生物活性

Staurosporine (broad spectrum protein kinase inhibitor) a microbial alkaloid with antifungal activity, has been shown to inhibit a variety of kinases including PKA (Ki=7.0 nM), PKG (Ki=8.5 nM), MYLK (MLCK, Ki=1.3 nM), PKC (Ki=0.7 nM), CaMK (IC50=20 nM), CAMKII (Ki=20nM), tyrosine kinases (IC50=70 nM) and phosphorylase kinase (IC50=0.5 nM). Inhibition is via interaction with the ATP binding site. It induces association of purified PKC with inside-out vesicles from erythrocyte membranes and augments PMA-induced ornithine decarboxylase. Staurosporine activates a bcl-2-regulated apoptosis pathway.

体外研究

体内研究

溶于DMSO,然后在盐水中稀释

激酶实验

In Vitro Kinase Inhibition Assay[1]

Asynchronously growing PC12 cells wereharvested from 15-cm plates and lysed in lysis buffer containing 10 mM Tris,150 mM sodium chloride, 1% NP40, 0.5% sodium deoxycholate, 50 mM sodiumflouride, 2 mM sodium vanadate, 0.5 units/ml aprotinin, 1mM PMSF, and 1 mg/mlleupeptin. The cells were passed through a 21-gauge needle to shear the DNA andthe cells were rocked on ice with the lysis buffer for 30 minutes. The lysatewas spun for 15 minutes at 4°C to remove the insoluble material. The p34cdc2kinase was collected on p13 agarose which specifically binds p34cdc2 kinase andkeeps it in a kinase-active form. The p33cdk2 protein was precipitated withanti-cdk2 bound to protein A agarose. The PC12 cell lysate (1 mg protein persample) was incubated overnight with 10 ml of p13 agarose or 1 mg/ml of anti-cdk2plus 20 ml of protein A agarose per sample at 4°C with constant rocking. Thep34cdc2-p13- or p33cdk2- antibody-protein A-agarose was then pelleted at 6,000 rpmat 4°C for 5 min and washed with Tris-buffered saline containing phosphataseinhibitors. The washed agarose-conjugate was incubated with histone substrate inkinase buffer (50 mM Tris pH 7.4, 1 mM dithiothreitol, 20 mM magnesiumchloride, histone 0.4 mg/ml) containing 20 mM coldATP and 2.5 mCi/sample γ³²P-ATP in the presence of variousconcentrations of staurosporine. After the kinase reaction was allowed to takeplace at 30°C for 10 min, the reaction was terminated by boiling in SDS samplebuffer. The phosphorylated histone was run on a SDS-PAGE gel aftercentrifugation and autoradiographed.

Each experiment was repeated independentlya minimum of two times.

InVivo Inhibition of Kinase Activity

PC12 cells were treated with 100 nMconcentration of staurosporine along with controls for 1–3 days and the cellswere harvested. Lysates were prepared as described above, and equal amounts oftotal protein were precipitated with p13 agarose or anti-cdk2 plus protein A agarose.The precipitates were then used in a kinase assay with histone substrate (nostaurosporine) and labeled withγ³²P-ATP as described above. Thereactions were stopped with SDS sample buffer, run on SDS-PAGE gels, andautoradiographed for 32P histone.

细胞实验

Cell lines and cell culture[2]

Human gastric mucinous adenocarcinoma cellline, MGC803, and human gastric carcinoma metastatic lymph node cell line,SGC7901, was obtained. The derived cell lines were grown in RPMI 1640 mediumsupplemented with 10% heat-inactivated fetal calf serum, 50U/ml penicillin and50μg/ml streptomycin. The cells were maintained at 37 in a humidifiedatmosphere containing 5% CO₂. Viability of the cells used in these experimentswas consistently more than 95% when evaluated by the trypan blue exclusionmethod.

Analysisof cell viability

Effect of ST on cell growth and viabilitywas measured by directly counting the number of cells by means of trypan blue dyeexclusion. Cells at a density of 2.5×10⁵/ml were seeded onto 24-wellplates, and then treated with ST at different concentrations for 24 and 48 h.Control cells were also cultured at the same time. Cell proliferation andinhibition curves were drawn, and the inhibitory concentration against 50%cells(IC50) was determined.

动物实验

Staurosporine (STS) treatment of mice[3]

Groups of male C57Bl6 mic were housedindividually and fed a low sodium diet (0.03% NaCl) for 2 weeks. Under halothaneanaesthesia, minipumps were then implanted subcutaneously to deliver vehicle orstaurosporine (1 mg/day). After treatment for 1 week with staurosporine orvehicle, mice were killed and blood was taken by cardiac puncture for theanalysis of plasma hormones and renin concentrations. During treatment, mice wereweighed regularly and were maintained in a temperature-(22 ℃) andlight-controlled (12 h light, 12 h dark) room with free access to food andwater.

Hormoneand renin measurements

Plasma corticosterone was measured byradioimmunoassay. Aldosterone was measured using a commercial kit. Plasma reninconcentration was measured as the generation of angiotensin I (ng per ml per h)when plasma samples were incubated with excess renin substrate (plasma with nointrinsic renin activity from a binephrectomised rat). Angiotensin I was measuredby radioimmunoassay.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Gollapudi L NK. Different mechanisms for inhibition of cell proliferation via cell cycle proteins in PC12 cells by nerve growth factor and staurosporine. J Neurosci Res. 1997;49(4):461-474.
[2] Ha MW HK, Liu YP, Yuan Y. Effect of staurosporine on cycle of human gastric cancer cells. World J Gastroenterol. 2004;10(2):161-166.
[3] Bureik M, Mion A, Kenyon CJ, Bernhardt R. Inhibition of aldosterone biosynthesis by staurosporine. Biol Chem. 2005;386(7):663-669.

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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