Apoptosis In Mantle Cell Lymphoma Cells Research Articles

Co-Treatment with Panobinostat Enhances Bortezomib-Induced Unfolded Protein Response, Endoplasmic Reticulum Stress and Apoptosis of Human Mantle Cell Lymphoma Cells

The 26S proteasome inhibitor bortezomib (BZ), which increases intracellular unfolded protein levels and toxicity through endoplasmic reticulum (ER) stress response, was shown to have a single agent activity in relapsed mantle cell lymphoma (MCL). Here we have determined that treatment with hydroxamic acid analogue (HA) pan-histone deacetylase (HDAC) inhibitor (HDI), e.g., panobinostat (LBH589, Novartis Pharmaceuticals Inc) induces the CDK inhibitors p21 and p27, and attenuates the levels of c-Myc, CDK4 and cyclin D1 in the cultured (Jeko-1, MO-2058 and Granta-519) and in primary patient-derived MCL cells. In a dose-dependent manner, panobinostat also induced Bax and Bak, and attenuated Bcl-xL, XIAP, survivin, AKT and c-Raf levels, resulting in growth inhibition and apoptosis of MCL cells. We have previously demonstrated that HDAC6 deacetylates heat shock protein (hsp) 90, as well as shuttles and sequesters misfolded and polyubiquitylated proteins into the protective perinuclear aggresome.. By inhibiting HDAC6, panobinostat (10 to 50 nM) induced acetylation of hsp90 in MCL cells. This inhibited the ATP binding and co-chaperone association, and abrogated the chaperone function of hsp90 for the MCL- relevant, hsp90 client proteins, e.g., cyclin D1, CDK4, c-Raf and AKT in the cultured and primary MCL cells. Panobinostat mediated inhibition of HDAC6 abrogated formation of the aggresome and augmented endoplasmic reticulum (ER)-based unfolded protein response (UPR). Treatment of MCL cells with BZ induced the formation of aggresome (as detected by confocal immuno-fluorescence microscopy and electron microscopy), as well as induced UPR and ER stress response. The latter was associated with BZ-mediated increased levels of GRP78, the spliced form of XBP1 (XBP1s) and p-eIF2α protein. As compared to the control siRNA treated cells, knockdown of GRP78 by siRNA markedly increased BZ-induced CHOP and Noxa levels and significantly augmented BZ-induced apoptosis of cultured MCL cells. Co-treatment of MCL cells with panobinostat abrogated BZ-induced aggresome formation, decreased the levels of ATF4, XBP1s and p-eIF2α, as well as increased the levels of CHOP, Noxa and GADD34. Ultrastructural analysis of Jeko-1 cells also revealed that co-treatment with panobinostat and BZ showed pronounced ER dilatation compared to panobinostat treatment alone, suggestive of enhanced ER stress. Higher and persistent CHOP and Noxa levels suggested a protracted ER-stress, associated with synergistic increase in apoptosis of MCL but not normal CD34+ bone marrow progenitor cells (p < 0.01). Conversely, knockdown of CHOP levels by siRNA significantly inhibited panobinostat and BZ-induced cell death of MCL cells. Results of ongoing in vivo studies of panobinostat and/or BZ in the NOD/SCID mouse xenograft model of Jeko-1 MCL cells will be presented. These findings strongly support further in vivo evaluation of the efficacy of the combination of panobinostat with BZ against human MCL. Additionally, the findings create the rationale to develop targeted knockdown of GRP78 as a novel strategy to augment lethal ER stress due to panobinostat and BZ and resulting activity against MCL cells.

Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteasome inhibitor bortezomib in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an incurable B cell lymphoma, and novel treatment strategies are urgently needed. We evaluated the effects of combined treatment with the proteasome inhibitor bortezomib and the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) on MCL. Bortezomib acts by targeting the proteasome, and--among other mechanisms--results in a reduced nuclear factor-kappa B (NF-kappaB) activity. HDACi promote histone acetylation, and also interfere with NF-kappaB signaling. Human MCL cell lines (JeKo-1, Granta-519 and Hbl-2) were exposed to bortezomib and/or SAHA. Cell viability and apoptosis were quantified by the MTT and annexin-V assay, respectively. Reactive oxygen species (ROS) were analyzed using the fluorophore H2DCFDA. In addition, activated caspases, proteasome- and NF-kappaB activity were quantified. Combined incubation with bortezomib and SAHA resulted in synergistic cytotoxic effects, as indicated by combination index values <1 using the median effect method of Chou and Talalay. The combination of both inhibitors led to a strong increase in apoptosis as compared to single agents and was accompanied by enhanced ROS generation, while each agent alone only modestly induced ROS. The free radical scavenger N-acetyl-L-cysteine blocked the ROS generation and reduced the apoptosis significantly. In addition, coexposure of bortezomib and SAHA led to increased caspase-3, -8 and -9 activity, marked reduction of proteasome activity and decrease of NF-kappaB activity. This is the first report giving evidence that SAHA and bortezomib synergistically induce apoptosis in MCL cells. These data build the framework for clinical trials using combined proteasome and histone deacetylase inhibition in the treatment of MCL.

