Effects of Triptolide Combined with Homoharringtonine on Proliferation and Apoptosis of KG-1α Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect and possible mechanism of low concentration of triptolide (TPL) combined with homoharringtonine (HHT) on the proliferation and apoptosis of KG-1α cells.</p><p><b>METHODS</b>CCK-8 method was used to detect the antiproliferating effects of different concentrations of TPL and HHT single-use and combined use on KG-1α cells, and the combined index (CI) was calculated. The colony formation ability was also determined by methylcellulose colony formation assay, cell surface molecules, apoptosis rate and cell cycle changes were detected by flow cytometry. Westerrn blot was used to detect the expression of Akt signaling pathway related proteins before and after low dose TPL combined with HHT using.</p><p><b>RESULTS</b>High expression of CD34 and CD123 were on KG-1a cells, which being lack expression of CD38. TPL and HHT dose-dependently inhibited the proliferation of KG-1α cells. Compared with low dosage TPL and HHT single-use groups, the cell proliferation and colony formation efficiency were lower, and the cell apoptosis rate was higher in the combined group. CI values also indicated that low concentration TPL combined with HHT possessed highly synergistic effect. After the combination of the 2 drugs, the expressions of P-Akt, P-Akt, BCL-2, PARP and survivin protein were down-regulated and the cleavage of PARP protein was increased.</p><p><b>CONCLUSION</b>Low concentration of TPL combined with HHT can synergistically inhibit KG-1α cell proliferation and induce its apoptosis through the PI3K/Akt signaling pathway and downstream protein.</p>

Combined Effect of Bortezomib and Homoharringtonine on K562 Cells and their Mechanisms / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of BTZ plus HHT on proliferation and apoptosis of K562 cells, and to clarify the relationship between the mechanism inderlying the effect of BTZ plus HHT on K562 cells and BCL-2, BAX, MCL-1 proteins.</p><p><b>METHODS</b>The K562 cells were divided into 4 groups by different treatment: BTZ(20 nmol/L), HHT(40 ng/ml), BTZ(20 nmol/L)+HHT(40 ng/ml) and control. The proliferation inhibition rates of K562 cells in each group were detected by using MTT, and the early apoptosis rates of K562 cells in each group were assayed by using flow cytometry with Annexin V-FITC/PI staining. The proteins level of BCL-2, BAX and MCL-1 in each group were examined by using Western blot.</p><p><b>RESULTS</b>The inhibition rate of K562 cell proliferation in combined group was higher than that in BTZ, HHT alone group(P<0.01). The early apoptosis rate of K562 cells in combined group was increased significantly in comparison with BTZ and HHT alone group(P<0.05). The BCL-2 protein level of K562 cells in combined group was significantly lower than that in BTZ and HHT alone group(P<0.05). BAX protein level of K562 cells in combined group was higher than that in BTZ and HHT alone group(P<0.05). The Orders of the MCL-1 protein level of K562 cells in 4 groups were BTZ>Control>BTZ plus HHT>HHT(P<0.05 ).</p><p><b>CONCLUSION</b>The combination of BTZ and HHT exerts the synergistic effect of anti-proliferative activity and induces apoptosis against K562 cells in vitro. The combination can induce apoptosis of K562 cells via suppression of BCL-2 protein and up-regulation of BAX protein. HHT can increase the sensitivity of K562 cells to BTZ by down-regulating the expression of MCL-1 protein.</p>

Long-term outcomes of homoharringtonine, cytarabine, daunorubicin or idarubicin (HAD/HAI) as induction chemotherapy in de novo acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To estimate the long-term outcomes and the prognostic factors of homoharringtonine, cytarabine, daunorubicin or idarubicin (HAD/HAI) as induction chemotherapy in de novo acute myeloid leukemia (AML).</p><p><b>METHODS</b>The CR rate, overall survival (OS) rate, relapse free survival (RFS) rate were retrospectively assayed in 143 de novo AML patients who received the HAD/HAI induction chemotherapy. The outcomes were compared among prognostic groups according to world health organization (WHO) classification, genetic prognosis and initial white blood cell (WBC) count. The role of consolidation chemotherapy consisting of middle-dosage Ara-C (MD-Ara-C) on long term survival was evaluated.</p><p><b>RESULTS</b>Of 143 patients, 112 (78.3%) achieved CR after the first course of HAD/HAI induction treatment, and early death occurred in only one case. Notably, the CR rate of patients with an initial WBC count ≥100×10(9)/L was not significantly different from those with an initial WBC count<100× 10(9)/L (70.4% vs 80.2%, P=0.266). The CR rate for the patients with favorable, intermediate and unfavorable integrated genetics risk factors was 93.7%, 71.4% and 61.3%, respectively, the difference between groups was statistically significant (P=0.001). Patients with FLT3-ITD mutation obtained similar CR rate (70.6%) to that of patients with FLT3 wild type (79.3%, P=0.528).The estimated 5-year OS rate and 5-year RFS rate for all patients was 40.0% and 37.0%, respectively, with a median follow-up of 24 (range 1-104) months. The median survival time was 30 [95%CI (12, 48)] months. 5-year OS and 5-year RFS of the 96 patients who achieved CR after first course chemotherapy without undergoing allo-HSCT in complete remission was 47.0% and 38.0%, respectively. 5-year OS was significantly higher in MD-Ara-C consolidation group than in no MD-Ara-C consolidation group among CR patients without allo-HSCT (58.0%, 19.0%, respectively, P=0.004). In patients who obtained CR after first course and received MD-Ara-C consolidation without allo-HSCT, the 5-year OS of patients with hyperleukocytosis was not significantly lower than that of patients without hyperleukocytosis (55.5%, 58.8%, respectively,P=0.419). FLT3-ITD mutation patients showed similar 5-year OS to that of wild type FLT3 patients (51.4%, 60.2%, respectively, P=0.482). And furthermore, 5-year OS of favorable, intermediate and unfavorable integrated genetics groups were 59.1%, 62.5%, 51.9%, respectively (P=0.332) in this subgroup.</p><p><b>CONCLUSION</b>HAD/HAI induction chemotherapy with sequential consolidation of MD-Ara-C could obtain satisfactory CR rate and long-term survival rate in de novo AML, especially for patients with hyperleukocytosis or FLT3-ITD mutation. It yet remains to be verified by large sample, prospective studies.</p>

