scholarly journals AAV8 vector expressing IL24 efficiently suppresses tumor growth mediated by specific mechanisms in MLL/AF4-positive ALL model mice

Blood ◽  
2012 ◽  
Vol 119 (1) ◽  
pp. 64-71 ◽  
Author(s):  
Hayato Tamai ◽  
Koichi Miyake ◽  
Hiroki Yamaguchi ◽  
Miyuki Takatori ◽  
Kazuo Dan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
C2c12 Cell ◽  
Caspase 8 ◽  
Apoptotic Pathway ◽  
High Relapse Rate ◽  
Tnf Α

Abstract Mixed-lineage leukemia (MLL)/AF4-positive acute lymphoblastic leukemia (ALL) is a common type of leukemia in infants, which is associated with a high relapse rate and poor prognosis. IL24 selectively induces apoptosis in cancer cells and exerts immunomodulatory and antiangiogenic effects. We examined the effects of adeno-associated virus type 8 (AAV8) vector-mediated muscle-directed systemic gene therapy in MLL/AF4-positive ALL using IL24. In a series of in vitro studies, we examined the effects of AAV8-IL24–transduced C2C12 cell-conditioned medium. We also examined the effects of AAV8-IL24 in MLL/AF4 transgenic mice. The results revealed the effects of AAV8-IL24 in MLL/AF4-positive ALL both in vitro and in vivo. With regard to the mechanism of therapy using AAV8-IL24 in MLL/AF4-positive ALL, we demonstrated the antiangiogenicity and effects on the ER stress pathway and unreported pathways through inhibition of S100A6 and HOXA9, which is specific to MLL/AF4-positive ALL. Inhibition of S100A6 by IL24 was dependent on TNF-α and induced acetylation of p53 followed by activation of the caspase 8–caspase 3 apoptotic pathway. Inhibition of HOXA9 by IL24, which was independent of TNF-α, induced MEIS1 activation followed by activation of the caspase 8–caspase 3 apoptotic pathway. Thus, gene therapy using AAV8-IL24 is a promising treatment for MLL/AF4-positive ALL.

scholarly journals Amlexanox As a Possible Breakthrough Drug for MLL/AF4-Positive Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1590-1590
Author(s):  
Hayato Tamai ◽  
Hiroki Yamaguchi ◽  
Koichi Miyake ◽  
Miyuki Takatori ◽  
Tomoaki Kitano ◽  
...  
Keyword(s):  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Control Diet ◽  
Hematopoietic Stem ◽  
Tnf Α ◽  
Breakthrough Drug

Abstract Background: MLL/AF4-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis even after allogeneic hematopoietic stem cell transplantation. Previously, we reported that this ALL shows resistance to TNF-α, which is the factor involved in the graft versus leukemia (GVL) effect or tumor immunity, by upregulation of S100A6 expression followed by interference with the p53-caspase pathway. Amlexanox, an anti-allergic drug, was reported to inhibit the translocation pathway of endogenous S100A6 in endothelial cells. Aims: This study was performed to examine the effects of Amlexanox on MLL/AF4-positive ALL. Methods: In vitro analysis, cell growth of MLL/AF4-positive ALL cell lines ( SEM and RS4;11) were analyzed with TNF-α (10 ng/mL) and Amlexanox (0, 10, and 100 µg/mL).The effect of Amlexanox on S100A6 and p53-caspase pathways were examined by Western blotting (WB) analysis. In vivo analysis MLL/AF4-positive transgenic mice model, which show CD45R/B220+leukemia by 12 months of age we established and human peripheral blood mononuclear cell (Hu-PBMC) NOD/SCID mice transplanted with SEM-Luc were examined to compare mice fed diet containing Amlexanox (0.02%) with mice fed control diet. Results: There were no significant differences between the growth of SEM or RS4;11 cells in the absence or presence of 10 µg/mL of Amlexanox in vitro under 10 ng/mL of TNF-α. However, both cells showed significant growth inhibition by 10 ng/mL of TNF-α in the presence of 100 µg/mL of Amlexanox (P = 0.0085 for SEM, P = 0.0196 for RS4;11) WB analysis showed that S100A6 was activated in the presence of 10 ng/mL TNF-α, and activated S100A6 was decreased and both acetyl-p53/p53 ratio and cleaved caspase 3/caspase 3 ratio were increased in cells treated with 100 µg/mL of Amlexanox under 10 ng/mL of TNF-α in the MLL/AF4-positive human ALL cell lines. In vivo, MLL/AF4-positive transgenic mice fed a diet containing Amlexanox (0.02%) developed significantly less volume of CD45R/B220+ leukemia at the age of 1 year in comparison with mice fed control diet (P<0.001 for BM and .P<0.001 for spleen). Hu-PBMC NOD/SCID mice transplanted with SEM-Luc in the Amlexanox group showed significantly longer survival than those in the control group (P < 0.014). Conclusions: Amlexanox may be a breakthrough drug for MLL/AF4-positive ALL because it inhibits the resistance of MLL/AF4-positive ALL to TNF-α by downregulating S100A6 expression followed by upregulating the p53-caspase pathway.Specifically, allogeneic hematopoietic stem cell transplantation is expected to show beneficial effects in combination with Amlexanox. Disclosures No relevant conflicts of interest to declare.

