Apoptosis In Human Erythroleukemia Cells Research Articles

Discovery and evaluation of ZT55, a novel highly-selective tyrosine kinase inhibitor of JAK2V617F against myeloproliferative neoplasms

BackgroundThe JAK2-STAT signaling pathway plays a critical role in myeloproliferative neoplasms (MPN). An activating mutation in JAK2 (V617F) is present in ~ 95% of polycythemia vera, essential thrombocythemia, and primary myelofibrosis cases. This study aims to explore the selective JAK2V617F inhibitor, evaluate the efficacy and possible mechanism of ZT55 on MPN.MethodsHTRF assays were conducted to evaluate the selective inhibition of ZT55 for JAKs. Cell apoptosis, proliferation, and cycle arrest assays were performed to examine the effect of ZT55 on HEL cell line with JAK2V617F mutation in vitro. Western analysis was used to monitor the expression and activity of proteins on JAK2/STAT pathway. A mice xenograft model was established to evaluate the antitumor efficacy of ZT55 in vivo. Peripheral blood samples from patients with the JAK2V617F mutation were collected to estimate the effect of ZT55 on erythroid colony formation by colony-forming assay.ResultsWe found that ZT55 showed a selective inhibition of a 0.031 μM IC50 value against JAK2. It exhibited potent effects on the cellular JAK-STAT pathway, inhibiting tyrosine phosphorylation in JAK2V617F and downstream STAT3/5 transcription factors. ZT55 inhibited the proliferation of the JAK2V617F-expressing HEL cell line, leading to cell cycle arrest at the G2/M phase and induction of caspase-dependent apoptosis. Notably, ZT55 also significantly suppressed the growth of HEL xenograft tumors in vivo. Further evaluation indicated that ZT55 blocked erythroid colony formation of peripheral blood hematopoietic progenitors from patients carrying the JAK2V617F mutation.ConclusionThese results suggest that ZT55 is a highly-selective JAK2 inhibitor that can induce apoptosis of human erythroleukemia cells by inhibiting the JAK2-STAT signaling.

Garmultins A-G, Biogenetically Related Polycyclic Acylphloroglucinols from Garcinia multiflora.

Garmultins A and B (1 and 2), two polycyclic polyprenylated acylphloroglucinols characterized by the coupling of two novel cages, 2,11-dioxatricyclo[4.4.1.03,9]undecane and tricyclo[4.3.1.03,7]decane, along with five biogenetically related analogues (3-7), were isolated from Garcinia multiflora. Their structures and absolute configurations were determined by extensive NMR analysis, X-ray crystallography, and electronic circular dichroism calculations. Three compounds were capable of inhibiting oncogene expression and inducing apoptosis in human erythroleukemia cells.

Regulation of apoptosis by cyclic nucleotides in human erythroleukemia (HEL) cells and human myelogenous leukemia (K-562) cells

The cyclic pyrimidine nucleotides cCMP and cUMP have been recently identified in numerous mammalian cell lines, in primary cells and in intact organs, but very little is still known about their biological function. A recent study of our group revealed that the membrane-permeable cCMP analog cCMP-acetoxymethylester (cCMP-AM) induces apoptosis in mouse lymphoma cells independent of protein kinase A via an intrinsic and mitochondria-dependent pathway. In our present study, we examined the effects of various cNMP-AMs in human tumor cell lines. In HEL cells, a human erythroleukemia cell line, cCMP-AM effectively reduced the number of viable cells, effectively induced apoptosis by altering the mitochondrial membrane potential and thereby caused changes in the cell cycle. cCMP itself was biologically inactive, indicating that membrane penetration is required to trigger intracellular effects. cCMP-AM did not induce apoptosis in K-562 cells, a human chronic myelogenous leukemia cell line, due to rapid export via multidrug resistance-associated proteins. The biological effects of cCMP-AM differed from those of other cNMP-AMs. In conclusion, cCMP effectively induces apoptosis in HEL cells, cCMP export prevents apoptosis of K-562 cells and cNMPs differentially regulate various aspects of apoptosis, cell growth and mitochondrial function. In a broader perspective, our data support the concept of distinct second messenger roles of cAMP, cGMP, cCMP and cUMP.

Efficacy of Vorinostat In a Murine Model of Polycythemia Vera

AbstractAbstract 629The JAK2V617F mutation is present in most patients with polycythemia vera (PV) and 50–60% patients with essential thrombocythemia (ET), and primary myelofibrosis (PMF). Inhibitors of histone deacetylases (HDAC), which regulate gene transcription, cell cycle progression and programmed cell death, are emerging as a new class of potential anticancer drugs. A number of HDAC inhibitors are in clinical trials for various neoplasms. Vorinostat (also known as SAHA or Zolinza) was the first FDA approved HDAC inhibitor for clinical use in treating cutaneous T cell lymphoma (CTCL). In this study, we have investigated the efficacy of vorinostat in hematopoietic cells expressing JAK2V617F and in a murine model of PV. We found that proliferation of Ba/F3-EpoR cells expressing JAK2V617F or human erythroleukemia (HEL) cells harboring JAK2V617F mutation was significantly reduced upon treatment with vorinostat at 0.5–1.0 μM. However, proliferation of wild type JAK2 expressing BCR-ABL-transformed K562 cells was only modestly affected by vorinostat. Vorinostat treatment also resulted in marked apoptosis in HEL cells but not in wild-type JAK2 expressing K562 or UT7 cells as determined by annexin V staining and flow cytometry. Moreover, vorinostat treatment (at 1.0 μM) completely inhibited the clonogenic growth of Ba/F3-EpoR-JAK2V617F and HEL cells in a colony assay. Clonogenic growth of K562 cells was only modestly affected by vorinostat at 1.0 μM. Biochemical analyzes revealed significant inhibition of phosphorylation of JAK2, STAT5, Akt, Erk1/2 in JAK2V617F-positive HEL cells but not in wild-type JAK2-expressing K562 cells. Expression of JAK2 and STAT5 was also significantly downregulated by vorinostat treatment in HEL cells but not in K562 cells. In addition, we observed a marked reduction in c-Myc and Pim-1, and cleavage of caspase 3 and PARP in HEL cells, but not in K562 cells, upon treatment with vorinostat. These results suggest that vorinostat selectively targets JAK2V617F mutated cells and inhibits the activation/expression of JAK2 and STAT5. We reported previously the generation of an inducible JAK2V617F knock-in mouse (Akada et al., Blood 2010; 115: 3589–3597). Expression of the JAK2V617F in the knock-in mice reproducibly produced all the features of human PV. We have utilized this JAK2V617F knock-in mouse model to test the in vivo effects of vorinostat. We found that vorinostat treatment (200 mg/kg/day by IP injection) for 2 weeks significantly reduced the increase in red blood cells (RBC), hemoglobin and hematocrit as well as spleen size in mice expressing JAK2V617F compared with vehicle-treatment. Furthermore, vorinostat markedly inhibited the clonogenic growth of primary erythroid progenitors expressing JAK2V617F without exhibiting any significant toxicity towards wild-type JAK2-expressing normal hematopoietic progenitors in mice. Thus, our in vitro and in vivo results suggest that vorinostat may have therapeutic potential for treatment of PV. Disclosures:No relevant conflicts of interest to declare.

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2: INDISPENSABILITY OF THE STILBENOID CORE

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.

Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells

Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 μM, diosgenin induced megakaryocytic differentiation, in contrast to 40 μM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.