Efficacy of HAA regimen in the treatment of 64 patients with refractory/relapsed acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of the HAA regimen (homoharringtonine,cytarabine and aclarubicin)as salvage chemotherapy in the treatment of refractory/relapsed acute myeloid leukemia (AML).</p><p><b>METHODS</b>We retrospectively analyzed 64 patients with refractory/relapsed AML who received the HAA regimen as salvage chemotherapy. The complete remission (CR)rate was analyzed. Kaplan-Meier method was used to estimate overall survival (OS) and relapse free survival (RFS), and the differences were compared by Log-rank test.</p><p><b>RESULTS</b>The overall CR rate was 70.1%, and 67.1% of the patients attained CR after the first induction course. The early death rate was 0. The median follow-up time was 61 (range:6-120) months. The estimated 3-year OS rate was 46.8% and the estimated 3-year RFS rate was 42.8%. The CR rates of patients with favorable/intermediate and unfavorable cytogenetics were 76.4% and 33.3%, respectively. The 3-year OS of favorable/intermediate and unfavorable group were 53.7% and 10.0%, respectively. The median survival time of unfavorable group was only 8 months. The side effects associated with the HAA regimen were tolerable, in which the most common toxicities were myelosuppression and infection.</p><p><b>CONCLUSION</b>HAA regimen is associated with a higher rate of CR and longer-term survival and its toxicity can be tolerated. The regimen is suitable for refractory/relapsed AML patients with favorable or intermediate risk .</p>

Caracterización de las causas de alopecia infantil / Aetiology of childhood alopecia

Introducción: La alopecia infantil es una afección poco frecuente en la consulta dermatológica pediátrica. Su etiología es variable según el grupo etario estudiado. El objetivo fue estudiar la causa de alopecia en niños en 2 hospitales pediátricos de referencia nacional en Chile. Pacientes y método: Análisis descriptivo de registros clínicos del total de pacientes atendidos entre enero de 2007 y junio de 2010 en los Servicios de Dermatología de los Hospitales Roberto del Río y Luis Calvo Mackenna. Se incluyeron pacientes con diagnóstico clínico de alopecia. Resultados: Se encontraron 345 registros clínicos, 179 varones (51,9%). La mediana de edad fue 72 meses. Los diagnósticos más prevalentes fueron alopecia areata (AA) (36,8%), tiña capitis (TC) (21%), nevo sebáceo (13,2%) y efluvio telógeno (8,7%). Según el grupo etario predominaron en recién nacidos: aplasia cutis y nevo sebáceo; en lactantes, preescolares y escolares: nevo sebáceo, AA y TC. En escolares se agregó tricotilomanía. En adolescentes nevo sebáceo, AA y efluvio telógeno. Se observó una correlación significativa entre AA con enfermedad autoinmune, enfermedad tiroidea, alteraciones ungueales, enfermedad psiquiátrica y síndrome de Down. En TC el agente etiológico más prevalente fue Microsporum Canis (86,6%). La tricotilomanía se correlacionó con enfermedad psiquiátrica significativamente. Conclusiones: Las principales causas de alopecia infantil fueron adquiridas y no cicatriciales. La etiología varía de acuerdo al grupo etario estudiado. Algunos tipos de alopecia infantil presentaron alta prevalencia de enfermedad psiquiátrica.

