D3-induced Differentiation Research Articles

Evaluation of cyclin D3 as a transcriptional regulator in a neutrophil differentiation model.

Abstract Cyclin D3 is known to regulate cell cycle entry during the G1 to S transition by associating with its binding partners CDK4/6 upon mitogenic stimulation. Cyclin D3 has also been implicated as a transcriptional regulator, but the indirect mechanism employed is not known. The HL60 cell line was used as a model to investigate cyclin D3’s role in altered gene transcription as DMSO-driven differentiation into neutrophils is induced. We have validated this system by evaluating changes in gene expression indicative of progression to the terminal neutrophil state. Interestingly, ectopic expression of cyclin D3 in undifferentiated HL60 (uHL60) cells causes changes in gene expression to mirror that seen in differentiated (dHL60) cells without the need to treat with DMSO. We have also assessed a cohort of target genes hypothesized to be sensitive to cyclin D3-impacted gene transcription as cells transition from uHL60 to dHL60. For those genes that are differentially expressed between uHL60 and dHL60, many are also sensitive to ectopic expression of cyclin D3. Forced expression of cyclin D3 mutants in uHL60 cells suggests that a C-terminal domain within cyclin D3 is responsible for this observed regulation of gene expression, as three mutations (L215M, Q244R, P278R) abrogate the wild-type cyclin D3-induced differentiation gene signature. Ongoing studies aim to elucidate transcription factor binding partners that interact in complex with cyclin D3 to cause differential gene expression.

Roles of Glutathione and AP-1 in the Enhancement of Vitamin D-Induced Differentiation by Activators of the Nrf2 Signaling Pathway in Acute Myeloid Leukemia Cells.

Active vitamin D derivatives (VDDs)-1α,25-dihydroxyvitamin D3/D2 and their synthetic analogs-are well-known inducers of cell maturation with the potential for differentiation therapy of acute myeloid leukemia (AML). However, their dose-limiting calcemic activity is a significant obstacle to using VDDs as an anticancer treatment. We have shown that different activators of the NF-E2-related factor-2/Antioxidant Response Element (Nrf2/ARE) signaling pathway, such as the phenolic antioxidant carnosic acid (CA) or the multiple sclerosis drug monomethyl fumarate (MMF), synergistically enhance the antileukemic effects of various VDDs applied at low concentrations in vitro and in vivo. This study aimed to investigate whether glutathione, the major cellular antioxidant and the product of the Nrf2/ARE pathway, can mediate the Nrf2-dependent differentiation-enhancing activity of CA and MMF in HL60 human AML cells. We report that glutathione depletion using L-buthionine sulfoximine attenuated the enhancing effects of both Nrf2 activators concomitant with downregulating vitamin D receptor (VDR) target genes and the activator protein-1 (AP-1) family protein c-Jun levels and phosphorylation. On the other hand, adding reduced glutathione ethyl ester to dominant negative Nrf2-expressing cells restored both the suppressed differentiation responses and the downregulated expression of VDR protein, VDR target genes, as well as c-Jun and P-c-Jun levels. Finally, using the transcription factor decoy strategy, we demonstrated that AP-1 is necessary for the enhancement by CA and MMF of 1α,25-dihydroxyvitamin D3-induced VDR and RXRα protein expression, transactivation of the vitamin D response element, and cell differentiation. Collectively, our findings suggest that glutathione mediates, at least in part, the potentiating effect of Nrf2 activators on VDDs-induced differentiation of AML cells, likely through the positive regulation of AP-1.

