Roscovitine Sensitizes Leukemia and Lymphoma Cells to TRAIL-Induced Apoptosis and Enhances Cell-Mediated Cytotoxicity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 596-596
Author(s):  
Jan Molinsky ◽  
Marie Markova ◽  
Magdalena Klanova ◽  
Michal Koc ◽  
Lenka Beranova ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Lines ◽  
Low Dose ◽  
Hematologic Malignancies ◽  
Additive Effect ◽  
Cytotoxic Agents ◽  
Potential Contribution ◽  
Primary Cells ◽  
Trail Receptors ◽  
Induced Apoptosis

Abstract Abstract 596 Roscovitine is a selective inhibitor of cyclin-dependent kinases (CDK) and it is under evaluation in several clinical trials in the treatment of diverse cancers. TNF-related apoptosis inducing ligand (TRAIL) is a death ligand important for tumor immunosurveillance with selective antitumor activity and minimal toxicity toward tissues. Soluble TRAIL is also under evaluation in several clinical trials. Unfortunatelly, many cancers are resistant to TRAIL. To circumvent TRAIL resistance, there is effort to combinate TRAIL with other cytotoxic agents. By measuring apoptosis and proliferation, we demonstrated that combination of low dose roscovitine and low dose TRAIL (low dose= up to 30% of apoptotic cells after 24h treatment) is synergistic in 20 of 21 tested hematologic cell lines including TRAIL resistant cell lines. Moreover, this combination was tested on primary cells from 9 patients with hematologic malignancies with synergism in 4 of 8 samples from patients with acute myeloid leukemia (AML) and 1 sample from patient with mantle cell lymphoma. Remaining 4 AML samples showed additive effect. Based on these results, we decided to explore molecular mechanisms responsible for the synergism between roscovitine and TRAIL using TRAIL-resistant K562 cells. Despite decreased mRNA, the surface expression of TRAIL receptors remained unaffected after 24h roscovitine treatment. Immunoprecipitation of death-inducing signaling complex (DISC) revealed distinct proapoptotic changes (enhanced CASP8 and 10, reduced FLIP at 12 and 24h). These proapoptotic changes suggested that roscovitine might synergize with other death ligands acting through the DISC, namely TNF and FASLG. Indeed, roscovitine significantly sensitized diverse cell lines (K562, DOHH2, RAMOS) to TNF or FASLG-induced apoptosis. We subsequently proved that pretreatment of the cells (K562, DOHH2, RAMOS) with roscovitine increased by approx. 20% the level of cell-mediated cytotoxicity (peripheral blood mononuclear cells from a healthy volunteer marked with carboxyfluorescein succinimidyl ester). Thus, proapoptotic changes of the DISC seem to play essential role in mediating roscovitine-induced sensitization to TRAIL. Despite detected alterations of the DISC, we decided to unveil additional potential changes in the protein levels of key apoptotic regulators by western blotting at 1.5, 3, 6, 12 and 24h timepoints. Like Ortiz-Ferron et al. we detected gradual downregulation of MCL1 that peaked at 12h, followed, however, by substantial upregulation at 24h. We proved that even at this point, i.e. at 24h exposure to roscovitine, the cells were sensitized to TRAIL-induced apoptosis. The role of MCL1 in mediating the proapoptotic change thus remains elusive. BCL-XL showed similar kinetics as MCL1. Several proapoptotic proteins were overexpressed (BAK and BAD at 1.5h, and PUMA at 1.5h and 24h). Gene-expression profiling unveiled additional changes that might contribute to sensitization to TRAIL, e.g. upregulation of proapoptotic death inducer-obliterator 1 (DIDO1) and downregulation of antiapoptotic DNA-damage-inducible transcript 4 (DDIT4). In contrast to TRAIL (and the other death ligands) roscovitine showed only additive effect or even antagonism with the tested genotoxic agents (cytarabine, doxorubicin, fludarabine, etoposide, cisplatin) probably due to the inhibition of CDK2 by roscovitine (Yu et al., Yanjun et al.). We demonstrated that combination of roscovitine and TRAIL is synergistic in hematologic cell lines and primary cells. In addition, roscovitine was shown to have potent immunostimulatory effect by increasing cell-mediated cytotoxicity. Based on our results we suggest that roscovitine-induced sensitization to TRAIL-triggered apoptosis was mediated by proapoptotic changes of the DISC with potential contribution of the proapoptotic changes in the protein expression of the apoptotic regulators (MCL1, BCL-XL, PUMA, BAK, BAD). We also suggest that roscovitine-induced increase in cell-mediated cytotoxicity, known to be mediated in part through death ligands, was also a consequence of the proapoptotic alteration of the DISC. Roscovitine, as a single agent, or in combination with TRAIL, might have a role in the experimental treatment of selected hematologic malignancies. Financial Support: LC 06044, MSM 0021620806, MSM 0021620808, GAUK 259211/110709, SVV-2010-254260507, IGA MZ NS/10287-3 Disclosures: No relevant conflicts of interest to declare.

