Anti-Leukemic Effect of a Synthetic Compound, (±)Trans-Dihydronarciclasione (HYU-01) Via Cell Cycle Arrest and Apoptosis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4017-4017
Author(s):  
Seoju Kim ◽  
Jinsun Yoon ◽  
Eun Shil Kim ◽  
Byoungbae PARK ◽  
Junghye Choi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myeloid Leukemia ◽  
Flow Cytometric Analysis ◽  
Cyclin B ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Flow Cytometric ◽  
Human Acute Myeloid Leukemia ◽  
Related Proteins ◽  
Dose Dependent

Abstract Over the past several decades, there has been considerable effort in the synthesis of narciclasine, lycoricidine, and pancratistatin. These naturally occurring isocarbostryls are known to have potent antitumoral and antiviral effects. Among them, trans-dihydronarciclasine isolated from the Chinese medicinal plant, Zephyranthes candida, exhibits even higher potency than pancratistatin against several human cancer cell lines and murine P388 lymphocytic leukemia cell line. However, much remains to be known about antitumoral mechanism of this natural product. In addition, the effect of transdihydronarciclasine in human acute myeloid leukemia (AML) has been not elucidated. The present study was undertaken to investigate the effect of novel synthetic (±)trans-dihydronarciclasine compound (code name; HYU-01) in human acute myeloid leukemia (AML). Treatment of HYU-01 for 72 hr inhibited the proliferation of human AML cell lines as well as primary leukemic blasts from AML patients in a dose-dependent manner with IC50 ranging from 1×10−7M to 5×10−8M. To address the mechanism of the antiproliferative effect of HYU-01, cell cycle analysis was performed in HL-60 cells. DNA flow cytometric analysis indicated that HYU-01 (2.5×10−7M) efficiently induced G1 arrest. Analysis of cell cycle-related proteins demonstrated that expression levels of CDK2, CDK4, CDK6, cyclin E and cyclin A were decreased in a time-dependent manner, and expression of cyclin D1 was up-regulated. In contrast, the level of cyclin B was not altered. In addition, HYU-01 (2.5×10−7M, 72 hr) increased the expression level of the CDKI p27kip1 and markedly enhanced the binding of p27 with CDK2, CDK4, and CDK6 compared to HYU-01-untreated cells. Furthermore, the activity of CDK2-associated kinase was decreased, which resulted in the hypophosphorylation of Rb protein. HYU- 01 also induced the apoptosis in HL-60 cells. The apoptotic process was associated with increased Bax and decreased Bid, Bcl-XL and poly(ADP-ribose) polymerase (PARP), primary leukemic blasts from AML patients in a dose-dependent manner with IC50 ranging from 1×10−7M to 5×10−8M. To address the mechanism of the antiproliferative effect of HYU-01, cell cycle analysis was performed in HL-60 cells. DNA flow cytometric analysis indicated that HYU-01 (2.5×10−7M) efficiently induced G1 arrest. Analysis of cell cycle-related proteins demonstrated that expression levels of CDK2, CDK4, CDK6, cyclin E and cyclin A were decreased in a time-dependent manner, and expression of cyclin D1 was up-regulated. In contrast, the level of cyclin B was not altered. In addition, HYU-01 (2.5×10− 7M, 72 hr) increased the expression level of the CDKI p27kip1 and markedly enhanced the binding of p27 with CDK2, CDK4, and CDK6 compared to HYU-01-untreated cells. Furthermore, the activity of CDK2-associated kinase was decreased, which resulted in the hypophosphorylation of Rb protein. HYU-01 also induced the apoptosis in HL-60 cells. The apoptotic process was associated with increased Bax and decreased Bid, Bcl-XL and poly(ADP-ribose) polymerase (PARP), and activation of caspase-8, -9, and -3, and release of cytochrome C from mitochondria into cytosol. In addition, the apoptosis by HYU-01 was accompanied with the down-regulation of ERK and P90RSK. These results suggest that HYU-01 inhibit the proliferation of AML cells via triggering the apoptosis as well as the induction of p27 and the reduction of CDK2 activity leading to G1 arrest.

