phenanthrenes and Leukemia--Promyelocytic--Acute

Acute promyelocytic leukemia (APL) is a distinctive subtype of acute myeloid leukemia (AML) in which the hybrid protein promyelocytic leukemia protein/retinoic acid receptor α (PML/RARα) acts as a transcriptional repressor impairing the expression of genes that are critical to myeloid cell mutation. We aimed at explaining the molecular mechanism of green tea polyphenol epigallocatechin-3-gallate (EGCG) enhancement of ATRA-induced APL cell line differentiation. Tumor suppressor phosphatase and tensin homolog (PTEN) was found downregulated in NB4 cells and rescued by proteases inhibitor MG132. A significant increase of PTEN levels was found in NB4, HL-60 and THP-1 cells upon ATRA combined with EGCG treatment, paralleled by increased myeloid differentiation marker CD11b. EGCG in synergy with ATRA promote degradation of PML/RARα and restores PML expression, and increase the level of nuclear PTEN. Pretreatment of PTEN inhibitor SF1670 enhances the PI3K signaling pathway and represses NB4 cell differentiation. Moreover, the induction of PTEN attenuated the Akt phosphorylation levels, pretreatment of PI3K inhibitor LY294002 in NB4 cells, significantly augmented the cell differentiation and increased the expression of PTEN. These results therefore indicate that EGCG targets PML/RARα oncoprotein for degradation and potentiates differentiation of promyelocytic leukemia cells in combination with ATRA via PTEN.

Topics: Catechin; Cell Differentiation; Chromones; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Leupeptins; Morpholines; Phenanthrenes; Promyelocytic Leukemia Protein; Proteolysis; PTEN Phosphohydrolase; Retinoic Acid Receptor alpha; Tretinoin

Our studies indicated that Tanshinone IIA (TanIIA), which is widely applied in the treatment of cardiovascular diseases with a rare occurrence of side effects, could promote APL cell differentiation and apoptosis. We found TanIIA induced the differentiation of NB4 and MR2 cells with elevated C/EBPβ and CHOP. When C/EBPβ was overexpressed in NB4 cells, the level of CD11b in the transfected cells was significantly elevated. When we used CHOP siRNA to suppress CHOP expression in NB4 cells and then treated these cells with a high concentration of TanIIA, the differentiation and apoptosis of these cells were both significantly increased. These data demonstrate that C/EBPβ is critical for APL cell differentiation and apoptosis induced by TanIIA, and that CHOP acts as a negative regulator of C/EBPβ activity. Our study suggested that TanIIA is a promising drug for treating newly diagnosed and ATRA-resistant APL, and a high concentration of TanIIA associated with inhibition of CHOP, maybe a potentially promising therapy strategy.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; CCAAT-Enhancer-Binding Protein-beta; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Transcription Factor CHOP

