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.

Rituximab-Mediated Sensitization of B-NHL Cells Lines to TRAIL-Induced Apoptosis: Involvement of YY1 Suppression and DR5 Upregulation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2387-2387
Author(s):  
Mario I. Vega ◽  
Sara Huerta-Yepez ◽  
Stavroula Baritaki ◽  
Cesar Bonilla-Gonzalez ◽  
Benjamin Bonavida
Keyword(s):  
Tumor Cells ◽  
Fas Ligand ◽  
Cell Lymphoma ◽  
Host Cells ◽  
Drug Resistant ◽  
Normal Tissues ◽  
Trail Receptors ◽  
Induced Apoptosis ◽  
Recombinant Trail

Abstract There has been considerable interest in the treatment of drug-resistant tumor cells with TRAIL or agonist monoclonal antibody directed against the TRAIL receptors DR4 and DR5. TRAIL has been shown to be largely non-toxic to normal tissues and cytotoxic to transformed tumor cells. However, many tumors, including B-NHL, are resistant to TRAIL-induced apoptosis. We have reported that rituximab signals B-NHL cells and inhibits several cell survival signaling pathways leading to chemosensitization (Jazirehi and Bonavida, Oncogene;2004:2121, 2005). In addition, we have recently reported that rituximab sensitizes B-NHL cells to Fas-ligand-induced apoptosis, via inhibition of the transcription repressor Yin Yang 1 (YY1) (Vega et al. The Journal of Immunology;175:2174 2005). We have found that YY1 negatively regulates DR5 transcription and expression and thus, we hypothesized that rituximab-mediated inhibition of YY1 may sensitize TRAIL-resistant B-NHL cells lines to TRAIL-induced apoptosis. The B-NHL cells lines, Ramos and Daudi, were treated with rituximab (20μg/ml for 6h) and were then exposed to various concentrations of recombinant TRAIL (2.5–10 ng/ml for 24h). Following incubation, the cells were examined for apoptosis by assessing activation of caspase-3 and by Annexin V/PI. The findings revealed that the cell lines were relatively resistant to TRAIL but following treatment with rituximab significant potentiation of apoptosis and synergy were achieved. Optimal apoptosis was observed with a concentration of TRAIL of 10ng/ml. The rituximab-treated cells showed a 2 fold upregulation of cell surface DR5 expression as compared to untreated cells. In addition, rituximab treated cells showed significant inhibition of YY1 expression as determined by Western and EMSA. We have also examined the expression of YY1 in tissue arrays containing formalin fixed, paraffin embedded sections from AIDS lymphoma, obtained from the Aids and Cancer Specimen Resource of the NCI. These arrays consisted of 21 Burkitt, 29 Large Cell Lymphoma and 6 Small Cell Lymphoma and were examined for YY1 by immunhistochemistry. The findings revealed that YY1 was overexpressed as compared to normal tissues. Currently, we are examining the effect of rituximab-mediated sensitization of patients derived B-NHL cells to TRAIL-induced apoptosis. The present findings demonstrate that drug-resistant and TRAIL-resistant B-NHL cells can be sensitized by rituximab to TRAIL-induced apoptosis. Further, the studies revealed a potential novel mechanism of rituximab-mediated effect in vivo by recruiting host cells expressing/secreting TRAIL to exert a cytotoxic activity on the rituximab-treated cells. The findings also suggest the potential therapeutic efficacy, in vivo, of combination of rituximab and either recombinant TRAIL or agonist monoclonal antibodies against DR4 or DR5 in the treatment of resistant cells. We propose that inhibitors of YY1 can serve as a sensitizing agent for TRAIL-induced apoptosis in rituximab-resistant B-NHL cells.

scholarly journals Protein kinase Cδ inactivation inhibits cellular proliferation and decreases survival in human neuroendocrine tumors

2011 ◽  
Vol 18 (6) ◽  
pp. 759-771 ◽  
Author(s):  
Zhihong Chen ◽  
Lora W Forman ◽  
Kenneth A Miller ◽  
Brandon English ◽  
Asami Takashima ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neuroendocrine Tumors ◽  
Small Molecule ◽  
Cellular Proliferation ◽  
Small Molecule Inhibitors ◽  
Cancer Therapeutics ◽  
Therapeutic Modality ◽  
Ras Signaling ◽  
Normal Tissues ◽  
Induced Apoptosis

