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This study summarizes the applications of monoclonal antibodies (MAbs) against bioactive natural compounds for the functional analysis of crude drugs. To date, we have established more than 40 kinds of MAbs against natural compounds and set up enzyme-linked immunosorbent assay (ELISA). The intracellular accumulation of harringtonine (HT) and its derivatives into cells was determined by ELISA using anti-HT MAb, and the uptake levels of these compounds conformed to their respective antiproliferative activities. Eastern blotting is an on-membrane quantitative analytical system that uses MAbs against natural compounds and it can also be used in a biological sample for pharmacokinetics study. Glycyrrhizin (GC) was detected in the serum of rats treated with GC by eastern blotting. Additionally, eastern blotting using MAb against 3-monoglucuronyl-glycyrrhetinic acid (3-MGA) could determine a potential causative agent of pseudoaldosteronism induced by excess intake of GC. The immunoaffinity column coupled with MAb made preparing an extract containing all components feasible except the target compound. Cell-based studies using GC-removed licorice extract, which was prepared by the immunoaffinity column conjugated with anti-GC MAb, revealed the potential functions of GC in licorice extract. Moreover, MAbs were used in determining cellular localization and target molecules of natural compounds. The combination of analysis using MAb and LC-MS/MS showed that aristolochic acid I (AA-I), a naturally occurring nephrotoxin and carcinogen, targets α-actinin-4 in the kidney cells. MAbs against natural compounds are useful tools to determine real pharmacological functions, cellular localization, and molecular targets of natural compounds in crude extracts.

Cancer is identified by the abnormal accumulation of rapidly dividing cells, serving as the collective term for a multitude of similar conditions categorized through cell growth and proliferation. The disease continues to lead death tolls worldwide and extends to communities across the board in terms of socioeconomic status.(^1) From 1991 to 2017, experts recorded a slight drop in cancer death rates, which can be accredited to an influx of scientific advancements within the past decade that allowed for the implementation of targeted therapy.(^1) In preclinical research, this therapy implies the conjugation of independent drugs retaining anti-carcinogenic properties. The current state of cancer pathology is observed to be non-specific and is, oftentimes, taxing on the patient, proving the need for localized treatment and more efficient drugs. This article analyzes the effectiveness of one compound under the harringtonine alkaloid family, cephalotaxine (CET).(^27) In this work, our team examines the biochemical profile of CET and congener homoharringtonine to hypothesize cephalotaxine’s efficacy as an alternative to invasive cancer therapy.

Herpes simplex virus type 1 (HSV-1) infection induces various clinical disorders, such as herpes simplex encephalitis (HSE), herpes simplex keratitis (HSK), and genital herpes. In clinical intervention, acyclovir (ACV) is the major therapeutic drug used to suppress HSV-1; however, ACV-resistant strains have gradually increased. In the present study, harringtonine (HT) significantly inhibited infection of HSV-1 as well as two ACV-resistant strains, including HSV-1 blue and HSV-1 153. Time-of-drug addition assay further revealed that HT mainly reduced the early stage of HSV-1 infection. We also demonstrated that HT mainly affected herpes virus entry mediator (HVEM) expression as shown by qPCR, Western Blot, and Immunofluorescence. Collectively, HT showed antiviral activity against HSV-1 and ACV-resistant strains by targeting HVEM and could be a promising therapeutic candidate for mitigating HSV-1-induced-pathogenesis.

In the search for treatment schemes of COVID-19, we start by examining the general weakness of coronaviruses and then identify approved drugs attacking that weakness. The approach, if successful, should identify drugs with a specific mechanism that is at least as effective as the best drugs proposed and are ready for clinical trials. All coronaviruses translate their non-structural proteins (∼16) in concatenation, resulting in a very large super-protein. Homo-harringtonine (HHT), which has been approved for the treatment of leukemia, blocks protein elongation very effectively. Hence, HHT can repress the replication of many coronaviruses at the nano-molar concentration. In two mouse models, HHT clears SARS-CoV-2 in 3 days, especially by nasal dripping of 40 ug per day. We also use dogs to confirm the safety of HHT delivered by nebulization. The nebulization scheme could be ready for large-scale applications at the onset of the next epidemics. For the current COVID-19, a clinical trial has been approved by the Ditan hospital of Beijing but could not be implemented for want of patients. The protocol is available to qualified medical facilities.

Mosquito-borne Zika virus (ZIKV) is a Flavivirus that came under intense study from 2014 to 2016 for its well-known ability to cause congenital microcephaly in fetuses and neurological Guillain&ndash, Barr&eacute, disease in adults. Substantial research on screening antiviral agents against ZIKV and preventing ZIKV infection are globally underway, but Food and Drug Administration (FDA)-approved treatments are not available yet. Compounds from Chinese medicinal herbs may offer an opportunity for potential therapies for anti-ZIKV infection. In this study, we evaluated the antiviral efficacy of harringtonine against ZIKV. Harringtonine possessed anti-ZIKV properties against the binding, entry, replication, and release stage through the virus life cycle. In addition, harringtonine have strong virucidal effects in ZIKV and exhibited prophylaxis antiviral ability prior ZIKV infection. The antiviral activity also observed in the treatment against Japanese encephalitis reporter virus (RP9-GFP strain). Overall, this study demonstrated that harringtonine would be a favorable potential candidate for the development of anti-ZIKV infection therapies.

Translation of problematic mRNA sequences induces ribosome stalling. Collided ribosomes at the stall site are recognized by cellular quality control machinery, resulting in dissociation of the ribosome from the mRNA and subsequent degradation of the nascent polypeptide and in some organisms, decay of the mRNA. However, the timing and regulation of these processes are unclear. We developed a SunTag-based reporter to monitor translation in real time on single mRNAs harboring difficult-to-translate poly(A) stretches. This reporter recapitulates previous findings in human cells that an internal poly(A) stretch reduces protein output ∼10-fold, while mRNA levels are relatively unaffected. Long-term imaging of translation indicates that poly(A)-containing mRNAs are robustly translated in the absence of detectable mRNA cleavage. However, quantification of ribosome density reveals a ∼3-fold increase in the number of ribosomes on poly(A)-containing mRNAs compared to a control mRNA, consistent with queues of many stalled ribosomes. Using single-molecule harringtonine runoff experiments, we observe the resolution of these queues in real-time by the cellular quality control machinery, and find that rescue is very slow compared to both elongation and termination. We propose that the very slow clearance of stalled ribosomes provides the basis for the cell to distinguish between transient and deleterious stalls, and that the human quality control apparatus predominantly targets the nascent protein rather than the mRNA.

Harringtonine (HT) and homoharringtonine (HHT), alkaloid esters isolated from the genus Cephalotaxus, exhibit antitumor activity. A semisynthetic HHT has been approved for treatment of chronic myelogenous leukemia. In addition to antileukemic activity, HT and HHT are reported to possess potent antiviral activity. In this study, we investigated the effects of HT and HHT on replication of varicella-zoster virus (VZV) in vitro. HT and HHT, but not their biologically inactive parental alkaloid cephalotaxine (CET), significantly inhibited replication of recombinant VZV-pOka luciferase. Furthermore, HT and HHT, but not CET, strongly induced down-regulation of VZV lytic genes and exerted potent antiviral effects against a VZV clinical isolate. The collective data support the utility of HT and HHT as effective antiviral candidates for treatment of VZV-associated diseases.

Babesia gibsoni is a tick-transmitted intraerythrocytic apicomplexan parasite that causes babesiosis in dogs. Due to the strong side effects and lack of efficacy of current drugs, novel drugs against B. gibsoni are urgently needed. Natural products as a source for new drugs is a good choice for screening drugs against B. gibsoni. The current study focuses on identifying novel potential drugs from natural products against B. gibsoni in vitro. Parasite inhibition was verified using a SYBR green I-based fluorescence assay. A total of 502 natural product compounds were screened for anti-B. gibsoni activity in vitro. Twenty-four compounds showed high growth inhibition (>80%) on B. gibsoni and 5 plant-derived compounds were selected for further study. The half-maximal inhibitory concentration (IC50) values of lycorine (LY), vincristine sulfate (VS), emetine·2HCl (EME), harringtonine (HT) and cephaeline·HBr (CEP) were 784.4 ± 3.3, 643.0 ± 2.8, 253.1 ± 1.4, 23.4 ± 1.2, and 108.1 ± 4.3 nM, respectively. The Madin-Darby canine kidney (MDCK) cell line was used to assess cytotoxicity of hit compounds. All compounds showed minimal toxicity to the MDCK cells. The effects of hit compounds combined with diminazene aceturate (DA) on B. gibsoni were further evaluated in vitro. VS, EME, HT or CEP combined with DA showed synergistic effects against B. gibsoni, whereas LY combined with DA showed an antagonistic effect against B. gibsoni. The results obtained in this study indicate that LY, VS, EME, HT and CEP are promising compounds for B. gibsoni treatment.

