Paclitaxel induces calcium oscillations via an inositol 1,4,5-trisphosphate receptor and neuronal calcium sensor 1-dependent mechanism

Abstract

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Despite a well documented tubulin-stabilizing effect, many side effects of taxol therapy cannot be explained by cytoskeletal mechanisms. In the present study submicromolar concentrations of taxol, mimicking concentrations found in patients, induced cytosolic calcium (Ca(2+)) oscillations in a human neuronal cell line. These oscillations were independent of extracellular and mitochondrial Ca(2+) but dependent on intact signaling via the phosphoinositide signaling pathway. We identified a taxol binding protein, neuronal Ca(2+) sensor 1 (NCS-1), a Ca(2+) binding protein that interacts with the inositol 1,4,5-trisphosphate receptor from a human brain cDNA phage display library. Taxol increased binding of NCS-1 to the inositol 1,4,5-trisphosphate receptor. Short hairpin RNA-mediated knockdown of NCS-1 in the same cell line abrogated the response to taxol but not to other agonists stimulating the phosphoinositide signaling pathway. These findings are important for studies involving taxol as a research tool in cell biology and may help to devise new strategies for the management of side effects induced by taxol therapy.

Fig. 1.

Taxol induces Ca²⁺ oscillations independent of extracellular and mitochondrial Ca²⁺. (A) Representative normalized Ca²⁺ changes induced by 800 ng/ml taxol (arrow). (B) Taxol-induced oscillations are concentration-dependent with a calculated EC₅₀ of 83 ng/ml (arrow). (C) Power spectral analysis of the cell shown in A reveals that the dominant peak of taxol-induced Ca²⁺ oscillations occurs at ≈12 mHz. (D–F) Ca²⁺-free solution (D) (10 mM EGTA), preincubation of cells with cyclosporin A (E), and treatment with the mitochondrial uncoupler FCCP (F) did not block oscillations induced by 800 ng/ml taxol (arrow). A, 10 μM ATP; T, 3 μM thapsigargin.

Fig. 2.

Taxol-induced Ca²⁺ oscillations depend on ER Ca²⁺, InsP₃R, and InsP₃ but not RyR. (A) Taxol-induced Ca²⁺ responses are abolished after depletion of ER–Ca²⁺ with thapsigargin. (B) Preincubation with 70 μM dantrolene decreases the response amplitude to 800 ng/ml taxol (arrow). (C and D) Preincubation with the InsP₃R inhibitors xestospongin C (5 μM; black trace) or 2-APB (20 μM; gray trace) (C) and treatment with the phospholipase C inhibitor U-73122 (5 μM) (D) abolish the response to 800 ng/ml taxol (arrow). A, 10 μM ATP; T, 3 μM thapsigargin.

Fig. 3.

Taxol binds to NCS-1, an interaction that enhances binding of NCS-1 to InsP₃R. (A) Coimmunoprecipitation of β-tubulin and InsP₃R from mouse cerebellar lysate. Lanes, from left to right, show mouse cerebellar lysate, beads treated with preimmune serum but no specific antibody, immunoprecipitate with anti-InsP₃R1, immunoprecipitate with anti-InsP₃R1 and 800 ng/ml taxol, immunoprecipitate with anti-β-tubulin, and immunoprecipitate with anti-β-tubulin and 800 ng/ml taxol. The immunoblot was probed with anti-InsP₃R in Upper and anti-β-tubulin in Lower. (B) Binding analysis of 7-bio-taxol 4 with NCS-1 phage. Phage rescue titer is reported in pfu as a function of probe concentration. (C) Taxol increases the binding of NCS-1 to the InsP₃R. Lanes, from left to right, show mouse cerebellar lysate, beads treated with preimmune serum, immunoprecipitate with anti-InsP₃R, and immunoprecipitate with anti-InsP₃R and 800 ng/ml taxol. The immunoblot was probed with anti-InsP₃R1 in Upper and anti-NCS-1 in Lower. (D) Cells transiently transfected with a vector expressing NCS-1 shRNA as well as GFP showed a significant reduction (≈80%) of the immunosignal compared with cells expressing scrambled shRNA. (E) Oscillations induced by 800 ng/ml taxol (arrow) were abrogated in NCS-1 knockdown cells (red trace) but unaffected in cells expressing scrambled shRNA (black trace). (F) Oscillations induced by 0.75 μM ATP were unaffected by NCS-1 knockdown (red trace) compared with scrambled shRNA-expressing cells (black trace). T, 3 μM thapsigargin.