| Objective:Acute myeloid leukemia(AML)is an aggressive hematologic malignancy. The development and progression of the leukemic disease invariably involve deregulation in the apoptotic response.Therefore,apoptosis-inducing therapy is one of the current primary treatments for AML.As2O3,as an apoptosis inducer,has shown substantial efficacy in treating both newly diagnosed and relapsed/refractory patients with acute promyelocytie leukemia(APL,a subset of AML).Moreover,As2O3 has clinical efficacy in patients with all-trans retinoic acid(ATRA)-resistant APL,for there seems to be no obvious cross-resistance between ATRA and As2O3.In addition, use of As2O3 generally does not lead to myelosuppression,providing a potential advantage over use of conventional cytotoxic agents.However,the reported chronic toxicities and carcinogenicity of As2O3 have hampered its acceptance as a first-choice drug.The major clinical limitation is a pronounced toxicity,which is dose dependent. Hence,one approach to overcoming the problem is combination therapy,which may allow for administration of lower doses of arsenic trioxide,minimizing toxicity and potential drug antagonism.Thus,it is of great importance to develop adjuvant agents, which have low toxicity,increase the apoptotic action of As2O3 and reduce the drug dosage to physiologically tolerable concentrations(＜5μM).Carnosic acid,the major rosemary polyphenol,is an antioxidant food additive with non-toxicity.It has many pharmacological effects,such as antioxidant,antitumor,antithrombotic and antiinflammatory effect.The purpose of our study was to evaluate the effects of carnosic acid on apoptosis,to specifically concentrate on the cooperated effects of carnosic acid with low concentration of As2O3 in HL-60 human myeloid leukemia cells,and to further investigate the molecular mechanisms involved in these effects in vitro and in vivo.Methods:In vitro:Cell growth was determined by counting cells using a Coulter counter.Cellular viability was measured by MTT assay,and cell cycle distribution and apoptosis were monitored by flow cytometry(FCM).The protein expression of cleaved caspase-9,BAD,p-BAD,p27,PTEN,Akt,p-Akt were assessed by western blot analysis and the lever of PTEN mRNA was tested by RT-PCR analysis. In vivo:(1)Establishment and Evaluation of Human Acute myeloid Leukemia HL-60 Model in SCID Mice.SCID mice were transplanted by tail vein injection with 1×107 HL-60 cells.Leukemic cells were detected by chromosome karyotype analysis and histopathologic methods.(2)In dose-response experiments,mice were treated for 4 w with daily i.g.of CA(200mg/Kg,300mg/Kg,500mg/Kg),after 7 days of leukemia engraftment.The average survival time was observed.(3)Treatment of carnosie acid and carnosie aeid-As2O3 combination.Mice were divided into 4 groups(①control group,②CA group,③As2O3 group,④CA+ As2O3 group) to receive treatment for 4 w after 7 days of leukemia engraftment.The changes of common condition and the average survival time were observed.After 4 w of leukemia engraftment,the mice were divided into 4 group(①control group,②CA group,③As2O3 group,④CA+ As2O3 group)to receive treatment for 10 d.At the end of the experiment,all mice were killed by the institutionally approved method. Spleen,liver,long bone and lymph node metastasis were excised and examined by microscopy and immunohistochemistry.The cell cycle progression and the apoptosis of spleen cells were examined using FCM.Results:In vitro:Camosic acid inhibited cell growth and decreased cell viability in a dose- and time- dependent manner,and induced G1 arrest and apoptosis.Carnosic acid also augmented these effects when they were induced by a low(physiological) concentration of arsenic trioxide,which was associated with upregulation of p27 and activation of caspace-9.These effects were apparently mediated by the induction of PTEN expression and by blocking Akt pathway.In vivo:(1)Transplantation was always successful,and all animals died generally in 24-32 d.(2)After 4 w treatment,the average survival time was as follows: 200mg/Kg.34.9±3.28 days,300mg/Kg:42.6±5.82 days,500mg/Kg:47.0±6.17 days. These results showed camosic acid treatment increased the average survival time in a dose- dependent manner(P＜0.05).(3)After 4 w treatment,the average survival time was as follows:control group:27.0±2.49 days,CA grpup:50.8±3.12 days,As2O3 group:62.2±2.66 days,CA+As2O3 group:80.1±5.02 days.The findings demonstrated that carnosic acid could particularly promote the average survival time induced by As2O3(P＜0.05).The cells in G1 phase(%)were as follows:control group: 36.0±4.53,CA group:46.4±6.47,As2O3 group:51.0±3.54,CA+As2O3 group: 75.2±8.53.These data showed that carnosic acid could particularly promote cell cycle arrest of HL-60 cells induced by As2O3(P＜0.05).The apoptotic cells(%)were as follows:control group:4.8±1.79,CA group:13.2±3.19,As2O3 group:58.2±5.97, CA+ As2O3 group:76.2±4.87.These observations showed that carnosic acid could enhance As2O3-induced apoptosis(P＜0.05).Microscopy indicated that in the tissue of untreated animals,leukemic blasts infiltrated the parenchyme as very large perivascular masses associated with smaller aggregates of leukemic cells that obstructed sinusoids.In the tissue of treated animals,some cells with a condensed nucleus had apoptotic-like features.After drugs treatments,immunohistochemistry showed that cleaved caspase-3,p27,PTEN level was increased.Conclusions:These findings indicate that PTEN/Akt pathway has an important role in the cooperated induction of apoptosis and G1 arrest by carnosic acid and As2O3. Carnosic acid may have potential as an adjuvant in As2O3 -induced apoptosis therapy for its anticipated safety and its great potency in enhancing apoptosis-inducing action of low concentration of As2O3.
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