scholarly journals Venetoclax Reverses Metabolic Reprogramming Induced By S1P Modulator FTY720, Suppresses Oxidative Phosphorylation and Synergistically Targets Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1195-1195
Author(s):  
Evgenia Rosenberg ◽  
Valeria Voevoda ◽  
Hila Magen ◽  
Olga Ostrovsky ◽  
Avichai Shimoni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Membrane Potential ◽  
Oxidative Phosphorylation ◽  
Metabolic Reprogramming ◽  
Mitochondrial Activity ◽  
Dependent Manner ◽  
Down Regulation ◽  
Mitochondrial Content ◽  
Bioenergetic Analysis

Abstract Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new therapeutic modalities that bypass these resistance mechanisms. FTY720, also known as fingolimod, is an S1P modulator approved by the FDA to treat the relapsing form of multiple sclerosis. Previously we reported that FTY720 exhibits potent anti-myeloma effect in vitro and in vivo in disseminated xenograft model of MM (Beider et al., Clin Cancer Res 2017). Cytotoxic activity of FTY720 was associated with down-regulation of anti-apoptotic protein MCL-1 while not affecting BCL-2 levels. It is therefore conceivable that BCL-2 inhibition using BH3-mimetic venetoclax may improve responses to FTY720. Incubation of human MM cell lines (n=8) and primary MM cells (n=3) with venetoclax and FTY720 combination synergistically potentiated cell death (CI<0.02), regardless of the MM cells t (11; 14) status. The robust apoptosis induced by venetoclax /FTY720 treatment was accompanied by cytochrome C release, activation of caspase-3 and extensive DNA damage, demonstrated by increased TUNEL staining and elevated levels of phosphorylated histone H2AX, respectively . These effects were associated with down-regulation of BCL-2 protein, stabilization of pro-apoptotic Bak protein, loss of mitochondrial membrane potential, ER stress induction, and inhibition of the AKT/mTOR signaling pathway. Furthermore, the venetoclax /FTY720 combination markedly induced mitochondrial calcium flux and mitochondrial ROS generation. Corresponding with mitochondrial destabilization, venetoclax/FTY720 combination promoted the release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol and subsequently increased AIF nuclear localization, suggesting its functional role in the execution phase of the apoptosis in response to the dual treatment. AIF is a mitochondrial oxidoreductase that contributes to cell death programs and participates in the assembly of the respiratory chain. Of note, single-agent treatment with FTY720 profoundly up-regulated mitochondrial AIF levels. Given the regulative role of AIF in mitochondrial bioenergetics, we could suggest that increased mitochondrial levels of AIF upon FTY720 exposure may support adaptive responses and promote MM survival upon mitochondrial stress. We thus investigated a possible effect of venetoclax and FTY720 separately or in combination on the metabolic activity of MM cells, observing distinct metabolic profiles of single versus combined exposures. FTY720 significantly suppressed glycolysis, down-regulating the transcript levels of the glycolytic enzymes HK2, PDK1, and LDHA. Glycolytic suppression may result in upregulation of mitochondrial content, which maintains cell survival. In accordance, increased mitochondrial activity was detected in FTY720-treated MM cells, detected by high uptake of MitoSpy Red, a dye that stains mitochondria in a membrane potential-dependent manner. To determine if the changes in the mitochondrial content also altered mitochondrial function, bioenergetic analysis was undertaken. FTY720-treated MM cells demonstrated increased levels of NDUFB8 and UQCRC2 (subunits of mitochondrial respiratory complexes I and III, respectively). Furthermore, FTY720 exposure up-regulated ATP production, suggesting an increase in tumor-protective oxidative phosphorylation (OXPHOS). In agreement, inhibition of mitochondrial electron transport chain using rotenone sensitized MM cells to FTY720, synergistically promoting cell death. Notably, co-treatment with venetoclax effectively reversed the metabolic changes mediated by FTY720, reducing mitochondrial mass, suppressing mitochondrial activity and strongly down-regulating the pathways related to OXPHOS. Furthermore, venetoclax/FTY720 combination significantly reduced glutathione (GSH) levels, therefore suppressing antioxidative cell responses. To conclude, we unveil venetoclax role in the metabolic regulation in MM cells. Venetoclax reverses metabolic reprogramming induced by FTY720, suppresses mitochondrial respiration, induces vigorous mitochondrial damage and preferentially targets MM cells in combination with FTY720. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Figure 1 Figure 1. Disclosures Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding.

