Understanding the Mechanism of Action of Rituximab in the Reversal of Multidrug Resistance in a Non-Hodgkins Lymphoma Cell Line

It has been previously demonstrated that an anti-CD20 monoclonal antibody (MAb) can reverse MDR in B lymphoma cells in vitro. However, the mechanisms underlying this effect are unknown. A recent study showed that anti-CD20 MAbs could induce rapid redistribution of CD20 into a detergent-insoluble embrane compartment (lipid rafts). By redistributing CD20 into rafts, Rituximab(tm) (RTX) modified their stability and organization. P-glycoprotein (P-gp) is the constituent protein of MDR tumor cells and is responsible for pumping chemotherapeutic agents out of cells. Because ~40% of P-gp is contained in lipid rafts, we hypothesized that when CD20 translocated into lipid rafts, it would displace P-gp. This displacement would lead to the reversal of MDR. To this end, we determined the function of the P-gp pump in the presence or absence of MAbs. In addition, the effects of dose and incubation time with MAbs and chemotherapeutic drugs were determined using Namalwa/MDR1 and three drug-sensitive cells. Finally, the iv distribution of P-gp and CD20 in membranes was monitored after treatment with MAbs by western blot analysis. We found that RTX inhibited the function of the P-gp pump while other anti-CD20 MAbs had no effect. RTX-mediated growth-inhibition of Namalwa/MDR1 cells was both dose- and time-dependent. Namalwa/MDR1 cells were resistant to doxorubicin and vincristine, but RTX rendered the Namalwa/MDR1 cells as chemosensitive as the parental Namalwa cells. RTX induced the translocation of CD20 into lipid rafts and the translocation of P-gp out of rafts. Our results supported our hypothesis that the ability of RTX to reverse MDR was initiated when CD20 was translocated into lipid rafts. This coincided with the translocation of P-gp out of rafts. When P-gp was no longer present in rafts, it lost activity and MDR was reversed.