芫花酯甲抗黑色素瘤的作用及其机理的初步研究
摘要
研究背景
芫花酯甲由瑞香科植物芫花中提取所得，可以抑制P-388，L1210，KB等多种细胞的增殖，抑制DNA，RNA和蛋白的合成，对拓扑异构酶Ⅰ的活性具有显著的抑制作用，而对肿瘤坏死因子TNF-α，白细胞介素IL-1α和IL-1β的生物合成则并无显著影响。动物体内实验的结果表明，芫花酯甲可抑制C57BL／6和BDF_1小鼠Lewis肺癌的发展和转移。
尽管许多研究证实芫花酯甲可抑制肿瘤细胞的增殖以及肿瘤的生长，但是其具体的作用途径目前尚未阐明。为此，我们检测了芫花酯甲对黑色素瘤A375细胞增殖的影响，肯定了其对黑色素瘤细胞增殖的抑制作用，并从细胞周期阻滞、诱导细胞凋亡以及对蛋白激酶C(PKC)活化作用三个方面解释了其可能的作用机理，并在小鼠黑色素瘤模型上验证了芫花酯甲的抗肿瘤活性，为今后研究芫花酯甲的作用机理及将其开发成为新的抗癌药物打下基础。
研究方法
我们采用台盼蓝染料排斥实验和CCK-8分析法来检测芫花酯甲对细胞存活率的影响。通过[~3H]-胸腺嘧啶掺入实验分析其对细胞DNA合成的影响。另外，对细胞进行碘化丙啶染色，利用流式细胞仪检测细胞的DNA含量，进而评价芫花酯甲对细胞周期分布的影响。使用annexin V-FITC／PI凋亡检测试剂盒，通过流式细胞仪定量分析磷脂酰丝氨酸外翻程度从而检测芫花酯甲作用后细胞凋亡比例的变化。采用Rhodamine 123染色，流式细胞技术检测芫花酯甲长期孵育对线粒体膜电势的影响。进行Western blot实验，分析细胞周期相关蛋白以及细胞凋亡相关蛋白表达量的变化。采用Ca~(2+)敏感的探针fluo-3·AM进行标记，激光共聚焦显微镜和流式细胞术检测芫花酯甲瞬时作用25min以及长期作用24h后对细胞内钙离子浓度的影响。制备红细胞膜，利用其对蛋白激酶C(PKC)的结合作用随Ca~(2+)浓度变化而改变的特性提取PKC，并经亲和柱层析进一步纯化PKC，通过检测γ~(32)p-ATP中~(32)p掺入底物组蛋白的量的多少反映细胞内及体外经纯化的PKC的活性变化。应用激光共聚焦显微镜观察转入pPKCγ-EGFP质粒的A375细胞在芫花酯甲作用下荧光位置的改变进而判断PKCγ的活性状态。建立C57BL／6小鼠黑色素瘤模型，通过测量黑色素瘤大小以及最后称重确定芫花酯甲对肿瘤生长的抑制作用。将经过药品处理的小鼠以及对照组小鼠的肺部包埋切片，HE染色，观察黑色素瘤的转移情况。
研究结果
1．在台盼蓝染料排斥试验中，我们观察道芫花酯甲能够抑制A375细胞的增殖，且呈现明显的剂量依赖关系和时间依赖关系。药物作用A375细胞24h的IC_(50)为6.78±2.4μM；处理48h的IC_(50)为1.23±0.85μM。
2．CCK-8分析实验显示，芫花酯甲能够显著抑制细胞活力水平，且呈剂量和时间依赖关系。10μM芫花酯甲作用12，24，36和48h，细胞活力抑制率分别为32.2±6.52％，43.51±4.69％，46.1±5.26％和55±2.28％。
3．[~3H]-胸腺嘧啶掺入实验表明芫花酯甲可以抑制A375细胞DNA合成，且呈时间和剂量依赖关系。0.1，1，10和50μM芫花酯甲孵育48h以后，A375细胞DNA的合成率分别降为对照组的51.6±2.57％，38.8±5.49％，32.3±6.53％，4.2±3.96％。
4．通过流式细胞术分析，我们得知，芫花酯甲能够诱导A375细胞发生G_2／M周期阻滞，而且这一效应具有剂量依赖性和时间依赖性。0.1μM，1μM和10μM芫花酯甲孵育48h后，A375细胞的G_2／M期比例由8.47±1.34％升高到11.42±2.49％。15.74±2.94％和22.4±2.48％，细胞与1μM芫花酯甲共同孵育12h，24h和48h后，G_2／M期细胞的比例由8.47±1.34％升高为10.62±1.96％，10.82±2.83％以及15.7±2.94％。
