Do Naturally Modified Nucleotides Contribute to Stabilizing Complexes between Ribosomes and Small Molecules? A Case Study with the Antitumor Drug Homoharringtonine.

Modified nucleotides are ubiquitous with RNAs, also in contact with drugs that target the ribosome. Whether this represents a stabilization of the drug-ribosome complex, thus affecting the drug's affinity and possibly also intrinsic efficacy, remains an open question, however. The challenge of answering this question has been taken here with the only human-ribosome-targeting small-molecule currently in clinical use, the antitumor plant alkaloid homoharringtonine (HHT). The approach consisted in dissecting HHT-nucleotide interaction energies from QM-MM simulations in explicit water. What emerged is a network of mostly weak interactions of the large, branched HHT with standard nucleotides and a single modified nucleotide, out of the four ones present at PCT's A site. This is unlike the case of the small, compact marine antitumor alkaloid agelastatin A, which displays only a few, albeit strong, interactions with site-A ribosome nucleotides. This should aid tailoring drugs targeting the ribosome.

Enantioselective semisynthesis of novel cephalotaxine esters with potent antineoplastic activities against leukemia.

Cephalotaxine-type alkaloids (CTAs), represented by homoharringtonine (HHT, 1), display potent efficacy against different types of leukemia cells. In this study, a method for hydrogenation of ß-substituted itaconic acid monoesters with chiral Ru[DTBM-SegPhos](OAc)2 was developed. This metal-catalyzed asymmetric hydrogenation enabled the convenient semisynthesis of novel cephalotaxine derivatives with chiral 2'-substituted-succinic acid 4-mono-methyl esters as side chains. The preliminary structure-activity relationship (SAR) of the compounds' antineoplastic activities was studied. Eventually, we discovered compound 10b with potent antineoplastic activities against leukemia and broadly anticancer activities against a panel of cancer cells. Our study provided a highly enantioselective process enabling the semisynthesis of cephalotaxine derivatives, which are interesting for further study on a scientific basis.

Hainanolide inhibits the progression of colon cancer via inducing the cell cycle arrest, cell apoptosis and activation of the MAPK signaling pathway.

Hainanolide (HN) is a norditerpenoid metabolite extract from Cephalotaxus fortunei Hook. f. C. fortunei Hook. f. is renowned for the active alkaloids, such as harringtonine (HT) and homoharringtonin (HTT), which have been clinically used to treat chronic myeloid leukemia. Nowadays, diterpenoids, another important metabolite, attracted the attention of chemists. Among them, Hainanolide (HN), a cephalotane-type diterpenoid, has been proven to possess potent antitumor activities. However, the underlying therapeutic mechanisms of HN in anti-tumor have not been investigated yet. Our present study demonstrated that HN inhibited HCT-116 and HCT-15 cell proliferation in a dose- and time-dependent manner. Further studies demonstrated that HN can induce G2/M phase arrest and alter the Cdc25C/Cdc2/CyclinB1 proteins. Western blot indicated that HN promoted apoptosis by up-regulating Bax and down-regulated Bcl-2. And the caspase-3 and caspase-9 activities of HCT-116 and HCT-15 cells were increased. Transcriptome analysis is used to reveal the possible mechanism. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested the genes were mainly enriched in the MAPK signaling pathway. Certainly, HN activates MAPK signaling pathway. In vivo, HN prevented the AOM/DSS-induced tumorigenesis of colon cancer in C57BL/6 mice. Our study indicated that HN inhibits the progression of colon cancer cells by blocking the cell cycle, inducing apoptosis, and activating the MAPK pathway. This study provides a theoretical and experimental scientific basis for future investigations of the antitumor effects of HN against colon cancer.

[Two novel cephalotaxine-type alkaloids from Cephalotaxus sinensis].