Targeting Histone Deacetylases and Unfolded Protein Mediated Endoplasmic Reticulum (ER) Stress as a Strategy Against Human Mantle Cell Lymphoma.

Poor clinical outcome of therapy of Mantle Cell Lymphoma (MCL) has generated the need to develop and test novel treatments for human MCL. Here we have determined that treatment with hydroxamic acid analogue (HA) pan-histone deacetylase (HDAC) inhibitor (HDI), e.g., LBH589 (Novartis Pharmaceuticals Inc) and vorinostat (Merck Pharmaceuticals), induces the CDK inhibitors p21 and p27, and attenuates the levels of c-Myc, CDK4 and cyclin D1 in the cultured (Jeko-1, MO-2058 and Granta-519) and in primary patient-derived MCL cells. In a dose-dependent manner, HA-HDI also induced Bax, Bak and Bim, and attenuated Bcl-xL, XIAP, survivin, AKT and c-Raf levels, resulting in growth inhibition and apoptosis of MCL cells. We have previously demonstrated that HDAC6 deacetylates heat shock protein (hsp) 90. By inhibiting HDAC6, both LBH589 (10 to 50 nM) and vorinostat (0.5 to 2.0 uM) induced acetylation of hsp90 in MCL cells. This inhibited the ATP binding and co-chaperone association, and abrogated the chaperone function of hsp90 for the MCL- relevant, hsp90 client proteins, e.g., cyclin D1, CDK4, c-Raf and AKT in the cultured and primary MCL cells. HDAC6 has been shown to shuttle and sequester misfolded and polyubiquitylated proteins into the protective perinuclear aggresome. Present studies demonstrate that inhibition of HDAC6 abrogates formation of the aggresome and augments the ER-based unfolded protein response (UPR). Treatment of MCL cells with the proteasome inhibitor bortezomib (BZ) induced the formation of aggresome (as detected by confocal immuno-fluorescence microscopy and electron microscopy), as well as induced UPR and ER stress response. The latter was associated with BZ-mediated increased levels of the spliced form of XBP1 (XBP1s) and p-eIF2α protein. It was also associated with increased levels of the protective ER chaperone protein GRP78, and increased expression of pro-death proteins, CHOP and Noxa. Treatment with BZ or HA-HDI also increased the expression of the transcriptional repressor, PRDM1. Co-treatment of MCL cells with LBH589 abrogated BZ-induced aggresome formation, but increased the levels of BZ-induced XBP1s and p-eIF2α, indicating increased ER stress response. Concomitantly, higher CHOP and Noxa levels suggested a protracted ER-stress, associated with significantly increased apoptosis of MCL cells (p < 0.01). These findings suggest that co-treatment with LBH589 accentuates BZ-induced ER-stress and cell death of MCL cells despite up-regulation of GRP78 levels. Next, we determined the effects of knocking down GRP78 on BZ-induced ER-stress response. As compared to the control siRNA treated cells, knockdown by siRNA to GRP78 markedly increased BZ-induced CHOP and Noxa levels and significantly augmented BZ-induced apoptosis of cultured MCL cells. Collectively, these findings strongly support the in vivo testing of the efficacy of the combination of HA-HDI with BZ in inducing protracted and lethal ER stress in MCL cells. These results also create the rationale to develop targeted knockdown of GRP78 as a novel strategy to augment the lethal ER stress in human MCL cells.

Bortezomib Is Synergistic with Rituximab and Cyclophosphamide in Inducing Apoptosis of Mantle Cell Lymphoma Cells In Vitro and In Vivo.

