DNR-induced Apoptosis Research Articles

CALCRL induces resistance to daunorubicin in acute myeloid leukemia cells through upregulation of XRCC5/TYK2/JAK1 pathway.

Chemotherapy is the main treatment option for acute myeloid leukemia (AML), but acquired resistance of leukemic cells to chemotherapeutic agents often leads to difficulties in AML treatment and disease relapse. High calcitonin receptor-like (CALCRL) expression is closely associated with poorer prognosis in AML patients. Therefore, this study was performed by performing CALCRL overexpression constructs in AML cell lines HL-60 and Molm-13 with low CALCRL expression. The results showed that overexpression of CALCRL in HL-60 and Molm-13 could confer resistance properties to AML cells and reduce the DNA damage and cell cycle G0/G1 phase blocking effects caused by daunorubicin (DNR) and others. Overexpression of CALCRL also reduced DNR-induced apoptosis. Mechanistically, the Cancer Clinical Research Database analyzed a significant positive correlation between XRCC5 and CALCRL in AML patients. Therefore, the combination of RT-PCR and Western blot studies further confirmed that the expression levels of XRCC5 and PDK1 genes and proteins were significantly upregulated after overexpression of CALCRL. In contrast, the phosphorylation levels of AKT/PKCε protein, a downstream pathway of XRCC5/PDK1, were significantly upregulated. In the response study, transfection of overexpressed CALCRL cells with XRCC5 siRNA significantly upregulated the drug sensitivity of AML to DNR. The expression levels of PDK1 protein and AKT/PKCε phosphorylated protein in the downstream pathway were inhibited considerably, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were upregulated. Animal experiments showed that the inhibitory effect of DNR on the growth of HL-60 cells and the number of bone marrow invasions were significantly reversed after overexpression of CALCRL in nude mice. However, infection of XCRR5 shRNA lentivirus in HL-60 cells with CALCRL overexpression attenuated the effect of CALCRL overexpression and upregulated the expression of apoptosis-related proteins induced by DNR. This study provides a preliminary explanation for the relationship between high CALCRL expression and poor prognosis of chemotherapy in AML patients. It offers a more experimental basis for DNR combined with molecular targets for precise treatment in subsequent studies.

NOXA-mediated degradation of MCL1 and BCL2L1 causes apoptosis of daunorubicin-treated human acute myeloid leukemia cells.

Daunorubicin (DNR) is used clinically to treat acute myeloid leukemia (AML), while the signaling pathways associated with its cytotoxicity are not fully elucidated. Thus, we investigated the DNR-induced death pathway in the human AML cell lines U937 and HL-60. DNR-induced apoptosis in U937 cells accompanied by downregulation of MCL1 and BCL2L1, upregulation of Phorbol-12-myristate-13-acetate-induced protein 1 (NOXA), and mitochondrial depolarization. DNR induced NOX4-mediated reactive reactive oxygen species (ROS) production, which in turn inactivated Akt and simultaneously activated p38 mitogen-activated protein kinase (MAPK). Activated p38 MAPK and inactivated Akt coordinately increased GSK3β-mediated cAMP response element-binding protein (CREB) phosphorylation, which promoted NOXA transcription. NOXA upregulation critically increased the proteasomal degradation of MCL1 and BCL2L1. The same pathway was also responsible for the DNR-induced death of HL-60 cells. Restoration of MCL1 or BCL2L1 expression alleviated DNR-induced mitochondrial depolarization and cell death. Furthermore, ABT-199 (a BCL2 inhibitor) synergistically enhanced the cytotoxicity of DNR in AML cell lines. Notably, DNR-induced DNA damage was not related to NOXA-mediated degradation of MCL1 and BCL2L1. Collectively, these results indicate that the upregulation of NOXA expression through the NOX4-ROS-p38 MAPK-GSK3β-CREB axis results in the degradation of MCL1 and BCL2L1 in DNR-treated U937 and HL-60 cells. This signaling pathway may provide insights into the mechanism underlying DNR-triggered apoptosis in AML cells.