The concept of targeting cancer therapeutics toward specific mutations or abnormalities in tumor cells, which are not found in normal tissues, has the potential advantages of high selectivity for the tumor and correspondingly low secondary toxicities. Many human malignancies display activating mutations in the Ras family of signal-transducing genes or over-activity of p21Ras-signaling pathways. Carcinoid and other neuroendocrine tumors have been similarly demonstrated to have activation of Ras signaling directly by mutations in Ras, indirectly by loss of Ras-regulatory proteins, or via constitutive activation of upstream or downstream effector pathways of Ras, such as growth factor receptors or PI3-kinase and Raf/mitogen-activated protein kinases. We previously reported that aberrant activation of Ras signaling sensitizes cells to apoptosis when the activity of the PKCδ isozyme is suppressed and that PKCδ suppression is not toxic to cells with normal levels of p21Rassignaling. We demonstrate here that inhibition of PKCδ by a number of independent means, including genetic mechanisms (shRNA) or small-molecule inhibitors, is able to efficiently and selectively repress the growth of human neuroendocrine cell lines derived from bronchopulmonary, foregut, or hindgut tumors. PKCδ inhibition in these tumors also efficiently induced apoptosis. Exposure to small-molecule inhibitors of PKCδ over a period of 24 h is sufficient to significantly suppress cell growth and clonogenic capacity of these tumor cell lines. Neuroendocrine tumors are typically refractory to conventional therapeutic approaches. This Ras-targeted therapeutic approach, mediated through PKCδ suppression, which selectively takes advantage of the very oncogenic mutations that contribute to the malignancy of the tumor, may hold potential as a novel therapeutic modality.

Tumor necrosis factor–related apoptosis-inducing ligand–mediated apoptosis in established and primary glioma cell lines

2002 ◽  
Vol 13 (3) ◽  
pp. 1-11 ◽  
Author(s):  
Jay Jaganathan ◽  
Joshua H. Petit ◽  
Barbara E. Lazio ◽  
Satyendra K. Singh ◽  
Lawrence S. Chin
Keyword(s):  
Cell Death ◽  
Tumor Cells ◽  
Cell Lines ◽  
Glioma Cell ◽  
Apoptotic Cell ◽  
Glioma Cells ◽  
Trail Receptors ◽  
Glioma Cell Lines ◽  
Caspase 7 ◽  
U373 Cells

