Effect of combination of all-trans retinoic acid and arsenic trioxide on apoptosis of acute promyelocytic leukemia cells.

To study the effect of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) combination treatment on apoptosis of acute promyelocytic leukemia cells (NB4), inflammation and prognosis. The effect of ATRA - ATO combination on the proliferation of NB4 was determined using MTT assay. Apoptosis of NB4 cells was assessed with TUNEL assay. The effect of ATRA-As2O3 combination on the expressions of IL-6 and TNF-α in NB4 cells was determined using ELISA kits, while its effect on the quality of life of 25 acute promyelocytic leukemia patients admitted to our hospital was scored, as an index of prognosis. The combination treatment with ATRA and ATO significantly inhibited the proliferation of NB4 cells and promoted their apoptosis, relative to the model group. In addition, the combination treatment reduced serum IL-6 and TNF-α levels in patients with acute promyelocytic leukemia, and improve their quality of life and survival. Combination treatment with ATRA and ATO significantly inhibits the proliferation of NB4 cells and promotes their apoptosis, and reduces inflammatory responses in patients with acute promyelocytic leukemia, while improving their quality of life and prognosis.

1,2-Benzenedithiol and Toluene-3,4-dithiol Arsenic(III) Complexes-Synthesis, Structure, Spectroscopic Characterization and Toxicological Studies.

A new group of arsenic(III) complexes with bidentate S,S-donor ligands, 1,2-benzenedithiol (Ph(SH)2) and toluene-3,4-dithiol (MePh(SH)2), were synthesized. The use of arsenic(III) iodide and bromide promoted the formation of neutral complexes (1-4) with the general formula AsX(LS2) (X = I or Br, L = MePh or Ph). The crystal structures of these compounds were determined using single-crystal X-ray diffraction (scXRD). Unlike other arsenic(III) complexes, AsBr(PhS2) complex (2) was found to crystallize with a rare 13 molecules in the asymmetric unit. The compounds were also characterized by conventional physico-chemical techniques (Fourier transform infrared (FT-IR) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), elemental analysis (EA) and electrospray ionization-mass spectrometry (ESI-MS)). The results from structural and spectroscopic studies were supported by DFT calculations using the B3LYP/LANL2DZ and (or) 6-31+G(d,p) approaches. The cytotoxicity of these complexes was estimated for human acute promyelocytic leukemia cell line (NB4). They exhibited remarkable cytotoxicities after 48 h of treatment with IC50 equal to about 10 µM and 40 µM for complexes with 1,2-benzenedithiolato and toluene-3,4-dithiolato ligand, respectively. Their toxicity was lower than that of commonly used chemotherapeutic As2O3 (IC50 = 1.4 µM).

Verteporfin Inhibits Cell Proliferation and Induces Apoptosis in Human Leukemia NB4 Cells without Light Activation.

Background and Aims: Verteporfin (VP), clinically used in photodynamic therapy for neovascular macular degeneration, has recently been proven a suppressor of yes-associated protein (YAP) and has shown potential in anticancer treatment. However, its anti-human leukemia effects in NB4 cells remain unclear. In this study, we investigated the effects of VP on proliferation and apoptosis in human leukemia NB4 cells. Methods: NB4 cells were treated with VP for 24 h. The effects of VP on cell proliferation were determined using a Cell-Counting Kit-8 assay (CCK-8) assay and colony forming assay. Apoptosis and cell cycle were evaluated by flow cytometry (FCM). The protein levels were detected by western blot. Results: We found that VP inhibited the proliferation of NB4 cells in a concentration and time-dependent manner. FCM analysis showed that VP induced apoptosis in a concentration dependent manner and that VP treatment led to cell cycle arrest at G0/G1 phase. Moreover, VP significantly decreased the protein expression of YAP, p-YAP, Survivin, c-Myc, cyclinD1, p-ERK, and p-AKT. In addition, VP increased the protein expression of cleaved caspase3, cleaved PARP, Bax, and p-p38 MAPK. Conclusions: VP inhibited the proliferation and induced apoptosis in NB4 cells.

Cholinergic activation enhances retinoic acid-induced differentiation in the human NB-4 acute promyelocytic leukemia cell line.