Puerarin attenuates the daunorubicin-induced apoptosis of H9c2 cells by activating the PI3K/Akt signaling pathway via the inhibition of Ca2+ influx.

Puerarin extracted from RadixPuerariae is well known for its pharmacological effects, including antioxidant, anti‑inflammatory, neuroprotective and cardioprotective properties. In this study, we aimed to investigate the effects of puerarin on the daunorubicin(DNR)-induced apoptosis of H9c2cells and to elucidate the potential mechanisms involved. MTT assay and flow cytometry were performed to evaluate cell cytotoxicity and apoptosis, respectively. Western blot analysis was used to assess changes in the expression levels of proteins, including caspase-3, Akt and phosphorylated Akt(p-Akt). Ratiometric imaging of intracellular calcium(Ca2+) using cells loaded with Fura-2 was also carried out. Our results revealed that puerarin pre-treatment protected the H9c2cells against DNR-induced cytotoxicity by inhibiting cell apoptosis, which was also confirmed by the decrease in the expression of cleaved caspase-3. Additionally, p-Akt activation was associated with the suppressive effects of puerarin. Following pre-treatment with puerarin, the extracellular Ca2+influx was restrained and this resulted in a reduction in the intracellular Ca2+levels; these effects were abrogated by LY294002[an inhibitor of phosphatidylinositol3-kinase (PI3K)]. The inhibition of Ca2+influx suggested that the PI3K/Akt signaling pathway participated in the suppressive effects of puerarin against H9c2cell apoptosis. Taken togher, our findings demonstrate that puerarin attenuates the DNR-induced apoptosis of H9c2cells by activating the PI3K/Akt signaling pathway via the inhibition of Ca2+influx, suggesting that puerarin may be a potential cardioprotective agent for use in the clinical treatment of cardiomyopathy triggered by DNR.

Raman spectrum reveals the cell cycle arrest of Triptolide-induced leukemic T-lymphocytes apoptosis

Triptolide (TPL), a traditional Chinese medicine extract, possesses anti-inflammatory and anti-tumor properties. Though some research results have implicated that Triptolide (TPL) can be utilized in the treatment of leukemia, it remains controversial about the mechanism of TPL-induced leukemic T-lymphocytes apoptosis. In this study, combining Raman spectroscopic data, principal component analysis (PCA) and atomic force microscopy (AFM) imaging, both the biochemical changes and morphological changes during TPL-induced cell apoptosis were presented. In contrast, the corresponding data during Daunorubicin (DNR)-induced cell apoptosis was also exhibited. The obtained results showed that Raman spectral changes during TPL-induced cell apoptosis were greatly different from DNR-induced cell apoptosis in the early stage of apoptosis but revealed the high similarity in the late stage of apoptosis. Moreover, above Raman spectral changes were respectively consistent with the morphological changes of different stages during TPL-induced apoptosis or DNR-induced apoptosis, including membrane shrinkage and blebbing, chromatin condensation and the formation of apoptotic bodies. Importantly, it was found that Raman spectral changes with TPL-induced apoptosis or DNR-induced apoptosis were respectively related with the cell cycle G1 phase arrest or G1 and S phase arrest.

Sodium Ferulate Prevents Daunorubicin - Induced Apoptosis in H9c2 Cells via Inhibition of the ERKs Pathway

Background: Daunorubicin (DNR)-induced cardiotoxicity, which is closely associated with cardiomyocyte apoptosis, limits the drug's clinical application. The activation of the extracellular regulated protein kinases (ERKs) pathway is responsible for the pro-apoptosis effect of DNR Sodium ferulate (SF) has recently been found to attenuate both DNR-induced cardiotoxicity and mitochondrial apoptosis in juvenile rats. Nonetheless, the precise mechanism underlying SF-induced cardio-protection remains unclear. Methods: The DNR-injured H9c2 cell model was prepared by incubating the cells in 1 µM DNR for 24 h. Amounts of 15.6, 31.3 or 62.5 µM SF were simultaneously added to the cells. The effect of SF on the cytotoxic and apoptotic parameters of the cells was studied by monitoring apoptosis regulation via the ERKs pathway. Results: SF attenuated DNR-induced cell death (particularly apoptotic death), cTnI and β-tubulin degradation, and cellular morphological changes. SF reduced mitochondrial membrane potential depolarization, cytochrome c leakage, and caspase-9 and caspase-3 activation. SF also decreased ERK1/2, phospho-ERK1/2, p53 and Bax expression and increased Bcl-2 expression. These effects were similar to the results observed when using the pharmacological ERKs phosphorylation inhibitor, AZD6244. Conclusion: We determined that SF protects H9c2 cells from DNR-induced apoptosis through a mechanism that involves the interruption of the ERKs signaling pathway.

