Targeting Aminopeptidases by Tosedostat (TST) (CHR2797), Alone and with LBH589, Induces Significant Cytotoxicity Against Human Multiple Myeloma (MM) Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1847-1847
Author(s):  
Chirag Acharya ◽  
Mike Y Zhong ◽  
Daniel Tannenbaum ◽  
Michelle Chen ◽  
Matt Ma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Caspase 3 ◽  
Combined Treatment ◽  
Single Agent ◽  
Annexin V ◽  
Novel Approach ◽  
Human Multiple Myeloma ◽  
Induced Apoptosis ◽  
G1 Cells

Abstract Abstract 1847 Aminopeptidases (AP) are necessary for the growth and development of malignant cells and have a selectively important role in the maintenance of intracellular amino acid (AA) levels in neoplastic cells. CHR2797 is a novel, low nanomolar inhibitor of the M1 family of AP, a group of metalloenzymes containing a central Zn2+ ion. CHR2797 has antiproliferative and apoptotic effects against MM in vitro by inducing the AA deprivation response (AADR). TST, an oral, chronically administered agent with a good safety profile has demonstrated activity in patients with relapsed/refractory AML and is currently under study as part of combination therapy for untreated elderly patients with AML. At the epigenetic regulatory level, Zn-dependent histone deacetylase (HDAC) cause the deacetylation of histone and non-histone cellular proteins which are critical for gene expression, inducing apoptosis and cell cycle arrest in cancer cells. LBH589 (Panobinostat) is an established pan-HDAC inhibitor with potent in vitro anti-cancer activity in many hematological malignancies. The clinical efficacy of Panobinostat is currently being studied in several Phase II/III clinical trials with particular promise seen in the treatment of MM. Here we examined the potential therapeutic effect of CHR2797, alone and with LBH589, against MM cells. Using MTS and CTG assays, CHR2797, at clinically achievable concentrations, decreased survival and proliferation in MM1S and IL-6-dependent ANBL6 cells, in the presence or absence of bone marrow stromal cells following 72 hours incubation. CHR2797 induces apoptosis in MM cells via activation of Caspase 3/7 and 9 but not Caspase 8. Significantly, CHR2797 (10 μM) induced apoptosis in patient MM cells, as seen by % of annexin V and PI from 22 + 1.5% to 39 + 2.3% after 48h incubation. Combined treatment with CHR2797 and LBH589 in MM cells (MM1S, ANBL6, and INA6) further reduced cell viability following 72 hour incubation when compared with CHR2797 treatment alone, as determined by CTG viability luminescent assay. Both drugs together also augmented growth inhibitory effects when compared with single agent alone, after 72 hours incubation followed by MTS assay. Importantly, the combination of both drugs increased caspase 3/7- & 9-mediated apoptosis than CHR2797 alone in these MM cells following 24h-treatment. Cell cycle analysis (CHR2797 at 1μM; LBH589 at 1 nM) showed an increased growth arrest in G0/G1 cells in MM1R cells treated with both drugs versus CHR2797 alone after 24 hours: 68.5±3.3% versus 36±2.5%. Furthermore, CHR2797 inhibited anti-apoptotic protein Mcl-1 in MM1R and U266 MM cells by immunoblottings. Combined treatment with CHR2797 and LBH589 further blocked Mcl-1 when compared with either treatment alone after 24 hours incubation. Together, these results show that the combination of CHR2797 and LBH589 enhanced anti-myeloma effects when compared with either drug alone. This combination, which also has the potential of being without overlapping clinical toxicities, provides a promising novel approach to anti-myeloma therapy. Disclosures: Singer: Cell Therapeutics, Inc: Employment, Equity Ownership. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees.

Design, Synthesis and In Vitro Anti-Cancer Evaluation of Novel Derivatives of 2-(2-Methyl-1,5-diaryl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-3- yl)acetamide

2020 ◽  
Vol 16 (3) ◽  
pp. 340-349
Author(s):  
Ebrahim S. Moghadam ◽  
Farhad Saravani ◽  
Ernest Hamel ◽  
Zahra Shahsavari ◽  
Mohsen Alipour ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Peripheral Neuropathy ◽  
Cell Line ◽  
Normal Cell ◽  
Caspase 3 ◽  
Annexin V ◽  
Normal Cell Line ◽  
Anti Cancer ◽  
Mcf 7

