The necrotic signal induced by mycophenolic acid overcomes apoptosis-resistance in tumor cells

Abstract

Background: The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42.

Methodology/principal findings: Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA-mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL-overexpressing cells). All tested cells remained sensitive to MPA-mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers.

Conclusions/significance: These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells.

Figure 1. IMPDH inhibition leads to a necrotic morphology.

Electron micrographs of activated PBLs. Cells were incubated with MPA (3 µg/mL) or with ribavirin (100 µg/ml) for 44 hours (Bars = 2 µm).

Figure 2. Cdc42 and actin are implicated in the MPA-mediated necrotic signal.

(A) Cells were pre-incubated with secramine A (40 µM) or DMSO for 1 hour and then incubated for 44 hours with MPA. Necrosis was quantified by assessing the loss of the plasma membrane integrity (entry of propidium iodide) and the cell morphology. (B) Upper panel: Jurkat cells transfected with plasmid encoding Q61L-Cdc42-GFP, Q61L-Rac1-GFP or GFP alone were treated with MPA for 44 hours. The percentage of dead cells (morphologic analysis) and the percentage of GFP-expressing cells among the living population are shown. Lower panel: Jurkat cells were transfected with the Cdc42-targeting shRNA or control shRNA (Scr) and pEGFP-N1 at a DNA weight ratio of 3∶1. 24 hours after transfection, living cells were isolated by Ficoll gradient and incubated for 44 hours with MPA. Cell death was assessed by analyzing plasma membrane damage (propidium iodide entry). The inset depicts the expression of Cdc42 and Rac1 in Jurkat cells untransfected (U) or transfected with scramble shRNA (Scr) or shRNA targeting Cdc42. Western blots were performed using the indicated antibodies. (C) The B-cell line Dab-1 and the T-cell line Jurkat were treated with Latrunculin (LtnA, 2.5 µM), Cytochalasin D (CytD, 5 µM) or DMSO (control) for 30 minutes. Cells were then fixed, permeabilized and F-actin was tagged using FITC-labeled phalloidin. The amount of F-actin was assessed by flow cytometry. (D) Cells were pre-incubated for 30 min with 2.5 µM of LtnA, 5 µM of CytD or DMSO and then treated in presence or not of MPA (5 µg/ml) for 14 hours. The amount of GTP (upper panel) and GDP (lower panel) was quantified as described in Materials and Methods. (E) Cells were pre-incubated for 30 min with LtnA (2.5 µM) or CytD (5 µM) and then treated with 5 µg/ml of MPA for the indicated times. ΔΨm was assessed using DiOC6 staining. Data represent the mean±SD of three independently performed experiments.

Figure 3. Apoptosis resistances are bypassed by the MPA–mediated necrotic signal.

(A) Parental CEM and doxorubicin-resistant CEM (CEM^DoxR) were incubated with indicated dose of doxorubicin, CD95L, TRAIL or MPA for 48 hours. Cell death was measured using a MTT assay. (B) Flow cytometry analysis of the CD95 expression on the doxorubicin-resistant CEM. (C) Parental Jurkat and the selected doxorubicin resistant clones (Jurkat^DoxR) were incubated for 48 hours with doxorubicin and MPA at the indicated concentrations. MTT assay was performed to quantify cell death. (D) CEM and the etoposide resistant CEM-VM1 were incubated with the etoposide or MPA for 48 hours and cell death was assessed using MTT assay. Results shown represent the mean±SD of three independent experiments.

Figure 4. MPA mediates necrosis in malignant cells resistant to apoptosis.

(A) Molecular characterization of the imatinib and nilotinib-resistant CML cell lines. MDR: multidrug-resistance related protein. (−) indicates an expression level similar to the untreated cells, (+) refers to the ectopic expression of BCR-Abl and (+++) indicates the over-expression of the indicated protein. (B) Imatinib or nilotinib-resistant CML cells were incubated with the indicated concentrations of imatinib (left panel), nilotinib (middle panel) or MPA (right panel) for 48 hours. Cell death was measured by MTT assay. (C) Expression of Bcl2 (endogenous and GFP-Bcl2) was assessed in GFP- and GFP-Bcl2-expressing clones by immunoblot. The indicated cells were incubated for 24 hours with soluble CD95L, staurosporine or MPA for 48 hours. Total cell death was measured by MTT.

Figure 5. MPA treatment efficiently kills B-CLL cells.

Leukemic B-cells isolated from CLL patients (n = 14) were treated with either the apoptotic inducers TRAIL (500 ng/ml) or CD95L (500 ng/ml) or with MPA (10 µg/ml) for 72 hours. Then total cell death was quantified using the metabolic assay MTT (total cell death). Statistical analysis was performed using a non-parametric Mann-Whitney two sample ranksum test (n = 14). N.S means non-significant and ** P≤0.01.