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-β-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.
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.
Jakub Golab and colleagues found that statins significantly decrease rituximab-mediated complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against B cell lymphoma cells.
Lymphomas are common cancers of the lymphatic system, the tissues and organs that produce and store the white blood cells (lymphocytes) that fight infections. In healthy people, the cells in the lymph nodes (collections of lymphocytes in the armpit, groin, and neck) and other lymphatic organs divide to form new cells only when the body needs them. Lymphomas form when a T or B lymphocyte starts to divide uncontrollably. The first sign of lymphoma is often a painless swelling in the armpit, groin, or neck caused by lymphocyte overgrowth in a lymph node. Eventually, the abnormal (malignant) lymphocytes, which provide no protection against infectious diseases, spread throughout the body. Treatments for lymphoma include chemotherapy (drugs that kill rapidly dividing cells) and radiotherapy. In addition, a drug called rituximab was recently developed for the treatment of some types of B cell lymphoma. Rituximab is a monoclonal antibody, a laboratory-produced protein. It binds to a protein called CD20 that is present on the surface of both normal and malignant B lymphocytes and induces cell killing through processes called “complement-dependent cytotoxity” (CDC) and “antibody-dependent cellular cytotoxity” (ADCC).
Why Was This Study Done?
Although rituximab lengthens the lives of patients with some types of B cell lymphoma, it is not a cure—the lymphoma usually recurs. Researchers are trying to increase the effectiveness of rituximab by combining it with other anticancer agents. One group of drugs that might be combined with rituximab is the “statins,” drugs that reduce the risk of heart disease by lowering the level of cholesterol (a type of fat) in the blood. In laboratory experiments, statins kill some cancer cells, in part by altering the fat composition of their outer (plasma) membrane. In addition, some population-based studies suggest that statin treatment might slightly decrease the risk of developing some kinds of cancer, including lymphoma. Statins are already undergoing clinical evaluation in combination with chemotherapy for the treatment of lymphoma, but in this study, the researchers investigate the influence of statins on rituximab-induced killing of B cell lymphomas.
What Did the Researchers Do and Find?
When the researchers tested the ability of rituximab and statin combinations to kill B cell lymphoma cells growing in dishes, they found that statins decreased rituximab-dependent CDC and ADCC of these cells. Statin treatment, they report, did not alter the total amount of CD20 made by the lymphoma cells or the amount of CD20 in their plasma membranes, but it did reduce the binding of another anti-CDC20 monoclonal antibody to the cells. Because both this antibody and rituximab bind to a specific three-dimensional structure in CD20 (a “conformational epitope”), the researchers hypothesized that statins might alter rituximab-induced killing by affecting the shape of the CD20 molecule on the lymphoma cell surface. To test this idea, they used two techniques—atomic force microscopy and limited proteolysis. The data obtained using both approaches confirmed that statins induce shape changes in CD20. Finally, the researchers took B cells from five patients who had taken statins for a short time and showed that this treatment had reduced the amount of anti-CD20 monoclonal antibody able to bind to these cells.
What Do These Findings Mean?
These findings indicate that statins change the shape of the CD20 molecules on the surface of normal and malignant B lymphocytes, probably by changing the amount of cholesterol in the cell membrane. This effect of statins has several clinical implications, which means that cancer specialists should check whether patients with known or suspected B cell lymphoma are taking statins to treat high cholesterol. First, the impaired binding of monoclonal antibodies to conformational epitopes of CD20 in patients being treated with statins might delay the diagnosis of B cell lymphomas (CD20 binding to lymphocytes is used during the diagnosis of lymphomas). Second, some patients with B cell lymphoma may receive an incorrect diagnosis and may not be offered rituximab. Finally, because statins impair the anti-lymphoma activity of rituximab, a possibility that needs to be investigated in clinical studies, cancer specialists should check that patients with B cell lymphoma are not taking statins before prescribing rituximab.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050064.
The MedlinePlus has an encyclopedia page on lymphoma and a list of links to other sources of information on lymphoma (in English and Spanish)
The US National Cancer Institute provides information about lymphoma and about statins and cancer prevention (in English and Spanish)
The UK charity Cancerbackup provides information for patients and caregivers on different types of B-cell lymphoma and on rituximab
The US Leukemia and Lymphoma Society also provides information for patients and caregivers about lymphoma