ABSTRACT
Leukemia can be a result of genetic changes associated with protein tyrosine kinase activity such as in MPL W515L and BCR/ABL genes. However, the current conventional treatment of leukemia produces severe side effects that urge the approach to use natural products. A medicinal mushroom, Lignosus rhinocerus shows potential as an anti-cancer treatment. To investigate the efficacy and mechanism of action of the L. rhinocerus cultivar (TM02®) extract on leukemogenic tyrosine kinase cell lines, a cold-water extract (CWE) was produced by using TM02® sclerotia powder at 4°C. The carbohydrate and protein contents were found to be 77.24% and 1.75% respectively. In comparison to the normal Ba/F3 cell, the CWE TM02® shows significant effects on exhibiting proliferation of Ba/F3 expressed MPL W515L and BCR/ABL, possibly due to the presence of phenolic compounds and antioxidant properties of TM02®, which contribute to act on various signaling pathways, and the reported apoptotic activity of CWE TM02®. In contrast, CWE TM02® significantly exhibited high scavenging activity of both Ba/F3 expressed MPL W515L and BCR/ABL. At concentrations of 125 µg/mL and 500 µg/mL of CWE TM02® decreased 49.5% and 67.5% of cell migration activity of Ba/F3 expressed MPL W515L and BCR/ABL respectively. Therefore, we postulate that CWE TM02® has the capability to mediate the migration route of the leukemogenic tyrosine kinase cell lines.
Subject(s)
Agaricales , Leukemia , Polyporaceae , Humans , Protein-Tyrosine Kinases , Agaricales/metabolism , Cell LineABSTRACT
The thrombopoietin receptor (MPL) has been shown to be mutated (MPL W515L) in myelofibrosis and thrombocytosis yet new approaches to treat this disorder are still required. We have previously shown that transcriptome and proteomic effects do not correlate well in oncogene-mediated leukemogenesis. We therefore investigated the effects of MPL W515L using proteomics. The consequences of MPL W515L expression on over 3300 nuclear and 3500 cytoplasmic proteins were assessed using relative quantification mass spectrometry. We demonstrate that MPL W515L expression markedly modulates the CXCL12/CXCR4/CD45 pathway associated with stem and progenitor cell chemotactic movement. We also demonstrated that MPL W515L expressing cells displayed increased chemokinesis which required the MPL W515L-mediated dysregulation of MYC expression via phosphorylation of the RNA transport protein THOC5 on tyrosine 225. In addition MPL W515L expression induced TGFß secretion which is linked to sphingosine 1-phosphate production and the increased chemokinesis. These studies identify several pathways which offer potential targets for therapeutic intervention in the treatment of MPL W515L-driven malignancy. We validate our approach by showing that CD34+ cells from MPL W515L positive patients display increased chemokinesis and that treatment with a combination of MYC and sphingosine kinase inhibitors leads to the preferential killing of MPL W515L expressing cells.
Subject(s)
Nuclear Proteins/metabolism , Receptors, Thrombopoietin/biosynthesis , Transforming Growth Factor beta/metabolism , Animals , Case-Control Studies , Cell Line, Tumor , Cell Movement/physiology , Cell Proliferation/physiology , Cells, Cultured , Humans , Mice , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/metabolism , Myeloproliferative Disorders/pathology , Nuclear Proteins/genetics , Phosphorylation , Receptors, Thrombopoietin/genetics , Receptors, Thrombopoietin/metabolism , Signal TransductionABSTRACT
Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.