Sodium Ferulate Protects Against Daunorubicin-induced Cardiotoxicity by Inhibition of Mitochondrial Apoptosis in Juvenile Rats

Daunorubicin (DNR) is a widely used chemotherapeutic agent; however, its clinical use is limited because of its cardiotoxicity. This study was aimed to investigate the protective effect of sodium ferulate (SF), an effective component from traditional Chinese herbs, against DNR-induced cardiotoxicity in juvenile rats. DNR was administered intraperitoneally to rats at the dosage of 2.5 mg·kg(-1)·wk(-1) for 5 consecutive weeks (cumulative dose of 12.5 mg/kg) or in combination with intraperitoneal injection of SF at 50 mg·kg(-1)·d(-1) over a period of 30 days. The animals were killed 6 days after the last injection of DNR. SF significantly ameliorated the DNR-induced cardiac dysfunction, structural damage of the myocardium, and release of lactate dehydrogenase and creatine kinase. Treatment with SF also reversed DNR-induced oxidative stress as evidenced by a decrease in malondialdehyde levels with a concomitant increase in myocardical superoxide dismutase activities. Furthermore, SF afforded significant cardioprotection against DNR-induced apoptosis in vivo and effectively suppressed the complex mitochondrion-dependent apoptotic signaling triggered by DNR. This study indicates that SF may improve cardiac function by inhibition of oxidative stress and apoptosis, thus providing a beneficial effect on the prevention of DNR-induced cardiotoxicity.

Functional p53 is required for rapid restoration of daunorubicin-induced lesions of the spleen

BackgroundThe tumour suppressor and transcription factor p53 is a major determinant of the therapeutic response to anthracyclines. In healthy tissue, p53 is also considered pivotal for side effects of anthracycline treatment such as lesions in haematopoietic tissues like the spleen. We used a Trp53null mouse to explore the significance of p53 in anthracycline (daunorubicin) induced lesions in the spleen.MethodsMice with wild type or deleted Trp53 were treated with the daunorubicin (DNR) for three consecutive days. Spleens were collected at various time points after treatment, and examined for signs of chemotherapy-related lesions by microscopic analysis of haematoxylin-eosin or tunel-stained paraffin sections. Expression of death-inducing proteins was analysed by immunoblotting. Changes between Trp53 wild type and null mice were compared by t-tests.ResultsSigns of cell death (pyknotic nuclei and tunel-positive cells) in the white pulp of the spleen occurred earlier following DNR exposure in wt-mice compared to Trp53-null mice. While the spleen of wt-mice recovered to normal morphology, the spleen of the Trp53-null animals still had lesions with large necrotic areas and disorganised histologic appearance eight days after treatment. Immunoblotting showed that only Trp53-wt mice had significant increase in p21 after DNR treatment. However, both wt and null mice had elevated p63 levels following DNR exposure.Conclusionsp53 protects against severe and enduring cellular damage of the spleen parenchyma after DNR treatment, and initial DNR-induced apoptosis is not predictive of tissue lesions in the spleen. Our data indicate that p53 induction following DNR treatment serves to protect rather than to destroy normal tissue.