Introduction: Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor clinical outcome. Although frontline therapy induces a high rate of complete remission, relapse is inevitable and new regimens are needed for relapsed MCL. The proteasome inhibitor bortezomib (BTZ) induces apoptosis and sensitizes MCL cells to chemotherapy in relapsed MCL, but as a single agent, response rate is low, duration of response is short and side effects are severe. Here we evaluated whether BTZ is additive or synergistic with cyclophosphamide (CTX) and rituximab (RTX).Material and Methods: Four human MCL cell lines SP53, MINO, Grant 519, and Jeko-1 and freshly isolated primary tumor cells from three MCL patients were treated with BTZ, CTX, RTX individually or in combination of RTX and CTX (RC), or BTZ plus RTX and CTX (BRC regimen). Cell proliferation and apoptosis were evaluated to determine if there was additive or synergistic effect of the BRC regimen. Western blot analysis was used to elucidate the molecular mechanism by which BTZ, RTX, CTX, RC and BRC induces apoptosis in MCL cells. In addition, in vivo experiments using severe combined immunodeficiency mice with human mantle cell lymphoma xenografts were performed to examine the in vivo efficacy of the regimen to control the growth of and eradicate MCL cells.Results: BTZ and CTX as single agents inhibited the growth of MCL cell lines in a dose-dependent manner (P < 0.01). The IC50 (inhibitory concentration at 50%) for BTZ and for CTX were between 10 and 20 nM and between 5 and 20 mM, respectively. Increasing doses of BTZ with a fixed dose of RTX (10 μg/mL) and CTX (10 mM) resulted in markedly synergistic growth inhibition of MCL cells (P < 0.01). The BRC regimen induced apoptosis in about 69.7% of MCL cell lines and 92.6% of primary tumor cells (P < 0.05 and P < 0.01, compared with those induced by BTZ, RTX, CTX or RC). Furthermore, western blotting analysis showed that BRC induced apoptosis earlier via activation and cleavage of caspases-8, -9, and -3, and PARP as compared with BTZ, RTX, CTX or RC. The pan-caspase inhibitor z-VAD-FMK completely blocked apoptosis induced by BRC. In vivo studies demonstrated that BRC regimen eradicated subcutaneous tumors in MCL-bearing SCID mice and significantly prolonged the long-term event-free survival up to 10 weeks in 70% of the mice, whereas all tumor-bearing mice receiving BTZ, RTX, CTX or RC or PBS (control) died of aggressive MCL within 6 weeks.Conclusion: Cytoreductive chemotherapy with both BTZ and anti-CD20 antibody effectively inhibited the growth and induced apoptosis of MCL cells in vitro and in vivo. Bortezomib-rituximab-cyclophosphamide (BRC) regimen may offer a better therapeutic modality for MCL patients. Thus, our data lay the basis for a clinical trial in relapsed MCL using the BRC combination treatment.

Atiprimod inhibits the growth of mantle cell lymphoma in vitro and in vivo and induces apoptosis via activating the mitochondrial pathways

AbstractAtiprimod is a novel cationic amphiphilic compound and has been shown to exert antimyeloma effects both in vitro and in mouse experiments. This study was undertaken to evaluate the therapeutic efficacy of atiprimod on mantle cell lymphoma (MCL) and elucidate the mechanism by which it induces cell apoptosis. Atiprimod inhibited the growth and induced apoptosis of MCL cell lines and freshly isolated primary tumor cells in vitro. More importantly, atiprimod significantly inhibited tumor growth in vivo and prolonged the survival of tumor-bearing mice. However, atiprimod also exhibited lower cytotoxicity toward normal lymphocytes. Atiprimod activated c-Jun N-terminal protein kinases (JNK) and up-regulated the level of Bax, Bad, and phosphorylated Bcl-2, resulting in release of apoptosis-inducing factor (AIF) and cytochrome c from mitochondria and activation and cleavage of caspase-9, caspase-3, and PARP. However, AIF, but not activation of caspases or PARP, was responsible for apoptosis in MCL cells because an AIF inhibitor, but not pan-caspase or paspase-9 inhibitors, completely abrogated atiprimod-induced apoptosis. Taken together, our results demonstrate that atiprimod displays a strong anti-MCL activity. Cell apoptosis was induced mainly via activation of the AIF pathway. These results support the use of atiprimod as a potential agent in MCL chemotherapy.

