Tetra-arsenic tetra-sulfide enhances NK-92MI mediated cellular immunotherapy in all-trans retinoic acid-resistant acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is liable to induce disseminated intravascular coagulation and has a high early mortality. Although the combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has significantly improved the complete remission rate, there are still some patients developed drug resistance. Growing evidence suggests that natural killer (NK) cell-mediated immunotherapy as a new treatment can help slow the progression of hematological malignancies. Previous studies also indicated that some tumors exhibited excellent responsiveness to NK cells in vitro. However, many clinical trial results showed that the anti-tumor effect of NK cells infusion alone was not ideal, which may be related to the inactivation of infiltrating NK cells caused by strong immunosuppression in tumor microenvironment, but the specific mechanism remains to be further explored. In the present study, we demonstrated that low doses of tetra-arsenic tetra-sulfide (As4S4) not only enhanced the in vitro killing of NK-92MI against ATRA-resistant APL cells, but also strengthened the growth inhibition of xenografted tumors in APL mouse model. Mechanistically, As4S4 altered the expression of natural killer group 2 member D ligands (NKG2DLs) and cytokines in APL cells, and PD-1 in NK-92MI cells. In addition, database retrieval results further revealed the relationship between the differentially regulated molecules of As4S4 and immune infiltration and its impact on prognosis. In conclusion, our findings confirmed the potential of As4S4 as an adjuvant for NK-92MI in the treatment of ATRA-resistant APL.

Realgar (As4S4), a traditional Chinese medicine, induces acute promyelocytic leukemia cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway in vitro.

Acute promyelocytic leukemia (APL) is a specific subtype of acute myelogenous leukemia (AML) characterized by the proliferation of abnormal promyelocytes. Realgar, a Chinese medicine containing arsenic, can be taken orally. Traditional Chinese medicine physicians have employed realgar to treat APL for over a thousand years. Therefore, realgar may be a promising candidate for the treatment of APL. Nevertheless, the underlying mechanism behind realgar therapy is largely unclear. The present study aimed to investigate the effect of realgar on cell death in the APL cell line (NB4) in vitro and to elucidate the underlying mechanism. In this study, after APL cells were treated with different concentrations of realgar, the cell survival rate, apoptotic assay, morphological changes, ATP levels and cell cycle arrest were assessed. The expression of Bcl-2, Bax, Cytochrome C (Cyt-C) and apoptosis-inducing factor (AIF) at the mRNA and protein levels were also measured by immunofluorescence, quantitative PCR (qPCR) and Western blotting. We found that realgar could significantly inhibit APL cell proliferation and cell death in a time- and dose-dependent manner. Realgar effectively decreased the ATP levels in APL cells. Realgar also induced APL cell cycle arrest at the S and G2/M phases. Following realgar treatment, the mRNA and protein levels of Bcl-2 were significantly downregulated, whereas the levels of Bax, Cyt-C, and AIF were significantly upregulated. In summary, realgar can induce APL cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway, suggesting that realgar may be an effective therapeutic for APL.

Cinobufagin induces acute promyelocytic leukaemia cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the ß-catenin signalling pathway.

CONTEXT: Acute promyelocytic leukaemia (APL) is a malignant hematological tumour characterized by the presence of promyelocytic leukaemia-retinoic acid receptor A (PML-RARA) fusion protein. Cinobufagin (CBG) is one of the main effective components of toad venom with antitumor properties. However, only a few reports regarding the CBG treatment of APL are available. OBJECTIVE: We explored the effect and mechanism of action of CBG on NB4 and NB4-R1 cells. MATERIALS AND METHODS: We evaluated the viability of NB4 and NB4-R1 cells treated with 0, 20, 40, and 60 nM CBG for 12, 24, and 48 h. After treatment with CBG for 24 h, Bcl-2 associated X (Bax), B-cell lymphoma 2 (Bcl-2), ß-catenin, cyclin D1, and c-myc expression was detected using western blotting and real-time polymerase chain reaction. Caspase-3 and PML-RARA expression levels were detected using western blotting. RESULTS: CBG inhibited the viability of NB4 and NB4-R1 cells. The IC50 values of NB4 and NB4-R1 cells treated with CBG for 24 h were 45.2 nM and 37.9 nM, respectively. CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner and inhibited the ß-catenin signalling pathway. DISCUSSION AND CONCLUSION: CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the ß-catenin signalling pathway. This study proposes a novel treatment strategy for patients with APL, particularly those with ATRA-resistant APL.

