丁香水提物对胰腺癌细胞增殖、迁移和侵袭的影响

Effects of Aqueous Extract of Clove on Proliferation, Migration and Invasion of Pancreatic Cancer Cells

关键词:  丁香水提物  胰腺癌  细胞增殖  迁移  侵袭

Key words:  aqueous extract of clove  pancreatic cancer  cell proliferation  migration  invasion

摘要:

目的 研究丁香水提物(aqueous extract of clove，AEC)对人胰腺癌细胞Panc-1和Panc-28增殖抑制和凋亡的影响，初步明确其分子机制。方法 Panc-1、Panc-28细胞经AEC作用后，用MTT法检测细胞增殖情况；流式细胞术检测细胞凋亡情况；克隆形成检测细胞群体依赖性情况；Transwell试验检测细胞迁移和侵袭的情况；Western blotting分析cleaved-PARP、Bax、Bcl-2和E-cadherin蛋白表达情况。结果 MTT试验结果显示，与对照组相比，在给药72 h时25 μg·mL^–1 AEC就能抑制Panc-1、Panc-28细胞的增殖，抑制率分别为(16.21±3.57)%和(22.44±4.92)%(P<0.01)。流式细胞术检测结果表明，与对照组相比，200 μg·mL^–1 AEC作用Panc-1、Panc-28细胞24 h显著诱导细胞凋亡，早期凋亡率分别为(11.52±0.34)%和(30.88±0.73)%(P<0.01)；晚期凋亡率分别为(23.59±1.04)%和(13.27±0.85)%(P<0.01)。克隆形成试验结果显示，50 μg·mL^–1AEC能显著抑制Panc-1、Panc-28细胞的集落形成(P<0.01)。Transwell试验结果表明100 μg·mL^–1AEC处理Panc-1、Panc-28细胞48 h后，迁移数目分别由对照组(275±7)个减少到(165±15)个，(227±25)个减少到(124±16)个(P<0.01)，侵袭数目分别由对照组(179±19)个减少到(106±7)个，(132±7)个减少到(61±5)个(P<0.01)。Western blotting试验结果表明，100 μg·mL^–1 AEC能显著诱导Panc-1、Panc-28细胞中cleaved-PARP(P<0.01)、E-Cadherin的蛋白表达(P<0.05)，提高Bax/Bcl-2比值(P<0.01)。结论 AEC能诱导Panc-1、Panc-28细胞凋亡，并抑制其增殖、克隆形成、迁移和侵袭的能力，其作用机制可能与细胞内E-cadherin蛋白表达升高及Bax/Bcl-2比值增加有关。

Abstract:

OBJECTIVE To investigate the effects of aqueous extract of clove(AEC) on proliferation and apoptosis of the human pancreatic cancer cells Panc-1 and Panc-28, and preliminarily clear the molecular mechanisms. METHODS MTT assay was used to detect the proliferation of Panc-1 and Panc-28 cells treated with AEC respectively, and flow cytometry was used to detect cells apoptosis, and clonal test was used to detect cells colony formation rate, and transwell assay was performed to detect cells migration and invasion. The level expression of cleaved-PARP, Bax, Bcl-2, E-cadherin protein were determined by Western blotting. RESULTS The results of MTT experiments revealed that 25 μg·mL^-1 of AEC was able to inhibit Panc-1 and Panc-28 cells growth compared with the control group at 72 h, and the inhibition rate reached (16.21±3.57)% and (22.44±4.92)% (P<0.01), respectively. The flow cytometry results indicated 200 μg·mL^-1 of AEC significantly induced Panc-1 and Panc-28 cells apoptosis compared with the control group at 24 h; the early apoptotic rates were (11.52±0.34)% and (30.88±0.73)%(P<0.01); the late apoptotic rates were (23.59±1.04)% and (13.27±0.85)%(P<0.01) respectively. The experiment of clonal formation ability showed 50 μg·mL^-1AEC could inhibit the formation of Panc-1 and Panc-28 cells colonies(P<0.01). Transwell assay revealed Panc-1 and Panc-28 cells were treated with 100 μg·mL^-1AEC for 48 h, respectively, the migration cells number decreased from 275±7 to 165±15, and 227±25 to 124±16(P<0.01); the invasion cell number decreased from 179±19 to 106±7 and 132±7 to 61±5 (P<0.01). Western blotting results confirmed that 100 μg·mL^-1 AEC up-regulated the expression of cleaved-PARP(P<0.01) and E-cadherin(P<0.05), and increased the ratio of Bax/Bcl-2(P<0.01) in Panc-1 and Panc-28 cells. CONCLUSION AEC can induce the apoptosis of Panc-1 and Panc-28 cells, and inhibit their proliferation, clone formation, migration and invasion, speculating that its mechanism may be related to the upregulation of E-Cadherin expression and increasing of ratio of Bax/Bcl-2.