title: Regulation of macroautophagy in ovarian cancer cells in vitro and in vivo by controlling glucose regulatory protein 78 and AMPK.
abstract: 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.
authors.affiliation: Department of Biomedical Sciences and Cancer Biology Center, Texas Tech University Health Sciences Center, Amarillo, TX, USA.
authors.forename: Prabodh K
authors.initials: PK
authors.lastname: Kandala
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Srivastava
mesh_terms: D000091162:AMP-Activated Protein Kinase Kinases; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D001343:Autophagy; D000071192:Autophagy-Related Protein 12; D020013:Calcium Signaling; D002277:Carcinoma / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress; D004722:Endoribonucleases; D005260:Female; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000378:metabolism* / Q000473:pathology; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering / Q000235:genetics; D000071456:Sequestosome-1 Protein; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism
chemical_list: C466624:ATG12 protein, human; D048868:Adaptor Proteins, Signal Transducing; D000071192:Autophagy-Related Protein 12; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D007211:Indoles; C502070:MAP1LC3B protein, human; D018942:Macrolides; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D025841:Small Ubiquitin-Related Modifier Proteins; C057620:bafilomycin A; D051742:Transcription Factor CHOP; D002738:Chloroquine; D011494:Protein Kinases; C403982:ERN2 protein, human; D017346:Protein Serine-Threonine Kinases; D000091162:AMP-Activated Protein Kinase Kinases; D004722:Endoribonucleases; C016392:3,3'-diindolylmethane