Inhibition of Wnt/β-Catenin Signaling by AV65 Treatment Caused Cell Cycle Arrest and Induced Caspase-Independent or -Dependent Apoptosis in CML Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2184-2184 ◽  
Author(s):  
Rina Nagao ◽  
Eishi Ashihara ◽  
Shinya Kimura ◽  
Hisayuki Yao ◽  
Miki Takeuchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Line ◽  
Cell Lines ◽  
K562 Cells ◽  
S Phase ◽  
G1 Phase ◽  
Cycle Arrest ◽  
Imatinib Resistant ◽  
Induced Apoptosis

Abstract Abstract 2184 Poster Board II-161 Imatinib has dramatically improved the management of CML, but cases of imatinib resistance have been reported. The second-generation ABL tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib overcome imatinib-resistant CML.These agents, however, are ineffective in CML cells harboring T315I mutation and in CML stem cells. Recently, loss of β-catenin has been reported to impair the renewal of CML stem cells (Chao et al, Cancer Cell 2007) and an in vivo study has showed that β-catenin is essential for survival of leukemic stem cells (Hu et al, Leukemia 2009). Thus, we hypothesized that the inhibition of β-catenin signaling may be efficacious in the treatment of CML. We have previously reported that a novel β-catenin inhibitor, AV65, suppresses the growth of imatinib resistant CML cell lines harboring Abl kinase domain mutations including T315I and hypoxia-adaptation (Nagao et al, ASH 2008). We herein examine the cell cycle and apoptotic effects of AV65 on CML cell lines and its therapeutic possibility for CML stem/progenitor cells. We observed that expression of β-catenin is increased 20 to 45-fold in K562, BV173, KT-1, and MYL CML cell lines compared with total bone marrow cells from healthy volunteers. We have previously demonstrated that AV65 induced apoptosis of CML cells. To investigate how AV65 inhibits β-catenin, we next analyzed the effects of AV65 using Western blotting and real time PCR. AV65 suppressed the expression of β-catenin in K562 in a time- and a dose-dependent manner in nuclear and cytosolic fractions as well as whole cell lysates. AV65 did not diminish the transcripts of β-catenin in K562 indicating the depletion of β-catenin due to an inhibition of its accumulation in CML cells. Next we examined the effects of AV65 on cell cycle. The fractions of G1 phase to S phase increased by AV65 treatment. TUNEL/PI staining showed that both K562 and BV173 began to be nicked by AV65 at 30 nM for 12 hours, resulting in the induciton of apoptosis from G1 phase to S phase 24 hours after AV65 treatment (Figure). In real-time PCR analysis, the transcripts of p21, p27, and p57 in CML cell lines increased by AV65 treatment, however, those of p53 were not altered. Taken together, it is suggested that CML cells first arrested from G1 phase to S phase and then induced apoptosis after AV65 treatment. Next we examined the mechanisms of apoptosis by AV65 treatment. AV65 treatment in the presence of Z-VAD did not induce cell death in BV173, indicating that AV65 induced caspase-dependent apoptosis in BV173. In K562 cells however, AV65 induced apoptosis with or without Z-VAD suggesting that AV65 induces apoptosis in CML cell lines in caspase-dependent or -independent pathways. Lastly, we investigated the effects on hypoxia-adapted CML cells. We established hypoxia-adapted K562 cell lines (K562/HA). This cell line shows characteristics of more primitive CML cells, including resistance to serial Abl TKIs and a higher transplantation efficacy compared to the parental K562 cells (Takeuchi, et al. ASH 2008). In Western blotting analysis, K562/HA cell line expressed more β-catenin than its parental K562 cell line. AV65 inhibited the growth of K562/HA at the similar concentration to K562. Taken together, AV65 is effective for primitive CML cells which overexpress β-catenin. This suggests that AV65 has a potential to eradicate CML stem/progenitor cells. In conclusion, AV65 inhibits the accumulation of β-catenin in CML cells and this causes cell cycle arrest from G1 to S phase, resulting in induction of caspase-independent or -dependent apoptosis in CML cells. The inhibition of Wnt/β-catenin signaling has great potential as a novel and attractive therapy for CML. Disclosures: No relevant conflicts of interest to declare.

Autophagy Inhibition Enhances CDK4/6 Inhibitor-Induced Apoptosis in t(8;21) Acute Myeloid Leukemia Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 50-50
Author(s):  
Kana Nakatani ◽  
Hidemasa Matsuo ◽  
Yutarou Harata ◽  
Moe Higashitani ◽  
Asami Koyama ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
G1 Phase ◽  
Therapeutic Approaches ◽  
Cycle Arrest ◽  
Induced Apoptosis ◽  
Autophagy Inhibition ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a genetically and clinically heterogeneous disease. Although t(8;21) AML patients have a more favorable prognosis than other cytogenetic subgroups, nearly 40% of t(8;21) AML patients experience relapse. Therefore, novel therapeutic approaches based on a better understanding of the biology of t(8;21) AML need to be developed. In this study, at first, we re-analyzed the sequencing data of 149 pediatric t(8;21) AML patients from St. Jude Children's Research Hospital tissue resource core facility and the JPLSG AML-05 study, and 134 adult t(8;21) AML patients from CALGB/Alliance trials and the University Hospital of Ulm. In pediatric patients, 13 CCND2 mutations were detected in 11 patients (11/149, 7.4%), and in adult patients, 14 CCND2 mutations were detected in 12 patients (12/134, 9.0%). In both cohorts, CCND2 mutations were located on the PEST domain, suggesting that the mutations stabilize the cyclin D2 protein. Next, we compared CCND2 mRNA expression between t(8;21) AML patients (n=24) and non-t(8;21) AML patients (n=163) using the TARGET AML cohort. In non-t(8;21) AML patients, CCND2 expression varied from low to high levels, whereas in t(8;21) AML patients, CCND2 expression was restricted to higher levels. Consistently, CCND2 expression was higher in t(8;21) AML cell lines (n=2: Kasumi-1 and SKNO-1), compared with non-t(8;21) AML cell lines (n=32). Kasumi-1 cells transfected with shCCND2 showed cell cycle arrest at G1 phase and impaired cell proliferation. These results suggest that the frequency of CCND2 mutations and CCND2 expression are increased in t(8;21) AML, and high CCND2 expression plays an important role in t(8;21) AML cell proliferation. Because CCND2 is not a druggable target, we examined the effect of CDK4/6 inhibitors (palbociclib and abemaciclib) on t(8;21) AML cells. Analysis of 19 AML cell lines showed that t(8;21) AML cells had lower IC50 values for CDK4/6 inhibitors than non-t(8;21) AML cells. CDK4/6 inhibitors caused cell cycle arrest at G1 phase and impaired cell proliferation in t(8;21) AML cells. To identify potential therapeutic approaches in combination with CDK4/6 inhibitors in t(8;21) AML, we performed microarray analysis and examined the effects of CDK4/6 inhibition. In addition to the pathways associated with the cell cycle (regulation of sister chromatid separation, retinoblastoma gene, and cell cycle), the MAP-ERK and PI3K-AKT-mTOR signaling pathways were downregulated by CDK4/6 inhibition. Because these pathways are involved in autophagy regulation via mTOR, we focused on examining autophagy in subsequent experiments. Assessment of the effect of CDK4/6 inhibition on autophagy in t(8;21) AML cells showed that the CDK4/6 inhibitor (abemaciclib) treatment induced LC3B-I to LC3B-II conversion in both Kasumi-1 and SKNO-1 cells. Transmission electron microscopic examination of autophagosome formation detected a large number of autophagosomes in the cytoplasm of Kasumi-1 and SKNO-1 cells treated with abemaciclib, whereas few autophagosomes were detected in control samples. These results suggest that autophagy is induced by CDK4/6 inhibition in t(8;21) AML cells. Autophagy is involved in the resistance to chemotherapy in cancer cells, therefore, we hypothesized that autophagy inhibition may be a promising therapeutic approach. Treatment of t(8;21) AML cells with the autophagy inhibitors chloroquine (CQ) or LY294002 in combination with abemaciclib significantly increased the frequency of apoptotic (Annexin V positive) cells compared with that in untreated cells, whereas CQ or LY294002 single treatment had no significant effect on apoptosis. Consistently, combinatorial inhibition of CDK4/6 and autophagy upregulated cleaved caspase 3 expression. The combinatorial effect was confirmed by silencing the autophagy-related protein ATG7 using small interfering RNA in abemaciclib-treated t(8;21) AML cells. These results suggest that autophagy inhibition enhances CDK4/6 inhibitor-induced apoptosis in t(8;21) AML cells. In conclusion, the present results indicate that inhibition of CDK4/6 and autophagy may be a novel and promising biomarker-driven therapeutic strategy for the treatment of t(8;21) AML. Disclosures: No relevant conflicts of interest to declare.

