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Homoharringtonine (HHT), a natural plant alkaloid derived from Cephalotaxus, has demonstrated to have a broad antitumor activity and efficacy in treating human chronic myeloid leukemia. An alternative source is required to substitute for the slow-growing and scarce Cephalotaxus to meet the increasing demand of the drug market. The objective of this study was to screen HHT-producing endophytic fungi from Cephalotaxus hainanensis Li. By screening 213 fungal isolates obtained from the bark parts of Cephalotaxus hainanensis Li, one isolate was found to be capable of biosynthesizing HHT. The fungus was identified as Alternaria tenuissima by morphological characteristics and internal transcribed spacer (ITS) sequence analysis and was named as CH1307. HHT obtained from CH1307 was analyzed through the HPLC and LC-MS/MS and NMR spectroscopy. The extract of the fermentation broth of CH1307 showed antiproliferative activities against K562 (chronic myelocytic leukemia), NB4 (acute promyelocytic leukemia), and HL-60 (promyelocytic leukemia) human cancer cell lines with IC50 values of 67.25 ± 4.26, 65.02 ± 4.75, and 99.23 ± 4.26 μg/mL, respectively. The findings suggest that HHT-producing endophytic fungus, Alternaria tenuissima CH1307 might provide a promising source for the research and application of HHT.

Background and Purpose: Homoharringtonine (HHT) is a natural alkaloid isolated from various Cephalotaxus species. HHT has been used to treat acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML), chronic lymphocyte leukaemia and myelodysplastic syndromes. Although HHT inhibits protein synthesis and promotes apoptosis of leukaemia cells in preclinical studies, its molecular target proteins remain unknown. The aim of this study was to identify target proteins of HHT., Experimental Approach: We have synthesized a biotinylated affinity column and used it to identify targets of HHT and confirmed the results by MS and Western blots. We also examined the effects of HHT on the target protein and determined roles of the target protein in anti-leukaemia activities of HHT through Western blots, flow cytometry and retrovirus transfection., Key Results: Myosin-9, a member of the myosin super-family, was identified as a direct interactor of HHT. Furthermore, HHT up-regulated the expression level of myosin-9 in both AML and CML cell lines in a time-dependent manner. Thus, HHT-induced apoptosis of leukaemia cells begins in 6 h and continues to increase for 24 h. There is a positive correlation between up-regulated myosin-9 expression level and increased percentage of apoptotic cells mediated by HHT. Overexpression of myosin-9 could increase the sensitivity of the leukaemia cells to the cytotoxicity of HHT and arrest cells in S and G2/M phases., Conclusions and Implications: Our results indicated that myosin-9 was the target protein of HHT and played an important role in the HHT-induced apoptosis of leukaemia cells., (© 2015 The British Pharmacological Society.)

Ribosomal protein S6 (rpS6), a component of the 40S ribosomal subunit, is phosphorylated on several residues in response to numerous stimuli. Although commonly used as a marker for neuronal activity, its upstream mechanisms of regulation are poorly studied and its role in protein synthesis remains largely debated. Here, we demonstrate that the psychostimulant d-amphetamine (d-amph) markedly increases rpS6 phosphorylation at Ser235/236 sites in both crude and synaptoneurosomal preparations of the mouse striatum. This effect occurs selectively in D1R-expressing medium-sized spiny neurons (MSNs) and requires the cAMP/PKA/DARPP-32/PP-1 cascade, whereas it is independent of mTORC1/p70S6K, PKC, and ERK signaling. By developing a novel assay to label nascent peptidic chains, we show that the rpS6 phosphorylation induced in striatonigral MSNs by d-amph, as well as in striatopallidal MSNs by the antipsychotic haloperidol or in both subtypes by papaverine, is not correlated with the translation of global or 5' terminal oligopyrimidine tract mRNAs. Together, these results provide novel mechanistic insights into the in vivo regulation of the post-translational modification of rpS6 in the striatum and point out the lack of a relationship between PKA-dependent rpS6 phosphorylation and translation efficiency., (Copyright © 2015 the authors 0270-6474/15/354113-18$15.00/0.)