PAEDERUS ALFIERI EXTRACT INDUCES APOPTOSIS IN HUMAN MYELOID LEUKEMIA K562 CELLS

2016 ◽  
Vol 10 (1) ◽  
pp. 72
Author(s):  
Amer Mohamed ◽  
Osama Rakha
Keyword(s):  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Insect Pests ◽  
Flow Cytometric Analysis ◽  
K562 Cells ◽  
Akt Pathway ◽  
Dependent Manner ◽  
Proliferative Effect ◽  
Induced Apoptosis ◽  
Dose Dependent

ABSTRACTObjective: The rove beetle Paederus alfieri Koch. (Coleoptera: Staphylinidae) is well-known among natural enemies in Egypt as an important predatorof agricultural insect pests, it used as an essential agent in the integrated pest management programs. Recent studies have revealed that Paederus mayhave anti-proliferative effect; however, its mechanisms remain unclear. The aim of the present study is to investigate the anticancer effect of P. alfieriextract (PAE) on K562 human myeloid leukemia cancer cells and elucidation of its mechanism.Methods: Human myeloid leukemia K562 cells were treated with PAE at different concentrations. Cell proliferation was measured using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was evaluated using flow cytometry analysis. The expressions ofBcl-2, Bax, active caspase-3, t-Akt, and p-Akt were evaluated by western blotting.Results: PAE has a dose-dependent antiproliferative effect against K562 cells. The half maximal inhibitory concentration was estimated as212±2.3 ng/ml. Flow cytometric analysis showed that PAE induces apoptosis in a dose-dependent manner in K562 cells. We also investigated themolecular mechanism of PAE-induced apoptosis. PAE downregulated Bcl-2 and upregulated Bax and cleaved caspase-3 proteins. Furthermore, thelevels of p-Akt are dose-dependently decreased in response to PAE, whereas the total Akt protein levels remained constant during PAE treatment.Conclusion: Taken together PAE-induced apoptosis in human myeloid leukemia K562 cells by modulating PI3K/Akt pathway. Our findings suggestthat may be PAE is a good extract for developing anticancer drugs for human myeloid leukemia cancer treatment.Keywords: Paederus alfieri, Pederin, K562, Apoptosis, PI3K/Akt pathway.

Mesenchymal Stromal Cells Expressing Interferon α Limit the Development of Acute Myeloid Leukemia, Inducing Apoptosis In Vitro and In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5216-5216
Author(s):  
Laura M Desbourdes ◽  
Adam J Guess ◽  
Suheyla Hasgur ◽  
Kathleen M Overholt ◽  
Minjun Yu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Flow Cytometric Analysis ◽  
Interferon Α ◽  
Flow Cytometric ◽  
Acute Myeloid

Abstract Introduction The 5-year survival for patients with acute myeloid leukemia (AML) has stagnated for over two decades at about 60% for children, 40% for young adults, and <15% for elderly patients. While most patients achieve remission, approximately 50% will relapse which is generally attributed to the persistence of leukemic stem cells. Interferon α (IFNα) is an effective therapy for patients with AML due to multiple mechanisms of action. However, high serum levels are associated with many adverse effects. In this proof-of-concept study, we used engineered mesenchymal stem/stromal cells (MSC) to deliver high concentrations of IFNα locally to an AML chloroma, potentially diminishing the poorly tolerated systemic side-effects. Methods Bone marrow MSCs from C57BL/6 mouse were isolated and transduced with a lentiviral vector expressing murine IFNα (IFNα-MSCs) and/or GFP (GFP MSCs). After measuring IFNα secretion by ELISA and confirming activity by the induction of the MHC I expression on the transduced cells, the anti-AML activity of these transduced MSCs was assessed by co-culture with the C57BL/6 AML cell line c1498 which expresses DsRed and firefly luciferase (FFluc). Apoptotic cell frequencies and cell cycle phase distributions of leukemia cells with or without MSCs were assessed by flow cytometry. The in vivo validation has been performed by subcutaneous injection of c1498 cells (chloroma) with or without GFP MSCs or IFNα MSCs in C57BL/6 mice. Tumor growth was monitored by bioluminescence imaging every 3 or 4 days. Results Flow cytometric analysis and ELISA confirmed the secretion of bio-active of IFNα by transduced MSCs (41.5 ng/1E06 MSCs/24h). In co-cultures, the presence of IFNα MSCs at the ratio 100:1 (c1498: MSC) significantly decreased the population of c1498 cells mainly by inducing apoptosis compared to MSC-free or GFP MSC co-cultures while no effect was observed on cell cycle distribution. The pro-apoptotic effect of IFNα MSCs was then investigated in vivo by subcutaneous injection of c1498 cells with or without MSCs (ratio 10:1) in C57BL/6 mice.The presence of IFNα MSCs significantly decreased leukemic cell mass as shown by the bioluminescence of the DsRed+ FFLuc+ c1498 cells. This result was confirmed by flow cytometric analysis of the percentage of DsRed + cells in the chloroma. Conclusions This study shows that IFNα MSCs present a strong anti-leukemic effect in vitro and in vivo promoting apoptosis and thus decreasing the leukemic burden. Further experiments will focus on a potential synergetic effect with Cytarabine treatment and a preclinical study using human IFNα MSCs in a xenograft murine model. Disclosures No relevant conflicts of interest to declare.