Differential gene regulation by a synthetic vitamin D receptor ligand and active vitamin D in human cells

Vitamin D (VD) exerts a wide variety of biological functions including calcemic activity. VD nutritional status is closely associated with the onset and development of chronic diseases. To develop a VD analog with the desired VD activity but without calcemic activity, we screened synthetic VDR antagonists. We identified 1α,25-dihydroxyvitamin D3-26-23-lactams (DLAM)-2a-d (DLAM-2s) as nuclear vitamin D receptor (VDR) ligands in a competitive VDR binding assay for 1α,25(OH)2 vitamin D3 (1α,25(OH)2D3), and DLAM-2s showed an antagonistic effect on 1α,25(OH)2 D3-induced cell differentiation in HL60 cells. In a luciferase reporter assay in which human VDR was exogenously expressed in cultured COS-1 cells, DLAM-2s acted as transcriptional antagonists. Consistently, DLAM-2s had an antagonistic effect on the 1α,25(OH)2D3-induced expression of a known VD target gene [Cytochrome P450 24A1 (CYP24A1)], and VDR bound DLAM-2s was recruited to an endogenous VD response element in chromatin in human keratinocytes (HaCaT cells) endogenously expressing VDR. In an ATAC-seq assay, the effects of 1α,25(OH)2 D3 and DLAM-2b on chromatin reorganization were undetectable in HaCaT cells, while the effect of an androgen receptor (AR) antagonist (bicalutamide) was confirmed in prostate cancer cells (LNCaP) expressing endogenous AR. However, whole genome analysis using RNA-seq and ATAC (Assay for Transposase Accessible Chromatin)-seq revealed differential gene expression profiles regulated by DLAM-2b versus 1α,25(OH)2D3. The upregulated and downregulated genes only partially overlapped between cells treated with 1α,25(OH)2D3 and those treated with DLAM-2b. Thus, the present findings illustrate a novel VDR ligand with gene regulatory activity differing from that of 1α,25(OH)2D3.

The effect of caspase-9 in the differentiation of SH-SY5Y cells

Neuroblastoma is the most common solid malignant tumor in infants and young children. Its origin is the incompletely committed precursor cells from the autonomic nervous system. Neuroblastoma cells are multipotent cells with a high potency of differentiation into the neural cell types. Neural differentiation leads to the treatment of neuroblastoma by halting the cell and tumor growth and consequently its expansion. Caspases are a family of proteins involved in apoptosis and differentiation. The present study aimed to investigate the potential role of caspase-9 activation on the differentiation of the human neuroblastoma SH-SY5Y cells. Here we investigated the caspase-9 and 3/7 activity during 1,25-dihydroxycholecalciferol (D3)-mediated differentiation of SH-SY5Y cells and took advantage of the inducible caspase-9 system in putting out the differentiation of the neuroblastoma cells. D3-induced differentiation of the cells could lead to activation of caspase-9 and caspase-3/7, astrocyte-like morphology, and increased expression of Glial fibrillary acidic protein (GFAP). By using the inducible caspase-9 system, we showed differentiation of SH-SY5Y cells to astrocyte-like morphology and increased level of GFAP expression. Furthered studies using a specific caspase-9 inhibitor showed inhibition of differentiation mediated by D3 or caspase-9 to astrocyte-like cells. These results show the potency of caspase-9 to direct differentiation of the human neuroblastoma SH-SY5Y cells into cells showing an astrocyte-like morphology.

Myosin Phosphatase Is Implicated in the Control of THP-1 Monocyte to Macrophage Differentiation.

Monocyte to macrophage differentiation is characterized by the activation of various signal transduction pathways, which may be modulated by protein phosphorylation; however, the impact of protein kinases and phosphatases is not well understood yet. It has been demonstrated that actomyosin rearrangement during macrophage differentiation is dependent on Rho-associated protein kinase (ROCK). Myosin phosphatase (MP) target subunit-1 (MYPT1) is one of the major cellular substrates of ROCK, and MP is often a counter enzyme of ROCK; therefore, MP may also control macrophage differentiation. Changes in MP activity and the effects of MP activation were studied on PMA or l,25(OH)2D3-induced differentiation of monocytic THP-1 cells. During macrophage differentiation, phosphorylation of MYPT1 at Thr696 and Thr853 increased significantly, resulting in inhibition of MP. The ROCK inhibitor H1152 and the MP activator epigallocatechin-3-gallate (EGCG) attenuated MYPT1 phosphorylation and concomitantly decreased the extent of phosphorylation of 20 kDa myosin light chain. H1152 and EGCG pretreatment also suppressed the expression of CD11b and weakened the PMA-induced adherence of the cells. Our results indicate that MP activation/inhibition contributes to the efficacy of monocyte to macrophage differentiation, and this enzyme may be a target for pharmacological interventions in the control of disease states that are affected by excessive macrophage differentiation.

