MAPK通路在红托竹荪多糖诱导人胃癌MKN-45细胞凋亡中的作用机制研究

Mechanism of MAPK Signaling Pathway in Apoptosis of Human Gastric Cancer MKN-45 Cells by Dictyophora Rubrovalvata Polysaccharide

目的:研究红托竹荪多糖对人胃癌MKN-45细胞增殖与凋亡的影响,并基于丝裂原激活的蛋白激酶(MAPK)信号通路探讨其可能的作用机制。方法:体外培养MKN-45细胞,分为对照组,红托竹荪多糖低、中及高剂量组,采用终浓度分别为0、4、6及8 mg/mL的红托竹荪多糖作用于细胞48 h后,采用细胞计数法检测红托竹荪多糖对人胃癌MKN-45细胞增殖的影响;采用倒置显微镜观察不同浓度的红托竹荪多糖作用于人胃癌MKN-45细胞48 h后细胞形态学的变化。设立实验组的红托竹荪多糖终浓度为8 mg/mL,采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶双染色流式细胞技术检测细胞的凋亡率;采用流式细胞术检测细胞周期分布;采用蛋白质印迹法检测红托竹荪多糖作用胃癌细胞后p38蛋白激酶(p38)、磷酸化p38蛋白激酶(p-p38)、细胞外调节蛋白激酶(ERK)、磷酸化细胞外调节蛋白激酶(p-ERK)、C-JUN氨基末端激酶(JNK)、磷酸化C-JUN氨基末端激酶(p-JNK)、B淋巴细胞瘤-2蛋白(Bcl-2)和活化的半胱氨酸蛋白酶-3(cleaved Caspase-3)蛋白的表达,即与3-磷酸甘油醛脱氢酶(GADPH)灰度值比值。结果:作用48 h后,不同浓度的红托竹荪多糖(4、6及8 mg/mL)对人胃癌MKN-45细胞增殖的抑制率分别为(8.27±5.99)%、(21.83±5.77)%和(56.95±5.21)%;在检测的浓度范围下,随着浓度的升高,红托竹荪多糖对MKN-45细胞增殖的抑制率逐渐升高。倒置显微镜下显示,经8 mg/mL的红托竹荪多糖作用48 h后,MKN-45细胞密度与对照组相比明显减少。流式细胞术检测结果显示,与对照组相比,实验组人胃癌MKN-45细胞凋亡率显著升高(P<0.05);细胞周期结果显示,实验组G_(1)期细胞比例明显减少(P<0.05),G_(2)期细胞比例明显增多(P<0.05),S期细胞比例明显减少(P<0.05);蛋白质印迹法结果显示,与对照组相比,实验组MKN-45细胞中cleaved Caspase-3的表达有升高趋势(P<0.05),Bcl-2的表达有降低趋势(P<0.05),p-P38/P38比值、p-ERK/ERK比值与p-JNK/JNK比值均低于对照组(P<0.05),上述差异均有统计学意义。结论:红托竹荪多糖在体外能抑制人胃癌MKN-45细胞增殖并诱导细胞凋亡,使肿瘤细胞阻滞在G_(2)/M期,其作用可能通过调控MAPK信号通路实现。

OBJECTIVE:To study the effect of dictyophora rubrovalvata polysaccharide on proliferation and apoptosis of human gastric cancer MKN-45 cells,and probe into the possible mechanism based on mitogen activated protein kinase(MAPK)signaling pathway.METHODS:MKN-45 cells were cultured in vitro,and divided into the control group,low-dose,medium-dose and high-dose dictyophora rubrovalvata polysaccharide group.The effect of dictyophora rubrovalvata polysaccharide with final concentrations of 0 mg/mL,4 mg/mL,6 mg/mL and 8 mg/mL on the proliferation of human gastric cancer MKN-45 cells was detected by cell counting(CCK-8)after 48 h of treatment.The morphological changes of MKN-45 cells were observed under inverted microscope after being treated with different concentrations of dictyophora rubrovalvata polysaccharide for 48 h.In the experimental group,the final concentration of polysaccharide was 8 mg/mL.Apoptosis rate was detected by Annexin V-FITC/PI double staining flow cytometry.Cell cycle was detected by flow cytometry.Western blotting was used to detect the expression of p38 protein kinase(p38),phosphorylated p38 protein kinase(p-p38),extracellular regulated protein kinase(ERK),phosphorylated extracellular regulated protein kinase(p-ERK),C-JUN N-terminal kinase(JNK),phosphorylated C-JUN N-terminal kinase(p-JNK),B-lymphomatoma-2 protein(Bcl-2)and the activated cysteine proteinase-3(cleaved Caspase-3)protein,by the ratio of gray value to glyceraldehyde 3-phosphate dehydrogenase(GADPH).RESULTS:After 48 h of treatment,the inhibition rates of dictyophora rubrovalvata polysaccharide under different concentration(4 mg/mL,6 mg/mL and 8 mg/mL)on the proliferation of human gastric cancer MKN-45 cells were(8.27±5.99)%,(21.83±5.77)%and(56.95±5.21)%,respectively.In the detected concentration range,the inhibition rates of dictyophora rubrovalvata polysaccharide on MKN-45 cell proliferation increased with the increase of concentration.Under the inverted microscope,the MKN-45 cell density decreased significantly compared with the control group after being treated with 8 mg/mL dictyophora rubrovalvata polysaccharide for 48 h.Results of flow cytometry showed that compared with the control group,the apoptosis rate of MKN-45 cells in the experimental group increased significantly(P<0.05);results of cell cycle showed that the proportion of cells in G 1 phase and S phase in cells in G_(1) in the experimental group decreased significantly(P<0.05,P<0.05),and the proportion of cells in G_(2) phase cells in G_(2) phase increased significantly(P<0.05);,and cells in S phase decreased significantly(P<0.05).Western blotting results showed that compared with the control group,the cleaved Caspase-3 in MKN-45 cells in the experimental group increased(P<0.05),Bcl-2 tended to decreased(P<0.05),p-P38/P38 ratio,p-ERK/ERK ratio and p-JNK/JNK ratio in the control group were lower than those in the control group(P<0.05),with statistically significant differences.CONCLUSIONS:Dictyophora rubrovalvata polysaccharide can inhibit the proliferation and induce apoptosis of human gastric cancer MKN-45 cells in vitro,and block tumor cells in G_(2)/M phase,which may be achieved by regulating MAPK signaling pathway.