To investigate the effect of tanshinone II A on the procoagulant activity (PCA) of human umbilical vein endothelial cells (HUVEC) induced by acute promyelocytic leukemia (APL) cell line NB4 cells.. The HUVEC were incubated for 6, 12, and 24 hours in different tanshinone II A conditioned medias (Tan II A-NB4-24h-CM, Tan II A-NB4-72h-CM, Tan II A-NB4-120h-CM). Then the HUVEC were incubated for 6, 12, 24, and 72 hours with Tan II A-NB4-120h-CM and different concentrations of Tan II A (0, 0.25, 0.5, 1.0 microg/mL). The HUVEC lysates were obtained by three repeated freezing and thrawing. Their PCA were tested using the one stage clotting assay. The activity of tissue factor (TF : act) was tested using the chromogenic substrate assay. The control groups included 0.3 microg/mL ATRA, 0.01% DMSO and RPMI 1640.. Tan II A-(72 h,120 h)-NB4-CM elevated PCA of HUVEC and six hours of incubation in the 120 h-NB4-CM had the greatest PCA. The PCA of HUVEC in the 1.0 microg/mL Tan II A-NB4-CM was the same as in the 0.3 microg/mL ATRA-NB4-CM. (2) The NB4-CM induced PCA of HUVEC decreased with 5.0 microg/mL of Tan II A, at a level similar to the decrease with 0.3 microg/mL of ATRA. Less than 5.0 microg/mL of Tan II did not reduce the NB4-CM induced PCA of HUVEC. (3) Both Tan II A 120 h-NB4-CM and ATRA 120 h-NB4-CM elevated the TF : act of HUVEC. The TF : act reached the peak after 6 hours of incubation. The Tan II A 120 h-NB4-CM maintained the peak level of TF : act at the 12th hour and fell to the base line at the 24th hour. The ATRA 120 h-NB4-CM induced TF:act dropped down with time after reaching its peak at the 6th hour. (4) The 1.0 microg/mL of Tan II A did not reduce the TF : act of HUVEC induced by the Tan II A 120 h-NB4-CM. But the 0.3 microg/mL of ATRA reduced the TF : act of HUVEC at the 6th hour.. TanIIA-NB4-CM increases PCA and TF : Act of HUVEC. TanIIA decreases PCA of HUVECs induces by TanIIA-NB4-CM.

Topics: Abietanes; Blood Coagulation Factors; Cell Line, Tumor; Endothelial Cells; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Thromboplastin; Tretinoin; Umbilical Veins

To evaluate the synergism of Tanshinone II A (Tan II A) and arsenic trioxide (As2O3) on the apoptosis of retinoic acid resistant acute promyelocytic leukemia (APL) cell line (MR2), and to investigate its effect on the expression of P-glycoprotein (Pgp) of MR2 cells.. As2O3 was added in the media of MR2 cells in a dose of 0.5 micromol/L, 2.0 micromol/L and 5.0 micromol/L, respectively, or combined with Tan II A in a dose of 1.0 microg/ml. The cell proliferation activity was assessed with MTT assay. The cell apoptosis was demonstrated by labeled Annexin V/PI method. The expression of Pgp was evaluated by immunocytochemical assay.. The MR2 cell proliferation activity was obviously inhibited in the two groups of As2O3 0.5 micromol/L, 2.0 micromol/L combined with Tan II A. The inhibitory effect gradually increased with the time extension. In 168 hours, the inhibitory rate of the two combination groups was (90.67+/-5.52)% and (86.70+/- 3.04)%, respectively, significantly stronger than that of corresponding dose of As2O3 alone group (P<0.01). The apoptosis effect of MR2 cell also gradually increased. In 168 hours, the apoptosis rate of the two combination groups was (81.52+/-7.23)% and (90.75+/-6.44)%, respectively, significantly stronger than that of corresponding dose of As2O3 alone group (P<0.01). At the same time, As2O3 alone and combination with Tan II A therapy can significantly reduce the MR2 cell Pgp expression (P<0.01).. There were apoptosis synergism on MR2 cell induced by Tan II A combined with As2O3, at the same time reduced the expression of Pgp in the cells.

Topics: Abietanes; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Arsenic Trioxide; Arsenicals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Humans; Leukemia, Promyelocytic, Acute; Oxides; Phenanthrenes; Tretinoin

To investigate the growth inhibiting and apoptosis inducing mechanisms of different matches of Tanshinone IIA and Salvianolic Acid B on Acute Promyelocytic Leukemia Cells (HL-60).. The HL-60s' growth inhibition and apoptosis-induced rates were detected by MTT reduction assay and flow cytometry with various matches of TanIIA and SalB.. The HL-60s' growth inhibition and apoptosis-induced rates were found higher in the group of TanIIA plus SalB than other single groups, and in the group TanIIA-SalB (10-5 microg/ml) they were the highest (P<0.05).. TanIIA and SalB both have obviously growth inhibiting and apoptosis inducing effect, and union groups show stronger effect than any single group, while different matching proportion results in different growth inhibiting and apoptosis inducing action.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Benzofurans; Cell Proliferation; Drugs, Chinese Herbal; Flow Cytometry; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Plants, Medicinal; Salvia miltiorrhiza