NLS-RARα contributes to differentiation block and increased leukemogenic potential in vivo

The fusion oncogene, promyelocytic leukemia (PML)-retinoic acid receptor-α (RARα), is crucial for acute promyelocytic leukemia (APL) pathogenesis. Previous studies have reported that PML-RARα is cleaved by neutrophil elastase (NE), an early myeloid-specific serine protease, leading to translocation of the nuclear localization signal (NLS) of the PML protein to the N-terminal of RARα. This study was designed to evaluate the value of NLS-RARα in the early diagnosis of APL. To investigate the potential functional role of NLS-RARα in leukemogenesis, HL-60 and U937 cell lines were transfected with NLS-RARα lentivirus and negative control (LVNC). The results showed that the induced expression of NLS-RARα down-regulated expressions of CD11b, CD11c, and CD14 compared to the LVNC group induced by 1α, 25-dihydroxyvitamin D3(1,25(OH)2D3). This suggested that NLS-RARα overexpression inhibited granulocytic and monocytic differentiation of myeloid leukemia cells. In addition, Wright-Giemsa staining, flow cytometry, respiratory burst assay, and NBT reduction assay all confirmed the importance of NLS-RARα in differentiation. The mechanistic investigations revealed that induced NLS-RARα expression inhibited 1,25(OH)2D3-induced granulocytic differentiation by regulating the cell cycle regulators p19INK4D, p21WAF1/CIP1, cyclinD1, cyclin E1, and pRB. Furthermore, the cleaved protein NLS-RARα enhanced the oncogenicity of U937 cells in NOD/SCID mice. These findings collectively demonstrated that NLS-RARα blocked granulocytic and monocytic differentiation of myeloid leukemia cells by inhibiting the downstream targets of the RARα signal pathway and the cell cycle. This may provide a promising new target and method for diagnosing and treating APL.

P-309 - Crosstalk between mitochondria and NOX2 in vitamin D3-induced differentiation in PLB-985 myeloid cells

To elucidate the role of reactive oxygen species (ROS) in immune enhancement, we focused on the redox signaling in vitamin D3-induced monocytic differentiation of leukemia cells. ROS generation in the cells was analyzed with ESR spin-trapping method. As a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used. Human myeloid PLB-985 cells were stimulated with 200 nM vitamin D3 and/or 100 nM phorbol 12-myristate 13-acetate. Immediately after vitamin D3-stimulation, ESR-spin trapping indicated the spectrum of DMPO-OH adduct. Flow cytometry showed the expression of CD11b/Mac1–alpha in the cells incubated with vitamin D3 for three days. Diphenyleneiodonium (NOX2 inhibitor:100 uM) and actinomycin A (mitochondrial electron transport chain complex III inhibitor:10 uM) have suppressed vitamin D3-induced the expression of CD11b and ROS generation. Our results indicate that vitamin D3-induced expression of CD11b have occurred through a close interaction between NOX2 and mitochondria.

The c-Raf modulator RRD-251 enhances nuclear c-Raf/GSK-3/VDR axis signaling and augments 1,25-dihydroxyvitamin D3-induced differentiation of HL-60 myeloblastic leukemia cells.

Differentiation therapy is used in cancer treatment. Epidemiologic studies showed that higher vitamin D levels are associated with reduced cancer risks. However, the therapeutic doses needed for differentiation are accompanied by hypercalcemia and intolerable pathological sequelae. In the present work we evaluated if RRD-251, a small-molecule, can enhance vitamin D3-induced differentiation of leukemic cells, in the hope of decreasing the needed vitamin D3-dose.We demonstrate that RRD-251 enhances vitamin D3-induced differentiation of leukemic cells, the enrichment of the c-Raf kinase in the nucleus, the binding of nuclear c-Raf to GSK-3, increased phosphorylation of GSK-3 ser 21/9 inhibitory sites, and the binding of GSK-3 to VDR, where GSK-3 inhibition is known to enhance transcriptional activation by VDR. Enhancement of D3-induced p-GSK-3 ser 21/9 by RRD-251 was associated with enhanced Akt-GSK-3 binding, Akt being a known GSK-3 inhibitor, and diminished Erk1/2 binding. Diminishing Erk interaction with GSK-3 was associated with enhanced interaction with Vav1, a known driver of myeloid differentiation. This is redolent of an ATRA/c-Raf/GSK-3/RARα axis we previously reported, although the phosphorylation effects to enhance transcriptional activation on RARα vs VDR diverge. Taken together this indicates potential therapeutic significance for a c-Raf/GSK-3/VDR or RARα axis for leukemic myelo-monocytic differentiation.

