thiourea and Pancreatic-Neoplasms

Calcium ions (Ca(2+)) are indispensable for the physiology of organisms and the molecular regulation of cells. We observed that CGK733, a synthetic chemical substance, induced non-apoptotic cell death and stimulated reversible calcium sequestration by vesicles in pancreatic cancer cells. The endoplasmic reticulum (ER) stress eukaryotic translation initiation factor 2-alpha kinase 3/C/EBP homologous protein (PERK/CHOP) signaling pathway was shown to be activated by treatment with CGK733. Ionomycin, an ER stress drug and calcium ionophore, can activate PERK/CHOP signaling and accelerate CGK733-induced calcium sequestration. Knockdown of CHOP diminished CGK733-induced vesicular calcium sequestration, but had no effects on the cell death. Proteomic analysis demonstrated that the ER-located calcium-binding proteins, calumenin and protein S100-A11, were altered in CGK733-treated cells compared to non-treated controls. Our study reveals that CGK733-induced intracellular calcium sequestration is correlated with the PERK/CHOP signaling pathway and may also be involved in the dysregulations of calcium-binding proteins.

Topics: Antineoplastic Agents; Benzeneacetamides; Calcium; Calcium Ionophores; Calcium-Binding Proteins; Cell Death; Cell Line, Tumor; Dose-Response Relationship, Drug; eIF-2 Kinase; Endoplasmic Reticulum; Humans; Pancreatic Neoplasms; Proteomics; RNA Interference; S100 Proteins; Signal Transduction; Thiourea; Time Factors; Transcription Factor CHOP; Transfection

Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II is essential for autophagosome formation and is widely used to monitor autophagic activity. We show that CGK733 induces LC3 II and LC3-puncta accumulation, which are not involved in the activation of autophagy. The treatment of CGK733 did not alter the autophagic flux and was unrelated to p62 degradation. Treatment with CGK733 activated the AMP-activated protein kinase (AMPK) and protein kinase RNA-like endoplasmic reticulum kinase/CCAAT-enhancer-binding protein homologous protein (PERK/CHOP) pathways and elevated the expression of p21Waf1/Cip1. Inhibition of both AMPK and PERK/CHOP pathways by siRNA or chemical inhibitor could block CGK733-induced p21Waf1/Cip1 expression as well as caspase-3 cleavage. Knockdown of LC3 B (but not LC3 A) abolished CGK733-triggered LC3 II accumulation and consequently diminished AMPK and PERK/CHOP activity as well as p21Waf1/Cip1 expression. Our results demonstrate that CGK733-triggered LC3 II formation is an initial event upstream of the AMPK and PERK/CHOP pathways, both of which control p21Waf1/Cip1 expression.

Topics: AMP-Activated Protein Kinases; Animals; Autophagy; Benzeneacetamides; Blotting, Western; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; eIF-2 Kinase; Humans; Mice; Microscopy, Fluorescence; Microtubule-Associated Proteins; NIH 3T3 Cells; Pancreatic Neoplasms; RNA Interference; Signal Transduction; Thiourea; Transcription Factor CHOP

To evaluate the anti-tumor effect of clobenpropit, which is a specific H3 antagonist and H4 agonist, in combination with gemcitabine in a pancreatic cancer cell line.. Three kinds of human pancreatic cancer cell lines (Panc-1, MiaPaCa-2, and AsPC-1) were used in this study. Expression of H3 and H4 receptors in pancreatic cancer cells was identified with Western blotting. Effects of clobenpropit on cell proliferation, migration and apoptosis were evaluated. Alteration of epithelial and mesenchymal markers after administration of clobenpropit was analyzed. An in vivo study with a Panc-1 xenograft mouse model was also performed.. H4 receptors were present as 2 subunits in human pancreatic cancer cells, while there was no expression of H3 receptor. Clobenpropit inhibited cell migration and increased apoptosis of pancreatic cancer cells in combination with gemcitabine. Clobenpropit up-regulated E-cadherin, but down-regulated vimentin and matrix metalloproteinase 9 in real-time polymerase chain reaction. Also, clobenpropit inhibited tumor growth (gemcitabine 294 ± 46 mg vs combination 154 ± 54 mg, P = 0.02) and enhanced apoptosis in combination with gemcitabine (control 2.5%, gemcitabine 25.8%, clobenpropit 9.7% and combination 40.9%, P = 0.001) by up-regulation of E-cadherin and down-regulation of Zeb1 in Panc-1 xenograft mouse.. Clobenpropit enhanced the anti-tumor effect of gemcitabine in pancreatic cancer cells through inhibition of the epithelial-mesenchymal transition process.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Cell Proliferation; Deoxycytidine; Epithelial-Mesenchymal Transition; Gemcitabine; Histamine Agonists; Histamine H3 Antagonists; Humans; Imidazoles; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Pancreatic Neoplasms; Receptors, G-Protein-Coupled; Receptors, Histamine; Receptors, Histamine H4; Thiourea; Tumor Burden; Xenograft Model Antitumor Assays

We recently reported that transforming growth factor-β (TGF-β) induces an increase in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pancreatic cancer cells, but the mechanisms by which TGF-β mediates [Ca(2+)](cyt) homeostasis in these cells are currently unknown. Transient receptor potential (TRP) channels and Na(+)/Ca(2+) exchangers (NCX) are plasma membrane proteins that play prominent roles in controlling [Ca(2+)](cyt) homeostasis in normal mammalian cells, but little is known regarding their roles in the regulation of [Ca(2+)](cyt) in pancreatic cancer cells and pancreatic cancer development. Expression and function of NCX1 and TRPC1 proteins were characterized in BxPc3 pancreatic cancer cells. TGF-β induced both intracellular Ca(2+) release and extracellular Ca(2+) entry in these cells; however, 2-aminoethoxydiphenyl borate [2-APB; a blocker for both inositol 1,4,5-trisphosphate (IP(3)) receptor and TRPC], LaCl(3) (a selective TRPC blocker), or KB-R7943 (a selective inhibitor for the Ca(2+) entry mode of NCX) markedly inhibited the TGF-β-induced increase in [Ca(2+)](cyt). 2-APB or KB-R7943 treatment was able to dose-dependently reverse membrane translocation of PKCα induced by TGF-β. Transfection with small interfering RNA (siRNA) against NCX1 almost completely abolished NCX1 expression in BxPc3 cells and also inhibited PKCα serine phosphorylation induced by TGF-β. Knockdown of NCX1 or TRPC1 by specific siRNA transfection reversed TGF-β-induced pancreatic cancer cell motility. Therefore, TGF-β induces Ca(2+) entry likely via TRPC1 and NCX1 and raises [Ca(2+)](cyt) in pancreatic cancer cells, which is essential for PKCα activation and subsequent tumor cell invasion. Our data suggest that TRPC1 and NCX1 may be among the potential therapeutic targets for pancreatic cancer.

Topics: Boron Compounds; Calcium; Carbazoles; Cell Line; Cell Movement; Enzyme Inhibitors; Homeostasis; Humans; Inositol 1,4,5-Trisphosphate Receptors; Pancreatic Ducts; Pancreatic Neoplasms; Phosphorylation; Protein Kinase C-alpha; Sodium-Calcium Exchanger; Thiourea; Transforming Growth Factor beta; Transient Receptor Potential Channels