To investigate molecular mechanism of tanshinone II A inducing differentiation and apoptosis in acute promyelocytic leukemia NB4 cells.. NB4 cells were cultured in vitro and treated with tanshinone II A and observed cellular morphology, cell category and the cellular proliferation. DNA microarray technique was used to analyze the gene expression profiles of NB4 cells induced by tanshinone II A.. 92.8% of NB4 cells treated with 0.5 mg x L(-1) tanshinone II A were induced into mature neutrophils, in which myetocytes and melamyetocytes were 27.0%, banded and segmented neutrophits 68.2%. Cell growth were inhibited. cDNA microarray showed the enormously expressed 183 genes including 23 differentiation associated genes, and other interrelated genes.. Tanshinone II A inducing differentiation in NB4 cells may be via regulation of many kinds of genes, especially differentiation associated genes expression. This partially explained the molecular mechanism of tanshinone II A inducing differentiation.

Topics: Abietanes; Cell Differentiation; Cell Line, Tumor; Drugs, Chinese Herbal; Gene Expression Regulation, Leukemic; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes

To investigate the effects of Tanshinone IIA (Tan IIA) on procoagulant activity (PCA) of human ECV304 cells induced by acute promyelocytic leukemia cell line NB4 cells.. ECV304 monolayers were respectively incubated for different hours at 37 degrees C in the conditioned media (CM) of NB4 cells treated with 0.5 microg/mL Tan IIA(Tan IIA-NB4-CM), 0.3 microg/mL all-trans retinoidic acid (ATRA)(ATRA-NB4-CM), DMSO(DMSO-NB4-CM) or the RPMI1640 medium. ECV304 lysates were tested for PCA using the one-stage clotting assay as well as for tissue factor activity (TF: Act) using the chromogenic substrate assay; ECV304 cell monolayers were incubated for different hours at 37 degrees C in a medium system including 0.5 microg/mL Tan IIA and Tan IIA-NB4-CM, and the ECV304 cell lysates were tested for PCA in the same way as above. Also they were controlled by 0.3 microg/mL ATRA, DMSO or RPMI1640 medium.. (1) The conditioned mediums from 0. 5 microg/mL Tan IIA that treated NB4 cells for 24, 72 and 120 hours respectively could elevate PCA of ECV cells, and this capability developed with the time of reaction. ATRA did the same as Tan IIA (P > 0.05). (2) 0.5 microg/mL Tan IIA down-regulated the PCA of ECV304 cells induced by Tan IIA-NB4-CM, and the inhibitory effects increased with time, reaching the highest at 120 hours. (3) Tan IIA120 h-NB4-CM up-regulated TF:Act of ECV304 cells, and the effect increased with time. (4) 0. 5 microg/mL Tan IIA down-regulated PCA and TF: Act of ECV304 cells induced by Tan IIA-NB4-CM, and the inhibitory effect increased with time; simultaneously, the test was controlled with 0.3 microg/mL ATRA, the effects on PCA and TF: Act were not significantly different (P > 0.05).. Tan IIA-NB4-CM can increase the levels of PCA and TF: Act of ECV304 cells through some unidentified factor; however, Tan IIA can obviously decrease the PCA and TF: Act levels of ECV304 cells induced by Tan IIA-NB4-CM.