Anti-Leukemia and Multiple Myeloma Selective Activity of CXCR4 Antagonist 4F-Benzoyl-TN14003 Involves Apoptotic Death Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3857-3857
Author(s):  
Katia Beider ◽  
Michal Begin ◽  
Michal Abraham ◽  
Hanna Wald ◽  
Ido Weiss ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Hematological Malignancies ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Jurkat Cells ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Hl60 Cells

Abstract Abstract 3857 Poster Board III-793 The chemokine receptor CXCR4 and its ligand CXCL12 are involved in the development and progression of a diverse number of hematological malignancies, including leukemia, lymphoma and multiple myeloma (MM). Binding CXCL12 to CXCR4 activates a variety of intracellular signal transduction pathways and effector molecules that regulate cell chemotaxis, adhesion, survival, apoptosis and proliferation. It was previously shown that CXCR4 signaling can directly induce caspase-independent cell apoptosis through the interaction with the HIV gp120 envelope protein. In the present study we investigated the effect of CXCR4 specific antagonists 4F-benzoyl-TN14003 (T140) and AMD3100 on the survival and proliferation of different human hematological cancer cells. Here, we demonstrate that T140, but not AMD3100, exhibits preferential cytotoxicity towards malignant cells of hematopoietic origin, as compared to primary normal cells or solid prostate and breast tumor cells. The in vitro treatment with T140, but not with AMD3100, significantly decreased the number of viable chronic myeloid leukemia K562 cells, acute T cell leukemia Jurkat cells, acute promyelocytic leukemia NB4 and HL60 cells, and four different MM cell lines (U266, NCI-H929, RPMI8226 and ARH77), demonstrating the highest sensitivity to T140 (p<0.01). Notably, T140 inhibited the growth of freshly isolated leukemia and MM cells obtained from consenting patients. T140 inhibits the growth of MM and leukemic cells by inducing their apoptotic cell death. The apoptotic changes in the cells were associated with morphological changes, phosphatidylserine externalization, sub-G1 arrest, DNA double-stranded breaks, decrease in mitochondrial membrane potential, release of cytochrome c, and caspase 3 activation. The important role of CXCR4 in T140-mediated cell death was confirmed by demonstrating that CXCR4 over-expression in NB4 and K562 cells increased their sensitivity to T140. Furthermore, pretreatment of NB4 and HL60 cells with AMD3100 abolishes the effect of T140 on these cells, indicating the involvement of CXCR4 in T140-induced apoptosis. Importantly, the combination with novel anti-myeloma agent bortezomib significantly augments anti-myeloma activity of T140. The anti leukemic and MM effect of T140 was confirmed in xenograft in vivo tumor models. Subcutaneous (s.c.) or intra-peritoneal (i.p.) injections of T140 (100 or 300 mcg/mouse) significantly reduced, in a dose-dependent manner, the tumor size in immuno-deficient mice that were previously inoculated s.c. with human acute leukemia cells NB4 or MM cells RPMI8226 (p<0.01). Tumors from animals treated with T140 had smaller sizes and weights, larger necrotic areas and high apoptotic scores. Taken together, these data support the unique anti-cancer effect of T140 in hematological malignancies and indicate the potential therapeutic role of T140 in MM and leukemia patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Arachidonic Acid Metabolism Controls Macrophage Alternative Activation Through Regulating Oxidative Phosphorylation in PPARγ Dependent Manner

2021 ◽  
Vol 12 ◽  
Author(s):  
Miao Xu ◽  
Xiaohong Wang ◽  
Yongning Li ◽  
Xue Geng ◽  
Xudong Jia ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Oxidative Phosphorylation ◽  
Acid Metabolism ◽  
Macrophage Polarization ◽  
Arachidonic Acid Metabolism ◽  
Metabolic Reprogramming ◽  
Alternative Activation ◽  
Dependent Manner ◽  
M2 Polarization

Macrophage polarization is mainly steered by metabolic reprogramming in the tissue microenvironment, thus leading to distinct outcomes of various diseases. However, the role of lipid metabolism in the regulation of macrophage alternative activation is incompletely understood. Using human THP-1 and mouse bone marrow derived macrophage polarization models, we revealed a pivotal role for arachidonic acid metabolism in determining the phenotype of M2 macrophages. We demonstrated that macrophage M2 polarization was inhibited by arachidonic acid, but inversely facilitated by its derived metabolite prostaglandin E2 (PGE2). Furthermore, PPARγ bridges these two seemingly unrelated processes via modulating oxidative phosphorylation (OXPHOS). Through inhibiting PPARγ, PGE2 enhanced OXPHOS, resulting in the alternative activation of macrophages, which was counterweighted by the activation of PPARγ. This connection between PGE2 biosynthesis and macrophage M2 polarization also existed in human and mouse esophageal squamous cell carcinoma. Our results highlight the critical role of arachidonic acid and metabolic PGE2 as immune regulators in modulating tissue homeostasis and pathological process.

