摘要
NG-18(N-(2-ethoxyethyl)gambogamide)是由中科院上海药物研究所段文虎课题研究组对藤黄中的有效成分藤黄酸(gambogic acid,GA)结构修饰而得到的新化合物。本研究首先评价该化合物体外抗肿瘤活性,进而对其诱导HL-60细胞凋亡作用机制进行初步探讨。
体外抑制细胞增殖实验中,悬浮细胞我们采用四氮唑盐还原法(3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide,MTT),贴壁细胞采用磺酰罗丹明B蛋白染色法(Sulforhodamine B,SRB)来观察NG-18对19株肿瘤细胞(包括2株白血病,2株肺癌,3株胃癌,3株结肠癌,3株乳腺癌,2株卵巢癌,1株宫颈癌和3株肝癌)体外增殖抑制作用。结果显示NG-18对这些肿瘤细胞均有显著地增殖抑制作用,具有广泛的细胞毒作用。NG-18和母核GA的平均IC_(50)值分别为0.86μM和1.24μM,因此NG-18的体外抗肿瘤能力要高于其母核GA。
接着,我们在人白血病细胞HL-60上运用PI染色-流式细胞分析、Annexin V-FITC结合实验、DNA片段化电泳分析等方法来观察NG-18对HL-60细胞凋亡和细胞周期的影响。结果表明NG-18有很强的诱导细胞发生凋亡的作用,并且呈时间和剂量依赖性。通过流式细胞分析,我们可以发现0.5μM NG-18作用6h和1μM NG-18作用3h,就能发生明显的细胞凋亡。DNA片段化电泳分析结果显示,0.5μM NG-18作用6h后,出现了明显的DNA梯状条纹,表明细胞发生了凋亡。但是NG-18在诱导HL-60细胞凋亡的过程中并没有影响其细胞周期的分布。
我们对NG-18诱导HL-60细胞凋亡的机制进行了初步的探讨。首先我们用碱性单细胞凝胶电泳来检测细胞凋亡早期,细胞核DNA是否发生了断裂。结果表明NG-18并非是通过诱导细胞核DNA发生断裂而引发细胞凋亡。在凋亡程序的启动及执行过程中,有一大类蛋白酶家族Caspases起着十分重要的作用。Caspases(Cystein-containing,aspartat-specific proteases)的本质是一类半胱氨酸蛋白酶,在正常的状态下,它们以无活性的酶原(proenaymes)形式存在。
用Western Blot的方法我们发现,在NG-18诱导HL-60细胞凋亡的过程中,胞外和胞内两条凋亡信号通路上的启动Caspases,Caspase-8和Caspase-9几乎能同时被激活。这一结果表明胞外和胞内两条凋亡途径都参与了NG-18诱导的HL-60细胞凋亡。接着,我们预先用Caspases泛抑制剂(Z-VAD-FMK)处理细胞,再加入NG-18,结果显示Z-VAD-FMK能够完全抑制NG-18诱导的细胞凋亡。我们用其它的Caspases抑制剂如Caspase-2抑制剂(Z-VDVAD-FMK)、Caspase-9抑制剂(Z-LEHD-FMK)和Caspase-8抑制剂(Z-IETD-FMK)相同的方法处理细胞,结果表明只有Caspase-8抑制剂(Z-IETD-FMK)才能大部分抑制NG-18诱导的细胞凋亡,抑制效果约为66.7%。以上结果提示,NG-18诱导细胞凋亡是依赖于Caspases途径,并且在此凋亡过程中,Caspase-8处于中心地位,起着关键性的作用。综合以上结果,我们得出以下结论:虽然胞外和胞内两条凋亡途径都参与了NG-18诱导的HL-60细胞凋亡,但是胞外途径是主要的凋亡途径。在NG-18诱导HL-60细胞凋亡的过程中,Bcl-2家族蛋白中的Bcl-2、Bax和Bid蛋白也参与其中。
综上所述,本研究阐明NG-18具有较强的体外抗肿瘤活性,并发现该化合物能够强烈的诱导肿瘤细胞凋亡的发生,而对肿瘤细胞的细胞周期无明显影响。NG-18诱导HL-60细胞凋亡是依赖于Caspases途径,并且Caspase-8在该凋亡过程中起着关键的作用,一些Bcl-2家族蛋白也参与了该凋亡过程。
NG-18 (N-(2-ethoxyethyl)gambogamide), a noval compound, is structurally modified from the gambogic acid (GA) which is the major active ingredient of gamboges by the Duan Wenhu group, Phytochemistry Department of Shanghai Institute of Materia Medica. The present study focused on evaluating the antitumor effect of NG-18 in vitro, and then the mechanism of apoptosis induced by NG-18 in HL-60 cells was investigated preliminary.