Targeting peroxiredoxin I potentiates 1,25-dihydroxyvitamin D3-induced cell differentiation in leukemia cells

Although 1,25‑dihydroxyvitamin D3 (VD3) is regarded as a promising inducing agent for leukemia cell differentiation, it is not as effective an agent as all‑trans‑retinoic acid, and its usefulness is also limited by the adverse effects of hypercalcemia. The aim of the present study was to determine whether combining VD3 with adenanthin, a peroxiresoxin I (Prx I)‑targeting natural compound, improves the efficacy of VD3. Cell viability was assessed using a trypan blue exclusion assay and flow cytometry was used to evaluate the expression of cell surface markers, CD11b/CD14, and the level of reactive oxygen species (ROS). Wright's staining was used to examine morphological changes and RNA‑interference was used to knockdown Prx I and p65 gene expression. Protein expression was determined by western blot analysis. The results demonstrated that adenanthin markedly enhanced VD3‑induced cell differentiation of leukemia NB4 cells, as evidenced by the increased percentage of CD11b‑ and CD14‑positive cells, the mature morphology of the monocytes and the increased phagocytic ability. Consistent with these results, knockdown of Prx I, but not nuclear factor‑κB (p65), enhanced VD3‑induced cell differentiation. The combinatorial effects of adenanthin and VD3 were shown to be associated with the ROS‑CCAAT‑enhancer‑binding protein (C/EBP)β axis, since N‑acetylcysteine, a ROS scavenger, was able to abrogate the differentiation‑enhancing effects of adenanthin, and the knockdown of C/EBPβ also inhibited the combinatorial effects of adenanthin and VD3. In addition, co‑treatment with adenanthin and VD3 was able to induce differentiation in other non‑acute promyelocytic leukemia cells and primary leukemia cells. In conclusion, the results of the present study revealed a novel role for Prx I in VD3‑induced cell differentiation, and suggested that targeting Prx I may represent a novel strategy to enhance VD3‑induced leukemia cell differentiation.

Abstract 4458: PDLIM2 is essential for feedback regulation of the B1 integrin-FAK-RhoA signalling pathway to maintain epithelial cell polarity and suppress transformation

Abstract PDLIM2 is a PDZ-LIM domain protein that regulates the stability of transcription factors (NFkB and STATs) in the nucleus. It is also associated with the cytoskeleton at cell adhesions and cell junctions and is required for polarized cell migration. PDLIM2 expression is suppressed by methylation in different cancers, but is strongly expressed in invasive breast cancer cells that have undergone an Epithelial Mesenchymal Transition (EMT). Moreover, PDLIM2 expression is associated with poor outcome in basal breast cancers and its expression can be enhanced by vitamin D-induced differentiation of breast cancer cells. Since PDLIM2 expression repression may facilitate oncogenic transformation and its expression is modulated by the differentiation status of breast cancer cells we asked whether it is essential for maintaining a non-transformed epithelial phenotype. To address this we suppressed PDLIM2 in MCF10A cells and assessed the phenotype and mechanism in 3D Matrigel cultures. We found that PDLIM2 suppression is sufficient to disrupt cell polarization and acinar formation in Matrigel cultures. MCF10A cells with suppressed PDLIM2 formed spheres of proliferating cells that exhibited reduced apoptosis in the luminal space, compared to control acini with hollow or apoptotic lumina. Spheroids with suppressed PDLIM2 had increased expression of cell-cell adhesion proteins including E cadherin and β-catenin. Proteins associated with cell-matrix adhesion were also up-regulated, including activated beta 1 (β1) integrin, laminin, and fibronectin production. Interestingly, both the IGF-1 Receptor and RACK1, which scaffolds IGF-1R to β1 integrin, were increased, indicating a transformed phenotype. FAK and cofilin phosphorylation, and RhoA activity were also enhanced in spheroids with PDLIM2 suppressed compared to controls. Importantly, pharmacological inhibition of either FAK or Rho Kinase (ROCK) was sufficient to rescue the polarity defect and abrogate all the phenotypic changes in MCF10A cell spheroids caused by PDLIM2 suppression. We conclude that PDLIM2 mediates feedback regulation of the β1-integrin signalling pathway through FAK and RhoA that is essential for maintenance of polarized breast epithelial acinar structures, and for suppression of uncontrolled proliferation and cellular transformation. Citation Format: Ravi Kiran-Deevi, Orla T. Cox, Rosemary O'Connor. PDLIM2 is essential for feedback regulation of the B1 integrin-FAK-RhoA signalling pathway to maintain epithelial cell polarity and suppress transformation. [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 4458. doi:10.1158/1538-7445.AM2014-4458

