Effects of Phorbol-12,13-dibuterate on Sodium Currents and Potassium Currents in Rat Trigeminal Ganglion Neurons

The effects of phorbol-12,13-dibuterate （PDBu） on total sodium current （INa-total）, tetrodotoxin-resistant sodium current （INa-TFXr）, 4-AP-sensitive potassium current （IA） and TEA-sensitive potassium current （IK） in trigeminal ganglion （TG） neurons were investigated. Whole-cell patch clamp techniques were used to record ion currents in cultured TG neurons of rats. Results revealed that 0.5μmol/L PDBu reduced the amplitude of INa-total by （38.3±4.5）% （n=6, P〈0.05）, but neither the G-V curve （control： V0.5 =-17.1±4.3 mV, k=7.4±1.3; PDBu： V0.5=-15.9±5.9 mV, k=5.9±1.4; n=6, P〉0.05） nor the inactivation rate constant （control： 3.6±0.9 ms; PDBu： 3.6±0.8 ms; n=6, P〉0.05） was altered. 0.5 μmol/L PDBu could significantly increase the amplitude of INa-TFXr by （37.2± 3.2）% （n=9, P〈0.05） without affecting the G-V curve （control： V0.5=-14.7±6.0 mV, k=6.9± 1.4; PDBu： V0.5=- 11.1±5.3 mV, k=8.1± 1.5; n=5, P〉0.05 ） or the inactivation rate constant （control： 4.6±0.6 ms; PDBu： 4.2±0.5 ms; n=5, P〉0.05）. 0.5 μmol/L PDBu inhibited IK by （15.6±5.0） % （n=16, P〈0.05）, and V0.5 was significantly altered from - 4.7±1.4 mV to -7.9 ±1.8 mV （n=16, P〈0.05）. IA was not significantly affected by PDBu, 0.5μmol/L PDBu decreased IA by only （0.3±3.2）% （n=5, P〉0.05）. It was concluded that PDBu inhibited INa-total ：.but enhanced INa-TFXr, and inhibited IK without affecting IA. These data suggested that the activation of PKC pathway could exert the actions.

Two potentially specific but relevant patterns of proteomic change Response of SH-SY5Y cells to differentiation with retinoic acid followed by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, and susceptibility of differentiated cells to dopamine

Dopamine （DA） exposure at a dose of 100 pmol/L for 24 hours causes oxidative stress in SH-SY5Y cells with induction of neuronal differentiation by retinoic acid （RA,10 pmol/L,72 hours） followed by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate （TPA,80 nmol/L,72 hours）. However,it remains unclear whether the alteration of phenotype observed in response to oxidative stress is associated with protein regulation in this cellular model for Parkinson＇s disease. The present study detected protein regulation affected by oxidative stress at a proteomic level：selection of differentially altered proteins using two dimensional difference in-gel electrophoresis and identification of these proteins using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated significant alterations in expression of six proteins in SH-SY5Y cells following the differentiation and fourteen proteins in the differentiated cells following the exposure,exemplified by an increase of tubulin alpha1 in the former but a decrease of tubulin alpha-ubiquitous chain in the latter. These results suggest that two potentially specific but relevant patterns of proteomic change may be produced in SH-SY5Y cells with the induction of differentiation by RA followed by TPA,and in the differentiated cells after DA exposure.

Sustained small and intermediate size proteins expression in phorbol 12-myristate 13-acetate/ionomycine prolonged stimulated human fibroblasts

Objective:To compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status.Methods:Dermal fibroblasts were stimulated with phorbol 12-myristate 13-acetate(PMA)/ionomycine for 72 h.MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern.Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8%–16%.Results:The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and78.8 k Da.These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 k Da band was appeared in unstimulated and 72 h PMA stimulated cells.Moreover,we found another seven small size(10–19.5 k Da) proteins in supernatants of48 h and 72 h unstimulated but not in PMA or PMA/Ionomycine stimulated fibroblasts.Most of these proteins expression were down regulated following fibroblast activation.This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death.Conclusions:Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation.Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.

The Role of Oxygen Radicals in Rat Acute Lung Injury Induced by Phorbol Myristate Acetate

We tried to clarify the role of oxygen radicals released from granulocytes stimulated byphorbol myristate acetate(PMA) in rat acute lung injury. It was found that DNA strand-breakdamage(DSBD) in peripheral white blood cells (WBC) was significantly increased 40 min after injec-tion of PMA. DSBD in lung tissue of rats treated with PMA was also markedly increased comparedwith the controls. The PMA-treated rats showed significantly higher lipid-peroxide (LPO) level inplasma and lung tissue hemogenate than the controls did. These results suggest that determination ofDSBD, a simple and sensitive indicator for oxygen radical damaging, might be useful in thediagnosis of adult respiratory distress syndrome (ARDS), when it is used together with themeasurement of plasma LPO.

