Pathway In HL-60 Cells Research Articles

Capsular polysaccharide from the marine bacterium Cobetia marina induces apoptosis via both caspase-dependent and mitochondria-mediated pathways in HL-60 cells

In the present study, we investigated the antiproliferative effect of the capsular polysaccharide (CPS) from marine Gram-negative bacterium Cobetia marina (formerly C. pacifica) KMM 3878 against human leukemia cells in vitro and the potential molecular mechanism underlying this activity. Our results showed that the CPS could inhibit the proliferation of HL-60 cells in a dose-dependent manner with no effect on normal PBMC cells. HL-60 cells treated with the CPS exhibited typical morphologic and biochemical signs of apoptosis. We found that the CPS caused the collapse of mitochondrial transmembrane potential (ΔΨm), activated caspases-8,-9, and − 3, decreased the ratio of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins, increased ROS production and TNF-α secretion, and stimulated phosphorylation of p38 MAPK and p53 in HL-60 cells. Taken together, these data suggest that both extracellular and intracellular signaling pathways contribute to the CPS-induced apoptosis in HL-60 cells.

Blocking exosomal secretion aggravates 1,4-benzoquinone-induced mitochondrial fission activated by the AMPK/MFF/Drp1 pathway in HL-60 cells.

There is in vivo and in vitro evidence that exposure to benzene or its metabolites could affect the mitochondrial function. However, the underlying molecular mechanism of mitochondrial damage remains to be elucidated. In this study, exposure of human promyelocytic leukemia cells (HL-60) to 1,4-benzoquinone (1,4-BQ; an active metabolite of benzene) increased the intracellular reactive oxygen species levels, decreased the mitochondrial membrane potential, adenosine triphosphate production and mitochondrial DNA (mtDNA) copy number, up-regulated the expression of mitochondrial fission proteins Drp1 and Fis1, and down-regulated the expression of mitochondrial fusion proteins Mfn2 and Opa1. Further study showed that 1,4-BQ mediated mitochondrial fission through activation of the AMP-activated protein kinase/mitochondrial fission factor/dynamin-related protein 1 pathway. Additionally, we also examined the role of exosomal secretion in mitochondrial damage under 1,4-BQ treatment. Results showed that 1,4-BQ increased the total protein level and mtDNA content in exosomes. Upon pre-treatment with the mitochondria-targeted antioxidant SS-31, there was attenuation of the mitochondrial damage induced by 1,4-BQ, accompanied by a change in the exosome release characteristics, while inhibition of exosomal secretion using GW4869 aggravated the 1,4-BQ-mediated mitochondrial fission. We concluded that exosomal secretion may serve as a self-protective mechanism of cells against 1,4-BQ-induced mitochondria damage and mitochondrial dynamics interference.

In vitro AND in silico EVALUATION OF THYMOQUINONE AS POTENTIAL ANTICANCER AGENT IN HUMAN ACUTE MYELOID LEUKEMIA HL-60 CELLS

This study aims to explore the cytotoxic, apoptotic and autophagic effects of thymoquinone on human acute myeloid leukemia. The cytotoxic effects of thymoquinone were determined with 3-(4, 5-dimethylthiazol-2-yl)-2 and 5-diphenyltetrazolium bromide (MTT) tests. B-cell lymphoma 2 associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), caspase 3, mammalian target of rapamycin (mTOR), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (AKT) gene expression analyzes were studied with quantitative real-time polymerase chain reaction (qRT-PCR). AutoDock Tools 4.2 software was applied to research the potential binding of thymoquinone in the active sites of Bax, Bcl-2, caspase 3, mTOR, PI3K, and AKT proteins. Thymoquinone caused a cytotoxic effect on HL-60 cells (Human leukemia cell line) with a value of 16.35 µM. Bcl-2 expression was decreased in all concentrations applied compared to the control. A decrease in caspase 3 expression level was detected in the cells treated with 10 µM, 15 µM, and 25 µM thymoquinone compared to the control. Thymoquinone induced an important decrease in mTOR and PI3K expressions compared to the control at all doses, while AKT decreased at a dose of 15 µM. The docking outcomes showed that thymoquinone interacts with the active site amino acids of apoptotic and autophagic proteins via hydrophobic interactions and hydrogen bonding. The present findings suggest that thymoquinone can stimulate autophagy by prevention of PI3K/AKT/mTOR pathway in HL-60 cells and may become a new target for the therapy of acute myeloid leukemia.