Retinoic Acid Therapy Resistance Progresses from Unilineage to Bilineage in HL-60 Leukemic Blasts

Emergent resistance can be progressive and driven by global signaling aberrations. All-trans retinoic acid (RA) is the standard therapeutic agent for acute promyelocytic leukemia, but 10–20% of patients are not responsive, and initially responsive patients relapse and develop retinoic acid resistance. The patient-derived, lineage-bipotent acute myeloblastic leukemia (FAB M2) HL-60 cell line is a potent tool for characterizing differentiation-induction therapy responsiveness and resistance in t(15;17)-negative cells. Wild-type (WT) HL-60 cells undergo RA-induced granulocytic differentiation, or monocytic differentiation in response to 1,25-dihydroxyvitamin D3 (D3). Two sequentially emergent RA-resistant HL-60 cell lines, R38+ and R38-, distinguishable by RA-inducible CD38 expression, do not arrest in G1/G0 and fail to upregulate CD11b and the myeloid-associated signaling factors Vav1, c-Cbl, Lyn, Fgr, and c-Raf after RA treatment. Here, we show that the R38+ and R38- HL-60 cell lines display a progressive reduced response to D3-induced differentiation therapy. Exploiting the biphasic dynamic of induced HL-60 differentiation, we examined if resistance-related defects occurred during the first 24 h (the early or “precommitment” phase) or subsequently (the late or “lineage-commitment” phase). HL-60 were treated with RA or D3 for 24 h, washed and retreated with either the same, different, or no differentiation agent. Using flow cytometry, D3 was able to induce CD38, CD11b and CD14 expression, and G1/G0 arrest when present during the lineage-commitment stage in R38+ cells, and to a lesser degree in R38- cells. Clustering analysis of cytometry and quantified Western blot data indicated that WT, R38+ and R38- HL-60 cells exhibited decreasing correlation between phenotypic markers and signaling factor expression. Thus differentiation induction therapy resistance can develop in stages, with initial partial RA resistance and moderate vitamin D3 responsiveness (unilineage maturation block), followed by bilineage maturation block and progressive signaling defects, notably the reduced expression of Vav1, Fgr, and c-Raf.

Porphyromonas gingivalis-derived Lysine Gingipain Enhances Osteoclast Differentiation Induced by Tumor Necrosis Factor-α and Interleukin-1β but Suppresses That by Interleukin-17A: IMPORTANCE OF PROTEOLYTIC DEGRADATION OF OSTEOPROTEGERIN BY LYSINE GINGIPAIN

Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159-166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1β in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1β were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.

Role of cell cycle regulatory molecules in retinoic acid- and vitamin D3-induced differentiation of acute myeloid leukaemia cells.