Evaluation of novel cell cycle inhibitors in mantle cell lymphoma

Signature abnormalities in the cell cycle and apoptotic pathway have been identified in mantle cell lymphoma (MCL), affording the opportunity to develop targeted therapies. In this study, we tested a novel class of kinase inhibitors, styryl sulfones, which differ from prior cell cycle inhibitors in that they are not related to purines or pyrimidines. We observed that two closely related compounds, ON013100 and ON01370, altered the growth and cell cycle status of MCL lines and potently inhibited the expression of several important molecules, including cyclin-dependent kinase 4, p53, mouse double minute 2 (MDM2), and cyclin D as well as increased cyclin B expression. Using both terminal deoxy transferase uridine triphosphate nick end-labelling and poly ADP-ribose polymerase assays, we found that these compounds caused apoptosis in MCL cells. In addition, using molecular analyses, we observed the modulation of caspase-3 activity but not the expression of B-cell lymphoma family molecules. Next, we investigated the cytotoxicity of the MCL lines upon treatment with styryl sulfone compounds in combination with other currently used chemotherapeutic agents, such as doxorubicin (DOX) or vincristine (VCR). We found that the combination of DOX plus styryl sulfone or VCR plus styryl sulfone increased cytotoxicity by one log scale, compared with the single styryl sulfone compound. Thus, styryl sulfones alone, or in combination with chemotherapeutic agents, present attractive opportunities for new drug development in MCL.

Atiprimod Inhibits Growth of Mantle Cell Lymphoma In Vitro and In Vivo and Induces Apoptosis Via Activation of the Mitochondrial Pathway.

Mantle cell lymphoma (MCL) has poor outcome and is a therapeutic challenge. Atiprimod, a cationic amphiphilic compound, inhibits the proliferation of most human tumor cell lines. Our study was undertaken to evaluate the therapeutic efficacy of Atiprimod on MCL cells and elucidate the mechanism by which it induces cell apoptosis. Four human MCL cell lines, SP53, MINO, Grant 519, and Jeko-1; freshly isolated primary MCL cells from three MCL patients; and normal peripheral blood mononuclear cells (PBMCs) from five healthy donors were treated with Atiprimod. A 3H-thymidine incorporation assay showed that Atiprimod not only inhibited the growth of the MCL cell lines SP53, MINO, Grant 519, and Jeko-1, but also freshly isolated patient MCL cells in a dose-dependent manner. Interestingly, Atiprimod also inhibited the proliferation of PHA-, PMA/ionomycin-, or anti-CD3 mAb/anti-CD28 mAb-activated, but not resting PBMCs from healthy donors. Flow cytometry analysis with fluorescence-labeled Annexin V and propidium iodide showed that Atiprimod induced apoptosis in both the cell lines and primary MCL cells in time-dependent and dose-dependent manners. Notably, Atiprimod did not induce apoptosis of normal T cells and B cells. Atiprimod was also effective and therapeutic in a MCL mouse model established in severe combined immunodeficient (SCID) mice. SP53 cells (5 × 106) were inoculated subcutaneously into the right leg of SCID mice. Three weeks later, after palpable tumors developed, mice were treated intraperitoneally with either vehicle alone (PBS) or Atiprimod (25 mg/kg per day) for 6 consecutive days. Tumor growth was significantly inhibited after Atiprimod treatment compared with vehicle control, and the survival time of tumor-bearing mice was significantly prolonged in the treatment group. Western blot analysis showed that apoptosis of MCL cells was induced through the activation of caspase-9, caspase-3, and PARP pathway. Furthermore, the expression of phosphorylated Bcl-2, Bax, Bad, Bax-xL, and cytochrome C was increased in Atiprimod-treated MCL cells. Pretreatment of cells with caspase-9 inhibitor (z-LEHD) or pan-caspase inhibitor (z-VAD) but not caspase-8 inhibitor (z-IETD) completely blocked Atiprimod-induced apoptosis. In conclusion, Atiprimod inhibits growth and induces apoptosis of MCL cells in vitro and in vivo. Cell apoptosis was induced via activation of the mitochondrial signaling pathway. The therapeutic efficacy of Atiprimod on MCL cells in the mouse model supports the use of Atiprimod as a potential agent in MCL chemotherapy.

Evaluation of Novel Cell Cycle Inhibitors in Mantle Cell Lymphoma.

