Effect of cordycepin triphosphate on in vitro RNA synthesis by picornavirus polymerase complexes.

Cordycepin triphosphate inhibited in vitro [3H]GMP incorporation by pricornavirus-specific polymerase complexes isolated from infected HeLa cells. The inhibition of [3H]GMP incorporation could be reversed with ATP added to the reaction mixture along with the inhibitor, but not with GTP so added or with ATP added 10 min after the inhibitor. Products synthesized in vitro in the presence of cordycepin triphosphate lacked full-length single-stranded viral RNA. These results support RNA chain termination by specific competition with ATP as the mechanism of inhibition of picornavirus-specific RNA synthesis by cordycepin triphosphate.     

Acid phosphatase of HeLa cells: properties and regulation of lysosomal activity by serum.

Although the subcellular distribution profile of acid phosphatase in HeLa cells is typical of a lysosomal enzyme, different lysosomal (70–80%) and supernatant forms (20–30%) have been demonstrated by their differences in pH activity curves, substrate specificities, thermal stability, sensitivity to inhibitors, and kinetics. Enzymes of the lysosomal fraction displayed anomalous kinetics in the hydrolysis of p-nitrophenyl phosphate. The major lysosomal acid phosphatase activity appears to be associated with the membrane.The total acid phosphatase activity in the cell is controlled by the concentration of serum in the medium. The specific activity in the homogenates of cells grown in high serum concentration (30%) is about twice that of cells grown in low serum concentration (1%). This doubling of specific activity holds for the lysosomal enzyme (or enzymes), but little change occurs in the supernatant form (or forms). Two other lysosomal enzymes, β-glucuronidase and N-acetyl-β-d-hexosaminidase, do not increase in specific activity. The serum-dependent formation of acid phosphatase is sensitive to cycloheximide, actinomycin D, and cordycepin. Cycloheximide blocks the increase in enzymatic activity immediately, whereas cordycepin and actinomycin D have no effect for at least 8 h. These findings suggest that de novo protein synthesis is involved in the induction of lysosomal acid phosphatase by serum and that the mRNA for this enzyme is relatively stable.     

THE SELECTIVE INTERRUPTION OF NUCLEOLAR RNA SYNTHESIS IN HELA CELLS BY CORDYCEPIN

Cordycepin is an analogue of adenosine lacking the 3'-OH. When incorporated into a growing RNA molecule, cordycepin prevents further elongation, thus producing a prematurely terminated RNA molecule. When HeLa cells are exposed to low concentrations of cordycepin, DNA and protein synthesis are unaffected during short exposure periods. The synthesis of completed ribosomal and ribosomal-precursor (45S) RNA is significantly depressed. Partially completed 45S ribosomal precursor molecules accumulate in the nucleolus. 18S ribosomal RNA can be cleaved from these incomplete precursors, while 32S ribosomal precursor cannot be produced from partially snythesized 45S molecules. The synthesis of transfer RNA is also reduced in the presence of cordycepin. The synthesis of the nuclear heterogeneous RNA species is unaffected by the drug while the cytoplasmic heterogeneous RNA is slightly reduced.     

3' deoxycytidine, like hydroxyurea, inhibits DNA synthesis without preventing the initiation of the cell cycle

3' Deoxycytidine, the cytidine analogue of cordycepin and selective inhibitor of pre-ribosomal RNA in HeLa cells, has been found to be a reversible inhibitor of DNA replication in RNA accumulation. Like other inhibitors of DNA replication such as hydroxyurea, it does not prevent serum-stimulated quiescent 3T3 cells from undergoing the random transition which is rate-limiting for entry into S phase.     

蛹虫草虫草素对ANIT诱导胆汁淤积性肝损伤的保护作用

本研究探讨虫草素对α-萘异硫氰酸酯(ANIT)诱导胆汁淤积性肝损伤的改善作用及保护机制.首先建立ANIT诱导胆汁淤积性肝损伤模型,通过检测血生化指标、HE染色观察肝脏组织病理的情况评价虫草素的保肝作用,进一步通过Western blot和实时定量PCR技术分析胆汁酸合成、分解、转运以及炎症相关通路的变化.结果 显示,与...     

Degradation of short-lived proteins is decreased by centrifugation

We have examined the effects of enucleation and of inhibitors of mRNA synthesis (actinomycin D and cordycepin) on protein turnover of HeLa cells. Enucleation markedly inhibited the rate of protein degradation for short-lived proteins. However, cells centrifuged in the absence of cytochalasin B at the speed required to obtain cytoplasts showed protein degradation rates identical to those of cytoplasts, while inhibitors of mRNA synthesis did not affect the process. Although enucleation may affect degradation of specific proteins, these results suggest that centrifugation is largely responsible for the inhibition of protein degradation in cytoplasts.     

Cordycepin inhibits migration of human glioblastoma cells by affecting lysosomal degradation and protein phosphatase activation

Cordycepin, a nucleoside-derivative-isolated form Cordyceps militaris, has been reported to suppress tumor cell proliferation and cause apoptosis. This study investigates the effect of cordycepin on the migration of human glioblastoma cells. Cordycepin suppressed the migration of the human glioblastoma cell lines U87MG and LN229 in transwell and wound healing assays. Cordycepin decreased protein expression of integrin α1, focal adhesion kinase (FAK), p-FAK, paxillin and p-paxillin. The lysosomal inhibitor NH₄Cl blocked the ability of cordycepin to inhibit focal adhesion protein expression and glioma cell migration. In addition, the protein phosphatase inhibitors calyculin A and okadaic acid blocked the cordycepin-mediated reduction in p-Akt, p-FAK and migration. Hematoxylin and eosin staining of mouse xenografts demonstrated that cordycepin reduced brain tumor size in vivo. In conclusion, cordycepin inhibited migration of human glioblastoma cells by affecting lysosomal degradation and protein phosphatase activation. This pathway may be a useful target for clinical therapy in the future.     