Hispolon from Phellinus linteus Induces G0/G1 Cell Cycle Arrest and Apoptosis in NB4 Human Leukaemia Cells

2013 ◽  
Vol 41 (06) ◽  
pp. 1439-1457 ◽  
Author(s):  
Yi-Chuan Chen ◽  
Heng-Yuan Chang ◽  
Jeng-Shyan Deng ◽  
Jian-Jung Chen ◽  
Shyh-Shyun Huang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Flow Cytometric Analysis ◽  
G1 Phase ◽  
Dependent Manner ◽  
Phellinus Linteus ◽  
Nb4 Cells ◽  
Cycle Arrest ◽  
Bax Protein ◽  
Flow Cytometric

Hispolon (a phenolic compound isolated from Phellinus linteus) has been shown to possess strong antioxidant, anti-inflammatory, anticancer, and antidiabetic properties. In this study, we investigated the antiproliferative effect of hispolon on human hepatocellular carcinoma NB4 cells using the MTT assay, DNA fragmentation, DAPI (4, 6-diamidino-2-phenylindole dihydrochloride) staining, and flow cytometric analysis. Hispolon inhibited the cellular growth of NB4 cells in a dose-dependent manner through the induction of cell cycle arrest at G0/G1 phase measured using flow cytometric analysis and apoptotic cell death, as demonstrated by DNA laddering. Exposure of NB4 cells to hispolon-induced apoptosis-related protein expressions, such as the cleavage form of caspase 3, caspase 8, caspase 9, poly (ADP ribose) polymerase, and the proapoptotic Bax protein. Western blot analysis showed that the protein levels of extrinsic apoptotic proteins (Fas and FasL), intrinsic related proteins (cytochrome c), and the ratio of Bax/Bcl-2 were increased in NB4 cells after hispolon treatment. Hispolon-induced G0/G1-phase arrest was associated with a marked decrease in the protein expression of p53, cyclins D1, and cyclins E, and cyclin-dependent kinases (CDKs) 2, and 4, with concomitant induction of p21waf1/Cip1 and p27Kip1. We conclude that hispolon induces both of extrinsic and intrinsic apoptotic pathways in NB4 human leukemia cells in vitro.

scholarly journals Morusin Functions as a Lipogenesis Inhibitor as Well as a Lipolysis Stimulator in Differentiated 3T3-L1 and Primary Adipocytes

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 2004 ◽  
Author(s):  
Mi Rim Lee ◽  
Ji Eun Kim ◽  
Jun Young Choi ◽  
Jin Ju Park ◽  
Hye Ryeong Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Insulin Receptor ◽  
Adipogenic Differentiation ◽  
Treated Group ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Glycerol Release ◽  
Insulin Receptor Signaling ◽  
Dose Dependent

