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


RESULTS
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.
Winiarska
Magdalena
M
Department of Immunology, Center of Biostructure Research, the Medical University of Warsaw, Warsaw, Poland.
Bil
Jacek
J
Wilczek
Ewa
E
Wilczynski
Grzegorz M
GM
Lekka
Malgorzata
M
Engelberts
Patrick J
PJ
Mackus
Wendy J M
WJ
Gorska
Elzbieta
E
Bojarski
Lukasz
L
Stoklosa
Tomasz
T
Nowis
Dominika
D
Kurzaj
Zuzanna
Z
Makowski
Marcin
M
Glodkowska
Eliza
E
Issat
Tadeusz
T
Mrowka
Piotr
P
Lasek
Witold
W
Dabrowska-Iwanicka
Anna
A
Basak
Grzegorz W
GW
Wasik
Maria
M
Warzocha
Krzysztof
K
Sinski
Maciej
M
Gaciong
Zbigniew
Z
Jakobisiak
Marek
M
Parren
Paul W H I
PW
Golab
Jakub
J
D000911:Antibodies, Monoclonal / Q000187:drug effects* / Q000378:metabolism / Q000627:therapeutic use*; D058846:Antibodies, Monoclonal, Murine-Derived; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects; D018951:Antigens, CD20 / Q000737:chemistry / Q000187:drug effects*; D000970:Antineoplastic Agents / Q000037:antagonists & inhibitors*; D001402:B-Lymphocytes / Q000378:metabolism; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000009:adverse effects*; D006937:Hypercholesterolemia / Q000097:blood / Q000188:drug therapy; D008148:Lovastatin / Q000494:pharmacology; D016393:Lymphoma, B-Cell / Q000188:drug therapy*; D021962:Membrane Microdomains / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D000069283:Rituximab
D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D000970:Antineoplastic Agents; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D000069283:Rituximab; D002784:Cholesterol; D008148:Lovastatin