Inhibition of the Serine/Threonine Kinase Pim-1 Has Anti-Proliferative Effects In Acute Myeloid Leukemia (AML) and Sensitizes Multidrug Resistant Cells to Chemotherapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1832-1832
Author(s):  
Karthika Natarajan ◽  
Mehmet Burcu ◽  
Maria R. Baer
Keyword(s):  
Cell Cycle ◽  
Drug Resistance ◽  
Cell Survival ◽  
Cell Lines ◽  
Colony Formation ◽  
Multidrug Resistant ◽  
Serine Threonine Kinase ◽  
Hl60 Cells ◽  
Threonine Kinase ◽  
Resistance Protein

Abstract Abstract 1832 Poster Board I-812 The serine/threonine kinase Pim-1, encoded by a proto-oncogene originally identified as the proviral integration site in Moloney murine leukemia virus lymphomagenesis, phosphorylates and thereby increases expression of multiple cellular proteins, including the pro-apoptotic protein BAD, the cell cycle regulatory proteins p21, p27, Cdc25A and Cdc25C, the transcription factors SOCS-1, RUNX3 and c-myc and, as we recently demonstrated, the drug resistance-associated ATP-binding cassette (ABC) proteins P-glycoprotein (Pgp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). Pim-1 is synthesized in an active form by virtue of its hinge structure, and its activity is therefore regulated solely by its level of expression. Pim-1 is overexpressed downstream of FLT3 in AML cells with FLT3-ITD, but less is known about its expression and role in AML with wild-type (wt) FLT3. We studied Pim-1 expression and the effects of Pim-1 inhibition on AML cell survival, proliferation, apoptosis and chemosensitivity. Cell lines studied included HL60, K562, U937, Kasumi-1 and EOL-1 FLT3-wt cells and MV4-11 and MOLM-14 FLT3-ITD cells, as well as Pgp+ HL60/VCR and BCRP+ 8226/MR20 and parental 8226 myeloma cells as a model for BCRP-mediated drug resistance. Expression of Pim-1 and of phospho-BAD at S112, a measure of Pim-1 activity, was studied by Western blot analysis, normalized to GAPDH expression. Effects of the Pim-1 inhibitor SGI-1776 (SuperGen, Inc., Dublin, CA) on survival, cell cycle, apoptosis and colony growth were measured in WST-1 cell survival, flow cytometric cell cycle and apoptosis, and methylcellulose colony formation assays, respectively. SGI-1776 inhibits Pim-1 at a concentration of 7 +/− 1.8 nM, but is more than 90% bound to human plasma protein, so that its Pim-1 inhibitory concentration in cell culture-based assays is in μM range. Of note, SGI-1776 also inhibits FLT3 in this concentration range. Pim-1 was expressed in all cell lines studied, and expression of Pim-1 and of phopho-BAD did not differ between FLT3-ITD and FLT3-wt cells, nor between drug-resistant and parental cells. SGI-1776 decreased viable cell numbers in 96-hour WST-1 cell viability assays, with IC50's of 5 to 7 μM in FLT-wt cells, while IC50's were 20 and 65 nM, respectively, in MV4-11 and MOLM-14 FLT3-ITD cells. SGI-1776 IC50's did not differ between Pgp+ or BCRP+ cells and parental cells. In FLT3-wt cells, SGI-1776 had no effect on cell cycle at concentrations up to 5 μM, and caused apoptosis at 10 μM, while in FLT3-ITD cells, G1 arrest and apoptosis occurred at 100 nM. HL60 colony formation was completely inhibited by 5 μM SGI-1776, while MOLM-14 colony formation inhibition occurred at 500 nM. Finally, SGI-1776 sensitized multidrug resistant, but not parental, cells to multidrug resistance protein substrate, but not non-substrate, drugs. SGI-1776 at 1 μM decreased the IC50 of the Pgp substrate chemotherapy drug daunorubicin in Pgp+ HL60/VCR cells 7-fold, but had no effect on daunorubicin IC50 in HL60 cells, nor on IC50 of the non-Pgp substrate cytarabine in either cell line. SGI-1776 at 1 μM also decreased the IC50 of the BCRP substrate chemotherapy drug mitoxantrone in BCRP+ 8226/MR20 cells 7-fold. SGI-1776 at 1 μM doubled the percentage of apoptotic cells among HL60/VCR, but not HL60, cells exposed to daunorubicin and 8226/MR20 cells exposed to mitoxantrone. Finally, SGI-1776 at 1 μM decreased HL60/VCR colony formation in the presence of daunorubicin, but not cytarabine, but had no effect in HL60 cells, and also decreased 8226/MR20 colony formation in the presence of mitoxantrone, but not cytarabine. Thus the Pim-1 inhibitor SGI-1776 has anti-proliferative effects in AML cells with wt FLT3 as well FLT3-ITD, and sensitizes Pgp+ and BCRP+ multidrug resistant cells to chemotherapy. These data support clinical trials of SGI-1776 in AML with wt FLT3 as well FLT3-ITD, as a single agent and in combination with chemotherapy in multidrug resistant AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Citron Rho-Interacting Serine/Threonine Kinase Promotes HIF1a-CypA Signaling and Growth of Human Pancreatic Adenocarcinoma