Objective: Several anti-tubulin agents were introduced for the cancer treatment so far. Despite successes in the treatment of cancer, these agents cause toxic side effects, including peripheral neuropathy. Comparing anti-tubulin agents, indibulin seemed to cause minimal peripheral neuropathy, but its poor aqueous solubility and other potential clinical problems have led to its remaining in a preclinical stage. Methods: Herein, indibulin analogues were synthesized and evaluated for their in vitro anti-cancer activity using MTT assay (on the MCF-7, T47-D, MDA-MB231 and NIH-3T3 cell lines), annexin V/PI staining assay, cell cycle analysis, anti-tubulin assay and caspase 3/7 activation assay. Results: One of the compounds, 4a, showed good anti-proliferative activity against MCF-7 cells (IC50: 7.5 μM) and low toxicity on a normal cell line (IC50 > 100 μM). All of the tested compounds showed lower cytotoxicity on normal cell line in comparison to reference compound, indibulin. In the annexin V/PI staining assay, induction of apoptosis in the MCF-7 cell line was observed. Cell cycle analysis illustrated an increasing proportion of cells in the sub-G-1 phase, consistent with an increasing proportion of apoptotic cells. No increase in G2/M cells was observed, consistent with the absence of anti-tubulin activity. A caspase 3/7 assay protocol showed that apoptosis induction by more potent compounds was due to activation of caspase 3. Conclusion: Newly synthesized compounds exerted acceptable anticancer activity and further investigation of current scaffold would be beneficial.

Priming Effect with G-CSF Enhances In Vitro Apoptosis by Treatment with Ara-C and VP-16 in Leukemic Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4333-4333
Author(s):  
Jun-ichi Kitagawa ◽  
Takeshi Hara ◽  
Hisashi Tsurumi ◽  
Nobuhiro Kanemura ◽  
Masahito Shimizu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Priming Effect ◽  
Low Dose ◽  
Combined Treatment ◽  
Annexin V ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Significant Difference

Abstract Introduction: We have recently reported that the effectiveness of low dose Ara-C, VP-16 and G-CSF (AVG therapy) for elderly AML patients who were ineligible for intensive chemotherapy (Hematol Oncol, in press). G-CSF has been reported to potentiate in vitro anti-leukemic effect of Ara-C. The mechanism of the potentiation is assumed to recruit quiescent G0 leukemic cells into cell cycle. We hypothesized that the enhanced cytotoxicity was due to the apoptosis by the effect of the priming of G-CSF, and the effect was depended on the cell cycle. In order to afford proof of this hypothesis, we assayed proliferation, apoptosis, and cell cycle in leukemic cell lines. Materials: Ara-C, VP-16, G-CSF was provided by Nippon Shinyaku, Nihonkayaku, Chugai pharmacy, respectively, Tokyo, Japan. 32D and HL-60 were obtained from RIKEN Bioresource Center Cell Bank (Ibaragi, Japan), Ba/F3 was generous gifts from Dr. Kume, Jichi medical school, Tochigi, Japan. Methods: 5 x 105/ml HL60, 32D and Ba/F3 were cultured with various concentrations of Ara-C and/or VP-16 in the presence or absence of G-CSF 50ng/ml for 3 days. At the end of the culture, cell proliferation and viability were determined by using the trypan blue. The Annexin V-binding capacity of treated cells was examined by flow cytometry using ANNEXIN V-FITC APOPTOSIS DETECTION KIT I purchased from BD Pharmingen™. Cell cycle analysis was done with BrdU Flow KIT purchased from BD Pharmingen™. The incorporated BrdU was stained with specific anti-BrdU fluorescent antibodies, and the levels of cell-associated BrdU are then measured by flow cytometory. Result: Ara-C and VP-16 inhibited proliferation and decreased viability of leukemic cell lines dose-dependently. Half killing concentration (IC50) was redused in combination of Ara-C and VP-16 than Ara-C or VP-16 alone. In G-CSF dependent cell line (32D), IC50 was redeced in the presence of G-CSF than absence of G-CSF at G-CSF, and there was no significant difference between with and without G-CSF in G-CSF independent cell lines (HL-60, Ba/F3) (p<0.05). In combined treatment of low dose Ara-C (10−7M) and VP-16 (10−7M), the percentage of apoptotic cells were increased to 20.67% from 13.04% by addition of G-CSF in 32D, and there was no significant differencebetween with and without G-CSF in HL-60 and Ba/F3 (p<0.05). At combined treatment of low dose Ara-C and VP-16, the percentage of G0/G1 phase cells were decreased to 43.94% from 35.63% and S phase cells were increased to 29.50% from 24.05% in 32D by addition of G-CSF, and there was no significant difference between with and without G-CSF in HL-60 and Ba/F3 (p<0.05). Discussion: We first showed a combination effect of Ara-C and VP-16. Next we demonstrated that the potentiation of the cytotoxicity was mediated through the mechanism of apoptosis, and apoptosis played an important role for eradicating leukemic cells by low dose Ara-C and VP-16. And G-CSF recruited cells G0/G1 phase into S phase in G-CSF dependent cells by addition of G-CSF. These results suggest that priming effect of G-CSF significantly potentiate the cytotoxicity mediated by AVG chemotherapy. Conclusion: The priming effect of G-CSF might be admitted at least of a part in AML cells.

