Diterpene promptly executes a non-canonical autophagic cell death in doxorubicin-resistant lung cancer.

16-hydroxycleroda-3,13-dien-15,16-olide (HCD) has antitumor activity reported in numerous types of cancers. However, the efficacy of HCD treatment in non-small-cell lung cancer (NSCLC) cells and doxorubicin-resistant (Dox-R)-NSCLC cells remains to be unraveled. The underlying anti-cancer mechanism of HCD on Dox-R and Dox-sensitive (Dox-S) of A549 cells was also investigated. Cytotoxicity of HCD against two cell lines (Dox-S and Dox-R) were determined via MTT assay, flow cytometry, and Western blot. A further examination of its anti-cancer efficacy was performed in A549-bearing xenograft mice via orthotopic intratrachea (IT) inoculation, which showed that HCD could arrest both Dox-S and Dox-R cells at G2/M phase without altering the sub-G1 cycle along with increasing of cleaved-PARP. HCD downregulated the mTOR/Akt/PI3K-p85 and PI3K-ClassIII/Beclin-1 signals and upregulated p62/LC3-I/II expressions to further confirm that the cell autophagy of NSCLC cells after being HCD-induced. Morphological observations of mouse lung sections illustrated that fewer cancer cells accumulated close to the trachea while less neoplastic activities were found in HCD orthotopic treated mice without liver, kidney, and spleen toxicity. Lastly, Dox, HCD, and target therapy medicines of EGFR and ALK were nicely docked with EGFR, ALK, and mTOR. Conclusively, HCD was demonstrated the chemotherapeutic potential regardless of Dox-R and Dox-S cells, suggesting natural autophagic inducer HCD provides a promising lead compound for new drug discovery and development of lung cancer therapies.

Doxorubicin metabolism moderately attributes to putative toxicity in prodigiosin/doxorubicin synergism in vitro cells.

Doxorubicin (Dox) is a widely neoplasm chemotherapeutic drug with high incidences of cardiotoxicity. Prodigiosin (PG), a red bacterial pigment from Serratia marcescens, has been demonstrated to potentiate Dox's cytotoxicity against oral squamous cell carcinoma cells through elevating Dox influx and identified as a Dox enhancer via PG-induced autophagy; however, toxicity of normal cell remains unclear. This study is conducted to evaluate putative cytotoxicity features of PG/Dox synergism in the liver, kidney, and heart cells and further elucidate whether PG augmented Dox's effect via modulating Dox metabolism in normal cells. Murine hepatocytes FL83B, cardio-myoblast h9c2, and human kidney epithelial cells HK-2 were sequentially treated with PG and Dox by measuring cell viability, cell death characteristics, oxidative stress, Dox flux, and Dox metabolism. PG could slightly significant increase Dox cytotoxicity in all tested normal cells whose toxic alteration was less than that of oral squamous carcinoma cells. The augmentation of Dox cytotoxicity might be attributed to the increase of Dox-mediated ROS accumulation that might cause slight reduction of Dox influx and reduction of Dox metabolism. It was noteworthy to notice that sustained cytotoxicity appeared in normal cells after PG and Dox were removed. Taken together, moderately metabolic reduction of Dox might be ascribed to the mechanism of increase Dox cytotoxicity in PG-induced normal cells; nevertheless, the determination of PG/Dox dose with sustained cytotoxicity in normal cells needs to be comprehensively considered.

8-chloro-adenosine activity in FLT3-ITD acute myeloid leukemia.

Nucleoside analogs represent the backbone of several distinct chemotherapy regimens for acute myeloid leukemia (AML) and combination with tyrosine kinase inhibitors has improved survival of AML patients, including those harboring the poor-risk FLT3-ITD mutation. Although these compounds are effective in killing proliferating blasts, they lack activity against quiescent leukemia stem cells (LSCs), which contributes to initial treatment refractoriness or subsequent disease relapse. The reagent 8-chloro-adenosine (8-Cl-Ado) is a ribose-containing, RNA-directed nucleoside analog that is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. In this report, we demonstrate antileukemic activities of 8-Cl-Ado in vitro and in vivo and provide mechanistic insight into the mode of action of 8-Cl-Ado in AML. 8-Cl-Ado markedly induced apoptosis in LSC, with negligible effects on normal stem cells. 8-Cl-Ado was particularly effective against AML cell lines and primary AML blast cells harboring the FLT3-ITD mutation. FLT3-ITD is associated with high expression of miR-155. Furthermore, we demonstrate that 8-Cl-Ado inhibits miR-155 expression levels accompanied by induction of DNA-damage and suppression of cell proliferation, through regulation of miR-155/ErbB3 binding protein 1(Ebp1)/p53/PCNA signaling. Finally, we determined that combined treatment of NSG mice engrafted with FLT3-ITD + MV4-11 AML cells with 8-Cl-Ado and the FLT3 inhibitor AC220 (quizartinib) synergistically enhanced survival, compared with that of mice treated with the individual drugs, suggesting a potentially effective approach for FLT3-ITD AML patients.

