Treatment Of Human Leukemia Research Articles

Multiple gene knockdown strategies for investigating the properties of human leukemia stem cells and exploring new therapies.

The past two decades have witnessed significant strides in leukemia therapies through approval of therapeutic inhibitors targeting oncogene-driving dysregulated tyrosine kinase activities and key epigenetic and apoptosis regulators. Although these drugs have brought about complete remission in the majority of patients, many patients face relapse or have refractory disease. The main factor contributing to relapse is the presence of a small subpopulation of dormant drug-resistant leukemia cells that possess stem cell features (termed as leukemia stem cells or LSCs). Thus, overcoming drug resistance and targeting LSCs remain major challenges for curative treatment of human leukemia. Chronic myeloid leukemia (CML) is a good example, with rare, propagating LSCs and drug-resistant cells that cannot be eradicated by BCR-ABL-directed tyrosine kinase inhibitor (TKI) monotherapy and that are responsible for disease relapse/progression. Therefore, it is imperative to identify key players in regulating BCR-ABL1-dependent and independent drug-resistance mechanisms, and their key pathways, so that CML LSCs can be selectively targeted or sensitized to TKIs. Here, we describe several easily adaptable gene knockdown approaches in CD34+ CML stem/progenitor cells that can be used to investigate the biological properties of LSCs and molecular effects of genes of interest (GOI), which can be further explored as therapeutic modalities against LSCs in the context of human leukemia.

Modulation of GILZ and Erg upon Non-Steroidal Treatment in Chicken Leukemic Cells

Background and aims: Glucocorticoids (GCs) have been successfully employed in the treatment of human leukemia; however, those effects can be deteriorated as the metabolic side effects occurred due to the activation glucocorticoid-induced leucine zipper (GILZ) leading to the induction of the GC resistance which is initiated via the Ets-related gene (Erg). In the present work, we recruited this antagonist influence on ERG for aiming to understand the physiological potential that might be of benefit in the treatment of leukemia using a non-steroidal compound (CPDA). Methods: We examined the effect of dexamethasone, as a steroid, and CPDA, as a non-steroid substance, on the GILZ and ERG receptor gene expression in The chicken leukemia cells (CLCs), DT40 cells, after treating them with the drug at 1μM followed by a 24-hour incubation. A real-time-polymerase chain reaction (RT-PCR) method was used to measure gene expression. Results: An increase (two-fold) in the expression of GILZ receptor was unveiled. This enhanced-GILZ model presents the steroidal positive actions and refers to the CPDA ability to mimic this action of steroids. The results also demonstrated a downregulation in the mRNA expression level of ERG, indicating of antagonism of steroids in responding cells and as a positive finding for the effect of the non-steroidal compound A tested. Conclusions: Steroid-induced upregulation in the GILZ receptor provides proof of GC undesirable effects leading to correlated ERG-initiated resistance to the steroids used in exposed cells. Interestingly, CPDA has a GC-based treating activity low GILZ expression.

Inhibition of the MiR-185-PAK6-Mediated Survival and Metabolic Pathways Selectively Targets Drug-Resistant CML Stem/Progenitor Cells