The important role of cell cycle regulatory molecules in all trans-retinoic acid (ATRA)- and vitamin D3-induced growth inhibition and differentiation induction has been intensively studied in both acute myeloid leukaemia primary cells and a variety of leukaemia cell lines. Cyclin-dependent kinases (CDK)-activating kinase has been demonstrated to interact with retinoic acid receptor (RAR)α in acute promyelocytic leukaemia cells, and inhibition of CDK-activating kinase by ATRA causes hypophosphorylation of PML-RARα, leading to myeloid differentiation. In many cases, downregulation of CDK activity by ATRA and vitamin D3 is a result of elevated p21- and p27-bound CDKs. Activation of p21 is regulated at the transcriptional level, whereas elevated p27 results from both (indirectly) transcriptional activation and post-translational modifications. CDK inhibitors (CKIs) of the INK family, such as p15, p16 and p18, are mainly involved in inhibition of cell proliferation, whereas CIP/KIP members, such as p21, regulate both growth arrest and induction of differentiation. ATRA and vitamin D3 can also downregulate expression of G1 CDKs, especially CDK2 and CDK6. Inhibition of cyclin E expression has only been observed in ATRA- but not in vitamin D3-treated leukaemic cells. In vitro, not only dephosphorylation of pRb but also elevation of total pRb is required for ATRA and vitamin D3 to suppress growth and trigger their differentiation. Finally, sharp reduction in c-Myc has been observed in several leukaemia cell lines treated with ATRA, which may regulate expression of CDKs and CKIs.

Kruppel-like factor 4 attenuates osteoblast formation, function, and cross talk with osteoclasts

Osteoblasts not only control bone formation but also support osteoclast differentiation. Here we show the involvement of Kruppel-like factor 4 (KLF4) in the differentiation of osteoclasts and osteoblasts. KLF4 was down-regulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in osteoblasts. Overexpression of KLF4 in osteoblasts attenuated 1,25(OH)2D3-induced osteoclast differentiation in co-culture of mouse bone marrow cells and osteoblasts through the down-regulation of receptor activator of nuclear factor κB ligand (RANKL) expression. Direct binding of KLF4 to the RANKL promoter repressed 1,25(OH)2D3-induced RANKL expression by preventing vitamin D receptor from binding to the RANKL promoter region. In contrast, ectopic overexpression of KLF4 in osteoblasts attenuated osteoblast differentiation and mineralization. KLF4 interacted directly with Runx2 and inhibited the expression of its target genes. Moreover, mice with conditional knockout of KLF4 in osteoblasts showed markedly increased bone mass caused by enhanced bone formation despite increased osteoclast activity. Thus, our data suggest that KLF4 controls bone homeostasis by negatively regulating both osteoclast and osteoblast differentiation.

Differentiation and Cell Survival of Myeloid Leukemia Cells

Differentiation and Cell Survival of Myeloid Leukemia Cells

Herba epimedii flavonoids suppress osteoclastic differentiation and bone resorption by inducing G2/M arrest and apoptosis

Accumulating evidences suggest that Herba epimedii has the potential benefits against osteoporosis. However, previous studies were focused on the crude extract, total flavonoids (TF) and icariin (ICA), and the detailed molecular mechanisms of action and structure-activity relationship (SAR) remain unclear. Herein we aimed to systematically investigate the effects of Herba epimedii flavonoids (HEF) on the activity of osteoclasts, and explore the potential SAR. Both ICA and baohuoside-1 (BS) significantly inhibited the proliferation of RAW 264.7 cells (IC(50) 25 μM and 67 μM, respectively). Treatment of ICA resulted in G2/M arrest and apoptosis in RAW 264.7 cells as early as 12 h. Besides, HEF remarkably suppressed vitamin D-induced differentiation of osteoclasts in rabbit bone marrow cells and the bone resorption of rabbit mature osteoclasts in vitro. It is notable that the inhibitory effect of 100 μM ICA and BS on osteoclast formation is almost 90%; and the inhibition rate on bone resorption is 50% and 80%, respectively. Besides, RANKL-induced osteoclast formation from RAW 264.7 cells and the expression of TRAP, CA II, CTSK and MMP-9 was significantly reduced by the treatment of 25 μM HEF and 17β-estradiol (ES), and the inhibitory strength increases in the order TF < ES < ICA < BS, which was blocked by ICI182780 suggesting that the regulation of osteoclast activity might be ER dependent. Furthermore, the free hydroxyl group at C-7 of BS played an important role in the SAR for anti-osteoclast action. To conclude, HEF could regulate the formation and activity of osteoclasts by inhibiting the proliferation and differentiation, inducing apoptosis and cell cycle arrest and suppressing bone resorption of osteoclasts. Changes in osteoclast activity are probably mediated predominantly by interaction with nuclear estrogen receptors and via mitochondrial pathway. HEF, especially BS, has great potential for the prevention and treatment of osteoporosis.

