Triptolide induces cell death in pancreatic cancer cells by apoptotic and autophagic pathways.
OBJECTIVE
Pancreatic adenocarcinoma, among the most lethal human malignancies, is resistant to current chemotherapies. We previously showed that triptolide inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. This study investigates the mechanism by which triptolide kills pancreatic cancer cells.


METHODS
Cells were treated with triptolide and viability and caspase-3 activity were measured using colorimetric assays. Annexin V, propidium iodide, and acridine orange staining were measured by flow cytometry. Immunofluorescence was used to monitor the localization of cytochrome c and Light Chain 3 (LC3) proteins. Caspase-3, Atg5, and Beclin1 levels were down-regulated by exposing cells to their respective short interfering RNA.


RESULTS
We show that triptolide induces apoptosis in MiaPaCa-2, Capan-1, and BxPC-3 cells and induces autophagy in S2-013, S2-VP10, and Hs766T cells. Triptolide-induced autophagy has a pro-death effect, requires autophagy-specific genes, atg5 or beclin1, and is associated with the inactivation of the Protein kinase B (Akt)/mammalian target of Rapamycin/p70S6K pathway and the up-regulation of the Extracellular Signal-Related Kinase (ERK)1/2 pathway. Inhibition of autophagy in S2-013 and S2-VP10 cells results in cell death via the apoptotic pathway whereas inhibition of both autophagy and apoptosis rescues cell death.


CONCLUSIONS
This study shows that triptolide kills pancreatic cancer cells by 2 different pathways. It induces caspase-dependent apoptotic death in MiaPaCa-2, Capan-1, and BxPC-3, and induces caspase-independent autophagic death in metastatic cell lines S2-013, S2-VP10, and Hs766T, thereby making it an attractive chemotherapeutic agent against a broad spectrum of pancreatic cancers.
Mujumdar
Nameeta
N
Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, Minnesota, USA.
Mackenzie
Tiffany N
TN
Dudeja
Vikas
V
Chugh
Rohit
R
Antonoff
Mara B
MB
Borja-Cacho
Daniel
D
Sangwan
Veena
V
Dawra
Rajinder
R
Vickers
Selwyn M
SM
Saluja
Ashok K
AK
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004224:Diterpenes / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004852:Epoxy Compounds / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000473:pathology*; D010616:Phenanthrenes / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases; D013997:Time Factors
C502778:ATG5 protein, human; D018906:Antineoplastic Agents, Alkylating; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; C491997:BECN1 protein, human; D000071186:Beclin-1; D004224:Diterpenes; D004852:Epoxy Compounds; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D010616:Phenanthrenes; C001899:triptolide; C546842:MTOR protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C505407:CASP3 protein, human; D053148:Caspase 3