scholarly journals Voruciclib, an Oral, Selective CDK9 Inhibitor, Enhances Cell Death Induced By the Bcl-2 Selective Inhibitor Venetoclax in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1361-1361 ◽  
Author(s):  
Daniel A Luedtke ◽  
Yongwei Su ◽  
Holly Edwards ◽  
Lisa Polin ◽  
Juiwanna Kushner ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Combination Index ◽  
Survival Rates ◽  
Annexin V ◽  
Antileukemic Activity ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Abstract Introduction: Patients with acute myeloid leukemia (AML) face overall 5-year survival rates of 65% and 27% for children and adults, respectively, leaving significant room for improvement. Relapse remains a major contributor to such low overall survival rates, and leukemic stem cells (LSCs) that survive treatment are believed to be responsible for AML relapse. The anti-apoptotic protein Bcl-2 is overexpressed in bulk AML cells and LSCs and is associated with poor clinical outcomes. Thus, Bcl-2 represents a promising therapeutic target for the treatment of AML. Venetoclax (ABT-199) is a selective Bcl-2 inhibitor that has shown great potential for treating a number of malignancies, including AML. Venetoclax inhibits Bcl-2, preventing it from sequestering pro-apoptotic Bcl-2 family protein Bim, leading to Bim activated Bax/Bak, resulting in apoptosis. However, Mcl-1 can also sequester Bim and prevent apoptosis. We previously showed that directly targeting Mcl-1 can enhance the antileukemic activity of venetoclax (Luedtke DA, et al. Signal Transduct Target Ther. Apr 2017). Alternatively, we proposed that indirect targeting of Mcl-1 may preserve or enhance the antileukemic activity of venetoclax, and prevent resistance resulting from Mcl-1. It has been reported that inhibition of CDK9 can downregulate cell survival genes regulated by superenhancers, including Mcl-1, MYC, and Cyclin D1. One CDK9 inhibitor in clinical development, flavopiridol (alvocidib), has progressed to phase II clinical trials in AML. However, off target effects and dose-limiting toxicities remain a concern. Voruciclib is an oral, selective CDK inhibitor differentiated by its potent inhibition of CDK9 as compared to other CDK inhibitors. This selectivity may potentially circumvent toxicities resulting from inhibition of non-CDK targets like MAK and ICK that are inhibited by flavopiridol. Voruciclib has been shown in vitro to promote apoptosis and decrease Mcl-1 expression levels in chronic lymphocytic leukemia (CLL) cells (Paiva C, et al. PLOS One. Nov 2015) and inhibit tumor growth in mouse xenograft models of diffuse large B-cell lymphoma (DLBCL) in combination with venetoclax (Dey J. et al Scientific Reports. Dec 2017). Based on these data, voruciclib may downregulate Mcl-1 in AML cells and therefore synergistically enhance the antileukemic activity of venetoclax. Methods/Results: Culturing AML cell lines (THP-1, U937, MOLM-13, MV4-11, and OCI-AML3) and primary patient samples with various concentrations of voruciclib resulted in a concentration-dependent increase in Annexin V+ cells (2 μM voruciclib induced 13.8-55.8% Annexin V+ cells) along with increased levels of cleaved caspase 3 and PARP, demonstrating that voruciclib induces apoptosis in AML cells. Next, we tested the combination of voruciclib and venetoclax in AML cell lines and primary AML patient samples at clinically achievable concentrations. Annexin V/PI staining, flow cytometry analysis, and combination index calculation (using CalcuSyn software) revealed synergistic induction of apoptosis by voruciclib and venetoclax combination (combination index values for MV4-11, U937, THP-1, and MOLM-13 cells were <0.73; treatment with 2 µM voruciclib and venetoclax for 24 h resulted in >80% apoptosis). Importantly, synergy was observed in both venetoclax sensitive and resistant cell lines. This was accompanied by increased cleavage of caspase 3 and PARP. Lentiviral shRNA knockdown of Bak and Bax partially rescued AML cells from voruciclib-induced apoptosis, showing that voruciclib induces apoptosis at least partially through the intrinsic apoptosis pathway. However, Bak and Bax knockdown had little to no effect on induction of apoptosis by the combination treatment, indicating that there might be other molecular mechanisms underlying the synergistic interaction between the two agents. Treatment with the pan-caspase inhibitor Z-VAD-FMK partially rescued cells from combination treatment induced-apoptosis. Discussion: Collectively, these results demonstrate that voruciclib and venetoclax synergistically induce apoptosis in AML cells in vitro and reverse venetoclax resistance. Further studies to determine the mechanism of action and in vivo efficacy of this promising combination in AML xenografts and PDX models are underway. Disclosures Ge: MEI Pharma: Research Funding.