Topics: Abietanes; Anticoagulants; Cell Differentiation; Cell Line; Cell Line, Tumor; Culture Media, Conditioned; Drugs, Chinese Herbal; Endothelial Cells; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Thromboplastin; Tretinoin; Umbilical Veins

Tanshinone IIA, a diterpene quinone extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, is used widely and successfully in clinics in China for treating inflammatory diseases. Recently tanshinone IIA has been reported to have apoptosis inducing effects on a large variety of cancer cells. In this study, the anti-proliferation and apoptosis inducing effects of tanshinone IIA as well as its influence on cell adhesion to and invasion through the extracellular matrix (ECM) on acute promyelocytic leukemia (APL) NB4 cells in vitro were studied. Cell proliferation was assessed by MTT assay, cell apoptosis was observed by Hoechst 33258 staining and flow cytometry (FCM); The variation of caspase-3 and apoptotic related genes were assayed by Western blotting, cell mitochondrial membrane potential as well as cell adhesive and invasive effects were also investigated by using standard methods. The results showed that tanshinone IIA exhibited induction of apoptosis by activation of caspase-3, downregulation of anti-apoptotic protein bcl-2 and bcl-xl and upregulation of pro-apoptotic protein bax, as well as disruption of the mitochondrial membrane potential. Furthermore, treatment by tanshinone IIA could reduce cell adhesion to and invasion through ECM in leukemia NB4 cells. These data provide a potential mechanism for tanshinone IIA-induced apoptosis and cell growth inhibition in leukemia NB4 cells, suggesting that tanshinone IIA may serve as an effective adjunctive reagent for the treatment of APL.

Topics: Abietanes; Apoptosis; Caspase 3; Cell Adhesion; Cell Line, Tumor; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Invasiveness; Phenanthrenes

A 30 years-old man was administrated with dizziness and fatigue for half month, and the big toe on his left foot got the prolonged bleeding of wound complicated with fever 7 days before the admission. The physical examination (PE) discovered that the case suffered from the anemic appearance, lower part tenderness of sternum, petechiae and purpura on skin of lower extremities, and with remaining not to be remarkable. The examination of blood routine showed WBC 2.3 x 10(9)/L, Hb 60/L, BPC 34 x 10(9)/L and blasts 0. 85. The bone marrow smear indicated markedly the hypercellularity, promyelocytes 89% and strongly positive myeloperoxidase (MPO). The PT and APTT were prolonged, and the FDP and D-dimer were positive. The acute promyelocytic leukemia (APL) with DIC was diagnosed. The patient was administered with all-trans retinoic acid (ATRA) with dosage of 20 mg three times per day. After 14 week treatment, the patient did not get complete remission. Then the tanshinone II A was taken orally with 30mg twice each day. After 8 week treatment of tanshinone II A, the blood routine was restored to normal. Four weeks later, the bone marrow also became normally, and the patient got a complete remission (CR). After more than 3 months of consolidation therapy with tanshinone II A, the patient was relapsed. When the homoharringtonine and cytarabine (HA) were given, the patient was got CR again. Three years later, he was relapsed secondarily, and then died of intracranial hemorrhage. The tanshinone II A could induce CR of APL with ATRA resistance, no side effect was observed; there is a reoccurring possibility from consolidation therapy with tanshinone II A.

Topics: Abietanes; Adult; Drug Resistance, Neoplasm; Humans; Leukemia, Promyelocytic, Acute; Male; Phenanthrenes; Remission Induction; Tretinoin