Introduction: Childhood alopecia is a relative rare event in general paediatric dermatology practice. Hair loss in children may have multiple causes, and there are different types of alopecia according to age groups. The aim of the study was to describe the clinical and epidemiological profile of alopecia in children from two Chilean paediatric hospitals. Patients and method: Descriptive analysis of clinical records of patients from the Dermatology Department of Roberto del Rio and Luis Calvo Mackenna Hospitals between January 2007 and June 2010. Patients with clinical diagnosis of alopecia were included. Results: A total of 345 clinical records were analysed, with 179 males (51.9%). The median age was 72 months. Overall, the most common diagnoses were: alopecia areata (AA), (36.8%), tinea capitis (TC), (21%), nevus sebaceous (13.2%), and tellogen effluvium (8.7%). According to age groups, in newborns, the most common causes were aplasia cutis and nevus sebaceous. In toddlers, pre-school and school children, the principal causes were nevus sebaceous, AA and TC. Trichotillomania was also significant in school children. In adolescents, nevus sebaceous, AA and tellogen effluvium were the most frequent diagnoses. AA was statistically associated with autoimmune disease, thyroid disease, nail disorder, psychiatric disease, and Down's syndrome. The most common aetiological agent in TC was M. canis (86.6%). Trichotillomania was also statistically associated to psychiatric disorders. Conclusions: In this study, the main causes of alopecia in children were acquired and non-scarring alopecia. In our results, the type of alopecia varies according to age group. Some types of childhood alopecia showed a close correlation to psychiatric disorders.

Cohort Study on GHA and New Combined Priming Chemotherapeutic Regimens in Treatment of Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the clinical efficacy and adverse effects of GHA(G-CSF+homoharringtonin+cytarabine C) and new combined priming chemotherapeutic regimens(GHAA/GHTA) with high efficacy and low toxicity for treatment of relapsed and refractory acute myeloid leukemia(AML) and myelodysplastic syndrome(MDS), and to analyze the relation of above-mentioned regimens with the expression of co-stimuolating molecule B7.1.</p><p><b>METHODS</b>Standard GHA regimen consisting of G-CSF: 100 µg/(m2·d) subcutaneous injection, d 0-14; homoharringtonine: 1.0 mg/(m2·d) intravenous drip, d 1-14; Ara-C: 7.5-10 mg/(m2·d) subcutaneous injection, q12h, d 1-14. Other regimens as GHAA/GHTA were combined respectively with aclarubicin 20 mg d 1-7, or pirarubicin 20 mg d 1-7. 74 patients with refractory AML and 46 patients with MDS received these priming chemotherapy. The clinical efficacy and toxicity of above-mentioned priming chemotherapy were compared with 56 patients received routine chemotherapy (MA/TAE) respectively. And the expression of costimulatory molecule B7.1 on leukemia cells in patients of different subtypes was also detected by immunofluoressence and its relationship with clinical efficiency was explored.</p><p><b>RESULTS</b>(1) for AML patients treated with priming chemotherapy, the total remission was 67.56% (CR 54.05%, PR 13.51%), which was much higher than that of patients received routine chemotherapy (P<0.05). The CR rate of AML-M2 and AML-M5 group (65.51%, 61.90% respectively) was much higher than that of AML other subtypes (P<0.05), and the longest remission period lasted for 4 years; (2) for MDS patients treated with priming chemotherapy, the total remission was 60.87% (CR 45.65%, PR 15.22%), which was also significantly higher than that of patients received routine chemotherapy (P<0.05); (3) in comparison with patients received standard GHA priming regimen, the remission rate of combined priming chemotherapy GHAA/GHTA was significantly higher both in patients with AML (85.18%) and MDS (81.25%); (4) side effects after chemotheropy, including granulocyte deficiency, thrombocytopenia and anemia etc, lasted for 7-14 days; the severe infection rate was 1%, there were no severe bleeding, digest effect and damage of function in heart, liver and kidney. The therapy-related mortality was zero. Compared with routine chemotherapy, priming chemotherapy proved significantly safe and effective (P<0.05); (5) the expression rate of costimulatory molecule B7.1 showed large variance between AML and MDS, it was higher in AML-M2/AML-M5 and lower in AML of other subtypes (P<0.05). In the same case, B7.1 expression was positive correlated with efficiency of priming chemotherapy.</p><p><b>CONCLUSION</b>GHA priming chemotherapy, as well as other combination regimens GHAA/GHTA, are well-tolerated, effective regimens for refractory AML and advanced MDS, without severe side effects and therapy-related mortality. Especially the new regimens GHAA/GHTA has better efficacy, which are proved more efficient than conventional GHA. Efficiency of priming chemotherapy is positive correlated with B7.1 expression, its mechanism will be further explored.</p>

Enhancing effect of beta-elemene emulsion on chemotherapy with harringtonine, aclacinomycin and Ara-c in treatment of refractory/relapsed acute myeloid leukemia

This study is to determine the curative effect of beta-elemene emulsion on chemotherapy in the treatment of refractory/relapsed acute myeloid leukemia [AML]. In the beta-elemene emulsion plus HAA chemotherapy [harringtonine, aclacinomycin, Ara-c group] group, 120 cases received beta-elemene emulsion [400 mg] plus the HAA treatment. A 14-day treatment was a course of treatment, followed by an 8-14 day pause, and then the next course of treatment. The HAA treatment included Ara-c, 100 mg/m2, once every 12 h from day 1 to day 7; aclacinomycin, 20 mg/m2, from day 1 to day 7; and homoharringtonine, 4 mg/m2, from day 1 to day 7. The patients in the control HAA group received HAA treatment only. For both groups, effective antibiotics were given to patients when it was necessary. The total effective rate in the beta-elemene emulsion plus HAA group was 80.8%. But the total effective rate in the HAA only group was 52.9%. These results suggest that the beta-elemene emulsion plus HAA treatment has a much better curative effect in comparison with the HAA only treatment [P < 0.05]. Furthermore, beta-elemene emulsion has slightly adverse response, without causing blood and bone marrow depression. Beta-elemene emulsion has a curative effect in treatment of refractory/relapsed AML in combination with harringtonine, aclacinomycin, and Ara-c