Harringtonine (HT) is a naturally occurring alkaloid isolated from the plant genus Cephalotaxus. It possesses antileukemic activity and has been clinically utilized for the treatment of acute leukemia and lymphoma. Sodium periodate (NaIO4) was reacted with HT to produce five HT derivatives including four novel compounds. Their antiproliferative activity against HL-60 acute promyelocytic leukemia cells revealed that the presence of the C-5′ methyl group enhances the antiproliferative activity because the IC50 values of the HT derivatives, including HT1 (5′-de-O-methylharringtonine), were at least 2000 times higher (>100 μM) than that of HT (∼47 nM). In addition, an indirect competitive enzyme-linked immunosorbent assay (icELISA) using a monoclonal antibody against HT (mAb 1D2) revealed that these antiproliferative activities were related to their cellular uptake. These results indicated that esterification of HT1 at the C-4′ carboxylic acid group may enhance the antiproliferative activity of HT.

Harringtonine (HT) is a natural compound, which is mainly produced by the genus Cephalotaxus, and has been clinically utilized in China for the treatment of acute leukemia and lymphoma. However, the amounts of HT in the Cephalotaxus species are very small; therefore, plant tissue cultures have been focused upon to enhance HT production. Qualitative/quantitative methods for HT detection are required to screen superior cell lines. We developed a one-step indirect competitive immunochromatographic assay (ICA) using colloidal gold nanoparticles conjugated with highly specific monoclonal antibodies against HT (MAb 1D2) for simple, rapid, and sensitive detection of HT in plant samples. This ICA can be completed in 15min after dipping the strip into analytes with a limit of detection of ∼313ng/mL. In developed ICA, fiber pad which is usually used for conventional ICA, was not used to shorten the time for preparing chromatographic strip, resulting in a decrease in the volume of valuable analytes (20μL). Considering simplicity, rapidity, and sensitivity of the developed ICA, this study could be applied to a fieldwork study for finding new natural resources containing HT.

Harringtonine (HT) is a promising natural product that is mainly isolated from plants of the genus Cephalotaxus. Due to its remarkable antileukemic activities, HT has been utilized clinically in China for the treatment of acute promyelocytic leukemia (APL). No antibody that recognizes free HT has been reported to date due to the difficulty of preparing antigen conjugates in which haptens bind to a carrier protein. To overcome this difficulty, we focused on sodium periodate (NaIO4), which catalyzes unique oxidative reactions; the resulting conjugates enabled the production of a highly specific monoclonal antibody (MAb) against HT (MAb 1D2) and the establishment of an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the determination of HT. Further analysis revealed that MAb 1D2 was produced by the HT3 (8-carbonyl HT)-based conjugate antigen; HT3 was synthesized by a NaIO4-mediated oxidative reaction. The minimum detectable concentration for HT in the icELISA system was found to be 0.76 ng mL−1, which is approximately 13 to 160 times more sensitive than a conventional HPLC system. Several validation analyses revealed that the icELISA using MAb 1D2 is sufficiently accurate, reliable, and sensitive to assess small amounts of HT in plant samples.

A series of homoerythrina alkaloid derivatives containing a 1,2,3-triazole moiety as PARP-1 inhibitors were designed and synthesized. And their anti-proliferative activity was further evaluated. Compound 10n had excellent activity to inhibit proliferation of A549 cells (IC50 = 1.89 μM), which was higher than harringtonine (IC50 = 10.55 μM), pemetrexed (IC50 = 3.39 μM), and rucaparib (IC50 = 4.91 μM). Furthermore, the selectivity index of compound 10n was higher than rucaparib and pemetrexed for lung cancer cells. Flow cytometry analysis showed that compound 10n significantly arrested the cell cycle in the S phase, then induced apoptosis of A549 cells (apoptosis rate is 46%), which effectively inhibited cell proliferation. Simultaneously, western blot analysis revealed that compound 10n could prevent the biosynthesis of PAR. Further analysis results revealed that compound 10n could inhibit the expression of cyclin A, down-regulate the expression of bcl-2/bax, activate caspase-3, and ultimately induce apoptosis of A549 cells. All the results indicated that compound 10n had potential research value as a novel PARP-1 inhibitor in antitumor, and it provided a new reference for further development of PARP-1 inhibitors.

The transmission of mosquito-borne Chikungunya virus (CHIKV) has large epidemics worldwide. Till date, there are neither anti-viral drugs nor vaccines available for the treatment of Chikungunya. Accumulated evidences suggest that some natural compounds i.e., Epigallocatechin gallate, Harringtonine, Apigenin, Chrysin, Silybin, etc. have the capability to inhibit CHIKV replication in vitro. Natural compounds are known to possess less or no side effects. Therefore, natural compound in its purified or crude extracts form could be the preeminent and safe mode of therapies for Chikungunya. Wet lab screening and identification of natural compounds against Chikungunya targets is a time consuming and expensive exercise. In the present study, we used in silico techniques like receptor-ligand docking, Molecular dynamic (MD), Three Dimensional Quantitative Structure Activity Relation (3D-QSAR) and ADME properties to screen out potential compounds. Aim of the study is to identify potential lead/s from natural sources using in silico techniques that can be developed as a drug like molecule against Chikungunya infection and replication. Three softwares were used for molecular docking studies. Potential ligands selected by docking studies were subsequently subjected 3D-QSAR studies to predict biological activity. Based on docking scores and pIC50 value, potential anti-Chikungunya compounds were identified. Best docked receptor-ligands were also subjected to MD for more accurate estimation. Lipinski’s rule and ADME studies of the identified compounds were also studied to assess their drug likeness properties. Results of in silico findings, led to identification of few best fit compounds of natural origin against targets of Chikungunya virus which may lead to discovery of new drugs for Chikungunya. Communicated by Ramaswamy H. Sarma

Harringtonine (HT) and homoharringtonine (HHT) are Cephalotaxus alkaloids with considerable antileukaemic activity. The objectives of this research were to (1) determine the content of HT and HHT present in Cephalotaxus griffithii needles alkaloid fraction (CGAF) and (2) compare the antiproliferative activity of CGAF, with that of HT and HHT on chronic myelogenous leukaemia K562 cell. The concentration of HT and HHT was found to be 122.14 and 16.79 mg/g of CGAF, respectively. Treatment of K562 cells with CGAF, HT and HHT decreased the viable cells in a dose- and time-dependent manner. Interestingly, the maximum cell death was found in CGAF, with IC50 value which was 3- to 4.6-fold lower than those of HT and HHT. Our results indicate that HT content in the needles of C. griffithii is higher than HHT, and alkaloids other than HT and HHT in CGAF are predominantly responsible for K562 cell death.

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic ligand from the TNF-alpha family that is under consideration, along with agonistic anti-TRAIL receptor antibodies, as a potential anti-tumor agent. However, most primary human tumors are resistant to monotherapy with TRAIL apoptogens, and thus the potential applicability of TRAIL in anti-tumor therapy ultimately depends on its rational combination with drugs targeting these resistances. In our high-throughput screening for novel agents/drugs that could sensitize TRAIL-resistant colorectal cancer cells to TRAIL-induced apoptosis, we found homoharringtonine (HHT), a cephalotaxus alkaloid and tested anti-leukemia drug, to be a very effective, low nanomolar enhancer of TRAIL-mediated apoptosis/growth suppression of these resistant cells. Co-treatment of TRAIL-resistant RKO or HT-29 cells with HHT and TRAIL led to the effective induction of apoptosis and the complete elimination of the treated cells. HHT suppressed the expression of the anti-apoptotic proteins Mcl-1 and cFLIP and enhanced the TRAIL-triggered activation of JNK and p38 kinases. The shRNA-mediated down-regulation of cFLIP or Mcl-1 in HT-29 or RKO cells variably enhanced their TRAIL-induced apoptosis but it did not markedly sensitize them to TRAIL-mediated growth suppression. However, with the notable exception of RKO/sh cFLIP cells, the downregulation of cFLIP or Mcl-1 significantly lowered the effective concentration of HHT in HHT + TRAIL co-treatment. Combined HHT + TRAIL therapy also led to the strong suppression of HT-29 tumors implanted into immunodeficient mice. Thus, HHT represents a very efficient enhancer of TRAIL-induced apoptosis with potential application in TRAIL-based, anti-cancer combination therapy.