Pharmacologic Doses of Amiloride Preferentially Induce Apoptosis and Growth Inhibition of Flt3-ITD Mutation Positive Acute Myeloid Leukemia Cell Lines

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5004-5004
Author(s):  
Yuliya Linhares ◽  
Jade Dardine ◽  
Siavash Kurdistani
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Cell Lines ◽  
K562 Cell ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
K562 Cell Line ◽  
Cycle Arrest

Abstract Abstract 5004 Introduction: Amiloride is an FDA approved potassium-sparing diuretic which targets Na+/H+ exchanger isoform 1 (NHE1). NHE1 is responsible for the regulation of the intracellular pH, as well as cell-cycle and apoptosis. In supra-pharmacologic concentrations, amiloride non-specifically inhibits protein kinases. Recent study demonstrated that proapoptotic effect of amiloride in CML cell lines is linked to the modulation of the alternative splicing of Bcl-x, HIPK3, and BCR/ABL genes and is independent of pHi. Here, we demonstrate that pharmacologic doses of amiloride preferentially induce growth inhibition, cell cycle arrest and apoptosis in Flt3-ITD positive acute myeloid leukemia cell lines as compared to Bcr-Abl positive leukemia cell line. Our data suggests that amiloride may have an effect on Flt3 signaling and that its treatment potential for Flt3-ITD positive acute myeloid leukemia needs to be explored. Methods: MV4-11, MOLM13 and K562 cells lines in log-phase growth were used for the experiments. Analysis of the baseline Flt3 expression and phosphorylation status was assessed via Flt3 immunoprecipitation and Western blotting for Flt3 and phosphotyrosine. Cells were incubated with various amiloride concentrations; equal volume dilutions of DMSO were used for control. Cell counting and trypan blue exclusion viability was performed on TC10 Bio-Rad automated cell counter. The cell cycle analysis was performed applying propidium iodide staining. To assess for apoptosis and cell death, we used annexin V/PI staining kit and flow cytometry. Results: MOLM13 and MV4-11 cell lines carry activating Flt3-ITD mutation. We confirmed the expression and constituative activation of Flt3 in MOLM13 and MV4-11 cells with Western blotting. Flt3 protein was not detectable in K562 cell line. Amiloride at 0.025 mM and 0.05 mM completely inhibited the growth of MV4-11 cells after 24 hrs of treatment with no significant increase in total or live cell numbers at 72 hrs, but only mildly affected K562 cell proliferation. While the above amiloride concentrations caused cell death in MV4-11 and MOLM13 cell lines, there was no increased cell death in K562 cells. Incubation of MOLM13 and MV4-11 cell lines with 0.05 mM amiloride for 20 hrs induced cell cycle arrest. In MV4-11 cell line, the proportion of S phase cells after amiloride treatment was 15.4% (SD=5.4%) as compared to 31.3% (SD=1.4%) in control. MOLM13 cell line demonstrated 15.3% (SD=4.7%) of cells in S after amiloride treatment as compared to 35.3% (SD=2.4%) cells in S phase in control treatment. In K562 cell line, there was less effect with 52% (SD=4.2%) of cells in S phase in control as compared to 37% (SD=8.9%) in amiloride treatment. MV4-11 and MOLM 13 cell lines were more sensitive than K562 cells to amiloride induced apoptosis with 28.8% (control 12.7%) of MV4-11 cells, 11.4% (control 7.4%) of MOLM13 cells, and 11.4% (control 8.6%) of K562 cells being apoptotic after 20 hr treatment with 0.05mM amiloride. At 72 hrs of amiloride treatment 34% (control 1.5%) of MV4-11 cells, 17% (control 5%) of MOLM13 cells and 11% of K562 cells (control 8.9%) were apoptotic. Amiloride had similar effect on the number of dead cells with no increase in total cell death in K562 cell line. Upon treatment with increasing amiloride concentrations, there was dose-dependent increase in cell death and apoptosis in all three cell lines with K562 line showing relative resistance to amiloride. Discussion: Our results demonstrate that amiloride induces cell cycle arrest and inhibits proliferation of Flt3-ITD positive cell lines MV4-11 and MOLM13 as well as K562 cell line at a pharmacologic concentration of 0.05 mM. Both, cell cycle arrest and antiproliferative effect are more pronounced in Flt3-ITD positive cells lines while it is mild in Bcr-Abl positive K562 cell line. Pharmacologic doses of amiloride induce cell death and apoptosis in Flt3-ITD positive cell lines but not in K562 cell line. Both, Bcr-Abl and Flt3 signaling stimulates proliferation and inhibits apoptosis in myeloid leukemia cells. Our study suggests that amiloride may induce cell cycle arrest and apoptosis via modulating Flt3 signaling cascade. We are currently investigating the effects of amiloride on Flt3 phosphorylation. In conclusion, our data suggests that amiloride presents a potential treatment option for Flt3-ITD positive acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Jellyfish extract induces apoptotic cell death through the p38 pathway and cell cycle arrest in chronic myelogenous leukemia K562 cells

