scholarly journals Ruxolitinib Combined with Gemcitabine against Cholangiocarcinoma Growth via the JAK2/STAT1/3/ALDH1A3 Pathway

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 885
Author(s):  
Shin-Yi Chung ◽  
Yi-Ping Hung ◽  
Yi-Ru Pan ◽  
Yu-Chan Chang ◽  
Chiao-En Wu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Translocation ◽  
Vital Role ◽  
First Line ◽  
Current Standard ◽  
Integrated Network ◽  
Jak2 Inhibitor ◽  
Therapeutic Drugs ◽  
Cholangiocarcinoma Cell ◽  
Malignant Behavior

Cholangiocarcinoma is the most common primary malignant tumor of the bile duct. The current standard first-line treatment for advanced or metastatic cholangiocarcinoma is gemcitabine and cisplatin. However, few effective treatment choices exist for refractory cholangiocarcinoma, and additional therapeutic drugs are urgently required. Our previous work demonstrated that the ALDH isoform 1A3 plays a vital role in the malignant behavior of cholangiocarcinoma and may serve as a new therapeutic target. In this study, we found a positive correlation between ALDH1A3 protein expression levels and the cell migration abilities of three cholangiocarcinoma cell lines, which was verified using ALDH1A3-overexpressing and ALDH1A3-knockdown clones. We also used ALDH1A3-high and ALDH1A3-low populations of cholangiocarcinoma cell lines from the library of integrated network-based cellular signatures (LINCS) program and assessed the effects of ruxolitinib, a commercially available JAK2 inhibitor. Ruxolitinib had a higher cytotoxic effect when combined with gemcitabine. Furthermore, the nuclear translocation STAT1 and STAT3 heterodimers were markedly diminished by ruxolitinib treatment, possibly resulting in decreased ALDH1A3 activation. Notably, ruxolitinib alone or combined with gemcitabine led to significantly reduced tumor size and weight. Collectively, our studies suggest that ruxolitinib might suppress the ALDH1A3 activation through the JAK2/STAT1/3 pathway in cholangiocarcinoma, and trials should be undertaken to evaluate its efficacy in clinical therapy.

scholarly journals Ruxolitinib Combined with Gemcitabine against Cholangiocarcinoma Growth via the jak2/stat1/3/aldh1a3 Pathway

2021 ◽  
Author(s):  
Shin-Yi Chung ◽  
Yi-Ping Hung ◽  
Yi-Ru Pan ◽  
Yu-Chan Chang ◽  
Chiao-En Wu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Translocation ◽  
Vital Role ◽  
First Line ◽  
Current Standard ◽  
Integrated Network ◽  
Jak2 Inhibitor ◽  
Therapeutic Drugs ◽  
Cholangiocarcinoma Cell ◽  
Malignant Behavior

Cholangiocarcinoma is the most common primary malignant tumor of the bile duct. The current standard first-line treatment for advanced or metastatic cholangiocarcinoma is gemcitabine and cisplatin. However, few effective treatment choices exist for refractory cholangiocarcinoma, and additional therapeutic drugs are urgently required. Our previous work demonstrated that the ALDH isoform 1A3 plays a vital role in the malignant behavior of cholangiocarcinoma and may serve as a new therapeutic target. In this study, we found a positive correlation between ALDH1A3 protein expression levels and cell migration abilities of three cholangiocarcinoma cell lines, which was verified using ALDH1A3-overexpressing and ALDH1A3-knockdown clones. We also used ALDH1A3-high and ALDH1A3-low populations of cholangiocarcinoma cell lines from the Library of Integrated Network-based Cellular Signatures (LINCS) program and assessed the effects of ruxolitinib, a commercially available JAK2 inhibitor. Ruxolitinib had a higher cytotoxic effect when combined with gemcitabine. Furthermore, the nuclear translocation STAT1 and STAT3 heterodimers were markedly diminished by ruxolitinib treatment, possibly resulting in decreased ALDH1A3 activation. Notably, ruxolitinib alone or combined with gemcitabine led to significantly reduced tumor size and weight. Collectively, our studies suggest that ruxolitinib might suppress the ALDH1A3 activation through the JAK2/STAT1/3 pathway in cholangiocarcinoma, and trials should be undertaken to evaluate its efficacy in clinical therapy.