Chikungunya virus (CHIKV) is a mosquito-transmitted virus that has reemerged as a significant public health threat in the last decade. Since the 2005-2006 chikungunya fever epidemic in the Indian Ocean island of La Réunion, millions of people in more than 40 countries have been infected. Despite this, there is currently no antiviral treatment for chikungunya infection. In this study, an immunofluorescence-based screening platform was developed to identify potential inhibitors of CHIKV infection. A primary screen was performed using a highly purified natural product compound library, and 44 compounds exhibiting ≥70% inhibition of CHIKV infection were identified as positive hits. Among these, four were selected for dose-dependent inhibition assays to confirm their anti-CHIKV activity. Harringtonine, a cephalotaxine alkaloid, displayed potent inhibition of CHIKV infection (50% effective concentration [EC 50 ] = 0.24 μM) with minimal cytotoxicity and was selected for elucidation of its antiviral mechanism. Time-of-addition studies, cotreatment assays, and direct transfection of viral genomic RNA indicated that harringtonine inhibited an early stage of the CHIKV replication cycle which occurred after viral entry into cells. In addition, quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses indicated that harringtonine affects CHIKV RNA production as well as viral protein expression. Treatment of harringtonine against Sindbis virus, a related alphavirus, suggested that harringtonine could inhibit other alphaviruses. This study suggests for the first time that harringtonine exerts its antiviral effects by inhibiting CHIKV viral protein synthesis.

Objective To investigate and compare the biological characteristics and sensitivity to chemotherapy and radiotherapy of intrahepatic and extrahepatic cholangiocarcinoma cells in vitro. Methods The intrahepatic and extrahepatic cholangiocarcinoma cell lines were established, and cells with steady passage were chosen to study the biological characteristics including morphology, growth dynamics, chromosome, and levels of cancer antigen (CA) 125, CA 19–9, alpha-fetoprotein (AFP), and carcino-embryonic antigen (CEA). Meanwhile, MTT assay was used to determine the sensitivity of both kinds of cells to 6 chemotherapeutic drugs, including cisplatin, paclitaxel, harringtonine, 5-fluorouracil, vincristine, and aclacimomycin, and the inhibitory rate of cells under the irradiation of 10 Gy ray was also measured. Results The intrahepatic cholangiocarcinoma cells were mostly fusiform in shape, and extrahepatic cholangiocarcinoma cells were mostly round or polygon in shape. Their doubling time was 26.3 hours and 23.1 hours, respectively. Their average number of chromosomes was 59 (range, 38–84) and 67 (range, 49–103), respectively. The chromosome karyotypes of most intrahepatic cholangiocarcinoma cells were hyperdiploid and hypotriploid, while hypertriploid was predominant in extrahepatic cholangiocarcinoma cells. The level of CA 125 in supernatant of extrahepatic cholangiocarcinoma cells increased obviously, while levels of other determined tumor markers in both kinds of cells were all within normal range. The intrahepatic cholangiocarcinoma cells were low sensitive to cisplatin and paclitaxel, but not sensitive to the other 4 chemotherapeutic drugs. The extrahepatic cholangiocarcinoma cells were high sensitive to cisplatin, but not sensitive to the other 5 drugs. Both kinds of cells had poor sensitivity to radiotherapy. Conclusion Intrahepatic and extrahepatic cholangiocarcinoma cells show differences in shape, doubling time, chromosome karyotype, tumor marker level, and chemosensitivity, whereas they both have poor radiosensitivity. Though they are similar in histopathology, they have different growth characteristics and have discrepancy in treatment and prognosis.

OBJECTIVE The anti-tumor drug, harringtonine (HT), has been extensively used with satisfactory results in the treatment of acute or chronic myeloid leukemia. Previous studies have shown that the anti-tumor activity of the drug is related to induced apoptosis of tumor cells, but the molecular mechanism still remains unclear. The main purpose of this research was to analyze the protein profiles formed during HT-induced apoptosis in K562 cells and to screen the apoptotic-related proteins. METHODS Annexin V and PI double staining was used in combination with flow cytometry to examine the early and the late stages of HT-induced apoptosis in K562 cells. In addition two-dimensional gel electrophoresis and computer-assisted image analysis were employed to separate and compare the HT-induced apoptotic proteins of the K562 cells and the controls. RESULTS When a concentration of 10 µg/ml HT was used to treat K562 cells, the percentage of the early-apoptotic cells (Annexin V+/PI-) was found to be 28.3% and 18.1% at 5 and 24 h, respectively (P

Dysregulated activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by self-DNA contributes to interferonopathy and promotes autoimmune diseases. To identify potential suppressors of STING-induced type I interferon (IFN) induction, ethanol extracts of medicinal plants were screened for inhibitory activity against IFN-ß promoter activation. Notably, 70% ethanol extract of Cephalotaxus koreana specifically down-regulated STING-induced, but not TBK1- or IRF3-induced, IFN-ß promoter activity. The compounds exerting inhibitory activity specifically against STING-mediated IFN-ß promoter activation were identified as ester alkaloids isolated from the genus, Cephalotaxus, homoharringtonine and harringtonine. Furthermore, these two compounds inhibited 2'3'-cGAMP-induced IFN-stimulated gene expression and interaction between STING and TBK1. These suppressive effects were not observed with cephalotaxine devoid of the ester side-chain. Our data support the potential utility of homoharringtonine and harringtonine to treat STING-associated interferonopathy and autoimmune diseases.

Most dinoflagellates in culture are bacterized, complicating the quantification of protein synthesis, as well as the analysis of its regulation. In bacterized cultures of Amphidinium carterae Hulbert, up to 80% of protein synthetic activity appears to be predominantly bacterial based on responses to inhibitors of protein synthesis. To circumvent this, axenic cultures of A. carterae were obtained and shown to respond to inhibitors of protein synthesis in a manner characteristic of eukaryotes. However, these responses changed with time in culture correlating with the reappearance of bacteria. Here we show that culture with kanamycin (50 μg/mL), carbenicillin (100 μg/mL), and streptomycin sulfate (50 μg/mL) (KCS), but not 100 units/mL of penicillin and streptomycin (PS), prevents the reappearance of bacteria and allows A. carterae protein synthesis to be quantified without the contribution of an associated bacterial community. We demonstrate that A. carterae can grow in the absence of a bacterial community. Furthermore, maintenance in KCS does not inhibit the growth of A. carterae cultures but slightly extends the growth phase and allows accumulation to somewhat higher saturation densities. We also show that cultures of A. carterae maintained in KCS respond to the eukaryotic protein synthesis inhibitors cycloheximide, emetine, and harringtonine. Establishment of these culture conditions will facilitate our ability to use polysome fractionation and ribosome profiling to study mRNA recruitment. Furthermore, this study shows that a simple and fast appraisal of the presence of a bacterial community in A. carterae cultures can be made by comparing responses to cycloheximide and chloramphenicol rather than depending on lengthier culture-based assessments.

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb Cephalotaxus hainanensis Li, has been used in the clinical treatment of human glanulocytic leukemia and chromic myelocytic leukemia. In this study, we investigated the effect of Bcl-2 on HT-induced apoptosis and Ca2+ mobilization in human leukemia HL-60 cells. 1 μg/ml HT induced the apoptosis of HL-60/Neo cells in a time-dependent manner; while 1 μg/ml HT failed to induce the apoptosis of HL-60/ Bcl-2 cells. HT-, A23187- (a Ca2+ ionophore), Carbonyl cyanide m-chlorophenylhydrazone (CCCP,a specific releaser of Ca2+ from mitochondria) and thapsigargin- (an inhibitor of endoplasmic reticulum Ca2+> -ATPase) induced changes in [Ca2+]i were monitored by using Fluo 3-AM with confocal laser scanning microscopy. The results demonstrated that HL-60 cells with enforced expression of Bcl-2 (HL-60/Bcl-2 cells) had increased Ca2+ permeability and increased intracellular Ca2+ store in comparison with HL-60 cells with negative control vectors (HL-60/Neo cells), suggesting that Bcl-2 might prevent HT-induced apoptosis by increased Ca2+ permeability and increased intracellular Ca2+ buffering capacity.

Traditional Chinese medicines have attracted great interest in recent researchers as alternative antineoplastic therapies. This review focuses on analytical approaches to various aspects of the antineoplastic ingredients of traditional Chinese medicines. Emphasis will be put on the processes of biological sample extraction, separation, clean-up steps and the detection. The problems of the extraction solvent selection and different types of column chromatography are also discussed. The instruments considered are gas chromatography, capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) connected with various detectors (ultraviolet, fluorescence, electrochemistry, mass, etc.). In addition, determinations of antineoplastic herbal ingredients, including camptothecin, taxol (paclitaxel), vinblastine. vincristine, podophyllotoxin, colchicine, and their related compounds, such as irinotecan, SN-38, topotecan, 9-aminocamptothecin, docetaxel (taxotere) and etoposide, are briefly summarized. These drugs are structurally based on the herbal ingredients, and some of them are in trials for clinical use. Evaluation of potential antineoplastic herbal ingredients, such as harringtonine, berberine, emodin, genistein, berbamine, daphnoretin, and irisquinone, are currently investigated in laboratories. Other folk medicines are excluded from this paper because their antineoplastic ingredients are unknown.