To investigate retinoic acid-resistant acute promyelocytic leukemia (APL) cell differentiation induced by tanshinone IIA (Tan IIA) and its molecular mechanism.. NB4 cells treated with 0.5 mg/L Tan IIA was regarded as positive control. After in vitro incubation of MR-2 cells with Tan IIA at the concentration of 1.0 mg/L for 4 days, the cell differentiation was observed by growth status, cytomorphology, and nitroblue tetrazolium test. Cell cycle, membrane cluster differentiation (CD) antigens (CD(33), CD(11b)) and expression of some oncogene (c-myc, c-fos, p53 and bcl-2) were analysed by flow cytometry.. The growth of MR-2 and NB4 cells was inhibited after Tan IIA treatment, the inhibition rate were 73.5% and 67.7% respectively (P < 0.01, P < 0.01) without significant difference. After Tan IIA treatment, MR-2 and NB4 cells were induced to undergo morphological differentiation, which exhibited small cell bulk decreased nucleus/cytoplasm proportion, rough chromatin, disappearance of nucleolus and formation of azurophil granules and anomalous nucleus. MR-2 cells could be induced to metamyelocyte while NB4 could be induced to band form. NBT reduction of MR-2 and NB4 cells treated with Tan IIA showed that positive cells accounted for (95.30 +/- 0.76)% and (93.20 +/- 1.04)% respectively; but the positive rate of either group of the treated positive cells was significantly higher than that of untreated, being (3.50 +/- 1.32)% and (2.80 +/- 0.29)% respectively (P < 0.01). Flow cytometry showed that the expression of CD(33) was reduced, while that of CD(11b) was increased. The quantity of treated cells in G(0)/G(1) phase increased but that in S phase decreased. The proliferous index was also decreased. After treated with Tan IIA, the expressions of anti-oncogene p53 and c-fos were up-regulated while those of oncogene bcl-2 and c-myc were down-regulated (P < 0.01).. 1.0 mg/L Tan IIA could inhibit proliferation of MR-2 cells and induce differentiation of MR-2 cells into mature granulocyte, the effectivity was the same as 0.5 mg/L Tan IIA treated NB4 cells. Its possible molecular mechanism might be related to modulation of oncogene expressions associated proliferation and differentiation as well as inhibition of DNA synthesis. Tan IIA probably can be applied to treat the patients with APL, particularly to the relapsed and drug resistant patients with broad prospect.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Tretinoin

To evalutate the synergistic effects of Tanshinone II A combined with all-trans retinoic acid (ATRA) on the differentiation and apoptosis of human acute promyelocytic leukemia (APL) cell line (NB4).. The NB4 cells were treated with 0.5 microg/ml Tanshinone II A combined with 0.5 microg/ml, 0.25 microg/ml and 0.125 microg/ml ATRA respectively in culture. Cells differentiation was demonstrated by morphology and NBT reduction assay. The expression of CD11b and CD33, cell cycle and apoptosis induced by these drugs were measured by flow cytometry (FCM).. The proliferative inhibition rate of the combination of Tan II A with ATRA was much higher. The differentiated cells accounted for over 90 percent, among them the band forms and neutrophils constituted more than 65 percent. NBT reduction and CD11b expression were much higher, and expression of CD33 was lower than that of Tan II A or ATRA alone (P<0.01). FCM analysis also showed that combination of Tan II A with ATRA arrested NB4 cells in G0/G1 phase and induced significantly apoptosis of NB4 cells (P<0.01). There were no significant dose dependent effects induced by ATRA in combination with Tan II A at 0.125 microg/ml to 0.5 microg/ml on differentiation and apoptosis of NB4 cells.. The combination of Tan II A with ATRA has synergistic effects on differentiation and apoptosis of NB4 cells. The effects do not increase with the dosage escalation of ATRA.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Differentiation; Cell Line, Tumor; Drug Synergism; Drugs, Chinese Herbal; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Tretinoin

To study the variation and role of Caspase3 activity in the process of Tanshinone (Tan II A) induced NB4 cells apoptosis.. NB4 cell apoptosis induced by Tan II A was demonstrated by cell morphology, DNA content analysis and DNA fragmentation assay. Caspase3 activity was determined by spectrofluorometry, and its inhibitory assay was performed using N-acetyl-Asp-Glu-Val-Asp-aldehyde(AC-DEVD-CHO).. Tan II A could induce NB4 cell apoptosis accompanied with increase of caspase3 activity. The induction of NB4 cell apoptosis by use of Tan II A could be partially inhibited by AC-DEVD-CHO.. The induction of NB4 cell apoptosis by Tan II A could be fulfilled by activating Caspase3.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspases; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Tumor Cells, Cultured