The non-neuronal cholinergic system (NNCS) has been shown to play a role in regulating hematopoietic differentiation. We determined the expression of cholinergic components in leukemic cell lines by Western blotting and in normal leukocyte subsets by flow cytometry and found a heterogeneous expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), choline transporter (CHT), M3 muscarinic acetylcholine receptor (M3-mAChR) and α7 nicotinic acetylcholine receptor (α7-nAChR). We then evaluated NNCS role in differentiation of human NB-4 acute promyelocytic leukemia cell line and discovered a dramatic induction of M3-mAChR after all-trans retinoic acid (ATRA) treatment (p<0.0001). Adding carbachol which is a cholinergic agonist to the ATRA treatment resulted in an increase of a granulocytic differentiation marker (CD11b) as compared with ATRA treatment alone (p<0.05), indicating that cholinergic activation enhanced ATRA in inducing NB-4 maturation. The combination of carbachol and ATRA treatment for 72h also resulted in decreased viability and increased cleaved caspase-3 expression when compared with ATRA treatment alone (p<0.05). However, this combination did not cause poly (ADP-ribose) polymerase (PARP) cleavage. Overall, we have shown that NB-4 cells expressed M3-mAChR in a differentiation-dependent manner and cholinergic stimulation induced maturation and death of ATRA-induced differentiated NB-4 cells.

Effects of LG268 on Cell Proliferation and Apoptosis of NB4 Cells.

AIMS: To investigate the effect of LG100268 (LG268) on cell proliferation and apoptosis in NB4 cells. METHODS: NB4 cells were treated with LG268 for 24 h or 48 h. The effect of LG268 on cell proliferation was assessed by the CCK-8 assay and colony-forming assay. Apoptosis and cell cycle were evaluated by flow cytometry. The protein expression levels of Survivin, PARP, c-Myc, cyclin D1, ERK, p-ERK, p38 MAPK, and p- p38 MAPK were detected by western blot. RESULTS: We found that LG268 inhibited the proliferation of NB4 cells in a dose-dependent manner. Flow cytometry analysis showed that LG268 accelerated apoptosis in NB4 cells in a time- dependent manner and that LG268 treatment led to cell cycle arrest at G0/G1 phase. Moreover, LG268 significantly decreased the protein levels of Survivin, c-Myc, and cyclinD1. Cleaved PARP was observed in the LG268 treatment group but not in the control group. In addition, LG268 increased the phosphorylation level of p38 MAPK and decreased the phosphorylation level of ERK. CONCLUSIONS: LG268 inhibited cell proliferation and promoted cell apoptosis in NB4 cells.

NLS-RARα Inhibits the Effects of All-trans Retinoic Acid on NB4 Cells by Interacting with P38α MAPK.

Nuclear localization signal retinoic acid receptor alpha(NLS-RARα), which forms from the cleavage of promyelocytic leukemia-retinoic acid receptor alpha(PML-RARα) protein by neutrophil elastase(NE), possesses an important role in the occurrence and development of acute promyelocytic leukemia(APL). However, the potential mechanism underlying the effects of NLS-RARα on APL is still not entirely clear. Here, we investigated the effects of NLS-RARα on APL NB4 cells and its mechanism. We found that all-trans retinoic acid(ATRA) could promote differentiation while inhibit proliferation of APL NB4 cells via upregulating the expression of phosphorylated p38α mitogen-activated protein kinase(p-p38α MAPK). We also found that NLS-RARα could inhibit differentiation while accelerate proliferation of NB4 cells via downregulating the expression of p-p38α protein in the presence of ATRA. Furthermore, immunofluorescence and co-immunoprecipitation assays confirmed NLS-RARα interacted with p38α protein directly. Finally, application of PD169316, an inhibitor of p38α protein, suggested that recruitment p38α-combinded NLS-RARα by ATRA eventually caused activation of p38α protein. In summary, our study demonstrated that ATRA cound promote differentiation while inhibit proliferation of APL NB4 cells via activating p38α protein after recruiting p38α-combinded NLS-RARα, while NLS-RARα could inhibit the effects of ATRA in the process.

Inhibition of autophagy enhances apoptosis induced by bortezomib in AML cells.

