Homoharringtonine induces apoptosis of endothelium and down－regulates VEGF expression of K562 cells

Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry, The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner, Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.

LONG-TERM EFFECT OF HOMOHARRINGTONINE ON CHRONIC GRANULOCYTIC LEUKEMIA

Objective: To observe the long-term effect of homoharringtonine (HHT) on chronic granulocytic leukemia (CGL) and its pharmacological mechanism. Methods: 76 patients with newly diagnosed early chronic phase CGL received treatment of merely 1.5 mg/m2 daily HHT for induction remission and long-term maintenance treatment. The apoptosis rate of bone marrow CD34+ cells induced by HHT was assayed with flow cytometer. Results: 86.8% patients achieved CHR, 13.2% patients PHR and 31.8% patients got cytogenetic response in HHT treatment group, which was longer than 31 (8-54) months in hydroxyurea (HU) group (P<0.05). The effect of apoptosis induction HHT was stronger on CGL-CP patients bone marrow CD34+ cells than on normal person bone marrow CD34+ cells. Conclusion: HHT is a very effective drug for remission induction and long-term maintenance treatment in early chronic phase CGL patients.

INDUCTION OF C-MYC GENE AMPLIFICATION BY HYDROXYUREA AND ITS INHIBITION BY HOMOHARRINGTONINE

Induction of c-myc gene amplification in L1210 cells by hydroxyurea and its inhibition by homohar-ringtonine were investigated using the DNA-DNA molecular hybridization technique. When the cells were treated with hydroxyurea 1.0 mM for 16 hours, and incubated a further 16 hours in a drug-free medium, the c-myc gene amplified 23.5-fold. If homohar-ringtonine 50 μM was used at the same time as hydroxyurea, gene amplification did not occur. Cycloheximide, an inhibitor of protein biosynthesis, produced a similar effect. Our results indicated that a (or some) protein factor(s) might be involved in gene amplification. Detailed analysis showed that the synthesis of this protein factor(s) started 4 hours before the initiation of the S phase but did not continue in the S phase. It was also found that this protein factor(s) was very labile and began to degrade 2 hours after its appearance.

A phase Ⅳ study of homoharringtonine, cytarabine, aclacinomycin and G-CSF(HCAG) regimen compared with traditional IA regimen in the treatment of newly diagnosed elderly acute myeloid leukemia patients

Objective · To compare the efficacy and prognostic factors of HCAG regimen with traditional IA regimen in the treatment of newly diagnosed elderly acute myeloid leukemia(AML) patients. Methods · Forty-one patients with AML(aged 55-71 years) were randomly divided into two groups(Group HCAG and Group IA) between 2014 and 2016 for induction and consolidation therapy. Multivariate analysis was applied to identify prognostic factors for relapse-free survival(RFS). Results · A total of 29 patients(70.7%) achieved complete remission(CR). The estimated 2-year overall survival(OS) was 66.8% in Group HCAG and 75.4% in Group IA(P=0.913). The estimated 2-year RFS was 61.8% in Group HCAG and 49.1% in Group IA(P=0.411). Age remained as the unfavorable prognostic factor, leading to significant differences in OS and RFS. In addition, RFS was influenced by cytogenetic/molecular risk stratification. Conclusion · Although HCAG seemed not to particularly benefit the group, the dose reduction of anthracyclines may be applied in elderly patients with comparable short-time outcome. Furthermore, the introduction of homoharringtonine resulted in an improvement of treatment response for more than 20% compared with CAG regimen.

Effect of Homoharringtonine on Corneal Haze after Excimer Laser Photorefractive Keratectomy in Rabbits

To evaluate the inhibiting effect of Homoharringtonine （HHT） on the corneal haze after excimer laser photorefractive keratectomy （PRK） in rabbits. 18 healthy rabbits which underwent PRK were randomly divided into three groups （A, B and C）. The refractive degree of ablation was - 10.0DS in each group. Group A was locally treated with a piece of filter paper soaked with 1 mg/ mL HHT for 5 min, and then the entire cornea was repeatedly irrigated with balance solution; Group B was dropped with 0.1 mg/mL HHT after PRK for 3 months; Group C was the control group, Corneal haze, histopathology, response, ect. were investigated. The corneal haze was significantly less in group A, while the difference between group B and group C was insignificant. Keratocytes and fibrocytes in corneal stroma were more active up to 3 months in group B and group C, Intraoperative use of topical HHT can reduce corneal haze after PRK in rabbits.

