2－甲氧基雌二醇诱导骨髓增生异常综合征MUTZ1 细胞凋亡机制的研究

　　作者：夏国华　陈宝安　邵泽叶　芦慧霞　 Dohner Konstanze　 Dohner Hartmut 作者单位：东南大学临床医学院/附属中大医院血液科，南京 210009; 1东南大学附属中大医院检验中心，南京 21009; 2乌尔姆大学医学院内科Ⅲ区, 德国 D89081

　　【摘要】为了研究2 甲氧基雌二醇(methoxyestradiol, 2ME)诱导骨髓增生异常综合征难治性贫血伴原始细胞增多型(MDSRAEB)细胞株MUTZ1细胞凋亡的机制，将不同浓度的2甲氧基雌二醇分别与MUTZ1细胞在体外培养，同时设二甲亚砜和空白对照组。采用四甲基偶氮唑蓝(MTT)比色法测定2ME对MUTZ1细胞的生长抑制率，瑞氏姬姆萨染色后观察2ME 引起细胞的形态学改变，流式细胞术分析细胞周期和凋亡率的变化，贝克曼全自动生化分析仪(synchron clinical system LX20)检测培养上清液中乳酸脱氢酶(lactate dehydrogenase， LD)的变化，DNA 凝胶电泳验证2ME 诱导的细胞凋亡。结果表明： 2ME 对MUTZ1细胞的增殖具有明显的抑制作用，该细胞凋亡率明显升高，并呈现时间和剂量依赖性，经统计学处理与对照组相比较有显著性差异(P< 0.05)。4 μmol/L 2ME 作用MUTZ1细胞12小时后，细胞呈现典型的凋亡细胞形态特征;2ME作用24小时后MUTZ1细胞出现G2/M期阻滞;培养上清液中LD 含量与对照组相比明显升高，差异具有显著性(P< 0.05); 4 μmol/L 2ME 作用MUTZ1细胞48小时后，DNA凝胶电泳可见明显的DNA 梯形条带。结论： 2ME对骨髓增生异常综合征细胞株 MUTZ1有较强的抗肿瘤效应，可能与细胞G2/M期阻滞引起的细胞凋亡有关;2ME 是一种有发展潜力的治疗骨髓异常综合征的药物。

　　【关键词】 2甲氧基雌二醇; 骨髓增生异常综合征; MUTZ1细胞株; 细胞凋亡

　　Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by ineffective hematopoiesis, translation into peripheral cytopenia of one, two or three lineages, together with normocytic or hyperplastic bone marrow because of the expansion of a malignant clone [1]. It is a potentially fatal hematological neoplasia whose incidence has significantly increased during the last three decades, twenty to thirty percent of MDS patients progress to overt myeloid leukemia with a poor prognosis, and the treatment approaches for MDS are not generally satisfactory, which prompted the investigation of new agents. J Exp Hematol 2007; 15(2) Mechanisms of 2MethoxyestradiolInduced Apoptosis in Myelodysplastic Syndrome MUTZ1 Cell Lines2Methoxyestradiol (2ME), an endogenous estrogen metabolite, has been shown to have potent tumor inhibiting effects in a number of cancer cell lines in vitro and in tumor xenograft models in vivo. It is presumed that 2ME has low systematic toxicity, and then considerable research efforts have been initiated lately to explore the possibility of 2ME as a lowtoxicity chemotherapeutic agent. Previous studies on anticancer mechanisms of 2ME had been suggested, including inhibition of superoxide dismutase[2], interference of spindle dynamics during mitosis[3] and interference of cell cycle progression[4], but the causal sequence and molecular mechanism of 2MEinduced apoptosis in MDS have not been elucidated.

　　In this report, we use a longterm cultured MDS cell line established from a patient with MDS at a stage of RAEB according to FAB classification[5] to further investigate the mechanism of 2ME on the induction of apoptosis.