Silica gel, octadecyl-silica(ODS), Sephadex LH-20, and semi-preparative high performance liquid chromatography(HPLC) was performed to isolate nine cephalotaxine-type alkaloids from Cephalotaxus sinensis: 8-oxodeoxyharringtonine(1), 8-oxonordeoxyharringtonine(2), cephafortunine A(3), 8-oxocephalotaxine(4), deoxyharringtonine(5), acetylcephalotaxine(6), cephalotaxine(7), epicephalotaxine(8), and cephalotaxinone(9). Compounds 1 and 2 were identified for the first time and their structures were determined by high-resolution-electrospray ionization-mass spectrometry(HR-ESI-MS), nuclear magnetic resonance(NMR), and electronic circular dichroism(ECD). Compounds 1-3 and 5 significantly inhibited the transcription of nuclear factor kappa B(NF-κB), with the half-maximal inhibitory concentration(IC_(50)) of(3.91±0.70),(2.99±0.45),(7.84±0.51), and(1.46±0.17) µmol·L~(-1), respectively.

Cephalotaxine homologous alkaloids from seeds of Cephalotaxus oliveri Mast.

Six undescribed isoquinoline alkaloids, named as cephaloliverines A-F, were isolated from the seeds of Cephalotaxus oliveri. They were identified by NMR and MS spectroscopic data analyses, combined with the time-dependent density functional theory ECD calculation for cephaloliverines A and B and also by X-ray crystal diffraction for cephaloliverine E. Biosynthetic considerations suggest that cephaloliverines A-D are homologous of cephalotaxine-, homoerythrina- and Erythrina-type alkaloids. The performed bioassay revealed no cytotoxic activity against cancer cells and no neuroprotective properties on HEI-OC-1 cells model.

Harringtonine Ester Derivatives with Enhanced Antiproliferative Activities against HL-60 and HeLa Cells.

Harringtonine (HT), produced from Cephalotaxus species, is known to exhibit potent antiproliferative activity against myeloid leukemia cells by inhibiting protein synthesis. A previous study using acute promyelocytic leukemia (HL-60) cells raised the possibility that the C-5' methyl group of HT plays an important role in regulating leukemia cell line antiproliferative activity. In order to investigate the effect of hydrocarbon chains at C-5' on the resultant activity, the C-5' methyl group was replaced with various straight- and branched-chain hydrocarbons using the corresponding alcohols, and their antiproliferative activity against HL-60 and HeLa cells was investigated. As a result, 4'-n-heptyl-4'-demethylharringtonine (1f, n-heptyl derivative) showed the most potent cytotoxicity among the HT ester derivatives produced, with IC50 values of 9.4 nM and 0.4 µM for HL-60 and HeLa cells, respectively. Interestingly, the cytotoxicity of derivative 1f against HL-60 and HeLa cells respectively was ∼5 (IC50 = 50.5 nM) and ∼10 times (IC50 = 4.0 µM) those of HT and ∼2 (IC50 = 21.8 nM) and ∼4 times (IC50 = 1.7 µM) more than homoharringtonine (HHT). These results demonstrate the potential of the derivative 1f as a lead compound against leukemia.

Cephalotaxine-type alkaloids with antiproliferation effects from the branches and leaves of Cephalotaxus fortunei var. alpina.

Eight cephalotaxine-type alkaloids (1-8), including two new compounds cephafortunines A and B (1-2), were isolated from the branches and leaves of Cephalotaxus fortunei var. alpina. Their structures were identified by a series of spectroscopic methods (MS, UV, IR, 1D, and 2D NMR) and comparison with the reported data of known analogs. The absolute configurations of 1 and 2 were determined by electronic circular dichroism (ECD) calculations. 1-8 were evaluated for their in vitro antiproliferation effects against two human leukemia cell lines (U937 and HL-60). All compounds showed different levels of antiproliferation effects against U937 cells with GI50 values of 4.21-23.70 µM. 4 and 5 were the most active against U937 cells with GI50 values of 4.21 and 6.58 µM and against HL-60 cells with GI50 values of 6.66 and 6.70 µM, respectively. 4 and 5 arrested HL-60 cell cycle in G0/G1 phase.

Cephalotaxine-type alkaloids from the seeds of Cephalotaxus fortunei and their cytotoxic activities.