Abstract 2739: Panobinostat potentiates the cytotoxic effects of ara-C and daunorubicin on acute myeloid leukemia cells through suppressing the DNA damage response

Abstract Acute myeloid leukemia (AML) remains a challenging disease to treat. Resistance to anthracycline [e.g., daunorubicin (DNR)] and ara-C-based chemotherapy is a major cause of treatment failure in this disease. Therefore, new therapies are urgently needed to improve treatment outcome of AML. Histone deacetylase (HDAC) inhibitors (HDACIs) are a promising new class of anti-cancer drugs, which induce differentiation, cell cycle arrest, and apoptosis in human leukemic cells, but not in normal cells. We previously demonstrated strikingly synergistic antileukemic interactions between valproic acid and ara-C in pediatric AML, accompanied by cooperative induction of DNA double-strand breaks (DSBs) and apoptosis. However, the precise molecular mechanisms remain to be identified. Recent studies with solid tumor cell lines established that HDACIs down-regulate expression of genes encoding critical DNA DSB repair proteins, which play critical roles in the DNA damage response (DDR), an important mechanism limiting chemotherapeutic efficacy. These results led to our hypothesis that HDACIs suppress the DDR in AML cells to enhance the cytotoxic effects of the DNA damaging chemotherapeutic drugs, DNR and ara-C. In four AML cell lines and ten diagnostic AML blast samples, treatment with the novel pan-HDACI, panobinostat, resulted in suppressed expression of BRCA1, CHK1, and RAD51 in a dose-dependent fashion, accompanied by dose-dependent induction of apoptosis or proliferation arrest. Treatments of the AML cell lines with ara-C or DNR resulted in induction of RAD51, BRCA1, and CHK1, which was abolished by co-administration of panobinostat. Importantly, panobinostat substantially enhanced ara-C- or DNR-induced DNA DSBs, as reflected in the induction of γH2AX, an established biomarker for DNA DSBs. This was accompanied by a dramatic synergistic induction of apoptosis. Whereas panobinostat alone resulted in G1 arrest, ara-C and DNR alone caused S-G2/M and G2/M arrest, respectively. The latter effects were abrogated when these agents were simultaneously administered with panobinostat. Interestingly, shRNA knockdown of BRCA1, CHK1, or RAD51 caused delayed repair of DNA DSBs induced by ara-C or DNR. Further, shRNA knockdown of BRCA1 or CHK1 resulted in significantly increased basal and ara-C- or DNR-induced apoptosis, along with substantially increased DNA DSBs and abrogated S and G2 checkpoints. In contrast, shRNA knockdown of RAD51 significantly enhanced DNR-induced DSBs and apoptosis, but had no impact on S and G2 checkpoints. These results support our hypothesis that suppression of the DDR by HDACIs represents a novel mechanism underlying the antileukemic activities of this class of drugs, and the synergy between HDACIs and DNA damaging agents in AML. Studies are underway evaluating in vivo efficacies of these drug combinations using xenograft NSG Mice. 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 2739. doi:1538-7445.AM2012-2739

MiR-181a sensitizes a multidrug-resistant leukemia cell line K562/A02 to daunorubicin by targeting BCL-2

The aim of this study was to investigate whether miR-181a could modulate the sensitivity of the leukemia drug-resistant cell line K562/A02 to the chemotherapeutic agent daunorubicin (DNR), and explore the mechanism of miR-181a on the DNR sensitivity of K562/A02 cells. MicroRNA microarray and stem-loop reverse transcription-polymerase chain reaction were used to detect the expression of miR-181a. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay was performed to quantify the effect of miR-181a on K562 cells growth and viability. Apoptotic cells were quantitatively detected using Annexin V/FITC and PI apoptosis detection kit. BCL-2 protein expression was measured by western blot. Luciferase reporter vector with the putative BCL-2 3' untranslated region was constructed to explore whether BCL-2 was a direct target gene of miR-181a. BCL-2 siRNA was transfected into the cell to explore the relationship between BCL-2 and DNR resistance. The miR-181a expression level was lower in the K562/A02 cells than in the K562 cells (P< 0.05). K562 cells that were transfected with miR-181a inhibitor had a significantly higher survival than K562 cells, and K562/A02 cells that were transfected with the miR-181a mimic had a significantly lower survival than K562/A02 cells (P< 0.05). miR-181a could enhance DNR-induced apoptosis in K562/A02 cells. BCL-2 siRNA transfected K562/A02 cells had decreased survival compared with the K562/A02 control group. In conclusion, miR-181a could play a role in the development of DNR resistance in K562/A02 cells and the over-expression of miR-181a could sensitize K562/A02 cells to DNR by targeting BCL-2.

Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR) than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1), DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells.MethodsPrimary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP) was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA) against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR.ResultsCurcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to DNR-induced apoptosis. More importantly, curcumin suppressed Bcl-2 expression, selectively inhibited proliferation and synergistically enhanced the cytotoxicity of DNR in primary CD34+ AML cells, while showing limited lethality in normal CD34+ hematopoietic progenitors.ConclusionCurcumin down-regulates Bcl-2 and induces apoptosis in DNR-insensitive CD34+ AML cell lines and primary CD34+ AML cells.

Magnetic nanoparticle of Fe3O4 and 5-bromotetrandrin interact synergistically to induce apoptosis by daunorubicin in leukemia cells

Apoptosis is a common pathway that finally mediated the killing functions of anticancer drugs, which is an important cause of multidrug resistance (MDR). The aim of this study was to investigate the potential benefit of combination therapy with magnetic nanoparticle of Fe(3)O(4) (MNP(Fe(3)O(4))) and 5-bromotetrandrin (BrTet). Analysis of the apoptosis percentage showed that combination of daunorubicin (DNR) with either MNP(Fe(3)O(4)) or BrTet exerted a potent cytotoxic effect on K562/A02 cells, while MNP(Fe(3)O(4)) and BrTet cotreatment can synergistically enhance DNR-induced apoptosis. Importantly, we confirmed that the distinct synergism effect of that composite on reverse multidrug resistance may owe to the regulation of various proliferative and antiapoptotic gene products, including P53 and caspase-3. Thus our in vitro data strongly suggests a potential clinical application of MNP(Fe(3)O(4)) and BrTet combination on CML.

Effect of Flavonoids on Daunorubicin‐induced Toxicity in H9c2 Cardiomyoblasts

Daunorubicin (DNR) is one of the most important antitumor agents belonging to the anthracycline group. However, its use is seriously limited by the development of cardiac toxicity. The present study was designed to investigate the effects of quercetin, pycnogenol and naringenin on daunorubicin-induced cytoxicity in H9c2 cells. Protection of H9c2 cardiomyocyte cells was concentration/dose dependent for quercetin > naringenin > pycnogenol = trolox. Quercetin (10(-4)-10(-5) mol/L) after 24 h of co-incubation with DNR significantly increased the cardiomyocyte survival (p < 0.001 and p < 0.05, respectively). A protective effect of other compounds was observed only in the highest concentration/dose used (p < 0.01). After 48 h of incubation quercetin and naringenin significantly decreased daunorubicin-induced cell death at concentrations of 10(-4)-10(-5) mol/L (p < 0.001 and p < 0.01, respectively). The protective effect of pycnogenol and trolox was weaker but significant in the two highest concentrations/doses (p < 0.001 and p < 0.05, respectively). This study also investigated DNR-induced apoptosis and it was shown that both quercetin and naringenin inhibit apoptosis of H9c2 cardiomyocytes cells in vitro. The findings provide evidence that quercetin and naringenin may act as survival factors. The protective effect of flavonoids was compared with that of trolox, a known cardioprotective antioxidant. These results are consistent with the notion that the use of flavonoids may be beneficial in modulating or preventing the cardiotoxicity associated with DNR therapy.

C-Jun N-Terminal Kinase (JNK) Activation Failure Confers a New Mechanism of Anthracycline Resistance in Acute Myeloid Leukemia (AML).

