Growth Of Human Leukemia Cells Research Articles

Ex Vivo Anti-Leukemic Effect of Exosome-like Grapefruit-Derived Nanovesicles from Organic Farming-The Potential Role of Ascorbic Acid.

Citrus fruits are a natural source of ascorbic acid, and exosome-like nanovesicles obtained from these fruits contain measurable levels of ascorbic acid. We tested the ability of grapefruit-derived extracellular vesicles (EVs) to inhibit the growth of human leukemic cells and leukemic patient-derived bone marrow blasts. Transmission electron microscopy and nanoparticle tracking analysis (NTA) showed that the obtained EVs were homogeneous exosomes, defined as exosome-like plant-derived nanovesicles (ELPDNVs). The analysis of their content has shown measurable amounts of several molecules with potent antioxidant activity. ELPDNVs showed a time-dependent antiproliferative effect in both U937 and K562 leukemic cell lines, comparable with the effect of high-dosage ascorbic acid (2 mM). This result was confirmed by a clear decrease in the number of AML blasts induced by ELPDNVs, which did not affect the number of normal cells. ELPDNVs increased the ROS levels in both AML blast cells and U937 without affecting ROS storage in normal cells, and this effect was comparable to ascorbic acid (2 mM). With our study, we propose ELPDNVs from grapefruits as a combination/supporting therapy for human leukemias with the aim to improve the effectiveness of the current therapies.

Atf7ip and Setdb1 interaction orchestrates the hematopoietic stem and progenitor cell state with diverse lineage differentiation

Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous group of cells with expansion, differentiation, and repopulation capacities. How HSPCs orchestrate the stemness state with diverse lineage differentiation at steady condition or acute stress remains largely unknown. Here, we show that zebrafish mutants that are deficient in an epigenetic regulator Atf7ip or Setdb1 methyltransferase undergo excessive myeloid differentiation with impaired HSPC expansion, manifesting a decline in T cells and erythroid lineage. We find that Atf7ip regulates hematopoiesis through Setdb1-mediated H3K9me3 modification and chromatin remodeling. During hematopoiesis, the interaction of Atf7ip and Setdb1 triggers H3K9me3 depositions in hematopoietic regulatory genes including cebpβ and cdkn1a, preventing HSPCs from loss of expansion and premature differentiation into myeloid lineage. Concomitantly, loss of Atf7ip or Setdb1 derepresses retrotransposons that instigate the viral sensor Mda5/Rig-I like receptor (RLR) signaling, leading to stress-driven myelopoiesis and inflammation. We find that ATF7IP or SETDB1 depletion represses human leukemic cell growth and induces myeloid differentiation with retrotransposon-triggered inflammation. These findings establish that Atf7ip/Setdb1-mediated H3K9me3 deposition constitutes a genome-wide checkpoint that impedes the myeloid potential and maintains HSPC stemness for diverse blood cell production, providing unique insights into potential intervention in hematological malignancy.

Case Report: 7-Ethyl-10-Hydroxycamptothecin, a DNA Topoisomerase I Inhibitor, Performs BRD4 Inhibitory Activity and Inhibits Human Leukemic Cell Growth.

7-Ethyl-10-hydroxycamptothecin (SN-38) is an active metabolite of CPT-11, which can inhibit DNA topoisomerase I, DNA synthesis and cause frequent DNA single-strand breaks. In our study, SN-38 was characterized as a potent and reversible BRD4 inhibitor [IC50 = 660.2 nM against BRD4 (BD1) and IC50 = 547.7 nM against BRD4 (BD2)] in biochemical assay using drug repurposing strategy. Additional cellular assay suggested that SN-38 can bind BRD4 in human leukemic cell K562 and inhibit cell growth with IC50 = 0.2798 μM in a BRD4 dependent manner partially. Additionally, mechanism study indicated that SN-38 can induce the accumulation of BRD4 substrate c-Myc and cleavage of caspase 3. In sum, our findings identified BRD4 as a new target of SN-38 and reveals SN-38 as a modifier of histone acetylation reader for the first time, which may provide a new insight for further optimization of dual target inhibitor.

Isoferulic acid inhibits human leukemia cell growth through induction of G2/M‑phase arrest and inhibition of Akt/mTOR signaling.

