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Statins impair antitumor effects of rituximab by inducing conformational changes of CD20.

Winiarska M, Bil J, Wilczek E, Wilczynski GM, Lekka M, Engelberts PJ, Mackus WJ, Gorska E, Bojarski L, Stoklosa T, Nowis D, Kurzaj Z, Makowski M, Glodkowska E, Issat T, Mrowka P, Lasek W, Dabrowska-Iwanicka A, Basak GW, Wasik M, Warzocha K, Sinski M, Gaciong Z, Jakobisiak M, Parren PW, Golab J - PLoS Med. (2008)

Bottom Line: Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells.An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells.These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Center of Biostructure Research, the Medical University of Warsaw, Warsaw, Poland.

ABSTRACT

Background: Rituximab is used in the treatment of CD20+ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas.

Methods and findings: Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a 51Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-beta-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells.

Conclusions: Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.

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Cholesterol Is Necessary for CD20 Binding and Rituximab-Mediated CDCRaji cells were incubated with either diluent or lovastatin (10 μM for 48 h [A and B]), simvastatin (10 μM for 48 h [C and D]) or MβCD (5 mg/ml for 30 min [E and F]). Water-soluble cholesterol (chol; 0.2 mg/ml) was added for 2 h prior to flow cytometry analysis (A, E) or addition of 10 μg/ml rituximab and 10% human AB serum (B and F). Mevalonic acid (MA) was added together with simvastatin at a 200 μM concentration (C and D). Then for flow cytometry studies (A, C, and E), 1 × 106/ml of cells were incubated with saturating amounts of FITC-conjugated anti-CD20 mAb (B9E9) or IgG1 (isotype control) for 30 min at room temperature in the dark. Cell viability was measured in a MTT assay (B, D, and F). The survival of cells is presented as percentage of corresponding diluent- or statin-pretreated cells without rituximab. *p < 0.001 (two-way Student's t-test) as compared to statin-treated (B and D) or MβCD-treated (F) cells.
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pmed-0050064-g006: Cholesterol Is Necessary for CD20 Binding and Rituximab-Mediated CDCRaji cells were incubated with either diluent or lovastatin (10 μM for 48 h [A and B]), simvastatin (10 μM for 48 h [C and D]) or MβCD (5 mg/ml for 30 min [E and F]). Water-soluble cholesterol (chol; 0.2 mg/ml) was added for 2 h prior to flow cytometry analysis (A, E) or addition of 10 μg/ml rituximab and 10% human AB serum (B and F). Mevalonic acid (MA) was added together with simvastatin at a 200 μM concentration (C and D). Then for flow cytometry studies (A, C, and E), 1 × 106/ml of cells were incubated with saturating amounts of FITC-conjugated anti-CD20 mAb (B9E9) or IgG1 (isotype control) for 30 min at room temperature in the dark. Cell viability was measured in a MTT assay (B, D, and F). The survival of cells is presented as percentage of corresponding diluent- or statin-pretreated cells without rituximab. *p < 0.001 (two-way Student's t-test) as compared to statin-treated (B and D) or MβCD-treated (F) cells.

Mentions: To further investigate the role of cholesterol in binding of anti-CD20 mAb to the target antigen and in rituximab-mediated CDC, we performed restitution experiments. Preincubation of Raji cells with 10 μM lovastatin followed by a 30-min incubation with exogenous cholesterol significantly restored or improved binding of anti-CD20 mAb (Figure 6A). Similarly, a 30-min incubation of Raji cells with cholesterol after a 48-h pretreatment with 10 μM lovastatin completely restored sensitivity of lymphoma cells to rituximab-mediated CDC (Figure 6B). Accordingly, co-treatment of Raji cells with mevalonic acid, a direct product of HMG-CoAR, completely restored binding of anti-CD20 mAb (Figure 6C) as well as rituximab-mediated CDC of statin-treated cells (Figure 5D). Similar results were obtained with exogenous cholesterol that significantly reversed MβCD-induced effects (Figure 6E and 6F).