MTT assay for suspend cells and SRB assay for attached cells were employed to evaluate the cytotoxic effects of NG-18 against 19 human tumor cell lines (including two human leukemia cell lines, two lung adenocarcinoma cell lines, three gastric adenocarcinoma cell lines, three colorectal carcinoma cell lines, three breast carcinoma cell lines, two ovarian carcinoma cell lines, one cervical adenocarcinoma cell line and three hepatocellular carcinoma cell lines) in vitro. The cell proliferation assay clarified its potent cytotoxic effects on various tumor cell lines. The mean IC_(50) values of NG-18 and its leading compound GA were 0.86μM and 1.24μM respectively. According to these results, we found that NG-18 had stronger inhibitory effect than its leading compound GA against those cell lines in vitro.
Next, PI staining-FACS analysis, Annexin VFITC binding assay and DNA fragmentation were employed.to observe apoptosis inducing capability of NG-18 on HL-60 cells. The results indicated that NG-18 possessed strong capability to induce apoptosis in HL-60 cells. The apoptosis induced by NG-18 was in time and does dependent manners. By the assay of PI staining-FACS analysis, we found that treatment with-0.5μM NG-18 for 6 h or 1μM NG-18 for 3 h, the apoptosis was induced significantly. DNA fragmentation assay showed that treatment with 0.5μM NG-18 for 6 h, DNA fragment could be detected obviously. But the cell cycle, distribution failed to respond to NG-18.
We investigated the mechanism of apoptosis induced by NG-18 preliminary. Firstly, alkaline signal cell gel electrophoresis techniqtie was employed to test whether nuclear DNA was damaged in the early apoptotic cells. The result indicated that the apoptosis induced by NG-18 was not result from the damage of nuclear DNA. At the beginning and in the process of apoptosis, a big protease family, Caspases, play a critical role in it. Caspases (Cystein-containing, aspartat-specific proteases) are cysterine proteases in essence and exist as inactive proenzymes in healthy cells. Using Western Blot assay, we found that the initiator Caspases, Caspase-8 and Caspase-9, in extrinsic and intrinsic apoptosis pathways respectively, could be activated almost at the same time in the apoptosis induced by NG-18. This result indicated that both extrinsic and intrinsic apoptosis pathways took part in the apoptosis induced by NG-18 in HL-60 cells. Next, Cells were pretreated with pan-Caspase inhibitor Z-VAD-FMK, and then exposed with NG-18. The result indicated that Z-VAD-FMK could inhibit apoptosis induced by NG-18 completely. Other Caspase inhibitors such as Z-VDVAD-FMK for Caspase-2, Z-LEHD-FMK for Caspase-9 and Z-IETD-FMK for Caspase-8 were used to treat with cells in the same way. The results showed that except Caspase-8 inhibitor Z-IETD-FMK, Z-VDVAD-FMK and Z-LEHD-FMK could not inhibit the apoptosis induced by NG-18. Z-IETD-FMK could inhibit NG-18 induced apoptosis almost by 66.7%. These results indicated that the apoptosis induced by NG-18 was in a Caspase-dependent manner in which Caspase-8 acted as a key executor. Integrating the results, we drew a conclusion: both extrinsic and intrinsic apoptosis pathways took part in the apoptosis induced by NG-18 in HL-60 cells, but the extrinsic pathway was the main apoptotic pathway. In the process of apoptosis induced by NG-18 in HL-60 cells, Bcl-2, Bax and Bid which belonged to Bcl-2 protein family also contributed to the apoptosis.
In conclusion, this study identifies that NG-18 exerts significant antitumor activity in vitro and it can induce apoptosis in tumor cells. Nevertheless, it doesn't cause evident change on cell cycle distribution. The apoptosis induced by NG-18 in HL-60 cells is in a Caspase-dependent manner in which Caspase-8 plays a very important role. Some Bcl-2 family proteins also take part in the apoptosis.
引文
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