Hematologic malignancy is a serious disease that develops quickly and aggressively, severely threatening human health owing to its high mortality. The current study aimed to evaluate the antitumor effects of isoferulic acid (IFA) on leukemia cells and investigate the possible molecular mechanisms. Hematologic cancer cell lines (Raji, K562 and Jurkat) were treated with IFA in a dose-dependent manner and proliferation was measured by a cell proliferation assay. Cell cycle arrest was detected via flow cytometry using propidium iodide (PI) staining. Cell apoptosis and apoptosis-associated signal pathways were analyzed via Annexin V/PI staining and western blot assays, respectively. IFA inhibited cell viability, induced cell apoptosis and triggered cell cycle arrest in G2/M phase in Raji, K562, and Jurkat cells in a dose-dependent manner. In response to IFA treatment, the levels of cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 were increased in Jurkat and K562 cells, which was associated with increased phosphorylation of Cdc2 and reduction of Cyclin B1 levels. IFA remarkably attenuated the phosphorylation of mTOR and Akt in Jurkat cells. Collectively, the present data suggested that IFA had therapeutic effects on Jurkat, K562, and Raji cells, indicating it as a promising candidate for the treatment of hematologic malignancy.

HDAC3 Activity is Essential for Human Leukemic Cell Growth and the Expression of β-catenin, MYC, and WT1.

Therapy of acute myeloid leukemia (AML) is unsatisfactory. Histone deacetylase inhibitors (HDACi) are active against leukemic cells in vitro and in vivo. Clinical data suggest further testing of such epigenetic drugs and to identify mechanisms and markers for their efficacy. Primary and permanent AML cells were screened for viability, replication stress/DNA damage, and regrowth capacities after single exposures to the clinically used pan-HDACi panobinostat (LBH589), the class I HDACi entinostat/romidepsin (MS-275/FK228), the HDAC3 inhibitor RGFP966, the HDAC6 inhibitor marbostat-100, the non-steroidal anti-inflammatory drug (NSAID) indomethacin, and the replication stress inducer hydroxyurea (HU). Immunoblotting was used to test if HDACi modulate the leukemia-associated transcription factors β-catenin, Wilms tumor (WT1), and myelocytomatosis oncogene (MYC). RNAi was used to delineate how these factors interact. We show that LBH589, MS-275, FK228, RGFP966, and HU induce apoptosis, replication stress/DNA damage, and apoptotic fragmentation of β-catenin. Indomethacin destabilizes β-catenin and potentiates anti-proliferative effects of HDACi. HDACi attenuate WT1 and MYC caspase-dependently and -independently. Genetic experiments reveal a cross-regulation between MYC and WT1 and a regulation of β-catenin by WT1. In conclusion, reduced levels of β-catenin, MYC, and WT1 are molecular markers for the efficacy of HDACi. HDAC3 inhibition induces apoptosis and disrupts tumor-associated protein expression.

USP39 regulates the cell cycle, survival, and growth of human leukemia cells.

Ubiquitin-specific peptidase 39 (USP39) is one member of the cysteine proteases of the USP family, which represents the largest group of DeUbiquitinases with more than 50 members in humans. The roles of USP39 in human cancer have been widely investigated. However, the roles of USP39 in human leukemia and the underlying mechanism remain unknown. Here we reported the function of USP39 in human leukemia. We observed that the expression of USP39 was up-regulated in human leukemia cells and the high expression of USP39 was correlated with poor survival of the patients with leukemia. Lentivirus-mediated knockdown of USP39 repressed the proliferation and colony formation of human leukemia cell lines HL-60 and Jurkat cells. Mechanism study showed that USP39 knockdown induced the arrest of cell cycle and apoptosis of leukemia cells. In addition, our microarray and bioinformatic analysis demonstrated that USP39 regulated diverse cellular signaling pathways that were involved in tumor biology, and several pivotal genes (IRF1, Caspase 8, and SP1) have been validated by quantitative real-time polymerase chain reaction. Knockdown or IRF1 partially restored the proliferation rate of leukemia cells with USP39 knockdown. Taken together, our findings implicate that USP39 promotes the development of human leukemia by regulating cell cycle, survival, and proliferation of the cells.

