homoharringtonine and Leukemia

Cephalotoxine esters, including homoharringtonine (HHT), have shown encouraging activity in leukemia in initial studies in China and in later studies in the U.S.. The authors conducted a review of the literature to examine the studies pertinent to HHT in relation to preclinical studies and Phase I-II trials in patients with hematologic malignancies and solid tumors.. HHT and analogues appear to induce differentiation and apoptosis. Studies from China reported high response rates in patients with leukemia. Trials in the U.S. using short HHT infusions (3-4 mg/m(2) daily for 5 days) resulted in a high incidence of cardiovascular complications that were reduced using continuous infusion schedules of 3-7 mg/m(2) daily for 5-7 days initially, and later lower dose schedules of 2.5 mg/m(2) daily for 7-14 days. Results in solid tumors were negative. However encouraging results were reported in patients with acute myeloid leukemia, myelodysplastic syndrome, acute promyelocytic leukemia, and, most important, chronic myeloid leukemia (CML). In CML patients, HHT has been investigated alone and in combination with interferon-alpha and low-dose cytarabine in late and early chronic phases, with positive results. Additional areas of interest include the potential use of HHT for the treatment of central nervous system leukemia, polycythemia vera, and other nonmalignant conditions such as malaria. New semisynthetic preparations and HHT derivatives that bypass multidrug resistance may improve the efficacy and toxicity profiles, and broaden the range of antitumor efficacy.. HHT and its derivatives appear to have promising activity in hematologic malignancies, a finding that needs to be pursued.

Topics: Antineoplastic Agents, Phytogenic; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Forecasting; Harringtonines; Homoharringtonine; Humans; Leukemia; Neoplasms; Research

Topics: Alkaloids; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Drug Evaluation; Drug Evaluation, Preclinical; Harringtonines; Homoharringtonine; Humans; Kinetics; Leukemia; Leukemia L1210; Neoplasms, Experimental; Protein Biosynthesis

Topics: Alkaloids; Clinical Trials as Topic; Harringtonines; Homoharringtonine; Humans; Leukemia; Plants, Medicinal; Taiwan; United States

Topics: Alkaloids; Arrhythmias, Cardiac; China; Clinical Trials as Topic; Drug Evaluation; Harringtonines; Homoharringtonine; Humans; Leukemia; United States

Cyclin-dependent kinase 2 (CDK2) complex is significantly over-activated in many cancers. While it makes CDK2 an attractive target for cancer therapy, most inhibitors against CDK2 are ATP competitors that are either nonspecific or highly toxic, and typically fail clinical trials. One alternative approach is to develop non-ATP competitive inhibitors; they disrupt interactions between CDK2 and either its partners or substrates, resulting in specific inhibition of CDK2 activities. In this report, we identify two potential druggable pockets located in the protein-protein interaction interface (PPI) between CDK2 and Cyclin A. To target the potential druggable pockets, we perform a LIVS in silico screening of a library containing 1925 FDA approved drugs. Using this approach, homoharringtonine (HHT) shows high affinity to the PPI and strongly disrupts the interaction between CDK2 and cyclins. Further, we demonstrate that HHT induces autophagic degradation of the CDK2 protein via tripartite motif 21 (Trim21) in cancer cells, which is confirmed in a leukemia mouse model and in human primary leukemia cells. These results thus identify an autophagic degradation mechanism of CDK2 protein and provide a potential avenue towards treating CDK2-dependent cancers.

Topics: Animals; Autophagy; CDC2-CDC28 Kinases; Cell Line, Tumor; Cyclin A; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; Homoharringtonine; Humans; Leukemia; Mice; Ribonucleoproteins

Cephalotaxine-type alkaloids (CTAs), represented by homoharringtonine (HHT, 1), display potent efficacy against different types of leukemia cells. In this study, a method for hydrogenation of β-substituted itaconic acid monoesters with chiral Ru[DTBM-SegPhos](OAc)

Topics: Antineoplastic Agents; Esters; Harringtonines; Homoharringtonine; Humans; Leukemia; Stereoisomerism

Cephalotaxine (CET) is a natural alkaloid with potent antileukemia effects. However, its underlying molecular mechanism has not been well understood. In this study, we verified that CET significantly inhibited the viability of various leukemia cells, including HL-60, NB4, Jurkat, K562, Raji and MOLT-4. RNA-sequencing and bioinformatics analysis revealed that CET causes mitochondrial function change. Mechanism research indicated that CET activated the mitochondrial apoptosis pathway by reducing the mitochondrial membrane potential, downregulating anti-apoptotic Bcl-2 protein and upregulating pro-apoptotic Bak protein. In addition, the autophagy signaling pathway was highly enriched by RNA-seq analysis. Then, we found that CET blocked the fluorescence colocation of MitoTracker Green and LysoTracker Red and upregulated the level of LC3-II and p62, which indicated that autophagy flow was impaired. Further results demonstrated that CET could impair lysosomal acidification and block autophagy flow. Finally, inhibiting autophagy flow could aggravate apoptosis of HL-60 cells induced by CET. In summary, this study demonstrated that CET exerted antileukemia effects through activation of the mitochondria-dependent pathway and by impairing autophagy flow. Our research provides new insights into the molecular mechanisms of CET in the treatment of leukemia.