Bortezomib is a novel proteasome inhibitor, which has been successfully used to treat mantle cell lymphoma and multiple myeloma. However, the direct effects of bortezomib on acute promyelocytic leukaemia (APL) have not been fully investigated. In the present study, the WST-8 assay, western blotting, flow cytometry, monodansylcadaverine staining and transmission electron microscopy were performed. It was demonstrated that bortezomib treatment induced a time- and dose-dependent decrease in the viability of NB4 cells. Bortezomib treatment induced cell apoptosis in NB4 cells, as assessed by Annexin V/propidium iodide analysis, and the detection of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase, Bax and Bcl-2 expression. Furthermore, bortezomib treatment induced autophagy in NB4 cells, as indicated by autophagosome formation, p62 degradation, LC3-I to LC3-II conversion and formation of acidic autophagic vacuoles. Notably, autophagy induced by bortezomib was initiated prior to apoptosis. Inhibition of autophagy by knocking down Beclin-1 expression increased bortezomib-induced apoptosis in NB4 cells. Therefore, the present study revealed that the combination of bortezomib and autophagy inhibition may be a potential treatment strategy for APL.

Suppression of proteasome induces apoptosis in APL cells and increases chemo-sensitivity to arsenic trioxide: Proposing a perception in APL treatment.

In the last three decades, the pathogenesis of acute promyelocytic leukemia (APL) has been mostly studied with regard to the oncogenic role of PML/RAR fusion protein; however, the latest discoveries have stated that the concerns with the treatment of APL patients would not be resolved until the role of aberrant networks is overlooked. The present study was designed to evaluate the anti-cancer property of second-generation of the proteasome inhibitors carfilzomib (CFZ) on APL-derived NB4 cells. Our results showed that pharmacologic targeting of proteasome in NB4 reduced the proliferative rate of malignant cells through a c-Myc-mediated G2/M cell cycle arrest. Moreover, we found that the suppression of proteasome was coupled with the induction of apoptotic NB4 cell death, which is probably mediated through down-regulation of anti-apoptotic target genes. Interestingly, our results suggested that the suppression of the autophagy system using chloroquine could serve as a mechanism through which the cytotoxicity of CFZ in APL cells was ameliorated. Finally, and consistent with the favorable efficacy of single agent of CFZ, we also noted an intensifying effect of the inhibitor on the anti-leukemic activity of arsenic trioxide (ATO) when it was used in combination. Overall, this study suggests that pharmaceutical targeting of proteasome using CFZ, either as a single agent or in combination with ATO, could be a promising mechanism through which the obstacle on the way of APL would be tackled; however, further investigations are needed to determine the advantages of the inhibitor in clinical applications.

HLX affects cell cycle and proliferation in AML cells via the JAK/STAT signaling pathway.

Acute myelogenous leukemia (AML) is a class of malignant tumors derived from hematopoietic stem or progenitor cells. The H2.0-like homeobox gene (HLX) encodes transcription factors that function in promoting normal hematopoietic cell proliferation and tumor immunity. The present study analyzed the effect of downregulating the HLX on cell cycle distribution and cell proliferation in AML. Moreover, the current study detected changes in the expression of genes and proteins in the Janus kinase (JAK)/STAT signaling pathway to investigate the mechanism of the action of HLX in tumor immunity in AML. HLX expression in AML cell lines was silenced using small interfering siRNA, and MTS/PMS-assay colorimetric assays were used to assess the effect of knockdown of HLX on AML cell proliferation. Flow cytometry was used to analyze changes in cell cycle distribution, while reverse transcription-quantitative PCR and western blotting were used to detect changes in the expression levels of key components of the JAK/STAT signaling pathway, such as p21-activated kinase 1 (PAK1), neuropilin 1 (NRP1), B-cell translocation gene 1 (BTG1) and STAT5. It was found that HLX was differentially expressed in AML cell lines of various subtypes, and HLX expression was higher in the AML/M3 subtype NB4 cell line compared with the control group. Knockdown of HLX in NB4 cells significantly inhibited cell proliferation and arrested cells in the G0/G1 phase. Moreover, STAT5 protein expression, as well as NRP1 and PAK1 expression levels were downregulated, while BTG1 expression was upregulated when HLX was knocked out by siRNA. Collectively, the results suggested that downregulation of HLX may cause G0/G1 phase arrest and inhibit the proliferation of AML cells by activating the JAK/STAT signaling pathway.

Effect of daunorubicin on acute promyelocytic leukemia cells using nuclear magnetic resonance spectroscopy-based metabolomics.

The aim of this study was to determine several key metabolites as potential biomarkers of daunorubicin (DNR) treatment of acute promyelocytic leukemia (APL) using APL blasts and NB4 cells. Samples which were obtained from 16 newly diagnosed APL patients and human APL NB4 cell lines were exposed to increasing concentrations of DNR (0 µM, 0.1 µM, 0.5 µM and 1.0 µM). Electron microscopy and Nuclear Magnetic Resonance (NMR) spectroscopy confirmed that there were clear differences between controls and DNR-treated groups, with the resultant models having excellent predictive and discriminative abilities. Four metabolites meeting the biomarker requirements were identified. KEGG analyses revealed that these biomarkers were associated with the metabolism of fat, choline, and glucose. These findings offered vital information about the effects of chemotherapies on the whole body biochemistry which might be important for monitoring apoptosis and injury to cells in order to reduce chemotherapies-induced side effects.