Overcoming drug resistance and targeting leukemic stem cells (LSCs) remain major challenges for curative treatment of human leukemia, including chronic myeloid leukemia (CML). Indeed, most patients with CML require life-long therapy with ABL1 tyrosine kinase inhibitors (TKIs), due to the persistence of residual LSCs that maintain the potential for relapse. Increasing evidence also indicates that LSCs are susceptible to cellular metabolic changes and seem to have a greater dependence on mitochondrial oxidative phosphorylation (OXPHOS) for survival. Previously, through global transcriptome profiling, we identified a key microRNA (miRNA), miR-185 as a predictive biomarker and it is also required for CML LSC survival. Its expression was significantly reduced in CD34+treatment-naïve CML cells and predictive of therapy response. Conversely, restored expression of miR-185 by lentiviral transduction in CD34+TKI-nonresponder cells significantly impaired survival of these cells, sensitizing them to TKIs in vitroand in pre-clinical xenotransplantation models, indicating that miR-185 acts as a tumor suppressor and is critical in regulating TKI response/resistance of CML stem/progenitor cells. PAK6, a serine/threonine-protein kinase, was identified as a target gene of miR-185; it is upregulated in CD34+TKI-nonresponder cells vs. TKI-responders, correlating with reduced miR-185 expression. To further investigate the molecular and biological roles of the miR-185-PAK6 axisin the regulation of survival of drug-resistant cells, including LSCs, we performed RNA-seq and gene set enrichment analysis (GSEA) in the same CD34+patient cells where miR-185 and PAK6 were identified as being differentially expressed. Interestingly, this analysis has now identified a significant gene set enrichment of OXPHOS, reactive oxygen species (ROS), and adipogenesis pathways in CD34+CML cells compared to healthy CD34+cells (Normalized Enrichment Scores (NES): 2.44, 1.65 and 1.8). Moreover, these changes were significantly higher in TKI-nonresponder cells than in TKI-responders (NES: 1.73, 0.49 and 0.34). We have thus hypothesized that the miR-185-PAK6 axis may contribute to the perturbation of specific metabolic pathways in TKI-nonresponder LSC/progenitor cells and confer therapy-resistance to these cells. Indeed, a pre-clinically validated pan-PAK inhibitor (PF-3758309) alone, or in combination with a TKI, greatly reduced mitochondrial activity in TKI-nonresponder cells, in MitoTracker analysis, an effect that was not seen in the same cells treated with a TKI. Notably, ROS production was also significantly reduced in these cells treated with PF-3758309 and further reduction was observed with a combination of PF-3758309 and TKIs. Notably, PF-3758309 significantly reduced the growth of IM-resistant cell lines (IC50 25-70 nM) and CD34+TKI-nonresponder cells, as assessed by viability and colony-forming cell assays, and increased their apoptosis; these effects were greatly enhanced by TKIs (2-fold, P<0.05). These results were further confirmed in TKI-resistant cells using a lentiviral CRISPR/Cas9 knockout system that specifically targets PAK6. In addition, specific molecular changes associated with PF-3758309 treatment were also investigated using the PharmacoDB database and PharmacoGx R-package. Several candidates were identified, including growth factor independent 1B transcriptional repressor (GFI1B), a myeloid-enriched transcription factor. Its expression was reduced by PF-3758309 treatment and significantly increased in CML compared to healthy controls (>2-fold). Interestingly, expression of GFI1B is further upregulated in CD34+TKI-nonresponders compared to responders. Taken together, these findings indicate that dual targeting of miR-185-PAK6-mediated survival and metabolic pathways, along with BCR-ABL, selectively eradicates therapy-resistant LSC/progenitors, providing a valuable therapeutic strategy for improved treatment and care. Disclosures No relevant conflicts of interest to declare.

Advances in the mouse models of myeloid leukemia

Mouse animal models are the most commonly used experimental tools in scientific research, which have been widely favored by researchers. The animal model of mouse leukemia appeared in the 1930s. During the past 90 years, researchers have developed various types of mouse leukemia models to simulate the development and treatment of human leukemia in order to promote effectively the elucidation of the molecular mechanism of leukemia' development and progression, as well as the development of targeted drugs for the treatment of leukemia. Considering that to myeloid leukemia, especially acute myeloid leukemia, there currently is no good clinical treatment, it is urgent to clarify its new molecular mechanism and develop new therapeutic targets. This review focuses on the various types of mouse models about myeloid leukemia used commonly in recent years, including mouse strains, myeloid leukemia cell types, and modeling methods, which are expected to provide a reference for relevant researchers to select animal models during myeloid leukemia research.