ZFX modulates the growth of human leukemic cells via B4GALT1.

ZFX modulates the growth of human leukemic cells via B4GALT1.

ZFX modulates the growth of human leukemic cells via B4GALT1.

Zinc finger protein X-linked (ZFX) is a key regulator of both embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs), which is required for both Notch intracellular domain (NotchIC)-induced acute T-cell leukemia and MLL-AF9-induced myeloid leukemia in mouse models. However, the role of ZFX and its underlying mechanism in human leukemic cells remain unclear yet, though accumulating data have demonstrated that ZFX is aberrantly expressed in various human tumors and plays an important role. Herein, we found that ZFX was aberrantly expressed in various human leukemic cell lines and primary cells from leukemia patients compared with control cells. The silence of ZFX led to the growth suppression through either the deregulated cell cycle or the induction of apoptosis in various cells including K562, Jurkat, Namalwa, and THP-1 cells. The gene expression analysis revealed that UDP-Gal:βGlcNAc β 1,4-galactosyltransferase, polypeptide 1 (B4GALT1) was significantly down-regulated upon ZFX silencing, which is implicated in the response of K562 cells to the treatment of imatinib mesylate (IM). In addition, lectin blot assay showed that the galactosylation of glycoproteins in K562 cells was suppressed upon ZFX silencing. Interestingly, overexpression of B4GALT1 restored the growth and conferred drug resistance to ZFX-silenced cells. Taken together, we have demonstrated that ZFX is aberrantly expressed in multiple human leukemic cells and it modulates the growth and drug response of leukemic cells partially via B4GALT1, which suggests that ZFX is a new regulator of leukemic cells and warrants intensive investigations on this 'stemness' regulator in these deadly diseases.

Ginsenoside 20(s)-Rh2 as potent natural histone deacetylase inhibitors suppressing the growth of human leukemia cells

Background and objectiveActivation and abnormal expression of histone deacetylase (HDAC) which is important target for cancer therapeutics are related to the occurrence of human leukemia. 20(s)-Ginsenoside Rh2 (20(s)-Rh2) may be a potential HDAC inhibitor (HDACi) of leukemia, but the mechanism has not been reported. MethodsThe cell proliferation and apoptosis was assessed in cultured K562 and KG-1α cells. The protein expression was measured with immunoblotting. The activities of HDAC and histone acetyltransferase (HAT) were measured with BCA. In vivo experiments were performed on naked mice carrying K562 cells for assessment of tumor growth, apoptosis, protein expression, and HDAC/HAT activities. Results20(s)-Rh2 effectively induced cell cycle arrest at G0/G1 phase and apoptosis in K562 and KG1-α cells, decreased the levels of proteins associated with cell proliferation (Cyclin D1, Bcl-2, ERK, p-ERK) and activated pro-apoptotic proteins (Bax, cleaved Caspase-3, p38, p-p38, JNK, p-JNK). 20(s)-Rh2 down-regulated HDAC1, HDAC2, HDAC6, increased histone H3 acetylation and HAT activity. Moreover, 20(s)-Rh2 inhibited the growth of human leukemia xenograft tumors in vivo. Conclusion20(s)-Rh2 inhibited the proliferation of K562 and KG1-α cell by reducing the expression and activity of HDACs, increasing histone acetylation, and regulating key proteins in the downstream signaling pathways. Therefore, 20(s)-Rh2 could become a potential natural HDACi for chemotherapy of leukemia.

P0099 Compound MacKay-03 inhibits growth and induces megakaryocytic differentiation in human leukaemia cells and augments megakaryocytopoiesis