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Lin Cong ◽  
Zhile Bai ◽  
Yang Du ◽  
Yong Cheng
Keyword(s):  
Cell Cycle ◽  
Colony Formation ◽  
Disease Free Survival ◽  
Cyclophilin A ◽  
Ductal Adenocarcinoma ◽  
Serine Threonine Kinase ◽  
Free Survival ◽  
Threonine Kinase ◽  
Cycle Arrest ◽  
Cypa Expression

In human pancreatic ductal adenocarcinoma (PDAC), the cyclophilin A (CypA) is overexpressed and promotes the development of PDAC. However, the mechanism underlying cyclophilin A expression remains elusive. Here, we reported that the citron Rho-interacting serine/threonine kinase (CIT) promotes the HIF1a-CypA signaling and growth of PDAC cells. CIT expression was higher in PDAC cells compared with the normal epithelial cells, and clinical data showed that CIT was overexpressed in PDAC tissues and high expression of CIT predicted poor overall and disease-free survival. In PDAC cells, knockdown of CIT expression repressed the rate of proliferation and capacity of colony formation, which were accomplished with an increased percentage of apoptotic cells and cell cycle arrest. The knockdown of CIT in PDAC cells reduced the expression of CypA while overexpression of CIT promoted the expression of CypA. We observed that the effects of CIT on the expression of CypA relied on the transcriptional factor HIF1a, which was previously reported to transcriptionally activate the expression of CypA in PDAC cells. Furthermore, the effects of CIT on apoptosis, cell cycle, proliferation, and colony formation of PDAC cells relied on its role in the regulation of CypA expression. Collectively, our data showed that CIT promoted the activation of HIF1-CypA signaling and enhanced the growth of PDAC cells.

The FLT3 Inhibitor PKC412 Interacts Synergistically with Both Daunorubicin and Cytarabine in Acute Myeloid Leukemia (AML) Cells by Heterogeneous Mechanisms.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1378-1378
Author(s):  
Maria R. Baer ◽  
Kareemah L. Sabur ◽  
Kieran L. O’Loughlin ◽  
Petr Starostik ◽  
Hans Minderman
Keyword(s):  
Cell Survival ◽  
Cell Lines ◽  
Drug Transport ◽  
Wild Type ◽  
Protein Substrate ◽  
Hl60 Cells ◽  
Abc Transport ◽  
Synergistic Cytotoxicity ◽  
Resistance Protein

