Ju Han Song

Resveratrol-mediated reversal of doxorubicin resistance in acute myeloid leukemia cells via downregulation of MRP1 expression

Chemo-resistance to anti-cancer drugs is a major obstacle in efforts to develop a successful treatment of acute myeloid leukemia (AML). In this study, we investigate whether resveratrol, a common ingredient in a broad variety of fruits and vegetables, can reverse drug resistance in AML cells. Three doxorubicin-resistant AML cell lines (AML-2/DX30, AML-2/DX100, AML-2/DX300) were prepared via long-term exposure to doxorubicin for more than 3 months. DNA microarray analysis demonstrated that many genes were differentially expressed in the resistant cells, as compared with the wild type AML-2/WT cells. In particular, the expression level of the MRP1 gene was significantly increased in the AML-2/DX300 cells, as compared to that detected in AML-2 cells. Importantly, the resveratrol was shown not only to induce cell growth arrest and apoptotic death in doxorubicin-resistant AML cells, but was also shown to downregulate the expression of an MRP1 gene. Furthermore, resveratrol treatment induced a significant increase in the uptake of 5(6)-carboxyfluorescein diacetate, a MRP1 substrate, into the doxorubicin-resistant AML-2/DX300 cells. The results of this study show that resveratrol may facilitate the cellular uptake of doxorubicin via an induced downregulation of MRP1 expression, and also suggest that it may prove useful in overcoming doxorubicin resistance, or in sensitizing doxorubicin-resistant AML cells to anti-leukemic agents.

Comparison of the gene expression profiles of monocytic versus granulocytic lineages of HL-60 leukemia cell differentiation by DNA microarray analysis

It is now recognized that precise patterns of differentially expressed genes ultimately direct a particular cell toward a given lineage. In this study, we compared the expression profiles of cancer-related genes by cDNA microarray analysis during the differentiation of human promyelocytic leukemia HL-60 cells into either monocytes or granulocytes. RNA was isolated at times 0, 6, 12, 24, 36, 48, and 72 h following stimulation of differentiation with all-trans retinoic acid (all-trans RA) or 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], and hybridized to the microarray gene chips containing 872 genes related to cell-cycles, oncogenes and leukemias. Several genes were commonly or differentially regulated during cell differentiation into either lineage, as demonstrated by both hierarchical and self-organizing map clustering analysis. At 72 h the expression levels of 45 genes were commonly up- or down-regulated at least a twofold in both lineages. Most importantly, 32 genes including alpha-L-fucosidase gene and adducin gamma subunit gene were up- or down-regulated only in all-trans RA-treated HL-60 cells, while 12 genes including interleukin 1beta and hypoxia-inducible factor 1alpha were up- or down-regulated only in 1,25-(OH)(2)D(3)-treated HL-60 cells. The expression of selected genes was confirmed by Northern blot analysis. As expected, some genes identified have not been examined during HL-60 cell differentiation into either lineage. The identification of genes associated with a specific differentiation lineage may give important insights into functional and phenotypic differences between two lineages of HL-60 cell differentiation.

Chemical Modification of Santonin into a Diacetoxy Acetal Form Confers the Ability to Induce Differentiation of Human Promyelocytic Leukemia Cells via the Down-regulation of NF-κB DNA Binding Activity

Many sesquiterpene lactone compounds either induce or enhance the cell differentiation of human leukemia cells. However, we reported in a previous study that santonin, a eudesmanolide sesquiterpene lactone, exerts no effects on the differentiation of leukemia cells. In this report, to evaluate the possibility of chemically modifying santonin into its derivatives with differentiation inducing activity, we synthesized a series of santonin derivatives, and determined their effects on cellular differentiation in the human promyelocytic leukemia HL-60 cell system. A diacetoxy acetal derivative of santonin (DAAS) was found to induce significant HL-60 cell differentiation in a dose-dependent manner, whereas santonin in its original form did not. The HL-60 cells were differentiated into a granulocytic lineage when exposed to DAAS. In addition, the observed induction in cell differentiation closely correlated with the levels of NF-κB DNA binding activity inhibited by DAAS. Both Western blot analyses and kinase inhibitor studies determined that protein kinase C, ERK, and phosphatidylinositol 3-kinase were upstream components of the DAAS-mediated inhibition of NF-κB binding activity in HL-60 leukemia cells. The results of this study indicate that santonin can, indeed, be chemically modified into a derivative with differentiation inducing abilities, and suggest that DAAS might prove useful in the treatment of neoplastic diseases.

