A role for transferrin receptor in triggering apoptosis when targeted with gambogic acid

Abstract

Transferrin receptor (TfR) has been shown to be significantly overexpressed in different types of cancers. We discovered TfR as a target for gambogic acid (GA), used in traditional Chinese medicine and a previously undiscovered link between TfR and the rapid activation of apoptosis. The binding site of GA on TfR is independent of the transferrin binding site, and it appears that GA potentially inhibits TfR internalization. Down-regulation of TfR by RNA interference decreases sensitivity to GA-induced apoptosis, further supporting TfR as the primary GA receptor. In summary, GA binding to TfR induces a unique signal leading to rapid apoptosis of tumor cells. These results suggest that GA may provide an additional approach for targeting the TfR and its use in cancer therapy.

Fig. 1.

GA induces apoptosis. (A) Electron microscopy of T47D cells treated with DMSO (Left) or GA(5 μM, Right). (Scale bar: 1 mm ≈ 0.01 μm.) (B) The structure of GA of which the tricyclic ring and α,β-unsaturated ring (boxed areas) were modified to obtain the derivatives.

Fig. 2.

GA binds to a cell surface receptor. (A) Biotin-GA conjugated to streptavidin FluoSpheres activates apoptosis. Jurkat cells were treated for 24 h with DMSO (C), 5 μM GA (F), or 5 μM biotin-GA (B) bound to streptavidin FluoSpheres (20). Propidium iodide viability staining was used to quantitate apoptosis. (B) Saturable receptor binding on cells. Jurkat cells were incubated with tritium-GA at 1 μM at 37°C, with or without 20 μM of unlabeled GA. At indicated time points, the amount of bound tritium-GA was determined by liquid scintillation counting. (C) GA binds to a biotinylated cell surface protein. Jurkat cells were surface-biotinylated and treated with either DMSO or 5 μM fluorescein-GA for 30 min at 37°C. Active-GA and inactive-GA were used as controls to determine the specificity of binding. (D) GA binds to TfR. Jurkat cells were treated with either DMSO or 5 μM fluorescein-GA, for 30 min at 37°C. Lysate was immunoprecipitated with anti-FITC antibody and detected by immunoblotting with anti-TfR antibody.

Fig. 3.

GA binds to soluble TfR protein. (A) GA binds TfR in vitro. TfR-coated wells were incubated with increasing concentrations of biotin-GA in binding/washing buffer for 20 min at 30°C, then incubated with Europium labeled-streptavidin (Eu-streptavidin). Amounts of bound Eu-Streptavidin were quantified by measuring time-delayed fluorescence. (B) IC₅₀ of active and inactive GA derivatives. In competition experiments, biotin-GA at 1 μM was premixed with increasing amounts of GA or the inactive-GA as a competitor. Amounts of bound Eu-Streptavidin were quantified by measuring time-delayed fluorescence. (C) GA bound to TfR in vitro can be displaced by active GA derivatives. TfR-coated wells were incubated with biotin-GA as described, washed, and incubated with GA or binding/washing buffer as a wash off control. (D) Binding of biotin-GA and tritium-GA to TfR is not inhibited by either apo-transferrin or holo-transferrin. Binding of biotin-GA and tritium-GA to Jurkat cells (hatched) or in vitro TfR-binding (solid) in the presence of 1 μM GA, 50 μg/ml apotransferrin, or 50 μg/ml holo-transferrin is shown in this graph.

Fig. 4.

Known mechanisms of iron regulation do not overlap with GA-mediated apoptosis. (A) The binding of holo-Tf to TfR has no effect on GA-induced apoptosis. Jurkat cells were pretreated with holo-transferrin for 30 min and subsequently treated with DMSO or 1, 2.5, or 5 μM GA for 4 h, after which cell viability was measured as described. (B) GA-induced apoptosis through TfR is not iron-dependent. Jurkat cells were treated with 10 μM desferrioxamine (DFO) or 50 μM ferric nitrate [Fe₃(NO)₃] for 1 h and treated as described in A. (C) GA interferes with TfR receptor internalization. T47D cells were treated with DMSO or 2 μM GA for 15 min and further treated with FITC-conjugated anti-TfR for 30 min at 37°C. The cells were then fixed with methanol at –20°C for 5 min, washed with PBS, and mounted with Vectashield mounting medium. Representative of three independently confirmed experiments. (D) GA interferes with TfR internalization as indicated by cell surface TfR expression. Jurkat cells were treated with holo-transferrin alone (50 μg/ml) for 3 or 5 min, GA alone (5 μM) for 2 min, or pretreated for 5 min with GA followed by 1, 3, 5, or 10 min of holo-transferrin treatment. Cells were then stained with FITC-conjugated anti-transferrin receptor antibody for 30 min at 4°C. After washing, cells were analyzed on a Becton Dickinson FACS Calibur. Data are shown as mean fluorescence units. Results were confirmed in three independent experiments.

Fig. 5.

GA displays differential apoptosis potential in normal versus tumor cell lines, and down-regulation of the TfR by using siRNA technology leads to a decrease in apoptosis with GA treatment. (A) Normal and tumor cell lines were treated with DMSO, 1 μM GA, or 1 μM inactive GA for 5 h and assessed for apoptosis. TfR cell surface expression is noted as follows: T47D (++++), 293T (+++), human umbilical vein endothelial cells (HUVEC, ±), and human mammary epithelial cells (HMEC, –). Results are independently confirmed in three experiments. (B) Real-time PCR showing the down-regulation of the TfR at the mRNA level. 293T cells were transfected for 48 h with lipid alone, Cph, TfR, or negative control siRNAs (NC). (C) Western blot representing the down-regulation of TfR in siRNA-transfected cells. Whole-cell lysates of 293T cells after transfection were subjected to SDS/PAGE and Western blotted with anti-TfR antibody. Actin was used as loading control (Lower). (D) Down-regulation of TfR protects cells from GA-induced apoptosis. 293T cells were transfected and treated as described in A. Cells were fixed and analyzed for cell death.

Fig. 6.

Signaling pathway of GA-induced apoptosis. (A) A time course of signaling events. Jurkat cells were treated with DMSO or GA (5 μM) for the indicated times. Western blotting was performed with anti-caspase-3, anti-caspase-8, anti-Bid, or anti-cytochrome c antibodies and detected by using ECL. (B) Apaf1 –/– MEFs show a decreased level of caspase activation. Dose–response of GA and methyl-GA induced caspase-3 activities in WT and Apaf –/– MEFs treated for 5 h.