Conflicting results for morusin activity during adipogenic differentiation are reported in 3T3-L1 adipocytes and cancer cells. To elucidate the influence of morusin on fat metabolism, their anti-obesity effects and molecular mechanism were investigated in 3T3-L1 cells and primary adipocytes. Morusin at a dose of less than 20 µM does not induce any significant change in the viability of 3T3-L1 adipocytes. The accumulation of intracellular lipid droplets in 3T3-L1 adipocytes stimulated with 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, 10 µg/mL insulin in DMEM containing 10% FBS (MDI)-significantly reduces in a dose-dependent manner after morusin treatment. The phosphorylation level of members in the MAP kinase signaling pathway under the insulin receptor downstream also decrease significantly in the MDI + morusin-treated group compared to MDI + vehicle-treated group. Also, the expression of adipogenic transcription factors (PPARγ and C/EBPα) and lipogenic proteins (aP2 and FAS) are significantly attenuated by exposure to the compound in MDI-stimulated 3T3-L1 adipocytes. Furthermore, the decrease in the G0/G1 arrest of cell cycle after culturing in MDI medium was dramatically recovered after co-culturing in MDI + 20 µM morusin. Moreover, morusin treatment induces glycerol release in the primary adipocytes of SD rats and enhances lipolytic protein expression (HSL, ATGL, and perilipin) in differentiated 3T3-L1 adipocytes. Overall, the results of the present study provide strong evidence that morusin inhibits adipogenesis by regulating the insulin receptor signaling, cell cycle and adipogenic protein expression as well as stimulating lipolysis by enhancing glycerol release and lipolytic proteins expression.

scholarly journals Morphological alterations and G0/G1 cell cycle arrest induced by curcumin in human SK-MEL-37 melanoma cells

2010 ◽  
Vol 53 (2) ◽  
pp. 343-352 ◽  
Author(s):  
Marcella Lemos Brettas Carneiro ◽  
Elaine Paulucio Porfírio ◽  
Andréia Hanada Otake ◽  
Roger Chammas ◽  
Sônia Nair Báo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometric Analysis ◽  
Melanoma Cells ◽  
G1 Arrest ◽  
Scanning Electronic Microscopy ◽  
Apoptotic Pathway ◽  
Morphological Alterations ◽  
Cycle Arrest ◽  
Flow Cytometric ◽  
G1 Cell Cycle Arrest

The aim of this work was to study the effect of curcumin on cell cycle in the human SK-MEL-37 melanoma cell line. In addition, morphological and structural analyses were also performed. Flow cytometric analysis showed a G0/G1 arrest at 5 µM after 24 h exposure and a concentration-dependent increase in the proportion of sub-G0 hypodiploid cells. Typical apoptotic events were also observed by the fluorescence microscopy, transmission and scanning electronic microscopy. Loss of mitochondrial membrane potential was not detected. Results suggested that curcumin could arrest human melanoma cells at G0/G1 phase and induce a mitochondrial-independent apoptotic pathway.

Activation of B-Cell Maturation Antigen (BCMA) on Human Multiple Myeloma Cells by a Proliferation-Inducing Ligand (APRIL) Promotes Myeloma Cell Function in the Bone Marrow Microenvironment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1503-1503
Author(s):  
Merav Leiba ◽  
Jiangchun Xu ◽  
Weihua Song ◽  
Xian-Feng Li ◽  
Peter Burger ◽  
...  
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Flow Cytometric Analysis ◽  
Cell Maturation ◽  
Signaling Cascades ◽  
Dependent Manner ◽  
B Cell Maturation ◽  
Flow Cytometric ◽  
Dose Dependent