Time rhythm of homoharringtonine inducing K562 cell apoptosis and its mechanism / 中国实验血液学杂志

This study was aimed to explore the change of K562 cell apoptosis at different time point after homoharringtonine (HHT) treatment and its mechanism. After treatment of K562 cells with 10 ng/ml HHT, the cell viability was tested with MTT assay; the expression of caspase-3 was detected with Western blot; the BCL-2 expression was analyzed with flow cytometry; the autophagosome was observed by electron microscopy. The results showed that the viability of K562 cells reduced gradually from day 1 to day 5 and ascended from day 6 to day 8 after HHT treatment. At the same time, the cleaved caspase-3 expression level of K562 cells increased gradually from day 1 to day 7, but reduced at the day 8 (P < 0.05). From day 1 to day 8 after HHT treatment, the BCL-2 expression level declined firstly and then went up (P < 0.05). Autophagosome was also seen remarkably at day 8 after HHT treatment. It is concluded that the apoptosis level of K562 cells after being treated with HHT enhances firstly and then declines , which may be associated with higher autophagy level in the late stage of HHT treatment.

Homoharringtonine combined arsenic trioxide induced apoptosis in human multiple myeloma cell line RPMI 8226: an experimental research / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To clarify the effects and mechanisms of homoharringtonine (HHT) monomer therapy or combination therapy with arsenic trioxide (ATO) on human multiple myeloma (MM) cell line RPMI 8226 in in vitro researches.</p><p><b>METHODS</b>Effects of HHT, ATO, and HHT combined ATO on the growth of MM cell line RPMI 8226 were detected using MTT assay. The morphological changes of cell apoptosis were detected by Hoechst staining. The early apoptosis rate was detected using flow cytometry. Expressions of Caspase-3, Caspase-9, poly-ADP-ribose polymerase (PARP), Bcl-2, Mcl-1, Bcl-xl, and AKT protein were detected by Western blot.</p><p><b>RESULTS</b>HHT and ATO inhibited the proliferation of RPM1 8226 cell line in a time- and dose-dependent manner (P < 0.05). Synergistic effects was shown in the combination group (Cl < 1). HHT and ATO induced the apoptosis of RPMI 8226 in a dose-dependent manner with typical morphological changes of apoptosis and higher early stage apoptosis rate. The enhancement in apoptotic induction was seen when two agents were combined. HHT activated expressions of Caspase-3 and PARP in a dose dependent manner at 24 h. HHT at 40 ng/mL and ATO at 8.5 micromol/L could significantly activate expressions of Caspase-3 and Caspase-9, and down-regulate expressions of anti-apoptotic proteins Bcl-xl and Mcl-1. In addition, the combination therapy of HHT at 40 ng/mL and ATO at 8.5 micromol/L inhibited phosphorylation of AKT in a time-dependent manner.</p><p><b>CONCLUSION</b>HTT, ATO, and combination therapy of HHT and ATO induced the apoptosis of RPMI 8226 cell line possibly through activating Caspase pathways, regulating expressions of Bcl-2 families, and inhibiting phosphorylation of AKT.</p>

Effect of homoharringtonine on expression of NF-κB and BCL-2 proteins in K562 cells / 中国实验血液学杂志

This study was aimed to investigate the effect of homoharringtonine (HHT) on K562 cell proliferation, apoptosis and expression of BCL-2 and NF-κB proteins. The cells proliferation was assayed with MTT method, the cell apoptosis, cell cycle and BCL-2 expression were analyzed with flow cytometry, NF-κB protein expression was detected with Western blot. The results showed that HHT concentration-dependently inhibited proliferation of K562 cells, the IC50 at 48 h was 43.89 ng/ml. Treated with HHT 10 ng/ml for 48 h, K562 cell apoptosis significantly increased, cell cycle was blocked at G0/G1, the expression level of BCL-2 and NF-κB proteins was lower than that in control group (P < 0.05). It is concluded that HHT may inhibit the proliferation of K562 cells, and down-regulating expression levels of BCL-2 and NF-κB may be one of its anti-CML mechanisms.