　　Materials and Methods

　　Cell line

　　MUTZ1, a myelodysplastic syndrome cell line , was established from a patient with MDS at a stage of RAEB according to FAB classification, the karyotype of the original MUTZ1 cells showing high frequency of chromosome 5q deletion was first detected and analyzed in 1997 [6];complex karyotype in MUTZ1 cell line was detected by metaphase fluorescence in situ hybridization [7]; the cell surface marker profiles of original MUTZ1 cells showed CD10,CD19,CyIgM positive[6].

　　Reagents

　　2Methoxyestradiol (2ME, MW 302.4), dimethyl sulfoxide (DMSO) and RMPI 1640 medium were purchased from Sigma Chemical (USA). 2ME was dissolved in DMSO at a concentration of 5 mmol/L. Stock aliquots were stored at 4℃ and the working concentrations of 1, 2，4, 8 and 16 μmol/L 2ME were prepared by dilution with culture medium RPMI 1640 immediately before use, the final concentration of DMSO in the culture system was less than 0.3%, which had no significant effect on the cell growth. AnnexinVFITC and propidine iodide were obtained from Pharmingen (USA).

　　Cell culture

　　MUTZ1 cell lines were cultured in RPMI 1640 medium supplemented with heatinactivated 10% fetal calf serum, 2 mmol/L Lglutamine, 100 U/ml streptomycin and 100 U/ml penicillin G. Incubation was carried out in a saturated humidified atmosphere with 5% CO2 at 37℃. The cells in logarithmic growth phase were used in all experiments.

　　Cell growth and viability assays

　　For cell growth and viability assays [8], 1×105 cells were plated in 96 wellplates and treated with 2ME. Cells were incubated with 20 μl of MTT reagent in each well at 37℃ in the dark for at least 4 hours.The formazan crystals were solubilized in 200 μl DMSO each well and the reduction of MTT was quantified by absorbance (A570 nm).

　　Morphological change

　　After being cultured in the medium containing 2ME, the cells were collected, stained with WrightGiemsa's staining and distilled water (1∶1) for 5 minutes, flushed with water, airdried and then observed with optical microscope, the photos were taken by OLYMPUS hunter imaging system (USPT, JAPAN).

　　Determination of lactate dehydrogenase in cell culture supernatant

　　After being treated with 0,1,2,4 and 8 μmol/L 2ME, MUTZ1 cells were collected at 12,24,36,48 and 72 hours and centrifuged. The lactate dehydrogenase in cell supernatant was determined by Beckman Counter (synchron clinical system LX20).

　　Assessment of cell cycle

　　1×106 cells were harvested, washed with icecold PBS, and resuspended with trypsin buffer. The cells were then washed again with icecold PBS, treated with trypsin inhibitor and RNase buffer, and stained with propidium iodied (PI). The cell cycle was determined with a computer programmed ModFit LT2.0 DNA assay by BectonDickinson FACSCalibur cytoflurometer (Mansfield, MA,USA ) .

　　Annexin VPI assays for apoptosis

　　The cells were stained with Annexin VFITC and PI, and evaluated for apoptosis by flow cytometry according to the manufacture's protocol. Briefly, 1×106 cells were stained with 5 μl Annexin VFITC and 10 μl PI (5 μg/ml) in 1×binding buffer (10 mmol/L HEPES, pH 7.4,140 mmol/L NaOH, 2.5 mmol/L CaCl2) for 20 minutes at room temperature in the dark. The apoptotic cells were determined using a BectonDickinson FACSCalibur cytoflurometer. Both early (Annexin Vpositive, PInegative) and late (Annexin Vpositive and PIpositive) apoptotic cells were included in cell death determinations.

　　DNA fragmentation assay to measure apoptosis

　　After treatment with 0,1,4 and 8 μmol/L 2ME, the cells were lysed in a buffer containing 10 mmol/L TrisHCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA and 0.5% TritonX100 for 30 minutes on ice. Lysates were vortexed and cleared by centrifugation at 10 000×g for 20 minutes. Fragmented DNA in the supernatant was extracted with an equal volume of neutral phenol∶chloroform∶isoamylalcohol (25∶24∶1,v/v/v) and analyzed by electrophonesis on 1.5% agarose gel containing 0.1 μg/ml ethidium bromide.