PML/RARA destabilization by hyperthermia: a new model for oncogenic fusion protein degradation?

In this issue, Maimaitiyiming and colleagues demonstrate thermic stress-induced PML/RARA oncogenic fusion protein destabilization driven by corepressor aggregation. Hyperthermia synergizes with PML/RARA degradation by ATO and may circumvent ATO-resistance in historical APL patients. This novel approach could be extended to other corepressor-associated oncogenic fusion proteins.

Towards plant-mediated chemistry - Au nanoparticles obtained using aqueous extract of Rosa damascena and their biological activity in vitro.

An eco-friendly, efficient, and controlled synthesis of gold nanoparticles with application of the aqueous extract of Rosa damascena (Au@RD NPs) without using any other reducing agents was studied. Au@RD NPs of narrow size distribution were characterized by UV-vis and FT-IR spectroscopies, transmission electron microscopy, X-ray powder diffraction, X-ray photoelectron spectroscopy, particle size analysis, and zeta potential measurements. In vitro stability experiments revealed that the Au@RD NPs were stable for over a year (pH ~ 3.5), proving a significant stabilizing potential of the aqueous RD extract. The high total content of polyphenols, flavonoids, and reducing sugars along with the powerful antioxidant activity of the RD extract was determined by spectroscopic and analytical methods. Colloids prepared from the purified and lyophilized Au@RD NPs (electrokinetic potential of ca. -33 mV) were stable for at least 24 h under terms similar to physiological conditions (pH = 7.4, PBS). The in vitro cytotoxicity of Au@RD NPs was investigated against peripheral blood mononuclear lymphocytes (PBML), acute promyelocytic leukemia (HL60), and human lung adenocarcinoma (A549). Selective cytotoxicity of Au@RD NPs towards cancer cells (HL60, A549) over normal cells (PBML) in vitro was explicitly demonstrated by viability assays. Comet assay revealed a higher level of DNA damages in cancer cells when compared with normal ones. Apoptotic death in cancer cells was proved by measuring caspases activity. Thus, the developed Au@RD NPs, obtained by the plant-mediated green synthesis, are attractive hybrid materials for the medical applications combining two active components - metal nanoparticles platform and plant-derived metabolites.

Metabolomics Reveals that Cysteine Metabolism Plays a Role in Celastrol-Induced Mitochondrial Apoptosis in HL-60 and NB-4 Cells.

Recently, celastrol has shown great potential for inducing apoptosis in acute myeloid leukemia cells, especially acute promyelocytic leukaemia cells. However, the mechanism is poorly understood. Metabolomics provides an overall understanding of metabolic mechanisms to illustrate celastrol's mechanism of action. We treated both nude mice bearing HL-60 cell xenografts in vivo and HL-60 cells as well as NB-4 cells in vitro with celastrol. Ultra-performance liquid chromatography coupled with mass spectrometry was used for metabolomics analysis of HL-60 cells in vivo and for targeted L-cysteine analysis in HL-60 and NB-4 cells in vitro. Flow cytometric analysis was performed to assess mitochondrial membrane potential, reactive oxygen species and apoptosis. Western blotting was conducted to detect the p53, Bax, cleaved caspase 9 and cleaved caspase 3 proteins. Celastrol inhibited tumour growth, induced apoptosis, and upregulated pro-apoptotic proteins in the xenograft tumour mouse model. Metabolomics showed that cysteine metabolism was the key metabolic alteration after celastrol treatment in HL-60 cells in vivo. Celastrol decreased L-cysteine in HL-60 cells. Acetylcysteine supplementation reversed reactive oxygen species accumulation and apoptosis induced by celastrol and reversed the dramatic decrease in the mitochondrial membrane potential and upregulation of pro-apoptotic proteins in HL-60 cells. In NB-4 cells, celastrol decreased L-cysteine, and acetylcysteine reversed celastrol-induced reactive oxygen species accumulation and apoptosis. We are the first to identify the involvement of a cysteine metabolism/reactive oxygen species/p53/Bax/caspase 9/caspase 3 pathway in celastrol-triggered mitochondrial apoptosis in HL-60 and NB-4 cells, providing a novel underlying mechanism through which celastrol could be used to treat acute myeloid leukaemia, especially acute promyelocytic leukaemia.

A novel realgar-indigo naturalis formula more effectively induces apoptosis in NB4 cells.