Opposite regulation of vitamin D receptor by ATRA in AML cells susceptible and resistant to vitamin D-induced differentiation

Some leukemic cell lines can be driven to differentiate to monocyte-like cells by 1,25-dihydroxyvitamin D3 (1,25D) and to granulocyte-like cells by all-trans retinoic acid (ATRA). Acute myloid leukemias (AMLs) are heterogeneous blood malignancies characterized by a block at various stages of hematopoietic differentiation and there are more than 200 known chromosome translocations and mutations in leukemic cells of patients diagnosed with AML. Because of the multiplicity in the genetic lesions causing the disease, AMLs are particularly difficult to treat successfully. In particular, various AML cells to a variable degree respond to 1,25D-based differentiation and only one type of AML undergoes successfully ATRA-based differentiation therapy. In this paper we describe that AML cell line KG-1 is resistant to 1,25D-induced monocytic differentiation, while sensitive to ATRA-induced granulocytic differentiation. We show that KG-1 cells have very low level of VDR protein and that expression of VDR mRNA is upregulated by ATRA. We show for the first time that this regulation is cell context-specific, because in another AML cell line, HL60, VDR mRNA is downregulated by ATRA. ATRA-induced VDR protein in cytosol of KG-1 cells can be further activated by 1,25D to induce monocytic differentiation of these cells.

Cell-Type-Specific Effects of Silibinin on Vitamin D-Induced Differentiation of Acute Myeloid Leukemia Cells Are Associated with Differential Modulation of RXRα Levels

Plant polyphenols have been shown to enhance the differentiation of acute myeloid leukemia (AML) cells induced by the hormonal form of vitamin D3 (1α,25-dihydroxyvitamin D3; 1,25D). However, how these agents modulate 1,25D effects in different subtypes of AML cells remains poorly understood. Here, we show that both carnosic acid (CA) and silibinin (SIL) synergistically enhancd 1,25D-induced differentiation of myeloblastic HL60 cells. However, in promonocytic U937 cells, only CA caused potentiation while SIL attenuated 1,25D effect. The enhanced effect of 1,25D+CA was accompanied by increases in both the vitamin D receptor (VDR) and retinoid X receptor alpha (RXRα) protein levels and vitamin D response element (VDRE) transactivation in both cell lines. Similar increases were observed in HL60 cells treated with 1,25D + SIL. In U937 cells, however, SIL inhibited 1,25D-induced VDRE transactivation concomitant with downregulation of RXRα at both transcriptional and posttranscriptional levels. These inhibitory effects correlated with the inability of SIL, with or without 1,25D, to activate the Nrf2/antioxidant response element signaling pathway in U937 cells. These results suggest that opposite effects of SIL on 1,25D-induced differentiation of HL60 and U937 cells may be determined by cell-type-specific signaling and transcriptional responses to this polyphenol resulting in differential modulation of RXRα expression.