Vertical Bridgman techniques to homogenize zinc composition of CdZn Te substrates

In order to improve the Zn homogeneity along the axial direction of CdZnTe boule, we have employed a modified Bridgman technique using a (Cd, Zn) alloy source in communication with the melt, whose temperature has been gradually changed from 800 to 840°C during growth. Electron probe microanalysis (EPMA) measurements of Zn composition in the boule shows an excellent homogeneity of Zn along the axis of the CdZnTe boule compared with results in a boule grown by using a fixed source temperature. We have performed a numerical simulation to obtain the approximate temperatures of additional heating and cooling needed to improve the radial Zn homogeneity. CdZnTe boule has been grown by seeded vertical Bridgman furnace with two zones of heater and cooler. Ultraviolet/visible spectroscopic measurements of Zn composition over the length of the boule indicate that the radial distribution of Zn composition is very homogeneous in the body region of the boule, where the radial variation of Zn composition is ±0.0005.

5-diphenylacetamido-indirubin-3′-oxime as a novel mitochondria-targeting agent with anti-leukemic activities

Current treatment for leukemia largely depends on chemotherapy. Despite the progress in treatment efficacy of chemotherapy, a poor outcome consequent upon chemoresistance against conventional anti‐cancer drugs still remains to be solved. In this study, we report 5‐diphenylacetamido‐indirubin‐3′‐oxime (LDD398) as a novel mitochondria‐targeting anti‐leukemic agent, which is a derivative of indirubin used in traditional medicine. Treatment with LDD398 resulted in caspase activation, cell death, and growth arrest at G2/M phases in leukemia cells. Interestingly, LDD398 quickly collapsed mitochondrial membrane potential (MMP) within 1 h, accompanied by cytochrome c release into cytosol and severe depletion of cellular ATP. However, the LDD398‐induced cellular events was significantly mitigated by blockage of mitochondrial permeability transition pore (MPTP) opening with chemical and genetic modifications, strongly supporting that LDD398 executes its anti‐leukemic activity via an inappropriate opening of MPTP and a consequent depletion of ATP. The most meaningful finding was the prominent effectiveness of LDD398 on primary leukemia cells and also on malignant leukemia cells resistant to anticancer drugs. Our results demonstrate that, among a series of indirubin derivatives, LDD398 induces leukemia cell death via a different mode from indirubin or conventional chemotherapeutics, and can be employed as a potent anti‐cancer agent in the treatment for newly diagnosed and relapsed leukemia.

Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 suppresses tumor growth in breast cancer-bearing mice by negatively regulating myeloid-derived suppressor cell functions

Myeloid-derived suppressor cells (MDSCs) are one of the most important cell types that contribute to negative regulation of immune responses in the tumor microenvironment. Recently, aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1), a novel pleiotropic cytokine, was identified as an antitumor protein that inhibits angiogenesis and induces antitumor responses. However, the effect of AIMP1 on MDSCs in the tumor environment remains unclear. In the present study, we demonstrated that AIMP1 significantly inhibited tumor growth in 4T1 breast cancer-bearing mice and reduced MDSCs population of tumor sites and spleens of tumor-bearing mice. AIMP1 reduced expansion of MDSCs from bone marrow-derived cells in the tumor-conditioned media. AIMP1 also negatively regulated suppressive activities of MDSCs by inhibiting IL-6 and NO production, and Arg-1 expression. Furthermore, treatment of breast cancer-bearing mice with AIMP1 decreased the capacity of MDSCs to suppress T cell proliferation and Treg cell induction. Western blot and inhibition experiments showed that downregulation of MDSCs functions by AIMP1 may result from attenuated activation of STATs, Akt, and ERK. These findings indicate that AIMP1 plays an essential role in negative regulation of suppressive functions of MDSCs. Therefore, it has a significant potential as a therapeutic agent for cancer treatment.

Induction of human leukemia cell differentiation via PKC/MAPK pathways by arsantin, a sesquiterpene lactone from Artemisia santolina

Abstract Sesquiterpene lactone compounds have received considerable attention in pharmacological research due to their therapeutic effects including anti-cancer and anti-inflammatory activities. In this report, we investigated the effect of arsantin, a sesquiterpene lactone compound present in Artemisia santolina, on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. Arsantin significantly induced HL-60 cell differentiation in a concentration-dependent manner. Cytofluorometric analysis indicated that arsantin induced HL-60 cell differentiation predominantly into granulocytes. Both PKC and MAPK inhibitors suppressed the HL-60 cell differentiation induced by arsantin. Moreover, treatment with arsantin increased protein levels of PKCα and PKCβII isoforms, and also induced increased protein levels and phosphorylation form of MAPKs in HL-60 cells. Importantly, arsantin synergistically enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)2D3 or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)2D3 or ATRA by arsantin may improve outcomes in the therapy of acute promyelocytic leukemia.

