withaferin-a and Carcinoma--Pancreatic-Ductal

Receptor tyrosine kinases (RTKs) are pharmaceutically attractive targets due to their fundamental role in tumor formation. The hallmark of pancreatic cancer is its high mortality rate attributed to the existence of cancer stem cell (CSC) subpopulations which result in therapy resistance and recurrence. c-Met is a known pancreatic CSC marker that belongs to the family of RTKs. To surmount the hurdles related to ligand-independent c-Met activation, we aimed to elucidate the inhibitory mechanisms of withaferin A (WA) and carnosol (CA) as two hit phytochemicals against c-Met kinase domain. Both tested compounds attenuated HGF-mediated proliferation across various established c-Met

Topics: Abietanes; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Cycle Checkpoints; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Mice; Neoplastic Stem Cells; NIH 3T3 Cells; Pancreatic Neoplasms; Phenotype; Phosphorylation; Phytochemicals; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Signal Transduction; Withanolides

The stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity, migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductal adenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little is known about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rate and reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestin significantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 status in PDAC cells; hence, nestin expression might be regulated by the TGF-β1/Smad4 pathway in PDAC. We examined nestin expression after TGF-β1 treatment in human pancreatic cancer PANC-1 and PANC-1 shSmad4 cells. The TGF-β1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4-dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-β1 signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxia-induced expression of nestin is mediated by the TGF-β1/Smad4 pathway. Finally, the antimicrotubule inhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potential antimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-β1-induced EMT. Anti-nestin therapeutics may serve as a potential treatment for PDAC metastasis.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Movement; Cytochalasin D; Disease Progression; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Humans; Male; Mice; Mice, SCID; Microtubules; Neoplasm Metastasis; Nestin; Pancreas; Pancreatic Neoplasms; Signal Transduction; Smad4 Protein; Stem Cells; Transforming Growth Factor beta1; Up-Regulation; Withanolides; Xenograft Model Antitumor Assays

Withaferin A (WA) (1) and two analogs [4-epi-withaferin A (2) and 4,27-diacetyl-4-epi-withaferin A (3)] were evaluated for antitumor activity in pancreatic cancer cells. IC(50) for 1, 2, and 3 were 0.87, 0.45, and 0.29 ?M (BxPC-3); 1.28, 1.53, and 0.52 ?M (MIAPaCa-2); and 0.59, 2.25, and 0.56 ?M (PANC-1), respectively. We chose WA analog 3 for functional studies with confirmatory RT-PCR and Western blotting. ANOVA identified 33 (MIAPaCa-2), 54 (PANC-1), and 48 (BxPC-3) gene expression changes. Fisher exact test demonstrated MAPK and glutathione pathways to be overexpressed with WA analog 3. WA analog 3 elicits a dose- and time-dependent apoptosis, activates MAPK and glutathione ?stress? pathways, and inhibits proliferation.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Gene Expression Profiling; Glutathione; Humans; Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Polymerase Chain Reaction; Withanolides