Isolation and Analysis of Mesenchymal Progenitors of the Adult Hematopoietic Niche.

Mesenchymal stromal cells are an important component of the adult hematopoietic stem cell niche. They are a diverse population of cells that include a hierarchy of primitive, intermediate, and mature osteoprogenitors that support HSCs and supply the bone with matrix producing osteoblast. To understand the different roles played by individual types of progenitors, it is necessary to separate individual populations and analyze them in a controlled environment. Here we describe two transplantation models, an ectopic bone forming assay and an intravenous injection assay, in which niche components can be isolated and manipulated to dissect their individual properties.

Bone marrow niche trafficking of miR-126 controls the self-renewal of leukemia stem cells in chronic myelogenous leukemia.

Leukemia stem cells (LSCs) in individuals with chronic myelogenous leukemia (CML) (hereafter referred to as CML LSCs) are responsible for initiating and maintaining clonal hematopoiesis. These cells persist in the bone marrow (BM) despite effective inhibition of BCR-ABL kinase activity by tyrosine kinase inhibitors (TKIs). Here we show that although the microRNA (miRNA) miR-126 supported the quiescence, self-renewal and engraftment capacity of CML LSCs, miR-126 levels were lower in CML LSCs than in long-term hematopoietic stem cells (LT-HSCs) from healthy individuals. Downregulation of miR-126 levels in CML LSCs was due to phosphorylation of Sprouty-related EVH1-domain-containing 1 (SPRED1) by BCR-ABL, which led to inhibition of the RAN-exportin-5-RCC1 complex that mediates miRNA maturation. Endothelial cells (ECs) in the BM supply miR-126 to CML LSCs to support quiescence and leukemia growth, as shown using mouse models of CML in which Mir126a (encoding miR-126) was conditionally knocked out in ECs and/or LSCs. Inhibition of BCR-ABL by TKI treatment caused an undesired increase in endogenous miR-126 levels, which enhanced LSC quiescence and persistence. Mir126a knockout in LSCs and/or ECs, or treatment with a miR-126 inhibitor that targets miR-126 expression in both LSCs and ECs, enhanced the in vivo anti-leukemic effects of TKI treatment and strongly diminished LSC leukemia-initiating capacity, providing a new strategy for the elimination of LSCs in individuals with CML.

Antileukemic activity and cellular effects of the antimalarial agent artesunate in acute myeloid leukemia.

The artimisinins are a class of antimalarial compounds whose antiparasitic activity is mediated by induction of reactive oxygen species (ROS). Herein, we report that among the artimisinins, artesunate (ARTS), an orally bioavailable compound has the most potent antileukemic activity in AML models and primary patients' blasts. ARTS was most cytotoxic to the FLT3-ITD+ AML MV4-11 and MOLM-13 cells (IC50 values of 1.1 and 0.82µM respectively), inhibited colony formation in primary AML and MDS cells and augmented cytotoxicity of chemotherapeutics. ARTS lowered cellular BCL-2 level via ROS induction and increased the cytotoxicity of the BCL-2 inhibitor venetoclax (ABT-199). ARTS treatment led to cellular and mitochondrial ROS accumulation, double stranded DNA damage, loss of mitochondrial membrane potential and induction of the intrinsic mitochondrial apoptotic cascade in AML cell lines. The antileukemic activity of ARTS was further confirmed in MV4-11 and FLT3-ITD+ primary AML cell xenografts as well as MLL-AF9 syngeneic murine AML model where ARTS treatment resulted in significant survival prolongation of treated mice compared to control. Our results demonstrate the potent preclinical antileukemic activity of ARTS as well as its potential for a rapid transition to a clinical trial either alone or in combination with conventional chemotherapy or BCL-2 inhibitor, for treatment of AML.

Identification of a CD133-CD55- population functions as a fetal common skeletal progenitor.

In this study, we identified a CD105+CD90.1-CD133-CD55- (CD133-CD55-) population in the fetal skeletal element that can generate bone and bone marrow. Besides osteoblasts and chondrocytes, the CD133-CD55- common progenitors can give rise to marrow reticular stromal cells and perivascular mesenchymal progenitors suggesting they function as the fetal common skeletal progenitor. Suppression of CXCL12 and Kitl expression in CD133-CD55- common progenitors severely disrupted the BM niche formation but not bone generation. Thus, CD133-CD55- common progenitors are the main source of CXCL12 and Kitl producing cells in the developing marrow.

Identification of a common mesenchymal stromal progenitor for the adult haematopoietic niche.