scholarly journals NeosedumosideIII induced Apoptosis of Human Hepatocellular Carcinoma HepG2 cells and SMMC-7721 Cells and Related Mechanism

2021 ◽  
Author(s):  
Xin-Yu Li ◽  
Xin Zhou ◽  
Yu- Liu ◽  
Feng Qiu ◽  
Qing-Qing Zhao
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Hepg2 Cells ◽  
Caspase 3 ◽  
Human Hepatocellular Carcinoma ◽  
Caspase 8 ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Induced Apoptosis

Abstract Purpose: NeosedumosideIII (Neo) is a megastigmanes and belongs to monocyclic sesquiterpenoids compound with antioxidant, anti-inflammatory and other pharmacological activities. In order to explore the anti-cancer effect and possible mechanism of Neo, the study examined the anti-proliferation and apoptosis effect of Neo against human hepatocellular carcinoma HepG2 cells and SMMC-772 cells and related mechanism in vitro. Methods ：The anti-proliferation effect of Neo was detected on HepG2 cells and SMMC-772 cells by MTT assay and IC50 with increasing dose and time. Cell cycle and apoptosis were detected by flow cytometer. The changes of Bcl-2, Bax, Caspase-3, Caspase-8 and Caspase-9 proteins were detected by western blotting.Results ：The results indicated that Neo could inhibited proliferation of HepG2 cells and SMMC-772 cells in vitro and promoted apoptosis, it significantly induced apoptosis of HepG2 cells and SMMC-772 cells arrested cell cycle at G0/G1 phase in a dose-dependent manner, reduce the expression of Bcl-2 protein, and increase the expression of Bax and Caspase-3, Caspase-8 and Caspase-9 proteins. Conclusion：Neo could inhibit proliferation and induce apoptosis of HepG2 cells and SMMC-7721 cells in vivo which suggested that it might be served as a promising candidate for the treatment of liver cancer.

Inhibition of mTOR Kinase Pathway Selectively Sensitizes Acute Myeloid Leukemia Cells to Cytarabine-Induced Apoptosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2474-2474
Author(s):  
Piotr Smolewski ◽  
Agnieszka Janus ◽  
Barbara Cebula ◽  
Anna Linke ◽  
Krzysztof Jamroziak ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Annexin V ◽  
Cyclin D3 ◽  
Leukemic Cells ◽  
G1 Phase ◽  
Mtor Kinase ◽  
Cycle Arrest ◽  
Induced Apoptosis ◽  
Kinase Pathway