Chemotherapy resistance remains a major challenge in the treatment of acute myeloid leukemia (AML). Besides the P-glycoprotein efflux of chemotherapeutics, additional cellular factors may contribute to drug resistance in AML. c- Jun N-terminal Kinase (JNK) is a protein kinase activated after exposure of cells to chemotherapeutic agents. Recently, studies in solid tumours have associated chemoresistance with failure of cancer cells to activate JNK. We asked whether drug resistance in AML is also attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative U937R cells showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. Inhibition of JNK in drug-sensitive U937 cells made them resistant to anthracyclines. First, JNK1-siRNA transfected U937 cells exhibited a 50.4% and 61.3% reduced daunorubicin- (DNR, 1μM 24hr) and doxorubicin- (DOX, 1.5 μM 24hr) induced apoptosis respectively; as compared to empty vector or untransfected U937 cells (P< 0.001). Second, pretreatment of U937 cells with the 420116 cell-permeable JNK inhibitor (1 μM) reduced to a less but yet significant extent DNR-induced apoptosis as compared to cells treated with a negative control peptide (P = 0.013<0.05). On the other hand, selective restoration of the inactive JNK pathway in resistant U937R cells by transfection with a mutant form of the SEK1/MKK4 upstream activator of JNK sensitized U937R cells to anthracyclines, compared to empty vector transfected cells (3.3-fold increase in DNR-induced apoptosis, 1μM DNR, 24hr and 3.1-fold increase in DOX-induced apoptosis, 1.5μM DOX, 24hr). Furthermore, we assessed the activation of JNK pathway in 29 primary AML bone marrow samples after short term (30-60 min) in vitro exposure to DNR (1 μM) and correlated it with clinical data. We found a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels in AML primary blasts and the response of the AML patients to standard induction chemotherapy (P = 0.012). In addition, the drug-induced JNK activation pattern correlated with AML evolving from antecedent MDS (P = 0.017) and patient age (P = 0.046). In summary, our in vitro and in vivo results suggest that JNK activation failure is another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.

Cytoprotective Effect of Glucosylceramide Synthase Inhibition against Daunorubicin-induced Apoptosis in Human Leukemic Cell Lines

Several studies have shown that ceramide (CER) glucosylation contributes to drug resistance in multidrug-resistant cells and that inhibition of glucosylceramide synthase sensitizes cells to various drug treatments. However, the role of glucosylceramide synthase has not been studied in drug-sensitive cancer cells. We have demonstrated previously that the anthracycline daunorubicin (DNR) rapidly induces interphasic apoptosis through neutral sphingomyelinase-mediated CER generation in human leukemic cell lines. We now report that inhibition of glucosylceramide synthase using d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) or 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) protected U937 and HL-60 cells from DNR-induced apoptosis. Moreover, blocking CER glucosylation did not lead to increased CER levels but to increased CER galactosylation. We also observed that pretreating cells with galactosylceramide (GalCER) significantly inhibited DNR-induced apoptosis. Finally, we show that GalCER-enriched lymphoblast cells (Krabbe's disease) were significantly more resistant to DNR- and cytosine arabinoside-induced apoptosis as compared with normal lymphoblasts, whereas glucosylceramide-enriched cells (Gaucher's disease) were more sensitive. In conclusion, this study suggests that sphingomyelin-derived CER in itself is not a second messenger but rather a precursor of both an apoptosis second messenger (GD3) and an apoptosis "protector" (GalCER).

Bcl-2 overexpression prevents daunorubicin-induced apoptosis through inhibition of XIAP and Akt degradation

Daunorubicin (DNR) induces apoptosis in the human myeloid leukemia cells by activation of neutral sphingomyelinease and ceramide production. In the present study, we determined the effect of the antiapoptosis protein Bcl-2 on caspase-3 activation, phospholipase C-γ1 (PLC-γ1) degradation and cytochrome c release during the DNR-induced apoptosis. Treatment with 3 μM DNR for 12 hr produced morphological features of apoptosis and DNA fragmentation in U937 cells, which was associated with caspase-3 activation and PLC-γ1 degradation. Induction of apoptosis was also accompanied by release of cytochrome c, down-regulation of X-linked inhibitor of apoptosis protein (XIAP), and inactivation of Akt, which was blocked by the pan-caspase inhibitor z-VAD-fmk. DNR-induced caspase-3 activation, PLC-γ1 degradation and apoptosis were significantly attenuated in Bcl-2 overexpressing U937/Bcl-2 cells. Ectopic expression of Bcl-2 appeared to inhibit DNR-induced apoptosis by interfering with inhibition of XIAP and Akt degradation.