Object Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is a member of the TNF cytokine family, which mediates programmed cell death (apoptosis) selectively in tumor cells. The selective tumoricidal activity of TRAIL is believed to be modulated by agonistic (DR4 and DR5) and antagonistic receptors (DcR1 and DcR2), which appear to compete for ligand binding. Because TRAIL is expressed in a wide range of tissues, including brain, kidney, and spleen, and seems consistently to induce cell death in tumor cells, the cytokine has been identified as a promising approach for selectively inducing tumor cell death. In this study, the authors examine the importance of TRAIL's receptors in both its selectivity for tumor cells and its ability to induce apoptosis. Methods The authors first examined sensitivity to TRAIL and expression of TRAIL receptors in four established and four primary cultured glioma cell lines by using viability and fluorescent apoptosis assays. They then evaluated DR5 expression and JNK, caspase 3, and caspase 7 activation by conducting immunoblot analyses. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed to study expression of DR4, DR5, DcR1, and DcR2. The DR5 transcripts from one TRAIL-sensitive, one partially TRAIL-resistant, and one TRAIL-resistant cell line were subsequently sequenced. The expression of TRAIL receptors in normal and glial brain tumor pathological specimens were then compared using immunohistochemistry. Finally, to study the direct effects of DR5 on glioma cells, the authors conducted transient and stable transfections of the fulllength DR5 transcript into glioma cells with and without preestablished overexpression of the antiapoptotic gene bcl-2. The established glioma cell lines T98G and U87MG, and all primary cell lines, were apoptotic at greater than or equal to 100 ng/ml TRAIL. The A172 cells, by contrast, were susceptible only with cycloheximide, whereas U373MG cells were not susceptible to TRAIL. The JNK, caspase 3, and caspase 7 activity evaluated after treatment with TRAIL showed that TRAIL-sensitive cell lines exhibited downstream caspase activation, whereas TRAIL-resistant cells did not. The DR5 sequences in T98G, A172, and U373MG cell lines were identical to published sequences despite these differences in sensitivity to TRAIL. The RT-PCR performed on extracts from the eight glioma cell lines showed that all expressed DR5. Immunohistochemistry revealed ubiquitous expression of DR5 in glioma specimens, with an associated lack of decoy receptor expression. Normal brain specimens, by contrast, stained positive for both DR5 and DcR1. Overexpression of DR5 under both transfection conditions resulted in cell death in all three cell lines. The previously seen resistance of U373 cells to TRAIL was not observed. Apoptotic cell death was confirmed using DNA fragmentation in T98G cell lines and fluorescent miscroscopy in all cell lines. The T98G cells stably transfected with bcl-2 before DR5 overexpression were protected from cell death. Conclusions The authors conclude that DR5 represents a promising new approach to directly activating the intrinsic caspase pathway in glioma cells. The fact that TRAIL-resistant gliomas do not express decoy receptors suggests a mechanism of resistance unique from that proposed for normal tissues. The overexpression of DR5 induced apoptotic cell death in glioma cells without TRAIL and was able to overcome the resistance to TRAIL demonstrated in U373 cells. The Bcl-2 protects cells from DR5 by acting downstream of the receptor, most likely at the level of caspase activation.

Regulation of Chemoresistance and Immune Resistance of B-NHL Cell Lines by Overexpression of YY1 and Bcl-xL, Respectively: Reversal of Resistance by Rituximab.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1517-1517
Author(s):  
Mario I. Vega ◽  
Ali R. Jazirehi ◽  
Sara Huerta-Yepez ◽  
Benjamin Bonavida
Keyword(s):  
Tumor Cells ◽  
Cell Lines ◽  
Drug Induced ◽  
Direct Role ◽  
No Donor ◽  
Immune Resistance ◽  
Apoptosis Inhibition ◽  
Immune Mediated ◽  
Induced Apoptosis

Abstract We have recently reported that treatment of B-NHL cell lines with rituximab sensitizes the tumor cells to both chemotherapy and Fas-induced apoptosis (Jazirehi and Bonavida, 2005, Oncogene, 24:2121–2145). This study investigated the underlying molecular mechanism of rituximab-mediated reversal of resistance. Treatment of B-NHL cell lines inhibited the constitutively activated NF- κB. Cells expressing dominant active IκB or treated with NF-κB specific inhibitors were sensitized to both drugs and FasL agonist mAb (CH-11)-induced apoptosis. Downregulation of Bcl-xL expression via inhibition of NF-κB activity correlated with chemosensitivity. The direct role of Bcl-xL in chemoresistance was demonstrated by the use of Bcl-xL overexpressing Ramos cells, Ramos HA-BclxL (gift from Genhong Cheng, UCLA), which were not sensitized by rituximab to drug-induced apoptosis. However, inhibition of Bcl-xL in Ramos HA-Bcl-x resulted in sensitization to drug-induced apoptosis. The role of Bcl-xL expression in the regulation of Fas resistance was not apparent as Ramos HA-Bcl cells were as sensitive as the wild type cells to CH-11-induced apoptosis. Several lines of evidence support the direct role of the transcription repressor Yin-Yang 1 (YY1) in the regulation of resistance to CH-11-induced apoptosis. Inhibition of YY1 activity by either rituximab, the NO donor DETANONOate, or following transfection with YY1 siRNA all resulted in upregulation of Fas expression and sensitization to CH-11-induced apoptosis. These findings suggest two complementary mechanisms underlying the chemo-sensitization and immuno-sensitization of B NHL cells by rituximab via inhibition of NF-κB. The regulation of chemoresistance by NF-κB is mediated via Bcl-xL expression whereas the regulation of Fas resistance by NF-κB is mediated via YY1 expression and activity. These findings suggest that drug-resistant NHL tumor cells may be sensitive to immune-mediated therapeutics.