Trail induced apoptosis and interaction with cytotoxic agents in soft tissue sarcoma cell lines

2001 ◽  
Vol 37 ◽  
pp. S111
Author(s):  
Sandra Tomek ◽  
Wolfgang Koestler ◽  
Thomas Brodowicz ◽  
Ingrid Pribill ◽  
Alexandra Budinsky ◽  
...  
Keyword(s):  
Soft Tissue ◽  
Soft Tissue Sarcoma ◽  
Cell Lines ◽  
Cytotoxic Agents ◽  
Sarcoma Cell ◽  
Induced Apoptosis

Bortezomib Sensitizes Non-Hodgkin’s Lymphoma Cells to Apoptosis Induced by Antibodies to TRAIL Receptors R1 and R2.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4614-4614
Author(s):  
Mitchell Reed Smith ◽  
Fang Jin ◽  
Indira Joshi
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Annexin V ◽  
Hematologic Malignancies ◽  
Caspase Inhibitor ◽  
Apoptosis Induction ◽  
Minimal Growth ◽  
Trail Receptors ◽  
Independent Mechanism ◽  
Induction Of Apoptosis

Abstract TRAIL-R1 and -R2 signaling induces apoptosis via a pathway that activates caspase 8. The proteosome inhibitor bortezomib may act via several pathways. Agonistic antibodies to TRAIL-R1 and -R2 and bortezomib are in clinical trials in solid tumors and hematologic malignancies. To develop rational combinations for future clinical studies, we investigated the actions of these agents on non-Hodgkin s lymphoma (NHL) cell lines. The t(14;18)+, EBV- NHL cell lines DoHH2 and WSU-FSCCL were treated with agonistic monoclonal antibodies to TRAIL-R1 (HGS-ETR1) and -R2 (HGS-ETR2) (Human Genome Sciences, Rockville, MD) and/or bortezomib. While HGS-ETR 1 and HGS-ETR 2 are effective inducers of apoptosis in FSCCL, DoHH2, which expresses dim TRAIL-R1 (DR4, HGS-ETR1 target) and TRAIL-R2 (DR5, HGS-ETR2 target), shows minimal growth inhibition or apoptosis induction by HGS-ETR1 or HGS-ETR2. Bortezomib has modest effects on DoHH2 cells in growth inhibition and apoptosis assays. HGS-ETR1 and HGS-ETR2 induction of apoptosis in WSU-FSCCL is efficiently blocked by the caspase inhibitor ZVAD. In contrast, bortezomib effects are not blocked by ZVAD, indicating an independent mechanism of action. To determine if these separate pathways would provide enhanced combination activity, DoHH2 cells were pre-treated with bortezomib for 30 min, followed by incubation with HGS-ETR1 or HGS-ETR2. This led to supra-additive induction of apoptosis (annexin V staining). We conclude that bortezomib sensitizes DoHH2 cells to the action of HGS-ETR1 and HGS-ETR2. Further, bortezomib induces apoptosis in DoHH2 cells by an independent mechanism, and the combination of TRAIL-receptor signaling and bortezomib may be a useful combination to explore.