Purpose: Proliferative vitreoretinopathy (PVR) is characterizedby the formation of cellular membranes on the detached retina and also in the vitreous. Glial cellscan be found in epiretinal and subretinal membranes from eyes with PVR, proliferative diabeticretinopathy (PDR), idiopathic macular pucker, uveitis and other diseases affecting theretina. Proliferation and contraction of glial cells appears to play a role in the pathogenesisof PVR. This study is designed to inspect the effectiveness of harringtonine, as well as colchicine,daunomycin and fluorouracil, against cellular proliferation of cultured human retinal glial cellsthat might be involved in the retinal and/or vitreous proliferation. Methods: Cultures of human retinal glial cells were preparedusing the enzyme digesting method. Cells that had been in culture for 2–5 passages were usedin this study. Harringtonine (0.063 μg/ml ∼ 2.0 μg/ml), colchicines(0.5 μg/ml ∼ 16.0 μg/ml), daunomycin (0.1 μg/ml ∼ 3.2 μg/ml) and 5-fluorouracil(0.5 μg/ml ∼ 16.0 μg/ml) were added to cultures of human retinal glial cellsand the proliferation rates of the cells were measured by the MTT method. Results: Harringtonine at the dosage of 0.063 μg/mlinduced suppression of cellular growth, but the changes were not statistically significant (p > 0.05).At a dosage ranging from 0.125 μg/ml to 2.0 μg/ml, harringtonine significantly suppressedcellular growth according to the test (p < 0.01). Likewise, other antiproliferativeagents inhibited cellular growth significantly at a dosage from 1.0 μg/ml to 16.0 μg/ml(colchicine), 0.2 μg/ml to 3.2 μg/ml (daunomycin) and 1.0 μg/ml to 16.0 μg/ml(5-fluorouracil), but not at 0.5 μg/ml (colchicine), 0.1 μg/ml (daunomycin) and0.5 μg/ml (5-fluorouracil). The ID50 were 0.33 μg/ml (harringtonine), 3.11 μg/ml (colchicine), 0.79 μg/ml (daunomycin) and 5.23 μg/ml (5-fluorouracil), respectively.Conclusions: Harringtonine was extremely effective ininhibiting human retinal glial cell proliferation, like other antiproliferative drugs such as colchicine,daunomycin and 5-fluorouracil. Harringtonine, therefore, may be a candidate for further studies regardingthe treatment of experimental PVR.

Harringtonine (HT), an anticancer drug with high chemotherapeutic efficiency to human chronic granulocytic/myelomonocytic leukemia, has been reported to rapidly induce apoptosis in HL-60 cells in a wide scope/range of dosage by investigators from our lab and others. In the present studies, by using video enhancement contrast (VEC) microscopy, we dynamically analyzed changes in intracellular calcium distribution in a single HL-60 cell over the period from the initiation of apoptosis to the obvious appearance of chromatin condensation. The results from this paper demonstrated the striking distinction of intracellular calcium distribution at different time points after treatment with HT. Before treatment in normal HL-60 cells the highest [Ca2+]i accumulation was observed in the peri-nuclear area and the lowest was observed in the nucleus; after treatment with 1 microg/ml HT for 30 min intracellular calcium diffused all over the cell compartments, while intranuclear calcium increased comparatively and significantly. The phenomenon of intranuclear calcium accumulation was further confirmed by using laser scanning confocal microscopy (LSCM). In addition, co-localization of the highest calcium region with condensed chromatin in apoptotic HL-60 cells was also observed by LSCM. Our results suggest that two sequential alterations of intracellular calcium distribution occurred in apoptotic HL-60 cells induced by HT, i.e. (a) accumulation of calcium in the nucleus and (b) regionalization in a specific nuclear region.

Chen and colleagues [1] in the current issue of Leukemia and Lymphoma have investigated a combination of homoharringtonine (HHT), aclarubicine, cytarabine and granulocyte colony-stimulating factor (G-CSF) (CHAG) in treating 36 patients (aged 16 – 61 years) with relapsed (19%) or refractory (67%) acute myeloid leukemia (AML) [2]. HHT was given at 1 mg/m 2 intravenously (IV) from day 1 to day 14. Complete remission (CR) was achieved in 81% and partial remission (PR) in 14% of the patients. Th e median duration to neutrophil recovery was 15 days. Cardiac toxicity was not noted in this study. One patient had liver dysfunction that spontaneously improved. Th e results are encouraging, with an excellent response rate that exceeds the expected 17 – 60% response rate for conventional therapies used in this setting [3 – 9]. However, longer follow-up and ultimately randomized trials are warranted to assess the actual eff ect of this or other combinations. In the early 1970s, HHT and other ester alkaloid cephalotaxins were isolated from the bark of the Cephalotaxus fortunei evergreen tree and found to have activity against murine leukemia by decreasing protein synthesis and inducing apoptosis [10 – 12]. In one of the earliest Chinese studies, mixtures of HHT and harringtonine (HT) at diff erent dosing schedules (IV or intramuscular [IM]) led to fi ve CRs in 41 evaluable patients (12%). Th e study indicated that the maximum tolerated therapeutic eff ective dose was 4 mg daily IV for 14 days. Major dose-limiting toxicities were myelosuppression and cardiovascular abnormalities, including hypotension and arrhythmias [13]. More Chinese phase I and II studies were summarized by Grem et al . [14] in a landmark article that showed a CR rate of 24% (65 of 274 patients) in patients with AML. As HHT is a G1/G2-phase specifi c drug that causes leukemic cell transformation into S-phase [15], this provided the theoretical basis to combine it with other agents that are S-phase specifi c such as cytarabine [16,17]. In 1980, the HOAP (HHT, vincristine, cytarabine and steroids) regimen resulted in CRs in 82% (23/28) of newly diagnosed patients with AML [18], but with more profound myelosuppression and cardiac toxicities. Similarly, HHT in combination with cytarabine and thioguanine was used in newly diagnosed patients with AML, resulting in a CR rate of 68% [19]. The success of the Chinese studies motivated US investigators to use HHT in acute leukemias. In 1984, the first US phase I trial by Legha et al . [20] investigated HHT daily bolus infusion over 10 – 360 min for 1 – 10 days, with dose escalation 0.2 – 8 mg/m 2 daily in 43 patients with advanced malignancies, including five patients with acute leukemia; CR was achieved in three of the five patients. Hypotension was the limiting toxicity for doses 4 mg/m 2 daily. Other phase I bolus studies showed similar results and recommended a phase II dose of 3 – 4 mg/m 2 daily for 5 days as a short infusion [21]. Since hypotension seemed to be related to vasodilatation following the bolus infusion, HHT was administered through continuous infusion (CIVI). In a phase I – II study, Warrell et al . [22] used CIVI HHT in 49 patients with relapsed or refractory AML and concluded that dosing at 5 mg/m 2 daily for 9 days is a reasonable option, as any higher doses caused significant hypotension as in the bolus schedule. CR was achieved in 25% of the patients (consisting of both relapsed and refractory AML) and lasted for a median of 6 months. HHT CIVI was also effective in myelodysplastic syndrome (MDS) and MDS transformed to AML as shown by Feldman et al . [23], where eight of 28 patients responded, but early death due to neutropenic complications was seen in 46%. Myelosuppression was an issue with CIVI, as full bone marrow recovery took up to 24 days. Subcutaneous (SQ) HHT was first used in a phase I trial in 2006 when Levy et al . [24] treated 18 adults patients with relapsed/refractory AML, accelerated myeloproliferative neoplasms or refractory MDS with dose-escalated twice-daily SQ HHT 0.5 – 6 mg/ m 2 for 9 days. The study defined a maximum tolerated dose as 5 mg/m 2 daily, resulting in two CRs in patients with AML (lasting 3 and 4 months) and hypoplastic bone marrow on day 10 in five of 14 patients, in addition to clearing peripheral blasts. Adverse events included hyperglycemia, fluid retention and infectious complications.

Trees have made an enormous phytochemical contribution in anticancer drugs' development more than any other life form. The contributions include alkaloids that are biosynthesized in various ways and yield. Lead alkaloids isolated from the trees are taxol and camptothecins that currently have annual sales in billion dollars. Other important alkaloids isolated from these life forms include rohitukine, harringtonine, acronycine, thalicarpine, usambarensine, ellipticine, and matrines. Studies on their mechanism of action and target on the DNA and protein of cancerous cells aided the development of potent hemisynthesized congeners. The molecules and their congeners passed/are passing a long period of historical development before approved as antineoplastic drugs for cancer chemotherapy. Some of them did not find the application as anticancer drugs due to ineffectiveness in clinical trials; others are generating research interest in the antineoplastic activity at the present and have reached clinical trial stages. Potentials in antineoplastic molecules from trees are high and are hoped to be commensurate with cancer types afflicting human society in the future.