JS-K, a GST-Activated Nitric Oxide Generator, Induces Apoptosis and Overcomes In Vitro Drug Resistance in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1593-1593
Author(s):  
Tanyel Kiziltepe ◽  
Kenji Ishitsuka ◽  
Teru Hideshima ◽  
Noopur Raje ◽  
Norihiko Shiraishi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Tumor Cells ◽  
Cell Lines ◽  
Biological Effects ◽  
Treatment Modalities ◽  
Cancer Drug ◽  
Induced Apoptosis

Abstract Multiple myeloma (MM) is currently an incurable hematological malignancy. A major reason for the failure of currently existing therapies is the chemotherapeutic resistance acquired by the MM cells upon treatment. Overexpression of glutathione S-transferases (GST) has been shown as one possible mechanism of anti-cancer drug resistance in a broad spectrum of tumor cells. JS-K (O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) belongs to a class of pro-drugs which are designed to release nitric oxide (NO) on reaction with GST. JS-K can possibly turn GST overexpression to the tumor’s disadvantage by (1) consuming intracellular GSH and preventing drug inactivation; and (2) by exposing tumor cells to high intracellular concentrations of NO. JS-K has potent in vitro and in vivo anti-leukemic activity. The purpose of the present study is to examine the biological effects of JS-K on human MM cells. We demonstrate that JS-K has significant in vitro cytotoxicity on MM cell lines, with an IC50 of 0.3-2 mM at 48 hours. JS-K also induces cytotoxicity on cell lines that are resistant to conventional chemotherapy (i.e., MM1R, RPMI-Dox40, RPMI-LR5, RPMI-MR20). Importantly, no cytotoxic effects of JS-K were detected on peripheral blood mononuclear cells (PBMNC) obtained from healthy volunteers at these doses. Moreover, JS-K could overcome the survival and growth advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells (BMSC). JS-K caused a transient G2/M arrest followed by apoptosis, as determined by flow cytometric analysis using PI, Annexin V and Apo2.7 staining. JS-K-induced apoptosis was associated with caspase 8, 7, 9 and 3 activation. Interestingly, Fas was upregulated by JS-K, suggesting the involvement of death receptor pathway in induction of apoptosis. JS-K also triggered Mcl-1 cleavage and Bcl-2 phosphorylation, suggesting the involvement of mitochondrial pathway. In addition, apoptosis inducing factor (AIF), endonuclease G (EndoG) and cytochrome c were released into the cytosol during apoptosis. Taken together, these findings suggest the involvement of both intrinsic and extrinsic apoptotic pathways in JS-K-induced apoptosis in MM cells. In summary, our studies demonstrate that JS-K induces apoptosis and overcomes in vitro drug resistance in MM cells. Therefore, JS-K is a novel compound which carries significant potential to be included in the repertoire of existing treatment modalities for MM. Ongoing studies are delineating the mechanism of action of JS-K to provide the preclinical rationale for combination therapies to overcome drug resistance and improve patient outcome.

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.

Effect of the specific P53 stabilizer CBS9106 on multiple myeloma (MM)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 8601-8601
Author(s):  
H. Ikeda ◽  
T. Hideshima ◽  
G. Perrone ◽  
Y. Okawa ◽  
N. Raje ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Protein Expression ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
P53 Protein ◽  
Wild Type ◽  
Cycle Arrest ◽  
P53 Protein Expression