Abstract Both FLT3 activating mutations and overexpression of ATP-binding cassette (ABC) transport proteins that confer multidrug resistance (MDR) are associated with inferior treatment outcomes in AML. The protein kinase C inhibitor PKC412 inhibits both FLT3 signaling and drug transport by the ABC protein P-glycoprotein (Pgp; ABCB1). The effects of PKC412 on other MDR-associated ABC proteins expressed in AML cells, including multidrug resistance protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; ABCG2), are not known. We studied the effects of PKC412 on transport of MDR protein substrate drugs and its interactions with the MDR protein substrate daunorubicin (DNR) and the non-MDR protein substrate cytarabine (AraC) in cell lines overexpressing Pgp (HL60/VCR), MRP-1 (HL60/ADR) and BCRP (8226/MR20), in wild-type HL60 cells, which express no MDR proteins, and in 8 AML patient bone marrow samples characterized for FLT3 mutations by a DNA-based assay and for expression and function of Pgp, MRP-1 and BCRP by flow cytometry using specific antibodies (MRK16, MRPm6 and BXP21) and modulators (PSC-833, probenecid and fumitemorgin C). in vitro drug sensitivity was assessed by cell survival measured by the wst-1 assay following 48- (AML samples) or 96- (cell lines) hour continuous drug exposure in 96-well microculture assays, and drug interactions were evaluated by the combination index approach of Chou and Talalay. PKC412 increased uptake of MDR protein substrate drugs in cell lines overexpressing MRP-1 and BCRP, as well as Pgp, in a concentration-dependent manner, with maximum effects at ≥5 μM. PKC412 was cytotoxic to all cell lines studied, and cell lines overexpressing Pgp, MRP-1 and BCRP were 20- to 40-fold resistant to PKC412 in relation to parental drug-sensitive cells, suggesting that PKC412 may be a substrate for Pgp, MRP-1 and BCRP and may thus modulate drug transport by competitive inhibition. The combination of PKC412 with DNR synergistically decreased cell survival (CI50 values <0.9) in cell lines overexpressing Pgp, MRP-1 and BCRP, but not in wild-type HL60 cells, whereas the combination of PKC412 and AraC synergistically decreased cell survival in wild-type HL60 cells as well as in the cell lines expressing Pgp, MRP-1 and BCRP. These data suggest that PKC412 interacts synergistically with the MDR protein substrate drug DNR at least in part by virtue of modulation of its transport, but interacts with AraC through alternative mechanisms in both wild-type and multidrug resistant cells. The eight AML patient samples studied included 4 each with FLT3 internal tandem duplication (ITD) and wild-type FLT3, and in vitro sensitivity to PKC412 did not differ between these two groups. All eight samples exhibited expression or function of at least one ABC transport protein. Synergistic cytotoxicity was observed for the combination of PKC412 with DNR in 7 of 8 AML patient samples, whereas the combination of PKC412 with AraC produced synergistic cytotoxicity in 4, including 3 with wild-type FLT3 and one with FLT3 ITD. Thus combinations of PKC412 with DNR and with AraC have the potential for synergistic activity in the treatment of AML, and synergy occurs both through modulation of Pgp, MRP-1 and BCRP and through other, likely heterogeneous, mechanisms. Interactions of PKC412 with both MDR protein substrate and non-substrate chemotherapy drugs have the potential to be exploited in novel combination regimens.

Knockdown of Adhesion-Regulating Molecule 1 Inhibits Proliferation in HL60 Cells

2015 ◽  
Vol 134 (2) ◽  
pp. 88-100 ◽  
Author(s):  
Xiaohui Zheng ◽  
Yafei Guo ◽  
Yingying Chen ◽  
Meilin Chen ◽  
Zhenxin Lin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Colony Formation ◽  
Nuclear Translocation ◽  
Leukemia Cell ◽  
26S Proteasome ◽  
Biological Behavior ◽  
Hl60 Cell ◽  
Hl60 Cells ◽  
Leukemia Cell Lines

Background/Aims: Adhesion-regulating molecule 1 (ADRM1), a receptor located on the 26S proteasome, is upregulated in many solid cancers. However, little is known about its role in acute leukemia (AL). Methods: We determined ADRM1 expression levels in both untreated AL samples and leukemia cell lines using real-time polymerase chain reaction or Western blot analysis. Growth curves, colony formation assays, cell cycle and apoptosis analyses, cell migration and invasion assays and NF-κB p65 nuclear translocation assays via Western blotting were used to examine the biological behavior of HL60 cells and the underlying mechanism. Results: ADRM1 was upregulated in both untreated AL samples and leukemia cell lines. ADRM1 knockdown significantly suppressed HL60 cell proliferation (48.82 ± 12.58%) and colony formation and caused cell cycle arrest in the G0/G1 phase. Furthermore, we confirmed that ADRM1 knockdown suppressed p65 nuclear translocation. Conclusion: Our study revealed that ADRM1 was overexpressed in AL, especially in CD34+ leukemia stem and progenitor cells. ADRM1 may play a role in AL via the proteasome-ubiquitin pathway by potentially sustaining the activation of NF-κB signaling.

scholarly journals Resveratrol promotes apoptosis and G2/M cell cycle arrest of fibroblast-like synoviocytes in rheumatoid arthritis through regulation of autophagy and the serine-threonine kinase-p53 axis

2021 ◽  
Author(s):  
Shu Li ◽  
Jinfeng Du ◽  
Haina Gan ◽  
Jinwei Chen ◽  
Yang Zhou ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Survival ◽  
Akt Pathway ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Proliferation And Apoptosis ◽  
Fibroblast Like Synoviocytes