The cyclohexene derivative MC-3129 exhibits antileukemic activity via RhoA/ROCK1/PTEN/PI3K/Akt pathway-mediated mitochondrial translocation of cofilin

The effects of MC-3129, a synthetic cyclohexene derivative, on cell viability and apoptosis have been investigated in human leukemia cells. Exposure of leukemia cells to MC-3129 led to the inhibition of cell viability and induction of apoptosis through the dephosphorylation and mitochondrial translocation of cofilin. A mechanistic study revealed that interruption of the RhoA/ROCK1/PTEN/PI3K/Akt signaling pathway plays a crucial role in the MC-3129-mediated dephosphorylation and mitochondrial translocation of cofilin and induction of apoptosis. Our in vivo study also showed that the MC-3129-mediated inhibition of the tumor growth in a mouse leukemia xenograft model is associated with the interruption of ROCK1/PTEN/PI3K/Akt signaling and apoptosis. Molecular docking suggested that MC-3129 might activate the RhoA/ROCK1 pathway by targeting LPAR2. Collectively, these findings suggest a hierarchical model, in which the induction of apoptosis by MC-3129 primarily results from the activation of RhoA/ROCK1/PTEN and inactivation of PI3K/Akt, leading to the dephosphorylation and mitochondrial translocation of cofilin, and culminating in cytochrome c release, caspase activation, and apoptosis. Our study reveals a novel role for RhoA/ROCK1/PTEN/PI3K/Akt signaling in the regulation of mitochondrial translocation of cofilin and apoptosis and suggests MC-3129 as a potential drug for the treatment of human leukemia.

Synthesis of Novel Class of <i>N</i>-Alkyl-isatin-3-iminobenzoic Acid Derivatives and Their Biological Activity in Zebrafish Embryos and Human Cancer Cell Lines

Isatin (1H-indole-2,3-dione) and many of its derivatives are reported to have pharmacological properties. In this study, we report the synthesis and biological activity of a new class of N-alkyl-isatin-3-iminobenzoic acid derivatives prepared via the condensation of N-alkyl isatin with 4-aminobenzoic acid by conventional, microwave, and ultrasonic methods. Microwave irradiation yielded the products in a shorter reaction time with higher yields and purities. The compounds were screened in zebrafish embryos, and also in three human cancer cell lines (MCF7, HepG2, and Jurkat) and one normal human cell line i.e., human foreskin cell line (HFF-1). Two compounds (3c, 3f) were found to be highly effective against hematopoiesis in live zebrafish embryo at 10 µM concentration. The developmental stage-dependent treatment indicated that these compounds interfered with the differentiation of hemangioblasts to hematopoietic cells in zebrafish embryos. The comparative screening of semaxanib (SU5416) (a known isatin derivatives), to compounds synthesized in this study, revealed the contrasting effects of these two classes of isatin derivatives on zebrafish hematopoiesis. Most of the N-alkyl-isatin-3-iminobenzoic acid derivatives were toxic on cancer and non-cancer tested human cells lines, however, the compounds 3c and 3f specifically affected the cell viability of Jurkat cells (human hematological cell line) with least IC50 values of 16.5 and 7.8 µM. The structure-activity relationship (SAR) analysis indicated that the substitution pattern of the isatin at the 5-position was vital for activity. The in vivo and in vitro biological activities of these compounds suggested their potential use as pharmaceutical compounds for human leukemia treatment.

In vitro and in vivo anti-leukemic effects of cladoloside C2 are mediated by activation of Fas/ceramide synthase 6/p38 kinase/c-Jun NH2-terminal kinase/caspase-8.

We previously demonstrated that the quinovose-containing hexaoside stichoposide C (STC) is a more potent anti-leukemic agent than the glucose-containing stichoposide D (STD), and that these substances have different molecular mechanisms of action. In the present study, we investigated the novel marine triterpene glycoside cladoloside C2 from Cladolabes schmeltzii, which has the same carbohydrate moiety as STC. We assessed whether cladoloside C2 could induce apoptosis in K562 and HL-60 cells. We also evaluated whether it showed antitumor action in mouse leukemia xenograft models, and its molecular mechanisms of action. We investigated the molecular mechanism behind cladoloside C2-induced apoptosis of human leukemia cells, and examined the antitumor effect of cladoloside C2 in a HL-60 and K562 leukemia xenograft model.Cladoloside C2 dose- and time-dependently induced apoptosis in the analyzed cells, and led to the activation of Fas/ceramide synthase 6 (CerS6)/p38 kinase/JNK/caspase-8. This cladoloside C2-induced apoptosis was partially blocked by specific inhibition by Fas, CerS6, and p38 siRNA transfection, and by specific inhibition of JNK by SP600125 or dominant negative-JNK transfection. Cladoloside C2 exerted antitumor activity through the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 without showing any toxicity in xenograft mouse models. The antitumor effect of cladoloside C2 was reversed in CerS6 shRNA-silenced xenograft models. Our results suggest that cladoloside C2 has in vitro and in vivo anti-leukemic effects due to the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 in lipid rafts. These findings support the therapeutic relevance of cladoloside C2 in the treatment of human leukemia.