Topics: Apoptosis; Autophagy; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Leukemic; Homeostasis; Homoharringtonine; Humans; Leukemia; Mitochondria; Signal Transduction; Transcriptome

In order to improve the in vivo safety and specific delivery efficiency of the antileukemic homoharringtonine (HHT) at the targets, the long-circulating PEGylated liposomes loaded with HHT (LCLipo-HHT) were prepared. Their physical characteristics, in vitro drug release, in vivo pharmacokinetic properties and elementary toxicity were evaluated. The mean diameter of the prepared LCLipo-HHT is 75.6 ± 3.2 nm and the zeta potential is -16.9 ± 2.5 mV. The entrapment efficiency of HHT in the liposomes is 69.5 ± 1.7%. In pharmacokinetic experiments, an increased plasma concentration as well as blood circulation time was obtained when distearoyl phosphoethanolamine-PEG 2000 lipid was added in the formulation, which results in enhancing drug delivery efficiency. Hemolysis test, vascular irritation test and acute toxicity test were used to demonstrate toxicity of LCLipo-HHT. Compared with clinical HHT injection dosage, LCLipo-HHT indicated no vascular irritation, good hemocompatibility, as well as much better safety. Therefore, the prepared LCLipo-HHT can be used as a promising anticancer formulation for antileukemic therapy in the future.

Topics: Animals; Antineoplastic Agents; Chemistry, Pharmaceutical; Drug Delivery Systems; Harringtonines; Homoharringtonine; Leukemia; Liposomes; Mice; Particle Size; Phosphatidylethanolamines; Polyethylene Glycols; Rabbits

Homoharringtonine combined with aclarubicin and cytarabine (HAA) is a highly effective treatment for acute myeloid leukemia (AML), especially for t(8;21) AML. However, the underlying mechanisms by which HAA kills t(8;21) AML cells remain unclear. In this study, SKNO-1 and Kasumi-1 cells with t(8;21) were used. Compared with individual or pairwise administration of homoharringtonine, aclarubicin, or cytarabine, HAA showed the strongest inhibition of growth and induction of apoptosis in SKNO-1 and Kasumi-1 cells. HAA caused cleavage of the AML1-ETO (AE) oncoprotein to form truncated AE (ΔAE). Pretreatment with the caspase-3 inhibitor caspase-3 inhibitor Q-DEVD-OPh (QDO) not only suppressed HAA-induced apoptosis but also abrogated the cleavage of AE and generation of ΔAE. These results suggest that HAA synergistically induces apoptosis in t(8;21) leukemia cells and triggers caspase-3-mediated cleavage of the AML1-ETO oncoprotein, thus providing direct evidence for the strong activity of HAA toward t(8;21) AML.

Topics: Aclarubicin; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 8; Core Binding Factor Alpha 2 Subunit; Cytarabine; Drug Synergism; Harringtonines; Homoharringtonine; Humans; Leukemia; Oncogene Proteins, Fusion; Proteolysis; RUNX1 Translocation Partner 1 Protein; Translocation, Genetic

Homoharringtonine (HHT) has been reported to be effective in a portion of patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). To investigate its mechanism of action, cell growth inhibition and cytotoxicity of HHT were investigated in three AML cell lines, HL-60, NB4, and U937, and in three CML cell lines, K562, KU812, and KCL22. AML cells were more sensitive than CML cells to HHT-induced cytotoxicity. Using HL-60 cells, it was revealed that HHT decreased the levels of myeloid cell leukemia 1 (Mcl-1), X-linked inhibitor of apoptosis protein (XIAP), survivin, and B-cell lymphoma 2 (Bcl-2)-homology domain 3 (BH3)-only proteins as well as the mitochondrial membrane potential. The levels of Bcl-2, Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist/killer (Bak) proteins in HL-60 cells were not changed after HHT treatment. U937, K562, KU812, and KCL22 cells expressed B-cell lymphoma-extra large (Bcl-xL) and were less responsive to HHT-induced apoptosis than HL-60 cells. Silencing Mcl-1 or Bcl-xL, but not XIAP or survivin, enhanced HHT-induced apoptosis in U937 cells. The levels of HHT-induced apoptosis in K562, KCL22, and KU812 cells were inversely correlated with the levels of Bcl-xL but not those of Bcl-2 or Mcl-1. K562 cells expressing high levels of Bcl-xL but no Bcl-2 were less responsive to HHT-induced apoptosis than KCL22 cells that expressed lower levels of Bcl-xL and higher levels of Bcl-2 protein. In K562 cells, knockdown of Bcl-xL, but not of Mcl-1, enhanced HHT-induced apoptosis. Transfection of Bcl-xL into KCL22 cells attenuated HHT-induced apoptosis. These data suggest that Bcl-xL plays a more important role than Bcl-2 and Mcl-1 in protecting against HHT-induced apoptosis.

