withanolides and Pancreatic-Neoplasms

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

In contrast to normal tissue, cancer cells display profound alterations in protein synthesis and degradation. Therefore, proteins that regulate endoplasmic reticulum (ER) homeostasis are being increasingly recognized as potential therapeutic targets. The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. However, interactions between autophagy, the proteasome, and ER stress pathways in cancer remain largely undefined. This study demonstrated that withaferin-A (WA), the biologically active withanolide extracted from Withania somnifera, significantly increased autophagosomes, but blocked the degradation of autophagic cargo by inhibiting SNARE-mediated fusion of autophagosomes and lysosomes in human pancreatic cancer (PC) cells. WA specifically induced proteasome inhibition and promoted the accumulation of ubiquitinated proteins, which resulted in ER stress-mediated apoptosis. Meanwhile, the impaired autophagy at early stage induced by WA was likely activated in response to ER stress. Importantly, combining WA with a series of ER stress aggravators enhanced apoptosis synergistically. WA was well tolerated in mice, and displayed synergism with ER stress aggravators to inhibit tumor growth in PC xenografts. Taken together, these findings indicate that simultaneous suppression of 2 key intracellular protein degradation systems rendered PC cells vulnerable to ER stress, which may represent an avenue for new therapeutic combinations for this disease.

Topics: Animals; Antineoplastic Agents; Apoptosis; Autophagosomes; Autophagy; Cell Death; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum Stress; Female; Homeostasis; Humans; Lysosomes; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Pancreatic Neoplasms; Plant Extracts; Proteasome Endopeptidase Complex; Proteasome Inhibitors; RNA Interference; Sequestosome-1 Protein; Ubiquitin; Withanolides

Application of oxaliplatin for the treatment of pancreatic cancer (PC) is restricted owing to its toxic side effects and drug resistance. We investigated how withaferin A (WA), a bioactive component isolated from the medicinal plant Withania somnifera, acts synergistically with oxaliplatin on human PC in vitro and in vivo. We found that WA enhanced oxaliplatin-induced growth suppression and apoptosis in PC cells dramatically through a mechanism involving mitochondrial dysfunction and inactivation of the PI3K/AKT pathway. Combination treatment resulted in significant accumulation of intracellular reactive oxygen species (ROS). Pretreatment of cells with the ROS scavenger N-acetylcysteine completely blocked the apoptosis induced by combination treatment, and recovered expression of AKT inactivation, which revealed the important role of ROS in apoptosis and AKT regulation. In vivo, combination therapy showed the strongest anti-tumor effects compared with single agents, without obvious additional toxicity. These results support the notion that combination treatment with oxaliplatin and WA could facilitate development of an effective strategy for PC treatment.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Drug Synergism; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Organoplatinum Compounds; Oxaliplatin; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; Random Allocation; Reactive Oxygen Species; Signal Transduction; Withanolides

Withaferin A (WA), a naturally occurring steroidal lactone, directly binds to Hsp90 and leads to the degradation of Hsp90 client protein. The purpose of this study is to investigate the structure activity relationship (SAR) of withanolides for their inhibition of Hsp90 and anti-proliferative activities in pancreatic cancer cells. In pancreatic cancer Panc-1 cells, withaferin A (WA) and its four analogues withanolide E (WE), 4-hydroxywithanolide E (HWE), 3-aziridinylwithaferin A (AzWA) inhibited cell proliferation with IC50 ranged from 1.0 to 2.8 μM. WA, WE, HWE, and AzWA also induced caspase-3 activity by 21-, 6-, 11- and 15-fold, respectively, in Panc-1 cells, while withaperuvin (WP) did not show any activity. Our data showed that WA, WE, HWE, and AzWA, but not WP, all directly bound to Hsp90 and induced Hsp90 aggregation,hence inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins Akt and Cdk4 through proteasome-dependent pathway in pancreatic cancer cells. However, only WA, HWE and AzWA disrupted Hsp90-Cdc37 complexes but not WE and WP. SAR study suggested that the C-5(6)-epoxy functional group contributes considerably for withanolide to bind to Hsp90, inhibit Hsp90 chaperone activity, and result in Hsp90 client protein depletion. Meanwhile, the hydroxyl group at C-4 of ring A may enhance withanolide to inhibit Hsp90 activity and disrupt Hsp90-Cdc37 interaction. These SAR data provide possible mechanisms of anti-proliferative action of withanolides.

Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Chaperonins; Ergosterol; HSP90 Heat-Shock Proteins; Humans; Pancreatic Neoplasms; Protein Binding; Proteolysis; Structure-Activity Relationship; Withanolides

β-catenin plays a pivotal role in organogenesis and oncogenesis. Alterations in β-catenin expression are common in pancreatic cancer, which is an extremely aggressive malignancy with a notably poor prognosis. In this report, we analyzed the apoptotic activity of withanolide-D (witha-D), a steroidal lactone that was purified from an Indian medicinal plant, Withania somnifera, and its underlying mechanism of action. Witha-D induced apoptosis in pancreatic ductal adenocarcinoma cells by prompting cell-cycle arrest at the G2/M phase. This lactone abrogated β-catenin signaling in these cells regardless of disease grade, mutational status, and gemcitabine sensitivity. Witha-D also upregulated E-cadherin in most cells, thereby supporting the inversion of the epithelial-mesenchymal transition. Furthermore, the Akt/Gsk3β kinase cascade was identified as a critical mediator of G2/M regulation and β-catenin signaling. Witha-D deactivated Akt, which failed to promote Gsk3β deactivation phosphorylation. Consequently, activated Gsk3β facilitated β-catenin destruction in pancreatic carcinoma cells. The knockdown of Chk1 and Chk2 further activated Akt and reversed the molecular signal. Taken together, the results of the current study represent the first evidence of β-catenin signal crosstalk during the G2/M phase by functionally inactivating Akt via witha-D treatment in pancreatic cancer cells. In conclusion, this finding suggests the potential identification of a new lead molecule in the treatment of pancreatic adenocarcinoma.

Topics: Apoptosis; beta Catenin; Cadherins; Carcinoma, Pancreatic Ductal; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; Cell Survival; Checkpoint Kinase 1; Checkpoint Kinase 2; Flow Cytometry; G2 Phase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Pancreatic Neoplasms; Protein Kinases; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA; Withanolides; Wnt Signaling Pathway

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

The purpose of this study is to investigate the efficacy and the mechanism of Hsp90 inhibition of Withaferin A (WA), a steroidal lactone occurring in Withania somnifera, in pancreatic cancer in vitro and in vivo. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro (with IC(50)s of 1.24, 2.93 and 2.78 microM) in pancreatic cancer cell lines Panc-1, MiaPaCa2 and BxPc3, respectively. Annexin V staining showed that WA induced significant apoptosis in Panc-1 cells in a dose-dependent manner. Western blotting demonstrated that WA inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins (Akt, Cdk4 and glucocorticoid receptor), which was reversed by the proteasomal inhibitor, MG132. WA-biotin pull down assay of Hsp90 using Panc-1 cancer cell lysates and purified Hsp90 showed that WA-biotin binds to C-terminus of Hsp90 which was competitively blocked by unlabeled WA. Co-immunoprecipitation exhibited that WA (10 microM) disrupted Hsp90-Cdc37 complexes from 1 to 24h post-treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6mg/kg) inhibited tumor growth in pancreatic Panc-1 xenografts by 30% and 58%, respectively. These data demonstrate that Withaferin A binds Hsp90, inhibits Hsp90 chaperone activity through an ATP-independent mechanism, results in Hsp90 client protein degradation, and exhibits in vivo anticancer activity against pancreatic cancer.

Topics: Animals; Cell Cycle Proteins; Cell Line, Tumor; Chaperonins; Drug Delivery Systems; Ergosterol; Female; HSP90 Heat-Shock Proteins; Humans; Mice; Mice, Nude; Pancreatic Neoplasms; Withanolides; Xenograft Model Antitumor Assays