New strategies for targeting leukemia stem cells

In this presentation, we will document our new strategies for the identification and ex vivo growth of primary leukemia stem cells. We will also show how we can exploit these newly developed methodologies to screen for compounds that specifically synergize to destroy leukemia stem cells. This includes cellular response to compounds and correlations of such responses to genetic anomalies found in each leukemia specimen. Redundancy of synthetic lethality between specimens appears to be predictable by companion tests which are being developed by our group. This chemogenomic project has the potential to bring paradigm shifts in the treatment of human leukemia.

Antileukemia multifunctionality of CD4(+) T cells genetically engineered by HLA class I-restricted and WT1-specific T-cell receptor gene transfer.

To develop gene-modified T-cell-based antileukemia adoptive immunotherapy, concomitant administration of CD4(+) and CD8(+) T cells that have been gene modified using identical HLA class I-restricted leukemia antigen-specific T-cell receptor (TCR) gene transfer has not yet been fully investigated. Here, using CD4(+) and CD8(+) T cells that had been gene modified with a retroviral vector expressing HLA-A*24:02-restricted and Wilms' tumor 1 (WT1)-specific TCR-α/β genes and siRNAs for endogenous TCRs (WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells), we examined the utility of this strategy. WT1-siTCR/CD4(+) T cells sufficiently recognized leukemia cells in an HLA class I-restricted manner and provided target-specific Th1 help for WT1-siTCR/CD8(+) T cells. By using a xenografted mouse model, we found that WT1-siTCR/CD4(+) T cells migrated to leukemia sites and subsequently attracted WT1-siTCR/CD8(+) T cells via chemotaxis. Therapy-oriented experiments revealed effective enhancement of leukemia suppression mediated by concomitant administration of WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells. Importantly, this augmented efficacy in the presence of WT1-siTCR/CD4(+) T cells was correlated with longer survival and enhanced formation of memory T cells by WT1-siTCR/CD8(+) T cells. Collectively, our experimental findings strongly suggest that this strategy would be clinically advantageous for the treatment of human leukemia.

Establishment of the human acute promyelocytic leukemia pathological model in the SCID beige mice by using NB4 cell line

This study was to establish a stable, effective and reproducible human acute promyelocytic leukemia model in severe combined immunodeficient (SCID) mice by using NB4 cell line, and to investigate the disease course character and biological behaviors. Three-five-week-old SCID beige mice were divided randomly into two groups: experimental and control group. SCID mice of experimental group were transplanted by tail vein (iv) injection of 5×10(6) NB4 cells. The WBC cell count and the positive rate of promyelocytes in peripheral blood were dynamically monitored by using smears. Morphological examination and histopathological assay were employed to confirm NB4 cell infiltration in organs (liver, spleen, lung, kidney and brain). The expression level of PML-RARα fusion protein was detected by Western blot. Within two weeks there was no significant difference in peripheral blood WBC count between two groups (P > 0.05), meanwhile, NB4 cells were not found. At the day 21 and 28 after inoculation, the peripheral blood white blood cell count of experimental group reached to (4.79 ± 1.13)×10(9)/L and (7.62 ± 2.24)×10(9)/L respectively, which were significantly higher than that in control group (P < 0.05); simultaneously, the positive rates of promyelocytes on smears were (2.14 ± 0.63)% and (6.6 ± 2.76)%, respectively. Morphological observation showed single or multiple tumor lumps at day 21 after inoculation; HE staining of tissue biopsies demonstrated a large number of promyelocyte in the liver, spleen, lung, kidney and brain tissue. Cell immunofluorescence results showed that the CD33 expression of bone marrow cells in mice of experimental group was strongly positive (P < 0.05). Western blot confirmed that the PML-RARα fusion protein was expressed variously in liver, kidney and brain tissue. The human acute promyelocytic leukemia SCID mouse model is succesfully established by tail vein injection of NB4 cells. This model can mimic the characters of involved bone marrow and diffuse growth of cells. This model is a useful tool to explore the pathogenic mechanism and experimental treatment of human leukemia.