scholarly journals Cannabidiol Induces Apoptosis and Perturbs Mitochondrial Function in Human and Canine Glioma Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Chase Gross ◽  
Dominique A. Ramirez ◽  
Stephanie McGrath ◽  
Daniel L. Gustafson
Keyword(s):  
Cell Death ◽  
Anticancer Agent ◽  
Glioma Cells ◽  
Fluorescent Imaging ◽  
Mitochondrial Activity ◽  
Dependent Manner ◽  
Trpv1 Channel ◽  
Pharmacologic Inhibitors ◽  
Dose Dependent

Cannabidiol (CBD), the major non-psychoactive compound found in cannabis, is frequently used both as a nutraceutical and therapeutic. Despite anecdotal evidence as an anticancer agent, little is known about the effect CBD has on cancer cells. Given the intractability and poor prognoses of brain cancers in human and veterinary medicine, we sought to characterize the in vitro cytotoxicity of CBD on human and canine gliomas. Glioma cells treated with CBD showed a range of cytotoxicity from 4.9 to 8.2 μg/ml; canine cells appeared to be more sensitive than human. Treatment with &gt;5 μg/ml CBD invariably produced large cytosolic vesicles. The mode of cell death was then interrogated using pharmacologic inhibitors. Inhibition of apoptosis was sufficient to rescue CBD-mediated cytotoxicity. Inhibition of RIPK3, a classical necroptosis kinase, also rescued cells from death and prevented the formation of the large cytosolic vesicles. Next, cellular mitochondrial activity in the presence of CBD was assessed and within 2 hours of treatment CBD reduced oxygen consumption in a dose dependent manner with almost complete ablation of activity at 10 μg/ml CBD. Fluorescent imaging with a mitochondrial-specific dye revealed that the large cytosolic vesicles were, in fact, swollen mitochondria. Lastly, calcium channels were pharmacologically inhibited and the effect on cell death was determined. Inhibition of mitochondrial channel VDAC1, but not the TRPV1 channel, rescued cells from CBD-mediated cytotoxicity. These results demonstrate the cytotoxic nature of CBD in human and canine glioma cells and suggest a mechanism of action involving dysregulation of calcium homeostasis and mitochondrial activity.

scholarly journals Oxidative Phosphorylation Regulated By PGC-1α Promotes Multiple Myeloma Progression

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5524-5524
Author(s):  
Yu Xiang ◽  
Bin Fang ◽  
Yilin Liu ◽  
Siqi Yan ◽  
Yicheng Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Oxidative Phosphorylation ◽  
Differential Expression Analysis ◽  
Gene Set Enrichment Analysis ◽  
Cell Counting ◽  
Mitochondrial Morphology ◽  
Dependent Manner ◽  
Gene Set ◽  
Oxphos Gene ◽  
Geo Database