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

scholarly journals TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

2021 ◽  
Author(s):  
Hongli Zhou ◽  
Minyu Zhou ◽  
Yue Hu ◽  
Yanin Limpanon ◽  
Yubin Ma ◽  
...  
Keyword(s):  
Cell Death ◽  
Enrichment Analysis ◽  
Angiostrongylus Cantonensis ◽  
Gene Set Enrichment Analysis ◽  
Caspase 8 ◽  
Cns Injury ◽  
Vitro Assay ◽  
Tnf Α

AbstractAngiostrongylus cantonensis (AC) can cause severe eosinophilic meningitis or encephalitis in non-permissive hosts accompanied by apoptosis and necroptosis of brain cells. However, the explicit underlying molecular basis of apoptosis and necroptosis upon AC infection has not yet been elucidated. To determine the specific pathways of apoptosis and necroptosis upon AC infection, gene set enrichment analysis (GSEA) and protein–protein interaction (PPI) analysis for gene expression microarray (accession number: GSE159486) of mouse brain infected by AC revealed that TNF-α likely played a central role in the apoptosis and necroptosis in the context of AC infection, which was further confirmed via an in vivo rescue assay after treating with TNF-α inhibitor. The signalling axes involved in apoptosis and necroptosis were investigated via immunoprecipitation and immunoblotting. Immunofluorescence was used to identify the specific cells that underwent apoptosis or necroptosis. The results showed that TNF-α induced apoptosis of astrocytes through the RIP1/FADD/Caspase-8 axis and induced necroptosis of neurons by the RIP3/MLKL signalling pathway. In addition, in vitro assay revealed that TNF-α secretion by microglia increased upon LSA stimulation and caused necroptosis of neurons. The present study provided the first evidence that TNF-α was secreted by microglia stimulated by AC infection, which caused cell death via parallel pathways of astrocyte apoptosis (mediated by the RIP1/FADD/caspase-8 axis) and neuron necroptosis (driven by the RIP3/MLKL complex). Our research comprehensively elucidated the mechanism of cell death after AC infection and provided new insight into targeting TNF-α signalling as a therapeutic strategy for CNS injury.

scholarly journals In Vitro and In Vivo Anticancer Activity of the Most Cytotoxic Fraction of Pistachio Hull Extract in Breast Cancer

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1776 ◽  
Author(s):  
Maryam Seifaddinipour ◽  
Reyhaneh Farghadani ◽  
Farideh Namvar ◽  
Jamaludin Bin Mohamad ◽  
Nur Airina Muhamad
Keyword(s):  
Breast Cancer ◽  
Caspase 3 ◽  
Active Agent ◽  
Suppressive Effect ◽  
Caspase 8 ◽  
Nuclear Staining ◽  
Active Components ◽  
Mcf 7