Thiamet-G-mediated inhibition of O-GlcNAcase sensitizes human leukemia cells to microtubule-stabilizing agent paclitaxel

Although the microtubule-stabilizing agent paclitaxel has been widely used for treatment of several cancer types, particularly for the malignancies of epithelia origin, it only shows limited efficacy on hematological malignancies. Emerging roles of O-GlcNAcylation modification of proteins in various cancer types have implicated the key enzymes catalyzing this reversible modification as targets for cancer therapy. Here, we show that the highly selective O-GlcNAcase (OGA) inhibitor thiamet-G significantly sensitized human leukemia cell lines to paclitaxel, with an approximate 10-fold leftward shift of IC50. Knockdown of OGA by siRNAs or inhibition of OGA by thiamet-G did not influence the cell viability. Furthermore, we demonstrated that thiamet-G binds to OGA in competition with 4-methylumbelliferyl N-acetyl-β-d-glucosaminide dehydrate, an analogue of O-GlcNAc UDP, thereby suppressing the activity of OGA. Importantly, inhibition of OGA by thiamet-G decreased the phosphorylation of microtubule-associated protein Tau and caused alterations of microtubule network in cells. It is noteworthy that paclitaxel combined with thiamet-G resulted in more profound perturbations on microtubule stability than did either one alone, which may implicate the underlying mechanism of thiamet-G-mediated sensitization of leukemia cells to paclitaxel. These findings thus suggest that a regimen of paclitaxel combined with OGA inhibitor might be more effective for the treatment of human leukemia.

Oxymatrine triggers apoptosis by regulating Bcl-2 family proteins and activating caspase-3/caspase-9 pathway in human leukemia HL-60 cells

With the objective of identifying promising antitumor agents for human leukemia, we carried out to determine the anticancer ability of oxymatrine on the human leukemia HL-60 cell line. In vitro experiments demonstrated that oxymatrine reduced the proliferation of HL-60 cells in a dose- and time-dependent manner via the induction of apoptosis and cell cycle arrest at G2/M and S phases. The proteins involved in oxymatrine-induced apoptosis in HL-60 cells were also examined using Western blot. The increase in apoptosis upon treatment with oxymatrine was correlated with downregulation of anti-apoptotic Bcl-2 expression and upregulation of pro-apoptotic Bax expression. Furthermore, oxymatrine induced the activation of caspase-3 and caspase-9 and the cleavage of poly(ADP-ribose) polymerase (PARP) in HL-60 cells. In addition, pretreatment with a specific caspase-3 (Z-DEVD-FMK) or caspase-9 (Z-LEHD-FMK) inhibitor significantly neutralized the pro-apoptotic activity of oxymatrine in HL-60 cells, demonstrating the important role of caspase-3 and caspase-9 in this process. Taken together, these results indicated that oxymatrine-induced apoptosis may occur through the activation of the caspase-9/caspase-3-mediated intrinsic pathway. Therefore, oxymatrine may be a potential candidate for the treatment of human leukemia.

Expression and Regulation of 11-β Hydroxysteroid Dehydrogenase Type 2 Enzyme Activity in the Glucocorticoid-Sensitive CEM-C7 Human Leukemic Cell Line

