scholarly journals PCI29732, a Bruton’s Tyrosine Kinase Inhibitor, Enhanced the Efficacy of Conventional Chemotherapeutic Agents in ABCG2-Overexpressing Cancer Cells

2018 ◽  
Vol 48 (6) ◽  
pp. 2302-2317 ◽  
Author(s):  
Chunlei Ge ◽  
Fang Wang ◽  
Chaochu Cui ◽  
Xiaodong Su ◽  
Kenneth Kin Wah To ◽  
...  
Keyword(s):  
Protein Expression ◽  
Atpase Activity ◽  
Kinase Inhibitor ◽  
Chemotherapeutic Agents ◽  
Atp Binding Site ◽  
Reversal Effect ◽  
Low Concentrations ◽  
Mrna And Protein Expression ◽  
High Concentrations ◽  
Substrate Binding Sites

Background/Aims: Multidrug resistance (MDR) induced by the ABC transporter subfamily B member 1 (ABCB1) and subfamilyG member 2 (ABCG2) limits successful cancer chemotherapy and no commercially available MDR modulator is used in the clinic. In the current study, we aimed to investigate the effects of PCI29732 on the enhancement of chemotherapeutic agents. Methods: Cell cytotoxicity and reversal effect were measured with MTT assay. Additionally, flow cytometry was employed to detect the accumulation and efflux of the drugs. We investigated the interaction of PCI29732 and the substrate binding sites of ABCG2 was investigated via the photo-labeling of ABCG2 with the [125I] iodoarylazidoprazosin. The vanadate-sensitive ATPase activity of ABCG2 was measured to identify whether the drug affected the ATPase activity. RT-PCR and Western blot were utilized to analyze mRNA and protein expression respectively. Results: Here, we found that PCI29732 significantly enhanced the efficacy of substrate chemotherapeutic agents in ABCG2-overexpressing cells and also in xenografts harboring the H460/MX20 cell that overexpress ABCG2, but not in their parental sensitive cells and ABCB1-overexpressing cells. Mechanistically, the intracellular accumulations of doxorubicin and Rhodamine 123 were increased in ABCG2-overexpressing S1-MI-80 cells with the presence of PCI29732. PCI29732 stimulated the ATPase activity of ABCG2 at low concentrations. However at the high concentrations, PCI29732 inhibited the ATPase activity, and competed with [125I]-iodoarylazidoprazosin for photo-affinity labeling of ABCG2. PCI29732 did neither alter the mRNA or protein expression levels of ABCG2 nor the phosphorylation levels of AKT and ERK1/2. Conclusion: Our study demonstrates that PCI29732 inhibits the function of ABCG2 by competitively binding to the ATP-binding site of ABCG2 and enhances the anti-tumor efficacy of substrate chemotherapeutic agents, This findings encourages the development of combinational chemotherapy for the treatment of ABCG2- overexpressing cancer patients.

NH4Cl affects the expression of Wnt/β-catenin pathway in hepatocytes

2018 ◽  
Vol 96 (3) ◽  
pp. 281-286
Author(s):  
Lu Bai ◽  
Jingjing Li ◽  
Xiaorui Liu ◽  
Shasha Li ◽  
Fulei Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Control Group ◽  
Cyclin D ◽  
Chang Liver Cell ◽  
Expression Levels ◽  
Low Concentrations ◽  
Short Period ◽  
Mrna And Protein Expression ◽  
High Concentrations ◽  
Chang Liver