Microenvironment cues received by haematopoietic stem cells (HSC) are important in regulating the choice between self-renewal and differentiation. On the basis of the differential expression of cell-surface markers, here we identify a mesenchymal stromal progenitor hierarchy, where CD45-Ter119-CD31-CD166-CD146-Sca1+(Sca1+) progenitors give rise to CD45-Ter119-CD31-CD166-CD146+(CD146+) intermediate and CD45-Ter119-CD31-CD166+CD146-(CD166+) mature osteo-progenitors. All three progenitors preserve HSC long-term multi-lineage reconstitution capability in vitro; however, their in vivo fates are different. Post-transplantation, CD146+ and CD166+ progenitors form bone only. While Sca1+ progenitors produce CD146+, CD166+ progenitors, osteocytes and CXCL12-producing stromal cells. Only Sca1+ progenitors are capable of homing back to the marrow post-intravenous infusion. Ablation of Sca1+ progenitors results in a decrease of all three progenitor populations as well as haematopoietic stem/progenitor cells. Moreover, suppressing production of KIT-ligand in Sca1+ progenitors inhibits their ability to support HSCs. Our results indicate that Sca1+ progenitors, through the generation of both osteogenic and stromal cells, provide a supportive environment for hematopoiesis.

CBFß-SMMHC creates aberrant megakaryocyte-erythroid progenitors prone to leukemia initiation in mice.

Acute myeloid leukemia (AML) arises through multistep clonal evolution characterized by stepwise accumulation of successive alterations affecting the homeostasis of differentiation, proliferation, self-renewal, and survival programs. The persistence and dynamic clonal evolution of leukemia-initiating cells and preleukemic stem cells during disease progression and treatment are thought to contribute to disease relapse and poor outcome. Inv(16)(p13q22) or t(16;16)(p13.1;q22), one of the most common cytogenetic abnormalities in AML, leads to expression of a fusion protein CBFß-SMMHC (CM) known to disrupt myeloid and lymphoid differentiation. Anemia is often observed in AML but is presumed to be a secondary consequence of leukemic clonal expansion. Here, we show that CM expression induces marked deficiencies in erythroid lineage differentiation and early preleukemic expansion of a phenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitor population. Using dual-fluorescence reporter mice in lineage tracking and repopulation assays, we show that CM expression cell autonomously causes expansion of abnormal Pre-Meg/E progenitors with compromised erythroid specification and differentiation capacity. The preleukemic Pre-Meg/Es display dysregulated erythroid and megakaryocytic fate-determining factors including increased Spi-1, Gata2, and Gfi1b and reduced Zfpm1, Pf4, Vwf, and Mpl expression. Furthermore, these abnormal preleukemic Pre-Meg/Es have enhanced stress resistance and are prone to leukemia initiation upon acquiring cooperative signals. This study reveals that the leukemogenic CM fusion protein disrupts adult erythropoiesis and creates stress-resistant preleukemic Pre-Meg/E progenitors predisposed to malignant transformation. Abnormality in Meg/E or erythroid progenitors could potentially be considered an early predictive risk factor for leukemia evolution.

Exosome-mediated microenvironment dysregulation in leukemia.

The hematopoietic stem cell (HSC) niche is composed of a complex set of stromal support cells that maintain HSCs and promote normal hematopoiesis. We now know that molecular changes within the hematopoietic niche contribute to leukemia development. Leukemia cells often reorganize the hematopoietic niche to promote and support their own survival and growth. Here we will summarize recent works that decipher the normal hematopoietic niche cellular components and describe how the leukemia-transformed niche contributes to hematological malignances. Finally, we will discuss recent publications that highlight a possible role for exosomes in the leukemia-induced niche reorganization. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.

Evidence that ß7 Integrin Regulates Hematopoietic Stem Cell Homing and Engraftment Through Interaction with MAdCAM-1.

α4ß7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis is controversial. We identified a subset of hematopoietic stem cells (HSCs) in the bone marrow (BM) that expressed ß7 integrin. These ß7(+) HSCs were capable of multilineage, long-term reconstitution and had an inherent competitive advantage over ß7(-) HSCs. On the other hand, HSCs that lacked ß7 integrin (ß7KO) had reduced engraftment potential. Interestingly, quantitative RT-PCR and flow cytometry revealed that ß7KO HSCs expressed lower levels of the chemokine receptor CXCR4. Accordingly, ß7KO HSCs exhibited impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4ß7 integrin ligand-mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on BM endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that ß7 integrin, when expressed by HSCs, interacted with its endothelial ligand MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment.

RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells.

Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.

CD150(-) Side Population Defines Leukemia Stem Cells in a BALB/c Mouse Model of CML and Is Depleted by Genetic Loss of SIRT1.

