Abstract
Radioimmunotherapy (RIT) is an emerging treatment option for non-Hodgkin lymphoma (NHL) producing higher overall response and complete remission rates compared with unlabelled antibodies. However, the majority of patients treated with conventional or myeloablative doses of radiolabelled antibodies relapse. The development of RIT with alpha-emitters is attractive for a variety of cancers because of the high linear energy transfer (LET) and short path length of alpha-radiation in human tissue, allowing higher tumour cell kill and lower toxicity to healthy tissues. In this study, we investigated the molecular effects of the alpha-emitter Bi-213 labelled to anti-CD20 antibodies ([Bi-213]anti-CD20) on cell cycle and cell death in sensitive and radio-/chemoresistant NHL cells. [Bi-213]anti-CD20 induced apoptosis, activated caspase-3, caspase-2 and caspase-9 and cleaved PARP specifically in CD20-expressing sensitive as well as in chemoresistant, beta-radiation resistant and gamma-radiation resistant NHL cells. CD20 negative cells were not affected by [Bi-213]anti-CD20 and unspecific antibodies labelled with Bi-213 could not kill NHL cells. Breaking radio-/ chemoresistance in NHL cells using [Bi-213]anti-CD20 depends on caspase activation as demonstrated by complete inhibition of [Bi-213]anti-CD20-induced apoptosis with zVAD.fmk, a specific inhibitor of caspases activation. This suggests that deficient activation of caspases was reversed in radioresistant NHL cells using [Bi-213]anti- CD20. Activation of mitochondria, resulting in caspase-9 activation was restored and downregulation of Bcl-x(L) and XIAP, death-inhibiting proteins, was found after [Bi- 213]anti-CD20 treatment in radio-/chemosensitive and radio-/chemoresistant NHL cells. [Bi-213]anti-CD20 seems to be a promising radioimmunoconjugate to improve therapeutic success by breaking radio- and chemoresistance selectively in CD20- expressing NHL cells via re-activating apoptotic pathways through reversing deficient activation of caspases and the mitochondrial pathway and downregulation of XIAP and Bcl-x(L).
Highlights
A quite novel and promising treatment option for B-cell non-Hodgkin lymphoma (NHL) are radioimmunotherapies (RIT) using monoclonal antibodies as vehicle to selectively target malignant cells with the coupled radionuclide [1]
RIT could be proven promising to overcome the limitations of unconjugated antibodies: The anti-CD20radioimmunoconjugates [I-131]tositumomab (Bexxar®) and [Y-90]ibritumomab-tiuxetan (Zevalin®) produce higher overall response and complete remission rates compared with unlabelled antibodies [7]
Immunotherapy using monoclonal antibodies like Rituximab targeting the B-cell-antigen CD20 has Figure 6: [Bi-213]anti-CD20 induced apoptosis depends on caspase activation. (A,B,C) The CD20-positive (CD20+/+) radiosensitive DoHH-2 (A), the CD20-positive (CD20+/+) beta-radiation resistant DoHH-2betaR (B), and the CD20-positive (CD20+/+) gamma-radiation resistant DoHH-2gammaR cells (C) pre-treated with 50μM of the broad-range caspase inhibitor zVAD.fmk (+zVAD; white columns) or without pre-treatment (-zVAD; black columns) were incubated with different activity concentrations of [Bi-213]anti-CD20 and time points as indicated
Summary
A quite novel and promising treatment option for B-cell non-Hodgkin lymphoma (NHL) are radioimmunotherapies (RIT) using monoclonal antibodies (mabs) as vehicle to selectively target malignant cells with the coupled radionuclide [1]. Chemo-/Radioresistance of NHL are often associated with the overexpression of different molecules like XIAP or Bcl-xL [22] Targeting these proteins by inhibition or downregulation is a novel approach in cancer therapy [23]. It was shown that the alpha-emitter Bi-213 labelled to an antiCD45-antibody has the capacity to abrogate chemo- and radioresistance in leukaemia cells via caspase activation and activation of the mitochondrial apoptotic pathway in vitro [24]. Our study demonstrates that after a G2-phase arrest, [Bi-213] anti-CD20 leads to apoptosis induction via activation of caspases using the mitochondrial pathway in sensitive as well as in radio- and chemoresistance in NHL B-cells. [Bi-213]anti-CD20 induces apoptosis in NHL which are resistant to anti-CD20 antibodies or to antibodies labelled with Y-90. [Bi-213] bound to antiCD20 seems to be a promising therapeutic strategy in the treatment of NHL especially if conventional therapeutic modalities failed
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