8601 Background: The mutations of P53 tumor suppressor protein are associated with progressive in Multiple Myeloma (MM), conversely, stabilization of P53 leads to cell cycle arrest and apoptosis. In this study, we examined p53 protein expression and demonstrated the effect of P53 stabilization using a novel specific P53 stabilizer CBS9106 in MM. Method: We examined P53 protein expression using Immunoblot analysis, as well as the growth inhibitory effect of CBS9106 in MM cell lines and primary tumor cells from MM patients. We also defined whether CBS9106 can overcome the growth promoting effect of exogenous cytokines and bone marrow stroma cells (BMSCs) using [3H]-thymidine uptake assay. Results: Expression of P53 protein was observed in 3/3 primary tumor cells from MM patients and 6/6 MM cell lines. CBS9106 at low nM levels triggered cytotoxicity against p53 wild type MM cell lines and primary tumor cells from MM patients, associated with phosphorylation of P53 (serine15 and 20). In contrast, CBS9106 did not affect the survival of normal peripheral blood mononuclear cells from healthy volunteers at concentrations as high as 10 μM. This agent also induced G1 cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, -8, -9 and PARP. Neither growth stimulating cytokines (IL-6 and IGF-1) nor BMSCs protected against apoptotic effect of CBS9106. Moreover, we demonstrate that combination of CBS9106 with MDM2 inhibitor Nutrin3 or proteasome inhibitor bortezomib induces synergistic anti-MM activity in both P53 wild type MM cell lines and primary tumor cells from MM patients. Conclusions: Stabilizing P53 by CBS9106 represents a novel promising p53-based therapy in MM. These results provide the preclinical framework supporting evaluation of CBS9106 in clinical trials to improve patient outcome in MM. No significant financial relationships to disclose.

scholarly journals mTOR Dual Inhibitor Induced Cytotoxicity Depends on 4EBP1/c-Myc/Puma and NFkB/Egr-1/Bim Pathways in Human Lymphoid Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3705-3705
Author(s):  
Seongseok Yun ◽  
Nicole D. Vincelette ◽  
Katherine L. B. Knorr ◽  
Luciana L. Almada ◽  
Paula A. Schneider ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Lymphoid Cells ◽  
Promoter Activation ◽  
Dual Inhibitor ◽  
Lymphoid Malignancies ◽  
Wide Range ◽  
Dual Inhibitors ◽  
Induced Apoptosis ◽  
Inhibitor Treatment

