OBJECTIVETo establish the best hydrolytic conditions from phorbol esters.

METHODThe orthogonal experiment was used to optimize 4 factors, which were reaction time, ratio of solid-to-liquid, hydrolytic times, and temperature. Diamonsil C18 column (4. 6 mm x 250 mm, 5 microm) was used and the mobile phase was consisted of acetonitrile and water for HPLC detection. The detection wavelength was set at 234 nm, the flow rate was 1 mL x min(-1), and the column temperature was 25 degrees C.

RESULTThe optimum conditions were 10 h of reaction time, 1:6 of solid-to-liquid (BaOH/MeOH) ratio, 25 degrees C of temperature, and one time of hydrolysis. There was a good linear relationship of phorbol in the range of 4.28-107 mg x L(-1) (r = 0.999 9), and the average recovery was 97.89%, with RSD 0.78%.

CONCLUSIONThe method is steady, reliable and reproducible, and it provides a mean for future study.

Chromatography, High Pressure Liquid ; Hydrolysis ; Phorbol Esters ; analysis ; chemistry

Tigliane type macrocyclic diterpenoids with special structures and diverse bioactivities are mainly extracted from plants of Euphorbiaceae and Thymelaeaceae. According to the different functional groups, they can be classified into types of phorbol esters, C-4 deoxyphorbol esters, C-12 deoxyphorbol esters, C-16 or C-17 substituted phorbol esters and others. Most of them present promising antiviral activities and cytotoxic activities and are expected to be developed as candidates for anti-AIDS, anti-tuberculosis, and anti-tumor clinical trials, demonstrating great potential for the application in healthcare. This paper reviews 115 novel tigliane-type diterpenoids discovered since 2013 and summarize their chemical structures and bioactivities, aiming to lay a foundation for further development and utilization of these compounds and provide new ideas for the development of clinical drugs.
Phorbols ; Molecular Structure ; Diterpenes/chemistry* ; Antiviral Agents ; Phorbol Esters

In order to investigate the action mechanism of protein kinase C on K+ channel in osteoblastic cell, effects of phorbol 12,13-dibutyrate on human osteoblast-like cells (G292) were studied by patch clamp technique with cell-attached configuration. In this experiment, 45pS ion channel was dominant in G292 cell line according to their approximate conductances in symmetrical 140mM KCl saline at holding potential of 60mV. In current-voltage relationship, reversal potential was 5.5mV at the condition of potassium enriched saline in the pipette and -27 mV at the condition of standard extracellular saline in the pipette. Phorbol 12, 13-dibutyrate 10 nM increased the open probability of 45 pS channel and staurosporine, an inhibitor of protein kinase C, suppressed this effect. Phorbol 12,13-dibutyrate moved the reversal potential of 45pS channel to more negative potential and increased the single channel current at the same membrame potential. In order to check the activation of protein kinase C in G292 cell by phorbol 12,13-dibutyrate, western blot of protein kinase C was performed. Phorbol 12,13-dibutyrate 0.1 micrometer translocated protein kinase C from cellular compartment to membrane compartment of the cell. These findings suggest that phorbol 12,13-dibutyrate, one of phorbol esters, activate 45pS channel in G292 cell and affect cell membrane potential, that regulate cellular function.
Blotting, Western ; Cell Line ; Cell Membrane ; Humans ; Ion Channels ; Membranes ; Osteoblasts ; Phorbol 12,13-Dibutyrate ; Phorbol Esters ; Potassium ; Protein Kinase C ; Staurosporine