Effect of Homoharringtonine on Bone Morrow CD34^+CD7^+ Cells in Chronic Granulocytic Leukemia

Objective： To explore the effect of homoharringtonine （HHT） on bone morrow CD34^＋CD7^＋ cells in chronic granulocytic leukemia （CGL）. Methods： The changes of bone morrow CD34^＋CD7^＋ cells were observed after the treatment of HHT in 23 cases with CGL. The proliferation and apoptosis of CD34^＋CD7^＋ cells treated with HHT in vitro were studied. Results： The proportion of CD34^＋CD7^＋ cells in CGL （0.145±0.021） was higher than that of normal control （0.052±0.013）. The proportion of CD34^＋CD7^＋ cells in patients who got cytogenetic responses to HHT （0.072±0.020） decreased remarkably, but not in those patients who did not got cytogenetic responses to HHT, （0.137±0.023）. the proliferation of CD34^＋ cells was inhibited and the proportion of CD34^＋CD7^＋ cells decreased after cultured with HHT （0.134 in 24 h, 0.126 in 48 h and 0.102 in 72）. The apoptosis rate of CD34^＋CD7^＋ cells was higher than that in CD34^＋CDT cells （35.39%±4.39% versus 24.57%±4.01%, P〈0.05） 72 h after culture with HHT. Conclusion： The proportion of CD34^＋CD7^＋ cells in CGL was higher than that of normal control and HHT may inhibit the proliferation and induce apoptosis of bone marrow CD34^＋CD7^＋ cells.

Homoharringtonine promotes heart allograft acceptance by enhancing regulatory T cells induction in a mouse model

Background:Homoharringtonine(HHT)is an effective anti-inflammatory,anti-viral,and anti-tumor protein synthesis inhibitor that has been applied clinically.Here,we explored the therapeutic effects of HHT in a mouse heart transplant model.Methods:Healthy C57BL/6 mice were used to observe the toxicity of HHT in the liver,kidney,and hematology.A mouse heart transplantation model was constructed,and the potential mechanism of HHT prolonging allograft survival was evaluated using Kaplan-Meier analysis,immunostaining,and bulk RNA sequencing analysis.The HHT-T cell crosstalk was modeled ex vivo to further verify the molecular mechanism of HHT-induced regulatory T cells(Tregs)differentiation.Results:HHT inhibited the activation and proliferation of T cells and promoted their apoptosis ex vivo.Treatment of 0.5 mg/kg HHT for 10 days significantly prolonged the mean graft survival time of the allografts from 7 days to 48 days(P<0.001)without non-immune toxicity.The allografts had long-term survival after continuous HHT treatment for 28 days.HHT significantly reduced lymphocyte infiltration in the graft,and interferon-γ-secreting CD4^(+)and CD8^(+)T cells in the spleen(P<0.01).HHT significantly increased the number of peripheral Tregs(about 20%,P<0.001)and serum interleukin(IL)-10 levels.HHT downregulated the expression of T cell receptor(TCR)signaling pathway-related genes(CD4,H2-Eb1,TRAT1,and CD74)and upregulated the expression of IL-10 and transforming growth factor(TGF)-βpathway-related genes and Treg signature genes(CTLA4,Foxp3,CD74,and ICOS).HHT increased CD4^(+)Foxp3^(+)cells and Foxp3 expression ex vivo,and it enhanced the inhibitory function of inducible Tregs.Conclusions:HHT promotes Treg cell differentiation and enhances Treg suppressive function by attenuating the TCR signaling pathway and upregulating the expression of Treg signature genes and IL-10 levels,thereby promoting mouse heart allograft acceptance.These findings may have therapeutic implications for organ transplant recipients,particularly those with viral infections and malignancies,which require a more suitable anti-rejection medication.

