Regulation of macroautophagy in ovarian cancer cells in vitro and in vivo by controlling glucose regulatory protein 78 and AMPK.
Regulation of macroautophagy in ovarian cancer cells in vitro and in vivo by controlling glucose regulatory protein 78 and AMPK.
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DOI：
10.18632/oncotarget.483
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发表时间：
2012-04
期刊：
影响因子：
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通讯作者：
Srivastava SK
中科院分区：
文献类型：
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作者：
Kandala PK;Srivastava SK
在这项研究中，我们发现二吲哚甲烷 (DIM) 通过调节内质网 (ER) 应激和 AMPK 诱导卵巢癌细胞自噬。通过流式细胞术测量，用不同浓度的 DIM 处理 SKOV-3、OVCAR-3 和 TOV-21G 卵巢癌细胞 24 小时，导致浓度依赖性的自噬诱导。电子显微镜证实 DIM 处理的细胞中存在自噬体。蛋白质印迹分析表明，DIM 处理增加了 LC3B（自噬的标志）以及自噬过程中积累的 p62 和 Atg 12 蛋白的表达。自噬抑制剂巴弗洛霉素或氯喹可抑制 DIM 诱导的自噬。此外，DIM 处理显着增加了 ER 应激调节因子的表达，例如 Grp78、IRE1 和 GADD153。放线菌酮或 ER 应激抑制剂光神霉素不仅可以阻断 DIM 激活的 ER 应激蛋白，还可以阻断自噬。沉默 Grp78 或 GADD 153 显着阻断 LC3B 和 p62 的表达，表明我们模型中的自噬是由 ER 应激介导的。敲除 LC3B 会抑制 DIM 诱导的自噬。 DIM 处理增加了细胞质钙水平，从而导致我们模型中 AMPK 的激活。用 BAPT-AM 螯合胞质钙不仅消除了 AMPK 的磷酸化，而且还阻止了 DIM 诱导的自噬。通过化学抑制剂或 siRNA 抑制 AMPK 会阻断 LC3B 或 p62 的诱导，表明 DIM 介导的自噬需要激活 AMPK。口服 DIM 显着抑制裸鼠体内 SKOV-3 肿瘤异种移植。在 DIM 处理的小鼠肿瘤中观察到 ER 应激和自噬的激活。综上所述，这些结果表明 DIM 在卵巢癌细胞中诱导自噬与 ER 应激和 AMPK 激活相关。
In this study we show that diindolylmethane (DIM) induces autophagy in ovarian cancer cells by regulating endoplasmic reticulum (ER) stress and AMPK. Treatment of SKOV-3, OVCAR-3 and TOV-21G ovarian cancer cells with varying concentrations of DIM for 24 hours resulted in a concentration dependent induction of autophagy as measured by flowcytometry. Electron microscopy confirmed the presence of autophagosomes in DIM treated cells. Western blot analysis showed that DIM treatment increased the expression of LC3B, a hall mark of autophagy as well as p62 and Atg 12 proteins that are accumulated during autophagy. Autophagy inhibitors bafilomycin or chloroquine inhibited DIM induced autophagy. Furthermore, DIM treatment significantly increased the expression of ER stress regulators such as Grp78, IRE1 and GADD153. Cycloheximide or ER stress inhibitor mithramycin not only blocked ER stress proteins that were activated by DIM but also autophagy. Silencing Grp78 or GADD 153 significantly blocked the expression of LC3B and p62 indicating that autophagy in our model is mediated by ER stress. Knocking out LC3B inhibited DIM induced autophagy. DIM treatment increased the cytosolic calcium levels which lead to the activation of AMPK in our model. Chelating cytosolic calcium with BAPT-AM abrogated not only the phosphorylation of AMPK but also prevented DIM induced autophagy. Inhibiting AMPK by a chemical inhibitor or siRNA blocked the induction of LC3B or p62, indicating that DIM mediated autophagy requires activation of AMPK. Oral administration of DIM significantly suppressed SKOV-3 tumor xenografts in nude mice. Activation of ER stress and autophagy were observed in the tumors of DIM treated mice. Taken together, these results suggest that induction of autophagy by DIM in ovarian cancer cells was associated with ER stress and AMPK activation.