Sensitization of Rituximab-Sensitive and Rituximab-Resistant B-NHL Cell Lines/Clones to TRAIL-Induced Apoptosis by Bortezomib and NF-κB Inhibitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1514-1514 ◽  
Author(s):  
Ali R. Jazirehi ◽  
Benjamin Bonavida
Keyword(s):  
Tumor Cells ◽  
Cell Lines ◽  
Wild Type ◽  
Normal Tissues ◽  
Survival Pathway ◽  
Trail Receptors ◽  
Wide Range ◽  
Significant Augmentation ◽  
Cytotoxic Molecule ◽  
Induced Apoptosis

Abstract Patients with B-NHL respond initially to conventional chemotherapy and/or to immunotherapy with rituximab (alone or in combination with chemotherapy). However, patients develop resistance to these modalities and novel approaches are needed. TRAIL is a cytotoxic molecule that exerts selective anti-tumor cytotoxic activity with minimal toxicity to normal tissues. Further, TRAIL or agonist monoclonal antibody (mAb) to TRAIL receptors, DR4 and DR5, are currently being tested clinically. The present study investigated the sensitivity of B-NHL cell lines to TRAIL-mediated apoptosis using the AIDS-related NHL (ARL) B-cell line, 2F7, and the B-NHL cell lines, Ramos and Daudi. Also, to recapitulate various aspects of acquired rituximab-resistance, we have generated rituximab-resistant (RR) clones from the parental wild type (wt) cells. Rituximab failed to chemo-sensitize the RR clones and the clones exhibited higher resistance to various drugs (e.g., CDDP, VP-16, ADR, Vincristine, Taxol) and to TRAIL (1–250 ng/ml-18 h) compared to the wt cells as analyzed by DNA fragment on assay. The findings demonstrate that the wild type and RR1 cells were resistant to TRAIL-mediated apoptosis at a wide range of TRAIL concentrations. We then examined means to reverse TRAIL resistance. We and others have reported that inhibition of NF-κB activity can sensitize TRAIL-resistant tumor cells to TRAIL-induced apoptosis. Hence, we examined the effect of the proteasome and NF-κB inhibitor, Bortezomib (Velcade), Bay 11–7085 and the specific NF-κB inhibitor DHMEQ (Kikuchi et. al, Cancer Research2003; 63:107). Pretreatment of the NHL tumor cells with Bortezomib, Bay 11–7085 or DHMEQ for 6 h followed by treatment with TRAIL for 18h resulted in significant augmentation of apoptosis and synergy was achieved. Both the rituximab-sensitive and rituximab-resistant tumor cells were sensitized by these inhibitors, though higher concentrations were required for sensitization of the RR clones. Interestingly, detailed analysis of the signaling pathways in the RR clones revealed constitutive hyper-activation of the NF-κB survival pathway leading to over-expression of anti-apoptotic gene products Bcl-2, Bcl-xL and Mcl-1. Based on the findings, we postulate that patients with resistant B-NHL can be treated with combination of TRAIL/anti-DR4 or DR5 mAb and NF-κB inhibitors. Alternatively, these patients can be treated with agents that up-regulate TRAIL expression on host effectors (e.g., T cells, NK cells) in combination with NF-κB inhibitors.

Inhibition of Histone Deacetylases 1 and 6 Enhances Ara-C-Induced Apoptosis In Pediatric Acute Myeloid Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3275-3275
Author(s):  
Xuelian Xu ◽  
Chengzhi Xie ◽  
Holly Edwards ◽  
Hui Zhou ◽  
Steven Buck ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Class I ◽  
Pediatric Aml ◽  
Induced Apoptosis ◽  
Class I Hdacs ◽  
The Impact ◽  
Acute Myeloid