Introduction: With the deeper understanding of metabolic reprograming, more and more studies highlight that many tumors heavily rely on mitochondrial oxidative phosphorylation (OXPHOS) for bioenergetics and biosynthesis, and targeting OXPHOS appears to be a promising approach for cancer therapy. Despite multiple metabolism alterations occur in multiple myeloma (MM), the role of OXPHOS in MM is rarely focused on. Many metabolic pathways are orchestrated by certain master co-regulators through transcriptional programs. The expression of OXPHOS gene set was reported to be regulated by peroxisome proliferator activated receptor gamma coactivator 1α (PGC-1α) in some other tissues, which serves as a transcriptional coactivator. Our previous studies demonstrated that PGC-1α was overexpression in MM cell line RPMI-8226 and knockdown of PGC-1α inhibited proliferation. Herein, we aimed to investigate the significance of OXPHOS in MM and clarify whether PGC-1α is the core regulator of OXPHOS in MM. Methods: Transcriptome data of GSE6477, GSE13591, GSE47552 were downloaded from the Gene Expression Omnibus (GEO) database and then removed batch effects. Gene set variation analysis (GSVA) were used to identify pathways enriched in MM. Differential expression analysis was performed and the PGC-1α expression value was extracted. Gene set enrichment analysis (GSEA) was adopted to explore biological functions involved by PGC-1α. RT-PCR was conducted to reflect the influence of PGC-1α inhibitor on OXPHOS gene set. Mitochondrial morphology was exhibited by transmission electron microscopy analysis. Cell counting kit-8 assay was employed to determine cell viability and flow cytometry analysis was used to assess apoptosis. Results: Integrated bioinformatics analysis revealed that OXPHOS pathway was significantly upregulated in MM than in normal donors (Figure A), and aberrantly overexpression of OXPHOS gene set was confirmed in our MM samples at mRNA level (Figure B-C). Moreover, we also demonstrated that aberrantly overexpression of OXPHOS genes in MM were generally associated with poorer survival (Figure D-F), indicating that OXPHOS appears as a potential oncogenic pathway for MM progression. Meanwhile, bioinformatics results showed that PGC-1α was upregulated in 247 MM patients from GEO database (Figure G). In addition, our results indicated that the overexpression of PGC-1α prevailed among 5 MM cell lines with different inherited backgrounds and our several MM samples (Figure H-I). GSEA analysis showed that gene sets of OXPHOS, TCA cycle, respiratory electron transport, ATP synthesis, were all significantly enriched in MM patients with high PGC-1α expression (Figure J-L). The inhibition of PGC-1α have exerted significant inhibition effect on the transcription of OXPHOS gene set in MM. In accordance with that OXPHOS is a major source of ATP, PGC-1α inhibitor resulted in the reduction of ATP levels of MM cells. Meanwhile, treatment of PGC-1α inhibitor triggered shriveled mitochondria with decreased volume, disorganized cristae, and increased electron density of matrix, indicative of impaired mitochondria OXPHOS. In vitro experiments suggested that PGC-1α inhibitor robustly inhibited proliferation of MM cells in a time and dose-dependent manner, with little effect on normal hematopoietic cells. Besides, PGC-1α inhibitor induced MM cells apoptosis significantly. Conclusions: Our investigations reported for the first time that OXPHOS regulated by PGC-1α may serve as a potential mechanism for MM progression. Besides, this study may provide new strategies for the treatment of MM from the perspective of OXPHOS and its core regulator. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Octadecaneuropeptide prevents toxicity induced by 6-hydroxydopamine in cultured rat astrocyte: involvement of the endogenous antioxidant systems and the intrinsic apoptotic pathway

2018 ◽  
Author(s):  
Hadhemi Kaddour ◽  
Yosra Hamdi ◽  
David Vaudry ◽  
Jérôme Leprince ◽  
Hubert Vaudry ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Apoptotic Cell ◽  
Antioxidant Systems ◽  
Mitochondrial Activity ◽  
Ros Generation ◽  
Potential Candidate ◽  
Dependent Manner ◽  
Antioxidant Defences ◽  
Apoptotic Pathway

AbstractOxidative stress, associated with various neurodegenerative diseases, induces imbalance in ROS generation, impairs cellular antioxidant defences and finally triggers both neurons and astroglial cell death by apoptosis. Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides, including the octadecaneuropeptide (ODN). We have previously reported that ODN is a potent neuroprotective agent that prevents 6-OHDA-induced apoptotic neuronal death. The purpose of the present study was to investigate the potential glioprotective effect of ODN on 6-OHDA-induced oxidative stress and cell death in cultured rat astrocytes. Incubation of astrocytes with graded concentrations of ODN (10−14 to 10−8 M) inhibited 6-OHDA-evoked cell death in a concentration- and time-dependent manner. In addition, ODN prevented the decrease of mitochondrial activity and caspase-3 activation induced by 6-OHDA. Toxin-treated cells exhibited high level of ROS associated with a generation of H2O2 and O2°-and a reduction of both SOD and catalase activities. Co-treatment of astrocytes with low concentrations of ODN dose dependently blocked 6-OHDA-evoked ROS production and inhibition of antioxidant enzymes activities. Taken together, these data demonstrate that ODN is a potent glioprotective agent that prevents 6-OHDA-induced oxidative stress and apoptotic cell death. ODN is thus a potential candidate to delay neuronal damages in various pathological conditions involving oxidative neurodegeneration.

