Rottlerin induces autophagy which leads to apoptotic cell death through inhibition of PI3K/Akt/mTOR pathway in human pancreatic cancer stem cells.
Multiple lines of evidence support the idea that autophagy plays an essential role in the development of drug resistance, self-renewal, differentiation, and tumorigenic potentials of cancer stem cells (CSCs). Rottlerin (ROT) is widely used as a protein kinase C-delta (PKC-δ) inhibitor. Recent studies revealed that ROT induces apoptosis through engagement of mitochondria. However, it is not known whether ROT-induced apoptosis is associated with other mechanisms such as autophagy. Here we found that ROT induced autophagy followed by induction of apoptosis via inhibition of PI3K/Akt/mTOR pathway and activation of caspase cascade in human pancreatic CSCs. ROT induced a dose- and time-dependent inhibition of cell survival and induction of cytoplasmic vacuolations. The conversion of microtubule-associated protein LC3-I to LC3-II, and increased accumulations of Atg7 and Beclin-1 were also observed in CSCs treated with ROT. Prolonged exposure of CSCs to ROT eventually caused apoptosis which was associated with the suppression of phosphorylated Akt (Ser473) and mTOR (Ser2448), downregulation of XIAP, cIAP-1, Bcl-2 and Bcl-X(L), induction of Bax, activation of caspase-3 and -9, and concomitant degradation of PARP. ROT-induced apoptosis was enhanced by dominant negative AKT, Akt1/2 inhibitor, and rapamycin. Our study also demonstrates that gene silencing of Atg7 and Beclin1, or cotreatment of the autophagosome inhibitor, 3-methyladenine, inhibited ROT-induced autophagy and accelerated ROT-induced apoptosis. The knockdown of PKC-δ did not block ROT-induced autophagy and cell death, suggesting these effects of ROT were exerted through PKC-δ-independent pathway. In summary, our data demonstrate that ROT can induce autophagy which leads to cell death in pancreatic CSCs.
Department of Pharmacology, Toxicology and Therapeutics, and Medicine, The University of Kansas Cancer Center, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.
Brahma N
BN
Singh
Dhruv
D
Kumar
Sharmila
S
Shankar
Rakesh K
RK
Srivastava
D000098:Acetophenones / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics; D001343:Autophagy / Q000187:drug effects; D000071193:Autophagy-Related Protein 7; D000071186:Beclin-1; D001578:Benzopyrans / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D010190:Pancreatic Neoplasms; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000502:physiology*; D044764:Ubiquitin-Activating Enzymes / Q000096:biosynthesis / Q000235:genetics
D000098:Acetophenones; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D000071193:Autophagy-Related Protein 7; D000071186:Beclin-1; D001578:Benzopyrans; D053148:Caspase 3; D053453:Caspase 9; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D058570:TOR Serine-Threonine Kinases; D044764:Ubiquitin-Activating Enzymes; C491997:BECN1 protein, human; C085746:rottlerin; C546842:MTOR protein, human; C509349:ATG7 protein, human