Background MacKay-03 (3,12-O-diacetyl-7-O-angeloyl-8-methoxyingol) is a small-molecule ingol diterpene compound isolated from the plant Euphorbia neriifolia. We isolated compounds according to screening of anticancer activity and found MacKay-03 preferentially inhibited growth of human leukaemic cells. The cellular and molecular mechanism of action was investigated in the present study. Methods The methods used were: cell viability by MTT assay, morphological observation by Liu’s and immunofluorescent stain, cell cycle analysis by DNA histogram, expression of surface antigens by flow cytometry, quantitation of secreted platelet factor 4 by ELISA, expression of regulatory proteins by western blotting, knockdown of stat3 mRNA by RNA interference, and megakaryocytopoiesis by colony forming unit-megakaryocytes (CFU-MK) assay. Findings MacKay-03 inhibited growth of leukaemia K562 and HEL cells in a dose-dependent and time-dependent manner. Morphological changes characteristic of megakaryocytes—e.g. enlarged cell size, multipolar spindles, and multi-nucleation—were noted in both cell lines treated with MacKay-03. A smaller population of apoptotic cells were also observed. MacKay-03 treatment caused polyploidy and hypoploidy populations. Surface expression of megakaryocytic marker CD61 was increased by MacKay-03, whereas the erythroid marker glycophorin A was decreased. The MacKay-03-treated HEL cells extensively secreted platelet factor 4, implicating a further platelet differentiation. MacKay-03, at optimum conditions for inducing megakaryocytic differentiation, upregulated the expression of JAK2 and phosphorylated STAT3 in K562 and HEL cells. Knockdown of stat3 mRNA partially blocked megakaryocytic differentiation. Interpretation These results indicate that MacKay-03 is able to inhibit growth, induce megakaryocytic differentiation, and to a lesser extent cause apoptosis in human leukaemia cells. Furthermore, MacKay-03 augmented the formation of CFU-MK in mouse bone marrow cells. We conclude that MacKay-03 could inhibit growth of human leukaemic cells, induce megakaryocytic differentiation, and promote megakaryocytopoiesis. MacKay-03 may have the potential to be developed as a candidate drug for management of leukaemia and thrombocytopenia.

Function of Growth Factor Independence 1 (GFI1) in the Polarization AML -Associated Stroma

The growth of various solid tumors, lymphoma and leukemias is not only the result of cell-specific changes at the genetic and epigenetic level, but is also affected by the surrounding stroma and the cells therein. It has been shown that tumor cells induce surrounding immune cells to express various cytokines and other factors, which promote further growth and spread of tumor cells. Most studies have been conducted with solid tumors, however, not much is known about the role of stroma cells with regard to haematopoietic tumors. In addition, our knowledge is limited with regard to the cell intrinsic factors governing the polarization of stroma cells. We intended to study the role of stroma cells in acute myeloid leukemia (AML), a malignant disease of the myeloid lineage and in myelodysplastic syndrome (MDS), a disease which is characterized by disturbed function of the bone marrow, which can often progress to AML.We focused our studies on two different components of the stroma: mesenchymal stroma cells (MSC) and monocytes.Bone marrow derived MSCs and monocytes originating from AML patients better supported the growth of human leukemia cells in vitrothan MSCs and monocytes from control healthy persons. Interestingly, after achieving a remission, MSCs and monocytes from the cured patients did not any more support the growth of leukemia cells to the same extent as monocytes and MSCs from leukemic patients.To better understand the mechanism behind this observation, we used different murine MDS and AML models. Similar to the finding in human patients, bone marrow derived MSCs and monocytes from MDS and AML mice better supported the growth of leukemic cells in vitro than monocytes and MDSc from healthy mice. In addition, we observed a higher number of non-malignant MSCs and monocytes in the bone marrow of leukemic mice than in the bone marrow of healthy mice. Furthermore, the higher the number of monocytes was in the bone marrow of the mice, the more aggressive was the course of the leukemia. On a molecular level we found that the transcription factor Gfi1 is 3-4 fold upregulated in the MSCs and monocytes of leukemic mice. To verify whether Gfi1 is indeed required for the polarization of MSCs and monocytes we used leukemic Gfi1-deficient mice. In these mice the accumulation of monocytes was less pronounced than in the bone marrow of Gfi1 wildtype mice. In addition MSCs and monocytes from Gfi1-deficient mice did not support to the same extent the growth of leukemic cell lines in vitro, as did the monocytes and MSCs of Gfi1 wildtype leukemic mice. Thus, we have first indications that MSCs and monocytes play an important role in the support of leukemic cells and that Gfi1 is involved in the polarization of these cells and thus Gfi1 could serve as an additional approach to treat leukemia and MDS. DisclosuresNo relevant conflicts of interest to declare.