Six new Cephalotaxus alkaloids, including five cephalotaxine-type alkaloids, and one homoerythrina-type alkaloid, along with six known analogues, were isolated from the seeds of Cephalotaxus fortunei. Their structures were elucidated by combination of spectroscopic data analyses, time-dependent density functional theory (TDDFT) ECD calculation, and single-crystal X-ray diffraction. Cephalofortine B represents the first example of C-5 epi-cephalotaxine-type alkaloid. All isolated compounds were tested for cytotoxicities against HCT-116, A375, and SK-Mel-28 cell lines. Cephalofortine E showed moderate activity against HCT-116 cell line, with an IC50 value of 7.46 ± 0.77 µM.

Translation elongation rate varies among organs and decreases with age.

There has been a surge of interest towards targeting protein synthesis to treat diseases and extend lifespan. Despite the progress, few options are available to assess translation in live animals, as their complexity limits the repertoire of experimental tools to monitor and manipulate processes within organs and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type-specific translation elongation rates in vivo. It is based on time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ more than 50% among mouse organs and determined them to be 6.8, 5.0 and 4.3 amino acids per second for liver, kidney, and skeletal muscle, respectively. We further found that the elongation rate is reduced by 20% between young adulthood and mid-life. Thus, translation, a major metabolic process in cells, is tightly regulated at the level of elongation of nascent polypeptide chains.

Isoharringtonine Induces Apoptosis of Non-Small Cell Lung Cancer Cells in Tumorspheroids via the Intrinsic Pathway.

Lung cancer is the major cause of cancer-associated death worldwide, and development of new therapeutic drugs is needed to improve treatment outcomes. Three-dimensional (3D) tumorspheroids offer many advantages over conventional two-dimensional cell cultures due to the similarities to in vivo tumors. We found that isoharringtonine, a natural product purified from Cephalotaxus koreana Nakai, significantly inhibited the growth of tumorspheroids with NCI-H460 cells in a dose-dependent manner and induced apoptotic cell death in our 3D cell culture system. On the other hand, A549 tumorspheroids displayed low sensitivity to isoharringtonine-induced apoptosis. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor known to regulate proliferation and apoptosis of cancer cells. We observed that knockdown of NR4A1 dramatically increased isoharringtonine-induced cancer cell death in A549 tumorspheroids by activating the intrinsic apoptosis pathway. Furthermore, treatment with combined isoharringtonine and iNR4A1 significantly inhibited multivulva formation in a Caenorhabditis elegans model and tumor development in a xenograft mouse model. Taken together, our data suggest that isoharringtonine is a potential natural product for treatment of non-small cell lung cancers, and inhibition of NR4A1 sensitizes cancer cells to anti-cancer treatment.

Harringtonine Inhibits Zika Virus Infection through Multiple Mechanisms.

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-Barré 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.

Apoptotic and cell cycle response to homoharringtonine and harringtonine in wild and mutant p53 hepatocarcinoma cells.

This study aimed to evaluate the modes of action of harringtonine (HT) and homoharringtonine (HHT) alkaloids in cell with wild (HepG2/C3A) and mutant p53 (HuH-7.5). We performed assays for cytotoxicity, genotoxicity, induction of apoptosis, cell cycle phase, and membrane integrity. Obtained data were compared with the relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. The relative expression of the genes showed an increase in apoptosis-inducing mRNAs, such as TNF and BBC3, as well as a reduction in BCL2 and BAK. The mRNAs of CYP2E1 and CYP2C19 xenobiotic metabolism genes increased in both lineages, while CYP3A4 increased only in the HuH-7.5 lineage. The mRNA expression of endoplasmic reticulum (ER) stress genes (ERN1 and EIF2AK3) was shown to increase in HHT and HT treatments. A similar increase was recorded in the mRNA expression of the TRAF2 gene. The changes observed in this study support the hypothesis that ER stress was more strongly associated with TNF induction, causing cell death by apoptosis in p53 mutant cells. This result with wild and mutant p53 cells may have clinical implications in the use of these compounds.

Ribosome profiling in archaea reveals leaderless translation, novel translational initiation sites, and ribosome pausing at single codon resolution.

High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.

Anti-varicella-zoster virus activity of cephalotaxine esters in vitro.

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.

Inhibition of Proliferation in U937 Cells Treated by Blue Light Irradiation and Combined Blue Light Irradiation/Drug.