Clinical investigation of homoharringtonine in combination with all-transretinoic acid and arsenic trioxide for acute promyelocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the clinical outcome, adverse effect and treatment cost of homoharringtonine (HHT) in combination with all-trans retinoic acid (ATRA) and arsenic trioxide (AS2O3) for newly diagnosed with patients acute promyelocytic leukemia (APL).</p><p><b>METHODS</b>Clinical data of treatment of newly diagnosed patients with APL in experimental group (HHT + ATRA + AS2O3, n = 14) and control group \[Idarubicin (IDA) + ATRA + AS2O3, n = 21\] were analyzed retrospectively. The therapeutic effects, side effects and costs during induction therapy were compared between the two groups.</p><p><b>RESULTS</b>(1) The complete remission (CR) rate were 92.9% (13/14) and 95.2% (20/21) in experimental group and control group, respectively. The time to achieve CR were (28.1 ± 3.8) and (31.7 ± 4.2) days, respectively (P > 0.05). The negative rate of PML-RARα fusion gene at the time of CR were 76.9% (10/13) and 75.0% (15/20), respectively, and that in CR patient at the end of the first cycle treatment were 100.0% (13/13) and 95.0% (19/20), respectively (P > 0.05). (2) 5-year overall survival (OS) rate were (92.6 ± 0.6)% and (89.9 ± 0.5)%, respectively (P > 0.05), 5-year disease free survival (DFS) rate were 100.0% and (86.8 ± 0.6)%, respectively (P > 0.05). (3) During induction therapy, the incidence of infection in experimental and control group were 23.1% (3/13), 60.0% (12/20), respectively (P < 0.05). The amount of platelet transfusion were (54.7 ± 29.6) and (76.5 ± 25.6) units, respectively (P > 0.05), and that of fresh frozen plasma were (1157.1 ± 238.4) and (1423.5 ± 324.6) ml, respectively (P > 0.05). The total medical costs (excluding HHT and IDA) in experimental and control group were (36074.9 ± 1245.6) and (50564.5 ± 3658.4)CNY, respectively (P < 0.05).</p><p><b>CONCLUSION</b>HHT in combination with ATRA and AS2O3 regimen for newly diagnosed APL has a better efficacy, a higher long-term survival rate, and a lower costs, which is one of the reasonable choice.</p>

Harringtonine induces apoptosis in NB4 cells through down-regulation of Mcl-1 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the growth inhibition effect, cytotoxicity and apoptotic induction of harringtonine (HT) in human acute promyelocytic leukemia (APL) NB4 cells,and the related mechanism.</p><p><b>METHODS</b>NB4 cells were treated with HT. Total cell numbers were counted by hemocytometer, and cell viabilities were determined by trypan blue exclusion. Apoptotic cells were determined by fluorescence microscopy and FACS after staining with AO and EB or PI, respectively. The cleavage of PARP and the activation of Bax and the expression of anti-apoptotic proteins were determined by Western Blot. siRNA was used to silence the expression of target genes. Primary cells were isolated following Ficoll-Hypaque density gradient centrifugation method.</p><p><b>RESULTS</b>HT inhibited cell growth and induced apoptosis of NB4 cells in a dose- and time-dependent manner. Apoptosis induced by HT was correlated with the down-regulation of Mcl-1 and the cleavage of PARP, while HT did not affect the protein level of Bax and Bak or change the protein level of Bcl-2. The silence of Bcl-XL sensitized HT-induced apoptosis in NB4 cells.Apoptosis induced by HT in primarily cultured APL cells was also correlated with the down-regulation of Mcl-1.</p><p><b>CONCLUSION</b>HT inhibits cell growth and induces apoptosis in NB4 cells and primarily cultured APL cells, which may be associated with down-regulation of Mcl-1.</p>

Establishment of human homoharringtonine-resistant SKM-1 cell line and its biological characteristics / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish a homoharringtonine (HHT)-resistant SKM-1 cell line and explore its biologic characteristics and mechanisms for drug resistance.</p><p><b>METHODS</b>The HHT-resistant SKM-1 cell line was established by repeatedly exposing the cells to comparatively large doses of HHT with a short-time duration, and gradually elevating the drug concentration to an endurable level. The morphology of the resistant and parental cell lines was observed through optical microscope. The MTT assay was used to determine the doubling time and the resistance index to draw growth curve. The immunophenotype, cell cycle distribution and DNR accumulation between SKM-1 and SKM-1/HHT were analyzed by flow cytometry, and the karyotypes by R-banding. Semi-quantitative real-time PCR was performed to evaluate the expression levels of mdr1, MRP and topo-IIa.</p><p><b>RESULTS</b>The HHT-resistant cell line SKM-1/HHT was eventually established following 7-month drug induction. Both the resistant and the parental cell lines were similar with regard to morphology and immunophenotype. The karyotypes of the former was more complicated with differences located in chromosome 20, X, 4, 5, 9 and 11. The resistant cell line had more G(1) phase cells (64.04% vs 41.91%), less S phase cells (34.92% vs 53.53%), and less G(2) phase cells (1.04% vs 4.56%) compared with the parental cell line. The SKM-1/HHT cell line showed significant drug resistance to HHT, VCR, DNR and etoposide, the resistance indices of HHT, VCR, DNR and etoposide were 17.94, 8.75, 5.99 and 13.76 respectively. DNR accumulation was impaired in SKM-1/HHT cell line as less fluorescence of DNR (698 ± 36 vs 858 ± 54). The expression of mdr1 increased dramatically in the resistant cell line, its 2(-ΔCt) value was 20.1 higher than that of the parental cell line \[(3.42 ± 0.46)×10(-2) vs (0.17 ± 0.01)×10(-2), P < 0.05\], while MRP also increased in the resistant by 3.56 folds \[(4.77 ± 0.87)×10(-3) vs (1.34 ± 0.56)×10(-3), P < 0.05\]; However there was a slightly decrease of topo-IIa, the ratio of the resistant to the parental calculated by their 2(-ΔCt) values was 0.619:1 \[(1.91 ± 0.30)×10(-4) vs (3.08 ± 0.21)×10(-4), P < 0.05\].</p><p><b>CONCLUSION</b>A HHT-resistant cell line SKM-1/HHT was established. The prominent overexpression of mdr1 may be the main cause for multidrug resistance.</p>