Topics: Apoptosis; bcl-X Protein; Cell Line, Tumor; Harringtonines; Homoharringtonine; Humans; Leukemia; RNA Interference

We study model sensitivity of the continual reassessment method (CRM). The context is that of dose-finding designs where certain design parameters are fixed by the investigator. Although our focus is on the CRM (O'Quigley et al., 1990), the essential ideas can be applied to any sequential dose-finding method. It is expected that different choices of a model family and particular parameterizations will have an impact on performance. Assuming that the constraints outlined in Shen and O'Quigley (1996) are respected, large sample performance is unaffected. However small sample performance will be affected by these choices, which are to some degree arbitrary. This work focuses on the retrospective robustness of the CRM in practice. The question is not of a general theoretical nature where, in the background, we would want to consider large numbers of true potential situations. Instead, the question is raised in the specific context of any actual completed study and is the following: Would we have come to the same conclusion concerning the MTD had we worked with a design specified differently? The sequential nature of the CRM means that this question cannot be answered in any definitive way. We can, though, by appealing to the retrospective CRM (O'Quigley, 2005), provide consistent estimates of the relationships between the MTD and the chosen model. If these estimates suggest that changes in different family model parameters will be accompanied by changes in final recommendation, then we would not be confident in the reliability of the estimated MTD and more work would be needed. Also, of course, at the planning stage, prospective robustness could be studied by simulating trials using particular models and parameterizations.

Topics: Algorithms; Antineoplastic Agents, Phytogenic; Biostatistics; Clinical Trials, Phase I as Topic; Computer Simulation; Dose-Response Relationship, Drug; Epidemiologic Research Design; Harringtonines; Homoharringtonine; Humans; Leukemia; Likelihood Functions; Maximum Tolerated Dose; Models, Statistical; Retrospective Studies

To investigate the synergistic effects of SG235-TRAIL, a novel oncolytic adenovirus expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and homoharringtonine (HHT) in human leukemia cell lines.. The combined effect of SG235-TRAIL and HHT was assessed using a crystal violet assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, followed by combination index analysis. Cell apoptosis was measured using flow cytometry combined with fluorescein-isothiocyanate-Annexin V staining. The activation of caspase pathway and the expression of Bcl-2 family proteins, TRAIL, and E1A were examined using Western blotting.. HHT synergized the cytotoxicity of SG235-TRAIL against leukemia cell lines Kasumi-1, KG-1, HL-60, and U937, concomitantly with increased apoptosis and enhanced activity of caspase-3 and -9. The combination therapy resulted in significantly lower levels of Bcl-2, Mcl-1, and Bid compared to treatment of cells with either HHT or SG235-TRAIL alone, suggesting that HHT sensitizes leukemia cells to SG235-TRAIL virus through alteration of anti-apoptotic signaling elements. Importantly, HHT combined with SG235-TRAIL did not show significant cytotoxicity to normal human mononuclear cells and mesenchymal stem cells.. Combining oncolytic adenovirus SG235-TRAIL and HHT synergistically enhances cytotoxicity in leukemia cells in vitro, suggesting that the combination therapy could represent a rational approach for the treatment of leukemia.

Topics: Adenoviridae; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Cell Line, Tumor; Combined Modality Therapy; Flow Cytometry; Harringtonines; Homoharringtonine; Humans; Leukemia; Leukocytes, Mononuclear; Mesenchymal Stem Cells; Oncolytic Virotherapy; Oncolytic Viruses; TNF-Related Apoptosis-Inducing Ligand