Six murine tumors, including ascetic tumors HepA, EC, P388 leukemia, S180 and solid tumor S180, and Lewis lung carcinoma, were employed in this work. The free sialic acid concentrations in both blood and ascites were measured in tumor-bearing mice. The results showed that the content of sialic acids in blood was increased in tumor growth and certain tumor types. Higher sialic acid content was observed in ascites than that present in blood. The influence of antineoplastic agents (vincristine, thiotepa, adriamycin, probimane, cisplatin, oxalysine, cortisone, nitrogen mustard, lycobetaine, Ara-C, harringtonine, and cyclophosphamide) on the content of sialic acids in mice blood bearing solid tumors of either S180 or Lewis lung carcinoma was observed. Different inhibitions of antineoplastic drugs on both tumor growth and serum sialic acid levels in mice bearing tumors were found. Among these antineoplastic drugs, probimane, cisplatin, nitrogen mustard, and lycobetaine were able to decrease the serum sialic acid levels in mice bearing tumors. Since these four antineoplastic drugs are all DNA chelating agents, it was proposed that the inhibition of tumor sialic acids by these drugs might be through the DNA template via two ways. Since we have found no effect of antineoplastic drugs on serum sialic acid levels in normal mice, this suggests that the inhibition of antineoplastic drugs on sialic acids is by tumor involvement.

The Chinese medicinal plant-derived antineoplastic alkaloids and their analogs are reviewed with emphasis on those discovered from the authors' laboratory. The active compounds include camptothecin, colchicine, ellipticine, harringtonine, Vinca alkaloids, indirubin, (-)-sophocarpine, lycobetaine, monocrotaline, d-tetrandrine, indicine N-oxide, maytansine, aporphine type alkaloids, palmatine, murrapanine, emarginatine alkaloids, and the alkaloids from Securinega virosa. The compounds are discussed briefly with the recent advances concerning their antitumor activity, structure-activity relationships and mechanisms of action.

Recent progress on the study of anticancer drugs originating from plants in China is reviewed in this paper. Guided by the experience of traditional Chinese medicine, several new drugs have been found. Indirubin from Indigofera tinctoria is useful for the treatment of chronic myelocytic leukemia. Irisquinone from Iris latea pallasii and 10-hydroxy camptothecin from Camptotheca accuminata have exhibited definite activity on rodent tumors. Recent studies indicate that ginsenoside Rh2 is an inducer of cell differentiation in melanoma B-16 cells in vitro. Pharmacological studies have demonstrated that curcumin from Curcuma longa is an antimutagen as well as an antipromotor for cancer. Daidzein and acetyl boswellic acid have been shown to be effective inducers of cell differentiation in HL-60 cells. Guided by the chemotaxonomic principle of plants, harringtonine and homoharringtonine isolated from Cephalotaxus hainanesis have exhibited significant antileukemia activity and are widely used in clinics in China. Taxol from Taxus chinensis has been shown to be an important new anticancer drug with unique chemical structure and mechanism of action. The continuous search for new anticancer drugs from plants will be a fruitful frontier in cancer treatment and chemoprevention.

Natural As4S4 and As2S3 are major components in realgar and orpiment, which also contain small amount of As2O3 and other toxic materials. Liu YF showed with us that NB4 cell line and its mice model were more sensitive to As4S4 than to As2O3 and As2S3. That was in agreement with the clinical data available from China. Wang FR and we proved in mice and in clinical pharmaceutical study that ground Seman platycladi as an excipient can appreciably increase the blood level of arsenic when taken P.O. together with As4S4. Our clinical study with 130 patients with t(15;17) APL showed both As4S4 and As2S3 alone were very effective in CR induction, including cytogenetic remission and PML-RAR reversion, yet the relapse rate was higher in the group treated by As4S4 alone than in the group treated together with other autileukemic agents, either together or in sequence. In the later group, LFS for 1 and 6 years was 96.7% and 87.4%, and the over-all survival for 1 and 6 years was 98.9% and 93.9%. High dose chemotherapy was proven to be not only unsafe but also unnecessary. In newly diagnosed APL patients with neutropenia, CR could be safely achieved by As4S4 P.O. and/with ATRA. Chemotherapy was then followed. In patients with leukocytosis, chemotherapeutic agents such as hydroxyurea or harringtonine was added at the beginning and then followed by anthracycline and asparaginase. The reasons of APL relapse after patients receiving As4S4 P.O. were: 1: emergence of a resistant cell clone showing additional chromosomal abnormalities other than t(15;17); 2: decrease of blood As level. The resistant APL case could respond favorably to combination of As4S4 P.O. together with ATRA. The blood level of As could be raised by modification of the preparation under investigation. Allogeneic stem cell transplantation (allo-SCT) is the last option in relapsed patients, became autologous stem cell transplantation (ASCT) has a significantly better outcome. In a few hospitals ASCT was performed early in the CR1. In rare cares, patients were referred with intra-cranial hemorrhage. CT or EMR scan was done urgently at this instance. Cranial surgery carried out without a minute of delay resulted in the rescue and long survival of patient, while delay of surgery had resulted in irreversible damage of Pons/Medulla oblongata and ended in death. As2O3 IV had been used with success in mentally unclear patients not suffering from intra-cranial hemorrhage. Although no parallel clinical trial has been done to compare the efficacy of As2O3 and As4S4, yet the fact was it was easier for doctors and patients to carry out and receive maintenance treatment with oral As4S4 rather than with As2O3. According to the data and principles above, most of the t(15;17) APL patients can be cured.

This study investigated the in vitro anti-cancer effects of four Chinese natural drugs on the human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780/CP70 (cisplatin-resistant). Cells were treated with series of concentrations of drug preparations for 24 h. Vincristine prepared by the same pharmaceutical firm in China; was used as an internal control. As assessed by colony formation assays, harringtonine induced similar growth inhibiting effects in A2780 and A2780/CP70 cell lines with a 50% inhibitory dose (IC50) of 0.195 mu g/ml in both. Rabdosia rubescens Hara demonstrated a cytotoxic effect on cisplatin-sensitive A2780 cells with an IC50 of 0.58 mg/ml; but there was no effect on A2780/CP70 cells. The data suggest direct anti-proliferative activity for at least two Chinese natural medicines used in the clinical treatment of cancer in China. Further investigation of Chinese natural medicines may be valuable in the identification of new and effective anti-cancer drugs with minimal side effects.

Several known clastogens and mutagens have been tested for their ability to induce micronuclei (MN) using the cytokinesis-block method in mouse splenocytes. The chemicals were harringtonine, cisplatin, cytosine arabinoside, vincristine sulfate, colchicine, potassium chromate, methyl methanesulfonate and 2-acetylaminofluorene. All chemicals tested induced a dose-dependent increase in MN and a delay in cell-cycle progression. The results suggest that the cytokinesis-block micronucleus method in mouse splenocytes is reliable, economical and sensitive enough for detecting mutagenic agents in vivo and in vitro.

To broaden the applications of magnetized water (MW) in medical science, the possible detoxicative effect of MW to anticancer drugs in vivo were studied. After being given ip with cyclophosphomide (CTX) 500 mg/kg, cisplatin (DDP) 40 mg/kg, harringtonine (HA) 20 mg/kg, mitomycin C (MMC) 8 mg/kg, lycobetaine (Lyc) 200 mg/kg, respectively, the mice were given MW ip 0.2 ml for 7 days. The average life span was calculated for each group. After being given subacutely lower doses of anticancer drugs (CTX 100 mg/kg, HA 3 mg/kg) ip 3 times, the mice were given MW ip 0.2 ml for 7 days and the blood white cells were counted as routine. It was shown that the mice in MW groups after ip anticancer drugs survived longer than those without MW. The effects of various anticancer drugs on life span were different. The white cell numbers of groups with MW were higher than that of the groups without MW. So it is possible that MW can remarkably extend the life span of mice and attenuate the leukopenia by mitigating the toxicity of anticancer drugs in vivo.

Harringtonine(H) level in serum was quantified by high-performance liquid chromatographic (HPLC) technique and its pharmacokinetic parameters were determined in 11 patients with ANLL after 4 hours continuous infusion of 4 mg of H. 74 serum samples over the range of H administered 1–6 mg/d were also determined, the dose of H administered paralleled the mean serum H concentration, but the highest concentration were 1.6–7 folds higher than the lowest under the same doses. It seems that the dosage of harringtonine must be individualized in order to achieve better antileukemia effect. It was found that under the similar serum H concentration the marrow blast decrease index (MBDI) in types M4 and M5 of ANLL is much more greater than in M2 after the first course of chemotherapy and in 3 patients of M2, the serum H level and the MBDI show positive correlation.

