Pancreatic cancer cells retain the epithelial-related phenotype and modify mitotic spindle microtubules after the administration of ukrain in vitro.
The aim of this study is to characterize the phenotype of pancreatic ductal adenocarcinoma (PDAC) cells in relation to the expression of epithelial-to-mesenchymal transition (EMT) markers and determine whether ukrain, an anticancer drug based on the alkaloids extracted from greater celandine, modulates in vitro the malignant behavior of PDAC cells in order to extend our understanding of its therapeutic potential. Three cell lines (HPAF-II, HPAC, and PL45) were treated with ukrain (5, 10, and 20 μmol/l) for 48 h or left untreated (control). Cell proliferation was assessed by growth curves. Apoptosis was determined by Hoechst nuclear staining and by cytochrome c and caspase-8 expressions. The EMT markers E-cadherin, β-catenin, and vimentin, as well as actin and tubulin cytoskeletons, were analyzed by immunofluorescence. Interphase and mitotic microtubules as well as abnormal mitotic figures were studied by fluorescence microscopy after tubulin immunolabeling. Ukrain strongly suppressed cell proliferation and induced apoptosis possibly through an extrinsic pathway as cytochrome c immunoreactivity suggested that the integrity of the mitochondria was not affected. Tubulin expression indicated an antiproliferative effect of ukrain on the basis of alterations in mitotic spindle microtubule dynamics, leading to abnormal mitosis. Membranous E-cadherin/β-catenin immunoreactivity was similarly expressed in control-treated and ukrain-treated cells, although the drug upregulated E-cadherin in cell lysates. Our results suggest that ukrain exerts its chemotherapeutic action on PDAC cells targeting mitotic spindle microtubules, leading to abnormal mitosis and apoptosis, and favoring cell cohesiveness. The differentiated epithelial phenotype of HPAF-II, HPAC, and PL45 cell lines concomitant with a highly invasive potential suggests that further experiments will be necessary to definitively clarify the role of EMT in PDAC progression.
Department of Biomedical Sciences for Health, EML-Extracellular Matrix Laboratory, Università degli Studi di Milano, Milan, Italy. nicoletta.gagliano@unimi.it
Nicoletta
N
Gagliano
Tatiana
T
Volpari
Marco
M
Clerici
Letizia
L
Pettinari
Isabella
I
Barajon
Nicola
N
Portinaro
Graziano
G
Colombo
Aldo
A
Milzani
Isabella
I
Dalle-Donne
Carla
C
Martinelli
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001600:Berberine Alkaloids / Q000494:pharmacology*; D015820:Cadherins / Q000096:biosynthesis; D021441:Carcinoma, Pancreatic Ductal / Q000378:metabolism / Q000473:pathology*; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D006801:Humans; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008870:Microtubules / Q000187:drug effects* / Q000473:pathology; D008938:Mitosis / Q000187:drug effects; D008940:Mitotic Index; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D010617:Phenanthridines / Q000494:pharmacology*; D010641:Phenotype; D008941:Spindle Apparatus / Q000187:drug effects* / Q000473:pathology; D013997:Time Factors; D051176:beta Catenin / Q000096:biosynthesis
D000972:Antineoplastic Agents, Phytogenic; D001600:Berberine Alkaloids; D015820:Cadherins; D010617:Phenanthridines; D051176:beta Catenin; C060921:ukrain