Abstract A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily that stimulates growth and survival of multiple myeloma (MM) cells, is mainly produced by MM patient bone marrow stromal cells (BMSCs) and osteoclasts in the BM microenvironment. Like BAFF, APRIL binds two receptors, B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator (TACI). Since BCMA has significantly higher affinity for APRIL than for BAFF (nM vs. mM range), APRIL/BCMA signaling may be more important than BAFF/BCMA in MM. Specifically, BCMA but not TACI is unequivocally expressed at mRNA level in the majority of CD138-expressing MM cell lines and patient MM cells. To date, it is unclear how APRIL signaling through BCMA contributes to BM microenvironment in support of MM cells. We here studied the functional sequelae of APRIL/BCMA pathway in MM cells, and further identified molecular mechanisms regulating these processes. Using flow cytometric analysis, cell surface BCMA expression was confirmed in the majority of MM cell lines. Microarray gene expression analysis showed significant expression of BCMA mRNA in CD138+ patient MM cells (n=105). Higher BCMA mRNA levels were observed in patient MM cells than in normal plasma cells (n=27) (p&lt; 0.03). Importantly, CD138+ patient MM cells express BCMA protein on the cell surface (n=15), as confirmed by CD38+BCMA+ dual immunofluorescence staining followed by flow cytometric analysis and immunohistochemical (IHC) study. Next, H929 MM cell line that expresses only BCMA, but not TACI, and Daudi B cells transduced with huBCMA (huBCMA-Daudi) were stimulated with APRIL. These cells were subjected to mRNA extraction at different time points followed by microarray analysis to identify downstream transcriptional targets. Stimulation of these two cell lines by APRIL activated the NF-kB signaling in a dose-dependent manner. More than 6-fold increase in NF-kB activity was induced by APRIL (100 ng/ml) using a p65 enzyme-linked DNA/protein interaction assay. IL-6 and PI3K/AKT signaling cascades were concurrently induced. Additionally, APRIL upregulated chemotactic/osteoclast activating factors, i.e., MIP-1a and MIP1b, in a dose-dependent manner, which was further validated by specific ELISA. Multi-Analyte Profiles also confirmed that angiogenesis and adhesion/chemoattractant factors, i.e., IL-8, CXCL10, RANTES, MDC (ccl22), were significantly induced upon APRIL stimulation. Conversely, a soluble BCMA-Fc protein inhibited APRIL binding to BCMA and blocked secretion of APRIL-induced chemokines, indicating specific functional interaction of BCMA with APRIL. Importantly, APRIL similarly induced these signaling cascades in CD138+ patient MM cells. Taken together, our studies are the first to demonstrate cell surface BCMA on the majority of CD138+ patient MM cells. Furthermore, APRIL induced osteoclasts-, migration-, and angiogenesis-associated factors in MM cells. These studies therefore establish a role of BCMA activation in the BM microenvironment that provides a niche for MM disease progression, supporting novel therapeutics specifically targeting APRIL/BCMA pathway in MM.

scholarly journals Arctigenin Inhibits Glioblastoma Proliferation through the AKT/mTOR Pathway and Induces Autophagy

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Yong’an Jiang ◽  
Jiayu Liu ◽  
Wangwang Hong ◽  
Xiaowei Fei ◽  
Ru’en Liu
Keyword(s):  
Cell Cycle ◽  
Western Blotting ◽  
Glioma Cells ◽  
Annexin V ◽  
Mtor Pathway ◽  
Migration And Invasion ◽  
Dependent Manner ◽  
Arctium Lappa ◽  
Related Proteins ◽  
Dose Dependent

Purpose. Arctigenin (ARG) is a natural lignan compound extracted from Arctium lappa and has displayed anticancer function and therapeutic effect in a variety of cancers. Arctigenin is mainly from Arctium lappa extract. It has been shown to induce autophagy in various cancers. However, as for whether arctigenin induces autophagy in gliomas or not, the specific mechanism is still worth exploring. Methods. Using CCK8, the monoclonal experiment was made to detect the proliferation ability. The scratch experiment and the transwell experiment were applied to the migration and invasion ability. PI/RNase and FITC-conjugated anti-annexin V were used to detect the cell cycle and apoptosis. Western blotting was used to determine the specified protein level, and constructed LC3B-GFP plasmid was used for analysis of autophagy.Results. Our research showed that ARG inhibited the growth and proliferation and invasion and migration of glioma cells in a dose-dependent manner (U87MG and T98G) and arrested the cell cycle and induced apoptosis. Interestingly, ARG induced autophagy in a dose-dependent manner. We applied Western blotting to measure the increase in the key autophagy protein LC3B, as well as some other autophagy-related proteins (increase in Beclin-1 and decrease in P62). In order to further explore the mechanism that ARG passed initiating autophagy to inhibit cell growth, we further found by Western blotting that AKT and mTOR phosphorylation proteins (P-AKT, P-mTOR) were reduced after ARG treatment, and we used AKT agonists to rescue, and the phosphorylated proteins of AKT and mTOR increased, and we found that the autophagy-related proteins were also reversed. And interestingly, the protein of apoptosis was also reversed along with autophagy. Conclusions. We thought ARG inhibited the proliferation of glioma cells by inducing autophagy and apoptosis through the AKT/mTOR pathway.