5．Western blot实验结果显示，0.1μM，1μM和10μM芫花酯甲作用于A375细胞48h后，细胞内CyclinB1和cdc2蛋白的表达量有了显著降低。
6．A375细胞经annexin V-FITC／PI双染后，流式细胞仪分析显示，0.1，1和10μM芫花酯甲处理细胞48h，发生早期凋亡细胞比例分别从3.7％升高到4.8％，8.5％和24.6％，晚期凋亡细胞数量没有明显变化。
7．Rhodamine 123染色后，经流式细胞仪分析荧光强度可知，芫花酯甲可引起A375细胞线粒体膜电势降低，且具有时间和剂量依赖性。0.1μM，1μM和10μM芫花酯甲孵育48h后，细胞的平均荧光强度由9.99±0.85分别降至8.02±1.45，6.1±0.15和4.23±0.7。而10μM芫花酯甲作用12，24和48h后，平均荧光强度值降低为对照组的69±7.4％，50±10.7％和43±4.23％。
8．Western blot实验分析显示，含有0.1μM，1μM和10μM芫花酯甲的培养基孵育A375细胞48h后，线粒体内细胞色素c含量降低，而胞浆中细胞色素c含量显著升高，同时，caspase-3水解为17kD片断增多，Bcl-2蛋白表达量降低而Bax蛋白表达量升高，使得Bax／Bcl-2比例明显升高。P53蛋白表达有所上升而mdm2蛋白表达量降低，同时我们还观测道p21蛋白表达也降低。
9．细胞经fluo-3·AM染色后，流式细胞术显示芫花酯甲急性和长期给药都可以引起细胞内Ca~(2+)浓度的升高，长期给药24h后升高为对照组的1.23倍，急性给药25min内Ca~(2+)缓慢升高。
10．芫花酯甲可引起细胞内PKC活性的增加，对于纯化后的PKC同样具有激活作用，具有剂量依赖性，而且该激活作用不依赖于Ca~(2+)。反应体系内不含Ca~(2+)和PMA时，1μM芫花酯甲使得cpm值升高为对照组的3.53±0.18倍，含有Ca~(2+)和PMA时升高为对照组的1.78倍。芫花酯甲的激活作用可被G06986完全抑制，而Rottlerin则部分抑制此活性。
11．激光共聚焦显微镜观察到芫花酯甲可直接引起PKCγ的活化，使其由胞浆部位转至胞膜，并且可以加速PMA引起的PKCγ转位。
12．芫花酯甲可以抑制C57BL／6小鼠黑色素瘤的生长，皮肤涂抹给药的抑制率为31％，皮下注射剂量为0.8mg／kg时，抑制率可达47％，腹腔注射浓度为0.4mg／kg抑制率达50.2％。芫花酯甲对于黑色素瘤在C57BL／6小鼠体内的转移并没有显著的影响。
结论
芫花酯甲能够抑制A375细胞的增殖，能够抑制A375细胞的活力水平以及DNA的合成，呈现剂量依赖关系和时间依赖关系。我们通过三个作用途径研究其抑制细胞增殖的机理。首先证实芫花酯甲可以使得A375细胞停滞于G_2／M期，该时期关键蛋白CyclinB1和cdc2蛋白表达降低；同时我们也证实芫花酯甲能够诱导细胞凋亡，此过程中线粒体膜电势降低，细胞色素c由线粒体释放至胞浆；caspase-3水解为17kD活性片断增多；抗凋亡的Bcl-2蛋白表达量下调而促进凋亡Bax蛋白表达量上升，导致Bax／Bcl-2比例升高；p53蛋白表达增多而p53抑制因子mdm2蛋白以及p21表达降低，在这些因素的共同作用下，使细胞进入凋亡前期。此外，芫花酯甲还能够增加细胞内钙离子浓度，可直接激活PKC。在这三个可能途径的作用下，细胞的增殖得以抑制。动物体内实验证实，芫花酯甲对于C57BL／6小鼠的黑色素瘤的生长有显著的抑制作用。总之，根据我们的研究结果可以推测，芫花酯甲作为一种植物提取成分，对于黑色素瘤的治疗具有潜在的应用价值。
Background.