Homoharringtonine (HHT), a natural alkaloid produced by various Cephalotaxus species, has antileukemic activity in acute and chronic myelogenous leukemia. However, HHT can also induce unanticipated effects in the gastrointestinal tract, such as diarrhea and nausea/vomiting, but the mechanism behind these adverse effects has not been clarified. In the present study, we show that HHT affects the epithelial permeability of intestinal Caco-2 cell monolayers. HHT reduced the transepithelial electrical resistance (TER) of Caco-2 cells in a dose- and time-dependent manner. The HHT effect was reversible and no cytotoxicity was observed at the concentrations used. HHT simultaneously increased the paracellular flux of the 4 kDa and 40 kDa FITC-dextrans associated with the TER reduction. Immunoblotting analysis revealed that HHT decreased the protein expression of TJ components such as claudin-3, -5, and -7. However, the transcription levels of these claudins were not repressed by HHT treatment. HHT also disturbed the cellular localization of claudin-1 and -4. These changes coincided with the reduced barrier function. Our findings suggest that HHT enhances the paracellular permeability of Caco-2 cell monolayers by modulating the protein expression and localization of claudin isoforms; these actions might be responsible for the gastrointestinal effects of HHT., (Copyright © 2014 Elsevier B.V. All rights reserved.)

It has been reported that the histamine H1 receptor (H(1)R) gene is up-regulated in patients with allergic rhinitis and H(1)R expression level strongly correlates with the severity of allergy symptoms. Drugs for therapy should avoid allergy symptoms, especially for patients with over-expressed H(1)R. Therefore, screening of the components which could induce H(1)R activation is urgently needed for drug safety evaluation. Homoharringtonine injection is a preparation for acute nonlymphocytic leukemia, which is approved by China Food and Drug Administration (CFDA) and US Food and Drug Administration. However, severely adverse reactions often occur with intravenous injection of the preparation. In present study, an H(1)R/CMC model was applied for capturing membrane retained components which could induce H(1)R activation. Retention components were enriched and analyzed by H(1)R/CMC-HPLC/MS. Homoharringtonine was recognized, separated and identified in homoharringtonine injection. Ca(2+) flux assay and p-IP3R expression founded that homoharringtonine retained by the H1 R/CMC model increased phosphorylation of IP3R and promoted cytosolic free Ca(2+) elevation in a dose-dependent manner which further verified the activity of homoharringtonine in activating the H1 R. In conclusion, homoharringtonine was screened and identified as a potential allergic factor. This provides an indication that a patient with over-expressed H1 R should be aware of possible allergic reaction when applying homoharringtonine injection., (Copyright © 2014 John Wiley & Sons, Ltd.)

Membrane-associated prostaglandin (PG) E synthase (mPGE synthase)-2 catalyzes the conversion of PGH(2) primarily to PGE(2). The enzyme is activated by various sulfhydryl reagents including dithiothreitol, dihydrolipoic acid, and glutathione, and it is different from mPGE synthase-1 and cytosolic PGE synthase, both of which require specifically glutathione. Recently, other investigators reported that their preparation of mPGE synthase-2 containing heme converted PGH(2) to 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) rather than to PGE(2) [T. Yamada, F. Takusagawa, Biochemistry 46 (2007) 8414-8424]. As we examined presently, the heme-bound enzyme expressed and purified according to their method synthesized HHT from PGH(2), but also PGE(2) in a decreased amount. Whereas the PGE synthase activity was completely lost at 50 degrees C for 5 min, the HHT synthase activity remained even at 100 degrees C for 5 min. In contrast, when the heme-bound enzyme was purified in the presence of dithiothreitol, only PGE(2) was produced, but essentially no HHT was detected. Thus, native mPGE synthase-2 enzymatically catalyzes only the conversion of PGH(2) to PGE(2), but not to HHT, and heme is not involved in this reaction.