Securinine Induces Differentiation of Human Promyelocytic Leukemic HL-60 Cells through JNK-Mediated Signaling Pathway

Acute myeloid leukemia is characterized by abnormal differentiation of hematopoietic stem cells, leading to the accumulation of immature myeloid cells. Differentiation therapy has been a successful treatment option for acute promyelocytic leukemia but suffers from adverse effects. Therefore, search for novel differentiation-inducing agents with minimal side effects is desirable. Securinine, a naturally-occurring alkaloid, induces differentiation in various leukemic cells and apoptosis in other types of cancers. However, the underlying molecular mechanism(s) remain elusive. Our study aimed to elucidate the possible molecular mechanism(s) and signaling events involved in securinine-induced differentiation of HL-60 cells. Securinine inhibited proliferation in a time- and dose-dependent manner and triggered differentiation. A higher CD14+ population indicated maturation toward monocytic lineage. Securinine caused cell cycle arrest at the G0/G1 phase and enhanced ROS generation. Quantitative gene expression analysis showed significant down-regulation of C/EBP-α, C/EBP-ε, GAΤΑ, and c-myc and up-regulation of the PU.1 gene. The expression of distinct protein kinases Lyn, Chk-2, Yes, FAK, c-Jun, and JNK were enhanced. Use of specific inhibitors of crucial intracellular signaling proteins indicated that JNK and ERK blockade resulted in a significant decline in differentiation. These data thus confirm that securinine induces differentiation through the activation of the JNK-ERK signaling pathway in HL-60 cells.

A comparative study of anti-leukemic effects of kaempferol and epigallocatechin-3-gallate (EGCG) on human leukemia HL-60 cells.

Objective:Acute promyelocytic leukemia (APL) is among the most threatening hematological malignant cancers. Defects in cell growth and apoptotic pathways lead to the pathogenesis of the disease as well as its resistance to therapy; therefore, it is a good model for examining pro-apoptotic agents. The present study compared the molecular mechanism induced by kaempferol and epigallocatechin gallate (EGCG) as well as all-trans retinoic acid (ATRA), in HL-60 leukemia cells during five days. Materials and Methods:Cell viability was determined by resazurin assay following treatment with ATRA (10 µM), EGCG, and kaempferol (12.5-100 µM), and apoptosis was detected by the ANX V/PI kit. Moreover, the levels of genes involved in apoptosis (PI3K, AKT, BCL2, BAX, P21, PTEN, CASP3, CASP8, and CASP9) and multi-drug resistance (MDR, ABCB1 and ABCC1) were assessed by using real-time PCR test. Results:Based on the findings, kaempferol decreased cell viability and increased apoptosis in HL60 cells more than EGCG. Apoptosis was induced via extrinsic and intrinsic pathways in HL60 cells by kaempferol and EGCG. In addition, kaempferol and EGCG increased apoptosis and inhibited MDR in a concentration- and time-dependent manner. Conclusion:Kaempferol at high concentrations can be taken into consideration for treating patients with APL as compared with EGCG.

Combustible Cigarette and Smokeless Tobacco Product Preparations Differentially Regulate Intracellular Calcium Mobilization in HL60 Cells