scholarly journals Biochanin A Induces S Phase Arrest and Apoptosis in Lung Cancer Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Yan Li ◽  
Haiyang Yu ◽  
Fengfeng Han ◽  
Mengmeng Wang ◽  
Yong Luo ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cancer Cells ◽  
Caspase 3 ◽  
Lung Cancer Cells ◽  
Biochanin A ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Related Proteins ◽  
Dose Dependent

Lung cancer is among the most common malignancies with a poor 5-year survival rate reaching only 16%. Thus, new effective treatment modalities and drugs are urgently needed for the treatment of this malignancy. In this study, we conducted the first investigation of the effects of Biochanin A on lung cancer and revealed the mechanisms underlying its potential anticancer effects. Biochanin A decreased cell viability in a time-dependent and dose-dependent manner and suppressed colony formation in A549 and 95D cells. In addition, Biochanin A induced S phase arrest and apoptosis and decreased mitochondrial membrane potential (ΔΨm) in A549 and 95D cells in a dose-dependent manner. Our results of subcutaneous xenograft models showed that the growth of Biochanin A group was significantly inhibited compared with that of control groups. Finally, P21, Caspase-3, and Bcl-2 were activated in Biochanin A-treated cells and Biochanin A-treated xenografts which also demonstrated that Biochanin A induced cell cycle arrest and apoptosis in lung cancer cells by regulating expression of cell cycle-related proteins and apoptosis-related proteins. In conclusion, this study suggests that Biochanin A inhibits the proliferation of lung cancer cells and induces cell cycle arrest and apoptosis mainly by regulating cell cycle-related protein expression and activating the Bcl-2 and Caspase-3 pathways, thus suggesting that Biochanin A may be a promising drug to treat lung cancer.

AZD6244 (ARRY-142886), a Potent and Selective MEK1/2 Inhibitor Blocks the ERK1/2 Signaling Pathway, Inhibits Osteoclast Differentiation and Activation in Multiple Myeloma: Clinical Implications.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3467-3467 ◽  
Author(s):  
Iris Breitkreutz ◽  
Sonia Vallet ◽  
Marc S. Raab ◽  
Xianfeng Li ◽  
Noopur Raje ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Resorption ◽  
Mononuclear Cells ◽  
Mapk Pathway ◽  
Osteoclast Differentiation ◽  
Flow Cytometric Analysis ◽  
Tartrate Resistant Acid Phosphatase ◽  
Dependent Manner ◽  
Flow Cytometric ◽  
Dose Dependent

