dihydropyridines and Lymphoma

In human lung cancer, Tripartite motif 65 (TRIM65) is documented as an important regulator in carcinogenesis. Knockdown of TRIM65 prevents the tumorigenesis of lung cancer cells, while TRIM65 overexpression presents the opposite effect. However, the roles of TRIM65 in human lymphocyte malignancies have reported little. Herein, we found that Jurkat (T-lymphocyte) and Raji (B-lymphocyte) expressed TRIM65. We aimed to investigate whether TRIM65 was a potential oncogenic protein that regulated the tumorigenesis of Jurkat and Raji cells. In our present study, cells were transfected with siRNA-TRIM65 or TRIM65 overexpression vector, Cell counting kit-8 (CCK-8), Flow cytometry and Annexin V-FITC/propidium iodide (PI) staining was carried out to detect cell viability, cell cycle profile and cell apoptosis, respectively. Extracellular signal-regulated kinases 1/2 (ERK1/2) pathway-associated proteins, such as Bcl2, cleaved-caspase 3, vascular endothelial growth factor (VEGF), and phosphorylated ERK1/2 (p-ERK1/2) were assessed. Our data indicated that knockdown of TRIM65 prevented the tumorigenesis of Jurkat and Raji cells. TRIM65 silencing inhibited cell proliferation, promoted cell apoptosis and arrested cell cycle, highly like through blocking ERK1/2 pathway. However, TRIM65 overexpression enhanced cell viability, increased the protein levels of Bcl2, VEGF, p-ERK1/2 while decreased cleaved-caspase 3 expression, suggesting the promoted effect of TRIM65 overexpression in the tumorigenesis of those two lymphoma cells. To validate the involvement of ERK1/2 pathway, ERK1/2 inhibitor AZD8330 (1 µmol/L) was introduced. We found that AZD8330 significantly prevented TRIM65 overexpression-induced tumorigenesis. We concluded that TRIM65 served as a potential oncogenic protein on Jurkat and Raji cells, and ERK1/2 pathway was the underlying mechanism. Approaches targeting TRIM65 provided a novel strategy for the treatment of lymphoma.

Topics: Apoptosis; Carcinogenesis; Cell Cycle Checkpoints; Cell Proliferation; Dihydropyridines; Extracellular Signal-Regulated MAP Kinases; Gene Knockdown Techniques; Gene Silencing; Humans; Jurkat Cells; Lymphoma; MAP Kinase Signaling System; Molecular Targeted Therapy; Protein Kinase Inhibitors; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

Series of novel N-acyloxy-1,4-dihydropyridines have been synthesized and evaluated as P-glycoprotein inhibitors in an in vitro assay to estimate their potential to act as multidrug resistance modulators in cancer cells. Structure-activity relationships are discussed and prove a significant and regiospecific influence of certain functional groups.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Dihydropyridines; Drug Resistance, Neoplasm; Lymphoma; Mice; Structure-Activity Relationship

The expression of P-glycoprotein (P-gp) in tumor cells causes a multidrug resistance (MDR) phenotype. P-gp has been shown to mediate the transport of structurally dissimilar drugs across the cell membrane in an energy-dependent manner. In this report, we show that BIBW22 BS, a phenylpteridine analog, reverses the MDR phenotype of CEM human lymphoma cells in a dose-dependent fashion. Using a photoactive analog of BIBW22 BS {[3H]azido-4-[N-(2-hydroxy-2-methylpropyl)-ethanolamino]-2, 7-bis(cis-2,6-dimethyl-morpholino)-6-phenylpteridine}, we show the photoaffinity labeling of a 170-kDa protein in drug-resistant cells immunoprecipitated with P-gp-specific monoclonal antibodies. The photolabeling of P-gp by [3H]azido-BIBW22 BS was specific and saturable. Furthermore, BIBW22 BS, vinblastine, and verapamil, but not colchicine, inhibited the photolabeling of P-gp by [3H]azido-BIBW22 BS. Drug binding studies showed that membranes from MDR cells bound more BIBW22 BS than parental drug-sensitive cells, and this binding was inhibited with vinblastine and, to a lesser extent, with uridine. However, drug transport studies demonstrated that BIBW22 BS is not a substrate for P-gp efflux pump. Interestingly, BIBW22 BS was shown to accumulate more in resistant cells. Also, BIBW22 BS accumulation in drug-sensitive and -resistant cells was not energy dependent. These results are in contrast with the observed decrease in accumulation or enhanced efflux of [3H]vinblastine seen in the same MDR cells. A comparison of [3H]azido-BIBW22 BS or [3H]azidopine photolabeled P-gp by Cleveland mapping with Staphylococcus aureus V8 protease showed differences in the photolabeled peptides. Taken together, the results of this study show that BIBW22 BS is a potent MDR-reversing agent that binds directly to P-gp but is not effluxed from drug-resistant cells.

Topics: Affinity Labels; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Binding Sites; Cell Line; Cell Survival; CHO Cells; Colchicine; Cricetinae; Dihydropyridines; Doxorubicin; Drug Resistance, Multiple; Humans; Kinetics; Lymphoma; Morpholines; Peptide Mapping; Protein Binding; Triamterene; Tumor Cells, Cultured; Verapamil; Vinblastine