Ligation of the CD44 adhesion molecule inhibits drug-induced apoptosis in human myeloid leukemia cells

AbstractAdhesion molecules can improve hematopoietic cell survival; however, their role in leukemic cell resistance to drug-induced apoptosis is poorly documented. The CD44 adhesion molecule is strongly expressed on acute myeloid leukemia (AML) blasts. Using 2 myeloid cell lines, HL60 and NB4, evidence is presented that prior incubation with the CD44-specific monoclonal antibody (mAb) A3D8, reported to induce differentiation of AML blasts, significantly decreases apoptosis induced by 3 drugs used in AML chemotherapy: daunorubicin (DNR), mitoxantrone, and etoposide. In addition, in HL60 cells, CD44 ligation with A3D8 mAb fully abrogates the DNR-triggered generation of ceramide, a lipid second messenger involved in the DNR apoptotic signaling pathway. Moreover, results show that the A3D8 mAb and Bcl-2 additively inhibit DNR-induced apoptosis in HL60 cells overexpressing Bcl-2. These results suggest that, to eradicate AML blasts, the differentiation-inducing anti-CD44 mAb A3D8 should not be administered prior to apoptosis-inducing drugs.

Ligation of the CD44 adhesion molecule inhibits drug-induced apoptosis in human myeloid leukemia cells

Adhesion molecules can improve hematopoietic cell survival; however, their role in leukemic cell resistance to drug-induced apoptosis is poorly documented. The CD44 adhesion molecule is strongly expressed on acute myeloid leukemia (AML) blasts. Using 2 myeloid cell lines, HL60 and NB4, evidence is presented that prior incubation with the CD44-specific monoclonal antibody (mAb) A3D8, reported to induce differentiation of AML blasts, significantly decreases apoptosis induced by 3 drugs used in AML chemotherapy: daunorubicin (DNR), mitoxantrone, and etoposide. In addition, in HL60 cells, CD44 ligation with A3D8 mAb fully abrogates the DNR-triggered generation of ceramide, a lipid second messenger involved in the DNR apoptotic signaling pathway. Moreover, results show that the A3D8 mAb and Bcl-2 additively inhibit DNR-induced apoptosis in HL60 cells overexpressing Bcl-2. These results suggest that, to eradicate AML blasts, the differentiation-inducing anti-CD44 mAb A3D8 should not be administered prior to apoptosis-inducing drugs.

Fas receptor-Fas ligand system is independent of both CD34 status and chemosensitivity in acute myeloid leukemia