We intended to explore whether NH4Cl influences the viability and regulates the expression of Wnt/β-catenin pathway in hepatocytes. The Chang liver cell line was used and cultured with different concentrations of NH4Cl (2.5, 5, 10, 20, 40, and 50 mmol/L) for 12, 24, and 48 h. The viability of hepatocytes was detected by MTT assay. The mRNA and protein expression level was analyzed with qRT–PCR and Western blotting, respectively. NH4Cl concentration significantly affects the viability of hepatocytes. With the increase of NH4Cl concentration, the viability of hepatocytes was decreased, accordingly. The mRNA and protein expression of Wnt1, β-catenin, and cyclin D was significantly increased after treatment with low concentrations of NH4Cl as compared with the control group, whereas their expression levels were decreased after treatment with high concentrations of NH4Cl. The mRNA and protein expression of Wnt1, β-catenin, and cyclin D was also significantly increased after treatment with NH4Cl for a short period as compared with the control group, whereas their expression levels were decreased after treatment with NH4Cl for a long period. In addition, we found NH4Cl treatment significantly reversed the results after RNA silencing of Wnt1 in hepatocytes. NH4Cl influences the viability of hepatocytes and affects the expression of Wnt/β-catenin pathway in hepatocytes.

scholarly journals Combined action of binase and bleomycin toward human lung adenocarcinoma cells

2016 ◽  
Vol 62 (3) ◽  
pp. 279-282 ◽  
Author(s):  
P.V. Zelenikhin ◽  
A.V. Makeeva ◽  
T.N. Nguen ◽  
Y.A. Siraj ◽  
O.N. Ilinskaya
Keyword(s):  
Lung Adenocarcinoma ◽  
Human Lung ◽  
A549 Cells ◽  
Chemotherapeutic Agents ◽  
Human Lung Adenocarcinoma ◽  
Low Concentrations ◽  
Wide Range ◽  
Bacillus Pumilus Rnase ◽  
High Concentrations ◽  
Lung Adenocarcinoma A549

Some microbial ribonucleases (RNases) demonstrate selective cytotoxic effect against a wide range of tumor cells. In this context combined use of cytotoxic RNases in complex therapy with other chemotherapeutic agents appears to be especially promising. In this study we have investigated the apoptosis-induced effect of Bacillus pumilus RNase (binase) in combination with known anti-tumor antibiotic bleomycin on human lung adenocarcinoma A549 cells. The combined effect of high concentrations of these agents did not have any mutual increase in their apoptosis-induced action, while a combination of non-apoptotic concentrations resulted in the increase of the proportion of apoptotic cells up to 22% as compared with individual effect of bleomycin (6%) and binase (12%) used separately. These results indicate that binase and bleomycin are effective in combination of their low concentrations and ineffective in combination of their high concentrations.

Temperature-induced functional deocclusion of thiols inhibitory for sheep erythrocyte K-Cl cotransport

1995 ◽  
Vol 269 (5) ◽  
pp. C1167-C1175 ◽  
Author(s):  
P. K. Lauf ◽  
N. C. Adragna
Keyword(s):  
Kinase Inhibitor ◽  
Sheep Erythrocyte ◽  
Inhibitory Effect ◽  
Temperature Treatment ◽  
Cell Swelling ◽  
Thiol Groups ◽  
Subsequent Removal ◽  
Low Concentrations ◽  
High Concentrations ◽  
Low K

In low-K sheep erythrocytes, K-Cl cotransport is activated by treatment with low concentrations of thiol reagents and by other interventions such as lowering of cellular free cytosolic Mg, hyposmotic cell swelling, the kinase inhibitor staurosporine, and hydroxylamine. High concentrations of N-ethylmaleimide or methylmethane thiolsulfonate reverse the activation through thiol groups and, as shown here, also the stimulation by non-thiol manipulations. The overriding inhibitory sites functionally associated with and different from those of the activating thiols (SHa) were further distinguished by temperature. Treatment with N-ethylmaleimide and its subsequent removal by dithiothreitol, both at 0 degrees C, prevented the inhibitory effect at 37 degrees C and thus the chemical modification of inhibitory thiols (SHi). Whereas stimulation through SHa closely followed the loss of glutathione, inhibition through SHi occurred only in glutathione-depleted cells. The reversal of K-Cl cotransport stimulation by all hitherto known interventions, which is strongest in metabolically depleted cells, suggests that the low temperature-protected SHi constitute crucial sites that, close to the transporter itself or at the cytoskeletal level, become functionally deoccluded upon temperature elevation.

scholarly journals A Novel Naphthyridine Derivative, 3u, Induces Necroptosis at Low Concentrations and Apoptosis at High Concentrations in Human Melanoma A375 Cells