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2895 ◽  
Author(s):  
Sun-Hyung Ha ◽  
Fansi Jin ◽  
Choong-Hwan Kwak ◽  
Fukushi Abekura ◽  
Jun-Young Park ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Chronic Myelogenous Leukemia ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
G1 Phase ◽  
Cycle Arrest ◽  
Anti Cancer ◽  
Induced Apoptosis ◽  
Cancer Activity

Jellyfish species are widely distributed in the world’s oceans, and their population is rapidly increasing. Jellyfish extracts have several biological functions, such as cytotoxic, anti-microbial, and antioxidant activities in cells and organisms. However, the anti-cancer effect of Jellyfish extract has not yet been examined. We used chronic myelogenous leukemia K562 cells to evaluate the mechanisms of anti-cancer activity of hexane extracts from Nomura’s jellyfish in vitro. In this study, jellyfish are subjected to hexane extraction, and the extract is shown to have an anticancer effect on chronic myelogenous leukemia K562 cells. Interestingly, the present results show that jellyfish hexane extract (Jellyfish-HE) induces apoptosis in a dose- and time-dependent manner. To identify the mechanism(s) underlying Jellyfish-HE-induced apoptosis in K562 cells, we examined the effects of Jellyfish-HE on activation of caspase and mitogen-activated protein kinases (MAPKs), which are responsible for cell cycle progression. Induction of apoptosis by Jellyfish-HE occurred through the activation of caspases-3,-8 and -9 and phosphorylation of p38. Jellyfish-HE-induced apoptosis was blocked by a caspase inhibitor, Z-VAD. Moreover, during apoptosis in K562 cells, p38 MAPK was inhibited by pretreatment with SB203580, an inhibitor of p38. SB203580 blocked jellyfish-HE-induced apoptosis. Additionally, Jellyfish-HE markedly arrests the cell cycle in the G0/G1 phase. Therefore, taken together, the results imply that the anti-cancer activity of Jellyfish-HE may be mediated apoptosis by induction of caspases and activation of MAPK, especially phosphorylation of p38, and cell cycle arrest at the Go/G1 phase in K562 cells.