Preferential Nuclear Accumulation of JAK2V617F In CD34+ but Not Granulocytic, Megakaryocytic or Erythroid Cells of Patients with Philadelphia-Negative Myeloproliferative Neoplasia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4089-4089
Author(s):  
Ciro R Rinaldi ◽  
Paola Rinaldi ◽  
Adele Alagia ◽  
Marica Gemei ◽  
Vitalyi Senyuk ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Localization ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Erythroid Cells ◽  
Jak2 Inhibitor ◽  
Preferential Accumulation ◽  
Cd34 Cells ◽  
V617f Mutation ◽  
Nuclear Signal

Abstract Abstract 4089 Constitutive activation of JAK2 by chromosomal translocations or a point mutation is a frequent event in hematological malignancies particularly in Philadelphia-negative MPN. Recently, Dawson et al. identified a novel nuclear role of JAK2 in the phosphorylation of Tyr 41 of histone H3 leading to chromatin displacement of HP1a in hematopoietic cell lines and in the CD34+ cells collected from the peripheral blood of one PMF patient with JAK2V617F mutation. To determine whether the V617F mutation observed in MPN patients affects the sub-cellular localization of JAK2, we first analyzed by confocal immunofluorescence (CIF) microscopy and Western Blot (WB), K562 cells stably transfected with pMSCV-puroJAK2V617F or pMSCV-puroJAK2. The results confirm the nuclear and cytoplasmic localization of JAK2 in K562 as reported by Dawson et al. However, we consistently observed a much stronger nuclear signal in the cells expressing JAK2V617F than in those carrying wt JAK2 suggesting that the mutation leads to a preferential accumulation of JAK2V617F in the nucleus. To determine whether there is a preferential nuclear translocation of JAK2V617F in vivo, we analyzed by CIF microscopy and WB the total BM cells of 10 JAK2V617F-positive MPN patients (ET n=4, PV n=3, PMF n=3, allele burden median: 56%, 70%, 72% respectively) and of 5 MPN patients with wt JAK2 (PMF n= 2, ET n=3). We found a strong nuclear signal in mononucleated cells of 10 of 10 JAK2V617F-positive patients but not in those with wt JAK2. The JAK2 signal was observed almost exclusively in the nucleus suggesting a predominantly nuclear homing of JAK2V617F. To identify the phenotype of these cells, we used fluorescence activated cell sorting (FACS) to isolate CD34+, CD15+, CD41+ and CD71+ fractions from the BM of three JAK2V617F-positive MPN patients (1 ET, 1 PV, 1 early PMF). We found nuclear JAK2 in CD34+ positive cells collected from BM only in V617F mutated patients. No obvious nuclear signal was detected in differentiated granulocytic, megakaryocytic and erythroid cells obtained from the patients (n=15). To determine whether the block of JAK2 activity could interfere with nuclear localization of JAK2, we incubated JAK2V617F and JAK2 expressing K562 with the selective JAK2 inhibitor AG490. At the IC50 dose (25 uM) and after 3 h of incubation, CIF images showed the JAK2 redistribution in the vast majority of V617F expressing K562 and the replacement in the cytoplasm but not in wt cells. By QRT-PCR we demonstrated that the V617F mutation strongly up-regulates LMO2 expression in K562 and in CD34+ cells. In our assay, the addiction of AG490 progressively and completely restore LMO2 levels in V617F expressing K562. Our data corroborate recently published results of a nuclear localization of JAK2 in hematopoietic cells and they also extend these findings by showing that in all subtypes of MPN patients JAK2V617F accumulates in the nucleus of progenitor CD34+ cells while remains mostly in the cytoplasm of their differentiated progeny. The chromatin alterations due to the preferential accumulation of JAK2V617F in the nucleus correlates with a significant increase in LMO2 expression in cell lines and in sorted CD34+ cells. The selective JAK2 inhibitor AG490 is able to revert nuclear JAK2 and normalize LMO2 levels in vitro, suggesting how the block in JAK2 nuclear translocation could be a new treatment strategy for JAK2 mutated patients. Disclosures: No relevant conflicts of interest to declare.