2018 ◽  
Vol 19 (10) ◽  
pp. 2975 ◽  
Author(s):  
Qinghong Kong ◽  
Jianxin Lv ◽  
Shengjiao Yan ◽  
Kwen-Jen Chang ◽  
Guanlin Wang
Keyword(s):  
Kinase Inhibitor ◽  
Chemical Substance ◽  
Serine Threonine Kinase ◽  
Human Malignant Melanoma ◽  
Threonine Kinase ◽  
Thiazolyl Blue Tetrazolium Bromide ◽  
Low Concentrations ◽  
Melanoma Treatment ◽  
High Concentrations ◽  
A375 Cells

Naphthyridine derivatives are a widely-used class of heterocycles due to their pharmacological activities. A novel compound (10-Methoxy-1,2,3,4-tetrahydrobenzo(g)(1,3) diazepino(1,2-a)-(1,8)naphthyridin-6-yl)(phenyl) methanone (named 3u), showed good anticancer activity in the human malignant melanoma cell line A375 via Thiazolyl Blue Tetrazolium Bromide (MTT) assay. After Western blotting confirmed, we found that 3u induces necroptosis at low concentrations and apoptosis at high concentrations via the upregulation of death receptors and scaffold protein in A375 cells. Furthermore, by combining 3u with the caspase inhibitor zVAD-fmk or Receptor Interacting Serine/Threonine Kinase 1 (RIP1) kinase inhibitor Necrostatin-1 (Nec-1), we found that the activity of caspase-8 was the crucial factor that determined whether either apoptosis or necroptosis occurred. The results indicate that 3u should be considered as a potential chemical substance for melanoma treatment.

Study on the Relevance of Inducing Apoptosis and Reversal Effect of Nilotinib In Combination with Tet on Multidrug Reststahoe of K562/A02 Cell Line

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4951-4951
Author(s):  
Bao-An Chen ◽  
Ting-yun Cui ◽  
Jia-hua Ding ◽  
Chong Gao ◽  
Xue-yun Shan ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Line ◽  
Combination Treatment ◽  
Expression Level ◽  
Survivin Mrna ◽  
Gray Scale ◽  
Protein Expression Level ◽  
Apoptosis Rate ◽  
Reversal Effect ◽  
Mrna And Protein Expression

Abstract Abstract 4951 Objective To investigate the relevance of Inducing apoptosis and reversal effect of nilotinib in combination with Tet on multidrug reststahoe of K562/A02 cell line and its mechanism. Methods Methyl-thiazol tetrazolium(MTT) assay is employed to examine the pharmacological effect of Nilotinib or Tet alone on K562/A02 cell line, to calculate the inhibitor concerntration 50(IC50) of daunorubicin(DNR) in K562/A02 cell line and the resistance multiple of DNR to K562/A02 cell line. Flow cytometry(FCM) was employed to comple the effect on the inducing apoptosis of K562/A02. The expression of bax/survivin mRNA was determined by RT-PCR, and the expression of bax/survivin protein was assessed by Western blot. Results After 48 h 5 nmol/L nilotinib or 1.0μmoL/L Tet treatment, IC50 of DNR to K562/A02 was 5.71mg/L or 6.52 mg/L respectively;While on combinative treatment, its IC50 decreased to 3.12 mg/L. Nilotinib or Tet alone was able to increase the DNR induced apoptosis rate of K562/A02 cell (P<0.05), while on combination treatment the apoptosis rate increased remarkably. After 48 h 5 nmoL/L nilotinib or 1.0μmoL/L Tet treatment alone, gray—scale vaule of bax mRNA Was 2.90±0.31 or 3.6±0.46, respectively Gwhile on combinative treatment the value increased to 5.9±0.98. The bax protein expression level in K562/A02 cells was 2.1±0.07 or 2.90±0.09 when treated with 5 nmoL/L nilotinib or 1.0μmoL/L Tet alone for 48 h, but on combination treatment, the level increased to 4.8±0.31. After 48 h 5 nmoL/L nilotinib or 1.0μmoL/L Tet treatment alone, gray—scale vaule of survivin mRNA was 0.70±0.01 or 0.55±0.02, respectively;while on combinative treatment the value decreased to 0.35±0.08. The survivin protein expression level in K562/A02 cells was 0.74±0.03or 0.61±0.04 when treated with 5 nmoL/L nilotinib or 1.0μmoL/L Tet alone for 48 h, but on combination treatment, the level decreased to 0.42±0.06. Conclusion Nilotinib or Tet alone can pattially reverse drug resistance of K562/A02 cells. There is resistance phenomena of DNR to K562/A02 cells, and Nilotinib or Tet alone alone is able to reserve the resistance of DNR and enhance the inducing apoptasis effect of DNR. The mechanism may be associated with the increase of bax mRNA and protein expression and decrease of survivin mRNA and protein expression and increase of the apoptosis rate. And there is a synergistic action with these two agants in combination. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MicroRNA 33 Potentially Participates in the Development of Goose Fatty Liver via Its Target Gene CROT