Abstract The mammalian target of rapamycin (mTOR), a kinase that regulates proliferation and apoptosis, has been extensively evaluated as a therapeutic target in hematologic malignancies. Rapamycin analogues, which partially inhibit mTOR complex 1 (mTORC1), showed limited anti-tumor activity due to feedback mechanisms involving mTORC2 and incomplete inhibition of mTORC1. Thus, attention has turned to agents targeting both mTOR complexes by binding the mTOR kinase domain. The purpose of this study was to delineate the mechanisms of mTOR dual inhibitor induced apoptosis in human neoplastic lymphoid cells in vitro. MTS assays and propidium iodide staining followed by flow cytometry for subdiploid populations demonstrated that OSI-027 and MLN0128 inhibited cell proliferation and induced apoptosis in a wide range of lymphoid cell lines including Jurkat, Nalm-6, Molt-4, and SeAX. Raptor and Rictor knockdown in Jurkat and Nalm-6 increased cell death, suggesting both mTOR complexes play a role in apoptosis. 4EBP1 phosphorylation was inhibited by mTOR dual inhibitors, but not by rapamycin. Expression of 4EBP1 T37A/T46A and 4EBP1 T37A/T46A/S65A/T70A, which mimic dephosphorylated 4EBP1, increased Puma mRNA and protein levels as well as apoptosis. Moreover, 4EBP1 knockdown abrogated mTOR dual inhibitor induced Puma upregulation and cell death, further supporting the role of 4EBP1 dephosphorylation in mTORC1 dependent apoptosis. In accord with the known dependence of c-Myc translation on the eIF4E/eIF4G complex, we also observed c-Myc downregulation after treatment with OSI-027, MLN0128 and 4EGI-1, but not rapamycin. Puma induction mirrored c-Myc downregulation under a variety of conditions, including expression of nonphosphorylatable 4EBP1 in parental Jurkat cells or wt 4EBP1 in 4EBP1 deficient cells. Furthermore, c-Myc knockdown induced Puma mRNA and protein as well as increased apoptosis. Collectively, these results support a model in which mTORC1 inhibition, acting through 4EBP1, induces Puma upregulation and apoptosis through c-Myc downregulation. In order to assess the parallel mTORC2-dependent Bim-mediated apoptotic mechanism, we utilized reporter assays and RNAseq experiments. OSI-027-induced Bim promoter activity decreased markedly when the nucleotides -29 to -18 were removed, suggesting that this response element is critical for OSI-027-induced promoter activation. In silico analysis identified eight transcription factors, including SP1, Egr-1, and Myb, that potentially bind this 12-bp region. In RNAseq experiments, we detected a 9-fold increase in Egr-1. Egr-1 upregulation was confirmed by qRT-PCR and immunoblotting after dual inhibitors treatment or Rictor knockdown. Moreover, dominant negative Egr-1 or Egr-1 knockdown diminished dual inhibitor-induced Bim promoter activation and Bim upregulation. Chromatin immunoprecipitation assays demonstrated that OSI-027 enhances binding of Egr-1 to a region of the Bim promoter including bp -29 to -18, further confirming that Egr-1 functions as a direct transcriptional activator for Bim upon mTOR dual inhibitor treatment. NFκB is a known transcription factor for Egr-1 and we observed increased p65 in the nucleus and increased NFκB transcriptional activity after dual inhibitor treatment. Overexpression of S32A/S36A IκB impaired the ability of dual inhibitors to induce NFκB transcriptional activation, Egr-1 mRNA and protein, Bim promoter activation and Bim mRNA and protein upregulation. Collectively, these results suggest that mTORC2 inhibition induces Bim upregulation and apoptosis through NFκB and Egr-1 transactivation. When fresh clinical ALL isolates were exposed to OSI-027 or MLN0128 ex vivo, inhibition of 4EBP1 phosphorylation along with upregulation of Egr-1 and Bim and/or c-Myc downregulation accompanied by Puma induction occurred, indicating that the pathways identified in ALL cell lines can also potentially be engaged in clinical ALLs. These observations not only provide new insight into the survival roles of mTOR in lymphoid malignancies, but also identify alterations that potentially modulate the action of mTOR dual inhibitors. Disclosures No relevant conflicts of interest to declare.

Effect of tivantinib on VEGF signaling pathways and apoptosis of gastric cancer cells with c-MET or VEGFA amplification.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14719-e14719
Author(s):  
Hyeong Su Kim ◽  
Dae Young Zang ◽  
Sung-Hwa Sohn ◽  
Bohyun Kim ◽  
Hee Jung Sul
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Growth Factor Receptor ◽  
Pcr Analysis ◽  
Gastric Cancer Cells ◽  
Migration Assay ◽  
Normal Tissues ◽  
Vegf Signaling ◽  
Induced Apoptosis

e14719 Background: VEGFA is the key mediator of angiogenesis in cancer and previous studies reported that VEGFA expression was significantly up-regulated in gastric cancer tissues compared with matched normal tissues. We showed that increased levels of VEGFA are significantly associated with expression of hepatocyte growth factor receptor (MET) (r = 0.6255, P < 0.0001). In addition, it is well known that c-MET is potentially a highly plausible target for cancer therapy in gastric cancer. In this study, cytotoxic activity of tivantinib were evaluated in gastric cancer cells with high c-MET expression or VEGFA amplification. Methods: In this study, Western blot and quantitative real-time PCR analysis were used to detect the expression of protein and genes after treatment of tivantinib. In addition, MTS, flow cytometry, and migration assay were used. Results: Tivantinib inhibited growths of a high c-MET-expressed or VEGFA-amplified cell lines. Furthermore, in migration and apoptosis analysis, tivantinib induced apoptosis of SNU620, MKN45 (carried VEGFB mutation), AGS, and MKN28 cells but not in KATO III (carried VEGFB and VEGFC mutation) cells. We also found that tivantinib inhibited the VEGF signaling pathway and MYC expression in VEGFA-amplified gastric cancer cell lines. Conclusions: The data indicate that tivantinib could be a potential therapeutic agent for the treatment of gastric cancer with high c-MET expression or VEGFA amplification.

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