Combination of quercetin and hyperoside inhibits prostate cancer cell growth and metastasis via regulation of microRNA‑21.

Previous studies have reported that hyperoside and quercetin in combination (QH; 1:1) inhibited the growth of human leukemia cells. The aim of the present study was to investigate the anti‑cancer effect of QH on prostate cancer cells. The results demonstrated that QH decreased the production of reactive oxygen species (ROS) and increased antioxidant capacity in PC3 cells at various concentrations (2.5‑60 µg/ml) with peak inhibition and augmentation changes of 3.22‑ and 3.00‑fold, respectively. Following treatment with QH for 48 and 72 h, the IC50-values on PC3 cells were 19.7 and 12.4 µg/ml, respectively. Western blot analysis revealed that QH induced apoptosis in human prostate cancer cells via activation of caspase‑3 and cleavage of poly(adenosine diphosphate ribose) polymerase. In addition, QH significantly inhibited the invasion and migration of PC3 cells as well as reduced the expression of numerous prostate tumor‑associated microRNAs (miRs), including miR‑21, compared to that of untreated human prostate cancer cells. QH was also found to enhance the expression of tumor suppressor programmed cell death protein 4, which was negatively regulated by miR‑21. Furthermore, induced overexpression of miR‑21 using pre‑miR‑21 oligonucleotides attenuated the beneficial effect of QH on prostate cancer cells. In conclusion, the results of the present study indicated that QH exerted an anti‑cancer effect on human prostate cancer cells, the mechanism of which proceeded, at least in part, via the inhibition of the miR‑21 signaling pathway.

Abstract 4788: MiR-509 inhibits human leukemia cell growth

Abstract Even though the majority of children with precursor B-cell acute lymphoblastic leukemia (B-ALL) can be cured, ALL is still the most frequent cause of cancer-related deaths and causes significant treatment-related toxicity in pediatrics. Thus, there is a need for more effective and less toxic therapies for B-ALL. Specific microRNAs (miRs) may be useful as therapeutics, or to identify downstream therapeutic targets, since miRs regulate essentially all cellular processes. We identified miR-509 via a human genome-wide screen (Cheng WC et al. BioTechniques 2013;54:77. PMC3671589) for miRs that inhibit growth of the NALM6 human B-ALL cell line (harboring the ETV6-PDGFRB fusion gene). MiR microarray expression analysis revealed low endogenous levels of miR-509 in multiple acute leukemia cell lines, CD34+ hematopoietic stem-progenitor cells from normal human donors, and normal B and T lymphocytes. Therefore, enforced expression of miR-509 was used for our mechanistic studies in leukemia cells. Since NALM6 cannot be easily transfected, the precursor miR-509 sequence was cloned downstream of green fluorescence protein (GFP) into a lentiviral vector construct. Enforced lentivector-mediated miR-509 expression was confirmed using RT-qPCR. In a confirmatory growth competition assay measuring the percent GFP-expressing cells by flow cytometry, NALM6 cells transduced with lentiviral miR-509 were progressively outnumbered by mock-transduced cells over five weeks. MiR-509's growth inhibitory effect in NALM6 was further confirmed by orthogonal assays employing trypan blue dye exclusion and alamarBlue fluorescence. MiR-509 similarly inhibited growth of another B-ALL cell line, RCH-ACV (harboring the TCF3-PBX1 fusion gene). In addition, NOD-SCID IL2rg-/- (NSG) mice transplanted miR-509-transduced NALM6 cells had increased survival as compared to empty vector. In order to elucidate the cellular mechanism by which miR-509 exerts its growth inhibitory effects, we performed cell cycle analysis using BrdU/7-AAD staining. MiR-509-transduced NALM6 cells had reduced numbers of S-phase cells, as compared to empty vector-transduced cells. We also observed an increase in apoptotic cell death in miR-509-transduced NALM6 cells, as compared to empty vector-transduced cells. To identify potential targets of miR-509, we used TargetScan to predict targets. We then focused on mRNAs that were expressed in NALM6 cells, as determined by gene expression data in the Cancer Cell Line Encyclopedia. RAB5C was reduced in miR-509 transduced cells as evidenced by qPCR and western blot; luciferase assays indicate that RAB5C is a direct miR-509-3p target. Knockdown of RAB5C by shRNA lentiviral constructs recapitulated the growth inhibitory phenotype induced by miR-509 overexpression in NALM6 cells. In summary, miR-509 suppresses B-ALL cell growth by inhibiting cell proliferation and inducing apoptosis, and miR-509 and RAB5C may be new therapeutic targets for B-ALL. Citation Format: Yee Sun Tan, Wen-Chih Cheng, Curt I. Civin. MiR-509 inhibits human leukemia cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4788. doi:10.1158/1538-7445.AM2014-4788

