The combination of TRAIL and MG-132 induces apoptosis in both TRAIL-sensitive and TRAIL-resistant human follicular lymphoma cells.

We have previously shown that the human follicular lymphoma cell line, HF28GFP, is sensitive to TRAIL-mediated apoptosis. Nevertheless, when the same cells overexpress anti-apoptotic Bcl-2 family protein, Bcl-xL (HF28Bcl-xL), they become resistant to TRAIL. Thus, these cell lines help us to investigate the action of novel apoptosis inducing candidate drugs. In the present study, we examined the effects of MG-132 (a proteasome inhibitor), LiCl (a glycogen synthase kinase-3 inhibitor) and/or TRAIL on pro-apoptotic Bcl-2 family proteins such as Bim and Bid. Here we demonstrate that the combination of MG-132 and TRAIL induced significant apoptotic cell death in both cell lines, HF28GFP and HF28BclxL. Apoptosis correlated with a decrease of phospho-ERK1/2, the accumulation of Bim and translocation of truncated Bid (tBid) and jBid. In addition, the combination of MG-132 and TRAIL seemed to target other apoptotic factors, which led to the accumulation of active capsase-3. Furthermore, co-stimulation of LiCl and TRAIL induced apoptosis in HF28GFP cells. However, HF28Bcl-xL cells were far less sensitive to the combinatorial effects of LiCl and TRAIL. Interestingly, we observed that LiCl did not target Bim and Bid proteins. In conclusion, these data show that targeting of pro-apoptotic Bcl-2 family proteins simultaneously through a selective proteasome inhibition might help to overcome TRAIL resistance caused by overexpression of anti-apoptotic Bcl-2 family proteins. Moreover, the data may provide new strategies to develop targeted therapies against lymphomas.

RIP1 has a role in CD40-mediated apoptosis in human follicular lymphoma cells.

CD40 is a cell surface receptor which belongs to tumor necrosis factor receptor (TNFR) family members. It transmits signals that regulate diverse cellular responses such as proliferation, differentiation, adhesion molecule expression and apoptosis. Unlike other TNFR family members (TRAIL-R, Fas-R and TNFR1), the CD40 cytoplasmic tail lacks death domain. However, CD40 is capable of inducing apoptosis in different types of cancer cells including lymphoma. The apoptotic effect of CD40 is linked to the involvement of Fas, TRAIL or receptor interacting protein 1 (RIP1) kinase. We have previously shown that CD40 activation has anti-apoptotic or apoptotic effect in follicular lymphoma (FL) cell lines. In this study, we investigated the mechanism by which CD40 mediates apoptosis in a follicular lymphoma cell line, HF4.9. We show here that CD40-induced apoptosis was dependent on caspase-8 activation because caspase-8 specific inhibitor, Z-IETD-FMK completely prevented apoptosis. Therefore, the involvement of TRAIL, Fas and RIP1 in caspase-8 activation was examined. The exogenous TRAIL-induced apoptosis was fully prevented by anti-TRAIL neutralizing antibody. However, the antibody had no effect on CD40-induced apoptosis indicating that CD40 did not induce the expression of endogenous TRAIL in HF4.9 cells. Moreover, the cells were not sensitive to Fas-mediated apoptosis. Interestingly, RIP1 specific inhibitor, necrostatin-1 decreased CD40-induced apoptosis, which showed that RIP1 has a role in caspase-8 activation. In conclusion, the survival or apoptotic effects of CD40-mediated signaling might be related to the differentiation stages of FL cells.

Advantages of targeting B cell receptor complex to treat B-cell derived autoimmune diseases and lymphomas.

Antibodies produced by B-cells provide protection from infectious agents. However, impaired cell death signaling pathways in B-cells can lead to cancer, immunodeficiency or autoimmune diseases. B-cell signaling molecules such as CD20, CD19, Btk, and BAFF-R are targeted by therapeutic drugs and used to treat B-cell derived lymphomas or autoimmune diseases. Nevertheless, B-cells could develop resistance to these therapeutic drugs or the therapeutic drugs may have off-target effects. For instance, repeated rituximab (anti-CD20 antibody) treatment may lead to the loss of its target cell surface molecule, CD20. In addition, in B-cell malignancies, loss of CD19 expression has been observed. Another target molecule, Btk is expressed not only in B-cells but also in mast cells, macrophages, and dendritic cells. Thus, targeting Btk could negatively regulate the functions of innate immunity. The expression of BAFF-R is thought to be restricted to B-cells but it is also expressed on T-cells. Targeting BAFF-R, therefore, may lead to depletion of T-cells in addition to B-cells. B cell receptor (BCR) expression and signaling, however, are critically important for development, differentiation and survival of B-cells. Moreover, BCR is exclusively expressed on B-cells, which makes it an excellent target to avoid off-target effects.

Timing determines dexamethasone and rituximab induced synergistic cell death.