Yuanhuacine (YHC) was isolated from D. genkwa and has been reported to have activity in the termination of early pregnancy by combination with testosterone propionate. YHC has been shown to be active against the growth of P-388 lymphocytic leukemia cells, L-1210 lympHoid leukemia, human KB carcinoma cell lines. In addition, YHC also suppresses DNA, RNA and protein synthesis of some tumor cells. Recent research showed that YHC has weak effect on tumor necrosis factor (TNF-a) and interleukin-1 (IL-la, IL-lb) biosynthesis, but exhibited potent inhibitory activities against DNA topo I. In the experiments in vivo, YHC significantly inhibited the growth of P-388 lympHocytic leukemia cells and suppressed tumor growth of Lewis lung carcinoma in BDF1 mice and in C57BL/6 mice.
However, although the antiproliferative effects of YHC have been well- documented, the cellular mechanisms underlying the antiproliferative activity of YHC are unclear. In present study, we demonstrated that yuanhuacine inhibited the growth of human malignant melanoma A 375 cells in a dose- and time-dependent manner. This growth arrest was shown to be because of cell cycle inhibition at G_2/M, apoptosis induction and activation of protein kinase C (PKC). The experiments in vivo confirmed that yuanhuacine suppressed the growth of melanoma. These results indicate that yuanhuacine can inhibit cancer cell growth not only in vitro but also in vivo, therefore it may ultimately be proved to be useful as a new potential antitumor medicine for malignant melanoma.
Methods.
Survival/proliferation of cell lines treated with YHC was determined by trypan blue dye exclusion assay or CCK-8 assay. [~3H]-thymidine incorporation assay was performed to assess DNA synthesis of cells. Cell cycle distribution was determined by flow cytometric analysis of DNA content of nuclei of cells following staining with propidium iodide. Apoptosis induction by YHC in A375 cells was assessed by quantification of phosphatidylserine exposure using annexin V-FITC kit. Alterations in mitochondrial membrane potential (△Ψm) were analyzed by flow cytometry using the△Ψm-Sensitive dye rhodamine123. Western blot was performed to investigate the expression of cell cycle and apoptosis related proteins. Cytosolic calcium level of cells after short-term and long-term YHC exposure was measured using a calcium probe fluo-3·AM by laser confocal microscopy and flow cytometry. Intracellular or purled PKC activity was determined by ~(32)p radioisotope incorporation. Confocal microscopy was used to observe fluorescence disposition to evaluate the translocation of PKCγ-EGFR The inhitition of tumor growth was confirmed by C57BL/6 melanoma modeling.
Results.
1. The treatment of A375 cells with 0.01-10μM of YHC resulted in a dose-dependent decrease in cell survival with an IC_(50) of 6.78±1.74μM for 24 h and 1.23±0.29μM for 48 h.
2. Viability of the cells was inhibited significantly upon treatment with YHC in a dose- and a time-dependent manner. The inhibition rate of cell viability was increased to 32.2±6.52%, 43.51±4.69%, 46.1±5.26% and 55±2.28% respectively which were treated with YHC for 12, 24, 36 and 48h in concentration of 10μM, and IC_(50) was 4.89μM for 48 h.
3. [~3H]-thymidine incorporation into A375 cells was decreased to 51.6±2.57%, 38.8±5.49%, 32.3±6.53%, 4.2±3.96% (as a percentage of control) respectively, after treatment with YHC in concentrations of 0.1, 1, 10 and 50 mM for 48h, and IC_(50) was 0.23μM.
4. Exposure of A375 cells to YHC for increasing time intervals produced a concentration and time-dependent G_2/M pHase cell cycle arrest. For example, after treatment with 0.1, 1 and 10μM YHC for 48 h, the percentage of cells in the G_2/M pHase increased to 11.42±2.49%, 15.74±2.94% and 22.4±2.48%, respectively, showing significant difference compared with the control group (8.47±1.34%). After incubation with 1μM YHC for 12 h, 24 h and 48 h, the percentage of cells in the G_2/M pHase increased to 10.62±1.96%, 10.82±2.83%and 15.7±2.94%, respectively.
5. When cells were treated with 0.1, 1 and 10μM YHC for 48 h, CyclinB 1 and cdc2 protein expression levels were decreased significantly.
