rottlerin and Pancreatic-Neoplasms

Pancreatic cancer (PaCa) is one of the most aggressive, apoptosis-resistant and currently incurable cancers with a poor survival rate. Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase, whose role in PaCa survival is not yet known. Here, we show that eEF-2K is overexpressed in PaCa cells and its down-regulation induces apoptotic cell death. Rottlerin (ROT), a polyphenolic compound initially identified as a PKC-δ inhibitor, induces apoptosis and autophagy in a variety of cancer cells including PaCa cells. We demonstrated that ROT induces intrinsic apoptosis, with dissipation of mitochondrial membrane potential (ΔΨm), and stimulates extrinsic apoptosis with concomitant induction of TNF-related apoptosis inducing ligand (TRAIL) receptors, DR4 and DR5, with caspase-8 activation, in PANC-1 and MIAPaCa-2 cells. Notably, while none of these effects were dependent on PKC-δ inhibition, ROT down-regulates eEF-2K at mRNA level, and induce eEF-2K protein degradation through ubiquitin-proteasome pathway. Down-regulation of eEF-2K recapitulates the events observed after ROT treatment, while its over-expression suppressed the ROT-induced apoptosis. Furthermore, eEF-2K regulates the expression of tissue transglutaminase (TG2), an enzyme previously implicated in proliferation, drug resistance and survival of cancer cells. Inhibition of eEF-2K/TG2 axis leads to caspase-independent apoptosis which is associated with induction of apoptosis-inducing factor (AIF). Collectively, these results indicate, for the first time, that the down-regulation of eEF-2K leads to induction of intrinsic, extrinsic as well as AIF-dependent apoptosis in PaCa cells, suggesting that eEF-2K may represent an attractive therapeutic target for the future anticancer agents in PaCa.

Topics: Acetophenones; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Benzopyrans; Cell Line, Tumor; Elongation Factor 2 Kinase; Humans; Pancreatic Neoplasms; Signal Transduction

The purpose of the study was to examine the molecular mechanisms by which rottlerin inhibited growth of human pancreatic tumors in Balb C nude mice, and pancreatic cancer cells isolated from Kras(G12D) mice. AsPC-1 cells were injected subcutaneously into Balb c nude mice, and tumor-bearing mice were treated with rottlerin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of components of Akt, Notch, and Sonic Hedgehog (Shh) pathways were measured by the immunohistochemistry, Western blot analysis, and/or q-RT-PCR. The effects of rottlerin on pancreatic cancer cells isolated from Kras(G12D) mice were also examined. Rottlerin-treated mice showed a significant inhibition in tumor growth which was associated with suppression of cell proliferation, activation of capase-3 and cleavage of PARP. Rottlerin inhibited the expression of Bcl-2, cyclin D1, CDK2 and CDK6, and induced the expression of Bax in tumor tissues compared to untreated control. Rottlerin inhibited the markers of angiogenesis (Cox-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9), thus blocking production of tumorigenic mediators in tumor microenvironment. Rottlerin also inhibited epithelial-mesenchymal transition by up-regulating E-cadherin and inhibiting the expression of Slug and Snail. Furthermore, rottlerin treatment of xenografted tumors or pancreatic cancer cells isolated from Kras(G12D) mice showed a significant inhibition in Akt, Shh and Notch pathways compared to control groups. These data suggest that rottlerin can inhibit pancreatic cancer growth by suppressing multiple signaling pathways which are constitutively active in pancreatic cancer. Taken together, our data show that the rottlerin induces apoptosis and inhibits pancreatic cancer growth by targeting Akt, Notch and Shh signaling pathways, and provide a new therapeutic approach with translational potential for humans.

Topics: Acetophenones; Angiogenesis Inhibitors; Angiogenic Proteins; Animals; Apoptosis; Apoptosis Regulatory Proteins; Benzopyrans; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Genes, ras; Hedgehog Proteins; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, Transgenic; Neovascularization, Pathologic; Pancreatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Notch; Signal Transduction; Time Factors; Tumor Burden; Tumor Microenvironment; Xenograft Model Antitumor Assays

Pancreatic ductal adenocarcinoma is one of the lethal cancers with extensive local tumour invasion, metastasis, early systemic dissemination and poorest prognosis. Thus, understanding the mechanisms regulating invasion/metastasis and epithelial-mesenchymal transition (EMT), is the key for developing effective therapeutic strategies for pancreatic cancer (PaCa). Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase that we found to be highly up-regulated in PaCa cells. However, its role in PaCa invasion/progression remains unknown. Here, we investigated the role of eEF-2K in cellular invasion, and we found that down-regulation of eEF-2K, by siRNA or rottlerin, displays impairment of PaCa cells invasion/migration, with significant decreases in the expression of tissue transglutaminase (TG2), the multifunctional enzyme implicated in regulation of cell attachment, motility and survival. These events were associated with reductions in β1 integrin/uPAR/MMP-2 expressions as well as decrease in Src activity. Furthermore, inhibition of eEF-2K/TG2 axis suppresses the EMT, as demonstrated by the modulation of the zinc finger transcription factors, ZEB1/Snail, and the tight junction proteins, claudins. Importantly, while eEF-2K silencing recapitulates the rottlerin-induced inhibition of invasion and correlated events, eEF-2K overexpression, by lentivirus-based expression system, suppresses such rottlerin effects and potentiates PaCa cells invasion/migration capability. Collectively, our results show, for the first time, that eEF-2K is involved in regulation of the invasive phenotype of PaCa cells through promoting a new signalling pathway, which is mediated by TG2/β1 integrin/Src/uPAR/MMP-2, and the induction of EMT biomarkers which enhance cancer cell motility and metastatic potential. Thus, eEF-2K could represent a novel potential therapeutic target in pancreatic cancer.