Signature abnormalities in the cell cycle and apoptotic pathway have been identified in mantle cell lymphoma (MCL), affording the opportunity to develop targeted therapies. Here, we report on a novel class of kinase inhibitors, styryl sulfones, that differ from prior cell cycle inhibitors in that they are not related to purines or pyrimidines. We observed that two closely related compounds, ON013100 and ON01370, altered the growth and cell cycle status of MCL lines and potently inhibited the expression of several important molecules, including CDK4, p53, MDM2, cyclin D, and cyclin B. Using both TUNEL and PARP assays, we found that these compounds caused apoptosis in MCL cells. In addition, using molecular analyses, we observed the modulation of caspase-3 activity but not the expression of Bcl family molecules in these cells. Next, we investigated the cytotoxicity of the MCL lines upon treatment with styryl sulfone compounds in combination with other currently used chemotherapeutic agents, such as doxorubicin (DOX) or vincristine (VCR). We found that the combination of DOX plus styryl sulfone or VCR plus styryl sulfone increased cytotoxicity by one log scale, compared with the single styryl sulfone compound. Thus styryl sulfones alone, or in combination with chemotherapeutic agents, present attractive opportunities for new drug development in MCL.

Bortezomib Synergizes with a Novel Jak2 Inhibitor, WP-1130, To Inhibit Cell Growth and Induce Apoptosis in “Classic” and “Blastoid-Variant” Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma B cells (NHL-B), a lymphoma with increasing incidence over the last few decades. MCLs are classified into at least two subgroups, “classic” type of MCL and “blastoid-variant”- an even more aggressive type of MCL. Both subgroups are incurable, unusually refractory to standard chemotherapy combinations and associated with poor prognosis. New therapeutic agents with greater efficacy and less toxicity are necessary in MCL. We have shown previously that in MCL cells, the key transcription factor NF-kB, is constitutively active and maintains lymphoma cells survival. We also demonstrated that treating the MCL cells with the proteasome inhibitor bortezomib (Velcade) inhibits constitutive NF-kB activation, leading to G1 cell cycle arrest and apoptosis. Recently, a novel small molecular weight compound called WP-1066, a derivative of AG490, was synthesized by screening a synthetic library for agents that block stat3 activation. WP-1066 has shown to have anti-tumor activity in MCL cells through the inhibition of IL-6 mediated stat3 activation and NF-kB inhibition. A more effective compound called WP-1130, a derivative of WP-1066, was synthesized and is shown to be more potent than WP-1066 in MCL cell lines. Single agents are rarely effective in treating a disease like MCL; therefore, we hypothesized that the combination of bortezomib and WP-1130 would likely be more effective in MCL. We showed that MCL cells, both “classic” and “blastoid-variant”, treated in-vitro with bortezomib in conjunction with WP-1130 resulted in a synergistic growth inhibition and apoptosis induction. The drug concentrations use for bortezomib and WP-1130 that produce the synergistic effects were in the low nanomolar (nM) and micromolar (uM) ranges, respectively. Bortezomib at a concentration of 10 nM induces approximately 15% MCL apoptosis after 48 hr treatment when compare to untreated controls, while WP-1130 at a concentration of 1 uM induces 5% apoptosis. The combination of bortezomib and WP-1130 at the same concentrations induces 60% of MCL cell apoptosis. Bortezomib and WP-1130 showed efficacy in both classic and blastoid-variant MCL cell lines. The apoptotic effects in these cells were correlated with the down-regulation of bcl-2 and the up-regulation of bax proteins. The status of constitutive NF-kB was also examined after drug treatments. While a single agent treatment with low drug concentrations had only minimal effect on NF-kB inhibition, the combination of the two drugs dramatically inhibits NF-kB activation. These two drugs also synergize to inhibit cyclin D1 (a molecular signature of MCL), and c-myc (an oncogene commonly over-expressed in lymphoma B cells). Agents such as bortezomib and WP-1130, that can pharmacologically modulate key intracellular targets such as constitutively expressed NF-kB and cyclin D1 in MCL cells, may be more effective therapeutic agents for the treatment of MCL.