Effects of interferon-α combined with homoharringtonine on K562 cell proliferation and β-catenin expression / 中国实验血液学杂志

The study was aimed to investigate the synergistically effect of interferon-α (IFN-α) and homoharringtonine (HHT) on the proliferation, apoptosis, cell cycle of K562 cells and the expression of β-catenin. The proliferation, apoptosis, cell cycle and β-catenin mRNA expression of K562 cells treated with IFN-α and/or HHT were assayed with MTT, flow cytometry or RT-PCR respectively. The results showed that HHT alone, but not IFN-α alone, displayed a proliferation inhibition, apoptosis induction, G(0)/G(1) phase block and down-regulation of β-catenin expression in K562 cells with concentration- and time-dependent manners. The expression level of β-catenin mRNA after being treated with HHT was 0.5576 ± 0.0373, which were lower than that in control group (0.9369 ± 0.0142). The down-regulation of β-catenin expression in group of IFN-α combined with HHT was higher significantly than that in HHT group (0.3737 ± 0.0529 vs 0.5576 ± 0.0373, P < 0.05). Otherwise, HHT combined with IFN-α did not demonstrate obvious toxicologic effect on bone marrow mononuclear cells. It is concluded that IFN-α combined with HHT can enhance the cytotoxic effect of HHT on K562 cells, which may be associated with down-regulation of β-catenin expression.

Cytotoxicity of homoharringtonine on leukemic stem-like cells in AML cell line KG-1 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of homoharringtonine (HHT) on leukemic stem-like cells (LSC) in human acute myeloid leukemia (AML) cell lines.</p><p><b>METHODS</b>The phenotypes of AML cell lines U937,Kasumi-1,and KG-1 cells were analyzed by flow cytometry (FACS). The effect of HHT on leukemia stem-like cells with immunophenotype of CD34(+)CD38(-)CD96(+) was detected with FACS. Cell growth was measured by MTT assay. Activation of Caspase pathway and expression of apoptosis-related regulator proteins were examined by Western blotting.</p><p><b>RESULTS</b>FACS demonstrated that the 69% of KG-1 cells expressed LSC phenotype CD34(+)CD38(-)CD96(+), while 26.7% on Kasumi-1 cells expressed this marker. In contrast,U937 cells showed CD96 negative. HHT significantly inhibited cell growth of KG-1 cells with an IC(50) of 16.9 ng/ml at 48 h. The ratio of CD34(+)CD38(-)CD96(+) cells decreased from 63.6% to 17.1% after HHT treatment. Enhanced apoptosis was demonstrated in HHT group evidenced by strong activation of Caspase-9,Caspase-3 and PARP.HHT treatment resulted in down-regulation of expression of anti-apoptotic protein BCL-2 and phosphorylated-Akt.</p><p><b>CONCLUSION</b>HHT can effectively kill the leukemic stem-like cells in human AML cell line KG1 by inhibiting cell growth and inducing apoptosis which is associated with activation of Caspase pathway and down-regulation of anti-apoptotic proteins and phosphorylated-Akt.</p>

Effect of homoharringtonine combined with AG490 on JAK2-STAT5 associated signal pathway in HEL cells / 中国实验血液学杂志

This study was aimed to explore the effect of homoharringtonine in combination with AG490 on JAK2-STAT5 associated signal pathway in HEL cells, and analyze its mechanism so as to provide theoretical basis for therapy of chronic myeloproliferative neoplasma by new program. The cell survival rates were tested by MTT, apoptosis was tested by flow cytometry after HEL cells were treated by 20 ng/ml HHT, 100 µmol/L AG490 and 20 ng/ml HHT in combination with 100 µmol/L AG490, while the signal proteins such as P-JAK2, P-STAT5 and BCL-xL activated by abnormal activated JAK2 were tested by Western blot. The results showed that both HHT and AG490 could inhabit the HEL cell proliferation after being treated for 24 hours, and Annexin V-PI double staining confirmed early apoptosis while HHT effect was more obvious, Western blot showed that the expressions of P-JAK2 and P-STAT5 were down-regulated, while the total protein levels of JAK2 and STAT5 were stable. It is concluded that HHT combined with AG490 can obviously inhibit the proliferation and induce early apoptosis of HEL cells, and there is synergistic effect between the two drugs. HHT possibly acts as a broad-spectrum PTK inhibitor and synergistically with AG490 inhibits the phosphorylation of signal proteins caused by JAK2V617F, thus down-regulating the transcription of STAT5.