IntroductionResveratrol, a polyphenol extracted from many plant species, has emerged as a promising pro-apoptotic agent in various cancer cells. However, the role of resveratrol in cell proliferation and apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis (RA-FLS) is not fully understood. The study was aimed at elucidating the role of resveratrol in cell proliferation and apoptosis of RA-FLS and the underlying molecular mechanism.Material and methodsCultured RA-FLSs were subjected to tumour necrosis factor  (TNF-). The cell proliferation was measured by Cell Counting Kit-8 assay. Cell apoptosis and cell cycle of RA-FLSs were determined by flow cytometry. The levels of apoptosis or autophagy or cell cycle-related protein were detected by immunoblot analysis.ResultsIn our study, we confirmed that resveratrol reversed TNF- mediated cell proliferation in RA-FLS. Meanwhile, resveratrol blocked cells at the G2/M stage and reduced the ratio of S phase cells through upregulation of p53 and consequently led to apoptotic cell death. Quite interestingly, we found that resveratrol reversed TNF--induced autophagy. Inhibition of autophagy by resveratrol or autophagy inhibitor or Beclin-1 siRNA suppressed TNF- mediated cell survival and promoted cell apoptosis. However, the autophagy inducer rapamycin (RAPA) reversed the effect of resveratrol on autophagy and cell proliferation. Mechanistic studies revealed that resveratrol inhibited the activation of the phosphoinositide 3-kinases/serine-threonine kinase (PI3K/AKT) pathway. Inhibition of PI3K/AKT pathway by inhibitor LY294002 or resveratrol increased the expression of p53 and decreased the expression of cycle protein (cyclin B1), which further led to block cells in the G2/M arrest.ConclusionsOur preliminary study indicated that resveratrol may suppress RA-FLS cell survival and promote apoptosis at least partly through regulation of autophagy and the AKT-p53 axis.

scholarly journals Death Receptor-Induced Activation of Initiator Caspase 8 Is Antagonized by Serine/Threonine Kinase PAK4

2003 ◽  
Vol 23 (21) ◽  
pp. 7838-7848 ◽  
Author(s):  
Nerina Gnesutta ◽  
Audrey Minden
Keyword(s):  
Cell Death ◽  
Cell Survival ◽  
Intracellular Signaling ◽  
Death Receptor ◽  
Caspase 8 ◽  
Death Domain ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Different Types ◽  
Promote Cell Survival

ABSTRACT Normal cell growth requires a precisely controlled balance between cell death and survival. This involves activation of different types of intracellular signaling cascades within the cell. While some types of signaling proteins regulate apoptosis, or programmed cell death, other proteins within the cell can promote survival. The serine/threonine kinase PAK4 can protect cells from apoptosis in response to several different types of stimuli. As is the case for other members of the p21-activated kinase (PAK) family, one way that PAK4 may promote cell survival is by phosphorylating and thereby inhibiting the proapoptotic protein Bad. This leads in turn to the inhibition of effector caspases such as caspase 3. Here we show that in response to cytokines which activate death domain-containing receptors, such as the tumor necrosis factor and Fas receptors, PAK4 can inhibit the death signal by a different mechanism. Under these conditions, PAK4 inhibits apoptosis early in the caspase cascade, antagonizing the activation of initiator caspase 8. This inhibition, which does not require PAK4's kinase activity, may involve inhibition of caspase 8 recruitment to the death domain receptors. This role in regulating initiator caspases is an entirely novel role for the PAK proteins and suggests a new mechanism by which these proteins promote cell survival.

scholarly journals Neurogenesis and Increase in Differentiated Neural Cell Survival via Phosphorylation of Akt1 after Fluoxetine Treatment of Stem Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Anahita Rahmani ◽  
Danial Kheradmand ◽  
Peyman Keyhanvar ◽  
Alireza Shoae-Hassani ◽  
Amir Darbandi-Azar
Keyword(s):  
Survival Rate ◽  
Cell Survival ◽  
Neural Cell ◽  
Neural Cells ◽  
Chain Reaction ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Fluoxetine Treatment ◽  
Polymerase Chain

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRI). Its action is possibly through an increase in neural cell survival. The mechanism of improved survival rate of neurons by FLX may relate to the overexpression of some kinases such as Akt protein. Akt1 (a serine/threonine kinase) plays a key role in the modulation of cell proliferation and survival. Our study evaluated the effects of FLX on mesenchymal stem cell (MSC) fate and Akt1 phosphorylation levels in MSCs. Evaluation tests included reverse transcriptase polymerase chain reaction, western blot, and immunocytochemistry assays. Nestin, MAP-2, andβ-tubulin were detected after neurogenesis as neural markers. TenμM of FLX upregulated phosphorylation of Akt1 protein in induced hEnSC significantly. Also FLX did increase viability of these MSCs. Continuous FLX treatment after neurogenesis elevated the survival rate of differentiated neural cells probably by enhanced induction of Akt1 phosphorylation. This study addresses a novel role of FLX in neurogenesis and differentiated neural cell survival that may contribute to explaining the therapeutic action of fluoxetine in regenerative pharmacology.