Abstract 1837: Cellular glutathione is an essential mediator of the enhancing effect of plant polyphenolic antioxidants on vitamin D-induced differentiation of acute myeloid leukemia cells

Abstract Acute myeloid leukemia (AML) is an aggressive malignancy with poor prognosis. Differentiation therapy may provide an alternative AML treatment, based on the induction of leukemic blasts to mature. The hormonal form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D), is a powerful inducer of monocytic differentiation of AML cells in vitro; however, it causes severe hypercalcemia in vivo at pharmacologically effective doses. We have previously shown that various plant polyphenolic antioxidants (PAOx), such as carnosic acid (CA), curcumin (CUR), and silibinin (SIL), strongly potentiate the differentiation of AML cells induced by a low, near physiologic dose of 1,25D (1 nM). Further, we have demonstrated that a functionally active transcription factor Nrf2 is required for the differentiation-enhancing effect of CA; however, the mechanism whereby Nrf2 contributes to this effect was unclear. Here we show that similar to CA, CUR and SIL transactivated the Nrf2 response element (antioxidant response element; ARE), induced Nrf2/ARE-responsive gene products, such as NADP(H)-quinone oxidoreductase, thioredoxin reductase-1 and the α-glutamylcysteine synthetase heavy subunit (≥GCSh), and elevated the total glutathione content in human AML cells (HL60 and U937). Interestingly, 1,25D markedly potentiated these effects of PAOx. Stable expression of dominant negative Nrf2 (dnNrf2) abolished the differentiation-enhancing effects of all the PAOx tested. Importantly, such inhibitory action of dnNrf2 was mimicked by treatment of untransfected cells with non-cytotoxic concentrations of L-buthionine-sulfoximine (BSO), the specific inhibitor of αGCS. Glutathione depletion in BSO-treated cells was associated with attenuation of both the transactivation of the transcription factor activating protein-1 (AP-1) and elevation of protein levels of the vitamin D receptor (VDR) and retinoid X receptor alpha (RXRα) induced by 1,25D/PAOx combinations. This may account for the reduction in the extent of 1,25D/PAOx-induced differentiation observed following BSO treatment. Collectively, our data suggest that cooperative elevation of glutathione levels by PAOx and 1,25D, as a result of Nrf2/ARE-dependent induction of αGCS, may mediate the enhancing effects of PAOx on 1,25D-induced differentiation of AML cells. These findings may provide a mechanistic basis for the therapy and/or prevention of AML using combinations of vitamin Dand plant antioxidants. (This research was supported by the Israel Science Foundation grant 635/11 to both M.D. and Y.S., and by the NIH-NCI grant RO1-CA117942-04 to both G.P.S. and M.D.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1837. doi:1538-7445.AM2012-1837

Dual role of hematopoietic progenitor kinase 1 (HPK1) as a positive regulator of 1α,25-dihydroxyvitamin D-induced differentiation and cell cycle arrest of AML cells and as a mediator of vitamin D resistance

Recent clinical trials aimed at improved treatment of AML by administration of vitamin D derivatives showed unremarkable results, suggesting development of vitamin D resistance in patients’ AML blasts. Since mechanisms of vitamin D resistance are not clear, we studied 40AF cells, a subline of HL60 cells that can proliferate in the presence of 1α,25-dihydroxyvitamin D (1,25D). We found that mRNA and protein levels of HPK1, an upstream MAP4 kinase, are dramatically increased in 40AF cells, and HPK1 protein is further increased when the 1,25D resistance of 40AF cells is partially reversed by the addition of carnosic acid and p38MAPK inhibitor SB202190 (DCS cocktail). Knockdown of HPK1 reduces 1,25D/DCS-induced differentiation of both 1,25D-sensitive HL60 and U937 cells and 1,25D-resistant 40AF cells, but the effect of HPK1 knockdown on differentiation-associated G1 arrest is more apparent in the resistant than the sensitive cells. To explain why 40AF and the intrinsically vitamin D-resistant KG-1a cells can proliferate in the presence of vitamin D, we found that the cleaved HPK1 fragment (HPK1-C) level is high in 40AF and KG-1a cells, but when differentiation is induced by DCS, HPK1-C decreases while full-length (FL)-HPK1 increases. Accordingly, inhibition of proteolysis with the pan-caspase inhibitor Q-VD-OPh reduced HPK1 cleavage and enhanced DCS-induced differentiation of 40AF cells. The results indicate that FL-HPK1 is a positive regulator of vitamin D-induced differentiation in AML cells, but the cleaved HPK1 fragment inhibits differentiation. Thus, high HPK1 cleavage activity contributes to vitamin D resistance, and HPK1 has a dual role in AML cell differentiation.