The Influence of Phorbol Ester on the Effect of Tamoxifen in Breast Cancer Cells

To investigate the correlation between the activity of kinases in the growth factor signal transduction pathway and the development of resistance of breast cancer to tamoxifen, reporter gene regulated by the regulating fragment of CCD1 was transfected into the MCF 7 cells, and the influence of tamoxifen on the reporter gene expression was examined under different conditions of TPA treatment. Our results showed that the reporter gene expression was inhibited by tamoxifen and promoted by TPA. Furthermore, tamoxifen exerts an agonist effect on the reporter gene expression when the cells was treated by TPA previously for 12 h. It is concluded that TPA could induce estrogen like effect of tamoxifen on estrogen receptor positive breast cancer cells and it may be one of the mechanisms responsible for the development of tamoxifen resistance.

Relaxin Inhibit Cardiac Fibrosis Induced by Phorbol 12-myristate 13-acetate

Relaxin is known to inhibit cardiac fibrosis. However, it is unclear whether relaxin could regulate the effects of Phorbol 12-myristate 13-acetate （PMA, PKC activator） on cardiac fibrosis. So the influence of relaxin on the cell proliferation and collagen expression induced by PMA in cultured cardiac fibroblasts was studied. It showed that PMA significantly increased cardiac fibroblasts proliferation, Type I pro-collagen protein expression, Type I pro-collagen mRNA expression, and rhRLX absolutely significantly decreased PMA induced effects on cardiac fibroblasts proliferation and Type I pro-collagen expressions, indicating that relaxin could inhibit cardiac fibrosis induced by PMA.

Experimental study on the inhibitory effect of arsenic trioxide combined with phorbol ester on the proliferation of acute promyelocytic leukemia cell line Kasumi-1

Objective:To study the effect of arsenic trioxide (As2O3) combined with phorbol ester (PMA) on the proliferation of acute promyelocytic leukemia cell line Kasumi-1.Methods:Acute promyelocytic leukemia cell lines Kasumi-1 were cultured and randomly divided into control group (treated with the RPMI1640 medium without drugs or serum), As2O3 group (treated with serum-free RPMI1640 medium containing 20 μmol/L As2O3), PMA group (treated with serum-free RPMI1640 medium containing 160 nmol/L PMA) and As2O3+ PMA group (treated with serum-free RPMI1640 medium containing 20 μmol/L As2O3 and 160 nmol/L PMA). After treatment, the cell proliferation activity, cell cycle ratio and the protein expression of related genes were measured.Results: 12 h, 24 h and 48 h after treatment, the cell proliferation activity of As2O3 group, PMA group and As2O3+PMA group were significantly lower than that of control group, and the cell proliferation activity of As2O3+PMA group was significantly lower than that of As2O3 group and PMA group;48 h after treatment, the G1 phase and S phase ratio as well as CDK1 and CyclinB1 expression of As2O3 group, PMA group and As2O3+PMA group were significantly lower than those of control group while the G2 phase ratio as well as Bax, Caspase-3, Caspase-9, p-Chk1 and p-Cdc25C9 expression were significantly higher than those of control group;the G1 phase and S phase ratio as well as CDK1 and CyclinB1 expression of As2O3+PMA group were significantly lower than those of As2O3 group and PMA group while the G2 phase ratio as well as Bax, Caspase-3, Caspase-9, p-Chk1 and p-Cdc25C9 expression was significantly higher than those of As2O3 group and PMA group.Conclusion:As2O3 combined with PMA can inhibit the proliferation of acute promyelocytic leukemia cell line Kasumi-1 by inducing apoptosis and blocking cell cycle.