Changes in the level of intracellular calcium ([Ca2+]i) are central to leukocyte signaling and immune response. Although evidence suggests that cigarette smoking affects inflammatory response via an increase in intracellular calcium, it remains unclear if the use of smokeless tobacco (e.g., moist snuff) elicits a similar response. In this study, we evaluated the effects of tobacco product preparations (TPPs), including total particulate matter (TPM) from 3R4F reference cigarettes, smokeless tobacco extract (STE) from 2S3 reference moist snuff, and nicotine alone on Ca2+ mobilization in HL60 cells. Treatment with TPM, but not STE or nicotine alone, significantly increased [Ca2+]i in a concentration-dependent manner in HL60 cells. Moreover, TPM-induced [Ca2+]i increase was not related to extracellular Ca2+ and did not require the activation of the IP3 pathway nor involved the transient receptor potential (TRP) channels. Our findings indicate that, in cells having either intact or depleted endoplasmic reticulum (ER) Ca2+ stores, TPM-mediated [Ca2+]i increase involves cytosolic Ca2+ pools other than thapsigargin-sensitive ER Ca2+ stores. These results, for the first time, demonstrate that TPM triggers [Ca2+]i increases, while significantly higher nicotine equivalent doses of STE or nicotine alone, did not affect [Ca2+]i under the experimental conditions. In summary, our study suggests that in contrast with STE or nicotine preparations, TPM activates Ca2+ signaling pathways in HL60 cells. The differential effect of combustible and non-combustible TPPs on Ca2+ mobilization could be a useful in vitro endpoint for tobacco product evaluation.

Curcumin and tetrahydrocurcumin induce cell death in Ara-C-resistant acute myeloid leukemia.

Most anticancer agents induce cancer cell death; however, multidrug-resistant cancers often lead to treatment failure. The effective use of curcumin as an anticancer agent has been demonstrated in clinical trials. Tetrahydrocurcumin, a major curcumin metabolite, exhibits pharmacological activities similar to those of curcumin. Curcumin induces cell death mainly through the apoptosis pathway, and tetrahydrocurcumin induces cell death mainly via an autophagy pathway in HL60 cells. Here, we investigated whether curcumin and tetrahydrocurcumin can induce apoptosis- and autophagy-mediated cell deaths in Ara-C-resistant cancer cells, respectively. The results demonstrated that curcumin and tetrahydrocurcumin induced cell death by apoptosis and autophagy, respectively, in Ara-C-resistant HL60 cells. Thus, curcumin and tetrahydrocurcumin have potential applications in the treatment of acute myeloid leukemia with Ara-C resistance.

MiR-183-5p Inhibits Occurrence and Progression of Acute Myeloid Leukemia via Targeting Erbin.

Erbin has been shown to have significant effects on the development of solid tumors. However, little is known about its function and regulatory mechanism in hematological malignancies. The biological function of Erbin on cell proliferation was measured in vitro and in vivo. The predicted target of Erbin was validated by dual-luciferase reporter assay and rescue experiment. We found that overexpression of Erbin could inhibit the cell proliferation and promote the cell differentiation of acute myeloid leukemia (AML) cells, whereas depletion of Erbin could enhance the cell proliferation and block the cell differentiation in AML cells in vitro and in vivo. Besides, miR-183-5p was identified as the upstream regulator that negatively regulated the Erbin expression. The results were confirmed by dual-luciferase reporter and RNA pull-down assay. Furthermore, we found that miR-183-5p negatively regulated Erbin, resulting in enhanced cell proliferation of AML cells via activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways. The activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways was mediated by Erbin interacting with Grb2. These results were also validated by rescue experiments in vitro and in vivo. All above-mentioned findings indicated that the miR-183-5p/Erbin signaling pathway might represent a novel prognostic biomarker or therapeutic target for treatment of AML.

Cell-specific regulation of Nrf2 during ROS-Dependent cell death caused by 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ)

2,3,5-tris(Glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic and nephroncarcinogenic metabolite of benzene and hydroquinone, retains the ability to redox cycle and create oxidative stress. We have previously detected that TGHQ induces ROS-dependent necrotic or apoptotic cell death in renal epithelial HK-2 and human leukemic HL-60 cells respectively. Herein, we sought to determine the nature of the Nrf2 regulation in HK-2 and HL-60 cells undergoing TGHQ-mediated ROS-dependent cell death, due to the key role of Nrf2 in oxidative stress. Intriguingly, Nrf2 was upregulated in HK-2, but not in HL-60 cells, despite the ROS-dependent nature of cell death in both cell types. The possibility that TGHQ targeted the GSK3β-dependent Nrf2 stabilization pathway in HL-60 cells was discounted, whereas TGHQ-induced decreases in Nrf2 phosphorylation at Ser40 site appears to partially underlie the inability of TGHQ to up-regulate Nrf2 expression in HL-60 cells. Moreover, whereas the TGHQ-induced post-translational stabilization of Nrf2 in HK-2 cells resulted in the expected upregulation of HO1 and NQO1 mRNA, TGHQ actually decreased Nrf2 mRNA in HL-60 cells, with a concomitant decrease in NQO1, but not HO1 mRNA. In summary, we define differences between the two cell types that might contribute to the engagement of the Nrf2 signaling pathways. By extension, these data provide evidence that Nrf2 is not necessarily activated in ROS-dependent cell death, and further delve into the knowledge that Nrf2 regulation sensing by cells might be achieved at solely transcriptional level, not related to its degradation.