Clinical study on combination of homoharringtonine, Ara-C and idarubicin induction for treatment of newly diagnosed acute myeloid leukemia patients / 中国实验血液学杂志

The purpose of this study was to assess the efficacy and toxicity of HAI regimen [(homoharringtonine 2.5 mg/(m(2)×d), days 1 - 7; cytarabine 150 mg/(m(2)×d), days 1 - 7; idarubicin 9 mg/(m(2)×d), days 1 - 7)] for induction treatment of newly diagnosed acute myeloid leukemia (AML) (except acute promyelocytic leukemia). 31 patients with newly diagnosed AML, aged 39 (14 - 58) years, were enrolled in this clinical study. The complete remission (CR) rate, especially after one course, the overall survival (OS) rate and relapse free survival (RFS) rate were estimated. The outcomes were compared between different prognostic groups according to World Health Organization (WHO) classification, genetics and initial WBC count. Safety was evaluated using standard WHO criteria. The results showed that 26 patients (84%) achieved CR after 1 course of induction. The CR rate for the patients with favorable, intermediate and unfavorable cytogenetics was 90%, 88% and 60% respectively. All 7 patients with a high initial WBC count (≥ 100×10(9)/L) obtained CR, while 19 out of 24 without a high initial WBC count obtained CR. With a median follow-up of 15(range 2-56) months, the estimated 3-year OS rate for all patients and the patients with CR was 44% and 52% respectively. The 3-year RFS rate was 51%. The patients receiving induction chemotherapy died of the chemotherapy. Profound myelosuppression was seen in all patients after the HAI induction with the median duration of neutropenia (ANC < 0.2×10(9)/L) of 16 (6 - 24) days. As the most common toxicity, severe infections (grade III-IV) involved in all the patients and the duration of febris was 6 (1 - 36) days. The incidence of septemia and invasive fungus infection were 19.4% and 45.2% respectively. The incidence of non-infection fever, increased glutamic-pyruvic transaminase (GPT), diarrhea, increased bilirubin and oral cavity mucositis were 6.5%, 6.5%, 3.2%, 3.2%, 3.2% respectively, as the more frequent severe non-hematological toxicities. It is concluded that HAI regimen is a high efficient induction schedule for the newly diagnosed AML, and archive the higher CR rate after one course than DNR/Ara-C standard induction regimen. Side effects are acceptable, except severe infection.

Outcome of acute promyelocytic leukemia with homoharringtonine (HHT) and ATRA / 中华血液学杂志

<p><b>OBJECTIVE</b>To assess complete remission (CR), the overall survival (OS), event-free survival (EFS) and adverse events of newly diagnosed acute promyelocytic leukemia (APL) with homoharringtonine (HHT) plus ATRA, to evaluate the therapeutic effect by comparing HHT plus ATRA with daunorubicin plus ATRA as induction regimen (HA with DA as post-remission regimen).</p><p><b>METHODS</b>115 APL patients (54 in HHT group, 61 in DNR group) after long-term follow-up were enrolled in the analyses of clinical feature, chromosome karyotype, molecular biology, OS and EFS.</p><p><b>RESULTS</b>The overall CR of 115 patients was 100%, the median interval to achieve hematological CR was 32 (22 - 43) days, the overall median OS was within 0.23 - 77.34 months, median EFS was within 0.23 - 77.34 months. 3-year OS rate was 93%, 5-year OS rate 93%, 3-year EFS rate 85% and 5-year RFS rate 75% respectively. Converting to PML-RARα PCR-negative after the induction therapy in the HHT and DNR group was 31.3% and 15.5% respectively, at the end of 1 consolidation course was 68.6% and 77.6% respectively, while the remaining 4 patients tested PML-RARα PCR-negative at the end of 2 consolidation courses in the DNR group. While both groups obtained the identical molecular biology relapse rate (9.8% and 8.6%, respectively). Survival analysis indicated that no significant difference was found on OS and EFS between the HHT group and the DNR group (P = 0.206 and 0.506). 5-year OS rate was 87% for the HHT group while 98% for the DNR group, 5-years EFS rate was 80% for the HHT group while 71% for the DNR group. And the risk group was not the factor affecting OS and EFS (P = 0.615 and 0.416). Grade 2 fever in the HHT group was less than in the DNR group during induction therapy. And no difference was found in terms of liver dysfunction, renal dysfunction, cardiac dysfunction, and hematologic toxicity between two groups.</p><p><b>CONCLUSION</b>Our study demonstrated comparable therapeutic effect of HHT or DNR on APL. HHT was also well tolerated and didn't cause serious adverse events.</p>

Efficiency of GHA priming therapy on patients with acute monocytic leukemia and its mechanism / 中国实验血液学杂志