Leukemia stem cells (LSCs) of chronic myeloid leukemia (CML) are refractory to tyrosine kinase inhibitor treatment, persist in the residual disease, and are important source for disease recurrence. Better understanding CML LSCs will help devise new strategies to eradicate these cells. The BALB/c mouse model of CML using retroviral bone marrow transduction and transplantation is a widely used mouse model system for CML, but LSCs in this model are poorly characterized. Here, we show that lineage negative CD150(-) side population (CD150(-)SP), but not CD150(+)SP, are CML LSCs in this model, although both CD150(-)SP and CD150(+)SP cells are enriched for long-term hematopoietic stem cells in normal BALB/c mice. We previously showed that BCR-ABL transformation activates protein lysine deacetylase SIRT1 and inhibition of SIRT1 sensitizes CML stem/progenitor cells to tyrosine kinase inhibitors by acetylating and activating p53. In this study, we demonstrate that SIRT1 homozygous knockout substantially reduces CD150(-)SP CML LSCs, and compromises the maintenance of CML LSCs in the BALB/c model. We identified several molecular alterations in CD150(-)SP LSCs that included the elevated expression of cyclin-dependent kinase Cdk6 facilitating LSC activation and significantly reduced p53 expression. SIRT1 knockout suppressed Cdk6 expression and likely increases p53 protein functions through deacetylation without increasing its expression. Our results shed novel insight into CML LSCs and support a crucial role of SIRT1 in CML LSCs. Our study also provides a novel means for assessing new agents to eradicate CML LSCs.

Altered lymphopoiesis and immunodeficiency in miR-142 null mice.

MicroRNAs (miRNAs) are a class of powerful posttranscriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is preferentially and abundantly expressed in immune cells, we created a mouse line with a targeted deletion of this gene. Our analysis of miR-142(-/-) mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, whereas the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142(-/-) animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142(-/-) B cells express elevated levels of B-cell-activating factor (BAFF) receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142(-/-) mice rescues the B-cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B-cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphopoiesis, and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.

Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development.

Hematopoietic stem cells (HSCs) reside in regulatory niches in the bone marrow (BM). Although HSC niches have been extensively characterized, the role of endosteal osteoblasts (OBs) in HSC regulation requires further clarification, and the role of OBs in regulating leukemic stem cells (LSCs) is not well studied. We used an OB visualization and ablation mouse model to study the role of OBs in regulating normal HSCs and chronic myelogenous leukemia (CML) LSCs. OB ablation resulted in increase in cells with a LSK Flt3(-)CD150(+)CD48(-) long-term HSC (LTHSC) phenotype but reduction of a more highly selected LSK Flt3(-)CD34(-)CD49b(-)CD229(-) LTHSC subpopulation. LTHSCs from OB-ablated mice demonstrated loss of quiescence and reduced long-term engraftment and self-renewal capacity. Ablation of OB in a transgenic CML mouse model resulted in accelerated leukemia development with reduced survival compared with control mice. The notch ligand Jagged-1 was overexpressed on CML OBs. Normal and CML LTHSCs cultured with Jagged-1 demonstrated reduced cell cycling, consistent with a possible role for loss of Jagged-1 signals in altered HSC and LSC function after OB ablation. These studies support an important role for OBs in regulating quiescence and self-renewal of LTHSCs and a previously unrecognized role in modulating leukemia development in CML.

In vitro multilineage differentiation and self-renewal of single pancreatic colony-forming cells from adult C57BL/6 mice.

In a previous study we established colony assays suitable for studying murine adult (2-4 months) pancreatic progenitor cells plated in semisolid media containing methylcellulose and extracellular matrix proteins. Using these assays, we found robust in vitro progenitor cell activities (multilineage differentiation and self-renewal) from pancreatic cells of adult mice in the CD-1 outbred background. However, it was not clear whether progenitor cell activities can be detected from inbred mice, a preferred mouse model for various genetic studies. It was also not clear whether a single cell is sufficient to self-renew. Here, we show that fluorescent activated cell sorting pancreatic CD133(+) but not CD133(-) cells from adult C57Bl/6 inbred mice are enriched for progenitor cells that self-renew and give rise to multilineage colonies in vitro. The number of cells in a colony is in proportion to its diameter. Around 60% of single handpicked 3-week-old colonies express trilineage markers, indicating most progenitors are tripotent for ductal, acinar, and endocrine lineage differentiation. Approximately 80% of primary (freshly sorted) colony-forming progenitor cells are capable of giving rise to secondary progenitors in vitro, indicating that a majority of the primary progenitors self-renew. A single cell is sufficient for self-renewal and a Wnt agonist, R-Spondin1, enhances the number of secondary progenitors from the primary progenitors. Together, our pancreatic colony assays allow quantitative analyses of progenitors at a single-cell level from inbred mice. These assays will be useful for elucidating in vitro mechanisms of pancreatic progenitor cell biology.

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells.

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.