Abstract Background: Rapamycin (RAPA) is an inhibitor of mTOR kinase pathway. In vitro low doses of this agent induce cell cycle arrest in G1 phase, whereas higher concentrations of RAPA exert proapoptotic effects. Aim: We assessed cytotoxicity of RAPA alone or in combination with cytarabine (cytosine arabinoside, ARA-C) in acute myeloblastic leukemia (AML) cells and in normal lymphocytes obtained from 10 healthy volunteers. Methods: AML cells (in vitro HL-60 cell line and ex vivo leukemic cells) and phytohemaglutynin (PHA)-stimulated normal lymphocytes were treated for 24 – 48 h with 1 ng/ml RAPA alone or in combination with 50 nM cytarabine (Ara-C). Moreover, cells was pre-incubated with RAPA for 24 h and then Ara-C was added for the next 24 h. Untreated cultures and those treated with RAPA, Ara-C or PHA alone served as respective controls. The proapoptotic effect was assessed by Annexin V assay and presented as a percentage of Annexin-V-positive cells (apoptotic index; AI). Cell cycle was analyzed by DNA distribution in propydium iodide/RN-ase stained cells. Cyclin D3, A and E expression was also measured using flow cytometry. Results: Median AI induced in HL-60 cells after 24 h treatment with RAPA+Ara-C (30.1%) was significantly higher than induced by RAPA (7.2%) or Ara-C (18.5%) alone (p=0.002 and p=0.03, respectively). The RAPA+Ara-C combination exerted additive effect (combination index 0.87) in that model. Additional 24 hour pretreatment with RAPA further increased apoptosis (median AI 41.5%, vs. 10.9% after 48 h-RAPA alone). In contrast to leukemic cells, pretreatment of normal PHA-stimulated lymphocytes with RAPA caused their G1 phase cell cycle arrest, with significant decrease in cyclin D3 expression (vs. untreated cells - p&lt;0.001). This resulted in prevention of Ara-C-induced cytotoxicity in healthy lymphocytes, when Ara-C was added for another 24 h. Importantly, that protective effect was reversible when RAPA-treated lymphocytes were rinsed and then cultured in fresh, RAPA-free medium for the next 24 h. In another set of experiments, cells from 12 de novo AML patients were treated with RAPA and Ara-C in above concentrations and time settings. RAPA and Ara-C were administrated to isolated peripheral blood mononuclear cells (PBMC). PBMC were immunophenotyped before and after treatment. Leukemic blasts were marked for individually chosen antigen, most characteristic for leukemic clone in particular patient. Normal CD3+ lymphocytes were also detected. Finally, Annexin V staining was performed. Based on that simultaneous three-color staining the proapoptotic effects of treatment could be measured by flow cytometry in both leukemic blasts and normal CD3+ cells. Thus, we found that pretreatment with RAPA protected majority of CD3+ cells (median of alive cells 85.5%) from Ara-C-induced apoptosis, whereas the leukemic blasts AI was higher than in samples treated with Ara-C. After Ara-C alone CD3+ rate decreased significantly (median 35.1%). Conclusions: Pretreatment with RAPA enhances cytotoxic effect of Ara-C on leukemic cells, but not on healthy lymphocytes. The phenomenon is probably due to reversible arrest of healthy cells in G1 phase of cell cycle by low doses of RAPA, what causes their transient resistance to proapoptotic action of cytostatic drugs. In contrast, the same RAPA doses selectively sensitizes leukemic cells to cytostatics. This suggests, that inhibition of mTOR kinase prior to cytostatics administration may result in selective anti-tumor treatment, with protection of normal cells.

Application of the nuclear factor-κB inhibitor BAY 11-7085 for the treatment of endometriosis: an in vitro study

2007 ◽  
Vol 293 (1) ◽  
pp. E16-E23 ◽  
Author(s):  
Kaei Nasu ◽  
Masakazu Nishida ◽  
Tami Ueda ◽  
Akitoshi Yuge ◽  
Noriyuki Takai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Caspase 3 ◽  
In Vitro Study ◽  
B Cell Lymphoma ◽  
Nuclear Factor Κb ◽  
Phase Cell ◽  
High Recurrence Rate ◽  
Induced Apoptosis

Most of the current medical treatments for endometriosis aim to downregulate estrogen activity. However, a high recurrence rate after medical treatment has been the most significant problem. BAY 11-7085, a soluble inhibitor of NK-κB activation, has been shown to inhibit cell proliferation and induce apoptosis of a variety of cells. To examine the potential application of BAY 11-7085 in the treatment of endometriosis, we investigated the effects of this agent on the cell proliferation and apoptosis of cultured ovarian endometriotic cyst stromal cells (ECSCs) by a modified methylthiazole tetrazolium assay, a 5-bromo-2′-deoxyuridine incorporation assay, and internucleosomal DNA fragmentation assays. The effect of BAY 11-7085 on the cell cycle of ECSCs was also determined by flow cytometry. The expression of apoptosis-related molecules was examined in ECSCs with Western blot analysis. BAY 11-7085 significantly inhibited the cell proliferation and DNA synthesis of ECSCs and induced apoptosis and the G0/G1 phase cell cycle arrest of these cells. Additionally, downregulation of the B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-XLexpression with simultaneous activation of caspase-3, -8, and -9 was observed in ECSCs after treatment with BAY 11-7085. These results suggest that BAY 11-7085 induces apoptosis of ECSCs by suppressing antiapoptotic proteins, and that caspase-3-, -8-, and -9-mediated cascades are involved in this mechanism. Therefore, BAY 11-7085 could be used as a therapeutic agent for the treatment of endometriosis.