Glucocorticoids are commonly used in the first-line treatment of hematological malignancies, such as acute lymphoblastic leukemia, due to the ability of these steroids to activate pro-apoptotic pathways in human lymphocytes. The goal of the current study was to examine the gene expression and enzyme activity of the microsomal enzyme, 11-β hydroxysteroid dehydrogenase type 2 (HSD11B2, HSD2), which is responsible for the oxidation of bioactive glucocorticoids to their inert metabolites. Using the glucocorticoid-sensitive human leukemic cell line, CEM-C7, we were able to detect the expression of HSD2 at the level of mRNA (via RT-PCR), protein (via immunohistochemistry and immunoblotting), and enzyme activity (via conversion of tritiated cortisol to cortisone). Furthermore, we observed that HSD2 enzyme activity is down regulated in CEM-C7 cells that were pretreated with the synthetic glucocorticoid, dexamethasone (100 nM, <15 hours), and that this down regulation of enzyme activity is blocked by the administration of the glucocorticoid receptor antagonist, RU-486. Taken collectively, these data raise the possibility that the effectiveness of glucocorticoids in the treatment of human leukemias may be influenced by: (1) the ability of these neoplastic cells to metabolize glucocorticoids via HSD2 and (2) the ability of these steroids to regulate the expression of this key enzyme.

Intraspecific variation and cytotoxic evaluation of the essential oils from Oxandra sessiliflora R. E. Fries

For the first time, the chemical composition and cytotoxic evaluation of essential oils from leaves of Oxandra sessiliflora R. E. Fries (Annonaceae) has been reported. Leaves of O. sessiflora were collected in four different periods during the years 2010 (September and December) and 2011 (March and June). The essential oils, obtained by hydrodistillation procedures, were analyzed by Gas chromatography (GC) and Gas chromatography-Mass spectrometry (GC-MS). Chemically, the predominance of hydrocarbon sesquiterpenes was detected (57.54 to 75.06%), with germacrene D (17.12 to 32.20%) and biclyclogermacrene (7.95 to 18.91%) as the main compounds. These oils were also composed of monoterpenes (9.65 to 17.02%) and oxygenated sesquiterpenes (4.42 to 11.43%). Additionally, the essential oils obtained from leaves collected in September, 2010 and March, 2011 were tested in vitro against murine (B16F10-Nex2) and human melanoma (A2058), breast adenocarcinoma (MCF7), leukemia (HL-60), and cervical carcinoma (HeLa) cell lines. Since values of IC50 to HL-60 cells were determined as approximately 6 µg/mL in both tested oils, the results suggested that the essential oils and their compounds could be used as prototypes for the development of new medicines for the treatment of human leukemia. Key words: Oxandra sessiliflora, essential oil composition, intraspecific variation, in vitrocytotoxic potential.

Anti-Proliferative Activities and Apoptosis Induction by Triterpenes Derived from Eriobotrya japonica in Human Leukemia Cell Lines

Eriobotrya japonica leaf is a traditional herbal medicine that contains numerous triterpenes, which have various pharmacological properties. In this study, we investigated the anti-proliferative activity of four triterpenes derived from E. japonica, including corosolic acid (CA), ursolic acid (UA), maslinic acid (MA) and oleanolic acid (OA), in human leukemia cell lines. CA showed the strongest anti-proliferative activity in all of the leukemia cell lines tested, but not in normal human skin fibroblast cell lines. To determine the mechanism underlying the anti-proliferative effect of CA, we examined the effect of CA on molecular events known as apoptosis induction. CA induced chromatin condensation, DNA fragmentation, sub-G1 phase DNA, activation of caspase-3, -8 and -9 and the cleavage of PARP in HL-60. CA also activated Bid and Bax, leading to the loss of mitochondrial membrane potential (Δψm) and cytochrome c release into the cytosol, whereas Bcl-2 and Bcl-xL were unaffected by CA. These results suggest that CA has an anti-proliferative effect on leukemia cells via the induction of apoptosis mediated by mitochondrial dysfunction and caspase activation. CA may be a potential chemotherapeutic agent for the treatment of human leukemia.

Chemical Constituents and Cytotoxic Evaluation of Essential Oils from Leaves of Porcelia macrocarpa (Annonaceae)