Efficacy and safety of homoharringtonine during consolidation therapy in chronic myeloid leukemia: a meta-analysis

In the present study, we aimed to assess the efficacy and safety of Homoharringtonine （HHT） for chronic myelogenous leukemia （CML）. Databases, such as PubMed, the Cochrane Library, EMbase, CENTRAL, VIP, WanFang Data, CBM and CNKI, were electronically searched from inception to May 2014 for clinical trials on HHT for CML. Literatures were independently screened by two reviewers based on the inclusion and exclusion criteria, data were extracted, and methodological quality was assessed accordingly. Meta-analysis was performed using RevMan 5.2. Five trials were included consisting of a total of 423 patients. The results of meta-analysis showed that the HHT group was superior to the hydroxycarbamide （HU） group in terms of complete hematologic response rate （CHR）, major cytogenetic responses （MCyR） rate, partial cytogenetic responses （PCyR） rate, blast rate and 4-year survival rate. There was no statistical difference in complete cytogenetic response （CCyR） and minor cytogenetic response （mCyR） rates between the HHT group and HU group. HHT caused less adverse reaction. Therefore, HHT alone showed considerable short-term and long-term efficacy in the treatment of late-phase CML. It could be a good choice for some CML. Since moderate selection bias might exist in the methodological quality of the included studies which might affect the authenticity of outcomes, our conclusions should be further proved by conducting more high-quality, large-scale and double-blinded randomized controlled trials （RCTs）.

Induction of apoptosis by homoharringtonine in G1 phase human chronic myeloid leukemic cells

Background Homoharringtonine (HHT) is a cephalotaxine ester derived from an evergreen tree found wildely throughout southern China, which has antileukemic activities against a variety of acute myeloid leukemic cells. For the sake of illustrating the mechanisms of HHT in the treatment of leukemia, we assessed the effect of HHT on the apoptosis of human chronic myeloid leukemic cell line K562.Methods The apoptosis of K562 cells induced by HHT was analyzed by transmission electron microscopy, agarose gel electrophoresis of DNA, flow cytometry and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick labeling.Results Characteristic apoptosis-related features emerged in K562 cells after exposed to HHT at a concentration 0.05-100 μg/ml. Transmission electron microscopy of HHT treated K562 cells displayed chromatin condensation and aggregation under the nuclear membrane, nuclear fragmentation and apoptosis body formation. Typical DNA ladder in agarose gel electrophoresis was observed in the cells exposed to HHT. The cell cycle analysis measured by flow cytometry showed G1 phase cells decreased with the increase of S phase cells while apoptosis was induced by HHT in K562 cells. The percentage of apoptotic cells in K562 cells treated with 50 μg/ml of HHT decreased significantly when pretreated with 1 μg/ml of cycloheximide, 0.05 μg/ml of Actinomycin D respectively.Conclusions HHT has apoptotic effects on K562 cells. The HHT induced apoptosis mainly of the cells in G1 phase and this process required RNA transcription and protein synthesis.

Homoharringtonine synergy with oridonin in treatment of t(8;21) acute myeloid leukemia

Collaboration of c-KIT mutations with AML1-ETO (AE) has been demonstrated to induce t(8;21) acute myeloid leukemia (AML).Targeted therapies designed to eliminate AE and c-KIT oncoproteins may facilitate effective treatment of t(8;21) AML.Homoharringtonine (HHT) features activity against tumor cells harboring c-KIT mutations,whereas oridonin can induce t(8;21) AML cell apoptosis and AE cleavage.Therefore,studies should explore the efficacy of combination therapy with oridonin and HHT in t(8;21) AML.In this study,we investigated the synergistic effects and mechanism of oridonin combined with HHT in t(8;21) AML cell line and mouse model.The two drugs synergistically inhibited cell viability and induced significant mitochondrial membrane potential loss and apoptosis.Oridonin and HHT induced significant downregulation of c-KIT and its downstream signaling pathways and promoted AE cleavage.HHT increased intracellular oridonin concentration by modulating the expressions of MRP1 and MDR1,thus enhancing the effects of oridonin.The combination of oridonin and HHT prolonged t(8;21) leukemia mouse survival.In conclusion,oridonin and HHT exert synergistic effects against t(8;21) leukemia in vivo and in vitro,thereby indicating that their combination may be an effective therapy for t(8;21) leukemia.