6. Flow cytometric quantification of apoptotic A375 cells stained with annexin V-FITC/PI showed that YHC treatment increased the percentage of apoptotic cells to 4.8%, 8.5% and 24.6%, respectively in concentration of 0.1, 1 and 10μM for 48 h.
7. The△Ψm of A375 cell was reduced from 9.99±0.85 to 8.02±1.45, 6.1±0.15 and 4.23±0.7 by exposure to 0.1, 1 and 10μM YHC.
8. Treatment with yuanhuacine resulted in cytochrome c releasing from mitochondria and activation of caspase-3, accompanied with significant down-regulation of the Bcl-2 and mdm-2 expression, up-regulation of Bax, p53 and p21 expression.
9. The cytosolic calcium level was 1.23 folds higher compared with control after treatment with 1μM YHC for 48 h, and the level's upregulation was observed within 25 min.
10. YHC induced protein kinase C activation both in cell and in tube, and this effect independented on Ca~(2+). PKC activity was 1.78 and 3.53 folds higher compared with or without Ca~(2+)/PMA.
11. YHC induced PKCγtranslocation from cytoplasm to plasma membrane and showed synergistic effect with PMA.
12. YHC suppressed the growth of malignant melanoma in C57BL/6, the inhibition rate was 31% for animal which were application with YHC, subcutaneous injection in dose of 0.8mg/kg induced 47% decrease and intraperitoneal injection 0.4mg/kg YHC resulted in the reduction of 50.2%.
Conclusion.
YHC inhibited A375 cell growth in a dose- and a time-dependent manner, and arrested cell cycle at the G_2/M phase through decreased expression of cyclin B1 and cdc2 and activation of p53. Simultaneously, our data also provide evidence for the induction of apoptotic cell death of YHC, which may be attributable to the dissipation of△Ψm, the up-regulation of Bax and down-regulation of Bcl-2, accompanied with cytochrome c releasing and caspase-3 cleavage. In addition, p53, p21 and mdm2 contribute to this process. YHC also induces cytosolic calcium increasing and PKC activation. Furthermore, YHC inhibits the growth of malignant melanoma in vivo. These results indicate that as an extraction from DapHne genkwae, YHC may ultimately prove useful for malignant melanoma.
芫花酯甲由瑞香科植物芫花中提取所得，可以抑制P-388，L1210，KB等多种细胞的增殖，抑制DNA，RNA和蛋白的合成，对拓扑异构酶Ⅰ的活性具有显著的抑制作用，而对肿瘤坏死因子TNF-α，白细胞介素IL-1α和IL-1β的生物合成则并无显著影响。动物体内实验的结果表明，芫花酯甲可抑制C57BL／6和BDF_1小鼠Lewis肺癌的发展和转移。
尽管许多研究证实芫花酯甲可抑制肿瘤细胞的增殖以及肿瘤的生长，但是其具体的作用途径目前尚未阐明。为此，我们检测了芫花酯甲对黑色素瘤A375细胞增殖的影响，肯定了其对黑色素瘤细胞增殖的抑制作用，并从细胞周期阻滞、诱导细胞凋亡以及对蛋白激酶C(PKC)活化作用三个方面解释了其可能的作用机理，并在小鼠黑色素瘤模型上验证了芫花酯甲的抗肿瘤活性，为今后研究芫花酯甲的作用机理及将其开发成为新的抗癌药物打下基础。
研究方法