Realgar as a kind of arsenic agent is currently used to treat APL in China. The effectiveness and low toxicity of realgar have been verified, lower than arsenic trioxide. Although the therapeutic efficacy of realgar is blocked severely by its poor insolubility in water. In our lab, we addressed this problem by obtaining realgar bioleaching solution (RBS) from microbiological leaching technique. To develop a tradition Chinese medicinal formula (TCMF) for clinical application realgar is usually used with other herbs. However, treated realgar with RBS has not been evaluated in TCMF contain realgar. In the present study we used NB4 to investigate the effects of novel Realgar-Indigo naturalis formula (FRBS) on cell proliferation and apoptosis. We used MTT assay to measure anti proliferative activity of FRBS. We further study the effects of FRBS on cell growth and apoptosis according flow cytometry, DNA fragmentation assay and Fluorescence microscopy and Western blot. The results revealed that FRBS significantly inhibited growth in a dose-dependent manner, and induced apoptosis in NB4 cells. NB4 cell inhibitory response to FRBS at 2µg ml-1 of arsenic concentration was twofold higher, dissimilar to RIF, and induced apoptosis more effectively. Further, a higher expression of caspase-3, caspase-9 and cytochrome C from increased from FRBS. RBS can substitute the traditional realgar powder in RIF in order to provide a novel and promising Realgar-Indigo naturalis formula to treat acute promyelocytic leukemia.

The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies.

Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.

Green tea polyphenol EGCG causes anti-cancerous epigenetic modulations in acute promyelocytic leukemia cells.

Green tea (Camellia sinensis) catechin epigallocatechin-3-gallate (EGCG) has been shown to possess diverse anti-cancerous properties. We demonstrated EGCG ability to inhibit acute promyelocytic leukemia (APL) cell proliferation and cause apoptosis. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) analysis revealed elevated expression of genes associated with cell cycle arrest and differentiation (p27, PCAF, C/EBPα, and C/EBPɛ). Furthermore, EGCG caused anti-cancerous epigenetic changes: downregulation of epigenetic modifiers DNMT1, HDAC1, HDAC2, and G9a was observed by RT-qPCR analysis. Reduced amount of H3K9me2 after treatment with EGCG confirmed G9a downregulation. Polycomb repressive complex 2 (PRC2) core components were also shown to be downregulated in gene and protein level. Chromatin immunoprecipitation (ChIP) analysis revealed that EGCG treatment enhanced hyperacetylated H4 and acetylated H3K14 histones binding to the promoter regions of p27, PCAF, C/EBPα, and C/EBPɛ and reduced binding effect to PRC2 core component genes EZH2, SUZ12, and EED. Our results indicate that EGCG, as cell proliferation inhibitor and epigenetic modifier, might be useful for APL treatment.

Berberine Induces Apoptotic Cell Death via Activation of Caspase-3 and -8 in HL-60 Human Leukemia Cells: Nuclear Localization and Structure-Activity Relationships.

Berberine (BBR), an isoquinoline alkaloid, is a well-known bioactive compound contained in medicinal plants used in traditional and folk medicines. In this study, we investigated the subcellular localization and the apoptotic mechanisms of BBR were elucidated. First, we confirmed the incorporation of BBR into the cell visually. BBR showed antiproliferative activity and promptly localized to the nucleus from 5[Formula: see text]min to 15[Formula: see text]min after BBR treatment in HL-60 human promyelocytic leukemia cells. Next, we examined the antiproliferative activity of BBR (1) and its biosynthetically related compounds (2-7) in HL-60 cells. BBR exerted strongest antiproliferative activity among 1-7 and the results of structures and activity relation suggested that a methylenedioxyl group in ring A, an [Formula: see text]-alkyl group at C-9 position, and the frame of isoquinoline may be necessary for antiproliferative activity. Moreover, BBR showed the most potent antiproliferative activity in HL-60 cells among human cancer and normal cell lines tested. Next, we examined the effect of BBR on molecular events known as apoptosis induction. In HL-60 cells, BBR induced chromatin condensation and DNA fragmentation, and triggered the activation of PARP, caspase-3 and caspase-8 without the activation of caspase-9. BBR-induced DNA fragmentation was abolished by pretreatment with inhibitors against caspase-3 and caspase-8, but not against caspase-9. ERK and p38 were promptly phosphorylated after 15 min of BBR treatment, and this was correlated with time of localization to the nucleus of BBR. These results demonstrated that BBR translocated into nucleus immediately after treatments and induced apoptotic cell death by activation of caspase-3 and caspase-8.

Zinc Depletion by TPEN Induces Apoptosis in Human Acute Promyelocytic NB4 Cells.