High TOP2B/TOP2A expression ratio at diagnosis correlates with favourable outcome for standard chemotherapy in acute myeloid leukaemia

Background:Cytosine arabinoside-based chemotherapy coupled with anthracycline is currently the first-line treatment for acute myeloid leukaemia (AML), but diverse responses to the regimen constitute obstacles to successful treatment. Therefore, outcome prediction to chemotherapy at diagnosis is believed to be a critical consideration.Methods:The mRNA expression of 12 genes closely involved in the actions of cytosine arabinoside and anthracycline was evaluated by real-time reverse transcriptase PCR (RT–PCR), in 54 diagnostic bone marrow specimens of M2-subtype AML.Results:Low expression levels of ribonucleotide reductase M2 (RRM2) and high expression levels of topoisomerase 2 beta (TOP2B) were correlated with longer survival in a univariate analysis. Another interesting finding is that high ratios of TOP2B/RRM2 and TOP2B/TOP2 alpha (TOP2A) in a combined analysis were also shown to have a prognostic impact for longer survival with improved accuracy. Among the four markers, when adjusted for the influence of other clinical factors in multivariate analysis, the TOP2B/TOP2A ratio was significantly correlated with treatment outcomes; patients with high ratios trended toward longer disease-free survival (HR, 0.24; P=0.002) and overall survival (HR, 0.29; P=0.005).Conclusion:Genes with distinct expression profiles such as TOP2B/TOP2A expression ratio at diagnosis can be employed for outcome prediction after the treatment with standard regimens in AML patients with M2 subtype.

Aminoacyl tRNA Synthetase–­Interacting Multifunctional Protein 1 Activates NK Cells via Macrophages In Vitro and In Vivo

Aminoacyl tRNA synthetase–interacting multifunctional protein 1 (AIMP1) has been reported to have antitumor effects in various tumor models. However, mechanisms by which AIMP1 ameliorates tumorigenesis are not well understood. As NK cells are a major cell type involved in antitumor activities and AIMP1 is known to activate professional APCs, we determined whether AIMP1 induced NK cell activation directly or via these APCs. AIMP1 induced the expression of surface activation markers on murine NK cells in total splenocytes, although AIMP1 did not directly induce these activation markers of NK cells. The inductive effect of AIMP1 on NK cell activation disappeared in macrophage-depleted splenocytes, indicating that macrophages were required for the AIMP1-induced activation of NK cells. Furthermore, coculture experiments showed that AIMP1 activated NK cells in the presence of isolated macrophages, but failed to activate NK cells when cultured alone or with dendritic cells or B cells. Although AIMP1 significantly promoted TNF-α production by macrophages, the secreted TNF-α partially affected the NK cell activation. Transwell cocultivation analysis revealed that direct contact between macrophages and NK cells was required for the AIMP1-induced NK cell activation. In addition, AIMP1 significantly enhanced cytotoxicity of NK cells against Yac-1 cells. Furthermore, the in vivo administration of AIMP1 also induced NK cell activation systemically with a macrophage-dependent manner. Importantly, AIMP1 dramatically reduced the lung metastasis of melanoma cells, which was mediated by NK cells. Taken together, our results show that AIMP1 induces antitumor responses by NK cell activation mainly via macrophages.

MST1 deficiency promotes B cell responses by CD4+ T cell-derived IL-4, resulting in hypergammaglobulinemia

MST1 deficiency causes T and B cell lymphopenia, resulting in combined immunodeficiency. However, MST1-deficient patients also exhibit autoimmune-like symptoms such as hypergammaglobulinemia and autoantibody production. Recent studies have shown that the autoimmune responses observed in MST1-deficient patients were most likely attributable to defective regulatory T (Treg) cells instead of intrinsic signals in MST1-lacking B cells. Nevertheless, it is not determined how MST1 deficiency in T cells breaks B cell tolerance and causes systemic autoimmune-like phenotypes. In this study, we confirmed that Mst1-/- mice developed hypergammaglobulinemia associated with increased levels of IgG, IgA, and IgE. We also showed that uncontrolled B cell responses were resulted from the IL-4-rich environment created by CD4+ T cells. Defective MST1-FOXO1 signaling down-regulated Treg cells, resulting in the collapse of immune tolerance where the populations of Th2 and T follicular helper cells expanded. In conclusion, we suggest that MST1 acts as a molecular brake to maintain immune tolerance by regulating T cell-mediated B cell activation.