Differential sensitivity of breast cancer and melanoma cells to proteasome inhibitor Velcade

Abstract

Abstract. Velcade (also known as PS-341 or Bortezomib) is a highly selective and reversible inhibitor of the 26S proteasome and is approved for the treatment of patients with advanced multiple myeloma. Here we investigated the antiproliferative effect of Velcade on 4T1 breast cancer and B16F10 melanoma cells and evaluated the mechanism of action. It was found that two cell lines are differentially sensitive to proteasome inhibitor Velcade. The IC 50 concentrations for B16F10 and 4T1 were 2.5 nM and 71 nM, respectively, indicating that B16F10 cells are more sensitive to proteasomal inhibition. Velcade was equally potent in inhibiting the chymotrypsin-like activity of the proteasome in both cell lines. It was determined that B16F10 cells proliferate more rapidly than 4T1 cells; doubling time (T d ) = 14.2 h versus T d =22.9 h, suggesting that a rapid proliferation rate may be an important factor in cellular resistance towards proteasomal inhibition. We observed for the first time that p53 and p21 proteins were increased in B16F10 cells but not in 4T1 following Velcade-treatment, demonstrating that p53 and p21 may enhance Velcade sensitivity. Furthermore, it was observed that caspase-3 proenzyme was reduced bỹ 20% in B16F10 melanoma cells, but not in 4T1 cells in response to 26S proteasomal inhibition by Velcade. Altogether, we concluded that p53 protein plays a central role in higher sensitivity of B16F10 cells to Velcade by inducing the accumulation of p21, a cell cycle inhibitor, as well as by stimulating the mitochondrial pathway of apoptosis through caspase-3 activation. IntroductionThe 26S proteasome, is an intracellular ATP-dependent multicatalytic protease involved in the degradation of shortlived proteins under normal metabolic conditions. It is also involved in the degradation of long-lived proteins, the processing of certain proteins (e.g., transcription factor NF-κB), and antigen presentation (1,2). The 26S proteasome has a molecular mass of ~2.5 MDa and is formed by the assembly of one 20S proteasome complex (the core particle) with 2 molecules of the 19S complex, the regulatory complex. The 19S regulatory complexes are attached to both ends of the 20S proteasome. The eukaryotic 20S proteasome is made up of 2 copies of 7 distinct α and 7 distinct ß-type subunits. The α subunits make up the 2 outer layers, and the ß subunits the 2 inner rings of the structure [i.e. (α 1 -α 7 )(ß 1 -ß 7 )(ß 1 -ß 7 )(α 1 -α 7 )] (3,4).Studies of the proteasome inhibitors MG132 and lactacystin indicated that the proteasome is responsible for the degradation of >80% of intracellular proteins. Among the key regulatory proteins degraded by the 26S proteasome are the transcription factor c-Fos, M-, S-, and G1-phase specific cyclins, cyclin-dependent kinase inhibitors, p53, polyamine biosynthetic enzymes ornithine decarboxylase, S-adenosylmethionine decarboxylase, and a variety of oncoproteins (1,(5)(6)(7)(8). For targeting the 26S proteasome, substrate proteins are usually conjugated to a 76 amino acid protein ubiquitin, a mul...

Estimation of intra-operator variability in perfusion parameter measurements using DCE-US

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A novel combination treatment for breast cancer cells involving BAPTA-AM and proteasome inhibitor bortezomib

“…Subsequently, the cells were incubated with 3% SDS (200 µl) + 40 mM HCl/isopropanol (1 ml) for 15 min in order to dissolve the MTT-formazan crystals. The absorbance of each sample was recorded at 570 nm (13)(14)(15). Cell survival was determined by analyzing the data with GraphPad Prism 3.03 software (GraphPad Software, Inc., La Jolla, CA, USA).…”

Bortezomib and etoposide combinations exert synergistic effects on the human prostate cancer cell line PC-3

“…Although, bortezomib has demonstrated strong antitumor activity in clinical settings against hematological malignancies (11), the drug has not been approved for the treatment of solid tumors to date. Bortezomib was previously indicated to cause apoptosis and growth inhibition through tumor protein p53 (p53)-dependent and p53-independent mechanisms in a number of cells in vitro (13,14). In the present study, the effect of bortezomib on the androgen-independent and p53-deficient cell line PC-3, alone and in combination with chemotherapeutic agents Cell culture and maintenance.…”

“…Following incubation, cells were lysed with 3% sodium dodecyl sulfate (SDS; 200 µl) plus 1 ml 40 mM HCl/isopropanol for 15 min. The homogenate was diluted 1:10 with the same solution used to lyse the cell (200 µl 3% SDS plus 1 ml 40 mM HCl/isopropanol), and the absorbance of each sample was recorded at 570 nm with a SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (13,15).…”

“…Stock cultures were maintained in 25-cm 2 flasks (Corning Incorporated, Corning, NY, USA). Cells were grown in 96-well cell culture plates (Corning Incorporated), and subcultured or seeded at ~70% confluence for subsequent experiments (13).…”