT fOr l5 hours. Thecytostatic ratio of these cells was examined by MTT assay. The results indicatedthat the inhibition of ANLL cells by HHT was significantly stronger than that of ALLcells (34.92 l l9.58 vs l9.68 t l8.83, P<0.05). Of 37 cases, 20 cases wereavailable to evaluate clinicaI effect. The apoptQsis of HHT-treated leukemic cellsfrom those 20 cases was analyzed by TUNEL (terminaI deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end Iabeling) method. The rate of apoptosisinduced by HHT was slightly lower in ALL cells than that in ANLL cells (17.34f9.60 vs 26.64l20.67, P>0.05).Furthermore, the rate of apoptosis of primary ALcells induced by chemotherapeutic agents(with HA or DA according to theirchemotherapy regimen, ALL with DNR)in vitro was correlated closely with themarrow blast cell decrease index (MBDI)(r=0.586,p<0.0l)in vivo. The rate ofapoptosis in AL cells was 27.6% (median value), and the complete remission (CR)rate in the group of apoptotic rate >27.6% was significantly higher than that in thegroup of apoptotic rate <27.6% [ 80.0%(8/l0) vs 30.0%(3/l0),p=0.03]. Among 9cases treated with HA regimen, the CR rate in cases with apoptotic rate>27.6% washigher than in those with apoptotic rate <27.6% [ 80.0%(4/5) vs25.0%(l/4) ].These results indicated that the apoptosis of AL cells in vitrocorresponded with the therapeutic effect in vivo. Apoptosis ofAL ceIls in vitro mayprognose clinical effect.The expression of hTERT mRNA in bone marrow cells was analyzed by semi-quntitative RT-PCR, from 30 patients with untreated AL,l2 CR patients and 6 norma1volunteers. The results suggested the expression of hTERT mRNA of the bonemarrow cells in AL patients was obviously higher than that in CR patients (0.7l f0.34 vs 0.43f0.25, P<0.05) and in normal volunteers(0.7l 10.34 vs 0.22f0.2l, P<0.0l).In the meantime, telomerase activity was examined by TRAP ELlSA.Telomerase activity of the bone marrow cells in AL patients was significantly higherthan that in CR patients (0.235 l0.395 vs 0.0l2l0.015,P=0.007). Moreover, therewas a correlation between the hTERT mRNA synthesis and telomerase activity in ALcelIs (r=0.42l,p<0.0l ).Human leukemic cells, HL60 were cultured with HHT at variableconcentration for l2 hours. Compared with the control, with HL60 cells treated withHHT at 0.005ug/ml ~ 0.03 ug/ml for l2 hours, the level of hTERT mRNA synthesisdidn't change significantly but it reduced obviously at 0.04ug/ml and wasundetectable at 0.05ug/ml. When HL60 cells were cultured with HHT at 0.02 ug/mlfor 6 hours to 30 hours, the expression of hTERT mRNA in HL60 ceIls began todecrease after 6 hours, especially after 18 hours and was undetectable at 30hours.These results demonstrated that the 1evel of hTERT mRNA could be down-regulated by HHT at optimal concentration and time. We also studied the effect ofHHT on telomerase activity and apoptosis of HL60 cells. The results suggested thattelomerase activity of HL60 cells reduced with the decrease of the expression ofhTERT mRNA. Thus,these findings suggested that down-regulating the transcriptionof hTERT and telomerase activity might be one of the mechenisms of HHT-inducedapoptosis.T...
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