Synthesis and anti-HIV activities of phorbol derivatives

In this study,37 derivatives of phorbol esters were synthesized and their anti-HIV-1 activities evaluated,building upon our previous synthesis of 51 phorbol derivatives.12-Para-electron-acceptor-trans-cinnamoyl-13-decanoyl phorbol derivatives stood out,demonstrating remarkable anti-HIV-1 activities and inhibitory effects on syncytia formation.These derivatives exhibited a higher safety index compared with the positive control drug.Among them,12-(trans-4-fluorocinnamoyl)-13-decanoyl phorbol,designated as compound 3c,exhibited the most potent anti-HIV-1 activity(EC_(50)2.9 nmol·L^(−1),CC50/EC_(50)11117.24)and significantly inhibited the formation of syncytium(EC_(50)7.0 nmol·L^(−1),CC50/EC_(50)4891.43).Moreover,compound 3c is hypothesized to act both as an HIV-1 entry inhibitor and as an HIV-1 reverse transcriptase inhibitor.Isothermal titration calorimetry and molecular docking studies indicated that compound 3c may also function as a natural activator of protein kinase C(PKC).Therefore,compound 3c emerges as a potential candidate for developing new anti-HIV drugs.

EFFECT OF PHORBOL ESTER ON cAMP－DEPENDENT PROTEIN KINASE ACTIVITY IN CARDIOMYOCYTES

ＥＦＦＥＣＴＯＦＰＨＯＲＢＯＬＥＳＴＥＲＯＮｃＡＭＰ－ＤＥＰＥＮＤＥＮＴＰＲＯＴＥＩＮＫＩＮＡＳＥＡＣＴＩＶＩＴＹＩＮＣＡＲＤＩＯＭＹＯＣＹＴＥＳＺｈｏｕＷｅｎｈｕａ（周文华），ＸｉａｏＤｉａｎｍｏ（肖殿模），ＺｈｅｎｇＣｈａｏｑｉａｎｇ（郑超强），...

Seco-cyclic phorbol derivatives and their anti-HIV-1 activities

Phorbol esters are recognized for their dual role as anti-HIV-1 agents and as activators of protein kinase C(PKC).The efficacy of phorbol esters in binding with PKC is attributed to the presence of oxygen groups at positions C20,C3/C4,and C9 of phorbol.Concurrently,the lipids located at positions C12/C13 are essential for both the anti-HIV-1 activity and the formation of the PKC-ligand complex.The influence of the cyclopropane ring at positions C13 and C14 in phorbol derivatives on their anti-HIV-1 activity requires further exploration.This research entailed the hydrolysis of phorbol,producing seco-cyclic phorbol derivatives.The anti-HIV-1 efficacy of these derivatives was assessed,and the affinity constant(Kd)for PKC-δprotein of selected seco-cyclic phorbol derivatives was determined through isothermal titration calorimetry.The findings suggest that the chemical modification of cyclopropanols could affect both the anti-HIV-1 activity and the PKC binding affinity.Remarkably,compound S11,with an EC_(50) of 0.27μmol·L^(−1) and a CC_(50) of 153.92μmol·L^(−1),demonstrated a potent inhibitory effect on the intermediate products of HIV-1 reverse transcription(ssDNA and 2LTR),likely acting at the viral entry stage,yet showed no affinity for the PKC-δprotein.These results position compound S11 as a potential candidate for further preclinical investigation and for studies aimed at elucidating the pharmacological mechanism underlying its anti-HIV-1 activity.

Yuanhuacin A Is a Selective Antagonist of Phorbol Ester Receptor in Protein Kinase C

Protein kinase C with a molecular weight of 82 kD has been purified to electrophoresis homogenous from rat brain through a series of chromatography columns including DE-52, Sepharose G-200 and phenyl-Sepharose. The enzyme possessed autophosphorylation activity. Yuanhuacin A inhibited the 3H-phorbol-12, 13-dibutyrate （3H-PdBu） binding of PKC with an IC50 value of 1.48±0.28×10-8 mol/L when the concentration of 3H-PdBu was 1.5×10-9 mol/L (Ki=1.2×10-8 mol/L). Yuanhuacin A inhibited the PdBu-stimulated PKC activity in the catalysis of the phosphorylation of Histone Ⅲ-S with an IC50 of 2.82±0.37×10-9 mol/L (PdBu=10-6 mol/L), while it had no effect on the basal and Ca2+-stimulated PKC activity in the same assay system. This result suggests that Yuanhuacin A is a selective antagonist of the phorbol ester receptor in protein kinase C.