scholarly journals 12-Epi-Napelline Inhibits Leukemia Cell Proliferation via the PI3K/AKT Signaling Pathway In Vitro and In Vivo

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Jia Han ◽  
Wei Hou ◽  
Bi-qing Cai ◽  
Fan Zhang ◽  
Jian-cai Tang
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Caspase 3 ◽  
Animal Experiments ◽  
Leukemia Cell ◽  
Tumor Model ◽  
Inhibitory Effect ◽  
Induced Apoptosis

This study aimed to investigate the inhibitory effect of 12-epi-napelline on leukemia cells and its possible mechanisms. The inhibitory effects of 12-epi-napelline on K-562 and HL-60 cells were evaluated using the CCK-8 assay, cell cycle arrest and apoptosis were detected by flow cytometry, and the expression of related proteins was measured by western blot. A K-562 tumor model was established to evaluate the antitumor effect of 12-epi-napelline in vivo. A reduction in leukemia cell viability was observed after treatment with 12-epi-napelline. It was determined that the cell cycle was arrested in the G0/G1 phase, and the cell apoptosis rate was increased. Moreover, caspase-3 and Bcl-2 were downregulated, whereas cleaved caspase-3 and caspase-9 were upregulated. Further study revealed that 12-epi-napelline could suppress the expression of PI3K, AKT, p-AKT, and mTOR. Insulin-like growth factor 1 (IGF-1) attenuated 12-epi-napelline-induced apoptosis and ameliorated the repression of PI3K, AKT, p-AKT, and mTOR by 12-epi-napelline. Animal experiments clearly showed that 12-epi-napelline inhibited tumor growth. In conclusion, 12-epi-napelline restrained leukemia cell proliferation by suppressing the PI3K/AKT/mTOR pathway in vitro and in vivo.

Arsenic Trioxide Plus ABT-737 Is Synergistic to Induce Apoptosis in AML Cells by Repression of Mcl-1 Protein and Inactivation of Bcl-2 Protein

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2653-2653
Author(s):  
Lijuan Xia ◽  
Rui Wang ◽  
Hao Qian ◽  
Janice Gabrilove ◽  
Samuel Waxman ◽  
...  
Keyword(s):  
Arsenic Trioxide ◽  
Cell Lines ◽  
Single Agent ◽  
Annexin V ◽  
K562 Cells ◽  
Parp Cleavage ◽  
Key Factor ◽  
Induced Apoptosis ◽  
Apoptotic Effects

Abstract Arsenic trioxide as a single agent induces remission in acute promyelocytic leukemia (APL) patients with minimal toxicity but not in other types of acute myeloid leukemia (AML). In vitro, APL as compared to AML cells are more sensitive to arsenic trioxide-induced apoptosis. Arsenic trioxide-induced apoptosis in APL cells results from activation of a mitochondria-mediated pathway. The Bcl-2 antiapoptotic protein family members Bcl-2, Bcl-XL, Bfl-1 and Mcl-1 block mitochondria-mediated apoptosis. In studies of several AML cell lines, we detected high levels of Bcl-2 and Mcl-1 protein, but lower or no expression of Bcl-XL and Bfl-1. Arsenic trioxide treatment decreased the levels of Mcl-1 without inducing apoptosis in AML cells suggesting that Bcl-2 would be a key factor causing arsenic trioxide resistance. We therefore hypothesize that inhibitors of Bcl-2 might restore arsenic trioxide-induced programmed cell death. In this study, the apoptotic effects of arsenic trioxide, ABT-737 (a potent Bcl-2 inhibitor) and their combination were investigated in NB4, HL-60, U937 and K562 cells. Arsenic trioxide at 1–2 μM induced apoptosis only in NB4 cells but decreased the levels of Mcl-1 in all of the four cell lines. ABT-737 at concentrations lower than 5 μM induced apoptosis in NB4, HL-60 and U937 cells but not in K562 cells which had undetectable Bcl-2 levels. Arsenic trioxide (2 μM) plus ABT-737 (0.05–0.5 μM) synergistically induced apoptosis in HL-60 and U937 but not in K562 cells as determined by PARP cleavage and Annexin V staining. Our data suggest that inhibition of Mcl-1 expression by arsenic trioxide and inactivation of Bcl-2 activity by ABT-737 leads to the synergistic apoptosis observed with this combination and is the basis for a novel treatment for AML.