Preclinical Study Of The Bromodomain Inhibitor OTX015 In Acute Myeloid (AML) and Lymphoid (ALL) Leukemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4218-4218 ◽  
Author(s):  
Thorsten Braun ◽  
marie Magdelaine Coude ◽  
Jeannig Berrou ◽  
Sibyl Bertrand ◽  
Eugenia Riveiro ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Caspase 3 ◽  
K562 Cell Line ◽  
Cytochrome C Release ◽  
Cycle Arrest ◽  
Ic50 Values ◽  
Induced Apoptosis ◽  
Lymphoid Cell Lines

Abstract Background Bromodomain and extra-terminal (BET) proteins, including the ubiquitous BRD2/3/4 proteins, are epigenetic readers implicated in c-MYC transcription, cellular proliferation, cell-cycle progression, RNA elongation and DNA damage response. Using shRNA screening and BRD inhibitors, BRD4 has been established as a promising therapeutic target in acute leukemia (Zuber, Nature 2011). In the present study, we investigated the in vitro anti-leukemic effects of the small-molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland). Methods Expression of BRD2/3/4 and c-MYC was assessed by RQ-PCR in 5 myeloid (HL60, KG1, KG1a, K562, NOMO1) and 4 lymphoid (Jurkat, RS4-11, BV173, TOM1) leukemia cell lines and by Western blotting (WB) using commercial antibodies in the HL60, K562, Jurkat and RS4-11 lines. Nineteen AML and ten ALL patient banked leukemic cells were assayed by RQ-PCR only. Cell viability and IC50 values were assessed in cell lines by MTT assays after exposure to OTX015 (0.1nM-10µM) for 72h. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation. Induction of apoptosis was evaluated in cell lines and patient cells by outer membrane phosphatidylserine exposure and PI incorporation at 72 hours with increasing doses of OTX015 (25nM-500nM). Caspase-3 activation and mitochondrial cytochrome c release were studied by immunofluorescence (IF). Maturation was assessed by morphological studies after MGG staining and detection of CD11b by FACS analysis. Modulation of BRD2/3/4 proteins was investigated by WB. Results OTX015 IC50 values were in the submicromolar range for KG1 and the MLL-driven NOMO1 cell lines (198.3 and 229.1nM, respectively), while K562 was the most resistant myeloid line, with an IC50 of 11.3µM. In contrast, in lymphoid cell lines tested, IC50 values ranged from 34.2 to 249.7nM, with the MLL-driven cell line RS4-11 being the most sensitive. Cell cycle arrest in subG1/G1 to S transition was observed in 8/9 cell lines and was most pronounced in RS4-11 and BV173. Significant apoptosis (up to 88% Annexin V positive cells) was only observed in KG1a and NOMO1 among myeloid cell lines, while OTX015 induced apoptosis in all lymphoid cell lines tested, ranging from 57% in RS4-11 to 90% in the BCR-ABL+ TOM1 cells. Similarly, OTX015 triggered caspase-dependent cell death, as NOMO1 and RS4-11 displayed significant caspase-3 activation and cytochrome c release, when compared to the resistant K562 cell line. Seven primary patient fresh samples (5 AML, 2ALL) were also analyzed. Ex vivo treatment induced apoptosis ranged from 35% to 87% in 6/7 patients. Exposure to OTX015 at 500nM for 7 days induced maturation in 51% and 65% of HL60 cells as detected by CD11b expression and morphology, respectively. Baseline expression of BRD2/3/4 varied among cell lines or patient samples, lower BRD2/3/4 expression levels were observed in the BCR-ABL+ K562 and BV173 cell lines, as well as in the 4 BCR-ABL+ ALL samples analyzed. Upon OTX015 exposure, down-regulation of the BRD4 target gene c-MYC was observed in all cell lines, without clear correlation with the proliferation inhibition rate and/or the intensity of induced apoptosis while no consistent BRD2/3/4 mRNAs down-regulation was seen. Interestingly, BRD2 protein was down-regulated in HL60, Jurkat and RS4-11 cell lines, but not in the K562 cell line. Conclusion OTX015 affects cell viability, induces cell cycle arrest in G1/S phase, and is able to induce significant apoptosis in leukemic cell lines and fresh AML and ALL samples at submicromolar drug concentrations. These concentrations were achieved in the serum of healthy volunteers after safe administration of the drug. With such characteristics, OTX015 appears to be an attractive anti-leukemic therapy, currently under early evaluation in a Phase Ib dose-escalation trial conducted in relapsed/refractory AML/ALL patients. Disclosures: Riveiro: Oncology Therapeutic Development: Employment. Herait:Oncoethix: Employment. Dombret:Oncoethix: Research Funding.