Superior activity of a new histone deacetylase inhibitor (ZYJ-34c) in inhibiting growth of human leukemia cells by inducing p21 WAF1 expression and cell cycle arrest

Histone deacetylase inhibitors are a new class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis and cell cycle arrest in various cancer cells. Recently, we identified ZYJ-34c, a modified histone deacetylase inhibitor that showed significantly higher antitumor activity in vivo than the FDA-approved drug suberoylanilide hydroxamic acid. We aimed to investigate its exact mechanism of action. Activity of ZY-34c against human erythroleukemic (K562) cells, human myeloid leukemia (HL-60) cells, and primary leukemic cells were investigated in vitro using proliferation assays, cell cycle assays, apoptosis assays, RNA interference, promoter acetylation assays, and assays of transcription of related molecules. ZYJ-34c strongly inhibited the proliferation of leukemia cells compared with suberoylanilide hydroxamic acid. Primary leukemic cells isolated from patients with acute myeloid leukemia were more sensitive to ZYJ-34c. The 50% growth-inhibitory concentrations of ZYJ-34c and suberoylanilide hydroxamic acid were 2.95±0.75 and 4.45±0.29 μmol/l for K562 cells treated for 48 h, respectively. We found that ZYJ-34c caused more significant G1 cell cycle arrest than suberoylanilide hydroxamic acid, in a time-dependent manner. ZYJ-34c-induced hyperacetylation of histone H3 and H4 around the promoter region of p21. P21 RNA interference markedly impaired ZYJ-34c-induced or suberoylanilide hydroxamic acid-induced G1 cell cycle arrest. In addition, levels of bcr-abl mRNA and protein in K562 cells decreased after treatment with either suberoylanilide hydroxamic acid or ZYJ-34c; moreover, ZYJ-34c had a higher inhibition activity. ZYJ-34c could represent a novel pharmacological agent with potential benefit for patients with leukemia.

Combination of quercetin and hyperoside has anticancer effects on renal cancer cells through inhibition of oncogenic microRNA-27a

Quercetin and hyperoside (QH) in combination (1:1 ratio) have previously been shown to inhibit the growth of human leukemia cells. Here, we investigated the anticancer activity of the same mixture in 786-O renal cancer cells. QH decreased the generation of reactive oxygen species (ROS) by up to 2.25-fold and increased the antioxidant capacity by up to 3-fold in 786-O cells (3.8-60 μg/ml), whereas IC50 values for viability were 18.2, 18.7 and 11.8 μg/ml, respectively. QH also induced caspase-3 cleavage (2-fold) and increased PARP cleavage. Specificity protein (Sp) transcription factors are overexpressed in cancer cells and regulate genes required for cell proliferation, survival and angiogenesis. QH treatment decreased the expression of Sp1, Sp3 and Sp4 mRNA and this was accompanied by decreased protein expression. Moreover, expression of the Sp-dependent anti-apoptotic survival gene survivin was also significantly reduced, both at the mRNA and protein levels. QH decreased microRNA-27a (miR-27a) and induced the zinc finger protein ZBTB10, an Sp-repressor, suggesting that interactions between QH and the miR-27a-ZBTB10 axis play a role in Sp downregulation. This was confirmed by transfection of cells with a specific mimic for miR-27a, which partially reversed the effects of QH. These findings are consistent with previous studies on botanical anticancer agents in colon cancer cells.

Biological and Structural Evaluation of 10R- and 10S-Methylthio-DDACTHF Reveals a New Role for Sulfur in Inhibition of Glycinamide Ribonucleotide Transformylase

Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC₅₀ = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC₅₀ = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase.