Abstract Multiple myeloma (MM)-associated bone disease is caused by upregulation of osteoclast (OCL) activity and constitutive inhibition of osteoblast function. The extracellular signal-regulated kinase 1/2 (ERK1/2) MAP kinase pathway contributes to cytokine-induced OCL differentiation and maturation. We hypothesized that inhibition of ERK1/2 could prevent OCL differentiation and downregulate OCL function. Here we investigate the effects of AZD6244, which blocks the ERK1/2 MAPK pathway via direct inhibition of MEK1/2, on OCL in MM. Peripheral blood mononuclear cells (PBMC) from healthy donors (n=3) and MM patients (n=11) were harvested and stimulated with RANKL (50ng/ml) and M-CSF (25ng/ml) for 2 weeks to induce OCL formation, in the presence or absence of AZD6244. OCL characteristics were measured by flow cytometric analysis of anti-alphaVbeta3 integrin expression. AZD6244 inhibited OCL differentiation in a dose-dependent manner (n=11, median control: 77.4% at 0 uM; 77% at 0.02 uM; 54% at 0.2 uM; 53% at 2 uM; 38% at 5 uM; 29% at 10 uM). TRAP staining (tartrate-resistant acid phosphatase) was performed to identify OCL and to confirm activity. Importantly, AZD6244 inhibited OCL in a dose-dependent manner, as evidenced by a marked loss of TRAP+ cells. To assess bone resorption activity, OCL were cultured with dentine discs in the presence or absence of AZD6244, followed by the measurement of soluble collagen I fragments in the supernatant. AZD6244 inhibited bone resorption in a dose-dependent manner. We next asked whether AZD6244 affects mature OCL. Mature OCL were induced by cytokine stimulation for 2 weeks and then AZD6244 was added for 3 days, followed by flow cytometric analysis. AZD6244 had no effect on total number of alphaVbeta3 integrin-expressing mature OCL (n=6). Two major myeloma growth and survival factors produced by OCL, B-cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL), were measured in OCL culture supernatants by ELISA. AZD6244 significantly inhibited secretion of BAFF and APRIL. In addition, macrophage inflammatory protein (MIP-1alpha), an important OCL differentiation factor and MM survival factor, was inhibited. These results indicate that AZD6244 inhibits OCL differentiation induced by M-CSF and RANKL, leading to reduced bone resorption activity. Moreover, AZD6244 downregulates MIP-1alpha and BAFF, APRIL secretion by OCL, which could inhibit MM cell survival in the bone marrow microenvironment. We have also demonstrated that AZD6244 inhibits proliferation and survival of human MM cell lines, either sensitive or resistant to conventional chemotherapy, as well as freshly isolated patient MM cells (Abstract #553572 and #553605, ASH 2006). In conclusion, the present study provides a preclinical rationale for the evaluation of AZD6244 (ARRY-142886) as a potential new therapy for patients with MM.

scholarly journals Autophagic degradation of CDK2 and CDK4 is responsible for NVP-BEZ235-induced G0/G1 cell cycle arrest in neuroblastoma

2021 ◽  
Author(s):  
Zhen Liu ◽  
Hong–Liang Zhang ◽  
Shan-Ling Liu ◽  
Chao Zhang ◽  
Qing-Xi Yue ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Interactions ◽  
Neuroblastoma Cell ◽  
Flow Cytometric Analysis ◽  
Neuroblastoma Cells ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Autophagic Degradation ◽  
Cell Arrest

Abstract Background Cyclin-dependent kinases (CDKs) are regarded as promising targets for cancer therapy. CDK2 and CDK4 are the core molecules in the G0 and G1 phases of the cell cycle. Exploring the molecular mechanism of CDK2 and CDK4 protein degradation will provide clues and solutions to control the G0 and G1 phases in tumor cells. Methods The expression levels of CDK2 and CDK4 were assessed by Western blotting and qRT-PCR. Cell viability, cell proliferation and growth were evaluated in CCK-8 and flow cytometric analysis. Protein interactions were analysed by immunoprecipitation and immunofluorescence methods. Results NVP-BEZ235 induced neuroblastoma cell arrest at the G0/G1 phase, and proliferation inhibition was associated with a significant reduction in the CDK2 and CDK4 proteins in a dose-dependent manner at nanomolar concentrations. Surprisingly, we found that the NVP-BEZ235-induced downregulation of CDK2 and CDK4 was dramatically rescued by autophagy-lysosome inhibitors. Additionally, the blockade of autophagy-related genes contributed to the remarkable rescue of CDK2 and CDK4, which thus strikingly improved NVP-BEZ235-induced G0/G1 arrest and growth inhibition of neuroblastoma cells. Subsequently, we observed the first evidence that NVP-BEZ235 induced the interaction of p62/SQSMT1 with CDK2 or CDK4 and the autophagic degradation of CDK2 and CDK4 in a cathepsin B-dependent manner. Analogous results regarding autophagy and the antiproliferative effects of NVP-BEZ235 were observed in a neuroblastoma xenograft mouse model in vivo. Conclusions These results not only established the pivotal role of the autophagy pathway in G0/G1 cell cycle-related protein turnover but also suggest the potential application of NVP-BEZ235 for cancer treatment by modulating elements of the autophagic machinery to promote degradation of G0/G1 cell cycle-related proteins.

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