冬凌草细胞的悬浮培养研究

以冬凌草愈伤组织为实验材料,研究了培养基组分、接种量等培养条件对冬凌草细胞悬浮培养的影响,结果表明MS基本培养基附加2,4-D 1.0 mg/L,NAA 0.5 mg/L,Vc 0.1 mg/L,30 g/L蔗糖为最佳培养基。同时选择适宜的接种量和合适的摇床转速,能够建立起良好的冬凌草细胞悬浮体系。

稀土元素对冬凌草细胞合成冬凌草甲素的影响

[目的]探讨稀土元素对冬凌草细胞合成冬凌草甲素的影响。[方法]以冬凌草悬浮培养细胞为材料,研究不同浓度的2种稀土离子镧和亚铈对冬凌草细胞分泌和释放冬凌草甲素的影响。[结果]添加不同种类、不同浓度的稀土离子均可促进冬凌草甲素的合成。添加镧离子的细胞合成的冬凌草甲素明显高于添加铈离子的细胞。当细胞处于延滞期时,添加稀土元素并未产生明显的影响;悬浮培养指数期和稳定期加入稀土元素,均可促进冬凌草细胞对冬凌草甲素的合成,且在稳定期添加稀土元素的效果更加显著。在稳定期加入500μl镧溶液的培养液,获得的冬凌草甲素含量最高,可达28.89 mg/g。[结论]该研究为冬凌草细胞的工业化生产奠定了基础。

Induction of apoptosis and G2/M cell cycle arrest by oridonin in human gastric cancer BGC-823 cells

Aim To investigate in vitro apoptosis-induction effects of oridonin on gastric tumor cells BGC-823 and its effects on cell cycle, mitochondrial membrane potential and intracellular Ca^2＋ to shed light on the mode of its anticancer action. Methods The MTT method was used to investigate the inhibitory effect of oridonin on BGC-823 cells. The apoptosis-induction effect was evaluated by confocal laser microscopy and flow cytometry. The change of mitochondrial membrane potential and the increase of intracellular Ca^2＋ were assessed by fluorescence probe rhodamine123 and Fluo 3-AM, respectively, with flow cytometry. The expression of apoptosis and cell cycle related proteins was studied using western blotting. Results Oridonin inhibited BGC-823 cells growth with IC50 of 22.21 p, mol.L^-1. It induced apoptosis in a dose-dependent manner. In addition, it decreased mitochondria membrane potential, increased intracellular Ca^2＋, and activated pro-caspase 3. BGC-823 cells were arrested in G2/M cell cycle phase with lower expression of cyclin A protein. The up-regulation of p53 was observed before apoptosis and cell cycle arrest occurred. Conclusion Oridonin inhibits the proliferation of BGC-823 cells through G2/M cell cycle arrest and apoptosis induction, which is mediated by influx of Ca^2＋, up-regulation of p53, activation of caspase-3, and down-regulation of cyclin A.

Inhibitory Effect of Oridonin on the Proliferation of NB4 Cells and Its Mechanism

Objective: To investigate the anti-proliferation e?ect of oridonin on leukemic NB4 cells and its mechanism. Methods: NB4 cells in culture medium in vitro were given di?erent concentrations of oridonin. The inhibitory rate of the cells were measured by MTT assay, cell apoptotic rate was detected by ?ow cytometry(FCM), morphology of cell apoptosis was observed by hoechst 33258 ?uorescence staining , and the activity of telomerase was detected using TRAP-PCR-ELISA before and after apoptosis occurred. Results: Oridonin (over 8 μmol/L) could decrease the telomerase activity, inhibit the growth of NB4 cells and induce apoptosis signi?cantly in a time- and dose-dependent manner. Marked morphological changes of cell apoptosis were observed by hoechst 33258 ?uorescence staining especially after the cells treated by oridonin for 48–60 h. Conclusion: Oridonin could inhibit the proliferation and induce the apoptosis of NB4 cells in vitro. One of the mechanisms may be the decrease of the telomerase activity of NB4 cells.

Oridonin induces apoptosis in gastric cancer through Apaf-1,cytochrome c and caspase-3 signaling pathway

AIM:To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.METHODS:The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide assay.After treatment with 10 μg/mL oridonin for 24 h and 48 h,the cells were stained with acridine orange/ethidium bromide.The morphologic changes were observed under an inverted fluorescence microscope.DNA fragmen-tation(a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay.After treated with oridonin(0,1.25,2.5,5 and 10 μg/mL),HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis,and oridonin-induced apoptosis in HGC-27 cells was detected.After treatment with oridonin for 24 h,the effects of oridonin on expression of Apaf-1,Bcl-2,Bax,caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction(RT-PCR) and Western blotting.RESULTS:Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose-and time-dependent manner.The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h(1.25,2.5,5 and 10 μg/mL) were 1.78% ± 0.36%,4.96% ± 1.59%,10.35% ± 2.76% and 41.6% ± 4.29%,respectively,which showed a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%,21.57% ± 3.75%,30.31% ± 4.91% and 61.19% ± 5.81%,with a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%,31.86% ± 3.86%,48.30% ± 4.16% and 81.80% ± 6.72%,with a significant difference(P < 0.05).Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining.After treatment with oridonin,the cells became round,shrank,and developed small buds around the nuclear membrane while forming apoptotic bodies.Lactate dehydrogenase(LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h,LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4%(P < 0.001).However,the change in the release of LDH caused by necrosis was insignificant,suggesting thatthe major cause of oridonin-induced HGC-27 cell death was apoptosis.Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls(P < 0.05).And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%,12.8% ± 2.53%,28.5% ± 4.23% and 49.6% ± 3.76%,which were in a dose-dependent manner(P < 0.05).After treatment for 24 h,DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dosedependent manner.RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3(0.917 ± 0.103 vs 0.357 ± 0.019,P < 0.05),cytochrome c(1.429 ± 0.111 vs 1.002 ± 0.014,P < 0.05),Apaf-1(0.688 ± 0.101 vs 0.242 ± 0.037,P < 0.05) and Bax(0.856 ± 0.101 vs 0.278 ± 0.027,P < 0.05)(P < 0.05),whereas down-regulated in Bcl-2(0.085 ± 0.012 vs 0.175 ± 0.030,P < 0.05).Western blotting analysis also confirmed this result.CONCLUSION:Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1,caspase-3 and cytochrome c,which are highly dependent upon the mitochondrial pathway.