This work reports the chemical composition and cytotoxic evaluation of the essential oils from three different samples of the leaves of Porcelia macrocarpa R. E. Fries (Annonaceae). The crude oils, obtained by hydrodistillation procedures, were chemically analyzed by GC/MS. The obtained data indicated the predominance of sesquiterpenes (89.8 +/- 0.7%), the main compounds being germacrene D (47 +/-+/- 1%) and bicyclogermacrene (37 +/- 1%). These oils also contained the monoterpene verbanyl acetate (0.5 +/- 0.06%) and the diterpene phytol (1.2 +/- 0.3%). The crude oils obtained from leaves were pooled and tested in vitro against six cancer cell lineages--murine melanoma (B16F10-Nex2), human glioblastome (U87), human cervical carcinoma (HeLa), human leukemia (HL-60), human colon carcinoma (HCT), human breast adenocarcinoma (SKBr), and human melanoma (A2058), as well as against a non-tumorigenic human cell line (HFF). Since the essential oil reduced more than 50% of the viability of several tumor cells at 100 microg/mL, indicating the presence of active compounds, the crude material was subjected to fractionation over a SiO2/AgNO3 column. This procedure afforded different fractions composed of pure as well as different mixtures of bicyclogermacrene and germacrene D, which were tested against the same tumor cell lines, indicating a significant cytotoxic potential against HL-60 cells. These results suggested that the crudeoils and their components, mainly germacrene D, could be used as prototypes for the development of new anti-cancer agents for the treatment of human leukemia.

Apoptosis triggered by vitexin in U937 human leukemia cells via a mitochondrial signaling pathway

Vitexin, a lignan compound, has been shown to exert apoptotic actions on human breast cancer cell lines and to have anti-inflammatory activities. Nevertheless, there is currently no study addressing the effects of vitexin on the induction of apoptosis in U937 human leukemia cells. The aim of this study was to determine the anticancer effects and molecular mechanisms of vitexin on U937 leukemia cells. We showed that vitexin can potently induce programmed cell death in U937 leukemia cell growth as well as morphological changes that were examined by MTT assay and phase contrast microscopy, respectively. The DNA content and the levels of mitochondrial membrane potential (∆Ψm) were determined by flow cytometric analysis. The cell cycle arrest-regulated and apoptosis-associated protein levels were measured by western blotting. Vitexin-triggered apoptosis was accompanied by a decrease of the level of ∆Ψm and the percentage of viability and provoked apoptosis in U937 cells. The downregulation of the protein level for Bcl-2 with the simultaneous upregulation of caspase-3 and -9 protein expression in U937 cells were observed after treatment with vitexin. Therefore, our data provide a potential mechanism for the chemopreventive activity of vitexin, and we suggest that vitexin may serve as a therapeutic agent for the treatment of human leukemia.

Synthesis and biological evaluation of novel AM80 derivatives as antileukemic agents

A series of novel AM80 derivatives as antileukemic agents were synthesized and their structures were confirmed by IR, 1H-NMR, and HR-MS spectra. All the target compounds were evaluated for in vitro antiproliferative activities against human leukemic HL-60, NB4, and K562 cell lines. Among these derivatives, compound 4g showed much stronger antiproliferative activities against all the three human leukemic cell lines than the positive control AM80, and compound 4b exhibited more active antiproliferative effects against HL-60 and K562 cells than AM80. Furthermore, the preliminary SAR analysis suggested that AM80 conjugating with HDAC inhibitors with small steric hindrance could give more effective antileukemic agents. These results would be helpful to design more potent antileukemic drugs for the treatment of human leukemia.

Aurora kinase A-specific T-cell receptor gene transfer redirects T lymphocytes to display effective antileukemia reactivity

AbstractAurora kinase A (AURKA) is overexpressed in leukemias. Previously, we demonstrated that AURKA-specific CD8+ T cells specifically and selectively lysed leukemia cells, indicating that AURKA is an excellent target for immunotherapy. In this study, we examined the feasibility of adoptive therapy using redirected T cells expressing an HLA-A*0201–restricted AURKA207-215-specific T-cell receptor (TCR). Retrovirally transduced T cells recognized relevant peptide-pulsed but not control target cells. Furthermore, TCR-redirected CD8+ T cells lysed AURKA-overexpressing human leukemic cells in an HLA-A*0201–restricted manner, but did not kill HLA-A*0201+ normal cells, including hematopoietic progenitors. In addition, AURKA207-215-specific TCR-transduced CD4+ T cells displayed target-responsive Th1 cytokine production. Finally, AURKA207-215-specific TCR-transduced CD8+ T cells displayed antileukemia efficacy in a xenograft mouse model. Collectively, these data demonstrate the feasibility of redirected T cell–based AURKA-specific immunotherapy for the treatment of human leukemia.