Genistein induces apoptosis in vitro and has antitumor activity against human leukemia HL-60 cancer cell xenograft growth in vivo.

Genistein, a major isoflavone compound in soybeans, has been shown to have biological activities including anti-cancer activates. In the present, we investigated the anti-leukemia activity of genistein on HL-60 cells in vitro. The percentage of viable cell, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS), and Ca2+ production and the level of ΔΨm were measured by flow cytometric assay. Cell apoptosis and endoplasmic reticulum (ER) stress associated protein expressions were examined by Western blotting assay. Calpain 1, GRP78, and GADD153 expression were measured by confocal laser microscopy. Results indicated that genistein-induced cell morphological changes, decreased the total viable cells, induced G2 /M phase arrest and DNA damage and fragmentation (cell apoptosis) in HL-60 cells. Genistein promoted ROS and Ca2+ productions and decreased the level of ΔΨm in HL-60 cells. Western blotting assay demonstrated that genistein increased ER stress-associated protein expression such as IRE-1α, Calpain 1, GRP78, GADD153, caspase-7, caspase-4, and ATF-6α at 20-50 μM treatment and increased apoptosis associated protein expression such as pro-apoptotic protein Bax, PARP-cleavage, caspase-9, and -3, but decreased anti-apoptotic protein such as Bcl-2 and Bid in HL-60 cells. Calpain 1, GRP78, and GADD153 were increased in HL-60 cells after exposure to 40 μM of genistein. In animal xenografted model, mice were intraperitoneally injected with genistein (0, 0.2, and 0.4 mg/kg) for 28 days and the body weight and tumor volume were recorded. Results showed that genistein did not affect the body weights but significantly reduced the tumor weight in 0.4 mg/kg genistein-treated group. Genistein also increased the expressions of ATF-6α, GRP78, Bax, Bad, and Bak in tumor. In conclusion, genistein decreased cell number through G2 /M phase arrest and the induction of cell apoptosis through ER stress- and mitochondria-dependent pathways in HL-60 cells and suppressed tumor properties in vivo.

ブナシメジ由来Hypsiziprenol A_9によるcAMP経路の抑制を介したHL-60細胞のアポトーシス誘導

ブナシメジ由来Hypsiziprenol A_9によるcAMP経路の抑制を介したHL-60細胞のアポトーシス誘導

Novel nitroaromatic compound activates autophagy and apoptosis pathways in HL60 cells

N-(2-butanoyloxyethyl)-4-(chloromethyl)-3-nitrobenzamide (NBCN) is a nitroaromatic bioreducible compound with cytotoxic effects in cancer cell lines. The aim of this work was to investigate the molecular mechanisms involved in cell death promoted by NBCN in HL60 cells. We observed that NBCN treatment increased intracellular ROS and reduced mitochondria membrane potential (ΔΨm). NBCN treatment also induced morphological changes, phosphatidylserine exposure, cell cycle arrest in G2/M-phase, DNA condensation and fragmentation, but it did not show cytotoxic effects on normal human peripheral blood mononuclear cells (PBMCs). NBCN-induced caspase 3- and 9-dependent DNA fragmentation, which was blocked by pretreatment with the broad-spectrum caspase inhibitor, z-VAD-fmk. Flow cytometry analysis demonstrated that NBCN also increased of the number of autophagic vesicles in HL60 cells, which was not observed when cells were pre-treated with bafilomycin A1. Taken together, these results indicate that NBCN triggered the mitochondrial apoptotic pathway and led to the onset of autophagic cell death, which contributed to its cytotoxic effects.