Pistacia (Pistacia vera) hulls (PV) is a health product that has been determined to contain bioactive phytochemicals which have fundamental importance for biomedical use. In this study, PV ethyl acetate extraction (PV-EA) fractions were evaluated with the use of an MTT assay to find the most cytotoxic fraction, which was found to be F13b1/PV-EA. After that, HPTLC was used for identify the most active compounds. The antioxidant activity was analyzed with DPPH and ABTS tests. Apoptosis induction in MCF-7 cells by F13b1/PV-EA was validated via flow cytometry analysis and a distinctive nuclear staining method. The representation of genes like Caspase 3, Caspase 8, Bax, Bcl-2, CAT and SOD was assessed via a reverse transcription (RT_PCR) method. Inhabitation of Tubo breast cancer cell development was examined in the BALB-neuT mouse with histopathology observations. The most abundant active components available in our extract were gallic acid and the flavonoid quercetin. The F13b1/PV-EA has antiradical activity evidence by its inhibition of ABTS and DPPH free radicals. F13b1/PV-EA displayed against MCF-7 a suppressive effect with an IC50 value of 15.2 ± 1.35 µg/mL. Also, the expression of CAT, SOD, Caspase 3, Caspase 8 and Bax increased and the expression of Bcl-2 decreased. F13b1/PV-EA dose-dependently inhibited tumor development in cancer-induced mice. Thus, this finding introduces F13b1/PV-EA as an effectual apoptosis and antitumor active agent against breast cancer.

Intracellular NAD+ Depletion Enhances Bortezomib-Induced Myeloma Cytotoxicity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 330-330
Author(s):  
Antonia Cagnetta ◽  
Michele Cea ◽  
Chirag Acharya ◽  
Teresa Calimeri ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Cell Death ◽  
Synergistic Effect ◽  
Cell Lines ◽  
Caspase 3 ◽  
Statistical Significance ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Nicotinamide Phosphoribosyltransferase

Abstract Abstract 330 Background: Our previous study demonstrated that inhibition of nicotinamide phosphoribosyltransferase (Nampt) acts by severely depleting intracellular NAD+ content and thus eliciting mitochondrial dysfunction and autophagic MM cell death. The proteasome inhibitor Bortezomib induces anti-MM activity by affecting a variety of signaling pathways. However, as with other agents, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we demonstrate that combining Nampt inhibitor and bortezomb induces synergistic anti-MM cell death both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. Material and Methods: We utilized MM.1S, MM.1R, RPMI-8226, and U266 human MM cell lines, as well as purified tumor cells from patients relapsing after prior therapies. Cell viability and apoptosis assays were performed using Annexin V/PI staining. Intracellular NAD+ level and proteasome activity were quantified after 12, 24, and 48h exposure to single/combination drugs by specific assays. In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, and tubulin. CB-17 SCID male mice (n = 28; 7 mice/EA group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum free RPMI-1640 medium. When tumors were measurable (3 weeks after MM cell injection), mice were treated for three weeks with vehicle alone, FK866 (30mg/kg 4 days weekly), Bortezomib (0.5 mg/kg twice weekly), or FK866 (30 mg/kg) plus Bortezomib (0.5 mg/kg). Statistical significance of differences observed in FK866, Bortezomib or combination-treated mice was determined using a Student t test. Isobologram analysis was performed using “CalcuSyn” software program. A combination index < 1.0 indicates synergism. Results/Discussion: Combining FK866 and Bortezomib induces synergistic anti-MM activity in vitro against MM cell lines (P<0.005, CI < 1) or patient CD138-positive MM cells (P< 0.004). FK866 plus Bortezomib-induced synergistic effect is associated with: 1)activation of caspase-8, caspase-9, caspase-3, and PARP; 2) improved intracellular NAD+ dissipation; 3) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteolytic activities; 4) inhibition of NF-kappa B signaling; and 5) inhibition of angiogenesis. Importantly, the ectopic overexpression of Nampt rescues this observed synergistic effect; conversely, Nampt knockdown by RNAi significantly enhances the anti-MM effect of bortezomib. In the murine xenograft MM model, low dose combination FK866 (30 mg/kg) and Bortezomib (0.5 mg/kg) is well tolerated, significantly inhibits tumor growth (P < 0.001), and prolongs host survival (2–2.5 months in mice receiving combined drugs, P = 0.001). These findings demonstrate that intracellular NAD+ levels represent a major determinant in the ability of bortezomib to induce apoptosis of MM cells, providing the rationale for clinical protocols evaluating FK866 together with Bortezomib to improve patient outcome in MM. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

