| 曾 舒,杜维民,叶珂帆,等.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[J].肿瘤学杂志,2024,30(7):586-598. |
| 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 |
| 投稿时间：2024-02-01 |
| DOI：10.11735/j.issn.1671-170X.2024.07.B009 |
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| 中文关键词: 大麻二酚 子宫内膜癌 内质网应激 活性氧 |
| 英文关键词:cannabidiol endometrial cancer endoplasmic reticulum stress reactive oxygen species |
| 基金项目:国家级妇科重点专科云南省妇产疾病临床研究中心开放课题（2022YJZX-FC09） |
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| 中文摘要: |
| 摘 要：［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 ～16 μmol/L时显著性抑制Ishikawa细胞的增殖能力（P＜0.05），CBD在浓度为6～16 μmol/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=0.007 4，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 RNA-like 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-eIF2α-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。 |
| 英文摘要: |
| Abstract： ［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 16 μM (P<0.05) and KLE cell proliferation at concentrations ranging from 6 to 16 μM (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=0.007 4， 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. |
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