scholarly journals The Novel CDK4/6-Inhibitor Abemaciclib Induces Early G1-Arrest in MCL Cell Lines, Sensitizes Cells to Cytarabine Treatment and Is Additive with Ibrutinib

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5124-5124
Author(s):  
Luca Fischer ◽  
Andrea Schnaiter ◽  
Bianca Freysoldt ◽  
Markus Irger ◽  
Yvonne Zimmermann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
S Phase ◽  
G1 Phase ◽  
G1 Arrest ◽  
The Novel ◽  
Sensitive Cell ◽  
Cycle Arrest ◽  
Arac Treatment

Abstract Introduction: Mantle cell lymphoma (MCL) is characterized by t(11;14) resulting in a constitutive cyclin D1 overexpression. The cyclin D1-CDK4/6 complex inactivates Rb through phosphorylation, leading to G1/S-phase transition. Therefore, inhibition of CDK4/6 is an efficient and rational approach to overcome cell cycle dysregulation in MCL. We evaluated the efficiency of the novel CDK4/6 inhibitor abemaciclib in various MCL cell lines and in primary MCL cells in combination with cytarabine (AraC) and ibrutinib. Material & Methods: MCL cell lines (Granta 519, JeKo-1, Maver-1, Mino) and primary MCL cells were exposed to abemaciclib alone and combined with AraC or ibrutinib. Cells were pretreated with abemaciclib and exposed to AraC or ibrutinib with or without consecutive wash-out of the CDK4/6 inhibitor. Proliferation and viability were measured by tryptan blue staining and Cell Titer Glo assay. Flow cytometry was used for cell-cycle (PI-staining) and apoptosis analysis (Annexin V PE/7AAD-staining). Western Blot analysis showed protein expression and phosphorylation status of various downstream proteins. Results: Abemaciclib inhibited cell proliferation by induction of early G1-arrest. Western Blot analysis revealed reduced phosphorylation of Rb on serine 795 without changes in CDK 4 and cyclin D1 expression, in line with reversible cell cycle arrest. IC50-values of sensitive cell lines (JeKo-1, Maver-1, Mino) were <30 nM after 72 h. We observed an almost complete and reversible G1-arrest in all sensitive cell lines by FACS analysis (JeKo-1: G1-phase +51,7 %; S/G2-phase -51,7 % at 31,25 nM after 24 h; G1-phase +35,4 %; S/G2-phase -34,8 % after 72 h), whereas cell viability was not reduced. Wash-out of abemaciclib after 24 h resulted in synchronized S-phase entry in all sensitive cell lines (e.g. Mino: G1-phase -20,4 %; S-phase +30,5 %). The sequential combination of abemaciclib followed by AraC showed strong synergy in Mino cells (CI=0,22 for 31,25 nM abemaciclib and 3,33 µM cytarabine). In contrast, simultaneous exposure to abemaciclib had a protective effect against AraC treatment in all sensitive cell lines, due to an ongoing G1-arrest (Mino: CI=-0,19 for 31,25 nM abemaciclib and 3,33 µM AraC). In primary MCL cells, 31,25 nM of abemaciclib had no impact on cell death. Moreover, no sensitization to AraC was observed as all cells were resting in G0-phase. The combination of abemaciclib induced G1 arrest and ibrutinib had additive or synergistic effects in sensitive cell lines (JeKo-1, Mino and Maver). Conclusion: The novel CDK4/6 inhibitor abemaciclib causes reversible G1 cell cycle arrest without loss of viability at low nanomolar doses. Rationale drug combinations exploiting the sequential effect may achieve major benefits, but drug interactions are complex: Pretreatment with abemaciclib sensitizes MCL cell line cells to AraC whereas simultaneous application protects them from AraC treatment. Further analyses explore the interaction with other targeted approaches (inhibitors of the B-cell receptor pathway) to better understand the underlying molecular mechanisms. Disclosures No relevant conflicts of interest to declare.

scholarly journals Silencing ZEB2 Induces Apoptosis and Reduces Viability in Glioblastoma Cell Lines