Camel Milk Exosomes Potentiate The Anticancer Effect of Doxorubicin on Multidrug-Resistant Human Leukemia Hl60 Cells in Vitro and in Vivo

2021 ◽  
Vol 15 (11) ◽  
pp. 3313-3320
Author(s):  
Rashad Qasem Ali Othman ◽  
Abdelnaser A. Badawy ◽  
Mohammed M. Alruwaili ◽  
Mohammed A. El-magd
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Multidrug Resistant ◽  
Chemotherapeutic Agents ◽  
Camel Milk ◽  
Human Leukemia ◽  
Anticancer Effect ◽  
Hl60 Cells ◽  
Cycle Arrest ◽  
Mdr Genes

Background: Multidrug resistance (MDR) is one of the strategies developed by cancer cells to inhibit the anticancer potential of the majority of chemotherapeutic agents and almost results in treatment failure. Objective: This study aimed to evaluate the therapeutic potential of camel milk exosomes (CME) on multidrug-resistant human acute promyelocytic leukemia HL60 cells (HL60/RS) and to investigate whether this CME could potentiate the anticancer effect of Doxorubicin (DOX) and decrease its side effects. Results: CME alone or combined with DOX significantly induced HL60/RS cell viability loss, apoptosis, and cell cycle arrest at the G0/G1 phase, and downregulated MDR genes (Abcb1, Abcc1, Abcg2) as compared to cells treated with DOX alone. Additionally, CME and DOX co-treated nude mice had the lowest tumor volume, Abcb1, Abcc1, Abcg2, and Bcl2 expression, and the highest Bax and caspase3 expression in HL60/RS xenografts. This combined therapy also decreased DOX adverse effects as revealed by decreased liver damage enzymes and lipid peroxide (MDA) and increased hepatic antioxidant enzymes (SOD, CAT, GPx). Conclusion: CME increased sensitivity of HL60/RS to DOX through, at least in part, reduction of MDR genes, induction of apoptosis, and cell cycle arrest. Thus, CME may be used as safe adjuvants to DOX during cancer treatment. Keywords: Camel milk exosomes; Myeloid leukemia; HL60; Apoptosis; MDR

scholarly journals Elucidating the expression and function of Numbl during cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma (MM)

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Yuejiao Huang ◽  
Xianting Huang ◽  
Chun Cheng ◽  
Xiaohong Xu ◽  
Hong Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Clinical Problem ◽  
Integrin Β1 ◽  
Cell Viability Assay ◽  
Adhesion Model

Abstract Background Cell adhesion-mediated drug resistance (CAM-DR) is a major clinical problem that prevents successful treatment of multiple myeloma (MM). In particular, the expression levels of integrin β1 and its sub-cellular distribution (internalization and trafficking) are strongly associated with CAM-DR development. Methods Development of an adhesion model of established MM cell lines and detection of Numbl and Integrinβ1 expression by Western Blot analysis. The interaction between Numbl and Integrinβ1 was assessed by a co-immunoprecipitation (CO-IP) method. Calcein AM assay was performed to investigate the levels of cell adhesion. Finally, the extent of CAM-DR in myeloma cells was measured using cell viability assay and flow cytometry analysis. Results Our preliminary date suggest that Numbl is differentially expressed in a cell adhesion model of MM cell lines. In addition to binding to the phosphotyrosine-binding (PTB) domain, the carboxyl terminal of Numbl can also interact with integrin β1 to regulate the cell cycle by activating the pro-survival PI3K/AKT signaling pathway. This study intends to verify and elucidate the interaction between Numbl and integrin β1 and its functional outcome on CAM-DR. We have designed and developed a CAM-DR model using MM cells coated with either fibronectin or bone marrow stromal cells. We assessed whether Numbl influences cell-cycle progression and whether it, in turn, contributes to activation of PI3K/AKT signal pathway through the adjustment of its carboxyl end. Finally, we showed that the interaction of Numbl with integrin β1 promotes the formation of CAM-DR in MM cells. Conclusions Our findings elucidated the specific molecular mechanisms of CAM-DR induction and confirmed that Numbl is crucial for the development of CAM-DR in MM cells.

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