Homoharringtonine (HHT), first isolated from the Chinese evergreen Cephalotaxus harringtonia, has been demonstrated to have a broad antitumor activity in rodents and antileukemic effects in humans. We found that HHT was metabolized to an acid product [HHT-acid; 2'-hydroxy-2'-(alpha-acetic acid)-6'-hydroxy-6'-methylheptanoyl cephalotaxine] when incubated with either human plasma or mouse plasma in vitro. The conversion was faster, however, in mouse plasma, and was both time- and temperature-dependent. Boiled plasma prevented the conversion of HHT to HHT-acid, suggesting that the conversion was enzymatically mediated. When mice were given an intravenous (i.v.) injection of HHT (4 mg/kg), the HHT-acid metabolite was found in both plasma and urine. In mice, HHT-acid was detected in the plasma within 5 min of the i.v. injection of HHT and declined rapidly thereafter. The initial half-lives (t 1/2 alpha) of HHT and HHT-acid were 9 and 17 min, respectively. Twenty-four hours after HHT dosing in mice, approximately 29% of the dose was excreted in the urine as HHT and 20% as HHT-acid. High-pressure liquid chromatography and mass spectrometry were used to confirm the identity and quantify HHT and its metabolite, HHT-acid. The HHT concentration inhibiting 50% of the growth of human leukemic HL-60 cells was 20 ng/ml, while for HHT-acid it was 14,500 ng/ml, indicating that the acid form was more than 700 times less cytotoxic than HHT. The lethal dose of HHT affecting 50% (LD50) of mice was 6.7 mg/kg, but HHT-acid produced no apparent toxic effects at doses up to 280 mg/kg.

The metabolism of HH07A in the rat has been investigated by GC-MS. After oral administration of HH07A, the rat urine was passed through a macroporous XAD-2 resin column and eluted with methanol. The methanol extract was hydrolyzed with glucuronidase, extracted with dichloromethane and concentrated for TMS derivatization with GC-MS. The mass spectra of HH07A and its four metabolites as well as their derivatives were presented. The structures of the metabolites were proposed and the in vivo metabolic pathway of HH07A was given in Figure 6.

The metabolism of hainanensine derivative HH07A has been studied in vitro with rat microsomes. A method of HPLC-DAD was developed for screening the metabolites from the microsomal incubation system. Two metabolites were found according to their UV spectra. One of them was extracted and purified with preparative HPLC and TLC, its chemical structure was identified by UV, IR, MS and NMR.

The development of resistance accounts for therapy failure in the majority of advanced cases of neuroblastoma in children. A new transplantable murine C-1300 neuroblastoma cell line was developed in vitro, by repeated exposure of a sensitive cell line to increasing, but sublethal, doses of Homoharringtonine (HHT). The ED50 of the highly resistant cells for HHT, using a standard agar colony assay, is 480 ng/ml, compared with 13 ng/ml for the sensitive parental line. The resistant cells have cross-resistance to a number of other agents, including adriamycin, vinca alkaloids, melphalan, and CCNU. Western blot analysis revealed progressive increases in P-glycoprotein, parallel to the graded development of resistance with a 29-fold elevation in the highest resistant cells. High-performance liquid chromatography (HPLC) indicated that resistant cells have a significantly lower uptake of HHT than parental sensitive cells. cyclosporine A (CsA) and dipyridamole (DPM) could modulate the acquired resistance and completely restore the cytotoxic effects of HHT and adriamycin as determined by the clonogenic assay. The reversal of resistance by CsA and DPM was dose dependent. With the relative low toxicity of dipyridamole and CsA in doses required for modulation of resistance, these agents may be candidates for clinical utilization in chemotherapy of resistant neuroblastoma.

In this paper, the metabolism of homoharringtonine has been studied in vitro with liver microsomes from rats and rabbits. A method of HPLC-DAD (photodiode array detector) with gradient elution was developed for screening the metabolites from the microsomal incubation system. Only one main metabolite, whose formation was independent of NADPH and NADH, was found. The metabolite was extracted and purified with preparative HPLC technique. Its chemical structure was identified as 2'-hydroxy-2'(alpha-acetic acid)-6'-hydroxy-6'-methyl-heptanoyl cephalotaxine by UV, IR, NMR, MS analysis, and confirmed further by comparison with the authentic compound, the spectra and chromatographic behavior were all the same. The rates of the metabolite formation was markedly increased in the phenobarbital pretreated liver microsome system.