Forskolin and Phorbol 12-myristate 13-acetate modulates the expression pattern of AP-1 factors and cell cycle regulators in estrogen-responsive MCF-7 cells

Activator protein-1(AP-1)transcription factor is a key component of many signal transduction pathways involved in the regulation of cellular processes and controls rapid responses of mammalian cells when exposed to the variety of stimulus.The phorbol 12-myristate 13-acetate and Forskolin(Fo)are well-known kinase activators/stimulators of Protein Kinase C(PKC)and Protein Kinase A(PKA)respectively.Importantly,these kinases are found to be present in transitional points of many cell signaling pathways,especially those involved in proliferation.The stimulating effect of PKC and PKA on the expression of AP-1 factors in MCF-7 breast cell proliferation is not well characterized.Hence,the role of PKC by PMA treatment and the role of PKA by using Fo in MCF-7 cells is investigated.Where,cells treated with PMA showed increased cell proliferation,while Fo had no effect,but inhibited the PMA induced proliferation.The RT-PCR results showed the PMA induced c-Jun,c-Fos and Fra-1 expressions compared to control and Fo.However,Fo in combination with PMA,inhibit the PMA induced above mRNA expressions where Fo alone has no effect.Western blot studies validated the c-Jun expressions in PMA treated MCF-7 cells.Further,PMA increases the mRNA expression of Cyclin-E1,Cyclin-D1,and CDK-4,whereas Fo decreases their expressions.Thus,mitogenic effect of PMA and inhibitory action of Fo on MCF-7 cells is probably enhanced via activation of AP-1 factors and concomitant action of cell cycle regulators in the downstream singling cascade.

佛波双酯对照品的制备研究

目的研究佛波双酯(TPA)对照品的制备方法及质量标准,为佛波双酯原料和相关制剂的质量控制提供依据。方法利用硅胶柱制备色谱和反相制备色谱对巴豆油的甲醇-水提取物进行分离纯化,分别以高效液相色谱法(HPLC)和气相色谱对佛波双酯对照品进行纯度检查和含量测定,用紫外光谱(UV)、红外图谱(IR)、质谱(MS)、核磁共振氢谱(1H-NMR)、碳-13核磁共振(13C-NMR)等方法进行图谱分析及结构确证,确认分子式为C36H56O8。结果纯化制备得到的佛波双酯对照品,紫外最大吸收波长为232 nm,MS结果为高分辨电喷雾质谱(HRESI-MS)m/z 615.4120(M-H)-,HPLC检查纯度>99.5%。结论本制备方法科学有效,制备的对照品纯度符合中药化学对照品的相关要求,可作为佛波双酯原料及相关制剂质量控制的化学对照品。

佛波酯化合物TPA诱导急性早幼粒细胞白血病细胞NB4增殖与凋亡的机制研究

目的:探讨佛波酯化合物TPA对急性早幼粒细胞白血病细胞NB4增殖、凋亡的影响及其分子机制。方法:CCK-8法检测不同浓度TPA在不同时间点对NB4细胞增殖的影响;瑞氏-吉姆萨染色观察NB4细胞形态学变化;流式细胞术检测TPA处理后NB4细胞周期、凋亡情况;高通量微阵列分析和实时荧光定量PCR法分析TPA处理后NB4细胞m RNA的表达;Western blot检测CDKN1A、CDKN1B、CCND1、MYC、Bax、Bcl-2、c-Caspase 3、c-Caspase 9、PIK3R6、AKT和p-AKT的蛋白表达。结果:与对照组相比,TPA能抑制NB4细胞增殖,诱导细胞向成熟粒-单核系分化;诱导细胞G_(1)期阻滞及凋亡;差异表达的m RNA显著富集在PI3K/AKT通路;TPA能提高NB4细胞中CCND1、CCNA1、CDKN1A的m RNA水平,降低MYC的m RNA水平;TPA能上调NB4细胞中CDKN1A、CDKN1B、CCND1、Bax、c-Caspase 3、c-Caspase 9、PIK3R6蛋白水平,下调MYC、Bcl-2、p-AKT蛋白水平。结论:TPA通过调控PIK3/AKT信号通路诱导NB4细胞周期阻滞于G_(1)期并促进其凋亡。