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 901
Author(s):  
Sahar Safaee ◽  
Masoumeh Fardi ◽  
Nima Hemmat ◽  
Neda Khosravi ◽  
Afshin Derakhshani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Brain Tumor ◽  
Brain Tumors ◽  
Cell Lines ◽  
Scratch Test ◽  
Cycle Arrest ◽  
U373 Cell ◽  
Brain Tumor Cell ◽  
Induced Apoptosis ◽  
Glioma Tumors

Background: Glioma is an aggressive type of brain tumor that originated from neuroglia cells, accounts for about 80% of all malignant brain tumors. Glioma aggressiveness has been associated with extreme cell proliferation, invasion of malignant cells, and resistance to chemotherapies. Due to resistance to common therapies, glioma affected patients’ survival has not been remarkably improved. ZEB2 (SIP1) is a critical transcriptional regulator with various functions during embryonic development and wound healing that has abnormal expression in different malignancies, including brain tumors. ZEB2 overexpression in brain tumors is attributed to an unfavorable state of the malignancy. Therefore, we aimed to investigate some functions of ZEB2 in two different glioblastoma U87 and U373 cell lines. Methods: In this study, we investigated the effect of ZEB2 knocking down on the apoptosis, cell cycle, cytotoxicity, scratch test of the two malignant brain tumor cell lines U87 and U373. Besides, we investigated possible proteins and microRNA, SMAD2, SMAD5, and miR-214, which interact with ZEB2 via in situ analysis. Then we evaluated candidate gene expression after ZEB2-specific knocking down. Results: We found that ZEB2 suppression induced apoptosis in U87 and U373 cell lines. Besides, it had cytotoxic effects on both cell lines and reduced cell migration. Cell cycle analysis showed cell cycle arrest in G0/G1 and apoptosis induction in U87 and U373 cell lines receptively. Also, we have found that SAMAD2/5 expression was reduced after ZEB2-siRNA transfection and miR-214 upregulated after transfection. Conclusions: In line with previous investigations, our results indicated a critical oncogenic role for ZEB2 overexpression in brain glioma tumors. These properties make ZEB2 an essential molecule for further studies in the treatment of glioma cancer.

scholarly journals Correction: Wogonin induces cell cycle arrest and erythroid differentiation in imatinib-resistant K562 cells and primary CML cells

Oncotarget ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 300-301
Author(s):  
Hao Yang ◽  
Hui Hui ◽  
Qian Wang ◽  
Hui Li ◽  
Kai Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Cycle Arrest ◽  
Imatinib Resistant

scholarly journals A Novel Methoxybenzyl 5-Nitroacridone Derivative Effectively Triggers G1 Cell Cycle Arrest in Chronic Myelogenous Leukemia K562 Cells by Inhibiting CDK4/6-Mediated Phosphorylation of Rb

2020 ◽  
Vol 21 (14) ◽  
pp. 5077
Author(s):  
Bin Zhang ◽  
Ting Zhang ◽  
Tian-Yi Zhang ◽  
Ning Wang ◽  
Shan He ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chronic Myeloid Leukemia ◽  
Cell Cycle Arrest ◽  
Chronic Myelogenous Leukemia ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
G1 Phase ◽  
Cycle Arrest ◽  
G1 Cell Cycle Arrest

Chronic myeloid leukemia (CML) is a malignant tumor caused by the abnormal proliferation of hematopoietic stem cells. Among a new series of acridone derivatives previously synthesized, it was found that the methoxybenzyl 5-nitroacridone derivative 8q has nanomolar cytotoxicity in vitro against human chronic myelogenous leukemia K562 cells. In order to further explore the possible anti-leukemia mechanism of action of 8q on K562 cells, a metabolomics and molecular biology study was introduced. It was thus found that most of the metabolic pathways of the G1 phase of K562 cells were affected after 8q treatment. In addition, a concentration-dependent accumulation of cells in the G1 phase was observed by cell cycle analysis. Western blot analysis showed that 8q significantly down-regulated the phosphorylation level of retinoblastoma-associated protein (Rb) in a concentration-dependent manner, upon 48 h treatment. In addition, 8q induced K562 cells apoptosis, through both mitochondria-mediated and exogenous apoptotic pathways. Taken together, these results indicate that 8q effectively triggers G1 cell cycle arrest and induces cell apoptosis in K562 cells, by inhibiting the CDK4/6-mediated phosphorylation of Rb. Furthermore, the possible binding interactions between 8q and CDK4/6 protein were clarified by homology modeling and molecular docking. In order to verify the inhibitory activity of 8q against other chronic myeloid leukemia cells, KCL-22 cells and K562 adriamycin-resistant cells (K562/ADR) were selected for the MTT assay. It is worth noting that 8q showed significant anti-proliferative activity against these cell lines after 48 h/72 h treatment. Therefore, this study provides new mechanistic information and guidance for the development of new acridones for application in the treatment of CML.