Abstract Abstract 3275 Acute myeloid leukemia (AML) accounts for one-fourth of acute leukemias in children, but is responsible for more than half of the leukemia deaths in this patient population. Resistance to cytarabine (ara-C)-based chemotherapy is a major cause of treatment failure in this disease. Therefore, new therapies for children with AML are urgently needed. Among the newer agents that have been recently investigated in high-risk AML in adults, histone deacetylase (HDAC) inhibitors [HDACIs, e.g., valproic acid (VPA) and Vorinostat (SAHA)] are particularly notable. The ability of HDACIs to induce cell differentiation, cell cycle arrest, and apoptosis in human leukemic cells, but not in normal cells, has stimulated significant interest in their potential as anti-leukemia agents. Numerous HDACIs have been developed during the last decade and the majority of these are in clinical trials including the novel class I-selective HDACIs, MS-275 and MGCD0103, and pan-HDACIs, LBH-589 and PXD101. Despite the well-characterized molecular and cellular effects of HDACIs, single-agent activity for this class of drugs has been modest. However, the clinical usefulness of HDACIs may be increased through rationally designed combination strategies including HDACIs with standard chemotherapy drugs. We previously hypothesized that VPA synergizes with ara-C, resulting in enhanced antileukemic activity in pediatric AML, by inducing apoptosis. We examined the impact of VPA on ara-C cytotoxicities in a panel of pediatric AML cell lines and diagnostic blast samples from children with de novo AML and demonstrated highly synergistic antileukemic activities of combined ara-C and VPA. This was especially pronounced in samples with t(8;21). Our mechanistic studies revealed that induction of DNA damage and Bim underlay the synergistic antileukemic activities of this drug combination. The present study was designed to identify members of the HDAC family which were deteminants of ara-C sensitivities, and to select the optimal HDACIs that were most efficacious when combined with ara-C for treating AML. Expression profiles of HDACs 1–11 in 4 clinically relevant pediatric AML cell lines (THP-1, Kasumi-1, MV4-11, and CMS) suggested that HDACs 5 and 11 were likely not involved due to marginal or lack of expression. The remaining class II HDACs and the entire class I enzymes could be relevant to HDACI anti-leukemic activities, based on the relationships between HDAC levels and HDACI cytotoxicities and responses to the combined VPA and ara-C, although the impact of class I HDACs seemed to predominate. Treatment of THP-1 cells with structurally-diverse HDACIs [SAHA (a pan-HDACI), VPA (a relatively class I selective-HDACI), and MS-275 (a class I selective-HDACI)] and enzymatic assays following immunoprecipitation of class I HDACs, revealed that inhibition of class I HDACs could augment ara-C-induced apoptosis. However, class II HDACs (e.g., HDAC6) were also implicated since SAHA was also effective. shRNA knockdown of HDACs 1 or 6 resulted in ∼2-fold increased apoptosis induced by ara-C in THP-1 AML cells (p<0.05). This was accompanied by substantially increased expression of Bim (2.3- and 1.4-fold, respectively). Down-regulation of HDAC2 resulted in ∼30% decreased ara-C-induced apoptosis. In contrast, shRNA knockdown of HDACs 3 and 4 had no effects on ara-C-induced apoptosis in THP-1 cells. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced ara-C-induced apoptosis in pediatric AML sublines including THP-1 and Kasumi-1. Our results further establish that HDACs are promising therapeutic targets for treating pediatric AML and identified HDACs 1 and 6 as the most relevant drug targets. Accordingly, treating pediatric AML patients with pan-HDACIs may be more beneficial than HDAC isoform-specific drugs. Based on our results, incorporation of pan-HDACIs (e.g., LBH-589 and PXD101) into ara-C-based clinical trials for treating pediatric AML should be strongly considered. Disclosures: No relevant conflicts of interest to declare.