2021 ◽  
Author(s):  
Xiao Lin ◽  
Ya Xing ◽  
Yihui Zhang ◽  
Biao Dong ◽  
Minmeng zhao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fatty Liver ◽  
Target Gene ◽  
Liver Cells ◽  
Acid Oxidation ◽  
Lipid Deposition ◽  
Primary Hepatocytes ◽  
Regulatory Relationship ◽  
Mrna And Protein Expression ◽  
High Concentrations

Abstract Background Previous studies indicate that microRNA33 (miR-33) and its target gene, CROT, are implicated in hepatic lipid metabolism, but it is unclear whether miR-33 participates in the development of goose fatty liver via CROT. Methods The expression of miR-33 in goose fatty liver, muscle and fat tissues, as well as the mRNA and protein expression of CROT in goose fatty liver was determined by q-PCR or Western-blot. The targeting regulatory relationship between miR-33 and CROT in goose liver cells was validated by miR-33 overexpression and interference assays. The effects of miR-33 mimic and CROT overexpression on lipid deposition and the expression of downstream genes were determined in goose primary hepatocytes. The treatment of high concentrations of glucose and insulin was performed to determine their regulation on the expression of miR-33 and CROT in goose primary hepatocytes. Results Here, data showed that miR-33 expression was significantly increased in the liver, muscle and fat tissues of overfed geese. Consistently, miR-33 mimic promoted lipid deposition in goose primary hepatocytes. Moreover, the regulatory targeting relationship between miR-33 and CROT was validated in goose primary hepatocytes. Consistently, the mRNA and protein expression of CROT were significantly reduced in goose fatty liver. Interestingly, CROT overexpression could induce the expression of fatty acid oxidation associated genes including CRAT, PEX5, EHHADH, CAT and ACOT8 in goose primary hepatocytes, but only the expression of PEX5 was significantly inhibited in goose fatty liver. However, it seemed conflicting that CROT overexpression increased lipid deposition and reduced lipid peroxidation in goose primary hepatocytes. Additionally, high glucose inhibited miR-33 expression and induced CROT expression in goose primary hepatocytes. Conclusions These findings suggest that miR-33 potentially participates in the development of goose fatty liver via CROT, and that miR-33/CROT may partially mediate the effect of glucose in goose liver cells.

Comparative Studies on the ATP-Binding Sites in Ca2+-ATPase and (Na+ + K+)-ATPase by the Use of ATP-Analogues

1982 ◽  
Vol 37 (7-8) ◽  
pp. 692-705 ◽  
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Sulfhydryl Groups ◽  
Atp Binding ◽  
Stable Form ◽  
Atp Binding Site ◽  
Atp Analogues ◽  
Transport Atpases ◽  
High Concentrations ◽  
Hydrolysis Of

