Sensitization of Rituximab-Resistant Ramos RR1 and Daudi RR1 Clones to Various Chemotherapeutic Drugs by a Novel Nuclear Factor-κB Activation Inhibitor.

Rituximab (chimeric anti-CD20 mAb) has been recently shown to signal NHL B-cell lines (e.g., Ramos and Daudi) and inhibits the constitutively activated NF-κB signaling pathway. This results in the selective downregulation of the anti-apoptotic Bcl-_xL expression and sensitization to various chemotherapeutic drug-induced apoptosis (Jazirehi et al., 2004. Submitted). The novel NF-κB factor inhibitor, dehydroxymethyl derivative of epoxyquinomicin (DHMEQ), inhibits NF-κB activity via inhibition of nuclear (translocation) accumulation of p65 (Ariga et al., JBC 277:24625, 2002). This study examined the similarities and differences observed in wild type (wt) parental and rituximab-resistant Ramos and Daudi cells following treatment with either rituximab (20 mg/ml) or DHMEQ (10 mg/ml). In the wt cells, both agents inhibited NF-κB DNA-binding activity, down-regulated Bcl-_xL expression and sensitized the cells to drug-induced apoptosis. Rituximab inhibited both the NIK—IκB-α—IKK—NF-κB and the Raf-1—MEK1/2—ERK1/2—AP-1 signaling pathways, while DHMEQ specifically inhibited NF-κB activity. We have generated rituximab-resistant Ramos and Daudi clones by continuous exposure of the cells to step-wise increasing concentrations of rituximab for several weeks and clones isolated by limiting dilution. The CD20⁺ expressing Ramos RR1 and Daudi RR1 clones were analyzed for their response to treatment with rituximab or DHMEQ. Compared to the wt cells, the resistant clones exhibited hyper-activation of the NF-κB pathway, increased expression of Bcl-_xL, and higher resistance to structurally and functionally distinct drugs; their treatment with DHMEQ (15 mg/ml), but not with rituximab, inhibited the activity of the hyper-activated NF-κB, reduced the over-expressed Bcl-_xL and sensitized the highly drug-resistant Ramos RR1 and Daudi RR1 clones to various chemotherapeutic drug (CDDP, ADR, VP-16, Taxol, etc.)-induced apoptosis. These findings demonstrate that DHMEQ can reverse the acquired drug-resistance phenotype in both parental and rituximab/drug-resistant NHL B-cell lines when used in combination with subtoxic concentrations of drugs. In vivo, DHMEQ inhibits subcutaneously transplanted prostate cancer xenografts in nude mice (Kikuchi et al., Cancer Res. 63:107, 2003). Consequently, the present findings suggest the potential therapeutic application of DHMEQ in combination with drugs, in the treatment of rituximab/drug resistant B-NHL.