Clinical pharmacokinetics of homoharringtonine (HHT) were studied in eight patients who received uniformly labeled HHT at 3-4 mg/m2 (150 mu Ci) by continuous 6-hour infusion. The drug and metabolites were quantified by radiochemical and high-performance liquid chromatographic techniques. Computerized nonlinear least-square regression and curve stripping were used to characterize HHT and total [3H]HHT equivalent pharmacokinetics. Unchanged HHT in the plasma declined biphasically, with an alpha-half-life of 0.5 +/- 0.1 hours and a beta-half-life of 9.3 +/- 1.4 hours. The total clearance of HHT was 177.4 +/- 27.7 ml X hour-1 X kg-1, and the apparent volume of distribution, estimated from the area under the drug concentration versus time curve, was 2.4 +/- 0.4 L X kg-1. Correspondingly, the total [3H]HHT equivalent disappeared from the plasma in a triphasic manner. Compared with the pharmacokinetic parameters of unchanged HHT, the terminal half-life of total 3H was 67.5 +/- 7.5 hours, 7.4 times longer; the total clearance was 30.9 +/- 3.1 ml X hour-1 X kg-1, 5.5 times slower; but the volume of distribution by area was 2.7 +/- 0.1 L X kg-1, nearly the same. The 72-hour cumulative urinary excretion of total tritium was 28.2% of the administered dose and only 38.3% of this resided in unchanged HHT. Thus, urinary excretion was not a major route of elimination of HHT. Moreover, HHT underwent extensive metabolism; one major and two minor unidentified products were detected in both plasma and urine.

Generally tritiated homoharringtonine ([3H]HHT, 150 microCi, 430 micrograms) was administered intravenously to seven patients at varying times before surgical resection of malignant brain tumor. Plasma, urine, cerebrospinal fluid (CSF), and tumor specimens were obtained during surgery, and the concentrations of HHT, its major metabolite, and [3H]HHT equivalent were determined chromatographically and radiochemically. For [3H]HHT equivalent, the concentration in tumor ranged from 0.6 to 4.3 ng/g and the ratio of tumor to plasma concentration from 0.5 to 1.8. In one patient who had CSF available for drug determination, the CSF to plasma ratio of total [3H]HHT was 0.3 at 45 minutes after drug administration and less than 0.2 ng/ml was unchanged HHT. For unchanged HHT, drug concentration in tumor ranged from undetectable (4 patients) to 1.8 ng/g. A major metabolite of HHT was detectable in the tumor specimens of all the patients. These results indicate that homoharringtonine can penetrate into brain tumors; in 3 patients with brain tumors, the ratios of HHT concentration in the tumor to that in the concurrent plasma were greater than one.

Ten human leukemia-lymphoma cell lines were tested for the growth-inhibitory effects of harringtonine (HT). HT was most active against HL-60 acute promyelocytic leukemia cells and least active against DND-41 acute lymphoblastic leukemia cells, with a 70-fold differential activity. Sensitivity of the cell lines is, in decreasing order: HL-60 greater than RPMI-8402 greater than DND-39A congruent to ML-2 congruent to MOLT-3 congruent to KG-1 greater than Daudi congruent to NALL-1 greater than BALM-2 greater than DND-41. The cell lines with rapid cell growth tended to be more sensitive to HT. To further elucidate the selectivity of the differential sensitivity, uptake and release of HT were compared in HL-60 and DND-41 cells. Uptake of [3H]HT into HL-60 and DND-41 cells showed no difference; however, the binding of [3H]HT to cellular components was greater than 16-fold higher in HL-60 cells than DND-41 cells. There were also minor, but significant differences in the inhibition of [3H]leucine incorporation into proteins of these two cell lines in the presence of 1 microgram/ml HT. To test whether the biological effects of HT are related to the concentration of, or exposure time to, HT, KG-1 cells were exposed to HT for different periods of time and the growth-inhibitory effects were compared. Increasing exposure time from 1 h to 3 h resulted in a 100-fold decrease in concentration X exposure time (c X t) at ID50; from 3 h to 6 h, in a 20-fold decrease at ID70; and from 6 h to 24 h, in a 16-fold decrease at ID90. HT was not inactivated by cells up to 24 h. These results indicate that (a) the sensitivity of different cell lines to HT may be related to the degree of HT binding; and (b) the effects of HT are more dependent on exposure time than concentration. Continuous infusion is thus rational for clinical trials of this drug, and the degree of HT binding to leukemic cells may be predictive of clinical response.