Induction of Apoptosis and Cell Cycle Arrest in Acute Myeloid Leukemia Cells by Histone Deacetylase Inhibitor, Suberoyl Hydroxamic Acid (SAHA): Lack of Cross-Resistance to Cytosine Arabinoside and Etoposide.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4362-4362
Author(s):  
Sabine Dreyer ◽  
Thomas Decker ◽  
Michaela Wagner ◽  
Christian Peschel ◽  
Thomas Licht
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Trichostatin A ◽  
Hdac Inhibitors ◽  
Constitutive Expression ◽  
Cross Resistance ◽  
Cycle Arrest ◽  
Induced Apoptosis ◽  
G1 Cells

Abstract Inhibitors of histone deacetylases (HDAC) such as SAHA are being introduced into the clinical treatment of hematopoietic neoplasms. These compounds can induce apoptosis or cell cycle arrest by modification of the chromatin structure of malignant cells, thereby modulating the expression of target genes. We investigated the effects of HDAC inhibitors, SAHA and trichostatin A, in twelve acute myeloid leukemia (AML) cell lines. Cytotoxicity was determined with the use of a tetrazolium-based colorimetric assay. The 50% inhibition concentrations (IC50) of SAHA were in the micromolar, of trichostatin A in the nanomolar range. Three cell lines were 3–5 times more resistant to both HDAC-inhibitors than the other leukemias, namely the myelomonocytic leukemia ML-2, and the erythroleukemias HEL and K562. All investigated AML cells were, however, more sensitive to SAHA than three patient samples of normal CD34+ progenitor cells mobilized into peripheral blood. A close association between the IC50 of TSA and SAHA was noted within the panel of AML lines (r=0.78). In contrast, chemosensitivity to HDAC-inhibitors was not significantly correlated with IC50 for etoposide, cytosine arabinoside, or staurosporine, respectively. To distinguish between growth arrest and induction of apoptosis by SAHA, we analyzed the cell cycle status by staining with propidium iodide, and exposure of phosphatidylserine by an Annexin V assay. Cell cycle arrest in G1 phase was observed in four AML cell lines with an increase of G1 cells by 20–49% in comparison with untreated cells. One cell line, KG-1a, displayed G2 arrest. SAHA induced apoptosis in eight cell lines with one line displaying both apoptosis and G1 cell cycle arrest simultaneously. KASUMI-1 cells bearing the AML1/ETO gene fusion, a target for HDAC’s, underwent apoptosis upon exposure to SAHA. Constitutive expression of the cell cycle inhibitor p27Kip1, determined by Western blotting, was associated with increased numbers of G1 cells following treatment with SAHA (p=0.036, one-tailed Mann-Whitney test). Conversely, constitutive expression of cyclins A, B1, D3 and E, as well as p53 and p16INK4 was not predictive for induction of cell cycle arrest. Expression of P-glycoprotein (ABCB1) as assessed by flow cytometry was not correlated with the IC50 for SAHA. The anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP were analyzed by Western blotting. No association with resistance to SAHA-induced apoptosis was noticable. In summary, AML cells from permanent lines were more sensitive to SAHA than normal hematopoietic progenitor cells. The potential role of p27Kip1 in modulation of the cytotoxicity of HDAC inhibitors requires further study. Lack of cross-resistance with drugs used in clinical treatment suggests that HDAC inhibitors may be useful for treatment of chemoresistant AML.

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