The cell viability and apoptosis of tumor U937 cells treated by blue light (BL) irradiation have been examined. BL irradiation can specially inhibit the proliferation and promote the apoptosis of U937 cells, relating to the production of reactive oxygen species (ROS) and the decline of mitochondrial membrane potential (ΔΨm). The apoptosis is further associated with varying downregulated B-cell lymphoma-extra large (Bcl-XL) and B-cell lymphoma 2 (Bcl-2) genes, upregulated Bcl-2-associated X (Bax) gene, the activation of caspase-3 and caspase-9, and the cleavage of poly (ADP-ribose) polymerase (PARP) by the BL irradiation process. Moreover, BL irradiation induced proliferation inhibition is higher than that treated by a common chemotherapeutic drug of homoharringtonine (HHT). When we synergize BL irradiation with HHT (BL-HHT), a higher proliferation inhibition is obtained than that treated by BL irradiation or HHT alone. These results are helpful for establishing a low toxicity and high efficiency strategy of BL irradiation for clinical treatment of acute myeloid leukemia, not limited to U937 cells.

Homoharringtonine regulates the alternative splicing of Bcl-x and caspase 9 through a protein phosphatase 1-dependent mechanism.

BACKGROUND: Homoharringtonine (HHT) is a natural alkaloid with potent antitumor activity, but its precise mechanism of action is still poorly understood. METHODS: We examined the effect of HHT on alternative splicing of Bcl-x and Caspase 9 in various cells using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The mechanism of HHT-affected alternative splicing in these cells was investigated by treatment with protein phosphatase inhibitors and overexpression of a protein phosphatase. RESULTS: Treatment with HHT downregulated the levels of anti-apoptotic Bcl-xL and Caspase 9b mRNA with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-xS and Caspase 9a in a dose- and time-dependent manner. Calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), significantly inhibited the effects of HHT on the alternative splicing of Bcl-x and Caspase 9, in contrast to okadaic acid, a specific inhibitor of PP2A. Overexpression of PP1 resulted in a decrease in the ratio of Bcl-xL/xS and an increase in the ratio of Caspase 9a/9b. Moreover, the effects of HHT on Bcl-x and Caspase 9 splicing were enhanced in response to PP1 overexpression. These results suggest that HHT-induced alternative splicing of Bcl-x and Caspase 9 is dependent on PP1 activation. In addition, overexpression of PP1 could induce apoptosis and sensitize MCF7 cells to apoptosis induced by HHT. CONCLUSION: Homoharringtonine regulates the alternative splicing of Bcl-x and Caspase 9 through a PP1-dependent mechanism. Our study reveals a novel mechanism underlying the antitumor activities of HHT.

[Combined Effect of Bortezomib and Homoharringtonine on K562 Cells and their Mechanisms].

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

Isoharringtonine inhibits breast cancer stem-like properties and STAT3 signaling.

OBJECTIVES: Breast cancer stem cells (BCSCs) contribute to breast cancer progression, relapse, and treatment resistance. Identification of the natural inhibitory components of BCSCs is therefore critical for clinical treatment. Here, we investigated whether isoharringtonine (IHT) had inhibitory effects on BCSCs in breast cancer cell lines. METHODS: HCC1806, HCC1937, and MCF7 cells were treated with IHT. The proliferation and the migration of cells were detected by MTS assay and wound healing migration assay, respectively. The proportions of BCSCs were determined by flow cytometry and tumor sphere formation assay. Using real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting, the expression of Nanog and activation of STAT3 were detected, respectively. RESULTS: Results showed that IHT inhibited the proliferation of HCC1806, HCC1937, and MCF-7 cells, and suppressed the migration of HCC1806 and HCC1937 cells in a dose-dependent manner. IHT treatment decreased the proportion of BCSCs in MCF-7, HCC1806, and HCC1937 cells. In addition, the mRNA level of Nanong was significantly downregulated after IHT treatment. We also found an inhibitory effect of IHT on STAT3 activation. CONCLUSION: IHT inhibited the proliferation, migration, and BCSC proportion of breast cancer cell lines via inhibition of the STAT3/Nanong pathway.

Sodium-Periodate-Mediated Harringtonine Derivatives and Their Antiproliferative Activity against HL-60 Acute Leukemia Cells.

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.