Winiarska, Magdalena, Bil, Jacek, Wilczek, Ewa, Wilczynski, Grzegorz M., Lekka, Malgorzata, Engelberts, Patrick J., Mackus, Wendy J.M., Gorska, Elzbieta, Bojarski, Lukasz, Stoklosa, Tomasz, Nowis, Dominika, Kurzaj, Zuzanna, Makowski, Marcin, Glodkowska, Eliza, Issat, Tadeusz, Mrowka, Piotr, Lasek, Witold, Dabrowska-Iwanicka, Anna, Basak, Grzegorz W., Wasik, Maria, Warzocha, Krzysztof, Sinski, Maciej, Gaciong, Zbigniew, Jakobisiak, Marek, Parren, Paul W.H.I., and Golab, Jakub
Background Rituximab is used in the treatment of [CD20.sup.+] 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 [sup.51]Cr 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 cholesteroldepleting agents (methyl-β-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximabmediated 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., Introduction Rituximab, a chimeric IgG1 monoclonal antibody (mAb) that binds the CD20 antigen, is approved for first-line treatment of follicular and diffuse large B cell lymphoma in combination with chemotherapy, [...]