In-Vitro Treatment of Chronic Lymphocytic Leukemia (CLL) Cells with Fludarabine, Etoposide and Alemtuzumab (Campath-1H) Reveals Different Rates and Mechanisms of Cell Death.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2112-2112 ◽  
Author(s):  
Dirk Winkler ◽  
Christof Schneider ◽  
Daniela Nitsch ◽  
Annett Habermann ◽  
Hartmut Doehner ◽  
...  
Keyword(s):  
Cell Death ◽  
High Risk ◽  
Caspase 3 ◽  
Annexin V ◽  
Chemotherapeutic Agents ◽  
Cross Linking ◽  
Serum Free ◽  
Induced Apoptosis ◽  
In Vitro Treatment

Abstract Chemotherapeutic agents such as fludarabine, etoposide and the monoclonal anti-CD52 antibody alemtuzumab induce cell death and clinical responses in CLL. However, the mechanisms by which these processes occur are not well understood. In an effort to gain better insight into these mechanisms CLL cells from 21 patients were collected and individually treated with fludarabine 500μM (n=19) and etoposide 60 μM (n=19) for 24 and 48 hours respectively, and 24 hours with alemtuzumab 10 μg/ml ± cross-linking F(ab’)2 fragments. Each sample treated with alemtuzumab was also cultured with and without serum as a source of complement. Enrichment for B-cells was also done in 4 cases using negative selection with anti-CD2 and anti-CD14 magnetic beads. Of 18 cases investigated 10 were unmutated VH and 6 of 19 had del11q and/or del17p. A FACS analysis with double staining for Annexin V/7AAD was used to measure rates of cell death and caspase-3 activation. Results: treatment with fludarabine and etoposide induced apoptosis in all cases. However, rates of apoptosis decreased in cells from patients with genetically high-risk CLL, and these cells showed higher caspase-3 activation in response to fludarabine. Response to alemtuzumab was significantly dependent on presence of serum in the culture: 8% Annexin-V/7AAD-positive cells in serum-free cultures vs 53% in cultures with serum. However, addition of F(ab’)2 fragments increased the percentage of Annexin-V/7AAD-positive cells even in serum-free cultures: 61% with serum vs 25% without serum. Response to alemtuzumab was found to be independent of the genetic subgroup of the case. Notably, treatment with alemtuzumab in serum cultures did not produce cells that stained Annexin-positive/7AAD-negative, a typical feature of early apoptosis, whereas treatment with fludarabine, etoposide and alemtuzumab in serum-free medium resulted in a significant number of Annexin-positive/7AAD-negative cells. This was also observed in CD19+ purified cultures. In the presence of serum alemtuzumab did not induce caspase-3 activation, neither did the addition of F(ab’)2 fragments. However, in 5 of 20 serum-free cell cultures, all of had unmutated VH, active caspase-3 was clearly detectable after alemtuzumab treatment, and caspase-3 activity was further up-regulated when F(ab’)2 fragments were also added. Summary: in CLL cells mechanism and rate of cell death dramatically differed depending on in-vitro treatment with fludarabine, etoposide and alemtuzumab, and differed between genetic subgroups. CLL cells from high-risk patients were more capable of caspase-3 activation when treated with fludarabine or alemtuzumab. Alemtuzumab killed CLL cells effectively and independently of serum as a source of complement, but the mechanism of response was different when serum was added. In serum-free CLL cultures, alemtuzumab induced apoptosis with activation of caspase-3, and addition of cross-linking F(ab’)2 fragments increased the rate apoptosis, whereas in the presence of serum treatment with alemtuzumab induced no typical features of apoptosis, even in B-cell enriched cultures. These findings favor CDC rather than apoptosis or ADCC as the major cell kill mechanism activated by in vivo alemtuzumab. mean % of cells AnnexinV+/7AAD+ caspase-3 activation etoposide (48 hrs) fludarabine (48 hrs) fludarabine (48 hrs) IgVH unmutated 33% 24% 32% IgVH mutated 74% 38% 20% del 11q/del 17p 39% 25% 37% del 13q/normal karyotype 61% 32% 21%