Redirected CD4+ T Cells Using WT1-Specific T-Cell Receptor Gene Transfer Can Supply Multifactorial Help to Enhance the Anti-Leukemia Reactivity Mediated by Similarly Redirected CD8+ T Cells Using the Identical Gene Transfer

Abstract 645[Purpose] Redirected T-cell-based anti-cancer adoptive therapy using cancer antigen-specific T-cell receptor (TCR) gene transfer has clinically shown promise, however there still remain considerable issues of achieving better clinical efficacy. For this purpose, helper function provided by concurrent CD4+ T cells should be evidently considerable. In this study, to achieve the enhanced anti-leukemia functionality mediated by redirected CD8+ T cells using WT1-specific TCR gene transfer, we in detail examined functionalities mediated by similarly redirected CD4+ T cells using the identical TCR gene transfer.[Methods] HLA-A*24:02-restricted and WT1235-243-specific codon-optimized TCR α/β genes were inserted into the novel retroviral vector encoding shRNAs for endogenous TCRs (WT1-siTCR vector). (1) Cognate antigen-responsive cytokine production mediated by WT1-siTCR transduced CD4+ T cells was assessed using cytokine beads array and ELISA assay. (2) Expression of CD40L and OX40 on redirected CD4+ T cells stimulated by WT1 peptide ligation was assessed using flow cytometer. (3) Impact caused by redirected CD4+ T cells on each magnitude of WT1-specific cytotoxicity, target-responsive proliferation and transition to central memory T-cell phenotype of WT1-siTCR transduced CD8+ T cells was measured using 51Cr-release assay, CD107a assay, intracellular IFN-γ assay and CFSE assay. (4) Chemokines produced by redirected CD4+ T cells stimulated using WT1 peptide was comprehensively assessed using real-time PCR. Consequent chemotaxis of redirected CD8+ T cells toward those stimulated redirected CD4+ cells was validated using transwell experiments. (6) Finally, anti-leukemia reactivity against autologous leukemia cells mediated by patients’ redirected CD8+ T cells was similarly examined in the presence or absence of such autologous CD4+ T cells. [Results]First, in this study, those redirected CD4+ T cells hardly became positive for intracellular FoxP3, a crucial marker for regulatory T cell phenotype. In response to the WT1 peptide, WT1-siTCR transduced CD4+ T cells produced Th1 cytokines; IL-2, IFN-γ and TNF-α, in the context of HLA-A*24:02, which also needed HLA class II molecules on target cells. Magnitudes of WT1-responsive CD107a expression, IFN-γ production and cytotoxicity mediated by WT1-siTCR transduced CD8+ T cells were efficiently enhanced in the presence of redirected, but not non-redirected CD4+ T cells. Similarly, in the presence of those redirected CD4+ T cells, redirected CD8+ T cells expressing WT1-specific TCR increased in number and the transition to central memory T-cell phenotype (CD45RA−CD62L+) of those CD8+ T cells was stimulated in response to the stimulation with WT1 peptide. WT1 peptide ligation stimulated those redirected CD4+ T cells to express membrane-bound OX40, which is involved in the formation of central memory CD8+ T cells. WT1 peptide ligation also stimulated the redirected CD4+ T cells to produce chemokines; CCL3 and CCL4. Redirected CD8+ T cells expressed the receptor for these chemokines, CCR5; efficient migration of redirected CD8+ T cells toward redirected CD4+ T cells stimulated with WT1 peptide was obviously observed in transwell experiments. Finally, redirected CD4+ T cells isolated from patients with leukemia successfully provided Th1 helper function to autologous redirected CD8+ T cells to enhance the anti-leukemia reactivity; cytotoxicity, proliferation and formation of central memory T cells, in response to autologous leukemia cells, in vitro. [Conclusion]Although further investigations are warranted, concurrently adopted WT1-siTCR introduced CD4+ T cells seems feasible to enhance the efficacy of WT1-targeting redirected T-cell-based adoptive therapy for treatment of human leukemia. Disclosures:No relevant conflicts of interest to declare.