Sonic Hedgehog Signaling in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is associated with poor clinical outcome among all the B-cell malignancies. MCL cells have a characteristic phenotype including IgM+, IgD+, CD5+, CD10−, CD19+, CD20+, Bcl2+, CD23−, CD24+, and have a (11:14) (q13:q32) translocation, involving the Bcl-1 locus and the IgH heavy chain joining region, resulting in up-regulation of Bcl-1/PRAD-1 with increased Cyclin D1 expression. In spite of our understanding of B cell development, we do not have effective therapeutic approach to treat MCL. This warrants an immediate understanding of the biology MCL cells, and key signaling pathways underlying the disease progression. Further, these key signaling pathways involved in MCL could be specifically targeted, which may have potential therapeutic value.Emerging evidences have revealed that sonic hedgehog (Shh) signaling promotes tumor cell proliferation, and protects them from apoptosis in different cancers such as pancreatic cancer, prostate cancer, gastric cancer, medulloblastoma, basal cell carcinoma, breast cancer, etc. In addition, targeting the Shh signaling has been shown as a potential therapeutic approach to treat some of these cancers. However, the role of Shh signaling has not been reported in any of B cell malignancies including MCL. Therefore, we studied the status of Shh signaling molecules in the proliferation and survival of MCL cells in vitro using JVM-2, Granta-519, Jeko-1 and Z138 cell lines and human primary MCL cells. Our results demonstrate that the molecules involved in the Shh signaling like patched and smoothened receptors, and the target transcription factors, GLI were over expressed in Granta-519, Jeko-1, Z-138 and JVM-2 MCL cell lines, and patients primary MCL cells compared to normal human B lymphocytes. Addition of exogenous Shh increased the proliferation of JVM-2 cells in vitro. There was an increased transcripts level of GLI1, BCL2 and Cyclin D1 (CCND1) in JVM-2 cells following the addition of Shh. Addition of Shh-signaling inhibitor Cyclopamine abrogated the increased proliferation and transcription of the above genes induced by Shh in JVM-2 cells (figure 1). Furthermore, the Gli2 anti-sense oligonucleotide decreased CCND1 and BCL2 transcript expression, as well as decreased the proliferation of JVM2 cells in vitro (figure 2). Taken together, these results suggest that Shh-Gli signaling may be one of the pathways that regulate the Bcl-2 and Cyclin D1 activation and thereby regulate the proliferation and apoptosis of MCL cells. Therefore, further dissecting of the Shh-Gli mediated regulation of Bcl2 signaling may have potential implications in advancing the treatment for MCL. [Display omitted] [Display omitted]

Arsenic Trioxide in Mantle Cell Lymphoma.

Abstract Background. Mantle cell lymphoma (MCL) is incurable for many patients. Arsenic trioxide (As2O3) has activity in vitro against lymphoid malignancies. The effects of As2O3 on MCL in vitro and in patients with refractory disease were investigated. Materials and methods. Mantle cell lymphoma (MCL) lines (Jeko-1, Granta-519) were treated with As2O3, in combination with mitoxantrone (MTZ) and ascorbic acid (AA). Consenting patients with refractory MCL were treated with oral-As2O3 (10 mg/day), AA (1 g/day) and chlorambucil (4 mg/day) as outpatients until maximum response or the disease judged refractory. Responses were defined by standard NCI criteria. In patients showing an initial response, vincristine (2 mg intravenously) and prednisolone (30 mg/day) might also be added. After achievement of maximum response, patients were maintained with As2O3 (10 mg/day) and AA (1 g/day) for two weeks every month, for a planned two years. Results. As2O3 and MTZ but not AA induced a dose dependent apoptosis in the MCL lines, as shown by flow cytometry and MTT assays. As2O3, MTZ and AA were tested in various combinations in MTT assays. Synergistic interactions were observed only in the combinations As2O3 (1 uM) + MTZ (0.2 mg/L), and As2O3 (1 uM) + MTZ (0.2 mg/L) + AA (100 uM). Western blotting showed that As2O3-induced apoptosis was associated with a dose and time dependent down-regulation of cyclin D1. However, quantitative polymerase chain reaction showed no change in cyclin D1 gene transcription during As2O3-induced apoptosis. Eleven patients (10 men, 1 women) at a median age of 69 (51–70) years with refractory MCL were studied, at a median of 33 (8–85) months from diagnosis. At the time of As2O3 treatment, they had already had a median of 2 (1 – 4) relapses managed with a median of 2 (1 – 6) previous chemotherapeutic regimens. Eight of eleven patients were evaluable (for the other three, one died after 4 days of treatment, and the two were still receiving therapy). At a median follow up of 9 (4 – 20) months, 4 patients had reached complete remission (CR) or probable CR (CRu), two were in good partial remission, and two had died with progressive disease. Conclusion. As2O3 induced apoptosis of MCL cells by post-transcription down-regulation of cyclin D1. Synergistic interactions were observed with AA and cytotoxics. Oral-As2O3, AA and chlorambucil were an active regimen for relapsed and therapy-refractory MCL, and treatment results compared favorably with other salvage regimens. This entirely oral regimen has several attractions, including outpatient treatment, low toxicity and cost.