OSU03012, a Novel Celecoxib Derivative, Induces Apoptosis in Multiple Myeloma Cells Independently of Caspase Activation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5075-5075
Author(s):  
Shuhong Zhang ◽  
Valerie L. White ◽  
Amy Johnson ◽  
Ching-Shih Chen ◽  
Sherif S. Farag
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Plasma Cells ◽  
Parp Cleavage ◽  
Caspase Inhibitor ◽  
High Concentration ◽  
Down Regulation ◽  
Vitro Effect

Abstract Multiple myeloma (MM) is a clonal disorder affecting terminally differentiated B cells, with the accumulation of plasma cells in the bone marrow. Previous studies showed that OSU03012 is a novel celecoxib derivative lacking cyclooxygenase-2 inhibitory activity that induces apoptosis in various types of cancer cells and is being developed as an anti-cancer therapy in the NCI Rapid Access to Intervention Therapy (RAID). Here, we examined the in vitro effect of OSU03012 in MM cell lines (U266, ARH-77, IM-9 and RPMI8226). Cytotoxicity data indicated that mean LC50 (lethal concentration 50%) of OSU03012 was 6.25±0.86 μM at 24 hours and 4.23±0.87 μM at 72 hours in these four cell lines. Using annexin V/PI (propidium iodide) flow cytometry assay, OSU03012 was shown to induce apoptosis in MM cells. OSU03012 activated caspases-8, -9, and -3, induced PARP (POLY ADP-RIBOSE Polymerase) cleavage, and reduced survivin and XIAP expression after 6 and 24 hour exposure. Although the caspase inhibitor Q-VD-OPH treatment strongly blocked OSU03012-induced PARP cleavage, it did not inhibit OSU03012-induced apoptosis of MM cells. The pan-caspase inhibitor z-VAD-fmk did not prevent OSU03012 mediated cell death. Cell death with OSU03012 treatment was associated with significant down-regulation of phospho-Akt. Several substrates of AKT, including phospho-GSK-3 beta (Ser9), phospho-FoxO1a (Ser256) and phospho-MDM2 (Ser166) were also down-regulated by OSU03012 drug. OSU03012 triggered both early (6h) and late (24h) down-regulation of cyclin D1 expression, but cyclin A and B1 expression was down-regulated only at 24h. There was no induction of p21 or p27 protein levels by OSU03012. After 24-hour exposure, low concentration (1–5 μM) OSU03012 arrested MM cell lines in the G1 phase of the cell cycle while high concentration (10 μM) OSU03012 induced G2 phase arrested. OSU03012 decreased both phospho-Stat3 (Ser727) and Stat3 expression. OSU03012 has on effect on phosphorylated MAP kinase kinase1/2 (pMEK1/2) but it decreased MEK1/2 expression at 24h. The expression levels of Bcl-2 family proteins, Bcl-2, Mcl-1, BAX, and BIM did not alter with OSU03012 treatment suggesting that Bcl-2 members may not play direct or significant roles in inducing cell death. Taken together, we conclude that OSU03012 is potently active against MM cells by predominantly caspase-independent mechanisms, and may involve downstream pathways consequent to phopho-Akt down-regulation. These studies provide preclinical rationale for investigating OSU03012 in the treatment of MM.

Nocodazole Induces Multiple Myeloma Cell Death and Reduces Tumor Growth through Sequential Microtubular Network Damage and C-Jun N-Terminal Kinase-Mediated Bcl-2 Phosphorylation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 296-296
Author(s):  
Rentian Feng ◽  
Huihui Ma ◽  
Noriyoshi Kurihara ◽  
Shirong Li ◽  
Judy A Ziegler ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Tumor Growth ◽  
Murine Model ◽  
Immunofluorescent Staining ◽  
Solid Cancer ◽  
Dependent Manner ◽  
Microtubular Network ◽  
Induced Apoptosis