　　Statistical analysis

　　The experiments data were presented as means± standard deviation (SD). Statistical differences between control and treated groups were determined by Student's ttest. P<0.05 was considered as statistically significant difference.

　　Results

　　Effect of 2ME on inhibition of MUTZ1 cell growth

　　The MTT assay revealed that treatment with 2ME inhibited the viability of MUTZ1 cells in a dosedependent manner, which reached a plateau at 4 μmol/L after treatment for 36 hours (Figure 1).

　　Figure 1. Growth suppression effect of 2ME on MUTZ1 cell. MUTZ1 cells were cultured and determined by MTT as described under Materials and Methods. The experiments were done in triplicates.

　　The level of lactate dehydrogenase in cell culture supernatant

　　The membrane integrity of MUTZ1 cells was assessed by monitoring the release of cytosolic lactate dehydrogenase as an index of cytotoxicity (Figure 2). MUTZ1 cells were incubated for 12,24,36,48 and 72 hours in medium containing 0,1,2,4 and 8 μmol/L 2ME, respectively, and the level of LDH had remarkably elevated after 36 hours in comparison to control group (P<0.05).

　　Figure 2. Level of lactate dehydrogenase in MUTZ1 cell culture supernatant after 2ME treatment. MUTZ1 cells were treated with different concentration of 2ME and harvested for assay as described under Materials and Methods.

　　Morphological change

　　To determine whether the subG1 population corresponds to apoptotic cells, light microscope was used to detect morphologic changes associated with the induction of apoptosis. After MUTZ1 cells being incubated with 4 μmol/L 2ME for 12 hours, significant changes in the morphologic characteristics of the cells were evident, such as cell shrinkage, "blebbing" of the cell membrane, nuclear fragmentation, nuclear condensation and presence of apoptotic bodies ( Figure 3).

　　Figure 3. Morphological changes of MUTZ1 cell shown after 2ME treatment. MUTZ1 cells were treated with dimethyl sulfoxide alone (A) or with 4 μmol/L 2ME for 12 hours (B, red arrow) (WrightGiemsa's staining, ×1000).

　　Distribution of MUTZ1 cells in different phases of the cell cycle after treatment with 2ME

　　Because of the inhibitory effect of 2ME, we further investigated the cell cycle distribution in 2MEtreated cells. As shown in Figure 4, in the absence of the 2ME, the majority of MUTZ1 cells were at the G1 or S phase. These cells had entered the G1 phase promptly after being released from a mitotic pause and moved progressively through the cell cycle into the next G1 phase. Once treated with 2ME, the percentage of cells in the G1 phase was significantly reduced and the majority of cells were arrested at the G2/M phase and an increase at the G2/M population from 9.87% to 37.45% and 46.36%, respectively.

　　Figure 4. Cell cycle analysis after 2ME treatment. MUTZ1 cells were treated with 0, 1, 4 μmol/L 2ME for 24 hours and evaluated for DNA content after PI staining. *P<0.05, compared with control group.

　　Induction of apoptosis of MUTZ1 cells by 2ME

　　Double staining assays with annexin VPI demonstrated that the apoptosis rate was significantly increased in 2MEtreated cells with the apoptosis rates of 14.40% ( Figure 5B), 20.11% ( Figure 5C), 68.21% ( Figure 5D) respectively in dosedependent manner, and were statistically significant in comparison with that in control (8.33%, Figure 5A) (P<0.05).

　　DNA fragmentation of 2ME induced apoptosis in MUTZ1 cells

　　Another hallmark of apoptosis is a degradation of chromosomal DNA at internucleosomal linkages. Accordingly, we analyzed whether DNA fragmentation was induced by 2ME in MUTZ1 cells. Following agarose gel electrophoresis of MUTZ1 cells treated with 0,1,4 and 8 μmol/L 2ME for 48 hours, a typical ladder pattern of internucleosomal fragmentation was observed (Figure 6).

　　Figure 5. Apoptosis assay by flow cytometry in MUTZ1 cells detected with AnnexinV and PI staining after treatment with 2ME for 36 hours. A: 0 μmol/L; B: 1 μmol/L; C: 4 μmol/L; D: 8 μmol/L.

