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栀子苷对结直肠癌细胞增殖和凋亡及Akt/ MDM2/p53通路的影响
The effect of geniposide on the proliferation and apoptosis of CRC cells and Akt/MDM2/p53 pathway
投稿时间:2021-07-28  修订日期:2021-11-30
DOI:
中文关键词:  栀子苷  Akt  增殖  凋亡  结直肠癌
英文关键词:Geniposide  Akt  Proliferation  Apoptosis  Colorectal cancer
基金项目:
作者单位邮编
于雅勤 浙江省荣军医院 314000
沈加熙 浙江省荣军医院药剂科 
张虹 浙江省荣军医院药剂科 
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中文摘要:
      目的 研究栀子苷(GEN)对结直肠癌(CRC)细胞的增殖和凋亡的影响,并探讨其潜在机制。方法 使用MTT测定不同浓度(0-200 μM)GEN对不同CRC细胞的抗增殖作用,筛选后续实验细胞及药物浓度。SW480细胞经GEN处理后,流式细胞术检测细胞凋亡及周期,采用蛋白质印迹法检测凋亡和周期相关蛋白和Akt/MDM2/p53的蛋白表达。建立稳定的AKT基因敲低细胞,MTT测定细胞增殖,免疫荧光法检测MDM2/p53表达。结果 GEN作用24?h和48?h后,CRC细胞增殖受到抑制,呈时间和剂量依赖性(P<0.05),而在该浓度范围内对正常细胞无显著毒性。与0 μM GEN处理组相比,GEN (5、100、150 μM)处理48h后,凋亡细胞百分率[(22.19±0.55)%、(48.57±0.57) %、(52.45±1.29)%比(10.44±0.48)%]、凋亡相关蛋白Bax/Bcl-2蛋白表达[(1.31±0.05)、(3.36±0.07)、(5.34±0.13)比(1.02±0.06)]显著增加,细胞周期阻滞于G2/M期[(15.66±0.59)%、(17.81±0.76)%、(19.65±0.89)%比(13.48±0.3)%],细胞周期蛋白cyclin B1表达[(0.89±0.07)、(0.77±0.08)、(0.42±0.11)比(1.07±0.08)]下降,且呈剂量依赖性(P<0.05)。此外,GEN下调了Akt[(0.84±0.04)、(0.67±0.04)、(0.59±0.09)比(1.00±0.02)]和MDM2蛋白[ (0.64±0.06)、(0.45±0.08)、(0.27±0.06)比(1.01±0.02)]表达,并激活p53蛋白表达[ (1.28±0.04)、(1.48±0.06)、(1.81±0.10)比(1.02±0.03)],而Akt敲低后细胞OD 值在48 h[(0.24±0.04)比(0.47±0.03)]和72 h[(0.28±0.02)比(0.63±0.04)]下降,GEN处理无法进一步抑制shRNA-AKT1/2细胞中的增殖能力,Akt敲低后MDM2被抑制,而p53被激活。结论 GEN抑制CRC细胞增殖和诱导G2/M期阻滞和凋亡,其作用机制可能通过AKT/MDM2/p53途径。
英文摘要:
      Objective To study the effects of geniposide (GEN) on the proliferation and apoptosis of colorectal cancer (CRC) cells, and explore its underlying mechanism. Methods MTT was used to determine the anti-proliferative effect of different concentrations (0-200 μM) of GEN on different CRC cells, and to screen subsequent experimental cells and drug concentrations. After SW480 cells were treated with GEN, flow cytometry was used to detect cell apoptosis and cycle. Western blotting was used to detect the protein expression of apoptosis and cycle-related proteins and Akt/MDM2/p53. Establish stable AKT gene knockdown cells, MTT assay cell proliferation, immunofluorescence assay MDM2/p53 expression. Results After 24?h and 48?h of GEN, the proliferation of CRC cells was inhibited in a time and dose-dependent manner (P<0.05), and there was no significant toxicity to normal cells within this concentration range. Compared with the 0 μM GEN treatment group, after GEN (5, 100, 150 μM) treatment for 48 hours, the percentage of apoptotic cells[(22.19±0.55)%, (48.57±0.57)%, (52.45±1.29)% vs (10.44±0.48)%] and the expression of apoptosis-related protein Bax/Bcl-2 protein [(1.31±0.05), (3.36±0.07), (5.34±0.13) vs (1.02±0.06)] increased significantly, and the cell cycle was blocked in G2/M phase[(15.66±0.59)%, (17.81±0.76)%, (19.65±0.89)% vs (13.48±0.3)%], The expression of cyclin B1[(0.89±0.07), (0.77±0.08), (0.42±0.11) vs (1.07±0.08)] decreased in a dose-dependent manner (P<0.05). In addition, GEN down-regulated Akt[(0.84±0.04), (0.67±0.04), (0.59±0.09) vs (1.00±0.02)] and MDM2[(0.64±0.06), (0.45±0.08), (0.27±0.06) vs (1.01±0.02)] protein expression, and activated p53 protein expression[ (1.28±0.04), (1.48±0.06), (1.81±0.10) vs (1.02±0.03)], while Akt knockdown, the cell OD value decreased at 48 h [(0.24±0.04) vs (0.47±0.03)] and 72 h [(0.28±0.02) vs (0.63±0.04)], and GEN treatment could not further inhibit the proliferation ability of shRNA-AKT1/2 cells. MDM2 was inhibited after Akt knockdown, while p53 was activated. Conclusion GEN inhibits the proliferation of CRC cells and induces G2/M phase arrest and apoptosis, and its mechanism of action may be through the AKT/MDM2/p53 pathway.
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