BACKGROUND/AIMS: The effects of zinc signaling on proliferation or apoptosis of leukemia cells remain elusive. In the present study, we used N, N, N', N'-tetrakis-(2-pyridylmethyl)-ethylene-diamine (TPEN), a membrane-permeable zinc chelator, to evaluate the effect of zinc depletion on survival and apoptosis of NB4 acute promyelocytic leukemia (APL) cells. METHODS: The pro-apoptotic effects of TPEN on NB4 cells were examined by flow cytometry, and observed using an optical microscope. Intracellular labile zinc, nitric oxide (NO) or reactive oxygen species (ROS) changes caused by TPEN were measured by flow cytometry. We then explored possible roles of the crosstalk between intracellular labile zinc signaling and nitric oxide signaling in TPEN-triggered apoptosis. RESULTS: we found that TPEN induced apoptosis in NB4 APL cells in a dosage-dependent manner. We further demonstrated that TPEN triggered apoptosis by attenuating intracellular zinc and nitric oxide signaling in NB4 cells. Both exogenous zinc supplement and the nitric donor sodium nitroprusside (SNP) pre-incubation reversed TPEN-mediated inhibition of intracellular NO and Zn2+ signaling, and rescued NB4 cells from apoptosis. CONCLUSION: These results suggest for the first time that crosstalk between zinc signaling and nitric oxide pathway is essential for the survival of NB4 cells. TPEN induces apoptosis in NB4 cells via negatively regulating intracellular NO and Zn2+ signaling. Our in vitro data suggest that zinc depletion by TPEN may be a potential therapeutic strategy for APL.

Xanthones from Garcinia paucinervis with in vitro anti-proliferative activity against HL-60 cells.

Three new xanthones, paucinervins H-J (1-3), as well as eleven known compounds (4-14), were isolated from the leaves of Garcinia paucinervis. The structures of the new compounds (1-3) were elucidated by 1D, 2D NMR spectra and HR ESIMS. In vitro antiproliferative activity against human promyelocytic leukemia HL-60 cells was tested, among which, compounds 2, 5, 6 and 7 exhibited strong growth inhibitory effects with GI50 values ranging from 1.30 to 9.08 µM, respectively. Preliminary SARs were also discussed.

DNA-mediated adjuvant immunotherapy extends survival in two different mouse models of myeloid malignancies.

We have previously shown that a specific promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) DNA vaccine combined with all-trans retinoic acid (ATRA) increases the number of long term survivors with enhanced immune responses in a mouse model of acute promyelocytic leukemia (APL). This study reports the efficacy of a non-specific DNA vaccine, pVAX14Flipper (pVAX14), in both APL and high risk myelodysplastic syndrome (HR-MDS) models. PVAX14 is comprised of novel immunogenic DNA sequences inserted into the pVAX1 therapeutic plasmid. APL mice treated with pVAX14 combined with ATRA had increased survival comparable to that obtained with a specific PML-RARA vaccine. Moreover, the survival advantage correlated with decreased PML-RARA transcript levels and increase in anti-RARA antibody production. In HR-MDS mice, pVAX14 significantly improved survival and reduced biomarkers of leukemic transformation such as phosphorylated mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1. In both preclinical models, pVAX14 vaccine significantly increased interferon gamma (IFNγ) production, memory T-cells (memT), reduced the number of colony forming units (CFU) and increased expression of the adapter molecule signalling to NF-κB, MyD88. These results demonstrate the adjuvant properties of pVAX14 providing thus new approaches to improve clinical outcome in two different models of myeloid malignancies, which may have potential for a broader applicability in other cancers.

Tetrandrine induces autophagy and differentiation by activating ROS and Notch1 signaling in leukemia cells.

All-trans retinoic acid (ATRA) is a differentiating agent for the treatment of acute promyelocytic leukemia (APL). However, the therapeutic efficacy of ATRA has limitations. Tetrandrine is a traditional Chinese medicinal herb extract with antitumor effects. In this study, we investigated the effects of tetrandrine on human PML-RARα-positive acute promyelocytic leukemia cells. Tetrandrine inhibited tumors in vivo. It induced autophagy and differentiation by triggering ROS generation and activating Notch1 signaling. Tetrandrine induced autophagy and differentiation in M5 type patient primary leukemia cells. The in vivo results indicated that low concentrations of tetrandrine inhibited leukemia cells proliferation and induced autophagy and then facilitated their differentiation, by activating ROS and Notch1 signaling. We suggest that tetrandrine is a potential agent for the treatment of APL by inducing differentiation of leukemia cells.