Introduction. Most acute myeloid leukemia (AML) patients with adverse prognosis either fail to achieve complete remission (CR) or relapse after short-term remission, new strategies are needed to improve therapeutic effect in these patients. Gene silencing via DNA methylation are frequent and reversible in high-risk AML, it provides the possibility to treat AML patients with DNA-hypomethylating agent. Decitabine alone has limited anti-leukemia effect and minimal toxicity. Studies demonstrated that decitabine prior to chemotherapy was likely to increase efficacy in AML. In this study, we investigated the effect of decitabine on susceptibility to cytotoxic drugs in chemoresistant AML cells. Efficacy and safety of decitabine prior to HAA regimen was evaluated in refractory, relapsed and high-risk AML patients. Methods. HL60, HL60/ADR, Kasumi-1, and primary AML cells were pre-treated with 1.0 μM decitabine for 72 hours, Sensitivities to harringtonine, adriamycin, cytarabine and the combination was determined by MTT, apoptosis was analyzed by flow cytometry. Protein expression was detected by western blotting. In clinic, Twenty-three patients were enrolled in decitabine prior to HAA regimen, and twenty-four patients in HAA alone. CR ratio was used to evaluate efficacy. Durations of neutrocytopenia or thrombcytopemia and infection incidence were observed to assess the safety. All of patients were followed up to 36 monthes, overall survival (OS) and disease-free survival (DFS) were calculated. Result. Decitabine increased sensitivity and apoptosis induced by harringtonine in HL60/ADR, as well as adriamycin and cytarabine in HL60/ADR and Kasumi-1 cells. But no change in sensitivity to each drug was observed in HL60, and sensitivity to harringtonine in Kasumi-1. The similar changes in sensitivity to adriamycin and cytarabine were also observed in primary AML cells from refractory patients (n=6). Cytotoxic effect of HAA was alsoincreaed by decitabine in HL60/ADR and Kasumi-1 cells (p=0.004, 0.018). Western blotting showed that decitabine decreased expression of DNMT1, activation of p53 and inhibition of c-Myc, Survivin and Bcl-2. In clinic, 16 (69.6%) patients in decitabine plus HAA regimen responded to the first course of induction therapy: 14 CR (60.9%) and 2 PR (8.7%). 1 in 2 patients with PR achieved CR after second course induction. It brought the overall CR was 65.2%. Otherwise, 10 (45.8%) patients in HAA regimen alone responded to the first induction: 7 CR (29.2%) and 4 PR (16.7%), 3 in 4 patients with PR achieved CR after the second induction, and overall CR reached 41.7%. The first-cycle CR ratio in decitabine plus HAA was higher than that in HAA alone (p=0.041). Patients continued to receive chemotherapy alternatively combined with decitabine. The median OS was 14 months in chemotherapy plus decitabine, 6 months in chemotherapy alone, and median DFS were 18 and 5 months in the former and latter. No significant difference was observed in the time of neutrophil (p=0.832, 0.631) or platelet recovery (p=0.798, 0.544) after induction and intensification therapy. The incidences of infection were also similar (p=0.724, 0.697). The clinic data indicated that decitabine plus HAA regimenit was efficacy and well-tolerated to treat refractory AML patients. Conclusion. Our results demonstrated that decitabine increased cytotoxic effect against chemoresistant AML cells. Combination of decitabine and HAA was safe and efficacy to treat refractory/relapsed AML. These findings support clinic protocols based on decitabine prior to chemotherapy to overcome resistance and improve therapeutic efficacy in AML patients. Disclosures Carter: PrismBiolab: Research Funding.

The current studies represent the first step in assessing the utility of harringtonine in combination with cisplatin as an improved approach for treating ovarian cancer. Three ovarian cancer cell lines, platinum-sensitive A2780, and platinum-resistant A2780/CP70 and OvCar-3, were exposed to their respective IC(50) dose of cisplatin for 1 h with or without a 24-h pretreatment with harringtonine. The level of platinum-DNA adducts was determined by atomic absorption spectrometry (AAS). These studies show for the first time that harringtonine pretreatment significantly increased the amount of platinum-DNA adducts in all ovarian cancer cell lines by 2-4 fold, immediately following 1-h exposure to cisplatin. Moreover, the level of cisplatin-DNA adducts in harringtonine-pretreated cells remained elevated by 3-4.7-fold for at least 6 h after cisplatin was removed, relative to cells only exposed to cisplatin. In all three cell lines the removal (repair) of platinum-DNA adducts was not significantly altered by harringtonine. In addition, the extent to which harringtonine altered the expression of select DNA damage response genes (p53, P16, ERCC1 and XPB) was determined using RT-PCR and Southern hybridization in A2780 and A2780/CP70 cells. The expression of ERCC1 and XPB RNAs were only modestly altered by harringtonine, consistent with a lack of effect of harringtonine on repair of cisplatin-DNA damage. However, harringtonine altered expression of p53 and P16 RNAs in both cell lines, although the down-regulation of p53 and P16 RNAs by harringtonine were more pronounced in A2780 cells. The novel observation that harringtonine augments platinum-DNA adducts in both platinum-sensitive and -resistant ovarian cancer cells indicates this combination of drugs may have utility in treating ovarian cancer and may be especially useful in managing platinum-resistant cancers. Additional studies are required to determine which sequence of these drugs is most beneficial, as well as the mechanism by which harringtonine increases cisplatin-DNA damage in ovarian cancer cells.

Introduction The loss of Apc, causing Wnt-mediated epithelial proliferation, is an early event in colorectal cancer (CRC) development. This hyperproliferative state requires signalling though the mTOR pathway, with the current paradigm suggesting that upregulation of translation initiation via phosphorylation of 4EBP1 is crucial. This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 function, would be ineffective in limiting development and progression of intestinal adenomas. Methods The inducible in vivo mouse models Lgr5Cre ER and VillinCre ER were used to selectively flox genes from intestinal stem cells and crypts respectively. mTOR complex 1 signalling was inhibited in Apc fl/fl mice either by rapamycin treatment or co-floxing the mTORC1 essential component Raptor. Translational status was assessed by sucrose gradient ultracentrifugation of intestinal epithelial extract from these mice and 35 S methionine incorporation and harringtonine chase assays on organoid cultures. The role of downstream mTORC1 effectors was established by assessing the intestinal regeneration following IR irradiation of 4EBP1/2 DKO , S6K1/2 DKO , rpS6 mut and eEF2k -/- mice. Survival studies for Apc fl/fl mice treated with rapamycin were performed both prior to, and on development of, symptoms Results mTORC1 activity is absolutely required for the proliferation of Apc deficient, but not wild type, intestinal crypts. Surprisingly, although protein synthesis is increased in Apc fl/fl crypts, it is translation elongation and not initiation that is the rate limiting step. Mechanistically, the inhibition of eukaryotic elongation factor (eEF2) kinase, to increase eEF2 activity downstream of mTORC1 and S6K is required for Wnt-mediated proliferation after IR irradiation. Treatment of established Apc fl/fl adenomas with rapamycin (which inhibits the mTORC1-S6K-eEF2k-eEF2 axis) arrests tumour growth and prolongs life. Furthermore, rapamycin treatment of mice immediately following homozygous Apc loss prevents the onset of symptoms. Conclusion These data show that intestinal adenoma formation and growth requires an mTOR mediated increase in translation elongation. Treatment of patients at high risk of developing CRC, such as those with Familial Adenomatous Polyposis, with Rapalogs may therefore be of therapeutic value. Disclosure of Interest None Declared.

The antiparasitic drug flubendazole and the antineoplastic compound harringtonine were studied for ability to induce chromosomal damage in Chinese hamster lung (CHL) cells and cytotoxicity and morphological transformation in C3H/10T1/2 Cl 8 (10T1/2) mouse embryo fibroblasts. Flubendazole caused a dose- and time-dependent induction of polyploidy in CHL cells. In cells treated with 0.78 micrograms/ml flubendazole, the yield of polyploid cells was 95%. Harringtonine caused a dose- and time-dependent induction of chromosome breaks, and 0.195 micrograms/ml harringtonine induced chromosome breaks in 47% of CHL cells. Both flubendazole and harringtonine caused dose-dependent cytotoxicity to 10T1/2 cells at concentration ranges of 0.04-1.60 and 0.05-0.8 micrograms/ml, respectively. Flubendazole and harringtonine at concentrations of 0.08-0.4 and 0.4-0.8 micrograms/ml, respectively, induced morphological transformation (predominantly type II foci) in 10T1/2 cells. Three of four harringtonine-transformed cell lines and two of four flubendazole-transformed cell lines formed foci in reconstruction experiments with non-transformed 10T1/2 cells. All four harringtonine-transformed and all four flubendazole-transformed cell lines formed colonies in soft agar. Similar concentrations of flubendazole and harringtonine induced chromosome damage in CHL cells and cytotoxicity and morphological transformation in 10T1/2 cells. The ability of flubendazole to induce polyploidy may be part of the mechanism by which this compound induces morphological transformation. Similarly, the ability of harringtonine to induce chromosomal aberrations may be part of the mechanism by which this compound induces morphological transformation. Therefore, flubendazole and harringtonine induce cytotoxicity and morphological and anchorage-independent transformation, harringtonine induces chromosome aberrations (breakage, translocation, and rings), and flubendazole induces polyploidy in cultured mammalian cells. The clastogenic and cell transformation-inducing properties of these compounds suggest that these drugs may have carcinogenic potential. This should be investigated rigorously in animal carcinogenesis bioassays. The genotoxicity of these drugs should be considered during their development as antiparasitic and antineoplastic agents.