Obinutuzumab (GA101) Significantly Enhances Cell Death and ADCC Compared to Rituximab Against CD20+ sensitive and Rituximab Resistant B-Cell Non-Hodgkin Lymphoma (NHL) and Lymphoblastic Leukemia (BLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4865-4865 ◽  
Author(s):  
Aradhana Awasthi Tiwari ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
Danielle Glassman ◽  
Anthony Sabulski ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Dismal Prognosis

Abstract Abstract 4865 Background: Patients who relapse with CD20+ B-NHL and B cell lymphoblastic leukemia (B-LL) have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012,Mils/Cairo et al. BJH,2012) and often require alternative therapeutic strategies. Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Cairo M.S. et al. ASCO, 2010). Resistance to RTX, however, may predispose patients with CD20+ NHL to an increase risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, mediates enhanced cell death vs RTX and has a glycoengineered Fc region that induces significantly enhanced ADCC (Mössner et al. Bld, 2010; Niederfellner G. et al. Bld, 2011; Bologna L et al. JI, 2012). Objective: To evaluate the in-vitro efficacy of GA101 compared to RTX against RTX sensitive and resistant CD20+ B-NHL and B-LL cell lines. Methods: Raji (CD20+,ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany), Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) and Raji-2R and Raji-4RH (generously supplied by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS and incubated with GA101 and/or RTX at 100 μg/ml for 24 hrs (n=6), 48 and 72 hrs (n=5). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. Loucy cells (CD20−) were used as the negative control. The caspase 3/7 activity was measured by FAM caspase 3/7 assay kit by FLICA™ methodology. RSCL, RRCL, U698-M and Loucy were incubated with GA101 and RTX treatment for 24, 48 and 72 hrs, and caspase3/7 activity was detected by FACS using 488 nm excitation and emission filter (n=3). ADCC were performed with K562-IL-15–41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) as well as IL-2 expanded NK cells, at 20:1 effector: target ratio (E: T, n=3) using europium release assay (Perkin-Elmer). Results: GA101 induced significantly more cell death compared to RTX in B-NHL and BLL cell lines. (Table-1) GA101 vs RTX shows a significantly increase in caspase 3/7 activity in Raji 16.92±0.84% vs 11.76±0.08% compared to Raji2R 6.7±0.62% vs 2.8±0.7%, Raji4RH 5.8±0.35% vs 2.0±0.3% and U698-M 12.54±0.44% vs 9.6±0.95% compared to Loucy 3.22±0.45% vs 2.59±0.05%, respectively, at 24 hrs of treatment (p<0.0001). GA101 vs RTX also elicited a significant increase a ADCC with K562-IL15–41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% compared to Raji-2R 38.0±2.0% vs 21.6±1.2%, Raji-4RH 40.0±1.6% vs 0.5±1.1% and U698-M 70.0±1.6% vs 45.56±0.1%, compared to Loucy 21.67±0.48% vs 15.92±0.52%, respectively (p<0.001) at day 7.The IL-2 alone expanded Hu-NK cells demonstrated a reduction of 10–20% cytotoxicity compared to K562-IL15–41BBL Hu-NK cells at day 7 against BLL, RSCL and RRCL, in-vitro. Conclusion: Obinutuzumab compared to RTX significantly enhanced cell death, caspase3/7 activity and NK mediated ADCC in sensitive and RTX resistant B-NHL and B-LL. Obinutuzumab represents a promising candidate for treating RTX sensitive and resistant CD20+ B-Cell Lymphomas and lymphoblastic leukemia. Further studies will investigate the combination of activated NK cells or chemotherapy that may enhance or synergize with the efficacy of GA101 (Obinutuzumab) both in -vitro and in-vivo in xenografted NOD/SCID mice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-α and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2