我们采用台盼蓝染料排斥实验和CCK-8分析法来检测芫花酯甲对细胞存活率的影响。通过[~3H]-胸腺嘧啶掺入实验分析其对细胞DNA合成的影响。另外，对细胞进行碘化丙啶染色，利用流式细胞仪检测细胞的DNA含量，进而评价芫花酯甲对细胞周期分布的影响。使用annexin V-FITC／PI凋亡检测试剂盒，通过流式细胞仪定量分析磷脂酰丝氨酸外翻程度从而检测芫花酯甲作用后细胞凋亡比例的变化。采用Rhodamine 123染色，流式细胞技术检测芫花酯甲长期孵育对线粒体膜电势的影响。进行Western blot实验，分析细胞周期相关蛋白以及细胞凋亡相关蛋白表达量的变化。采用Ca~(2+)敏感的探针fluo-3·AM进行标记，激光共聚焦显微镜和流式细胞术检测芫花酯甲瞬时作用25min以及长期作用24h后对细胞内钙离子浓度的影响。制备红细胞膜，利用其对蛋白激酶C(PKC)的结合作用随Ca~(2+)浓度变化而改变的特性提取PKC，并经亲和柱层析进一步纯化PKC，通过检测γ~(32)p-ATP中~(32)p掺入底物组蛋白的量的多少反映细胞内及体外经纯化的PKC的活性变化。应用激光共聚焦显微镜观察转入pPKCγ-EGFP质粒的A375细胞在芫花酯甲作用下荧光位置的改变进而判断PKCγ的活性状态。建立C57BL／6小鼠黑色素瘤模型，通过测量黑色素瘤大小以及最后称重确定芫花酯甲对肿瘤生长的抑制作用。将经过药品处理的小鼠以及对照组小鼠的肺部包埋切片，HE染色，观察黑色素瘤的转移情况。
研究结果
1．在台盼蓝染料排斥试验中，我们观察道芫花酯甲能够抑制A375细胞的增殖，且呈现明显的剂量依赖关系和时间依赖关系。药物作用A375细胞24h的IC_(50)为6.78±2.4μM；处理48h的IC_(50)为1.23±0.85μM。
2．CCK-8分析实验显示，芫花酯甲能够显著抑制细胞活力水平，且呈剂量和时间依赖关系。10μM芫花酯甲作用12，24，36和48h，细胞活力抑制率分别为32.2±6.52％，43.51±4.69％，46.1±5.26％和55±2.28％。
3．[~3H]-胸腺嘧啶掺入实验表明芫花酯甲可以抑制A375细胞DNA合成，且呈时间和剂量依赖关系。0.1，1，10和50μM芫花酯甲孵育48h以后，A375细胞DNA的合成率分别降为对照组的51.6±2.57％，38.8±5.49％，32.3±6.53％，4.2±3.96％。
4．通过流式细胞术分析，我们得知，芫花酯甲能够诱导A375细胞发生G_2／M周期阻滞，而且这一效应具有剂量依赖性和时间依赖性。0.1μM，1μM和10μM芫花酯甲孵育48h后，A375细胞的G_2／M期比例由8.47±1.34％升高到11.42±2.49％。15.74±2.94％和22.4±2.48％，细胞与1μM芫花酯甲共同孵育12h，24h和48h后，G_2／M期细胞的比例由8.47±1.34％升高为10.62±1.96％，10.82±2.83％以及15.7±2.94％。
5．Western blot实验结果显示，0.1μM，1μM和10μM芫花酯甲作用于A375细胞48h后，细胞内CyclinB1和cdc2蛋白的表达量有了显著降低。
6．A375细胞经annexin V-FITC／PI双染后，流式细胞仪分析显示，0.1，1和10μM芫花酯甲处理细胞48h，发生早期凋亡细胞比例分别从3.7％升高到4.8％，8.5％和24.6％，晚期凋亡细胞数量没有明显变化。
7．Rhodamine 123染色后，经流式细胞仪分析荧光强度可知，芫花酯甲可引起A375细胞线粒体膜电势降低，且具有时间和剂量依赖性。0.1μM，1μM和10μM芫花酯甲孵育48h后，细胞的平均荧光强度由9.99±0.85分别降至8.02±1.45，6.1±0.15和4.23±0.7。而10μM芫花酯甲作用12，24和48h后，平均荧光强度值降低为对照组的69±7.4％，50±10.7％和43±4.23％。
8．Western blot实验分析显示，含有0.1μM，1μM和10μM芫花酯甲的培养基孵育A375细胞48h后，线粒体内细胞色素c含量降低，而胞浆中细胞色素c含量显著升高，同时，caspase-3水解为17kD片断增多，Bcl-2蛋白表达量降低而Bax蛋白表达量升高，使得Bax／Bcl-2比例明显升高。P53蛋白表达有所上升而mdm2蛋白表达量降低，同时我们还观测道p21蛋白表达也降低。
9．细胞经fluo-3·AM染色后，流式细胞术显示芫花酯甲急性和长期给药都可以引起细胞内Ca~(2+)浓度的升高，长期给药24h后升高为对照组的1.23倍，急性给药25min内Ca~(2+)缓慢升高。
10．芫花酯甲可引起细胞内PKC活性的增加，对于纯化后的PKC同样具有激活作用，具有剂量依赖性，而且该激活作用不依赖于Ca~(2+)。反应体系内不含Ca~(2+)和PMA时，1μM芫花酯甲使得cpm值升高为对照组的3.53±0.18倍，含有Ca~(2+)和PMA时升高为对照组的1.78倍。芫花酯甲的激活作用可被G06986完全抑制，而Rottlerin则部分抑制此活性。
11．激光共聚焦显微镜观察到芫花酯甲可直接引起PKCγ的活化，使其由胞浆部位转至胞膜，并且可以加速PMA引起的PKCγ转位。
12．芫花酯甲可以抑制C57BL／6小鼠黑色素瘤的生长，皮肤涂抹给药的抑制率为31％，皮下注射剂量为0.8mg／kg时，抑制率可达47％，腹腔注射浓度为0.4mg／kg抑制率达50.2％。芫花酯甲对于黑色素瘤在C57BL／6小鼠体内的转移并没有显著的影响。
结论
芫花酯甲能够抑制A375细胞的增殖，能够抑制A375细胞的活力水平以及DNA的合成，呈现剂量依赖关系和时间依赖关系。我们通过三个作用途径研究其抑制细胞增殖的机理。首先证实芫花酯甲可以使得A375细胞停滞于G_2／M期，该时期关键蛋白CyclinB1和cdc2蛋白表达降低；同时我们也证实芫花酯甲能够诱导细胞凋亡，此过程中线粒体膜电势降低，细胞色素c由线粒体释放至胞浆；caspase-3水解为17kD活性片断增多；抗凋亡的Bcl-2蛋白表达量下调而促进凋亡Bax蛋白表达量上升，导致Bax／Bcl-2比例升高；p53蛋白表达增多而p53抑制因子mdm2蛋白以及p21表达降低，在这些因素的共同作用下，使细胞进入凋亡前期。此外，芫花酯甲还能够增加细胞内钙离子浓度，可直接激活PKC。在这三个可能途径的作用下，细胞的增殖得以抑制。动物体内实验证实，芫花酯甲对于C57BL／6小鼠的黑色素瘤的生长有显著的抑制作用。总之，根据我们的研究结果可以推测，芫花酯甲作为一种植物提取成分，对于黑色素瘤的治疗具有潜在的应用价值。
Background.