Objective To determine whether the Fas receptor-Fas ligand (FasR-FasL) system, which triggers apoptosis in sensitive cells, is an important mechanism of cytotoxicity in acute myeloblastic leukemia (AML). Materials and Methods We investigated FasR expression in primary AML cells and its upregulation by tumor necrosis factor (TNF), as well as the apoptosis induced by anti-Fas antibody and the potential interaction between the FasR-FasL system and the cytotoxic drug daunorubicin (DNR). Results FasR was expressed on all 25 AML samples and three normal bone marrow harvests. The intensity of expression was variable (range 1.6–2.1 in normal bone marrow CD34 + cells and 1.5–5.1 in AML cells, median 2.4) and was related to the morphologic FAB classification, with the highest expression in FAB types M4 and M5 (range 1.6–5.1, median 3.2). No relationship was found between FasR expression and expression of the CD34 antigen. FasR was heterogeneously upregulated in all AML cells on treatment with TNF-α. The degree of FasR upregulation induced was found to be related to the FAB subtype, with the greatest response observed in immature FAB types M1, M2, and M6 (range 11.0–207.1%, median 48.7%). Apoptosis could be induced in all AML samples, but not in normal bone marrow CD34 + ve cells, by the CH11 anti-FasR antibody, although the response was variable (range 4.1–37.6%, median 16.5%). The monocytic differentiated M4 and M5 AML cells exhibited the greatest sensitivity to Fas-mediated apoptosis (range 4.4–37.6, median 20.65%); however, no relationship was found between sensitivity to Fas-mediated apoptosis and FasR expression or CD34 positivity. Apoptosis in response to DNR was observed in all AML cases; however, sensitivity was heterogeneous and found to be unrelated to FasR expression or sensitivity to Fas-mediated apoptosis. The blocking anti-FasR antibody ZB4 blocked anti-FasR-mediated apoptosis but had no inhibitory effect on DNR-induced apoptosis in AML blasts. No cytotoxic synergistic effect was demonstrated when anti-FasR antibody was used in combination with DNR. Conclusion In AML, DNR induces apoptosis through an Fas-independent pathway. However, the induction of apoptosis through the Fas pathway might be a novel and effective approach for leukemia immunotherapy, particularly because Fas-mediated apoptosis was noted in CD34 + and CD34 − cases.

Intraclonal Heterogeneity in the In Vitro Daunorubicin-Induced Apoptosis in Acute Myeloid Leukemia

Leukemic cells from ten patients with acute myeloid leukemia (AML) were sorted on the basis of in vitro daunorubicin (DNR) uptake. The obtained subpopulations with high and low DNR accumulation were compared with regard to induction of apoptosis, expression of bcl-2 and p53. Heterogeneous induction of apoptosis, confined to subpopulations with high DNR uptake, was observed. The size of the DNR-induced apoptotic fraction (4% to 16%) within a given AML blast population was determined by intracellular drug accumulation and was not related to the level of bcl-2 expression. All tested leukemic samples displayed expression of p53 in a growth promoter orientation, i.e. PAb1620-/PAb240+. In two samples, however, sub-populations expressing a growth suppressor orientation of p53, i.e. PAb1620+/PAb240-, were also present. These subpopulations were confined to high-DNR-uptake fractions and associated with the induction of apoptosis. We conclude that intraclonal heterogeneity in the intracellular drug accumulation and subsequently in DNR-induced apoptosis might allow the selection of inherently drug-resistant AML clones thus contributing to relapse of leukemia.

ダウノルビシンのヒト白血病細胞アポトーシス誘導に対するＰＧＥ１の抑制効果

Daunorubicin (DNR), one of the chemotherapeutic agents for the treatment of leukemia, is known to induce apoptotic cell death of leukemia cells. Apoptosis of leukemia cells in vivo accompanies phagocytic response by macrophages, which is now known to produce various mediators including PGE2. In the present study, we studied the effect of PGE1, a PGE2 analog, on the DNR-induced apoptosis of human leukemia cell line, U937.PGE1 itself had no effect on the cell cycle of U937, but it inhibited DNR-induced apoptosis in a dose dependent manner. This inhibitory effect was mimicked by a membrane-permeable cAMP analog, db-cAMP, and forskolin. Furthermore, pretreatment of U937 cells with H-89, a protein kinase A inhibitor, reversed the inhibiting effect of PGE1. When co-cultured with LPS-activated peripheral blood monocytes, DNR-treated U937 cells showed less apoptosis, which was augmented by inhibiting endogenous production of PGE2 from LPS-activated monocytes when they are cultured with indomethacin. Therefore, it suggested that PGE1 inhibited DNR-induced apoptosis through activated cAMP, PKA signaling pathway. These results clearly show that PGE1 inhibition of DNR-induced apoptosis of U937 is mediated by cAMP/PKA signaling pathway and that endogenous production of PGE2 by macrophages, which eliminate apoptotic cells by phagocytosis in vivo, attenuates DNR-induced apoptosis of U937 cells. The present study suggests that administration of cyclooxygenase inhibitor, such as indomethacin, enhances chemotherapeutic effect of DNR-induced apoptosis of leukemia cells.