Alisertib, an Aurora a Kinase Inhibitor, Induces Synthetic Lethality and Overcomes Chemoresistance in Myc-Overexpressing Lymphoma Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1551-1551
Author(s):  
Steven I. Park ◽  
Carolina P. Lin ◽  
Dirk P. Dittmer ◽  
Steven P. Angus ◽  
Greg G. Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Synthetic Lethality ◽  
Single Agent ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Raji Cells ◽  
Induced Apoptosis

Abstract Background: The proto-oncogene Myc is a key regulator of cell growth and survival, and aberrant Myc expression plays a significant role in various tumors, including non-Hodgkin lymphoma (NHL). Myc-associated lymphoma is clinically aggressive, more resistant to standard therapies, and associated with a significantly higher rate of mortality. Novel treatment paradigms are needed to improve survival of patients with Myc-associated NHL. Expression of Aurora Kinase (Aurk) has been associated with Myc, and Aurk is thought to be essential for the maintenance of Myc-driven lymphoma. Aurk is required for assembly of the mitotic spindle and plays key roles in cell proliferation. Amplification and overexpression of Aurk have been observed in various human tumors, including lymphoma, and are frequently associated with tumor progression as well as resistance to chemotherapy. Inhibition of Aurk may overcome resistance to chemotherapy and improve clinical outcomes in patients with Myc-overexpressing lymphoma. Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines resistant or sensitive to a conventional chemotherapeutic drug cyclophosphamide. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. The Multiplexed Inhibitor Beads and quantitative Mass Spectrometry (MIB/MS) assays were used to profile kinome changes in response to Aurk inhibition. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined therapy. Results: Two Myc-overexpressing cell lines were identified as resistant (Raji) or sensitive (Ramos) to cyclophosphamide, with IC50 of ~ 400 µM and ~ 250 µM, respectively. Raji cells were characterized by increased expression of multidrug resistant protein 1 (MDR1) and mutated p53. There were no significant differences in baseline Aurk or Myc expressions between Raji and Ramos cells. Both cell lines were sensitive to alisertib, an aurora A kinase inhibitor, with maximum cytotoxicity achieved at ~ 100 nM. Combined treatment with alisertib and cyclophosphamide induced more significant cell growth inhibition as compared to treatment with the single agent alone. The combination index (CI) values were less than 1, indicating that alisertib was synergistic to cyclophosphamide in terms of inhibitory effect on tumor cell viability. Alisertib induced apoptosis and pronounced cell cycle arrest, resulting in polyploidy, in Raji cells. Alisertib had little to no effect on Myc, p53, or the total aurora A kinase protein expression in Raji cells although p-Histone-3-Ser10, a downstream target of Aurk, and p-Src levels were significantly decreased at 24 hours of treatment in vitro. Nocodazole-treated cells had reduced p-Aurk level and increased p-Rb as well as increased Mdm2 when treated with alisertib for 24 hours. Athymic nude mice bearing Ramos or Raji lymphoma xenografts were treated with cyclophosphamide, alisertib, or the combination. As expected, all mice bearing Ramos xenograft had complete tumor regression by day 35 of treatment while all mice bearing Raji xenograft had rapid disease progression with median survival of ~ 35 days when treated with cyclophosphamide alone. In contrast, when treated with the combination of cyclophosphamide and alisertib, all mice bearing Raji xenograft had complete regression of tumor by day 35 and had significant improvement in survival (median survival not reached by day 100) compared to the single agent control (p=0.022). Lastly, kinome analysis of Raji xenograft tumors treated with alisertib showed suppression of various kinases involved in Aurk, Src, and PI3K pathways. Western blot of the Raji tumors treated with a prolonged course (25 days) of alisertib showed significant decrease in p-Src and p53 protein levels. Conclusion: Our data demonstrates that alisertib induces synthetic lethality and overcomes chemoresistance in Myc-overexpressing tumors even in the presence of MDR1 overexpression and p53 mutation. The synergistic effect was largely independent of depletion of cytoplasmic level of Myc. Alisertib, when combined with a conventional chemotherapy drug, induced apoptosis and cell cycle arrest of Myc-overexpressing tumor cells in vitro and showed promising anti-tumor activity in mice bearing chemoresistant Myc-overexpressing lymphoma. Disclosures Park: Janssen: Other: travel; Seattle Genetics: Research Funding; Teva: Research Funding.

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