Influence of Nucleostemin Expression Down-Regulation on PI3K/AKT/mTOR Signal Pathway in HL-60 Cells

To explore the effect of nucleostemin(NS) RNAi on the expression of signal molecules in PI3K/AKT/mTOR pathway, a candidate of p53-independent signal pathway in the leukemia HL-60 cells. The expression of NS was interfered by transfection of P53-deficient HL-60 cells with the recombinant lentivirus expression vector NS-RNAi-GV248. The exression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by Western blot. The fluorescence microscopy showed that the recombinant lentivirus vector NS-RNAi-GV248 transfected HL-60 cells successfully with a 80% transfection rate. Western blot showed that the expression of NS protein was inhibited obviously in HL-60 cells, and the expression levels of AKT, p-AKT, p70s6k and p-p70s6k were not statistically different(t1=2.31,P>0.05;t2=3.62,P>0.05;t3=1.60,P>0.05;t4=2.72,P>0.05) in comparison with control; the expression of GβL protein was statistically down-regnlated (t=15.01,P=0.002). The changes of GβL protein correlats with NS knockdown. The PI3K/AKT/mTOR pathway may be one of nucleostemin p53-independent signal pathways.

Synergistic effects of phenylhexyl isothiocyanate and LY294002 on the PI3K/Akt signaling pathway in HL-60 cells.

The aim of the present study was to investigate the synergistic effect of phenylhexyl isothiocyanate (PHI) and LY294002 [an inhibitor of phosphoinositide 3-kinase (PI3K)] on the PI3K/protein kinase B (Akt) signaling pathway, modulating histone acetylation, inhibiting cell viability and inducing apoptosis in HL-60 cells. The inhibition of HL-60 cell viability was monitored using an MTT assay. Cell apoptosis was measured using flow cytometry. Expression of acetylated histone H3 and histone H4, and the Akt signaling pathway proteins phosphorylated Akt (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR) and phosphorylated ribosomal protein S6 kinase (p-p70S6K) was detected using western blotting. The results of the present study identified that PHI and LY294002 were able to inhibit cell viability and induce cell apoptosis in HL-60 cells. The combination exhibited a synergistic effect on cell viability and apoptosis. PHI treatment led to an accumulation of acetylated histone H3 and histone H4, but LY294002 treatment had no effect on histone acetylation. However, LY294002 was identified to enhance the effect of PHI on histone acetylation in HL-60 cells. PHI and/or LY294002 were identified to dephosphorylate proteins in the PI3K/Akt signaling pathway, with a synergistic effect observed when used in combination. The results of the present study indicated that the combination of PHI and LY294002 may offer a novel therapeutic strategy for acute myeloid leukemia.

Hinesol, a compound isolated from the essential oils of Atractylodes lancea rhizome, inhibits cell growth and induces apoptosis in human leukemia HL-60 cells.

Hinesol is a unique sesquiterpenoid isolated from the Chinese traditional medicine, Atractylodes lancea rhizome. In a previous study, we screened various natural products in human leukemia HL-60 cells and identified an essential oil fraction from A. lancea rhizome that exhibited apoptosis-inducing activity in these cells; hinesol was subsequently shown to be the compound responsible for this apoptosis-inducing activity. In this study, we describe the cytotoxic effects and molecular mechanisms of hinesol in HL-60 cells. The antitumor effect of hinesol was associated with apoptosis. When HL-60 cells were treated with hinesol, characteristic features of apoptosis, such as nuclear fragmentation and DNA fragmentation, were observed. These growth-inhibitory and apoptosis-inducing activities of hinesol in leukemia cells were much stronger than those of β-eudesmol, another compound isolated from the essential oil fraction. Furthermore, hinesol induced activation of c-Jun N-terminal kinase (JNK), but not p38, prior to the onset of apoptosis. These results suggested that hinesol induced apoptosis through the JNK signaling pathway in HL-60 cells. Therefore, hinesol may represent a novel medicinal drug having indications in the treatment of various cancers, including leukemia.