Reversal of Drug/TRAIL-Resistant B-NHL Cells to Apoptosis by the Combination of Rituximab (anti-CD20) and Either Mapatumumab or Lexatumumab

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4931-4931
Author(s):  
Mario I. Vega ◽  
Sara Huerta-Yepez ◽  
Melisa Martinez-Paniagua ◽  
Stavroula Baritaki ◽  
Haiming Chen ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Tumor Cells ◽  
Neutralizing Antibodies ◽  
Conflicts Of Interest ◽  
Phase 1 ◽  
Death Receptor ◽  
Hematologic Malignancies ◽  
Direct Role ◽  
Induced Apoptosis ◽  
Anti Cd20

Abstract Abstract 4931 Conventional treatments of non-Hodgkin's lymphoma (B-NHL) consist primarily of chemotherapy. Currently, rituximab is used alone or in combination with chemotherapy. However, there are subsets of patients who do not respond initially or develop resistance to further treatment. Therefore, there is an urgent need to develop other immunotherapies with less toxicities. At present, both TRAIL and agonist antibodies directed against TRAIL-R1 and -R2 have been explored for various cancer treatments in various phase 1 and phase 2 clinical trials. We have recently demonstrated that rituximab sensitizes TRAIL-resistant B-NHL cells to TRAIL-induced apoptosis. Sensitization was the result of rituximab-induced inhibition of the constitutively activated NF-κB pathway and downstream the DR5 transcription repressor Yin Yang 1 (YY1). The direct role of YY1 in the regulation of resistance to TRAIL was demonstrated in cells transfected with YY1 siRNA and that became sensitive to TRAIL- apoptosis. Treatment with rituximab did not have any observed effects on the expression of DR4. Based on these findings, it was possible that rituximab-mediated sensitization to TRAIL may invoke either TRAIL-R1 (DR4) or TRAIL-R2 (DR5), or both; thus, this possibility is currently being examined by the use of either neutralizing antibodies against each death receptor or by the use of silencing RNA. Currently, clinical trials are being conducted with both mapatumumab (anti-TRAIL-R1,) and lexatumumab (anti-TRAIL-R2) against a variety of cancers. These agonist antibodies have been evaluated clinically as single agents and in combination with standard therapy in solid and hematologic malignancies. It is not clear whether tumors can develop resistance to agonism of either one or both death receptors and thus, may not respond to monotherapy alone. Combination therapies may be required and we have hypothesized that the combination treatment of rituximab and agonist antibodies may be complementary or synergistic. This hypothesis was based on our findings that rituximab inhibits survival pathways and downregulates anti-apoptotic gene products and, thus, significantly reducing the threshold of resistance. Thus, this rituximab-mediated effect will facilitate the direct cytotoxicity of the agonist death receptor antibodies. The present study investigated whether rituximab can sensitize TRAIL-resistant tumor cells by either agonist TRAIL-R1 or TRAIL-R2 antibodies To address this question, we have examined the effect of agonist antibodies directed against either TRAIL-R1 (mapatumumab) or TRAIL-R2 (lexatumamab). Treatment of the TRAIL-resistant Ramos B-NHL cells with rituximab for 24h and followed with treatment with non-toxic concentrations of mapatumumab (12 μg/ml) or lexatumumab (12 μg/ml) for 18h resulted in significant sensitization to apoptosis as assessed by activation of caspase 3. The mechanism of the sensitization by rituximab for each antibody was also examined. These findings demonstrated that rituximab sensitizes tumor cells to apoptosis by activation of either DR4 or DR5. Although there is heterogeneous expression of TRAIL-R1 and TRAIL-R2 in B-NHL cells, such cells may still be sensitive to rituximab-mediated sensitization to apoptosis by the corresponding agonist death receptor antibody. Recent findings demonstrated that some tumors expressing both DR4 and DR5 were shown to respond to TRAIL by preferential activation of DR4 and not DR5. Therefore, preclinical findings obtained with the use of TRAIL may not be predictive of outcome compared to the use of TRAIL-receptor specific agonist antibodies; mapatumumab or lexatumumab. Disclosures: No relevant conflicts of interest to declare.