Abstract 4912 We report here three acute myeloid leukemia cases with novel clonal non-characteristic chromosome aberrations and morphologically resembling acute promyelocytic leukemia (APL). Case No.1: A 57-year-old woman came with mucosal bleeding, ecchymoses, and fever. Blood tests showed hemoglobin (Hb) of 9.3g/dL, platelet count (Plt) of 29×109/L and white blood cell count (WBC) of 1.3×109/L. D-dimer was 3.6ug/ml and Fibrin degradation product (FDPs)>5ug/ml. Bone marrow (BM) morphology showed 91% atypical hypergranular cells with Auer rods and resembling APL. The immunophenotype of peripheral blood (PB) blasts showed 83% positive for CD9, CD13, CD15, CD33, CD64, CD117 and negative for CD34, HLA-DR and B, T-cell markers. PML-RARA and t(15;17) negative but clonal t(1;16)(q44;q22) positive. The patient was initially treated with all-trans retinoic acid (ATRA), As4S4 and mitoxantrone and then switched to harringtonine and ara-C treatment but couldn't get remission. Case No.2: A 39-year-old woman came with asthenia, mucosal bleeding and ecchymoses. Blood tests showed Hb of 10g/dL, Plt of 49×109/L and WBC of 6.2×109/L. D-dimer >20ug/ml and FDPs >20ug/ml. BM morphology showed 84% atypical hypergranular cells with Auer rods and resembling APL. The immunophenotype of PB blasts showed 76% positive for CD13, CD33, CD64, CD117, cMPO and negative for CD9, CD34, HLA-DR and B, T-cell markers. PML-RARA and t(15;17) negative but clonal t(7;17)(p10;q10) positive. The patient was initially treated with ATRA and As4S4 and mitoxantrone but couldn't get a good response. He was then switched to idarubicin and ara-C treatment and got partial response. He is now waiting for allogeneic hematopoietic stem cell transplantation. Case No.3: A 62-year-old man came with asthenia. Blood tests showed Hb of 68 g/dL, Plt of 61×109/L and WBC of 3.3×109/L. D-dimer was 10.2ug/ml and FDPs was not tested. Bone marrow (BM) morphology showed 63.5% atypical hypergranular cells with Auer rods and resembling APL. The immunophenotype of Bone marrow showed 71.2% positive for CD13, CD15, CD33, CD117, cMPO and negative for CD34, HLA-DR,and B, T-cell markers. PML-RARA and t(15;17) negative but clonal (47,XY,+mar1) positive. The patient was initially treated with ATRA and As4S4 but couldn't get a good response. He was then swiched to Daunorubicin and ara-C treatment and got partial remission. APL should be distinguished from other subtypes of acute myeloid leukemia (AML) because of the increased risk of disseminated intravascular coagulation (DIC) and its response to ATRA and arsenic compounds. Some cases of AML seem morphologically similar to APL but lack the characteristic t(15;17) or other RARA-related translocations, and they generally didn't response to ATRA and arsenic compounds treatment. The 3 cases reported here reiterate the lack of specificity of morphologic and Immunophenotyping findings in APL-like AMLs. Karyotypic analysis, as well as FISH and RT-PCR, must be conducted on these cases to rule out APL and other chemotherapy regimens should be considered. APL-like AMLs that do not demonstrate RARA-related translocations may constitute a heterogeneous population of AML with diverse clonal non-characteristic chromosome aberrations, and the molecular mechanisms is worthy of further study. Disclosures: No relevant conflicts of interest to declare.

The treatment of chronic myelogenous leukemia (CML) has been revolutionized by the introduction of tyrosine kinase inhibitors in the therapeutic arsenal. Disease response and survival have been dramatically improved with these agents, however a significant cohort of patients may resist to such inhibitors. In tyrosine kinase inhibitors (TKIs)-resistant CML patients, the identification of the BCR-ABLT315I mutation is a rare event, but usually translates into poor survival rates and therapeutic options remain few in the absence of a suitable allogeneic donor. In this study, within the CGX-635 CML 202 trial or not, we investigated the impact of cycles of sub-cutaneous Homo-harringtonine (HHT) [Omacetaxine mepesuccinate] as monotherapy on wild-type and mutated transcripts in 7 chronic phase CML patients resistant to TKIs, harbouring a BCR-ABLT315I mutation. Patients were monthly monitored in a centralized manner for total disease burden (RQPCR) and BCR-ABLT315I transcripts (PCR-RFLP, sensitivity threshold 1%) for up to 14 cycles. There were 4 M and 3 F, median age: 51 (38–79) at HHT start. All patients were in chronic phase (CP) at diagnosis, Sokal score was high for 5, intermediate for 1 and unknown for 1 patient. One patient had a Ph1 duplication at diagnosis. Two patients had short term IFN before imatinib. Median time of imatinib treatment was 27 (15–54) months with, as best responses: CHR for 2, a minor CyR for 1, PCyR for 2, CCyR without MMR for 2. Three patients had a second generation TKI for a median of 1 (1–22) month for unsatisfactory response to imatinib. At T315I identification, all patients were in CP CML, 4 in hematological relapse, 3 in CHR (1 molecular progression in CCyR, 1 in cytogenetic progression, 1 with no cytogenetic response). The proportion of T315I transcripts among total BCR-ABL transcripts was 95 (20–100) % and the BCR-ABL/ABL ratio (IS) was 67 (26–112) % at T315I discovery. Three patients had clonal evolution (Ph1 duplication, -7, -Y+variant Ph1). The median interval imatinib start-T315I was 27 (10–65) months. After T315I discovery, imatinib was withdrawn and, if necessary, patients went on hydroxyurea. The median time between T315I detection and HHT start was 2 (1–4.3) months. All patients demonstrated hematologic or cytogenetic improvements to HHT (7 patients in CHR, 1 in CCyR, after a median follow-up of 11 months). A rapid decline and a sustained disappearance of T315I mutated transcripts was observed in 6 out of 7 patients, median proportion of T315I transcripts was 65 % at Cycle 2, 19 % at cycle 3, 1 % at cycle 4, < 1 % at cycle 5 and beyond, The total tumour burden reduction was moderate with one patient converted to a CCyR after 3 cycles, but with no other cytogenetic improvement. A moderate decline was observed in BCR-ABL/ABL ratios from a median of 67 before HHT to 39 % at cycle 10, suggesting a differential activity of HHT on wild-type versus BCRABLT315I cells. Patients received a median of 8 (4–17) cycles of HHT at last follow-up and none of the patients have progressed. Transient grade 3–4 hematological toxicities after the first cycle and grade 0–2 after maintenance cycles were observed in all patients. Nonhematological toxicities were grade1–2 (site pain after injections, nausea, diarrhea). Since high levels of BCR-ABLT315I transcripts in CML are thought to be associated with disease progression, HHT (i. e. a non-targeted therapy) in these cases seems to exert a somewhat preferential activity on T315I mutated CML cells through an unknown mechanism yet. Therefore, by reducing the T315I+ tumor burden, this treatment is expected to decrease progression rates, and improve survival.

To investigate the effect of bortezomib alone or combined with harringtonine (HT) or arsenic trioxide (As2O3) on the proliferation activity and apoptosis of HL60/ADM cell line and refractory/relapse acute leukemia primary cell. HL60/ADM cells or refractory/relapse acute leukemia primary cells were incubated with bortezomib at different doses alone and in combination with HT or As2O3. The proliferation activity was abserved by MTT assay, cell apoptosis was studied by fluorescence microscopy and flow cytometry. Intracellular concentration of daunorubicin (DNR) was determined by flow cytometry. In bortezomib-treated tumor cells, inhibition rate enhanced and apoptotic cells increased in mumber time- and dose-dependently. 40nM Bortezomib can best inhibit the proliferation activity of HL60/ADM cells when incubated for 48 hours. 15uM As2O3 or 752nM HT combined with different doses of bortezomib can inhibit the proliferation and induce the apoptosis of HL60/ADM cells, The dual inhibition of As2O3 plus bortezomib or HT plus bortezomib may present a superior anticancer efficancy to either inhibition of the drugs above alone (P