Natural Killer Cells Generated from Cord Blood Hematopoietic Progenitor Cells Efficiently Target Bone Marrow-Residing Human Leukemia Cells in NOD/SCID/IL2Rgnull Mice

Natural killer (NK) cell-based adoptive immunotherapy is an attractive adjuvant treatment option for patients with acute myeloid leukemia. Recently, we reported a clinical-grade, cytokine-based culture method for the generation of NK cells from umbilical cord blood (UCB) CD34+ hematopoietic progenitor cells with high yield, purity and in vitro functionality. The present study was designed to evaluate the in vivo anti-leukemic potential of UCB-NK cells generated with our GMP-compliant culture system in terms of biodistribution, survival and cytolytic activity following adoptive transfer in immunodeficient NOD/SCID/IL2Rgnull mice. Using single photon emission computed tomography, we first demonstrated active migration of UCB-NK cells to bone marrow, spleen and liver within 24 h after infusion. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Thereafter, we showed that low dose IL-15 mediates efficient survival, expansion and maturation of UCB-NK cells in vivo. Most importantly, we demonstrate that a single UCB-NK cell infusion combined with supportive IL-15 administration efficiently inhibited growth of human leukemia cells implanted in the femur of mice, resulting in significant prolongation of mice survival. These preclinical studies strongly support the therapeutic potential of ex vivo-generated UCB-NK cells in the treatment of myeloid leukemia after immunosuppressive chemotherapy.

Anti-Leukemic Activity of Ubiquinone-Based Compounds Targeting Trans-plasma Membrane Electron Transport

Trans-plasma membrane electron transport (tPMET) is a ubiquinone-dependent cell survival pathway for maintaining intracellular redox homeostasis in rapidly dividing cells. To target this pathway, fifteen ubiquinone-based compounds were designed and synthesized to position at the plasma membrane and disrupt tPMET. We established that quaternary ammonium salt moieties carrying highly hindered, positive electronic charges located to the plasma membrane. A ten-carbon chain linked to these moieties was effective at positioning the redox-active ubiquinone-like function within the lipid bilayer to disrupt tPMET in human leukemic cells (IC50 9 ± 1 μM). TPMET inhibition alone was not sufficient to induce significant cell death, but positively charged compounds could also enter the cell and disrupt intracellular redox balance, distinct from their effects on mitochondrial electron transport. The synergistic effect of tPMET inhibition plus intracellular redox disruption gave strong antiproliferative activity (IC50 2 ± 0.2 μM). Positively charged ubiquinone-based compounds inhibit human leukemic cell growth.

Anticancer Effect in HL-60 Human Leukemia Cells and Other Helath-Beneficial Functions of Cheese

With regard to the aim of cancer prevention and/or treatment, a considerable number of basic studies have indicated that dairy and other plant-originated natural food products may possess anticancer activity. On the growth of human leukemia cells, for example, enzymatically digested skim milk or fermented milk cultured with various bacteria can exhibit differential suppressive activities. Our research team has previously revealed that highly ripened cheese was capable of demonstrating strong growth inhibition and induction of apoptotic DNA damage on HL-60 human promyelocyticleukemia cells. In this short review, the available information concerning potent anticancer effects of cheese was summarized. From the stand point of Food Science, functional implications for cancer prevention as well as multifaceted function of cheese are discussed.

Study on the Immunomodulation Effect of Isodon japonicus Extract via Splenocyte Function and NK Anti-Tumor Activity

Here we investigated the potential immune-enhancing activity of Isodon japonicus on murine splenocyte and natural-killer (NK) cells in vitro. The ethanol extract of I. japonicus significantly enhanced the proliferation of splenocyte and induced the significant enhancement of NK cells’ activity against tumor cells (YAC-1). In addition, I. japonicus increased the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, suggesting that the increase in NK cell cytotoxicity could be due to the enhancement of the NK cell production of both cytokines. Taken together, I. japonicus extract inhibited the growth of human leukemia cells (K562) by 74%. Our observation indicated that the anti-tumor effects of I. japonicus may be attributed to its ability to serve as a stimulant of NK anti-tumor activity. In addition, our results support the development of functional food studies on I. japonicus.