Yuanhuacine (YHC) was isolated from D. genkwa and has been reported to have activity in the termination of early pregnancy by combination with testosterone propionate. YHC has been shown to be active against the growth of P-388 lymphocytic leukemia cells, L-1210 lympHoid leukemia, human KB carcinoma cell lines. In addition, YHC also suppresses DNA, RNA and protein synthesis of some tumor cells. Recent research showed that YHC has weak effect on tumor necrosis factor (TNF-a) and interleukin-1 (IL-la, IL-lb) biosynthesis, but exhibited potent inhibitory activities against DNA topo I. In the experiments in vivo, YHC significantly inhibited the growth of P-388 lympHocytic leukemia cells and suppressed tumor growth of Lewis lung carcinoma in BDF1 mice and in C57BL/6 mice.
However, although the antiproliferative effects of YHC have been well- documented, the cellular mechanisms underlying the antiproliferative activity of YHC are unclear. In present study, we demonstrated that yuanhuacine inhibited the growth of human malignant melanoma A 375 cells in a dose- and time-dependent manner. This growth arrest was shown to be because of cell cycle inhibition at G_2/M, apoptosis induction and activation of protein kinase C (PKC). The experiments in vivo confirmed that yuanhuacine suppressed the growth of melanoma. These results indicate that yuanhuacine can inhibit cancer cell growth not only in vitro but also in vivo, therefore it may ultimately be proved to be useful as a new potential antitumor medicine for malignant melanoma.
Methods.
Survival/proliferation of cell lines treated with YHC was determined by trypan blue dye exclusion assay or CCK-8 assay. [~3H]-thymidine incorporation assay was performed to assess DNA synthesis of cells. Cell cycle distribution was determined by flow cytometric analysis of DNA content of nuclei of cells following staining with propidium iodide. Apoptosis induction by YHC in A375 cells was assessed by quantification of phosphatidylserine exposure using annexin V-FITC kit. Alterations in mitochondrial membrane potential (△Ψm) were analyzed by flow cytometry using the△Ψm-Sensitive dye rhodamine123. Western blot was performed to investigate the expression of cell cycle and apoptosis related proteins. Cytosolic calcium level of cells after short-term and long-term YHC exposure was measured using a calcium probe fluo-3·AM by laser confocal microscopy and flow cytometry. Intracellular or purled PKC activity was determined by ~(32)p radioisotope incorporation. Confocal microscopy was used to observe fluorescence disposition to evaluate the translocation of PKCγ-EGFR The inhitition of tumor growth was confirmed by C57BL/6 melanoma modeling.