　　Figure 6. Apoptosis of MUTZ1 cells detected by agarose gel electrophoresis after treatment with various concentrations of 2ME for 48 hours. M: marker. Lane 1: blank. Lane 2: 1 μmol/L; Lane 3: 4 μmol/L; Lane 4: 8 μmol/L.

　　Discussion

　　MDS is still an incurable disease, even with intensive cytotoxic therapy[9]. Therefore, new treatment approaches are needed to improve patients' outcome, if possible with a safe program good enough to allow treatment in unfit or old patients. 2ME is an endogenous metabolite of estrogen, which has recently been shown to have unique antiproliferative and apoptotic activities mediated independently of estrogen α and β receptor[10]. Also, the potential of 2ME to affect estrogen target tissues independently of the estrogen receptor has recently been illustrated[11], but the mechanism of tumor inhibition by 2ME is not yet fully understood[12]. Previous studies suggested the anticancer mechanism of 2ME, including inhibition of superoxide dismu tase[2,13], interference with spindle dynamics during mitosis [3] and interference with cell cycle progression [4]. Although 2ME has been studied in clinical trials for acute and chronic leukemia[2], the causal sequence and molecular mechanism of 2MEinduced apoptosis in MDS have not yet been elucidated.

　　In this report, we found that treatment of MDS cell line MUTZ1 with 2ME resulted in a significant changes. MTT assay revealed that the treatment with 2ME inhibited the viability of MUTZ1 cells in a dosedependent manner, but in a cultured medium supplemented with 4 μmol/L to 16 μmol/L concentrations of 2ME, the MUTZ1 cell showed a similar concentrationdependent antiproliferative effect, quite in agreement with the results reported by others[10]. In animal experiments, 2ME has been shown only to inhibit the growth of dividing cells but not of resting cells and the high dosages of 2ME necessary for its inhibiting effect possess little toxicity[14] and resistance has not been observed[15], the concentrations of 2ME used in the present experiments are higher than human serum levels of 2methoxyestrogens, ranging from 10 (adult males) to 3700 pg/ml (pregnant females)[16], the effects of 2ME are concentrationdependent[11] and the effects at physiological concentration may exist but would be rare.

　　Significant changes in the morphologic characteristics of the cells were evident after treatment with 2ME, such as cell shrinkage, "blebbing" of the cell membrane, nuclear fragmentation, nuclear condensation and presence of apoptotic bodies highly suggestive of apoptosis. Another hallmark of apoptosis is a degradation of chromosomal DNA at internucleosomal linkages[17]. Following agarose gel electrophoresis of MUTZ1 cells treated with different concentrations of 2ME for 48 hours, a typical ladder pattern of internucleosomal fragmentation was observed (Figure 6). To quantify the degree of apoptosis, we analyzed the amount of DNA by flow cytometry of fixed nuclei,as shown in Figure 4, the appearance of a subG1 population of cells is a potential indicator of apoptosis, thus showing that 2ME may inhibit the growth of cells by inducing apoptosis, and the addition of 2ME to MUTZ1 cells resulted in markedly increased accumulation of the G2/M phase in a dosedependent manner. Therefore, it is most likely that 2ME acts through the reduction of the cell G1 phase and the cells are arrested at the G2/M phase to reduce apoptosis.

　　Further more, our study has shown that MUTZ1cells are susceptible to the cytotoxic effects of 2ME through monitoring the membrane integrity of MUTZ1 cells by assessing the release of cytosolic lactate dehydrogenase. These findings are consistent with recent findings in human leukemia HL60 cells [18], this process appears to be mediated by a common set of downstream elements that act as regulators and effectors of cell death, which needs to be further studied for elucidation of the mechanism.

　　Taken together, our data suggest that 2ME is an effective agent inhibiting cell proliferation via enhancing apoptosis; 2ME also upregulates the level of LD in cell culture supernatant and causes a block of cell cycle at the G2/M phase. Therefore, 2ME may be an adjunctive anticancer drug potentially useful to treat myelodysplastic syndrome.

　　Acknowledge: We are grateful to Chen JunHao, Song Ping and Du HaiZhen for their skillful technical assistance.

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