Cytotoxic effects of tetracycline analogues (doxycycline, minocycline and COL-3) in acute myeloid leukemia HL-60 cells.

Tetracycline analogues (TCNAs) have been shown to inhibit matrix metalloproteinases and to induce apoptosis in several cancer cell types. In the present study, the cytotoxic effects of TCNAs doxycycline (DOXY), minocycline (MINO) and chemically modified tetracycline-3 (COL-3) were investigated in the human acute myeloid leukemia HL-60 cell line. Cells were incubated with TCNAs in final concentrations of 0.5–100 µg/ml for 24 h. Viability of the leukemic cells was inhibited in a concentration-dependent manner using resazurin assay. The estimated IC50s were 9.2 µg/ml for DOXY, 9.9 µg/ml for MINO and 1.3 µg/ml for COL-3. All three TCNAs induced potent cytotoxic effects and cell death. Apoptosis, which was assessed by morphological changes and annexin V positivity, was concentration- and time-dependent following incubation with any one of the drugs. TCNAs induced DNA double strand breaks soon after treatment commenced as detected by γH2AX and western blot. The loss of mitochondrial membrane potential (Δψm), caspase activation and cleavage of PARP and Bcl-2 were observed; however, the sequence of events differed among the drugs. Pancaspase inhibitor Z-VAD-FMK improved survival of TCNAs-treated cells and decreased TCNAs-induced apoptosis. In summary, we demonstrated that TCNAs had a cytotoxic effect on the HL-60 leukemic cell line. Apoptosis was induced via mitochondria-mediated and caspase-dependent pathways in HL-60 cells by all three TCNAs. COL-3 exerted the strongest anti-proliferative and pro-apoptotic effects in concentrations that have been achieved in human plasma in reported clinical trials. These results indicate that there is a therapeutic potential of TCNAs in leukemia.

Gene expression profiling of HL-60 cells following knockdown of nucleostemin using DNA microarrays.

Nucleostemin (NS) plays an important role in tumorigenesis and progression. Most studies consider that NS plays its role through combining with p53 and inhibiting it, however our previous studies revealed that NS could also function without the existence of p53. To date, few studies have focused on the p53-independent pathway of NS, and its molecular mechanism remains unknown. The aim of the present study was to investigate the p53-independent pathway of NS in the human acute myeloid leukemia cell line HL-60 which was p53-null by using the DNA microarray technique. Lentivirus-mediated RNA interference technique was used to knock down NS expression in HL-60 cells, and then DNA microarray and bioinformatics were used to analyze the gene expression profiling changes. The microarray data showed that after knocking down NS in HL-60 cells, 2,628 differentially expressed genes were identified through ≥ 2 or ≤ 0.5-fold-change, in which 818 genes were upregulated and 1,810 genes were downregulated. Real-time quantitative polymerase chain reaction (qPCR) validated the reliability of DNA microarray data. Pathway analysis showed extensive signal pathways in HL-60 cells were influenced by inhibiting NS expression. In particular, the inhibition of PI3K-AKT pathway, JAK-STAT pathway, RAS-RAF-MEK-ERK1/2 pathway and activation of JNK pathway, p38 MAPK pathway may associate with the apoptosis of HL-60 cells after knocking down NS. The findings of this study provide insight to further explore the specific molecular mechanism of NS function in p53-null leukemia and they also lay the foundations for exploring new therapeutic targets for p53-null leukemia and even p53-null tumors.

Cytotoxic and Melanogenesis‐Inhibitory Activities of Limonoids from the Leaves of Azadirachta indica (Neem)