The aim of this study was to investigate whether Tanshinone II A (Tan II A) can induce human acute promyelocytic leukemia (APL) cells to differentiate or not in primary culture. The APL cells from 5 cases were cultured respectively with Tan II A at the concentration of 0.5 microgram/ml for 7 days in vitro. The differentiations of these leukemia cells were observed cytomorphologically and examined by nitroblue tetrazolium (NBT) test. The cell DNA cycle and membrane cluster differentiation (CD) antigens (CD33, CD11b) were analyzed by flow cytometry. The results showed that 82.5% +/- 4.8% of APL cells were induced into morphologically and functionally differentiated cells. The cell growth curve showed that the growth of APL cells was inhibited. The degree of differentiation and growth inhibition induced by Tan II A was not different from that by ATRA (P > 0.05). Flow cytometry analysis showed that Tan II A arrested APL cells in G0/G1 phase and inhibited cellular DNA synthesis. This study demonstrates that Tan II A can induce differentiation of APL cells in vitro, and hence it is worthy of further studies for clinical use.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Cell Differentiation; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Tumor Cells, Cultured

To investigate APL cell differentiation induced by tanshinone II (Tan II A) and its molecular mechanism.. In vitro incubation of NB4 cells with Tan II A at the concentration of 0.5 microg/ml for 5 days, the cell differentiation was observed by cytomorphology, and nitroblue tetrazolium (NBT) test. Cell cycle, membrane CD(33), CD(11b) antigens and gene expressions (c-myc, c-fos, p53 and bcl-2) were analysed by flow cytometry.. (91.3 +/- 2.1)% of NB4 cells were induced into morphologically and functionally more differentiated cells including 0.26 of myelocytes and metamyelocytes, and 0.68 of band form and neutrophils. Cell growth curve showed that growth of NB4 cells were inhibited. NBT reduction was significantly increased. Expression of CD(33) decreased and CD(11b) increased. The degrees of cell differentiation and growth inhibition induced by Tan II A or ATRA were no difference. Flow cytometry analysis showed that Tan II A arrested NB4 cell in G(0)/G(1) phase, inhibited cellular DNA synthesis, down-regulated c-myc and bcl-2 genes expression, and up-regulated c-fos and p53 genes expression.. Tan II A can induce differentiation and growth inhibition of NB4 cells. Its possible molecular mechanism might relate to modulation of gene expressions associated proliferation and differentiation, and to inhibition of DNA synthesis.

Topics: Abietanes; Cell Differentiation; Cell Division; Genes, p53; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes

Triptolide (Tri) is a diterpenoid triepoxide isolated from Tripterygium wilfordii Hook F. The effects of Tri on the colony formation of breast cancer cell lines MCF-7 and BT-20, stomach cancer cell lines MKN-45, MKN-7, and KATO-III, and promyelocytic leukemia cell line HL-60 were reported. Using Hamburger-Salmon's double layer agar technique with certain modifications, cancer cells were cultured in 0.3% agar in a highly humidified atmosphere of 5% CO2 at 37 degrees C for 14-21 d. Colonies were counted on d 14 (occasionally d 21) with the colony analyzer system CA-7A. Of the 5 solid tumor cell lines tested, 4 showed diminished colony formation in soft agar by greater than 70% of control value in Tri 10(-8) mol.L-1 (continuous exposure). The magnitudes of the inhibitory effect of Tri on most breast and stomach cancer cell lines were similar to that on the leukemia cell line HL-60. IC50 were 0.504-1.22 micrograms.L-1. The clinically achievable peak plasma concentration (PPC) of Tri was estimated as 0.15 mg.L-1, being 72-126 times higher than the IC70 of the cancer cell lines except KATO-III. The results suggest that Tri might have a potential therapeutic effect on some types of solid tumors, e.g., breast and stomach cancers.

Topics: Antineoplastic Agents; Breast Neoplasms; Diterpenes; Epoxy Compounds; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Stomach Neoplasms; Tumor Cells, Cultured; Tumor Stem Cell Assay