To study the resistant related molecules of human leukemia drug resistant K562 cells (K562/HHT) induced by homoharringtonine (HHT).. Gene expression profiles on K562/HHT, K562 and K562/HHT/RU486 (K562/HHT reversed by RU486) cells were detected by DNA microarray. The bone marrow tyrosine kinase gene in chromosome X (BMX) which changed dynamically among the three cells was confirmed by RT-PCR and Western blot. Then, BMX was transfected into K562 and K562/HHT cells, and the changes of daunorubicin (DNR) concentrations in these two cells were observed for BMX overexpression.. As compared with K562, there were changes in 117 gene expressions in K562/HHT, 57 of which were up-regulated and 60 down-regulated. The mdrl gene was significantly up-regulated. When compared with K562/HHT, 50 significantly differently expressed genes were screened out in the K562/HHT/RU486 cells, of which up- and down-regulated genes were 13 and 37 respectively. These genes involved in drug resistance, cell signaling, cell differentiation, cell proliferation, transcription regulator, ion transport and so on. Four genes [NM-001721 (BMX), NM-031459 (SESN2), NM-033642 (FGF13) and AL-049309 (SFRS12)] expressed significantly differently in the two group cells, BMX gene expression was higher in K562/HHT, than in K562, but lower than in K562/HHT/RU486 as confirmed by RT-PCR and Western blot. After the plasmid pCI-neo-BMX was transfected into K562 and K562/HHT cells, DNR concentration was significantly lower (79.28 +/- 4.04, 29.84 +/- 2.67) than those before transfection (158.52 +/- 8.08, 58.58 +/- 6.53).. BMX is associated with multi-drug resistance of K562/HHT cell line.

Topics: Drug Resistance, Multiple; Drug Resistance, Neoplasm; Gene Expression Profiling; Harringtonines; Homoharringtonine; Humans; K562 Cells; Leukemia; Protein-Tyrosine Kinases

Homoharringtonine has been shown to lead to apoptosis of leukemic cells in several studies. Here we showed that the endoplasmic reticulum (ER) may be the initial site of apoptotic signal induced by homoharringtonine in MUTZ-1 cells. After incubation with homoharringtonine, the percentage of apoptotic MUTZ-1 cells increased in a time-dependent manner, Ca(2+) translocated from ER pool to cytosol, the mitochondrial membrane potential decreased, and Bid protein translocated from ER to mitochondria. The activation of ER stress-associated proapoptotic factor CHOP and ER chaperones BiP and XBP1 genes followed by cleavage of caspase-3 but not caspase-4 protein were also observed.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Calcium; Cell Line, Tumor; Cytosol; Endoplasmic Reticulum; Harringtonines; Homoharringtonine; Humans; K562 Cells; Leukemia; Membrane Potentials; Mitochondria; Myelodysplastic Syndromes; Time Factors

The inhibitor of apoptosis (IAP) gene family is involved in the suppression of apoptotic cell death as well as an increasing number of seemingly unrelated cellular functions. It is not known, however, whether IAP expression in malignant hematopoietic cells is affected by chemotherapeutic agents such as homoharringtonine (HHT). In this study, we investigated mRNA expression levels of IAPs, especially survivin, in various hematopoietic cell lines in relation with apoptosis induced by HHT.. Semiquantitative reverse transcriptase polymerase chain reaction was used to determine survivin mRNA levels. Cell apoptosis was examined by flow cytometry. Cell viability and proliferation assay was evaluated by MTT. The experiments were performed on the malignant hematopoietic cell lines MUTZ-1, K562, Jurkat, RMPI and HL60, with or without survivin antisense-oligodeoxynucleotides (AS-ODN) and HHT.. The expression levels of survivin mRNA were variable in the cell lines and negatively correlated to HHT induced cell apoptosis. Survivin AS-ODN significantly decreased mRNA level of survivin, but not those of bax and bcl-2. Survivin also inhibited MUTZ-1 cell growth and induced apoptosis in a dose dependent manner. AS-ODN and HHT showed synergistic effect on MUTZ-1 cell growth.. The apoptotic effect of HHT on the hematopoietic cell lines is associated with decreased level of survivin expression. Survivin could be a new marker for drug sensitivity and a new target for cancer treatment.

Topics: Anemia, Refractory, with Excess of Blasts; Apoptosis; bcl-2-Associated X Protein; Cell Cycle; Cell Line; Harringtonines; Homoharringtonine; Humans; Inhibitor of Apoptosis Proteins; Leukemia; Microtubule-Associated Proteins; Neoplasm Proteins; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Survivin