Effects of lapatinib on cell proliferation and apoptosis in NB4 cells.

Acute promyelocytic leukemia (APL), characterized by the presence of the promyelocytic leukemia (PML)-retinoic acid α receptor (RARα) fusion protein, responds to treatment with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, drug resistance and side effects restrict the application of these reagents. Hence, the development of novel therapeutic drugs for APL treatment is critical. Lapatinib, a small-molecule tyrosine kinase inhibitor, has been used in the treatment of different tumors. However, it is unclear whether lapatinib exerts antitumor effects on APL. The present study investigated the antitumor effects and potential mechanisms of lapatinib on NB4 cells derived from APL. Cell Counting Kit-8 assay and colony forming analysis indicated that lapatinib inhibited NB4 cell proliferation in a dose-dependent manner. Flow cytometry analysis revealed that lapatinib induced cell cycle arrest at the S phase and promoted cell apoptosis. Furthermore, Liu's staining and Hoechst 33258 staining revelaed that lapatinib treatment induced an apoptotic nuclear phenomenon. Furthermore, lapatinib induced apoptosis by decreasing Bcl-2 and PML-RARα levels, and by increasing the levels of Bax, cleaved PARP, cleaved caspase-3 and cleaved caspase-9. In addition, lapatinib increased the levels of phospho-p38 MAPK and phospho-JNK, and decreased the levels of phospho-Akt. The p38 inhibitor PD169316 partially blocked lapatinib-induced proliferation inhibition and apoptosis, whereas the JNK inhibitor SP600125 had no such effects. Therefore, treatment with lapatinib may be a promising strategy for APL therapy.

Location of NLS-RARα protein in NB4 cell and nude mice.

In the majority of acute promyelocytic leukemia (APL) cases, translocons produce a promyelocytic leukemia protein-retinoic acid receptor α (PML-RARα) fusion gene. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RAα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. NLS-RARα promotes cell growth and inhibits differentiation in response to ATRA. However, the mechanisms by which NLS-RARα affects cell biological characteristics are yet to be fully elucidated. The present study found that the location of RARαwas altered after it was cleaved by NE. Firstly, NE was overexpressed during the preparation of recombinant plasmid NB-4/pCMV6-NE-Myc to cleave PML-RARα. The total protein expression levels of myc and NE and expression levels of NLS-RARα in nucleoprotein were detected by western blotting. Location of NLS-RARα protein was detected by immunofluorescence and confocal laser scanning. Secondly, a nude mice model was constructed and NE protein, NLS-RARα and RARα protein assays, and the location of NLS-RARα and RARα proteins were assessed as described. The present results showed that, compared with the control groups, the location of NLS-RARα protein was predominantly detected in the nucleus, whereas RARα was mainly distributed in the cytoplasm. These findings were consistent with those of the nude mice model, and these may be used as a foundation to explain the occurrence mechanism of APL.

Aryl Hydrocarbon Receptor Antagonist StemRegenin 1 Promotes the Expansion of Human Promyelocytic Leukemia Cell Line, NB4.

BACKGROUND/AIM: StemRegenin 1 (SR1), an antagonist of aryl hydrocarbon receptor (AHR), reportedly promotes expansion of hematopoietic stem cells but its effect on leukemia cells is unclear. This study focused on the role of SR1 in leukemia cell proliferation. MATERIALS AND METHODS: AHR expression was compared in the cell lines Jurkat, Kasumi-1, NB4 and K562, using real-time polymerase chain reaction. Highly AHR-expressing NB4 cells were cultured with SR1 for 2 and 4 days, and evaluated for viability and gene expression. DNA microarray was also performed. RESULTS: The viability of NB4 cells treated with 1.5 µM SR1 increased at day 4. Expression of B-cell CLL/lymphoma 2 (BCL2) was up-regulated, while that of BCL2 associated X protein (BAX) was down-regulated at day 2. Increased cyclin D1 (CCND1), CCND2 and v-myc avian myelocytomatosis viral oncogene homolog (MYC) expressions were observed at day 4. Global gene expression profiles showed up-regulation of splice variant-related genes and down-regulation of inflammation-related genes. CONCLUSION: SR1 promotes the expansion of NB4 cells in vitro, implying the need for caution regarding in vivo use of R1.