Abstract Abstract 296 Background: Benzimidazoles, originally categorized as broad-spectrum anthelmintic drugs, have been recently reported to induce growth arrest and apoptosis in some solid cancer models (e.g. colorectal and lung). We performed a multiplex drug-screening assay that identified benzimidazoles as potential anti-multiple myeloma (MM) agents. Methods and Results: In this study, we demonstrate that one of the benzimidazole members, nocodazole, inhibited proliferation and induced apoptosis in MM cell lines and primary MM cells alone and in co-culture with bone marrow stromal cells. The resistant phenotype of those MM cells resistant to conventional therapies could be completely reversed by nocodazole. The IC50 values were 60 nM (RPMI8226-S), 25 nM (RPMI8226-Dox40), 80 nM (RPMI8226-MR20), 60 nM (RPMI8226-LR5), 65 nM (MM.1S) and 60 nM (MM.1R). Viability of primary cells decreased by 66% in CD138+ cells and 7% in CD138− mononucleated bone marrow cells after 48 hour treatment. Cell cycle analysis revealed a G2/M arrest and subsequent cell death induced by nocodazole. Nocodazole also caused morphologic elongation in MM cells in a dose-dependent manner during prometaphase. The morphologically changed cells exhibited a microtubular network disarray as evidenced by microtubular immunofluorescent staining. Signaling studies indicated that increased expression of Bim protein and reduced XIAP and Mcl-1 levels were involved in nocodazole-induced apoptosis. Further investigation showed Bcl-2 phosphorylation as a critical mediator of cell death, which was triggered by the activation of JNK, instead of p38 kinase or ERKs. Treatment with JNK inhibitor SP600125 completely inhibited Bcl-2 phosphorylation at Ser70 and Thr56 induced by nocodazole. Nocodazole-induced cell death subsequently decreased from 79% to 28% after pretreatment with SP600125. Combination of nocodazole with dexamethasone induced significantly stronger induction of cell death at either drug dose. Dexamethasone at 20 nM, nocodazole at 15 or 30 nM could only induce 19%, 10.3% and 16% cell death, respectively. However, their combinations resulted in 67% and 92% nuclear fragmentation, respectively. Based on our in vitro data, we analyzed nocodazole in a SCID xenograft murine model. Nocodazole alone (5 and 20 mg/kg) or combined with dexamethasone (2 mg/kg) at a lower dose of 12 mg/kg significantly inhibited H929 tumor growth and prolonged survival in a SCID xenograft murine model. Conclusions: Our studies demonstrate that nocodazole has a potent anti-MM activity and might be a promising new treatment approach for MM. * Supported by a grant from the Multiple Myeloma Research Foundation. Disclosures: Roodman: Acceleron: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.

TM-233 Exerts Anti-Myeloma Activity and Overcomes Bortezomib-Resistance In Human Multiple Myeloma Cells By Targeting NF-κB and JAK/STAT Dual-Signaling Pathways

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4427-4427
Author(s):  
Morihiko Sagawa ◽  
Tatsuki Tomikawa ◽  
Tomoe Anan ◽  
Takayuki Tabayashi ◽  
Reiko Watanabe ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Signaling Pathways ◽  
Conflicts Of Interest ◽  
Subcutaneous Administration ◽  
Cellular Growth ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Grade 3

Although the introduction of bortezomib and immunomodulatory drugs (IMiDs) has led to improved outcomes in patients with multiple myeloma (MM), the disease remains incurable. Bortezomib, a proteasome inhibitor, is widely used in the treatment of MM and has resulted in marked therapeutic effects; however, this therapy is often complicated by peripheral neuropathy (PN), of which grade ≥3 PN is dose-limiting toxicity and can necessitate cessation of therapy. Subcutaneous administration of bortezomib can reduce the incidence of PN; however, among cases of PN that still occur, 24% are grade 2 PN and 6% are grade 3 PN. These data suggest that the incidence of PN higher than grade 2 is not attenuated by the subcutaneous delivery of bortezomib. In addition, patients often become refractory to bortezomib after long-term use. In an effort to identify potent and well-tolerated agents, clinical trials of novel agents (e.g., carfilzomib, pomalidomide, and monoclonal antibody against CS-1) are being conducted both in patients with newly diagnosed MM and in those with relapsed/refractory disease. We previously reported that 1’-acetoxychavicol acetate (ACA) obtained from the rhizomes of the plant Languas galanga induces cell death of MM cells in vitro and in vivo through inhibition of NF-κB-related functions (Cancer Res, 2005; 65: 4417). Subsequently, we developed several ACA analogs based on quantitative structure-activity relationship (QSAR) analysis to develop more potent NF-κB inhibitors, and successfully synthesized a novel benzhydrol-type analog of ACA, named TM-233, that exerted potent growth inhibition against various MM cells (U266, RPMI8226, and MM-1S cells) in a dose- and time-dependent manner when compared with ACA (Chem Pharm Bull., 2008; 56: 1490). Further, TM-233 inhibited constitutive phosphorylation of JAK2 and STAT3 and down-regulated the expression of anti-apoptotic Mcl-1 protein. TM-233 directly bound and activated the transcription of the Mcl-1 gene promoter. Mcl-1 is the downstream molecule of STAT3; therefore, these results suggest that TM-233 induces cell death in MM cells with down-regulated Mcl-1 via modulation of the JAK/STAT pathway. In addition, we examined the DNA-binding activity of NF-κB in TM-233-treated MM cells and found that NF-κB was inhibited by TM-233. Further, Western blotting showed that TM-233 rapidly decreased the nuclear expression of NF-κB but increased the accumulation of NF-κB in the cytosol, suggesting that TM-233 inhibits the translocation of NF-κB from the cytosol to the nucleus. Immunohistochemical analysis confirmed that the p50/RelA dimer of NF-κB was located in the cytosol and not in the nucleus in TM-233-treated MM cells. We then examined the effects of TM-233 on bortezomib-resistant MM cells. Bortezomib-resistant MM cell lines (i.e., KMS-11/BTZ and OPM-2/BTZ) were established by limiting dilution. We found that these cells have a unique point mutation, G322A, in the gene encoding the proteasome β5 subunit (Leukemia 2010; 24: 1506). TM-233, but not bortezomib, inhibited cellular growth and induced cell death in KMS-11/BTZ and OPM-2/BTZ cells in a time- (0-48 hours) and dose- (0-5 μM) dependent manner. Furthermore, the combination of low-dose TM-233 (less than 2 μM) and bortezomib (10 nM) significantly induced cell death in bortezomib-resistant MM cells via inhibition of NF-κB activity. These results indicate that TM-233 could overcome bortezomib resistance in MM cells by acting via different mechanisms from those of bortezomib. In conclusion, TM-233 induced cell death in MM cells, and this effect was mediated through the JAK/STAT and NF-κB dual-signaling pathways. These data indicate that TM-233 might be a more potent and more specific NF-κB inhibitor than that of original compound (ACA), and might be able to overcome bortezomib-resistance in MM cells. Therefore, further studies investigating clinical approaches, including combination therapy, are warranted. Disclosures: No relevant conflicts of interest to declare.