The Inhibitory Effect of Oridonin on the Growth of Fifteen Human Cancer Cell Lines

OBJECTIVE To study the inhibitory effect of oridonin on the growth of cancer cells. METHODS Fifteen human cancer cell lines were subjected to various concentrations of oridonin in culture medium. The inhibitory rate of cell growth was measured by the MTT assay, and compared with a negative control and 5-Fu-positive control. RESULTS The 50% inhibiting concentration （IC50） and maximal inhibition （Imax） of oridonin shown by studying the growth of the cancer cell lines were as follows： leukemias （HL60 cells： 3.9 μg/ml and 73.8%, K562 cells： 4.3 μg/ml and 76.2%）; esophageal cancers（SHEEC cells： 15.4 μg/ml and 99.2%, Eca109 cells： 15.1 μg/ml and 84.6%, TEl cells： 4.0 μg/ml and 70.2%）; gastric cancers （BGC823 cells： 7.6 μg/ml and 98.7%, SGC7901 cells： 12.3 μg/ml and 85.7%）; colon cancers （HT29 cells： 13.6 μg/ml and 97.2%, HCT cells： 14.5 μg/ml and 96.5%）; liver cancers （Bel7402 cells： 15.2 μg/ml and 89.2%, HepG2 cells： 7.1 μg/ml and 88.3%）; pancreatic cancer （PC3 cells： 11.3 μg/ml and 68.4%）; lung cancer （A549 cells： 18.6 μg/ml and 98.0% ）; breast cancer （MCF7 cells： 18.4 μg/ml and 84.7%）; uterine cervix cancer （Hela cells： 13.7 μg/ml and 98.5%）. CONCLUSION Oridonin had a relatively wide anti-tumor spectrum, and a relatively strong inhibitory effect on the growth of the 15 human cancer cells. Inhibitory effects were concentration dependent.

Altered gene expression reveals molecular mechanisms underlying oridonin-induced apoptosis of multiple myeloma LP-1 cells

Objective： To investigate the effect of oridonin on proliferation and invasion of human multiple myeloma LP-1 ceils and the underlying mechanism. Methods： LP-1 cells in culture medium in vitro were treated with oridonin at the different concentration Cell proliferation was measured by Microwave Theory and Techniques （MTT） assay and cell apoptotic rate was detected by flow cytometry. Morphology of cell apoptosis was observed by transmission electron microscope. Expressions of Bax, Bcl-2, Caspase-3, NFqcB as well as I-~B mRNA were detected by real-time PCR. Results： The MTT assays and flow cytometry revealed that oridonin could inhibit the growth of LP-1 cells and cause apoptosis significantly; the suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis were found under a transmission electron microscope after the cells were treated with oridonin at 25 ~rnol/L for 24 h. Along with the apoptotic process, Bcl-2, Caspase-3,NF-r,.B gene expressions were down-regulated （P〈0.05）. On the contrast, the Bax and I-~zB gene expressions were up-regulated （P〈0.05）. Conclusion： Oridonin could inhibit the proliferation of LP-1 cells via inducing apoptosis. We concluded that oridonin induces apoptosis in LP-1 cells via activation of caspase-3 as well as down-regulation of Bcl-2 and up-regulation of Bax expression. The results suggested that oridonin could induce apoptosis of LP-1 cells through mitochondria- and caspase3-dependent pathways. Meanwhile, the inhibition of NF-r,_B and the activation of I-~B indicate pro-apoptotic stimuli. In one word, oridonin might be an important potential anti-myeloma reagent.

Inhibitory effect of oridonin on proliferation of RPMI8226 cells and the possible underlying mechanism

OBJECTIVE:To observe the effects of oridonin on proliferation and apoptosis of myeloma RPMI8226cells and to investigate the potential underlying mechanisms.METHODS:RPMI8226 cells were treated with various concentrations of oridonin.Cell proliferation was analyzed using the thiazolyl blue tetrazolium bromide method.Ultramicrostructure was observed by transmission electron microscopy.Annexin-V/PI staining and flow cytometry was performed to determine cell apoptosis.Expression of apoptosis-related proteins was evaluated by western blot analysis.RESULTS:Oridonin suppressed the proliferation of RPMI8226 cells and induced apoptosis in a timeand dose-dependent manner.Transmission electron microscopy confirmed apoptotic morphologyupon treatment with 20μmol/L oridonin and western blot revealed decreased expressions of the apoptosis suppressors survivin,Bcl-2 and pro-caspase-3 proteins,and the increased expression of the apoptosis inducer Bax.CONCLUSION:Our results show that oridonin exhibits an inhibitory effect on the proliferation of RPMI8226 cells and induces apoptosis.This is associated with altering the balance between Bcl-2 and Bax protein expressions and decreased survivin and pro-caspase-3 expressions.