Multidrug resistance (MDR) was the main cause of treatment failure in leukemic chemotherapy. P170, the protein expressed from MDR-1 gene, play an important role in MDR. P27Kip1 was a member of cyclin dependent kinase inhibitor family. It was also a negative regulator in cell generation cycle. It also promoted cells apoptosis. Valproic acid (VPA) was a kind of histone deacetylace inhibitor which could inhibit cell cycle and induce apoptosis. In the current study, we investigated the effects of VPA on the expression of P27Kip1 and P170 of HL60 cells in vitro. HL60/HT cells were induced from HL60 cells by harringtonine (HT) in gradient concentrations. The P27Kip1 expression and P170 expression were tested in flow cytometry. We tested the expression of P27Kip1 and P170 in normal mononuclear cells, HL60 cells and HL60/HT cells, also the effects of VPA on P27Kip1 expression and P170 expression in HL60 cells and HL60/HT cells. The 50% inhibiting concentration (IC50) of vincristine for HL60/HT cells was 5.2 times higher than that for HL60 cells (0.078ug/ml vs. 0.015ug/ml). The IC50 of HT for HL-60/HT cells was 9.3 times higher than that for HL-60 cells (0.14μg/ml vs. 0.015μg/ml). The IC50 of Cytarabine for HL60/HT cells was 3.65 times higher than that for HL60 cells (0.73ug/ml vs. 0.20ug/ml). The expression of P27Kip1 in normal mononuclear cells was the highest, while that in HL60 cells was higher than HL60/HT cells (96.18±3.13% vs. 85.83±4.65% vs. 35.91±3.13%, one way ANOVA, P0.05). But the P170 expression of HL60/HT cells was 6.71±1.61%, which was much higher than that of normal mononuclear cells or HL60 cells (P0.05). In HL60/HT cells, the expression of P27Kip1 increased after cultured with VPA (66.20±5.07% vs. 35.91±3.13%, P0.05). These results indicate that the P27Kip1 expression in normal mononuclear cells were higher than that in leukemia cells (HL60 cells), and the P27Kip1 expression in common HL60 cells were higher than that in MDR HL60/HT cells. VPA could increase the expression of P27Kip1 in HL60 cells and HL60/HT cells. VPA had no effect on the expression of P170 in HL60 cells or HL60/HT cells. That means, The MDR-reverse effect of VPA may not come from the P170 descent mechanism.

Objective To explore the apoptosis effect induced by bortezomib combined with homoharringtonine or arsenious acid in HL-60 cell line and the mechanism.Methods Cell' s apoptosis was demonstrated by MTT assay and Hoechst33342 staining.Expression of bcl-2,Caspase-9,Caspase-3 and PARP protein was detected by Western blot.Results HL-60 cells' apoptosis could be induced by bortezomib,homoharringtonine and arsenious acid respectively.Proliferation inhibition of HL-60 cells could be enhanced significantly when treated by bortezomib combined with homoharringtonine or arsenious acid compared with treated by any of the three drugs alone (P ＜ 0.05).At the same time morphology shows the apoptosis induced by drugs combined is more obviously than by one drug.Western blot showed bcl-2 protein was down-regulated and Caspase-9,Caspase-3 and PARP proteins were all cleaved activation when cells were treated by 15 μmol/ L arsenious acid alone,but only cleaved activation of PARP and down-regulation of bcl-2 protein be detected when cells were treated with 30 nmol/L homoharringtonine alone,expression of Caspase-9 and Caspase-3 had no change compared with the control.The changes of associated proteins were paralleled with the cell' s apoptosis when treated with combined drugs.Conclusion HL-60 cells' apoptosis effect is inhanced significantly when bortezomib combined with homoharringtonine or arsenious acid.Arsenious acid and bortezomib can inhibit caspase signaling pathway and down-regulate the expression of bcl-2 protein together,but homoharringtonine and bortezomib can only down-regulate the expression of bcl-2 protein and induce cleaved activation of PARP together. Key words: Cells, HL-60; Bortezomib; Homoharringtonine; Arsenious acid; Apoptosis

Background Arsenic trioxide provides significant benefits in newly diagnosed and relapsed acute promyelocytic leukemia (APL) respectively. However, the high relapsed rate is still threatened the life of APL patients. Which regimen should be used to overcome or reduce the relapse in consolidated treatment is a key problem at present. We performed a pilot study about that. Objective To Compare the effectiveness and security of cocktail therapy with single arsenic trioxide therapy in APL consolidated treatment. Methods Sixty-Five APL patients, who once received arsenic trioxide treatment and obtained complete remission, were enrolled in this study. Patients were divided into two groups according to the different consolidated regimens. After reinforced treated with DA (daunomycin and cytarabine) or HOAP (harringtonine, vincristin, cytarabine and prednisone) for two course, Group A involved twenty cases received single arsenic trioxide consolidated, Group B included forty-five cases treated with the cocktail therapy, alternatively treated with arsenic trioxide, all trans-retinoic acid and chemotherapy (DA or HOAP). The relapse rates, the survival rates and the central nervous system infiltration rates in 3 years followed up were compared. Results The relapsed rate of Group A was 55%, which was higher than that of Group B(17.8%). The re-remission rate after the first relapse in Group A was 22%, which was lower than that of Group B(42.8%). The central nervous system infiltration rate of Group A was 28%, which was higher than that of Group B(6%). The average survival time of Group A was 10.5±4.2months, which was shorter than that of Group B (22.5±5.5 months). The three-year survival rate of Group A was 15%, which was less than that of Group B (65.8%). Conclusions Cocktail therapy —alternatively treated with arsenic trioxide, all trans-retinoic acid and chemotherapy will be the reasonable regimen for APL consolidated treatment. Which provided benefited on inhibiting relapse and central nervous system infiltration of APL.

Harringtonine (HT) is a new antitumor agent reported to be active in patients with leukemia and lymphoma. The interaction of HT with various antitumor agents was studied in vitro using a human acute myelogenous leukemia cell line KG-1. For the analysis of the drug - drug interaction at the cellular level, Steel proposed the concept of an envelope of additivity. Using this concept, the effect of a two drug combination can be classified as supraadditive (enhancement of the effect), non-interactive (additive), subadditive, and protective (antagonistic). Combination of HT and cytosine arabinoside or HT and dexamethasone produced only additive effects. Combination of HT and methotrexate was subadditive. For HT plus adriamycin or HT plus 5-fluorouracil, data points indicated both subadditive and protective interaction. HT plus acivicin or HT plus L-asparaginase combinations were found to be protective of each other. None of the seven agents produced supraadditive interaction. These results may provide the basis for selecting sequential rather than concurrent combinations which include HT for the treatment of leukemia in man.

The effect of different cytostatic chemotherapeutic drugs on the frequency of sister chromatid exchanges and growth kinetics of the bone marrow cells in normal adults was studied by means of an in vitro bromodeoxyuridine chromosome labeling method. Cytosine arabinoside, harringtonine, methotrexate, vincristine, and 6-mercaptopurine were tested. Three of these agents induced significantly high frequency of sister chromatid exchange (p less than 0.01 or p less than 0.05). The sister chromatid exchange frequency induced by cytosine arabinoside and that induced by 6-mercaptopurine were higher than those of the control group. The increased frequency of sister chromatid exchange predominated in C and A group chromosomes. At the concentration adopted by the authors, the growth kinetics were little influenced by these drugs.

Antitumor activities of harringtonine(HA) and homoharringtonine(HO) both belong to cephalotaxus alkaloids were compared with those of vinca alkaloids, vincristine (VCR) and vinblastine (VLB). HA and HO had significant activities against P388 leukemia, L1210 leukemia and B16 melanoma by intraperitoneal injection comparable to VCR and VLB. The therapeutic index (LD10/ED50) of HA and HO in B16 melanoma system was 1.90 and 2.31, while those of VCR and VLB were 1.38 and less than 1.00, respectively. The oral administration of these cephalotaxus alkaloids evoked a significant antitumor activity but that of vinca alkaloids induced no activity against P388 and B16 melanoma. The efficacy of HO on various tumor systems was equal or greater than that of HA when comparison was made between these two cephalotaxus alkaloids. In contrast to VCR and VLB, HA and HO demonstrated moderate activity against P388/VCR, a subline of P388 resistant to vincristine. The intraperitoneal injection of VCR or VLB produced an increase in the mitotic index of P388 cells as compared with the control values, but HA or HO decreased it. HA and HO, cephalotaxus alkaloids are a new class of active compounds of plant origin which may differ from the vinca alkaloids in the mechanism of antitumor activity.

Homoharringtonine and harringtonine are esters of cephalotaxine and are naturally occurring alkaloids in certain coniferous trees in China. These compounds have been shown to have activity against certain types of leukemia in cell cultures, experimental animals, and initial clinical trials in China, and will undergo Phase I trials in several institutions. A gas chromatographic mass spectrometric technique was developed for the quantification of both drugs in serum. One drug serves as internal standard for the other. The drugs are extracted from serum with dichloromethane and are quantified by monitoring the protonated molecular ions of the trimethylsilyl derivatives obtained by chemical ionization (methane). Masses monitored are: m/z = 690 for homoharringtonine and m/z = 676 for harringtonine. Detection limit: 10 ng ml-1 (20 nmol); reproducibility: 1.6-15.0% in the 0-200 ng ml-1 range. Serum levels of harringtonine reached 145 ng mg-1 upon intraperitoneal injection of 3 mg kg-1 in BDF-3 mice.