Glivec was approved by Food & Drug Administration (FDA) in May 2001 as a gene target drug for treatment of chronic myeloid leukemia (CML) and showed a good curative effect for patients with chronic myeloid leukemia in chronic phase. But its effectiveness was poor in patients with CML blast phase treated with Glivec alone. Glivec was reported having synergetic effect with other chemical agents in vitro, but there is few report in clinical combined application. In this paper, we analyzed effectiveness of Glivec in combination with homoharringtonine (HHT) and cytarabine (Ara-C) for patients with Ph chromosome positive acute leukemia (Ph(+)-AL), and investigated patients' tolerance to side effects of this trial.. A total of 20 patients (16 males and 4 females, median age was 43 years) were eligible. Blasts in peripheral blood (PB) or bone marrow (BM) were both more than 30%, bcr/abl fusion genes were detected positive in 90% cells by analysis of karyotype or fluorescence in situ hybridization (FISH). Five patients showed t(9;22) and other 15 patients showed more complicated chromosome abnormality. Of these 20 patients, 17 patients developed Ph(+)-ANLL from CML, 1 case developed Ph(+)-ALL, and other 2 cases were primary Ph(+)-ALL. The median interval from diagnosis to Glivec treatment was 4 months. Eighteen of 20 patients received different chemotherapy regimens for 2-4 treatment cycles, but no one reached hematological complete remission (HCR). All patients were given oral Glivec daily at the doses of 0.3-0.6 g in a median treatment time of 2.5 months (range, 1-6.5 months). Ph(+)-ANLL patients were infused with HHT over 6-24 hours daily at the doses of 1-2 mg intravenously and Ara-C 30-50 mg daily subcutaneously for 10-14 days; 3 patients with Ph(+)-ALL received HOAP or DOP combination treatment regimens (One cycle consists of HA with the same dosage described above for Ph(+)-ANLL patients for 7 days, daunorubicin at the dose of 40 mg/d intravenously for 3 days, vincristine at the dose of 2 mg/wk for two weeks, and prednisone at the doses of 60-80 mg/d for 14 days). Median treatment cycle was 2 (range, 1-3). The dosage of Glivec could be reduced or treatment was suspended when bone marrow inhibition happened. G-CSF was used when necessary. The curative effect was evaluated by international hematology and cytogenetics standards, in which bone marrow was examined every chemotherapy cycle and chromosome was analyzed 3 months later.. Among the 20 patients receiving Glivec, 40% achieved HCR, and 25% achieved hematological partial remission (HPR), but only 15% patients approached a partial cytogenetic remission and no cytogenetic responses were found in other 85% patients. White blood cells (WBC) in peripheral blood reduced from 41+/-31 x 10(9)/L to normal level within 1 week. The blasts decreased from (50+/-30)% to(1.9+/-2.9)% (P< 0.001) in median time of 21.0+/-16.8 days. Three patients with high fever recovered normal temperature after 3 days treatment. When follow-up median time at 8 months, the total survival rate reduced to 40%, the rate of death and lost follow-up number of patients added to 60%.. Regimen of Glivec in combination with HA could increase chemotherapy effect for the patients with Ph(+)-AL, prolong their life time and the side-effects were tolerable.

Topics: Acute Disease; Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Female; Follow-Up Studies; Harringtonines; Homoharringtonine; Humans; Imatinib Mesylate; Leukemia; Male; Middle Aged; Philadelphia Chromosome; Piperazines; Pyrimidines

The BCR/ABL tyrosine kinase has been implicated in the pathogenesis of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL). STI571 is a novel anticancer agent that selectively inhibits the BCR/ABL tyrosine kinase. The cytotoxic effects of STI571 were studied in combination with antileukemic agents against Ph(+) leukemia cell lines, KU812, K-562, TCC-S, and TCC-Y. The cells were exposed to STI571 and to other agents simultaneously for 5 or 7 days. Cell growth inhibition was determined by MTT assay. The cytotoxic effects in combinations at the inhibitory concentration of 80% level were evaluated by the isobologram. STI571 produced synergistic effects with recombinant and natural alpha-interferons in 2 of 3 and 3 of 3 cell lines, respectively. STI571 produced additive effects with hydroxyurea, cytarabine, homoharringtonine, doxorubicin, and etoposide in all 4 cell lines. STI571 with 4-hydroperoxy-cyclophosphamide, methotrexate, or vincristine produced additive, antagonistic, and synergistic effects in 3 of 4 cell lines, respectively. These findings suggest that the simultaneous administration of STI571 with other agents except methotrexate would be advantageous for cytotoxic effects against Ph(+) leukemias. Among them, the simultaneous administration of STI571 and alpha-interferons or vincristine would be highly effective against Ph(+) leukemias and these combinations would be worthy of clinical trials. In contrast, the simultaneous administration of STI571 with methotrexate would have little therapeutic efficacy. Although there are gaps between in vitro studies and clinical trials, the present findings provide useful information for the establishment of clinical protocols involving STI571. (Blood. 2001;97:1999-2007)

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Cytarabine; Dose-Response Relationship, Drug; Drug Interactions; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Inhibitors; Fusion Proteins, bcr-abl; Harringtonines; Homoharringtonine; Humans; Hydroxyurea; Imatinib Mesylate; Interferon-alpha; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Methotrexate; Microbial Sensitivity Tests; Neoplasm Proteins; Neoplasms; Piperazines; Pyrimidines; Tumor Cells, Cultured; Vincristine