Dysregulation of cell death signaling pathways in many cell types such as B lymphocytes (B-cells) can lead to cancer, for example to B-cell lymphomas. Rituximab (RTX) and glucocorticoids such as dexamethasone (Dex) are widely used to treat hematological malignancies including B-cell lymphomas. Although the combination of Dex and RTX improves the treatment outcome of lymphoma patients, most lymphomas remain incurable diseases. Therefore, a detailed investigation of Dex- and RTX-induced signaling might provide new insights into the therapeutic benefits of these drugs. In this paper, we describe Dex- and RTX-induced signaling pathways and their downstream target proteins/cells. In addition, we also overview how the signaling initiated by Dex and RTX modulate the outcome of Dex- and RTX-mediated cell death in lymphoma cells. The combination of Dex and RTX results in massive cell death in lymphoma cells. However, pretreatment of lymphoma cells or mononuclear cytotoxic cells with Dex followed by RTX leads to a decrease in apoptosis or it impairs antibody-dependent cellular cytotoxicity (ADCC). RTX-mediated ADCC is impaired by Dex-induced depletion of cytotoxic cells, whereas RTX-mediated short-term ERK1/2 activation decreases Dex-induced apoptosis. Therefore, the timing of the combination of Dex and RTX is a determining factor for the synergistic effect of these cell death inducing agents.

Differential Expression of Bcl-2 Family Proteins Determines the Sensitivity of Human Follicular Lymphoma Cells to Dexamethasone-mediated and Anti-BCR-mediated Apoptosis.

Bcl-2 family comprises proapoptotic and antiapoptotic proteins. The balance between these proteins is critical for the survival of the cells. Overexpression of the antiapoptotic protein, Bcl-2, is the hallmark of follicular lymphoma (FL). High expression of Bcl-2 provides survival advantage and may facilitate chemotherapeutic resistance in FL. In the present study, we examined expression profile of Bcl-2 family proteins such as Bcl-2, Bcl-xL, and Bim in human FL cell lines, HF1A3 and HF28. We assessed the correlation between the expression levels of these proteins and cells' sensitivity to dexamethasone (Dex)-mediated and B-cell receptor (BCR)-mediated apoptosis. Here, we show that Dex and anti-BCR-induced synergistic apoptosis which correlated with significant downregulation of Bcl-xL, inhibition of ERK1/2 phosphorylation and accumulation of nonphosphorylated Bim. However, HF28 cells were less sensitive than HF1A3 cells to Dex-induced and anti-BCR-induced apoptosis due to high Bcl-2 protein level. It is interesting to note that, a Bcl-2-specific inhibitor, ABT-199, sensitized HF28 cells to Dex-induced or anti-BCR-induced apoptosis. In addition, overexpression of Bcl-xL prevented Dex-mediated, anti-BCR-mediated, and ABT-199-mediated apoptosis, indicating that mitochondria were involved. In conclusion, these data show that the expression levels of Bcl-2 family proteins may serve to predict tumor response to BH3 mimetics and the sensitivity of FL cells to Dex-induced and anti-BCR-induced apoptosis. Moreover, our results show that BCR-targeted apoptosis might have therapeutic benefit against FL and B-cell lymphomas.

ERK1/2 has an essential role in B cell receptor- and CD40-induced signaling in an in vitro model of germinal center B cell selection.

Germinal center (GC) B cells undergo apoptosis after B cell receptor (BCR) ligation, unless they receive CD40-mediated survival signal from helper T cells. In the present study, we used a human follicular lymphoma cell line HF1A3, as an in vitro model to study the selection process in germinal centers. We show here that BCR ligation led to immediate ERK1/2 activation and phosphorylations of its downstream targets, Bim EL/L and Bcl-2 (at Ser70) which resulted in short-term survival. On the other hand, during the late phase of BCR signaling, ERK1/2 phosphorylation was inhibited which resulted in apoptosis. In addition, CD40 signaling led to sustained ERK1/2 activation and up-regulation of Bcl-xL in BCR-primed HF1A3 GC B cells. In conclusion, MEK-ERK pathway and Bcl-2 family proteins are crucial players in BCR-mediated survival/apoptosis and CD40-mediated survival.

Rituximab-induced early and late signaling have opposite effects on dexamethasone-induced apoptosis in human follicular lymphoma cells.

The addition of rituximab (RTX) to standard chemotherapy has improved the treatment of B-cell malignancies. We show here that RTX and dexamethasone (Dex) induced synergistic apoptosis in follicular lymphoma cell lines. However, apoptosis was delayed by RTX-induced early protective signaling. RTX-induced early signaling also decreased Dex-induced apoptosis and led to phosphorylation of ERK1/2, Bcl-2 (at serine 70) and phosphorylation/degradation of BimL/EL. All these events were prevented by the MEK inhibitor, UO126. Therefore, we suggest that RTX-induced ERK-mediated signaling events lead to protection from apoptosis during early signaling and that blocking of Bim and Bcl-2 phosphorylation might be used as a novel strategy for lymphoma treatment.