The aim of the present study was to investigate the influence of osmotic pressure on myocardial contractility and the possible mechanism. Electrical stimulation was used to excite papillary muscles of the left ventricle of Sprague-Dawley (SD) rats. The contractilities of myocardium in hyposmotic, isosmotic, and hyperosmotic perfusates were recorded. The influences of agonist and antagonist of the transient receptor potential vanilloid 4 (TRPV4) on the contractility of myocardium under hyposmotic, isosmotic and hyperosmotic conditions were observed. The results were as follows: (1) Compared with that under isosmotic condition (310 mOsm/L), the myocardial contractility was increased by 11.5%, 21.5% and 25.0% (P<0.05) under hyposmotic conditions when the osmotic pressure was at 290, 270 and 230 mOsm/L, respectively; and was decreased by 16.0%, 23.7% and 55.2% (P<0.05) under hyperosmotic conditions when the osmotic pressure was at 350, 370 and 390 mOsm/L, respectively. (2) When ruthenium red (RR), an antagonist of TRPV4, was added to the hyposmotic perfusate (270 mOsm/L), the positive inotropic effect of hyposmia was restrained by 36% (P<0.01); and when RR was added to the hyperosmotic perfusate (390 mOsm/L), the inhibitory effect of hyperosmia on myocardial contractility was increased by 56.1% (P<0.01). (3) When 4-α-phorbol-12,13-didecanoate (4α-PDD), an agonist of TRPV4, was added to the isosmotic perfusate (310 mOsm/L), the myocardial contractility did not change; and when 4α-PDD was added to the hyperosmotic perfusate (390 mOsm/L), the inhibition of myocardial contractility by hyperosmia was increased by 27.1% (P<0.01). These results obtained indicate that TRPV4 is possibly involved in the osmotic pressure-induced inotropic effect.
Animals ; Heart ; physiology ; Myocardial Contraction ; physiology ; Osmotic Pressure ; Phorbol Esters ; pharmacology ; Rats ; Rats, Sprague-Dawley ; TRPV Cation Channels ; physiology

The effects of phorbol esters were studied in rabbit carotid artery to evaluate the action of protein kinase C on the regulation of vascular tone by isoproterenol. The vascular rings, 2 mm in width, were myographied isometrically in an isolated organ bath and the effects of phorbol 12,13-dibutyrate(PDBu) and phorbol 12-myristate 13-acetate(PMA) were determined. Isoproterenol, a beta adrenergic agonist, relaxed the vessel which was precontracted by phenylephrine, but not that by phorbol esters. The action of isoproterenol was attenuated by removal of endothelium or pretreatment with methylene blue or nitro-L-arginine. The pretreatment with phorbol esters at concentrations which did not induce contraction, decreased isoproterenol-induced relaxation of vascular rings with or without endothelium. The action of PDBu on isoproterenol-induced relaxation was less effective than that of PMA, unlike those observed in contractile response, but the contractile effect of the former was more potent than that of the latter. PMA did not affect relaxant effect of forskolin, an activator of adenyl cyclase. Staurosporine, a protein kinase C inhibitor, inhibited the action of these drugs on both isoproterenol-induced relaxation and the contractile response. These results suggest that the relaxation induced by isoproterenol was reducd by the activation of protein kinase C, which may be isozyme different from that involved in contractile response.
Adenylyl Cyclases ; Adrenergic beta-Agonists ; Baths ; Carotid Arteries ; Colforsin ; Endothelium ; Isoproterenol* ; Methylene Blue ; Muscle, Smooth, Vascular* ; Phenylephrine ; Phorbol Esters* ; Protein Kinase C ; Relaxation ; Staurosporine

OBJECTIVE: Activation of protein kinase C(PKC) may play a certain role in the development of cerebral vasospasm. However, this mechanism still elusive. This study was undertaken to investigate the action of protein kinase C on calcium-dependent K+ channels(KCa) and internal calcium concentration [Ca2+]i in freshly isolated smooth muscle cells. METHODS: Whole-cell patch clamp and calcium microfluorimetry technique were used for measuring the K Ca and internal calcium concentration. RESULTS: In patch-clamp studies, depolarization evoked an outward KCa current which is sensitive to caffeine and A23187, and shown to be blocked by TEA(tetraethylammonium) but not by glibenclamide. Activation of protein kinase C by phorbol 12-myristate 13-acetate(PMA:1-100nmol/1) and phorbol 12, 13-dibutyrate(PDB:1-100nmol/1) dose-dependently enhanced KCa current. Subsequent application of TEA(10-30mmol/1) but not glibenclamide(3-6nmol/1), in the presence of phorbol esters, reduced the potassium current activated by phorbol esters. Preincubation with 1-(5-isoquinoline sulphonyl)-2-methylpiperazine(H-7:10nmol/1), a protein kinase C inhibitor, prevented the effect of phorbol esters on KCa. In calcium microfluorimetric studies, PMA(100nmol/1) increased intracellular calcium concentration and this effect of PMA was prevented by pre-incubation of cells with H-7(10nmol/1). CONCLUSION: These results indicate that activation of PKC increases intracellular calcium concentration and elevation of internal calcium concentration activates KCa in cerebral vascular smooth muscle cells.
Animals ; Caffeine ; Calcimycin ; Calcium ; Glyburide ; Muscle, Smooth* ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle* ; Phorbol Esters ; Potassium ; Protein Kinase C* ; Protein Kinases* ; Rats* ; Vasospasm, Intracranial

OBJECTIVETo investigate the inhibitory effect of arsenic trioxide (As₂O₃) combined with tetradecanoylphorbol acetate (TPA) on the proliferation of Kasumi-1 cell line and its mechanism.

METHODSKasumi-1 cells were treated with 200 nmol/L TPA, different concentrations of As₂O₃ alone and combined with 200 nmol/L TPA. The proliferative inhibition rates were determined with CCK-8. Annexin V was adopted to detect apoptosis. Colony formation assay was used to determine the cloning efficiency. Flow cytometry was used to detect the cell differentiation and cell cycle changes. Western blot was employed to detect the expression of P38 and p-P38.

RESULTSThe proliferation inhibition rates of Kasumi-1 cells by TPA combined with different concentrations of As₂O₃ (0.2, 2.0 and 20.0 mmol/L)for 48 h were (25.56 ± 7.29)%, (60.63 ± 6.64)%, and (73.37 ± 2.15)%, the apoptosis rates were (61.65 ± 2.62)%, (75.39 ± 1.04)%, and (89.95 ± 1.46)%, and the colony formation rates were (76.17 ± 2.06)%, (38.50 ± 1.87)%, and (18.53 ± 2.20)%, respectively, compared with the different concentrations of As₂O₃ alone groups, the difference was statistically significant (P<0.05). Cells treated with both TPA and As₂O₃ expressed more CD11b antigens compared with the cells exposed to As₂O₃ alone. TPA treated Kasumi-1 cells were arrested at G1 phase compared with the control group, while As₂O₃ increased the percentage of Kasumi-1 cells in the G2 phase. Combination treatment increased the expression of p-P38 of Kasumi-1 cells compared with the cells exposed to As₂O₃ alone.

CONCLUSIONTPA can enhance the effect of As₂O₃ on inducing apoptosis and regulating cell cycle, thereby enhancing its anti-leukemia effect.

Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Oxides ; pharmacology ; Phorbol Esters ; pharmacology

OBJECTIVETo explore the changes in expression of WT1 gene and ration of its isomers during phorbol ester (TPA) induced differentiation of leukemia cell line K562 by fluorescence quantitative RT-PCR and analysis the relationship between different isomers and hematogenic cell differentiation.

METHODSThe degree of cellular maturation were verified by NBT reduction test and immunophenotyping. Expression of WT1 gene was determined by fluorescence quantitative RT-PCR during differentiation of K562 cell line. The relative ratio of the four splicing variants WT1 ( + / + ), WT1 ( + / - ), WT1 ( - / + ), WT1 ( - / - ) were calculated.

RESULTSDuring the differentiation of K562 cell, the NBT reduction rate and the CD9 positive rate both increased significantly (P < 0. 05). The expression of WT1 gene decreased immediately to (1.67 +/- 0.45) x 10(-3) from (4.67 +/- 1.11) x 10(-3), and then increased again to (4.64 +/- 1.53) x 10(-3) at 96 hours. The ratio of WT1 ( + / + ) was decreased gradually, from 0 hour (39.65 +/- 19.46)% to 96 hour (15.25 +/- 7.27)%. While the ratio of WT1( - / - ) was increased, from 0 hour (15.38 +/- 11.34)%, to 96 hour (37.60 +/- 11.90)%. The other two isomers ratios did not change significantly.

CONCLUSIONDuring the TPA induced differentiation of K562 cell, there are two high expression levels of WT1 gene. Before differentiation, the majority is WT1 ( + / + ), and after differentiation, is WT1 ( - / - ). It indicates that WT1 gene may activate or inhibit cell differentiation by regulating the ratio of its four splicing variants.

Cell Differentiation ; drug effects ; genetics ; Gene Expression ; drug effects ; Humans ; K562 Cells ; Phorbol Esters ; pharmacology ; Protein Isoforms ; genetics ; metabolism ; WT1 Proteins ; genetics ; metabolism

BACKGROUND: Oxygen free radicals have generally been considered as cytotoxic agents. On the other hand, recent results suggest that small nontoxic amounts of these radicals may act a role in intracellular signal transduction pathway and many efforts to reveal the role of these radicals as secondary messengers have been made. It is evident that the oxygen radicals are released by various cell types in response to extracellular stimuli including LPS, TNF, IL-1 and phorbol esters, all of which translocate the transcription factor NFkappaB from cytoplasm to nucleus by releasing an inhibitory protein subunit, MB. Activation of NFkappaB is mimicked by exposure to mild oxidant stress, and inhibited by agents that remove oxygen radicals. It means the cytoplasmic form of the inducible tanscription factor NFkappaB might provide a physiologically important target for oxygen radicals. At the same time, it is well known that LPS induces the release of oxygen radicals in neutrophil with the activation of NFkappaB. From above facts, we can assume the expression of IL-8 and IL-1beta gene by LPS stimulation may occur through the activation of NFkappaB, which is mediated through the release of MB by increasing amounts of oxygen radicals. But definitive evidence is lacking about the role of oxygen free radicals in the expression of IL-8 and IL-1beta gene in mononuclear phagocytic cells. We conducted a study to determine whether oxygen radicals act a role in the expression of IL-8 and IL-1beta gene in mononuclear phagocytic cells. METHOD: Human peripheral blood monocytes were isolated from healthy volunteers. Time and dose relationship of H2O2-induced IL-8 and IL-1beta mRNA expression was observed by Northern blot analysis. To evaluate the role of oxygen radicals in the expression of IL-8 and IL-1beta mRNA by LPS stimulation, pretreatment of various antioxiants including PDTC, TMTU, NAC, ME, Desferrioxamine were done and Northern blot analysis for IL-8 and IL-1beta mRNA was performed. RESULTS: In PBMC, dose and time dependent expression of IL-8 and IL-1beta mRNA by exogenous H2O2 was not observed. But various antioxidants suppressed the expression of LPS-induced IL-8 and IL-1beta mRNA expression of PBMC and the suppressive activity was most prominant when the pretreatment was done with TMTU. CONCLUSION: Oxygen free radical may have some role in the expression of IL-8 and IL-1beta mRNA of PBMC but that radical might not be H2O2.
Antioxidants ; Blotting, Northern ; Cytoplasm ; Cytotoxins ; Deferoxamine ; Free Radicals ; Hand ; Healthy Volunteers ; Humans ; Interleukin-1 ; Interleukin-8* ; Monocytes ; Neutrophils ; Oxygen* ; Phagocytes* ; Phorbol Esters ; Protein Subunits ; Reactive Oxygen Species ; RNA, Messenger ; Signal Transduction ; Transcription Factors

OBJECTIVETo investigate the effect of Euphorbia fischeriana extract on latent HIV reactivation and the pathway involved in this process and discuss the value of Euphorbia fischeriana extract in eliminating HIV.

METHODSFresh tissues of Euphorbia fischeriana root were crushed into powder after quick freezing with liquid nitrogen and extracted with acetone followed by a three-day vacuum freeze-drying for dehydration of the extract. The extract (EFE) was separated using RP-C18 column with high-performance liquid chromatography (HPLC) and identified with mass spectrometry (MS). The activity of reactivated latent HIV was analyzed by fluorescence-activated cell sorting in a J-Lat 10.6 cell model treated with EFE (50 µg/mL) for 24 h, using TNF-α (10 ng/mL) as the positive control. The effect of a NF-κB pathway inhibitor (Bay 11-7082) on EFE activity was tested. The changes in P65 expression in the cell nuclei within 2 h and HIV protein p24 expression within 24 h were analyzed by Western blotting in cells treated with EFE.

RESULTSEFE was obtained by one-step acetone extraction, and the concentration of prostratin in the extract was around 0.53 mmol/L. About 50% of the cells showed HIV reactivation after treatment with 50 µg/mL EFE for 24 h accompanied by a significantly increased p24 expression. The activity of EFE in reactivating latent HIV was inhibited by Bay 11-7082 in a concentration-dependent manner, and p65 accumulation was detected in the cell nuclei within 2 h.

CONCLUSIONEFE we obtained contains the active compounds of prostratin and its analogues and shows a strong capacity to reactivate latent HIV through classical NF-κB pathway.

Euphorbia ; chemistry ; Flow Cytometry ; HIV ; drug effects ; HIV Infections ; Humans ; NF-kappa B ; metabolism ; Nitriles ; Phorbol Esters ; chemistry ; Plant Extracts ; pharmacology ; Signal Transduction ; Sulfones ; Tumor Necrosis Factor-alpha ; Virus Latency ; drug effects