A Single Pitx1 Binding Site Is Essential for Activity of the LHβ Promoter in Transgenic Mice

2001 ◽  
Vol 15 (5) ◽  
pp. 734-746 ◽  
Author(s):  
Christine C. Quirk ◽  
Kristen L. Lozada ◽  
Ruth A. Keri ◽  
John H. Nilson
Keyword(s):  
Transgenic Mice ◽  
Binding Site ◽  
Cell Lines ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Vital Role ◽  
Proximal Region ◽  
Expression Vectors ◽  
Homeodomain Protein

Abstract Reproduction depends on regulated expression of the LHβ gene. Tandem copies of regulatory elements that bind early growth response protein 1 (Egr-1) and steroidogenic factor 1 (SF-1) are located in the proximal region of the LHβ promoter and make essential contributions to its activity as well as mediate responsiveness to GnRH. Located between these tandem elements is a single site capable of binding the homeodomain protein Pitx1. From studies that employ overexpression paradigms performed in heterologous cell lines, it appears that Egr-1, SF-1, and Pitx1 interact cooperatively through a mechanism that does not require the binding of Pitx1 to its site. Since the physiological ramifications of these overexpression studies remain unclear, we reassessed the requirement for a Pitx1 element in the promoter of the LHβ gene using homologous cell lines and transgenic mice, both of which obviate the need for overexpression of transcription factors. Our analysis indicated a striking requirement for the Pitx1 regulatory element. When assayed by transient transfection using a gonadotrope-derived cell line (LβT2), an LHβ promoter construct harboring a mutant Pitx1 element displayed attenuated transcriptional activity but retained responsiveness to GnRH. In contrast, analysis of wild-type and mutant expression vectors in transgenic mice indicated that LHβ promoter activity is completely dependent on the presence of a functional Pitx1 binding site. Indeed, the dependence on an intact Pitx1 binding site in transgenic mice is so strict that responsiveness to GnRH is also lost, suggesting that the mutant promoter is inactive. Collectively, our data reinforce the concept that activity of the LHβ promoter is determined, in part, through highly cooperative interactions between SF-1, Egr-1, and Pitx1. While Egr-1 can be regarded as a key downstream effector of GnRH, and Pitx1 as a critical partner that activates SF-1, our data firmly establish that the Pitx1 element plays a vital role in permitting these functions to occur in vivo.

scholarly journals Functional Stratification of Cancer Drugs through Integrated Network Similarity

2022 ◽  
Author(s):  
Nurcan Tuncbag ◽  
Seyma Unsal Beyge
Keyword(s):  
Cell Lines ◽  
Drug Targets ◽  
Hdac Inhibitors ◽  
Treatment Strategies ◽  
Network Models ◽  
Drug Combinations ◽  
Multiple Cancer ◽  
Integrated Network ◽  
Drug Molecules ◽  
The Difference

Abstract Heterogeneity across tumors is the main obstacle in developing treatment strategies. Drug molecules not only perturb their immediate protein targets but also modulate multiple signaling pathways. In this study, we explored the networks modulated by several drug molecules across multiple cancer cell lines by integrating the drug targets with transcriptomic and phosphoproteomic data. As a result, we obtained 236 reconstructed networks covering five cell lines and 70 drugs. A rigorous topological and pathway analysis showed that chemically and functionally different drugs may modulate overlapping networks. Additionally, we revealed a set of tumor-specific hidden pathways with the help of drug network models that are not detectable from the initial data. The difference in the target selectivity of the drugs leads to disjoint networks despite sharing the exact mechanism of action, e.g., HDAC inhibitors. We also used the reconstructed network models to study potential drug combinations based on the topological separation, found literature evidence for a set of drug pairs. Overall, the network-level exploration of the drug perturbations may potentially help optimize treatment strategies and suggest new drug combinations.

scholarly journals CYP 3A5*3 Gene Polymorphism Among Sudanese Patients With Chronic Myeloid Leukemia 

2020 ◽  
Author(s):  
Elharam Ibrahim Abdallah ◽  
Nazic Ibrahim M.Alamin ◽  
Wala Eldin Osman Elradi ◽  
Salah Eldin G. Elzaki ◽  
NassrEldin Mohamed Ahmed Shrif
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Vital Role ◽  
Chi Square ◽  
Mutant Genotype ◽  
Therapeutic Drugs ◽  
Pcr Rflp ◽  
Chi Square Test ◽  
Healthy Control