Novel Therapeutic Approach to Overcome Both Bortezomib- and Lenalidomide-Resistant Multiple Myeloma By Targeting TOPK/PBK

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1763-1763
Author(s):  
Takayuki Tabayashi ◽  
Yasuyuki Takahashi ◽  
Yuta Kimura ◽  
Tatsuki Tomikawa ◽  
Tomoe Nemoto-Anan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Protein Kinase ◽  
Cell Growth ◽  
Research Funding ◽  
Apoptotic Cell ◽  
Mapk Signaling ◽  
Apoptotic Cell Death ◽  
Dependent Manner ◽  
Dose Dependent

Abstract Multiple myeloma (MM) is a neoplasm of plasma cells that often remains fatal despite the use of high-dose chemotherapy with hematopoietic stem cell transplantation. In the clinical setting, the introduction of novel agents, such as proteasome inhibitors and immunomodulatory drugs, has improved the clinical outcomes of both patients with newly diagnosed MM and patients with advanced MM. However, most patients eventually relapse and develop drug resistance. T-LAK cell-originated protein kinase (TOPK), also known as PDZ-binding kinase (PBK), is a mitogen-activated protein kinase kinase (MAPKK)-like serine/threonine kinase that plays a critical role in many cellular functions, such as cell proliferation, apoptotic cell death, and inflammation, in normal tissues. Because the expression of TOPK is up-regulated during mitosis and is activated by the Cdk1/cyclin B1 complex, TOPK is thought to have a role in cytokinesis. While the expression of TOPK is very low in most normal human tissues except for testis and placenta, it is overexpressed in various malignant neoplasms, indicating its crucial role in tumorigenesis. Phosphorylation of TOPK leads to the activation of the MAPK signaling pathway including p38 and Ras extracellular signal-regulated kinase (ERK). Moreover, TOPK interacts with p53 tumor suppressor protein and inhibits its function. Ribosomal protein S6 kinase (RSK2) is a downstream target of the ERK/MAPK signaling cascade and it has a pivotal role in cell survival and proliferation. Recent studies suggest that RSK2 inhibition induces apoptotic cell death and sensitizes MM cells to lenalidomide. Suppression of p53 function is also involved in MM progression. Taken together, these data suggest that TOPK might be an attractive target for new therapeutic agents against this incurable hematological malignancy. HI-TOPK-032, which is a potent and specific inhibitor of TOPK, occupies the ATP-binding site of TOPK and thereby suppresses TOPK kinase activity. In the present study, we investigated the role of TOPK/PBK in MM as a potential therapeutic target by using HI-TOPK-032. MTSand trypan blue dye exclusion assays showed that HI-TOPK-032 inhibited the proliferation of various MM cell lines, including U266, RPMI8226, MM1.S, OPM-2, and KMS-11, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. To examine the mechanisms behind the growth inhibition effect induced by HI-TOPK-032, assays for apoptotic cell death were performed; these assays demonstrated that HI-TOPK-032 induced both early and late apoptosis in MM cells. To investigate the molecular mechanisms of HI-TOPK-032-induced cell death in MM cells, the expression of various cell death-associated proteins and down-stream molecules of TOPK was examined. Western blotting analysis showed that HI-TOPK-032 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by reducing t he phosphorylation of ERK and RSK2, thereby reducing the expression of the target molecules of RSK2, i.e., MCL1 and c-Myc. Moreover, HI-TOPK-032 induced p53 expression in a dose-dependent manner. We next examined the effects of HI-TOPK-032 on bortezomib (BTZ)-resistant MM cells, which represent an urgent issue in clinics and for which a therapeutic solution is important. Interestingly, HI-TOPK-032 inhibited the proliferation of both BTZ-sensitive wild-type KMS cells and BTZ-resistant KMS cells, suggesting that BTZ resistance can be overcome by targeting TOPK. Because our results showed that HI-TOPK-032 reduced the phosphorylation of RSK2, and previous studies have suggested that RSK2 inhibition sensitized MM cells to lenalidomide, we next studied the effects of HI-TOPK-032 in combination with lenalidomide on MM cell growth. HI-TOPK-032 and lenalidomide synergisticallyinduced growth arrest in not only lenalidomide-sensitive MM cells, but also in lenalidomide-resistant cells. To determine whether HI-TOPK-032 can re-sensitize BTZ-resistant cells to the anti-MM activity of BTZ, the effects of the combination of HI-TOPK-032 and BTZ were tested using an MTS assay. Interestingly, HI-TOPK-032 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that the inhibition of TOPK may serve as an attractive therapeutic option for both patients with BTZ- or lenalidomide-resistant MM. In conclusion, these data suggest that TOPK/PBK can be a promising molecular target for the treatment of MM. Disclosures Kizaki: Nippon Shinyaku Co., Ltd.: Research Funding; Ono Phranacutical Co., Ltd.: Consultancy; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Phrarmaceutical Co., Ltd.: Research Funding.