MNNG和佛波酯诱导建立结肠癌的恶性转化细胞模型

目的建立结肠癌的恶性转化细胞模型。方法以1-甲基-3-硝基-1-亚硝基胍(MNNG)为启动剂、佛波酯(PMA)为促癌剂,对永生化的正常人结肠上皮细胞(NCM460细胞)进行多代诱导处理。采用MTT实验、细胞克隆形成实验和细胞划痕实验鉴定NCM460细胞恶性转化的倾向性和恶性生物学特征,采用Western blot检测神经钙黏附蛋白(N-cadherin)、转化生长因子β1(TGF-β1)和表皮生长因子受体(EGFR)的蛋白表达水平。结果正常NCM460细胞形态为梭形,排列有序,呈单层生长;诱导后的第30代NCM460细胞形态发生显著变化,大小不一,排列紊乱,呈团块状生长。正常NCM460细胞、第17代NCM460细胞、第30代NCM460细胞的增殖能力依次升高,克隆形成数量依次增多,48 h迁移距离和迁移率依次增加或升高(P<0.05)。第30代NCM460细胞中N-cadherin、EGFR、TGF-β1的蛋白表达水平高于正常NCM460细胞(P<0.05)。结论经过MNNG和PMA连续多代诱导处理后,NCM460细胞的生物学功能(增殖、克隆形成和迁移能力)发生明显改变,具备恶性细胞的生物学特征。该模型可用于结肠癌的发病机制和治疗方法研究。

千年桐根的化学成分研究

研究千年桐Vernicia montana根的化学成分。采用各种现代色谱手段对其化学成分进行分离,测定各种波谱数据并结合文献研究以鉴定化合物的结构。分离到5化合物,分别鉴定为二萜12-O-palmityl-13-O-acetyl phorbol（1）,三萜maslinic acid（2）,木脂素syringaresinol（3）,棕榈酸palmitic acid（4）和单棕榈酸甘油酯glycerol monopalmitate（5）。五个化合物（1~5）均为首次从该植物中分得。

不同产地麻疯树种仁的含油量及脱油种仁的佛波酯含量

选用国内4种不同产地的麻疯树种仁,对其含油量以及脱油后的佛波酯含量进行了研究.采用GB/T 5512-85粮食、油料检验粗脂肪测定法进行麻疯树种仁含油量的测定;脱油后种仁经二氯甲烷超声提取佛波酯,采用高效液相色谱(HPLC)对其含量进行检测.结果表明,小金河、攀枝花、仁里、片角乡麻疯树种仁的含油量分别为58.29%、60.84%、58.83%和56.12%,脱油后种仁中佛波酯含量分别为2.43mgg-1、2.25mgg-1、2.03mgg-1和1.21mgg-1.不同产地的麻疯树种仁含油量均有差异,在深入开发麻疯树资源时,种仁含油量的高低可作为其选种依据之一.4种不同产地的麻疯树种仁脱油后的佛波酯含量均高于无毒麻疯树品种,要利用麻疯树的脱油种仁这一潜在饲料资源,对其进行进一步脱毒处理是必要的.

肝素增强佛波酯对人鼻咽癌CNE2细胞的作用 :上调c-jun ,p53 ,p21的表达(英文)

目的 :观察肝素联合佛波酯对人鼻咽癌细胞的增殖与凋亡的影响及其可能的分子机制。方法 :用细胞记数法、流式细胞术观察肝素联合佛波酯对人鼻咽癌细胞的增殖及细胞周期的影响 ;而用TUNEL、琼脂糖凝胶电泳、Westernblot等方法观察肝素与佛波酯联合应用对人鼻咽癌细胞凋亡的作用。结果 :肝素与佛波酯联合应用后对鼻咽癌细胞的生长抑制及其凋亡具有显著增强的作用 ,同佛波酯单独应用于诱导细胞凋亡相比 ,低剂量的肝素与佛波酯联合应用后发现 :TUNEL阳性细胞明显增多 ;G1 期细胞阻止 ,S期细胞明显减少 ,凋亡率增加 ;DNA“梯形”变化更加明显 ;c-jun ,p53 ,p2 1基因表达明显升高 ,而c-fos在整个用药过程中没有改变。结论 :肝素增强佛波酯对人鼻咽癌细胞的抗增殖及促凋亡 ,这可能与c-jun ,p53 ,p2

佛波脂对体外培养Th细胞表面CD4分子表达的影响

目的 探索佛波脂(PMA)对Th细胞表面CD4分子表达的影响。方法 用荧光标记的抗CD3 4 8抗体同经10、15、2 0、2 5ng mL的PMA和1μg mL的Ionomycin刺激培养2、3、4h的正常人外周全血细胞中的Th细胞上的CD4分子结合,溶血后用流式细胞仪检测CD4通道的荧光强度(MFI)。结果 CD4通道的MFI随PMA质量浓度的升高和刺激时间的延长而降低。结论 Th细胞表面CD4分子在刺激培养过程中随PMA质量浓度的升高和刺激时间的延长而减少。