title: Dendrimer-Based Selective Proteostasis-Inhibition Strategy to Control NSCLC Growth and Progression.
abstract: Elevated valosin containing protein (VCP/p97) levels promote the progression of non-small cell lung carcinoma (NSCLC). Although many VCP inhibitors are available, most of these therapeutic compounds have low specificity for targeted tumor cell delivery. Hence, the primary aim of this study was to evaluate the in vitro efficacy of dendrimer-encapsulated potent VCP-inhibitor drug in controlling non-small cell lung carcinoma (NSCLC) progression. The VCP inhibitor(s) (either in their pure form or encapsulated in generation-4 PAMAM-dendrimer with hydroxyl surface) were tested for their in vitro efficacy in modulating H1299 (NSCLC cells) proliferation, migration, invasion, apoptosis and cell cycle progression. Our results show that VCP inhibition by DBeQ was significantly more potent than NMS-873 as evident by decreased cell proliferation (p<0.0001, MTT-assay) and migration (p<0.05; scratch-assay), and increased apoptosis (p<0.05; caspase-3/7-assay) as compared to untreated control cells. Next, we found that dendrimer-encapsulated DBeQ (DDNDBeQ) treatment increased ubiquitinated-protein accumulation in soluble protein-fraction (immunoblotting) of H1299 cells as compared to DDN-control, implying the effectiveness of DBeQ in proteostasis-inhibition. We verified by immunostaining that DDNDBeQ treatment increases accumulation of ubiquitinated-proteins that co-localizes with an ER-marker, KDEL. We observed that proteostasis-inhibition with DDNDBeQ, significantly decreased cell migration rate (scratch-assay and transwell-invasion) as compared to the control-DDN treatment (p<0.05). Moreover, DDNDBeQ treatment showed a significant decrease in cell proliferation (p<0.01, MTT-assay) and increased caspase-3/7 mediated apoptotic cell death (p<0.05) as compared to DDN-control. This was further verified by cell cycle analysis (propidium-iodide-staining) that demonstrated significant cell cycle arrest in the G2/M-phase (p<0.001) by DDNDBeQ treatment as compared to control-DDN. Moreover, we confirmed by clonogenic-assay that DDNDBeQ treatment significantly (p<0.001) inhibits H1299 colony-formation as compared to control/DDN. Overall, encapsulation of potent VCP-inhibitor DBeQ into a dendrimer allows selective VCP-mediated proteostasis-inhibition for controlling NSCLC-tumor growth and progression to allow tumor-targeted sustained drug delivery.
authors.affiliation: College of Medicine, Central Michigan University, Mount Pleasant, Michigan, United States of America.
authors.forename: Kyla
authors.initials: K
authors.lastname: Walworth
authors.affiliation: College of Medicine, Central Michigan University, Mount Pleasant, Michigan, United States of America.
authors.forename: Manish
authors.initials: M
authors.lastname: Bodas
authors.affiliation: College of Medicine, Central Michigan University, Mount Pleasant, Michigan, United States of America.
authors.forename: Ryan John
authors.initials: RJ
authors.lastname: Campbell
authors.affiliation: Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, Michigan, United States of America.
authors.forename: Doug
authors.initials: D
authors.lastname: Swanson
authors.affiliation: Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, Michigan, United States of America.
authors.forename: Ajit
authors.initials: A
authors.lastname: Sharma
authors.affiliation: College of Medicine, Central Michigan University, Mount Pleasant, Michigan, United States of America.|Department of Pediatric Respiratory Sciences, The Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Neeraj
authors.initials: N
authors.lastname: Vij
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001980:Bronchi / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053179:Caspase 7 / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D050091:Dendrimers / Q000737:chemistry / Q000378:metabolism; D004337:Drug Carriers; D004339:Drug Compounding; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D059748:Proteolysis / Q000187:drug effects; D011799:Quinazolines / Q000737:chemistry / Q000494:pharmacology*; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism; D057149:Ubiquitinated Proteins / Q000235:genetics / Q000378:metabolism; D000074405:Valosin Containing Protein
chemical_list: D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D050091:Dendrimers; D004337:Drug Carriers; D004791:Enzyme Inhibitors; C489304:KDELR1 protein, human; C558357:N2,N4-dibenzylquinazoline-2,4-diamine; C104700:PAMAM Starburst; D011799:Quinazolines; D018000:Receptors, Peptide; D057149:Ubiquitinated Proteins; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein