STAT3 (inhibits phosphorylation at Tyr705) [1]
JAK1 (inhibits phosphorylation at Tyr1022/1023) [1]

Homoharringtonine exhibits antagonistic effects on both dose and time in inhibiting IL-6-induced STAT3 phosphorylation. Through mechanisms, homoharringtonine (HHT) causes apoptosis while suppressing colony formation, cell proliferation, and survival. Homoharringtonine's cytotoxicity was investigated using gefitinib-controlled human non-small cell lung cancer (NSCLC) cell lines, A549 (wild-type EGFR) and NCI-H1975 (type EGFR of H1975, L858R, and T790M). Using an IC50 of 3.7 μM, the MTT experiment revealed that homoharringtonine was mildly hazardous to A549 cells. With an IC50 of 0.7 μM, homoharringtonine more highly sensitively affected H1975 cells. Using the MTT assay, homoharringtonine exhibited dose- and time-dependent inhibition of A549 and H1975 cell growth and proliferation. Homoharringtonine dramatically reduced the clonogenic potential of A549 and H1975 cells at dose and time [1] according to the trypan blue intermittent test.

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HHT inhibited cell growth, cell viability and colony formation in A549 (wild-type EGFR) and NCI-H1975 (EGFR L858R/T790M) NSCLC cells. [1]
IC50 values for HHT: A549 cells = 3.7 μM; NCI-H1975 cells = 0.7 μM (by MTT assay). [1]
HHT rapidly reduced viable A549 and H1975 cells in a dose- and time-dependent manner (trypan blue exclusion assay). [1]
HHT significantly inhibited the clonogenic ability of A549 and H1975 cells (soft-agar colony formation assay). [1]
Unlike Gefitinib, HHT had no effect on EGFR phosphorylation (Y1173) in A549 cells; neither HHT nor Gefitinib downregulated EGFR phosphorylation in H1975 cells (western blot). [1]
HHT induced nuclear condensation and fragmentation (Hoechst 33258 staining). [1]
HHT treatment resulted in increased cytochrome C release into cytoplasm, decreased full-length Caspase 9 and Caspase 3, and increased PARP cleavage in A549 and H1975 cells in a dose-dependent manner (western blot). [1]
HHT disrupted mitochondrial transmembrane potential (JC-1 staining, reduced red fluorescence). [1]
Intracellular Ca2+ levels peaked at 4h after HHT treatment then decreased to basal level (FLUO-4 flow cytometry). [1]
HHT had no effect on STIM1 and Orai1 expression (western blot). [1]
HHT-induced apoptosis was significantly suppressed by pre-treatment with Z-VAD-FMK (20 μM), a general caspase inhibitor (MTT assay). [1]
MCL1 and Survivin expression decreased with HHT treatment in a concentration-dependent manner in both A549 and H1975 cells; BCL2 and BAX were not affected (western blot). [1]
HHT decreased STAT3 phosphorylation at Tyr705 (p-STAT3 Y705), but not at Ser727, and decreased Pim-1 and c-Myc expression (western blot). [1]
HHT significantly decreased JAK1 phosphorylation (Tyr1022/1023) but did not change PDK1, AKT, ERK, Smad2 or Smad3 phosphorylation (western blot). [1]
Combination of pan-JAK inhibitor Pyridone 6 (P6, 1 μM) and HHT (1 μM) for 12h further inhibited phosphorylated STAT3, activated Caspase 3, and augmented cell inhibition rates (western blot, MTT assay). [1]
Overexpression of constitutively active mutant STAT3 (STAT3C) exhibited resistance to HHT treatment; inhibition rate significantly decreased compared to control group (MTT assay). [1]
IL-6 production was elevated in H1975 cells (3723 pg/mL) compared to A549 and MCF-10A cells (ELISA). [1]
HHT pretreatment (4h) repressed IL-6 (5 ng/mL)-induced STAT3 phosphorylation and nuclear translocation (western blot, immunofluorescence, nuclear/cytoplasmic fractionation). [1]
HHT inhibited STAT3 transcription activity in H1975 cells (luciferase reporter assay). [1]
HHT inhibition of IL-6-induced STAT3 phosphorylation was dose-dependent (0-4 μM in A549; 0-2 μM in H1975) and time-dependent (0-4h pretreatment). [1]
HHT inhibition of IL-6-induced STAT3 phosphorylation was reversible; after 6h in HHT-free medium, STAT3 phosphorylation almost recovered (western blot). [1]
Recovery of STAT3 phosphorylation was not due to newly synthesized STAT3 protein (CHX treatment experiment). [1]
HHT and Docetaxel (DTX) combination showed synergistic effect (CI < 1) in A549 and H1975 cells (MTT assay, CalcuSyn software). [1]
HHT (2 μM or 0.25 μM) and DTX (8 nM) combination resulted in elevated Caspase 3 activation, increased PARP cleavage, and decreased STAT3 phosphorylation (western blot). [1]

Homoharringtonine (10 mg/kg) successfully stopped the growth of tumors as compared to gefitinib or vehicle control (P<0.05). Furthermore, mice treated with homoharringtonine (HHT) did not lose body weight, suggesting that homoharringtonine has no discernible effect. Every mouse was put to sleep, separated, examined, and tumor samples were gathered. Western blot of tumor-expressing cells will be used to ascertain whether homoharringtonine reduces STAT3 phosphorylation. When compared to the vehicle control or gefitinib treatment groups, the homoharringtonine-treated group exhibited a substantial decrease in both STAT3 phosphorylation and MCL1 levels. In parallel, treatment with harringtonine suppressed the phosphorylation amplifiers for ERK1/2 and AKT1/2/3, which is in line with the results above. Tumor tissues were frozen, and sections were diluted at 10 μM for fluorescence histochemistry in order to investigate STAT3 phosphorylation in deeper detail in xenograft tumor samples that were treated differently. therapy with homoharringtonine suppressed STAT3 phosphorylation more than either gefitinib therapy or tumor immune control [1].

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HHT (10 mg/kg) efficiently repressed tumor growth in nude mice bearing H1975 xenografts compared to vehicle control or Gefitinib (30 mg/kg) (P < 0.05); tumor volumes calculated every two days for 3 weeks, 5 times per week. [1]
HHT treatment did not reduce mouse body weight, suggesting no apparent side effect. [1]
Levels of STAT3 phosphorylation and MCL1 from HHT treatment group were significantly decreased compared to vehicle control or Gefitinib treatment (western blot of tumor lysates). [1]
AKT1/2/3 and ERK1/2 phosphorylation was not inhibited by HHT treatment (western blot of tumor lysates). [1]
HHT treatment inhibited STAT3 phosphorylation compared to vehicle control or Gefitinib treatment (fluorescent immunohistochemistry of tumor sections). [1]

Cytotoxicity was determined by MTT assay. Cells were seeded into 96-well plates and precultured for 24h, then treated with HHT for 24h or 48h. Absorbance was measured at 570nm. Cell death rate was calculated as: (average A570 of control group - average A570 of experimental group)/(average A570 of control group - average A570 of blank group). [1]
Cell viability was estimated by trypan blue dye exclusion assay. Cell suspension (0.5 mL, 1×10^6 cells/mL) was mixed with 0.1 mL of 0.4% trypan blue dye, incubated for 3min at room temperature, and counted in a hemacytometer. Cell viability (%) = (number of viable cells/number of total cells) × 100%. [1]
For Hoechst 33258 staining, cells were stained at 10 mg/mL for 10min and observed by fluorescence microscope. [1]
Soft-agar colony formation assay: Cells (1000 cells/plate) were suspended in 1 mL medium containing 0.3% low-melting-point agarose and 10% FBS with indicated HHT concentration, plated on a bottom layer containing 0.6% agarose and 10% FBS in 6-well plates. After 2 weeks, plates were stained with 0.5 mL of 0.005% crystal violet for >1h and colonies counted under light microscope. [1]
For intracellular Ca2+ measurement, H1975 cells were treated with HHT, harvested with cell dispersant, incubated with FLUO-4 for 60s, and analyzed by flow cytometry using the FL1 channel. [1]
ELISA for IL-6: Conditioned medium was collected after 12h culture, centrifuged at 3,000 rpm for 15min, and analyzed with Human IL-6 Quantikine ELISA Kit. Culture cells were harvested and counted for normalization. [1]
Cytoplasmic fractionation for cytochrome C detection: Cells were incubated with cytosol lysis buffer (0.01% digitonin, 2mM EDTA, 1mM PMSF, protease inhibitors in 1× PBS) for 3min at room temperature, centrifuged at 16,000×g for 2min, and supernatant (cytoplasmic fractions) collected for western blot. [1]
Nuclear and cytoplasmic fractionation: Cells were scraped in cold PBS, centrifuged at 2,000 rpm for 4min at 4°C, resuspended in Buffer A (10mM HEPES pH7.9, 10mM KCl, 0.1mM EDTA, 1mM DTT, protease inhibitors) on ice for 10min, then 10 μL of 10% NP-40 added. Supernatant collected by centrifugation at 6,000 rpm for 4min at 4°C (cytoplasmic fraction). Pellet resuspended in Buffer B (20mM HEPES pH7.9, 0.4M NaCl, 1mM EDTA, 1mM DTT, protease inhibitors) on ice for 15min (nuclear fraction). Isolation effect assessed by western blot using Lamin B and GAPDH. [1]
JC-1 apoptosis detection for mitochondrial transmembrane potential: After HHT treatment for 24h, cells were washed with PBS, 100 μL JC-1 work solution added, cultured for 20min, washed with incubation buffer, and observed by fluorescence microscope. [1]
Immunofluorescence staining: Cells fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, primary antibody against pSTAT3 (Y705) (1:50) added overnight at 4°C, FITC-conjugated secondary antibody used, nucleus stained with DAPI, observed by confocal microscope. [1]
STAT3 luciferase reporter assay: H1975 cells were transfected with pSTAT3-TA-luc plasmids using Lipofectamine 2000. After 4h, medium changed, cells treated with HHT for 4h followed by IL-6 for 20h. Firefly luciferase activities assayed using Luciferase Assay System. [1]
Drug combination assay: Combination index (CI) calculated by Chou-Talalay equation. CI = (D)HHT/(Dx)HHT + (D)DTX/(Dx)DTX. CI < 1 indicates synergism; CI = 1 additive; CI > 1 antagonism. Calculated using CalcuSyn software (Version 2.1). [1]

All animal studies were conducted according to protocols approved by the Tsinghua University Animal Care and Use Committee, complying with the rules of REGULATIONS FOR THE ADMINISTRATION OF AFFAIRS CONCERNING EXPERIMENTAL ANIMALS (Approved by the State Council of China). [1]
Female and male nude immunodeficient mice (nu/nu), 6-8 weeks old, were purchased from Guangdong Province Medical Animal Center and fed/monitored in a specific pathogen-free environment. [1]
Mice were injected subcutaneously with NSCLC H1975 cells (2.5×10^6) suspended in 100 μL RPMI-1640 medium into the right flank of each mouse. [1]
Treatments were started when tumors reached a palpable size. Mice were randomly divided into three groups (n=10) and treated with HHT (10 mg/kg), Gefitinib (30 mg/kg), or vehicle control for 3 weeks, 5 times per week. [1]
Tumor volume was estimated using vernier caliper measurements of the longest perpendicular tumor diameters with the formula: 4π/3 × (width/2)^2 × (length/2). [1]
Animals were sacrificed when tumors reached 2 cm or if mice appeared moribund. Tumors were excised; cells were lysed for western blot and immunohistochemistry. [1]
For fluorescent immunohistochemistry of tumor tissue: Frozen sections (10 μm) were fixed with 4% paraformaldehyde for 1h, dehydrated in 10%, 20%, and 30% sucrose solutions, wrapped in Tissue-Tek O.C.T. compound, frozen at -80°C, sectioned (4-10 μm), blocked with 3% BSA/0.2% Triton X-100 in PBS for 1h, incubated with anti-pSTAT3 (Y705) (1:50) at 4°C overnight, FITC-conjugated secondary antibody used, nucleus stained with DAPI, observed by confocal microscope. [1]

Absorption, Distribution and Excretion
Absorption of homoharringtonine was not quantitatively analyzed, but maximum concentration was reached approximately 30 minutes later. The primary elimination pathway of homoharringtonine is unclear, but renal elimination was less than 15%. The steady-state volume of distribution (Vd) of homoharringtonine was 141 ± 93.4 L. The clearance of homoharringtonine was not quantitatively analyzed. Metabolism/Metabolites Homoharringtonine is minimally metabolized in the liver, primarily through hydrolysis by plasma esterases to 4'-dimethylhomoharringtonine (4'-DMHHT). Biological Half-Life Following subcutaneous injection, the half-life of homoharringtonine is approximately 6 hours.

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Protein Binding
Plasma protein binding rate ≤50%. Toxicity Data Mice (intraperitoneal injection): LD50: 1960 μg/kg Mice (intraperitoneal injection): LD50: 6.5 mg/kg

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HHT treatment did not reduce mouse body weight, suggesting no apparent side effect (in nude mice bearing H1975 xenografts, HHT 10 mg/kg, 5 times per week for 3 weeks). [1]

[1]. Homoharringtonine induces apoptosis and inhibits STAT3 via IL-6/JAK1/STAT3 signal pathway in Gefitinib-resistant lung cancer cells. Sci Rep. 2015 Jul 13;5:8477.

Omacetaxine mepesuccinate is an alkaloid ester derived from cephalotaxine (Cephalotaxus harringtonia) and used to treat chronic or accelerated phase chronic myeloid leukemia. It possesses antitumor, protein synthesis inhibitory, apoptosis-inducing, and anticoronavirus activities. It is an alkaloid ester, tertiary alcohol, organic heteropentane compound, and enol ether. Its function is similar to that of cephalotaxine. Omacetaxine mepesuccinate (formerly known as HHT or high-harringtonine) is a cephalotaxine ester and protein synthesis inhibitor, and its clinical activity as a single agent in hematologic malignancies has been demonstrated. Omacetaxine mepesuccinate is synthesized from cephalotaxine, which is extracted from the leaves of plants in the genus Taxus. In October 2005, omaxitabine mepiquat succinate received Orphan Drug Designation from the European Medicines Agency (EMEA) for the treatment of chronic myeloid leukemia (CML). Subsequently, in March 2006, it received Orphan Drug Designation from the U.S. Food and Drug Administration (FDA) for the treatment of CML. In November 2006, omaxitabine mepiquat succinate received Fast Track Designation from the FDA for the treatment of CML. Most recently, in October 2012, omaxitabine methyl ester succinate, marketed under the brand name Synribo, was approved by the FDA for the treatment of patients with accelerated or chronic phase CML who are intolerant and/or resistant to two or more tyrosine kinase inhibitors. Omaxitabine methyl ester succinate has been reported to be found in Taxus chinensis (Cephalotaxus fortunei) and Taxus harringtonia, and relevant data are available. Omaxistatin methyl succinate is a semi-synthetic preparation of goharingtonine, a cytotoxic plant alkaloid isolated from the evergreen tree Taxus, and possesses potential antitumor activity. Omaxistatin binds to the 80S ribosome in eukaryotic cells, inhibiting protein synthesis by interfering with chain elongation. This drug can also induce differentiation and apoptosis in certain cancer cell types. A semi-synthetic derivative of haringtonine, it acts as a protein synthesis inhibitor, inducing apoptosis in tumor cells. Used to treat chronic myeloid leukemia. See also: Omaxistatin (with the active moiety).

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Drug Indications
For patients who are intolerant and/or resistant to two or more tyrosine kinase inhibitors used to treat accelerated or chronic phase chronic myeloid leukemia.
FDA Label
Philadelphia chromosome-positive chronic myeloid leukemia, for patients carrying the T315I Bcr-Abl kinase domain mutation and resistant to prior imatinib treatment. Mechanism of Action Homoharringtonine inhibits protein synthesis by not directly binding to Bcr-Abl. It binds to the A-site cleft of the large ribosomal subunit, affecting chain elongation and preventing protein synthesis. Pharmacodynamics The pharmacodynamics of homoharringtonine is not fully elucidated. Homoharringtonine is known to participate in protein synthesis inhibition, which contributes to its antitumor activity.

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Homoharringtonine (HHT; 4-methyl (2R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl)butanedioate) is a cephalotaxine ester isolated from Cephalotaxus harringtonia (a plant widely distributed in China and Japan). C. harringtonia seeds are toxic to humans and used for Chinese traditional medicine. [1]
Clinically, HHT has exhibited efficient inhibition activity against acute myelocytic leukemia (AML) and chronic myeloid leukemias (CML) alone or combined with granulocyte colony-stimulating factor, cytarabine, or interferon-α. [1]
Previous studies showed that HHT could inhibit protein synthesis by preventing aminoacyl-tRNAs binding to the peptidyl-transferase A-site cleft in the ribosome. [1]
HHT was more efficient in cancer cells with wild-type p53 in a high-throughput screening assay with 55 NCI cell lines. [1]
Possible mechanisms of HHT in anti-myeloma may be inhibition of AKT phosphorylation and several AKT target genes including NF-κB, XIAP, cIAP and Cyclin D1, and inhibition of MCL1 protein synthesis and induction of apoptosis in chronic lymphocytic leukemia. [1]
This study demonstrated that HHT has potential in Gefitinib-resistant NSCLC treatment by inducing apoptosis and inhibiting STAT3 via IL-6/JAK1/STAT3 signal pathway. [1]

C29H39NO9

545.6213

545.262

26833-87-4

285033

Off-white to yellow solid powder

1.3±0.1 g/cm3

713.1±60.0 °C at 760 mmHg

144-146ºC

385.1±32.9 °C

0.0±2.4 mmHg at 25°C

1.605

2.7

2

10

11

39

968

4

CC(C)(CCC[C@@](CC(=O)OC)(C(=O)O[C@H]1[C@H]2C3=CC4=C(C=C3CCN5[C@@]2(CCC5)C=C1OC)OCO4)O)O

HYFHYPWGAURHIV-JFIAXGOJSA-N

InChI=1S/C29H39NO9/c1-27(2,33)8-5-10-29(34,16-23(31)36-4)26(32)39-25-22(35-3)15-28-9-6-11-30(28)12-7-18-13-20-21(38-17-37-20)14-19(18)24(25)28/h13-15,24-25,33-34H,5-12,16-17H2,1-4H3/t24-,25-,28+,29-/m1/s1

(S)-1-((11bS,12S,14aR)-13-methoxy-2,3,5,6,11b,12-hexahydro-1H-[1,3]dioxolo[4',5'

Omacetaxine mepesuccinate Synribo CGX-635 Myelostat CGX635Ceflatonin homoharringtonine, 3H-labeled, (3(R))-isomer

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 50 mg/mL (~91.64 mM)
H2O : ~1.4 mg/mL (~2.57 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.58 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.58 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1.67 mg/mL (3.06 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)