Glutathione Depletion Enhances Arsenic Trioxide-Induced Apoptosis in Lymphoma Cells through Mitochondrial and Caspase-Independent Mechanisms.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2708-2708 ◽  
Author(s):  
Savita Bhalla ◽  
Leo I. Gordon ◽  
Amareshwar Singh ◽  
Sheila Prachand ◽  
Shuo Yang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Membrane Potential ◽  
Follicular Lymphoma ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Primary Cells ◽  
Wild Type ◽  
Induced Apoptosis

Abstract Abstract 2708 Poster Board II-684 Introduction: Arsenic trioxide (ATO) induces apoptosis in cancer through several mechanisms including modulation of the redox system and targeting the BCL-2 family and mitochondria, resulting in cytochrome c release and activation of caspases. However, the mechanisms of arsenic-induced cell death in lymphoma remain to be elucidated, while methods to enhance ATO-related apoptosis have not been fully explored. In the present study, we evaluated the cytotoxic activity and pathways of cell death induced by ATO alone and in combination with a glutathione (GSH)-depleting agent in non-Hodgkin lymphoma (NHL) cell lines and primary cells from patients with lymphoma. We hypothesized that depletion of GSH would enhance ATO induced apoptosis through mitochondrial and caspase-mediated pathways. Methods: We treated Ramos (Burkitt lymphoma), HF1 (follicular lymphoma), and SUDHL4 (diffuse large B-cell lymphoma) NHL cell lines, and primary follicular lymphoma and CLL/SLL cells with increasing concentrations (10–100μM) of ATO for 24–72 hours (hrs). Apoptosis was determined by Annexin V-Propidium iodide staining followed by flow cytometry and Z-VAD-FMK was used as a pan-caspase inhibitor. ROS was measured by H2DCFDA staining followed by flow cytometry. We further studied the mechanism of apoptosis by measuring alteration in mitochondrial membrane potential (MMP), Bax translocation, and caspase and PARP activation. In addition, we treated wild type and Bax−/−/Bak−/− double knockout mouse endothelial fibroblasts (MEFS) with ATO or ATO/BSO and determined apoptosis and mitochondrial membrane potential compared with MEF wild type (MEF-wt). Results: Treatment of cells with ATO resulted in dose- and time-dependent apoptosis, although high concentrations were necessary for effect (ED50 >10–20μM). ATO treatment redistributed Bax from the cytosol to mitochondria and there were significant changes in MMP, as well as increased PARP, cleaved caspse-3 and -9, while BID was downregulated. Furthermore, ATO-induced apoptosis was caspase-dependent. Addition of the GSH-depleting agent, buthionine sulfoximine (BSO), to ATO induced strong synergistic cell death (combination indices <0.1 in all cell lines), resulting in apoptosis that was ROS-related, but mitochondrial- and caspase-independent. Using immortalized wild-type MEFS and Bax−/−Bak−/− double knockouts, ATO-induced alteration in MMP and apoptosis that was Bax-dependent (see figure below), while targeted disruption of the Bax/Bak genes had no enhancing effects on ATO/BSO-induced MMP or apoptosis. Interestingly, ATO alone induced appreciable apoptosis in primary CLL/SLL and follicular lymphoma cells, while BSO had minimal additive effects in these fresh cells. This appeared due to markedly lower intracellular GSH content within primary cells compared with cell lines (p<0.001). Conclusion: Altogether, our findings establish that ATO has anti-lymphoma activity and approaches to enhance its cytotoxic activity are feasible via engagement of distinct cell death mechanisms. Continued investigation of arsenic-based therapy, including new arsenical compounds and methods of tissue delivery that are able to overcome mechanisms of cellular resistance, for the treatment of lymphoma is warranted. Disclosures: Gordon: Cure Tech: Membership on an entity's Board of Directors or advisory committees.

Rational for the Use of a Targeted-Therapy Using ABT-737 In Mantle-Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 770-770
Author(s):  
Cyrille Touzeau ◽  
Christelle Dousset ◽  
Lynda Bodet ◽  
Stephanie Bonnaud ◽  
Patricia Gomez-Bougie ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Targeted Therapy ◽  
Western Blot ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Down Regulation ◽  
Mantle Cell ◽  
Induced Apoptosis ◽  
20 Nm