Seventeen limonoids (tetranortriterpenoids), 1-17, including three new compounds, i.e., 17-defurano-17-(2,5-dihydro-2-oxofuran-3-yl)-28-deoxonimbolide (14), 17-defurano-17-(2ξ-2,5-dihydro-2-hydroxy-5-oxofuran-3-yl)-28-deoxonimbolide (15), and 17-defurano-17-(5ξ-2,5-dihydro-5-hydroxy-2-oxofuran-3-yl)-2',3'-dehydrosalannol (17), were isolated from an EtOH extract of the leaf of neem (Azadirachta indica). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines, seven compounds, i.e., 1-3, 12, 13, 15, and 16, exhibited potent cytotoxicities with IC50 values in the range of 0.1-9.9 μM against one or more cell lines. Among these compounds, cytotoxicity of nimonol (1; IC50 2.8 μM) against HL60 cells was demonstrated to be mainly due to the induction of apoptosis by flow cytometry. Western blot analysis suggested that compound 1 induced apoptosis via both the mitochondrial and death receptor-mediated pathways in HL60 cells. In addition, when compounds 1-17 were evaluated for their inhibitory activities against melanogenesis in B16 melanoma cells, induced with α-melanocyte-stimulating hormone (α-MSH), seven compounds, 1, 2, 4-6, 15, and 16, exhibited inhibitory activities with 31-94% reduction of melanin content at 10 μM concentration with no or low toxicity to the cells (82-112% of cell viability at 10 μM). All 17 compounds were further evaluated for their inhibitory effects against the EpsteinBarr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells.

Down-regulatory effect of Nucleostemin expression on signal molecule of PI3K/AKT/mTOR pathway in HL-60 cells

This study was purpose to explore the down-regulatory effect of nucleostemin (NS) expression on signal molecules of PI3K/AKT/mTOR pathway belonged to candidate ways of p53-independent signal pathway in the leukemia cells. The expression of NS was interfered by using recombinant lentivirus expression vector NS-RNAi-GV248 to transfect HL-60 cells of p53 deficiency. The expression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by using Real-time PCR. The results of showed that the HL-60 cells were transfected by recombinant lentivirus vector NS-RNAi-GV248 successfully and with transfection rate up to 80%. According to results of Real-time PCR detection, the inhibition rate of NS gene was 56.5% in HL-60 cells. And the expression levels of PI3K,AKT and GβL mRNA (0.491 ± 0.084,0.398 ± 0.164, 0.472 ± 0.097 respectively) were obviously down-regulated by silencing NS, and showed statistical difference (P < 0.05) in comparison with control (1.002 ± 0.171, 1.000 ± 0.411, 1.001 ± 0.206 respectively) . It is concluded that the changes of signal molecules of PI3K/AKT/mTOR pathway positively correlate with NS down-regulation, which provides evidence for confirming PI3K/AKT/mTOR signal pathway possible as a type of NS p53-independent pathway.

The anticancer potential of flavonoids isolated from the stem bark of Erythrina suberosa through induction of apoptosis and inhibition of STAT signaling pathway in human leukemia HL-60 cells

Erythrina suberosa is an ornamental tall tree found in India, Pakistan, Nepal, Bhutan, Burma, Thailand and Vietnam. We have isolated four known distinct metabolites designated as α-Hydroxyerysotrine, 4′-Methoxy licoflavanone (MLF), Alpinumisoflavone (AIF) and Wighteone. Among the four isolated metabolites the two flavonoids, MLF and AIF were found to be the most potent cytotoxic agent with IC50 of ∼20μM in human leukemia HL-60 cells. We are reporting first time the anticancer and apoptotic potential of MLF and AIF in HL-60 cells. Both MLF and AIF inhibited HL-60 cell proliferation and induce apoptosis as measured by several biological endpoints. MLF and AIF induce apoptosis bodies formation, enhanced annexinV-FITC binding of the cells, increased sub-G0 cell fraction, loss of mitochondrial membrane potential (Δψm), release of cytochrome c, Bax, activation of caspase-9, caspase-3 and PARP (poly ADP Ribose polymers) cleavage in HL-60 cells. MLF and AIF also increase the expression of apical death receptor, Fas, with inhibition of anti-apoptotic protein Bid. All the above parameters revealed that these two flavonoids induce apoptosis through both extrinsic and intrinsic apoptotic pathways in HL-60 cells. In spite of apoptosis, these two flavonoids significantly inhibit nuclear transcription factor NF-κB and STAT (Signal Transducer and Activator of Transcription) signaling pathway, which are highly expressed in leukemia. The present study provide an insight of molecular mechanism of cell death induced by MLF and AIF in HL-60 cells which may be useful in managing and treating leukemia.