Abstract The effects of ATP-analogues on Ca2+-ATPase and (Na+ + K+)-ATPase have been studied. The participation of sulfhydryl groups in the recognition of ATP by both transport ATPases is indicat ed by the fact, that the disulfide of thioinosine triphosphate inactivates both enzymes. The reactivity of rapidly and slowly reacting sulfhydryl groups in the ATP binding sites of both enzymes is altered by the presence of transport substrates. At least in (Na+ + K+)-ATPase Na+ and Mg2+ appear to alter the structure of the ATP binding site, which conclusion is fortified by the fact, that the photoinactivation of the enzyme by 3′-O-[3-(2-nitro-4-azidophenyl)-propionyl]-ATP need Mg2+. Chromium(III)ATP, a MgATP analogue, inactivated both transport ATPases by the formation of a stable chromo-phosphointermediate. In the case of Ca2+-ATPase this was concomited by the occlusion of Ca2+ in a stable form. No occlusion of Na+ was observable so far in the (Na++ K+)-ATPase. Contrary to the expectation of the Albers-Post-scheme the hydrolysis of the phosphointermediate formed from chromium(III)ATP was protected by K+, but activated by high concentrations of Na+. Consequently, despite of the inhibition of (Na+ + K+)-ATPase activity chromium(III)-ATP supported the Na+ - Na+ -exchange reaction in everted red bood cells.

The Modification of Actomyosin ATPase Activity by Tropomyosin-Troponin and its Dependence on Ionic Strength, ATP-Concentration, and Actin-Myosin Ratio

1974 ◽  
Vol 29 (9-10) ◽  
pp. 496-505 ◽  
Author(s):  
Peter Daneker
Keyword(s):  
Ionic Strength ◽  
Atpase Activity ◽  
Actin Filaments ◽  
Regulatory Proteins ◽  
High Affinity ◽  
Low Concentrations ◽  
Dependence On Ionic Strength ◽  
High Concentrations ◽  
Low Ionic Strength ◽  
Better Than

Abstract At millimolar concentrations of ATP the ATPase activity of regulated actomyosin (which consisted of myosin and of actin containing the regulatory proteins tropomyosin and troponin) was lower than that of unregulated actomyosin (containing actin devoid of the regulatory proteins) when the ionic strength was high (> 0 .0 3 ᴍ KCl). At low ionic strength (0.03 ᴍ KCl) the ATPase activity of regulated actomyosin was similar to or even higher than that of unregulated acto­ myosin. Besides increasing ionic strength an increasing actin-myosin ratio tended to depress the ATPase activity of regulated actomyosin below that of unregulated one. At lower ATP concen­ trations (0.1 mᴍ or lower) the ATPase activity of regulated actomyosin was higher than that of unregulated actomyosin at any ionic strength and at any actin-myosin ratio. EGTA inhibited the ATPase of regulated actomyosin under any conditions at high ATP concentrations. At lower ATP concentrations EGTA inhibited either at higher ionic strength or at a higher actin-myosin ratio. The inhibition of the ATPase activity of acto-HMM by increasing ionic strength was not in­ fluenced by the regulatory proteins. - For the interpretation of these results it has been assumed that in actomyosin regulated actin can adopt three states: A low-affinity state which activates the ATPase of myosin only slightly (occurring at high ATP concentrations and in the absence of Ca2+), a high affinity state which activates the ATPase of myosin better than does unregulated actin (occurring at low concentrations of ATP and in the presence of Ca2+), and an intermediate state. This latter state (occurring at high concentrations of ATP and in the presence of Ca2+ or at low concentrations of ATP and in the absence of Ca2+) activates the ATPase of myosin less than does unregulated actin when the actin-myosin ratio is high (wide spacing of myosin on the actin filaments) but activates more (or at least not less) when the actin-myosin ratio is low (dense spacing of myosin on the actin filaments)

Bone sialoprotein mRNA and protein expression in human multiple myeloma cell lines and patients

2000 ◽  
Vol 111 (4) ◽  
pp. 1118-1121 ◽  
Author(s):  
A. Bellahcene ◽  
I. Van Riet ◽  
C. de Greef ◽  
N. Antoine ◽  
M. F. Young ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Bone Sialoprotein ◽  
Multiple Myeloma Cell ◽  
Human Multiple Myeloma ◽  
Mrna And Protein Expression

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

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