Abstract Abstract 770 Mantle-cell lymphoma (MCL) is still considered as incurable, thus new therapeutic approaches are needed. The anti-apoptotic protein Bcl-2 is known to be over-expressed in MCL and implicated in drug resistance. Therefore, there is a strong rational for the use of a Bcl-2-targeted therapy in MCL. The BH3-only mimetic ABT-737 (Abbott) is a potent specific inhibitor of Bcl-2, Bcl-xL and Bcl-w but not of Mcl-1. ABT-737 have been investigated in several hematologic malignancies cell lines, such as chronic lymphoid leukemia (CLL) and diffuse large B cell lymphoma, and showed promising results. In this study, we investigated the anti-tumoral effect of ABT-737 in the setting of MCL using MCL cell lines and MCL patients' samples. Our aims were also to identify prognostic biomarkers that may predict MCL tumor cells sensitivity to ABT737 and to study the anti-MCL activity of ABT737 alone or in combination. Five well characterized MCL cell lines were used: JEKO-1, MINO, REC-1, GRANTA-519 and UPN-1. Cytotoxicity of ABT-737 was assessed by flow cytometry using APO 2.7 staining. MINO and GRANTA 519 cell lines were highly sensitive (EC50= 20 nM after 24 hours of treatment) and apoptosis occurred rapidly within 2 hours following treatment as demonstrated by caspase 3 cleavage. In contrast, the three other cell lines (JEKO-1, REC-1 and UPN-1) were resistant (EC50> 4μM) to ABT-737. Western Blot analysis revealed that a major difference between sensitive cell lines (MINO and GRANTA-519) compared to other cell lines was their Bcl-2high/Mcl-1low profile. We also investigated ABT-737-induced apoptosis in primary MCL tumor cells (n=12). Seven patients' samples were sensitive to ABT-737 (median EC50 of 20 nM), whereas the 5 others samples were resistant (EC50 not achieved). As observed in MCL cell lines, western blot analysis revealed that primary tumor cells showing a Bcl-2high/Mcl-1low profile were associated with sensitivity to ABT-737. In contrast a Mcl-1high profile was associated with resistance to ABT-737. Taken together our investigation in both MCL cell lines and primary tumor cells from patients suggested that expression level Mcl-1 could influence ABT-737-induced apoptosis. Flavopiridol is a cyclin dependant kinase inhibitor and known to induce a transcriptional down-regulation of Mcl-1 in multiple myeloma and CLL. We therefore hypothesized that addition of Flavopiridol to ABT-737 could enhance induced-apoptosis. We first confirmed that two hours of treatment with flavopiridol induced a strong down-regulation of Mcl-1 at both mRNA and protein levels. Using suboptimal dose of ABT-737 (25 nM) and Flavopiridol (100 nM) for 24 hours, we observed a strong synergistic anti-MCL as evidence by a combination indice <1 according to the Chou-Talalay method. In conclusion, ABT-737 at low nanomolar concentration induces a strong apoptosis in the subgroup of Bcl-2high/Mcl-1low MCL cells profile which represents around 50% of the MCL patients. Thus, this subgroup of patients appears to be good candidate for clinical trials evaluating ABT-737 alone. In the subgroup of Mcl-1high patients, a specific down-regulation of Mcl-1 overcomes Mcl-1-induced resistance and synergizes with ABT-737. Indeed, our results strongly support the use of ABT-737 in MCL based on the concept of a targeted therapy according to the Bcl-2/Mcl-1 tumor cell profile. ABT-263, an orally bioavailable BH3 mimetic compound of the same class than ABT-737, is currently under investigation in various hematological malignancies, thus our investigation provides a biological rational for future clinical trials evaluating ABT-263 in combination or not with Flavopiridol in MCL patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An inhibitor of proteasome β2 sites sensitizes myeloma cells to immunoproteasome inhibitors

2018 ◽  
Vol 2 (19) ◽  
pp. 2443-2451 ◽  
Author(s):  
Sondra Downey-Kopyscinski ◽  
Ellen W. Daily ◽  
Marc Gautier ◽  
Ananta Bhatt ◽  
Bogdan I. Florea ◽  
...  
Keyword(s):  
Cell Lines ◽  
Active Sites ◽  
Proteasome Inhibitors ◽  
Specific Inhibitor ◽  
Hematologic Malignancies ◽  
Interferon Γ ◽  
Primary Cells ◽  
The Us