scholarly journals Pectolinarigenin Induced Cell Cycle Arrest, Autophagy, and Apoptosis in Gastric Cancer Cell via PI3K/AKT/mTOR Signaling Pathway

Nutrients ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 1043 ◽  
Author(s):  
Ho Lee ◽  
Venu Venkatarame Gowda Saralamma ◽  
Seong Kim ◽  
Sang Ha ◽  
Suchismita Raha ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Cycle ◽  
Cell Death ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Phase Cell ◽  
Dependent Manner ◽  
Down Regulation ◽  
Cycle Arrest ◽  
Anti Cancer

Pectolinarigenin (PEC), a natural flavonoid present in Cirsium chanroenicum and in some species of Citrus fruits, has various pharmacological benefits such as anti-inflammatory and anti-cancer activities. In the present study, we investigated the anti-cancer mechanism of PEC induced cell death caused by autophagy and apoptosis in AGS and MKN28 human gastric cancer cells. The PEC treatment significantly inhibited the AGS and MKN28 cell growth in a dose-dependent manner. Further, PEC significantly elevated sub-G1 phase in AGS cells and G2/M phase cell cycle arrest in both AGS and MKN28 cells. Apoptosis was confirmed by Annexin V and Hoechst 33342 fluorescent staining. Moreover, Immunoblotting results revealed that PEC treatment down-regulated the inhibitor of apoptosis protein (IAP) family protein XIAP that leads to the activation of caspase-3 thereby cleavage of PARP (poly-ADP-ribose polymerase) in both AGS and MKN28 cells in a dose-dependent manner. The autophagy-inducing effect was indicated by the increased formation of acidic vesicular organelles (AVOs) and increased protein levels of LC3-II conversion in both AGS and MKN28 cells. PEC shows the down regulation of PI3K/AKT/mTOR pathway which is a major regulator of autophagic and apoptotic cell death in cancer cells that leads to the down-regulation of p-4EBP1, p-p70S6K, and p-eIF4E in PEC treated cells when compared with the untreated cells. In conclusion, PEC treatment might have anti-cancer effect by down-regulation of PI3K/AKT/mTOR pathway leading to G2/M phase cell cycle arrest, autophagic and apoptotic cell death in human gastric cancer cells. Further studies of PEC treatment can support to develop as a potential alternative therapeutic agent for human gastric carcinoma.

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