Statins impair antitumor effects of rituximab by inducing conformational changes of CD20.

Winiarska M, Bil J, Wilczek E, Wilczynski GM, Lekka M, Engelberts PJ, Mackus WJ, Gorska E, Bojarski L, Stoklosa T, Nowis D, Kurzaj Z, Makowski M, Glodkowska E, Issat T, Mrowka P, Lasek W, Dabrowska-Iwanicka A, Basak GW, Wasik M, Warzocha K, Sinski M, Gaciong Z, Jakobisiak M, Parren PW, Golab J - PLoS Med. (2008)

Cholesterol Is Necessary for CD20 Binding and Rituximab-Mediated CDCRaji cells were incubated with either diluent or lovastatin (10 μM for 48 h [A and B]), simvastatin (10 μM for 48 h [C and D]) or MβCD (5 mg/ml for 30 min [E and F]). Water-soluble cholesterol (chol; 0.2 mg/ml) was added for 2 h prior to flow cytometry analysis (A, E) or addition of 10 μg/ml rituximab and 10% human AB serum (B and F). Mevalonic acid (MA) was added together with simvastatin at a 200 μM concentration (C and D). Then for flow cytometry studies (A, C, and E), 1 × 106/ml of cells were incubated with saturating amounts of FITC-conjugated anti-CD20 mAb (B9E9) or IgG1 (isotype control) for 30 min at room temperature in the dark. Cell viability was measured in a MTT assay (B, D, and F). The survival of cells is presented as percentage of corresponding diluent- or statin-pretreated cells without rituximab. *p < 0.001 (two-way Student's t-test) as compared to statin-treated (B and D) or MβCD-treated (F) cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2270297&req=5

pmed-0050064-g006: Cholesterol Is Necessary for CD20 Binding and Rituximab-Mediated CDCRaji cells were incubated with either diluent or lovastatin (10 μM for 48 h [A and B]), simvastatin (10 μM for 48 h [C and D]) or MβCD (5 mg/ml for 30 min [E and F]). Water-soluble cholesterol (chol; 0.2 mg/ml) was added for 2 h prior to flow cytometry analysis (A, E) or addition of 10 μg/ml rituximab and 10% human AB serum (B and F). Mevalonic acid (MA) was added together with simvastatin at a 200 μM concentration (C and D). Then for flow cytometry studies (A, C, and E), 1 × 106/ml of cells were incubated with saturating amounts of FITC-conjugated anti-CD20 mAb (B9E9) or IgG1 (isotype control) for 30 min at room temperature in the dark. Cell viability was measured in a MTT assay (B, D, and F). The survival of cells is presented as percentage of corresponding diluent- or statin-pretreated cells without rituximab. *p < 0.001 (two-way Student's t-test) as compared to statin-treated (B and D) or MβCD-treated (F) cells.
Mentions: To further investigate the role of cholesterol in binding of anti-CD20 mAb to the target antigen and in rituximab-mediated CDC, we performed restitution experiments. Preincubation of Raji cells with 10 μM lovastatin followed by a 30-min incubation with exogenous cholesterol significantly restored or improved binding of anti-CD20 mAb (Figure 6A). Similarly, a 30-min incubation of Raji cells with cholesterol after a 48-h pretreatment with 10 μM lovastatin completely restored sensitivity of lymphoma cells to rituximab-mediated CDC (Figure 6B). Accordingly, co-treatment of Raji cells with mevalonic acid, a direct product of HMG-CoAR, completely restored binding of anti-CD20 mAb (Figure 6C) as well as rituximab-mediated CDC of statin-treated cells (Figure 5D). Similar results were obtained with exogenous cholesterol that significantly reversed MβCD-induced effects (Figure 6E and 6F).

Bottom Line: Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells.An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells.These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Center of Biostructure Research, the Medical University of Warsaw, Warsaw, Poland.

ABSTRACT

Background: Rituximab is used in the treatment of CD20+ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas.

Methods and findings: Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a 51Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-beta-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells.

Conclusions: Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.

Show MeSH
Related in: MedlinePlus