Results.
1. The treatment of A375 cells with 0.01-10μM of YHC resulted in a dose-dependent decrease in cell survival with an IC_(50) of 6.78±1.74μM for 24 h and 1.23±0.29μM for 48 h.
2. Viability of the cells was inhibited significantly upon treatment with YHC in a dose- and a time-dependent manner. The inhibition rate of cell viability was increased to 32.2±6.52%, 43.51±4.69%, 46.1±5.26% and 55±2.28% respectively which were treated with YHC for 12, 24, 36 and 48h in concentration of 10μM, and IC_(50) was 4.89μM for 48 h.
3. [~3H]-thymidine incorporation into A375 cells was decreased to 51.6±2.57%, 38.8±5.49%, 32.3±6.53%, 4.2±3.96% (as a percentage of control) respectively, after treatment with YHC in concentrations of 0.1, 1, 10 and 50 mM for 48h, and IC_(50) was 0.23μM.
4. Exposure of A375 cells to YHC for increasing time intervals produced a concentration and time-dependent G_2/M pHase cell cycle arrest. For example, after treatment with 0.1, 1 and 10μM YHC for 48 h, the percentage of cells in the G_2/M pHase increased to 11.42±2.49%, 15.74±2.94% and 22.4±2.48%, respectively, showing significant difference compared with the control group (8.47±1.34%). After incubation with 1μM YHC for 12 h, 24 h and 48 h, the percentage of cells in the G_2/M pHase increased to 10.62±1.96%, 10.82±2.83%and 15.7±2.94%, respectively.
5. When cells were treated with 0.1, 1 and 10μM YHC for 48 h, CyclinB 1 and cdc2 protein expression levels were decreased significantly.
6. Flow cytometric quantification of apoptotic A375 cells stained with annexin V-FITC/PI showed that YHC treatment increased the percentage of apoptotic cells to 4.8%, 8.5% and 24.6%, respectively in concentration of 0.1, 1 and 10μM for 48 h.
7. The△Ψm of A375 cell was reduced from 9.99±0.85 to 8.02±1.45, 6.1±0.15 and 4.23±0.7 by exposure to 0.1, 1 and 10μM YHC.
8. Treatment with yuanhuacine resulted in cytochrome c releasing from mitochondria and activation of caspase-3, accompanied with significant down-regulation of the Bcl-2 and mdm-2 expression, up-regulation of Bax, p53 and p21 expression.
9. The cytosolic calcium level was 1.23 folds higher compared with control after treatment with 1μM YHC for 48 h, and the level's upregulation was observed within 25 min.
10. YHC induced protein kinase C activation both in cell and in tube, and this effect independented on Ca~(2+). PKC activity was 1.78 and 3.53 folds higher compared with or without Ca~(2+)/PMA.
11. YHC induced PKCγtranslocation from cytoplasm to plasma membrane and showed synergistic effect with PMA.
12. YHC suppressed the growth of malignant melanoma in C57BL/6, the inhibition rate was 31% for animal which were application with YHC, subcutaneous injection in dose of 0.8mg/kg induced 47% decrease and intraperitoneal injection 0.4mg/kg YHC resulted in the reduction of 50.2%.
Conclusion.
YHC inhibited A375 cell growth in a dose- and a time-dependent manner, and arrested cell cycle at the G_2/M phase through decreased expression of cyclin B1 and cdc2 and activation of p53. Simultaneously, our data also provide evidence for the induction of apoptotic cell death of YHC, which may be attributable to the dissipation of△Ψm, the up-regulation of Bax and down-regulation of Bcl-2, accompanied with cytochrome c releasing and caspase-3 cleavage. In addition, p53, p21 and mdm2 contribute to this process. YHC also induces cytosolic calcium increasing and PKC activation. Furthermore, YHC inhibits the growth of malignant melanoma in vivo. These results indicate that as an extraction from DapHne genkwae, YHC may ultimately prove useful for malignant melanoma.
引文
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