Department of Thyroid and Breast Surgery, Zhejiang Provincial People's Hospital, Hangzhou 310014, Zhejiang Province, China.
World J Gastroenterol. 2012 Dec 28;18(48):7166-74. doi: 10.3748/wjg.v18.i48.7166.
To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.
The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. After treatment with 10 μg/mL oridonin for 24 h and 48 h, the cells were stained with acridine orange/ethidium bromide. The morphologic changes were observed under an inverted fluorescence microscope. DNA fragmentation (a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay. After treated with oridonin (0, 1.25, 2.5, 5 and 10 μg/mL), HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis, and oridonin- induced apoptosis in HGC-27 cells was detected. After treatment with oridonin for 24 h, the effects of oridonin on expression of Apaf-1, Bcl-2, Bax, caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction (RT-PCR) and Western blotting.
Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose- and time-dependent manner. The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h (1.25, 2.5, 5 and 10 μg/mL) were 1.78% ± 0.36%, 4.96% ± 1.59%, 10.35% ± 2.76% and 41.6% ± 4.29%, respectively, which showed a significant difference (P < 0.05). The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%, 21.57% ± 3.75%, 30.31% ± 4.91% and 61.19% ± 5.81%, with a significant difference (P < 0.05). The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%, 31.86% ± 3.86%, 48.30% ± 4.16% and 81.80% ± 6.72%, with a significant difference (P < 0.05). Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining. After treatment with oridonin, the cells became round, shrank, and developed small buds around the nuclear membrane while forming apoptotic bodies. Lactate dehydrogenase (LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h, LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4% (P < 0.001). However, the change in the release of LDH caused by necrosis was insignificant, suggesting that the major cause of oridonin-induced HGC-27 cell death was apoptosis. Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls (P < 0.05). And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%, 12.8% ± 2.53%, 28.5% ± 4.23% and 49.6% ± 3.76%, which were in a dose-dependent manner (P < 0.05). After treatment for 24 h, DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dose-dependent manner. RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3 (0.917 ± 0.103 vs 0.357 ± 0.019, P < 0.05), cytochrome c (1.429 ± 0.111 vs 1.002 ± 0.014, P < 0.05), Apaf-1 (0.688 ± 0.101 vs 0.242 ± 0.037, P < 0.05) and Bax (0.856 ± 0.101 vs 0.278 ± 0.027, P < 0.05) (P < 0.05), whereas down-regulated in Bcl-2 (0.085 ± 0.012 vs 0.175 ± 0.030, P < 0.05). Western blotting analysis also confirmed this result.
Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1, caspase-3 and cytochrome c, which are highly dependent upon the mitochondrial pathway.
研究冬凌草甲素对体外培养的胃癌细胞株 HGC-27 的作用及其机制。
采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）比色法检测冬凌草甲素对 HGC-27 细胞的抑制作用。用 10 μg/mL 冬凌草甲素处理细胞 24 h 和 48 h 后，吖啶橙/溴化乙锭染色观察细胞形态变化。DNA 梯状电泳检测 DNA 片段化（凋亡的标志性改变），乳酸脱氢酶（LDH）释放实验检测 LDH 活性。用不同浓度（0、1.25、2.5、5 和 10 μg/mL）冬凌草甲素处理 HGC-27 细胞后，采用 Annexin V-藻红蛋白/7-氨基放线菌素 D 双染法进行流式细胞术分析，检测冬凌草甲素诱导 HGC-27 细胞凋亡情况。用冬凌草甲素处理细胞 24 h 后，采用逆转录聚合酶链反应（RT-PCR）和 Western blot 法检测凋亡相关蛋白天冬氨酸蛋白酶激活因子-1（Apaf-1）、B 细胞淋巴瘤-2（Bcl-2）、Bcl-2 相关 X 蛋白（Bax）、半胱氨酸天冬氨酸蛋白酶-3（caspase-3）和细胞色素 c 的表达。
冬凌草甲素呈时间和剂量依赖性抑制 HGC-27 细胞增殖。用浓度分别为 1.25、2.5、5 和 10 μg/mL 的冬凌草甲素处理 HGC-27 细胞 24 h 的抑制率分别为 1.78%±0.36%、4.96%±1.59%、10.35%±2.76%和 41.6%±4.29%，差异有统计学意义（P<0.05）。用浓度分别为 1.25、2.5、5 和 10 μg/mL 的冬凌草甲素处理 HGC-27 细胞 48 h 的抑制率分别为 14.77%±4.21%、21.57%±3.75%、30.31%±4.91%和 61.19%±5.81%，差异有统计学意义（P<0.05）。用浓度分别为 1.25、2.5、5 和 10 μg/mL 的冬凌草甲素处理 HGC-27 细胞 72 h 的抑制率分别为 25.77%±4.85%、31.86%±3.86%、48.30%±4.16%和 81.80%±6.72%，差异有统计学意义（P<0.05）。吖啶橙/溴化乙锭染色显示，冬凌草甲素处理后的细胞呈典型的凋亡特征，细胞变圆、皱缩，核膜周围出现小芽，形成凋亡小体。LDH 释放实验显示，用 1.25 μg/mL 和 20 μg/mL 冬凌草甲素处理 24 h 后，HGC-27 细胞因凋亡导致的 LDH 释放量由 22.94%±3.8%增加至 52.68%±2.4%（P<0.001）。但坏死引起的 LDH 释放变化不明显，提示冬凌草甲素诱导 HGC-27 细胞死亡的主要原因是凋亡。流式细胞术分析也显示，与对照组相比，冬凌草甲素诱导了明显的细胞凋亡（P<0.05）。4 种不同浓度冬凌草甲素诱导的 HGC-27 细胞凋亡率分别为 5.3%±1.02%、12.8%±2.53%、28.5%±4.23%和 49.6%±3.76%，呈剂量依赖性（P<0.05）。用冬凌草甲素处理 24 h 后，DNA 梯状电泳显示冬凌草甲素诱导 DNA 片段化呈剂量依赖性增加。RT-PCR 显示与对照组相比，caspase-3（0.917±0.103 比 0.357±0.019，P<0.05）、细胞色素 c（1.429±0.111 比 1.002±0.014，P<0.05）、凋亡相关因子-1（Apaf-1）（0.688±0.101 比 0.242±0.037，P<0.05）和 Bax（0.856±0.101 比 0.278±0.027，P<0.05）的 mRNA 表达水平上调，而 Bcl-2（0.085±0.012 比 0.175±0.030，P<0.05）的 mRNA 表达水平下调。Western blot 分析也证实了这一结果。
冬凌草甲素诱导的 HGC-27 细胞凋亡可能与 Apaf-1、caspase-3 和细胞色素 c 的差异表达有关，这些差异表达高度依赖于线粒体途径。