In the present study, we investigated the in vitro apoptotic response of leukemic cells to the cellular stress induced by homoharringtonine (HHT), a plant alkaloid with antileukemic activity which is currently being tested for treatment of acute and chronic leukemias. A comparison of leukemic cell lines with different p53 gene status revealed a considerably higher sensitivity to HHT-induced apoptosis in the cells with a wt p53, and apoptotic events in wt p53 leukemia cells (MOLT-3 cell line) were studied in more detail. To this end, we examined components of apoptotic cascades including Bax expression and its intracellular localization, changes of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) levels, cytochrome c release from mitochondria and activation of caspases. Bax protein levels did not increase despite an up-regulation of bax at mRNA level. However, Bax translocation from cytosol towards mitochondria was observed. In addition, we observed a release of cytochrome c from the mitochondria, and the localization changes of both Bax and cytochrome c were found already at the early, annexin V-negative stage of HHT-induced apoptosis. HHT-treated MOLT-3 cells revealed loss of MMP as well as activation of caspases demonstrated by DEVD-, IETD- and LEHD-tetrapeptide cleavage activity in the cell lysates. ROS levels only slightly increased in HHT-treated cells and antioxidants did not prevent apoptosis and MMP changes. Therefore, wt p53 leukemic cells respond to HHT-specific cellular stress by induction of ROS-independent apoptotic pathway characterized by translocation of Bax, mitochondrial cytochrome c release and activation of caspases.

Topics: Antioxidants; Apoptosis; bcl-2-Associated X Protein; Biological Transport; Caspases; Cytochrome c Group; Dose-Response Relationship, Drug; Enzyme Activation; Harringtonines; Homoharringtonine; Humans; Leukemia; Membrane Potentials; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Investigation of the role of mitochondrial membrane potential (MMP) in the homoharringtonine (HHT)-induced apoptosis.. Annexin V staining, flow cytometry and confocal laser scan microscopy were used to observe the relationship between Bax, cytochrome C and MMP in the HHT-induced apoptosis of leukemic T lymphocytic line Molt-3.. The induction of apoptosis by HHT resulted in the translocation of Bax from cytosol to mitochondrial membrane and the decrease of cellular MMP, followed by the release of cytochrome C from mitochondria to cytosol.. Changes of mitochondrial membrane potential might play a critical role in the HHT-induced apoptosis of leukemic T-cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cytochromes c; Cytosol; Harringtonines; Homoharringtonine; Humans; Leukemia; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Protein Transport; T-Lymphocytes

To investigate the mechanism of resistance to an antineoplastic natural product homoharringtonine (HHT) in leukemic cells, we have established 5 sub-lines of human myeloid leukemia K562 cells, designated as K-H30, K-H100, K-H200, K-H300 and K-H400, which showed progressive resistance to different concentrations of HHT. These sub-lines were cross-resistant to daunorubicin, vincristine, etoposide and mitoxantrone, but not to melphalan. Immunofluorescence with monoclonal anti-Pgp antibody MRK16 and Northern-blot analysis demonstrated that resistance to HHT is related to the sequential emergence of MRP- and MDR1-gene over-expression. In the low-level-resistant K-H30 sub-line, the MDR1 gene was not over-expressed, but the MRP gene was over-expressed 2.1-fold. In the intermediate-level-resistant K-H100 and K-H200 sublines, both the MRP and the MDR1 genes were over-expressed. However, in the high-level-resistant K-H300 and K-H400 sublines, MDR1-gene over-expression predominated (20- and 21-fold respectively). On the other hand, GST pi-gene expression was decreased in all 5 sub-lines. Southern-blot analysis revealed no MRP-gene amplification in any of the 5 sub-lines, whereas the MDR1 gene was amplified in the high-level-resistant K-H300 and K-H400 sub-lines. The most interesting observation is a homogeneously staining region (HSR) found in chromosome 2 of the K-H300 and K-H400 sub-lines. Chromosome painting and in situ hybridization demonstrated that this HSR was translocated from chromosome 7 and consisted of the amplified MDR1 gene, suggesting that there is a relationship between MDR1-gene, translocation and MDR1-gene amplification.

Topics: Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Chromosome Mapping; Chromosomes, Human, Pair 7; Drug Resistance; Harringtonines; Homoharringtonine; Humans; Leukemia; Multidrug Resistance-Associated Proteins; Translocation, Genetic; Tumor Cells, Cultured

Liposomes encapsulating homoharringtonine were prepared using soybean phospholipids, egg yolk phospholipids and phospholipids from human red cell membrane, respectively. The quantity of homoharringtonine encapsulated by phospholipids from human red cell membrane was greater than that encapsulated by the other two types of liposomes, so this kind of phospholipid was used for further studies. The liposomes containing homoharringtonine (HH) were covalently coupled with HI30 (CD45). The mixture was separated by gel column chromatography. HI30-HH-liposomes demonstrated a sensitive targeting function with an immune activity equal to that observed with original HI30 in indirect immunofluorescence tests using the human cell line CEM-M3.