Homoharringtonine is a safe and effective substitute for anthracyclines in children younger than 2 years old with acute myeloid leukemia

Homoharringtonine (HHT),a plant alkaloid from Cephalotaxus harringtonia,exhibits a unique anticancer mechanism and has been widely used in China to treat patients with acute myeloid leukemia (AML) since the 1970s.Trial SCMC-AML-2009 presented herein was a randomized clinical study designed based on our previous findings that pediatric AML patients younger than two years old may benefit from HHT-containing chemotherapy regimens.Patients randomized to arm A were treated with a standard chemotherapy regimen comprising mainly of anthracyclines and cytarabine (Ara-C),whereas patients in arm B were treated with HHTcontaining regimens in which anthracyclines in all but the initial induction therapy were replaced by HHT.From February 2009 to November 2015,59 patients less than 2 years old with de novo AML (other than acute promyelocytic leukemia) were recruited.A total of 42 patients achieved a morphologic complete remission (CR) after the first course,with similar rates in both arms (70.6% vs.72.0%).At the end of the follow-up period,40 patients remained in CR and 5 patients underwent hematopoietic stem cell transplantation in CR,which could not be considered as events but censors.The 5-year event-free survival (EFS) was 60.2%± 9.6% for arm A and 88.0%± 6.5% for arm B (P=0.024).Patients in arm B experienced shorter durations of leukopenia,neutropenia,and thrombocytopenia and had a lower risk of infection during consolidation chemotherapy with high-dosage Ara-C.Consequently,the homoharringtonine-based regimen achieved excellent EFS and alleviated hematologic toxicity for children aged younger than 2 years with de novo AML compared with the anthracycline-based regimen.

Homoharringtonine in combination with cytarabine and aclarubicin as induction therapy improves remission and survival of patients with higher risk myelodysplastic syndromes

Myelodysplastic syndromes （MDS） are a heterogeneous group of clonal disorderscharacterized by ineffective hematopoiesis; they manifest clinically as anemia, neutropenia, and thrombocytopenia.1＇2 Higher risk MDS include patients in the intermediate-2 and high-risk categories of the International Prognostic Scoring System （IPSS）, as well as patients with MDS secondary to radiation or chemical exposure.3 The patients with higher risk MDS have a peculiar propensity to evolve into acute myeloid leukemia （AML）, and have a median survival of 〈12 months.4 Management of higher risk MDS varies from supportive care with transfusions or hematopoietic cytokines to high-intensity3 chemotherapy or hematopoietic stem cell transplantation. The impact of intensive myelosuppressive chemotherapy, similar to AML induction therapy, has been investigated. The results are comparable to the reported outcome of patients with AML, i.e. complete remission （CR） rates of 40%-60% and median overall survival （OS） of 8-12 months,s Thus, innovative approaches are needed for the treatment of higher risk MDS.

Prolonged chronic phase in chronic myelogenous leukemia after homoharringtonine therapy

Background Homoharringtonine （HHT） is effective in treating late stage chronic myelogenous leukaemia （CML）, but little is known about long term maintenance during complete cytogenetic response. Long term efficacy and toxicity profiles of low dose HHT were evaluated in this study. Methods One hundred and six patients with CML received 1.5 mg/m^2 of HHT alone by continuous daily infusion for seven to nine days every four weeks. Of 79 patients in the control group, 31 were treated with interferon α （IFN-α） and 48 with hydroxycarbamide. For 17 patients who failed to achieve cytogenetic response within 12 months＇ treatment of IFN-α, HHT was administered. Quantitative RT-PCR was used to detect the BCR-ABL mRNA expression in 36 Philadelphia positive CML patients enrolled after 2007. Haematological and cytogenetic responses were evaluated in all patients at the 12th month of follow-up. Long term efficacy was assessed in a follow-up with a median time of 54 months （12 months-98 months）. Results After 12 months of therapy, cytogenetic response rate of the HHT, IFN-α and hydroxycarbamide groups were 39/106, 14/31 and 3/48, and corresponding molecular cytogenetic response rates 6/18, 3/8 and 0. Of the 17 patients who received HHT as salvage treatment, 6 achieved cytogenetic response （3 major）. At the 48 months＇ follow-up, cytogenetic response was maintained in 32/39 patients treated with HHT. Patients who had cytogenetic response in HHT group or treated with IFN-α also showed longer median chronic durations, which were 45 months （12 months-98 months） and 49 months （12 months-92 months） respectively, indicating a longer survival time. Conclusions Low dose HHT alone showed considerable short term and long term efficacy in the treatment of late stage CML. It may also be a good choice for patients who have failed imatinib, IFN-α treatment or haematopoietic stem cell transplantation or cannot afford these treatments.

Correlation between survivin mRNA expression and homoharringtonine induced apoptosis of malignant hematopoietic cells

Background 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. Methods 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.Results 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.Conclusion 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.