Nardostachys chinensis induces granulocytic differentiation with the suppression of cell growth through p27(Kip1) protein-related G0/G1 phase arrest in human promyelocytic leukemic cells.

CONTEXT: Nardostachys chinensis Batalin (Valerianaceae) has been used in Korean traditional medicine to elicit stomachic and sedative effects. However, the anti-leukemic activities of N. chinensis have not been well examined. OBJECTIVE: To investigate the effect of N. chinensis on differentiation and proliferation in the human promyelocytic leukemic HL-60 cells. MATERIALS AND METHODS: The dried roots and stems of N. chiensis are extracted using hot water and then freeze-dried. The yield of extract was 12.82% (w/w). The HL-60 cells were treated with 25-200 µg/ml of N. chinensis for 72 h or 100 µg/ml of N. chinensis for 24-72 h. RESULTS: Nardostachys chinensis significantly inhibited cell viability dose dependently with an IC50 of 100 µg/ml in HL-60 cells. Nardostachys chinensis induced differentiation of the cells as measured by reduction activity of NBT and expression of CD11b but not of CD14 as analyzed by flow cytometry, which indicates a differentiation toward the granulocytic lineage. Nardostachys chinensis also induced growth inhibition through G0/G1 phase arrest in the cell cycle of HL-60 cells. Among the G0/G1 phase in the cell cycle-related protein, the expression of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) was increased in N. chinensis-treated HL-60 cells, whereas the expression levels of CDK2, CDK4, CDK6, cyclin D1, cyclin D3, cyclin E, and cyclin A were decreased. Interestingly, N. chinensis markedly enhanced the binding of p27(Kip1) with CDK2 and CDK6. DISCUSSION AND CONCLUSION: This study demonstrated that N. chinensis is capable of inducing cellular differentiation and growth inhibition through p27(Kip1) protein-related G0/G1 phase arrest in HL-60 cells.

[Significance of PLSCR1 in Matrine Induced Differentiation of ATRA Resistant APL Cells].

OBJECTIVE: To observe the expression of phospholipid scramblase 1 (PLSCR1) in matrine (MAT) induced differentiation of all-trans retinoic acid (ATRA) resistant acute promyelocytic leukemia (APL) cells, and to explore its correlation to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signal pathway. METHODS: NB4 (an APL cell line sensitive to ATRA) and NB4-R1 (a resistant strain of ATRA) were observed as subjects in this study. Effects of combined treatment of 0.1 mmol/L MAT and 1 [mol/L ATRA on the differentiation of two cell lines were detected using nitroblue tetrazolium (NBT) reduction test and flow cytometry (CD11b). Expressions of PML/RARot and PLSCR1 protein/gene were detected using Western blot and Real-time fluorescence quantitative PCR assay. Meanwhile, H89, PKA antagonist, was used to observe cell differentiation antigen and changes of aforesaid proteins and genes. RESULTS: MAT combined ATRA could significantly elevate positive rates of NBT and CD11 b in NB4-R1 cells, and significantly down-regulate the expression of PML/RARapha-fusion protein/gene (P < 0.05, P < 0.01). ATRA used alone could obviously enhance the expression of PLSCRI in NB4 cells at protein and mRNA levels (P < 0.01). But the expression of PLSCR1 was up-regulated in NB4-R1 cells, but with statistical.difference only at the protein level (P <0. 01). In combination of MAT, PLSCR1 protein expression was further elevated in the two cell lines (P < 0.01). Besides, there was statistical difference in mRNA expressions in NB4-R1 cells (P < 0.05). All these actions could be reversed by treatment of 10 micromol/L H89 (P < 0.05, P < 0.01). CONCLUSION: MAT combined ATRA could significantly induce the differentiation of NB4-R1 cells, and inhibit the expression of PML/RARalpha fusion gene/protein, which might be associated with up-regulating PLSCR1 expression.

Antiproliferative activity and induction of apoptosis by Annona muricata (Annonaceae) extract on human cancer cells.

