大麻二酚通过PERK-eIF2α-ATF4信号通路调节子宫内膜癌细胞的作用机制

Cannabidiol Regulates Biological Behaviors of Endometrial Cancer Cells Through PERK-eIF2α-ATF4 Signaling Pathway

[目的]探究大麻二酚(cannabidiol,CBD)对子宫内膜癌细胞生物学行为具体作用及可能机制。[方法]将Ishikawa和KLE细胞分为对照组、二甲基亚砜(dimethyl sulfoxide,DMSO)组、CBD+DMSO组及CBD+DMSO+GSK2606414(蛋白激酶R样内质网激酶抑制剂)组,通过CCK-8实验、细胞划痕愈合实验、Transwell实验、流式细胞术等实验检测CBD对子宫内膜癌细胞增殖、迁移、侵袭及凋亡的影响,通过活性氧(reactive oxygen species,ROS)探针检测细胞内ROS的水平,并通过蛋白质印迹分析(Western blot)检测子宫内膜癌细胞中C/EBP同源蛋白(CCAAT-enhancer-binding protein homologous protein,CHOP)蛋白、PERK-真核翻译起始因子2α(eukaryotic translation initiation factor 2α,eIF2α)、转录活化因子4(activating transcription factor 4,ATF4)信号通路相关蛋白的表达,并使用PERK信号通路抑制剂GSK2606414对通路进行验证。[结果]CBD呈浓度及时间依赖性抑制Ishikawa和KLE细胞的增殖能力,CBD在浓度为2~16μmol/L时显著性抑制Ishikawa细胞的增殖能力(P<0.05),CBD在浓度为6~16μmol/L时显著性抑制KLE细胞的增殖能力(P<0.05)。在CBD干预Ishikawa和KLE细胞≥24 h后显著性抑制Ishikawa及KLE细胞的增殖能力(P均<0.05)。CBD抑制Ishikawa和KLE细胞的细胞迁移能力、侵袭能力及诱导细胞凋亡(P均<0.05)。Ishikawa细胞CBD+DMSO组24 h愈合率(37.54%±0.97%),48 h愈合率(47.87%±1.46%);CBD+DMSO组24 h KLE细胞愈合率(41.93%±2.85%),48 h愈合率(51.29%±0.75%)。CBD提高细胞内ROS水平(Ishikawa P=0.0074,KLE P<0.001)。CBD作用于Ishikawa和KLE细胞后,CHOP、磷酸化蛋白激酶R样内质网激酶(phosphorylated protein kinase RNAlike endoplasmic reticulum kinase,p-PERK)、磷酸化真核翻译起始因子2α(phosphorylated eukaryotic translation initiation factor 2α,p-eIF2α)、ATF4表达量较对照组及DMSO组明显增加(P<0.001)。Ishikawa和KLE细胞在加入GSK2606414后p-PERK(P均<0.001)、p-eIF2α(P<0.001)、ATF4(P<0.001)表达量较CBD+DMSO组下调,但仍高于对照组及DMSO组。[结论]在子宫内膜癌细胞中,CBD通过激活PERK-e IF2α-ATF4信号通路抑制细胞增殖、迁移、侵袭能力,诱导细胞凋亡,从而发挥抗肿瘤作用,其抗肿瘤作用可能与内质网应激的激活及ROS积累有关。

[Objective]To investigate the effect and mechanism of cannabidiol(CBD)on biological behaviors of endometrial cancer cells.[Methods]Endometrial cancer Ishikawa and KLE cells were divided into control group,dimethyl sulfoxide(DMSO)group,CBD+DMSO group,and CBD+DMSO+GSK2606414[a protein kinase RNA-like endoplasmic reticulum kinase(PERK)inhibitor]group.The effects of CBD on proliferation,migration,invasion and apoptosis of endometrial cancer cells were evaluated by CCK-8 assay,scratch healing assay,Transwell assay and flow cytometry,respectively.The level of reactive oxygen species(ROS)in cells was measured with ROS probes.The expression of CCAAT-enhancer-binding protein homologous protein(CHOP)and PERK-eukaryotic translation initiation factor 2α-activating transcription factor 4(eIF2α-ATF4)signaling pathway-related proteins in endometrial cancer cells was detected with Western blot.The PERK signaling pathway inhibitor GSK2606414 was used to validate the pathway.[Results]Cannabidiol exhibited concentration-and time-dependent inhibition of proliferation of Ishikawa and KLE cells.Cannabidiol significantly inhibited Ishikawa cell proliferation at concentrations ranging from 2 to 16μM(P<0.05)and KLE cell proliferation at concentrations ranging from 6 to 16μM(P<0.05).After Cannabidiol intervention for≥24 h,there was a significant inhibition of proliferation in both Ishikawa and KLE cell(P<0.05).Cannabidiol also inhibited the migration,invasion,and induced apoptosis of Ishikawa and KLE cell(P all<0.05).The 24 h healing rate of Ishikawa cell in the CBD+DMSO group was 37.54%±0.97%,and the 48 h healing rate was 47.87%±1.46%,for KLE cells,the 24 h healing rate was 41.93%±2.85%,and the 48h healing rate was 51.29%±0.75%.Cannabidiol increased intracellular ROS levels(Ishikawa P=0.0074,KLE P<0.001).Following Cannabidiol treatment in Ishikawa and KLE cell,the expression levels of CHOP,phosphorylated PERK(p-PERK),phosphorylated elF2α(p-eIF2α),and ATF4 were significantly increased compared to the control and DMSO groups(P<0.001).After the addition of GSK2606414,the expression levels of p-PERK(P<0.001),p-eIF2α(P<0.001),and ATF4(P<0.001)were downregulated compared to the CBD+DMSO group but was higher than the control and DMSO groups.[Conclusion]In endometrial cancer cells,cannabidiol inhibits cell proliferation,migration,and invasion capabilities,induces apoptosis,and thus exerts anti-tumor effects by activating the PERK-eIF2α-ATF4 signaling pathway.Its anti-tumor effects may be associated with the activation of endoplasmic reticulum stress and the accumulation of ROS.