Abstract Introduction: Interaction of environmental and genetic elements plays a vital role in the pathogenesis of CML and other types of cancer. CYP 3A5 is enzyme responsible of metabolizing of approximately 50 % of therapeutic drugs and xenobiotics. So polymorphism variation may genetically predispose individuals to CML.Objective: The purpose of this study was to determine CYP3A5*3 Polymorphism among Sudanese patients with Chronic Myeloid Leukemia (CML).Materials and Methods: This case control study was conducted among 50 patients with chronic myeloid leukemia among both genders at different ages and 42 apparently healthy control subjects. The CYP3A5*3 genotype was determined using (PCR-RFLP) method.Results: Paerson`s chi-square test was used to assess the possible link between CYP3A5*3 genotype and CML. The percentage of CYP3A5*3*3 genotype in CML patients was significantly higher than in control (OR = 11.71, 95% CI: 4.30 –31.83; p =0.000). The frequency of mutant genotype was higher among CML patients with advance phase compared with chronic phase, 100% versus 80% respectively.Conclusion: CYP3A5*3/*3 genotype associated with CML development and progression.

scholarly journals Internalization of prolactin receptor and prolactin in transfected cells does not involve nuclear translocation

1997 ◽  
Vol 110 (9) ◽  
pp. 1123-1132 ◽  
Author(s):  
M. Perrot-Applanat ◽  
O. Gualillo ◽  
H. Buteau ◽  
M. Edery ◽  
P.A. Kelly
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Map Kinases ◽  
Nuclear Translocation ◽  
Short Form ◽  
Confocal Laser Microscopy ◽  
Confocal Laser ◽  
Laser Microscopy ◽  
Transfected Cells

Prolactin (PRL) interacts with a specific, well characterized plasma membrane receptor (PRLR) that is coupled to signal transduction pathways involving Jak2, Fyn, and MAP kinases, and signal transducers and activators of transcription (STAT). Although a few previous studies have indicated nuclear translocation of PRL in IL-2 stimulated T lymphocytes, PRL-dependent Nb2 lymphoma cell lines and 235–1 lactotrophs, the mechanisms of nuclear targeting remain unknown and conflicting results have been reported concerning the putative nuclear translocation of the PRLR. We therefore decided to investigate nuclear translocation of PRLR and PRL in various cell lines transfected with an expression plasmid encoding PRLR, using confocal laser microscopy. We have constructed various cDNAs of the long and short forms of the rat PRLR containing an oligonucleotide encoding a Flag epitope inserted either just before the N-terminal amino acid or in the C-terminal end of the mature receptor (named N-terminal or C-terminal Flag-tagged PRLR). The corresponding receptors function as the PRLR in transfected cells: they are expressed at the plasma membrane and in compartments of the secretory pathway, they bind PRL with normal affinity (Kd= 4x10(−10) M) and have the same capacity to stimulate the transcriptional activity of a milk protein (beta-casein) gene as wild-type PRLR. In addition, the tagged receptors are much more efficiently immunodetected using anti-Flag antibodies, as compared to anti-PRL antibodies (U5 or U6). Immunofluorescence combined with detailed confocal laser microscopy showed that addition of PRL (0 to 12 hours) to COS-7, CHO and NIH-3T3 transfected fibroblasts induces rapid internalization of the receptor (long form), without any translocation to the nucleus. Using PRL-R tagged both in the N-terminal or C-terminal regions of the mature receptor excludes the possibility of a cleaved fragment which could have been subsequently imported into the nucleus. An absence of nuclear translocation of PRLR was also observed in a 293 cell line stably expressing the receptor, and in physiological targets for PRL, i.e. in Nb2 lymphoma cells expressing the Nb2 form of the receptor or in BGME mammary gland epithelial cells upon overexpression of a Flag-tagged PRLR. Similarly, the short form of the PRLR was not detected in nuclei of transfected COS cells upon PRL treatment. Clearly, our results provide evidence that internalization of the plasma membrane PRLR does not lead to nuclear translocation of the receptor, or part of it, in most fibroblasts and epithelial cells at physiological concentrations of PRL. Also, in co-localization experiments, PRL was internalized without nuclear translocation. Activation of STATs transcription factors and MAP kinases, as well as translocation of these proteins to the nucleus following their phosphorylation, probably remains the intracellular mechanism coupling stimulation to nuclear events.

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