BACKGROUND: Annona muricata (A. muricata) is widely distributed in Asia, Africa and South America. Different parts of this plant are used to treat several diseases in Cameroon. The aim of this study is to determine the in vitro anti-proliferative effects and apoptotic events of A. muricata extracts on HL-60 cells as well as to quantify its phenols content. METHODS: The cell viability was measured by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay while the changes in morphology of HL-60 cells, membrane mitochondrial potential (MMP) and the cell cycle were used for assessment apoptosis induction. RESULTS: The results show that the concentration of phenols, flavonoids and flavonols in the extracts varied depending on the part of the plant. All the extracts tested inhibited the proliferation of HL-60 cells in a concentration dependent manner with IC50 varied from 6-49 µg/mL. The growth inhibition of the cells by extracts was associated with the disruption of MMP, reactive oxygen species (ROS) generation and the G0/G1 cell arrest. CONCLUSION: These findings suggest that the extracts from A. muricata have strong antiproliferation potential and can induce apoptosis through loss of MMP and G0/G1 phase cell arrest.

The anti-promyelocytic leukemia mode of action of two endophytic secondary metabolites unveiled by a proteomic approach.

As a result of a program to find antitumor compounds of endophytes from medicinal Asteraceae, the steroid (22E,24R)-8,14-epoxyergosta-4,22-diene-3,6-dione (a) and the diterpene aphidicolin (b) were isolated from the filamentous fungi Papulaspora immersa and Nigrospora sphaerica, respectively, and exhibited strong cytotoxicity against HL-60 cells. A proteomic approach was used in an attempt to identify the drugs' molecular targets and their respective antiproliferative mode of action. Results suggested that the (a) growth inhibition effect occurs by G2/M cell cycle arrest via reduction of tubulin alpha and beta isomers and 14-3-3 protein gamma expression, followed by a decrease of apoptotic and inflammatory proteins, culminating in mitochondrial oxidative damage that triggered autophagy-associated cell death. Moreover, the decrease observed in the expression levels of several types of histones indicated that (a) might be disarming oncogenic pathways via direct modulation of the epigenetic machinery. Effects on cell cycle progression and induction of apoptosis caused by (b) were confirmed. In addition, protein expression profiles also revealed that aphidicolin is able to influence microtubule dynamics, modulate proteasome activator complex expression, and control the inflammatory cascade through overexpression of thymosin beta 4, RhoGDI2, and 14-3-3 proteins. Transmission electron micrographs of (b)-treated cells unveiled dose-dependent morphological characteristics of autophagy- or oncosis-like cell death.

MicroRNAs contribute to promyelocyte apoptosis in As2O3-treated APL cells.

BACKGROUND: Arsenic trioxide (As2O3), an ancient drug used in traditional Chinese medicine, has substantial anticancer activities, especially in the treatment of patients suffering from acute promyelocytic leukemia (APL); however the underlying mechanisms are not well understood. METHODS: MTT assay was used to detect the cell viability. Flow Cytometry analysis and caspase-3 activity assay were used to measure apoptosis of APL cells. Caspase-3 and Bax levels were analyzed by western blot and let-7d and miR-766 levels were determined by real-time RT-PCR. RESULTS: As2O3 significantly inhibited cell viability and induced apoptosis in APL cells. Several microRNAs, including let-7d and miR-766, were dysregulated in APL cells treated with As2O3. The expression of caspase-3 and Bax, which are targets of let-7d and miR-766, respectively, were up-regulated in As2O3 treated cells. Transfection of let-7d and miR-766 into NB4 cells decreased the expression of caspase-3 and Bax, respectively. Correspondingly, transfection of these microRNAs increased NB4 cell viability. As2O3 induced degradation of promyelocytic leukemia (PML), and then induced the down-regulation of both let-7d and miR-766 in NB4 cells. CONCLUSIONS: We construct a dysregulated microRNA network involved in As2O3-induced apoptosis in APL. Targeting this network may be a new strategy for the prevention of side effects associated with APL treatment with As2O3.

Sequence-specific electrochemical detection of double-strand PCR amplicons of PML/RARα fusion gene in acute promyelocytic leukemia.

A novel electrochemical method for the sequence-specific detection of double-stranded polymerase chain reaction (PCR) products of PML/RARα fusion gene in acute promyelocytic leukemia (APL) was described in detail. Based on a "sandwich" sensing mode involving a pair of locked nucleic acids probes (capture probe and reporter probe), this DNA sensor exhibited excellent selectivity and specificity. The direct and quantitative analysis of double-stranded complementary was firstly performed by our sensor without the use of alkali, helicase enzymes, or denaturants. Finally, combining PCR technique with electrochemical detection scheme, PCR amplicons (191 bp) of the PML/RARα fusion gene were obtained and rapidly identified with a low detection limit of 79 fmol in the 100-µL hybridization system. The results clearly showed the power of sensor as a promising tool for the sensitive, specific, and portable detection of APL and other diseases.