The aim of this study was to explore the clinical efficiency and side effects of GHA-priming therapy on patients with acute monocytic leukemia, and to analyze its mechanism. 37 patients with refractory, relapse, hypocellular acute monocytic leukemia and elderly patients with AML-M(5) were treated with GHA-priming therapy (G-CSF, homoharringtonine and low dosage of cytarabine). Clinical efficiency, side effects, and therapy-relevant mortality were observed. By using U937 cell line as in vitro model, effect of G-CSF on cell cycle was determined by propidium iodide staining method. The inhibition rate, apoptosis rate of U937 cell line treated with various combination of G-CSF, homoharringtonine and cytarabine were detected by flow cytometry. The expression of MLAA34 on U937 before or after treating with chemotherapy was analyzed by immunohistochemical method. The results showed that in all the 37 patients, the total remission rate was 62.2% [complete remission rate was 45.95% (17/37) and partial remission rate was 16.2% (6/37)]. The incidence of granulocyte deficiency was 18.92% (2/37) with median time of 4 days. The severe infection occurred in 2 cases. No severe bleeding, no mild digestive effect occurred. Other non-hematological toxicities were low in vitro when incubated with G-CSF for 24 hours, the S-phase cells obviously increased. The inhibition rate, apoptosis rate and expression of MLAA34 of U937 cells treated by GHA significantly decreased as compared with cells treated with HA. It is concluded that the GHA priming therapy can be used to treat patients with refractory, relapse, senile and hypocellular acute monocytic leukemia with satisfied response rate and low hematological and non-hematological toxicities. G-CSF can enhance cytotoxicity of drugs such as Ara-C and HHT by promoting G(0) phase cells into the reproductive cycle. GHA and HA therapy can inhibit cell proliferation, induce apoptosis, and the former has a more significant function. GHA priming therapy can down regulate the expression of MLAA 34. MLAA-34 is a novel anti-apoptotic factor of acute monocytic leukemia.

Efficacy of induction chemotherapy for patients with high-risk myelodysplastic syndrome (MDS) or MDS-transformed acute myeloid leukemia with CHG regimen and its comparison with regimen GAG and HA / 中国实验血液学杂志

This study was aimed to investigate the efficacy of moderate intensity regimen, CHG (homoharringtonine, cytarabine and granulocyte colony-stimulating factor (G-CSF)) on the patients with high-risk MDS or MDS-transformed acute myeloid leukemia. 30 newly diagnosed adult patients with high-risk MDS or MDS-transformed AML were enrolled in this clinical trial to evaluate the efficacy of sequential low-dose homoharringtonine/cytarabine chemotherapy combined with G-CSF priming. Homoharringtonine and Ara-C were injected intravenously at doses of 1 mg and 25 mg daily for 14 consecutive days respectively, G-CSF was injected subcutaneously once daily at a dose of 300 microg on 12 hours prior to chemotherapy and continued given until the end of chemotherapy or when the peripheral WBC count reached > 20 x 10(9)/L. This regimen was given only for one course, and followed by conventional chemotherapy as maintenance or consolidation therapy when CR achieved. 33 patients with high- risk MDS and MDS-transformed AML were injected aclarubicin/Ara-C intravenously at doses of 10 mg and 25 mg for 8 and 14 consecutive days respectively, G-CSF was used at the same dose and the same way as the CHG regimen. 33 patients with high-risk MDS and MDS-transformed AML were treated with HHT/Ara-C intravenously at doses of 2 - 3 mg and 100 - 150 mg daily for 7 consecutive days respectively, G-CSF was injected when WBC count was below 4 x 10(9)/L, and it was stopped to be used when WBC count was > 4 x 10(9)/L. The results showed that (1) 14 patients achieved complete remission (CR) (46.67 %) and 7 patients achieved partial remission (PR) (23.33 %) with one course of CHG regimen, total effective rate was 70%; 14 patient achieved CR (42.4%) and 9 patients achieved PR (27.3%) with one course of CAG regimen, total effective rate was 69.7%; 7 patient achieved CR (33.3%) and 3 patients achieved PR (9.1%) with one course of HA regimen, total effective rate was 42.4%. There was no statistical difference between the effective rate of CHG and CAG, but difference was significant between CHG and HA. (2) Agranulocytosis (neutrophil < 0.5 x 10(9)/L) occurred in 12 cases (40%) of CHG-treated patients with a mean 8 days of agranulocytic period, so the infectious complications were less serious and tolerable without treatment-related death. (3) Among 14 out of 30 patients with CR, 9 relapsed, the mean duration from CR to replace was 8.2 months. All relapsed patients reusing CHG regimen did not achieved CR again. (4) Among 13 cases with CR, 6 patients just received HA or DA regimens as consolidatory and intensive chemotherapy after CR have relapsed, the mean CR time was only 6.1 months. Otherwise, the mean CR time of 7 CR patients received alternative succeeded chemotherapy containing mitoxantrone/idarubicin/THP/homoharringtonine/daunorubicin/aclarubicin after CR was 10.6 months; and among them 4 are still in continuous CR. It is concluded that the CHG chemotherapy regimen has a similar effect with CAG but better than HA, and in a saft chemotherapy regimen with slight myelosuppression in clinical application, strong and alternative succeeded chemotherapy is necessary for CR patients to keep longer CR and survival.