Topics: Antibodies, Monoclonal; Antigens, CD; Antineoplastic Agents, Phytogenic; Erythrocyte Membrane; Harringtonines; Histocompatibility Antigens; Homoharringtonine; Humans; Immunotoxins; Leukemia; Leukocyte Common Antigens; Liposomes; Phospholipids; Tumor Cells, Cultured

Homoharringtonine (HHT) is a cephalotaxine ester derived from an evergreen tree of southern China. We studied the effect of HHT on the clonal proliferation and differentiation of human leukemic cells from cell lines and patients. Dose-response studies found that HHT inhibited colony formation of myeloid cell lines (50% inhibitory dose range, 7 to 12 ng/ml), lymphocytic cell lines (50% inhibitory dose range, 4 to 7 ng/ml), and fresh leukemic cells (50% inhibitory dose range, 2 to 25 ng/ml). Pulse-exposure studies showed that colony formation of HL-60 cells was inhibited 50% by HHT (10 to 20 ng/ml) at 45 h and completely inhibited at 72 h. Radioactive precursor studies using HL-60 cells showed that HHT predominantly inhibited protein synthesis as compared with RNA and DNA synthesis. Taking advantage of this, we have found that the combination of HHT with 1-beta-D-arabinofuranosylcytosine (inhibitor of DNA synthesis) was synergistic in the inhibition of HL-60 clonal growth. HHT (2 to 20 ng/ml) also was found to induce up to 28% of HL-60 cells to differentiate toward macrophage-like cells.

Topics: Alkaloids; Antineoplastic Agents; Cell Differentiation; Cell Division; Cell Survival; Colony-Forming Units Assay; Dactinomycin; Dose-Response Relationship, Drug; Harringtonines; Homoharringtonine; Humans; In Vitro Techniques; Leukemia; Tumor Cells, Cultured

A high-performance liquid chromatographic procedure was developed for the quantitation of homoharringtonine in plasma. Harringtonine was used as an internal standard, and 1 ml of sample was required. The single-step extraction with dichloromethane resulted in almost 100% recovery for homoharringtonine and harringtonine. Analysis was performed on a reversed-phase CN column with amperometric detection. Chromatography was completed in 12 min. At an oxidation potential of +1.0 V, the detection limit was 1 ng/ml at a signal-to-noise ratio of 2. The mean analytical recovery for homoharringtonine was 99.5%. The within-run precision and between-run precision were both less than 11%. The method is equally applicable for plasma or serum, and it has been demonstrated to be applicable for study of the pharmacokinetics of homoharringtonine in patients suffering from acute non-lymphocytic leukaemia.

Topics: Alkaloids; Antineoplastic Agents, Phytogenic; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Electrochemistry; Harringtonines; Homoharringtonine; Humans; Leukemia; Oxidation-Reduction

Homoharringtonine (HHT) has been reported to induce hyperglycemia. This report describes a study conducted to characterize the effect of HHT on insulin production and action. Our data indicate that HHT-induced hyperglycemia results from the development of insulin resistance. A review of the literature suggests that patients receiving HHT continuous infusions of 5 mg/m2/d or greater and patients greater than 10 years of age may be at increased risk for the development of HHT-induced hyperglycemia. We recommend that patients with these risk factors, as well as diabetic patients and patients concurrently receiving asparaginase and/or prednisone, have their blood glucoses routinely monitored for hyperglycemia.

Topics: Acute Disease; Alkaloids; Antineoplastic Agents, Phytogenic; Blood Glucose; C-Peptide; Drug Evaluation; Harringtonines; Homoharringtonine; Humans; Hyperglycemia; Infusions, Intravenous; Insulin; Insulin Resistance; Leukemia; Risk Factors; Time Factors

Continuous infusion of homoharringtonine was administered to 17 children with refractory leukemia. Ten children with acute lymphoblastic leukemia received a total of 18 courses and seven children with acute nonlymphoblastic leukemia had a total of 13 courses. Doses were escalated from 1.65 to 8.5 mg/m2 for 5-10 consecutive days. Side effects included mild nausea and vomiting and transient changes in liver enzymes. Mucositis and diarrhea were more frequently seen at higher dose levels. Grade 3 hypotension and pain were seen at doses of 7 mg/m2 for 10 days. This is considered to be the maximum tolerated dose in this limited phase I trial. None of these previously heavily treated patients achieved a marrow remission.