PLoS ONE ◽  
2013 ◽  
Vol 8 (4) ◽  
pp. e61136 ◽  
Author(s):  
Li-Ping Chai ◽  
Zhang-Feng Wang ◽  
Wei-Ying Liang ◽  
Lei Chen ◽  
Dan Chen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Line ◽  
Laryngeal Carcinoma ◽  
Carcinoma Cell ◽  
Suicide Gene ◽  
Tnf Α ◽  
Combined Gene Therapy ◽  
Human Laryngeal Carcinoma

Analysis of caspase-3, caspase-8 and caspase-9 enzymatic activities in mouse oocytes and zygotes

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 57-64 ◽  
Author(s):  
Adrian Papandile ◽  
David Tyas ◽  
Donald M. O'Malley ◽  
Carol M. Warner
Keyword(s):  
Caspase 3 ◽  
Enzymatic Activities ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Mouse Oocytes ◽  
Apoptotic Pathways

The current consensus in the literature is that ovulated oocytes that are not fertilized die by apoptosis, but the details of the proteins involved in the apoptotic pathways have not been elucidated. In this paper we confirm that caspase-3, the executioner of apoptosis, is expressed in mouse oocytes, and show that two initiators of apoptosis, caspase-8 and caspase-9, are expressed in mouse oocytes. Comparisons were made of caspase-3, -8, and -9 activities in superovulated oocytes that were freshly collected or allowed to age in vivo or in vitro. We found that caspase-3 activity significantly increased in aged oocytes compared with young oocytes (p<0.001), and that both caspase-8 activity and caspase-9 activity decreased in aged oocytes compared with young oocytes (p<0.001 for caspase-8 and p<0.05 for caspase-9 activity). A comparison of superovulated with naturally ovulated oocytes showed the same amount of caspase-8 activity in each, but a significant (p<0.001) decrease in caspase-9 activity in naturally ovulated compared with superovulated oocytes. There was no difference in caspase-3, -8, or -9 activity in oocytes compared with zygotes. Finally, we showed that culture of oocytes in staurosporine increased the activity of caspase-8 and caspase-9. In conclusion, the finding of both caspase-8 and caspase-9 activity in oocytes shows that unfertilized oocytes have the machinery to undergo apoptosis by using either the extrinsic (caspase-8 dependent) or intrinsic (caspase-9 dependent) pathways.

Combination of a Novel Proteasome Inhibitor NPI-0052 and Lenalidomide Trigger in Vivo Synergistic Cytotoxicity in Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3662-3662 ◽  
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Mohan Brahmandam ◽  
Giada Bianchi ◽  
Klaus Podar ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Low Dose ◽  
Caspase 3 ◽  
Caspase 8 ◽  
Caspase 9