Topics: Acetophenones; Benzopyrans; Cell Line, Tumor; Elongation Factor 2 Kinase; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; Integrin beta1; Neoplasm Invasiveness; Pancreatic Neoplasms; Protein Glutamine gamma Glutamyltransferase 2; Receptors, Urokinase Plasminogen Activator; RNA, Small Interfering; Signal Transduction; src-Family Kinases; Transglutaminases

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.

Topics: Acetophenones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 7; Beclin-1; Benzopyrans; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Enzyme Activation; Humans; Membrane Proteins; Neoplastic Stem Cells; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Signal Transduction; TOR Serine-Threonine Kinases; Ubiquitin-Activating Enzymes

Rottlerin is a polyphenolic compound derived from Mallotus philipinensis. In the present study, we show that rottlerin decreased tumor size and stimulated apoptosis in an orthotopic model of pancreatic cancer with no effect on normal tissues in vivo. Rottlerin also induced apoptosis in pancreatic cancer (PaCa) cell lines by interacting with mitochondria and stimulating cytochrome c release. Immunoprecipitation results indicated that rottlerin disrupts complexes of prosurvival Bcl-xL with Bim and Puma. Furthermore, siRNA knockdown showed that Bim and Puma are necessary for rottlerin to stimulate apoptosis. We also showed that rottlerin and Bcl-2 and Bcl-xL inhibitor BH3I-2' stimulate apoptosis through a common mechanism. They both directly interact with mitochondria, causing increased cytochrome c release and mitochondrial depolarization, and both decrease sequestration of BH3-only proteins by Bcl-xL. However, the effects of rottlerin and BH3I-2' on the complex formation between Bcl-xL and BH3-only proteins are different. BH3I-2' disrupts complexes of Bcl-xL with Bad but not with Bim or Puma, whereas rottlerin had no effect on the Bcl-xL interaction with Bad. Also BH3I-2', but not rottlerin, required Bad to stimulate apoptosis. In conclusion, our results demonstrate that rottlerin has a potent proapoptotic and antitumor activity in pancreatic cancer, which is mediated by disrupting the interaction between prosurvival Bcl-2 proteins and proapoptotic BH3-only proteins. Thus rottlerin represents a promising novel agent for pancreatic cancer treatment.

Topics: Acetophenones; Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-Associated Death Protein; bcl-X Protein; Benzamides; Benzopyrans; Cell Line, Tumor; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Humans; Membrane Proteins; Mice; Mice, Nude; Mitochondria; Neoplasm Transplantation; Pancreatic Neoplasms; Protein Kinase C-delta; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

Elevated expression of tissue transglutaminase (TG2) in cancer cells has been implicated in the development of drug resistance and metastatic phenotypes. However, the role and the mechanisms that regulate TG2 expression remain elusive. Here, we provide evidence that protein kinase Cdelta (PKCdelta) regulates TG2 expression, which in turn inhibits autophagy, a type II programmed cell death, in pancreatic cancer cells that are frequently insensitive to standard chemotherapeutic agents. Rottlerin, a PKCdelta-specific inhibitor, and PKCdelta small interfering RNA (siRNA) down-regulated the expression of TG2 mRNA and protein and induced growth inhibition without inducing apoptosis in pancreatic cancer cells. Inhibition of PKCdelta by rottlerin or knockdown of TG2 protein by a TG2-specific siRNA resulted in a marked increase in autophagy shown by presence of autophagic vacuoles in the cytoplasm, formation of the acidic vesicular organelles, membrane association of microtubule-associated protein 1 light chain 3 (LC3) with autophagosomes, and a marked induction of LC3-II protein, important hallmarks of autophagy, and by electron microscopy. Furthermore, inhibition of TG2 by rottlerin or by the siRNA led to accumulation of green fluorescent protein (GFP)-LC3-II in autophagosomes in pancreatic cancer cells transfected with GFP-LC3 (GFP-ATG8) expression vector. Knockdown of Beclin-1, a specific autophagy-promoting protein and the product of Becn1 (ATG6), inhibited rottlerin-induced and TG2 siRNA-induced autophagy, indicating that Beclin-1 is required for this process. These results revealed that PKCdelta plays a critical role in the expression of TG2, which in turn regulates autophagy. In conclusion, these results suggest a novel mechanism of regulation of TG2 and TG2-mediated autophagy in pancreatic cancer cells.

Topics: Acetophenones; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Benzopyrans; Cell Line, Tumor; Cell Proliferation; Down-Regulation; GTP-Binding Proteins; Humans; Membrane Proteins; Pancreatic Neoplasms; Protein Glutamine gamma Glutamyltransferase 2; Protein Kinase C-delta; Protein Kinase Inhibitors; RNA, Small Interfering; Transglutaminases