Topics: Adolescent; Alkaloids; Blast Crisis; Child; Child, Preschool; Female; Harringtonines; Homoharringtonine; Humans; Infant; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Male

Topics: Acute Disease; Adolescent; Adult; Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cells, Cultured; Female; Harringtonines; Homoharringtonine; Humans; Leukemia; Male; Middle Aged; Retrospective Studies; Tumor Stem Cell Assay

Homoharringtonine (HHT) is a new plant alkaloid originally isolated in the People's Republic of China. Preliminary studies have suggested antitumor activity in several neoplastic diseases. We treated 49 patients with relapsed or resistant acute leukemia with escalating doses of homoharringtonine administered by continuous infusion. Three dose levels were examined: 5 mg/m2 for seven days, 7 mg/m2 for seven days, and 5 mg/m2 for nine days. Of 28 patients with acute nonlymphoblastic leukemia who received cumulative doses of 45 to 49 mg/m2, seven patients (25%) achieved complete remission. Four of these remissions occurred in a subset of ten patients previously resistant to two or more induction attempts with conventional chemotherapy. There were no remissions in three patients with secondary leukemia or in seven patients with acute lymphoblastic leukemia. Reversible hypotension, fluid retention, diarrhea, and tumor lysis syndrome were the major toxic effects of this treatment. Our results indicate that homoharringtonine is an effective new drug for the treatment of acute nonlymphoblastic leukemia and that this drug does not share cross-resistance with conventional antileukemic agents. The recommended dose is 5 mg/m2/d administered by continuous infusion for nine days.

Topics: Acute Disease; Adolescent; Adult; Aged; Alkaloids; Antineoplastic Agents; Bone Marrow; Bone Marrow Transplantation; Drug Evaluation; Harringtonines; Homoharringtonine; Humans; Hyperglycemia; Hypotension; Infusions, Parenteral; Leukemia; Leukocyte Count; Middle Aged; Platelet Count

Topics: Acute Disease; Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Female; Harringtonines; Homoharringtonine; Humans; Leukemia; Lymphocytes; Male; Middle Aged; Sister Chromatid Exchange

Homoharringtonine is a cephalotaxine ester derived from Cephalotaxus harringtonia, which is a Chinese evergreen tree. A limited clinical evaluation of this drug in China revealed antileukemic activity, which prompted clinical trials in the United States. We have treated 43 patients with a variety of refractory malignancies using a daily iv treatment for 5 days at 3-4-week intervals. The starting dose of homoharringtonine was 0.2 mg/m2/day and it was escalated to a maximum of 8 mg/m2/day. The dose-limiting toxic effect was hypotension, which was generally mild with daily dose levels of 3-4.5 mg/m2/day and required no specific treatment besides iv fluid supplements in some patients. Hypotension became increasingly severe at the higher dose levels and resulted in cardiovascular collapse in four of 16 patients treated with dose levels of 5-6 mg/m2/day. A moderately severe degree of myelosuppression was observed with homoharringtonine doses of greater than or equal to 3 mg/m2. Myelosuppression was clearly related to the extent of prior treatment and was minimal in patients who had not received extensive prior treatment. Gastrointestinal toxic effects of nausea, vomiting, and diarrhea were observed in approximately two-thirds of the patients but these side effects were generally mild and self-limited. Drug-related fever and alopecia were also observed in some patients. No major responses were observed, although three patients with solid tumors evidenced minor responses and three of five patients with acute leukemia showed some degree of antileukemic activity. For phase II studies of homoharringtonine in solid tumors, a daily dose of 3 mg/m2 for 5 days in patients with extensive prior treatment and 4 mg/m2/day for 5 days in patients with good bone marrow reserve will be utilized. The daily dose must not exceed 4 mg/m2 to avoid serious hypotension; further dose escalations should be accomplished by extending the number of days of treatment beyond 5 days.

Topics: Adult; Aged; Alkaloids; Bone Marrow Diseases; Dose-Response Relationship, Drug; Drug Evaluation; Female; Harringtonines; Homoharringtonine; Humans; Hypotension; Leukemia; Male; Middle Aged; Neoplasms

Topics: Acute Disease; Adolescent; Alkaloids; Antineoplastic Agents; Child; Child, Preschool; Drug Therapy, Combination; Female; Harringtonines; Homoharringtonine; Humans; Leukemia; Male; Prednisone; Vincristine

Topics: Adolescent; Adult; Alkaloids; Brain Neoplasms; Child; Child, Preschool; Female; Harringtonines; Homoharringtonine; Humans; Injections, Spinal; Leukemia; Male; Middle Aged

Topics: Acute Disease; Adolescent; Adult; Antineoplastic Agents; Child; Cytarabine; Drug Therapy, Combination; Female; Harringtonines; Homoharringtonine; Humans; Leukemia; Male; Medicine, Chinese Traditional; Middle Aged; Prednisone; Vincristine