Abstract Background: Our previous study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib in its chemical structure, effects on proteasome activities, and mechanisms of action, and importantly, triggers apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib therapies. These preclinical data provided the basis for the ongoing phase-I clinical trial of NPI-0052 in relapsed/refractory MM patients. Recently, a Phase-1/2 clinical trial of bortezomib with Lenalidomide and low dose dexamethasone demonstrated safety and remarkable efficacy in newly diagnosed MM patients. Given that the combination of bortezomib with Lenalidomide has proven a successful treatment strategy, coupled with our findings that NPI-0052 is a potent proteasome inhibitor, we determined whether combining NPI-0052 with Lenalidomide triggered synergistic/additive anti-MM activity. Material and Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from patients relapsing after prior therapies including Lenalidomide or bortezomib. Informed consent was obtained from all patients in accordance with the Helsinki protocol. Cell viability and apoptosis assays were performed using MTT and Annexin V staining. In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, BIM, p-JNK or tubulin. In vitro and in vivo proteasome activity assays were performed using fluorogenic peptide substrates. All animal studies were approved by the DFCI Institutional Animal Care and Use Committee. CB-17 SCID male mice (n = 30; 5 mice/EA group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum free RPMI-1640 medium. When tumors were measurable (~150 mm3) three weeks after MM cell injection, mice were treated with oral doses of vehicle alone, NPI-0052 (0.15 mg/kg), Lenalidomide (2.5 mg/kg), Lenalidomide (5.0 mg/kg), NPI-0052 (0.15 mg/kg) plus Lenalidomide (2.5 mg/kg) or NPI-0052 (0.15 mg/kg) plus Lenalidomide (5.0 mg/kg) on a twice weekly schedule for NPI-0052 and four consecutive days weekly for Lenalidomide for four weeks. Statistical significance of differences observed in NPI-0052, Lenalidomide or NPI-0052 plus Lenalidomide-treated mice was determined using a Student t test. Isobologram analysis was performed using “CalcuSyn” software program. A combination index < 1.0 indicates synergism. Results: Combining NPI-0052 and Lenalidomide induces synergistic/additive anti-MM activity in vitro using MM cell lines (P<0.005, n=3, CI < 1) or patient CD138-positive MM cells (5 patients, P< 0.004). NPI- 0052 plus Lenalidomide-induced synergistic apoptosis is associated with: activation of caspase-8, caspase-9, caspase-3, and PARP; induction of c-Jun-NH2-terminal kinase; activation of BH-3 protein BIM; inhibition of migration of MM cells and angiogenesis; suppression of chymotrypsin-like, caspase-like and trypsin-like proteolytic activities in an additive manner; and inhibition of NF-kappa B signaling. Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus Lenalidomide-induced apoptosis (61 ± 7.1% decrease in cell death; P < 0.003, n=2). Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus Lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low dose combination NPI-0052 (0.15 mg/kg) and Lenalidomide (2.5 or 5.0 mg/kg) is well tolerated, significantly inhibits tumor growth (P < 0.03), and prolongs survival (4–5 months in mice receiving combined drugs, P = 0.001). Immununohistochemistry analysis of MM tumors excised from NPI-0052 plus Lenalidomide-treated mice showed growth inhibition (Ki-67), apoptosis (TUNEL assay, caspae-3 activation), a decrease in associated angiogenesis (Factor VIII and VEGF receptor), and additive inhibition of proteasome activity. Taken together, our study provides the preclinical rationale for clinical protocols evaluating Lenalidomide together with NPI-0052 to improve patient outcome in MM.

Ginkgetin inhibits proliferation of human leukemia cells via the TNF-α signaling pathway

2017 ◽  
Vol 72 (11-12) ◽  
pp. 441-447 ◽  
Author(s):  
Ling-Ling Pan ◽  
Wen-Jun Wu ◽  
Gao-Feng Zheng ◽  
Xiao-Yan Han ◽  
Jing-Song He ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
K562 Cell ◽  
Lymphoblastic Leukemia ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Human Leukemia ◽  
Dependent Manner ◽  
Tnf Α

AbstractGinkgetin is known to be an anticancer agent in many studies. However, its effectiveness in treating chronic lymphoblastic leukemia (CLL) remains unknown. The present study aimed to evaluate the effects of ginkgetin on the growth of the K562 cell line. The MTT assay was employed to examine the proliferation of K562, and a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining was conducted to detect the apoptotic rates. Furthermore, changes of tumor necrosis factor-α (TNF-α) were detected by Western blot analysis. Ginkgetin inhibited the proliferation of K562 cells in a dose- and time-dependent manner. Concentrations of ginkgetin required to induce 50% death of K562 at 24, 48 and 72 h were 38.9, 31.3 and 19.2 μM, respectively. Moreover, treatment of ginkgetin increased K562 apoptosis in vitro along with increased levels of TNF-α. Interestingly, anti-TNF-α antibody prevented ginkgetin-induced K562 cell apoptosis and growth inhibition via deactivation of caspase-8, caspase-9 and caspase-3. Concomitantly, downregulation of TNF-α by etanercept in vivo attenuated ginkgetin-induced inhibitory effects on the tumor growth in an xenograft mouse model. Our results indicate that ginkgetin effectively inhibits K562 cell proliferation, and TNF-α plays a key role in ginkgetin-induced cell apoptosis.

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