Abstract Proteasome inhibitors bortezomib, carfilzomib and ixazomib (approved by the US Food and Drug Administration [FDA]) induce remissions in patients with multiple myeloma (MM), but most patients eventually become resistant. MM and other hematologic malignancies express ubiquitous constitutive proteasomes and lymphoid tissue–specific immunoproteasomes; immunoproteasome expression is increased in resistant patients. Immunoproteasomes contain 3 distinct pairs of active sites, β5i, β1i, and β2i, which are different from their constitutive β5c, β1c, and β2c counterparts. Bortezomib and carfilzomib block β5c and β5i sites. We report here that pharmacologically relevant concentrations of β5i-specific inhibitor ONX-0914 show cytotoxicity in MM cell lines similar to that of carfilzomib and bortezomib. In addition, increasing immunoproteasome expression by interferon-γ increases sensitivity to ONX-0914 but not to carfilzomib. LU-102, an inhibitor of β2 sites, dramatically sensitizes MM cell lines and primary cells to ONX-0914. ONX-0914 synergizes with all FDA-approved proteasome inhibitors in MM in vitro and in vivo. Thus, immunoproteasome inhibitors, currently in clinical trials for the treatment of autoimmune diseases, should also be considered for the treatment of MM.

Fenretinide-Induced Apoptosis of Human Head and Neck Squamous Carcinoma Cell Lines

1998 ◽  
Vol 118 (4) ◽  
pp. 464-471 ◽  
Author(s):  
Richard L. Scher ◽  
Wilfred Saito ◽  
Richard K. Dodge ◽  
William J. Richtsmeier ◽  
Robert L. Fine
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Clinical Trials ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Gel Electrophoresis ◽  
Deoxyribonucleic Acid ◽  
Induced Apoptosis ◽  
Hnscc Cell

BACKGROUND: Squamous cell carcinoma of the head and neck (HNSCC) has a high incidence of recurrence and associated second primary malignancy. The retinoid 13- cis-retinoic acid has been shown to be effective as both a chemopreventive and chemotherapeutic agent for HNSCC, but often with treatment-limiting toxicity. The synthetic retinoid fenretinide (N-(4-hydroxyphenyl)retinamide) (HPR) has significant antiproliferative activity against a number of animal and human malignancies and has been used in clinical trials as a chemopreventive agent in patients with breast and prostate cancer and oral leukoplakia. HPR has been shown to have a toxicity profile lower than that for other retinoids used in clinical trials. PURPOSE: The aim of this study was to investigate the effect of HPR on the growth of HNSCC cell lines in vitro. METHODS: Four HNSCC cell lines (JHU-011-SCC, JHU-020-SCC, JHU-022-SCC, and FaDu) were treated with a range of concentrations of HPR for various times. After HPR exposure, cell viability was determined by tetrazolium dye (MTT) colorimetric assay, comparing cell survival with that of untreated control cells. HPR-induced apoptosis was determined by flow-cytometric deoxyribonucleic acid cell-cycle analysis, ultrastructural analysis with electron microscopy, and deoxyribonucleic acid fragmentation detected by gel electrophoresis. RESULTS: HPR caused significant growth inhibition in three of the four HNSCC cell lines in a dose- and time-dependent fashion. In two cell lines (JHU-011-SCC, JHU-020-SCC) a significant antiproliferative effect was achieved between 1 and 2.5 μ mol/L HPR after 72 hours of treatment. By deoxyribonucleic acid cell-cycle analysis, electron microscopy, and gel electrophoresis, HPR was shown to induce apoptosis in the JHU-011-SCC and JHU-020-SCC cell lines, but not in the FaDu cell line, which was insensitive to the growth inhibitory effect of HPR. CONCLUSIONS: This study has demonstrated that HPR reduces cell viability in HNSCC cells in vitro at clinically relevant doses, with the growth inhibition occurring through the induction of apoptosis.

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