Effects of As2O3 nanoparticles on cell growth and apoptosis of NB4 cells.

The aim of the present study was to explore the preparation of arsenic trioxide (As2O3) nanoparticles and examine the antitumor effects of these nanoparticles on NB4 cells. As2O3 nanoparticles were prepared using the sol-gel method and characterized using transmission electron microscopy and energy dispersive spectroscopy. The results indicated that the As2O3 nanoparticles prepared in the present study were round or elliptical, well dispersed and had an ~40-nm or <10-nm diameter. The antitumor effects of As2O3 nanoparticles at various concentrations were analyzed by flow cytometry and the MTT assay, and were compared with those of traditional As2O3 solution. At the same concentration and incubation time (48 h), the survival rate of cells treated with As2O3 nanoparticles was significantly lower than that of cells treated with the As2O3 solution. The growth inhibition rate under both treatments was time- and dose-dependent. In addition, at the same concentration and incubation time, the apoptosis rate of the cells treated with As2O3 nanoparticles was significantly higher than that of the cells treated with the As2O3 solution. Furthermore, As2O3 nanoparticles resulted in a greater reduction in the expression of the anti-apoptotic protein B-cell lymphoma 2 compared with the As2O3 solution. In conclusion, As2O3 nanoparticles, prepared using the sol-gel method, were found to produce a stronger cytotoxic effect on tumor cells than that produced by the As2O3 solution, possibly by inhibiting Bcl-2 expression.

cAMP protects acute promyelocytic leukemia cells from arsenic trioxide-induced caspase-3 activation and apoptosis.

More recently, arsenic trioxide (ATO), was integrated into acute promyelocytic leukemia (APL) treatment, showing high efficacy and tolerability in patients with both ATRA-sensitive and ATRA-resistant APL. ATO could induce apoptosis at relatively high concentrations (0.5 to 2.0 micromol/L) and partial differentiation at low concentrations (0.1 to 0.5 micromol/L) in leukemic promyelocytes. It is known that cAMP agonists enhance low-dose ATO-induced APL cells differentiation. Less well appreciated was the possible interaction between relatively high-doses of ATO and enhanced levels of cAMP in APL cells. Here, we show that elevation of cAMP levels by forskolin inhibited ATO-mediated apoptosis in APL-derived NB4 cells, and this inhibition could be averted by cell permeable cAMP-dependent protein kinase inhibitor (14-22) amide. Inactivating phosphorylation of the proapoptotic protein Bad at Ser118 and phosphorylation of the CREB proto-oncogene at Ser133 were observed upon elevation of cAMP levels in NB4 cells. Phosphorylation of these PKA target proteins is known to promote cell survival in AML cells. The ability of cAMP to endow the APL cells with survival advantage is of particular importance when cAMP agonists may be considered as adjuncts to APL therapy.

Zoledronic acid exerts antitumor effects in NB4 acute promyelocytic leukemia cells by inducing apoptosis and S phase arrest.

The aim of this study was to investigate the antitumor effect of zoledronic acid (ZOL) in the NB4 human acute promyelocytic leukemia (APL) cell line and explore the potential mechanism of action of this compound. NB4 cells were exposed to various concentrations (0-200µM) of ZOL. Cell viability was measured by MTS assay. The extent of cell apoptosis and distribution of cells in the different phases of the cell cycle were analyzed with flow cytometry. The expression of apoptosis- and cell cycle-related proteins was assayed by Western blot. The combined effect of ZOL and arsenic trioxide (ATO) on the proliferation of NB4 cells was also determined. The results of this study indicate that ZOL inhibits cell proliferation in a time- and dose-dependent fashion and also induces apoptosis and S phase arrest in a dose-dependent manner. The Western blot analysis confirmed the induction of apoptosis and S phase arrest, revealing that the pro-apoptosis proteins Bax, Puma and activated caspase-9 were upregulated and the anti-apoptosis proteins Bcl-2 and Bcl-xL were downregulated. ZOL at a concentration of 50µM synergized with 0.5µM ATO on the growth inhibition of NB4 cells. Taken together, our data indicate that ZOL exerts a potent antitumor effect on NB4 cells by inducing apoptosis and cell cycle arrest, and that ZOL can synergize with the traditional chemotherapy drug ATO.