Cordycepin. An inhibitor of newly synthesized globin messenger RNA.

The effect of cordycepin (3'-deoxyadenosine) on newly synthesized globin mRNA in cultured mouse fetal liver erythroid cells is investigated. At cordycepin concentrations that do not inhibit amino acid incorporation into acid-precipitable material, the quantity of pulse-labeled (radioactive) globin mRNA nucleotide sequences is reduced by 90%, as compared to adenosine-treated controls. The reduction of radioactivity in globin-specific RNA sequences is greater than the inhibition of total RNA synthesis in experiments in which the labeling times range from 6 to 60 min. Control experiments demonstrate that cordycepin does not reduce the recovery of total cell RNA or steady state (unlabeled) globin mRNA. The hybridization assay used to detect radioactive globin mRNA sequences is independent of the cellular location or the number of 3'-terminal adenylate residues in the mRNA-containing molecules. These data thus indicate that cordycepin inhibits newly synthesized mRNA as effectively as it inhibits ribosomal and transfer RNA synthesis.     

Cordycepin activates AMP‐activated protein kinase (AMPK) via interaction with the γ1 subunit

Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP‐activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin‐induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)‐elicited intracellular lipid accumulation and increased AMPK activity in a dose‐dependent manner. Cordycepin‐induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin‐dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit.     

虫草素对肺癌细胞生长及迁移的影响

探讨虫草素对非小细胞肺癌细胞株H1781细胞凋亡及迁移的影响及作用机制。培养H1781细胞并分组,对照组用不含药物的培养基处理,虫草素组用含有10、20、30和40 μmol/L虫草素处理,处理24 h后测定细胞活力,通过显微镜观察细胞形态学,HE染色观察虫草素对细胞整体的影响,细胞免疫荧光技术检测细胞中MMP-9和DAPI核染色情况观察细胞凋亡,Western blotting检测凋亡等相关蛋白表达。与对照组相比,虫草素处理24 h后,H1781细胞系活力显著降低;细胞数量明显减少;HE染色观察发现随着虫草素浓度增加,细胞数量及细胞集团明显变少,免疫荧光技术检测发现药物处理后细胞凋亡明显促进;划痕实验发现虫草素明显降低细胞迁移能力;Western blotting实验中Bax、cleaved caspase-3蛋白表达明显上调,MMP-9、Bcl-2蛋白表达明显下调。虫草素对肺癌H1781细胞迁移有抑制作用、对凋亡有促进作用,推测其作用机制为上调促凋亡蛋白表达、下调抗凋亡蛋白表达。     

从蛹虫草小麦培养基残渣分离虫草素及提取物对肝癌细胞的抑制效果

从蛹虫草Cordyceps militaris小麦培养基残渣中分离和纯化虫草素,并进行了提取物体外人肝癌细胞Hep G2 (human hepatoma cell line,Hep G2)的抑制作用研究。以蛹虫草小麦培养基残渣为原料,热超声水提取虫草素,探究pH值、温度、浸提时间和超声功率对虫草素的提取效果,用苯乙烯型弱极性大孔吸附树脂(AB-8)柱层析分离、重结晶纯化虫草素,并用高效液相色谱法(high performance liquid chromatography,HPLC)、红外光谱法(infrared spectroscopy,IR)鉴定。虫草素处理肝癌细胞Hep G2后,倒置相差显微镜下观察肝癌细胞形态变化,并用四甲基偶氮唑蓝(methylthiazolyltetrazolium,MTT)法测定细胞生长情况。结果表明：固液比1:50、pH值6.0、温度60 ℃、浸提时间3 h和超声功率300 W时提取效果好。经鉴定,分离提取物的虫草素纯度达到99%以上。不同浓度的虫草素对肝癌细胞Hep G2有明显的抑制作用,且与虫草素浓度和作用时间呈正相关,被抑制的细胞凝集、变圆、碎裂增加。本提取工艺得到的虫草素纯度高,工艺简单易扩大,且提取物对肝癌细胞增殖作用具有明显的抑制作用,为其在功能性食品添加剂方向的应用提供了数据基础。     

虫草素高产菌株的筛选及不同添加物对虫草素产量的影响研究

通过对14株蛹拟青霉菌株进行摇瓶液体发酵培养试验,筛选虫草素产量最高的蛹虫草菌株,并确定了虫草素的最佳收获期;比较8种营养添加物对虫草发酵过程中虫草素积累的影响,筛选出最佳的营养添加物,以提高液体发酵生产虫草素的产量。结果表明：蛹虫草CM001号菌株的虫草素产量最高,蛹虫草菌在第10天细胞量达到最大值20.44mg/mL,虫草素含量在第13天达到最大值73.81mg/L,70%以上的虫草素分布在发酵液中。其中分别添加腺苷、腺嘌呤、丙氨酸、L-天冬氨酸、甘氨酸5种物质,对虫草素合成的促进作用均较明显,均能有效提高蛹虫草液体发酵虫草素的产量,特别是添加1g/L的腺嘌呤能使10号菌株的虫草素产量提高7.09倍。     

Acceleration of CHO cells into mitosis and reduction of X-ray-induced G2 delay by cordycepin

The mitotic cell selection technique was used to monitor the effect of cordycepin and/or 100 rad of X-rays on the entry of asynchronous or synchronous Chinese hamster ovary cells into mitosis. Continuous exposure of asynchronous cells to 5–50 μg/ml of cordycepin caused a rapid increase in the relative numbers of cells entering mitosis. In irradiated cells, cordycepin also reduced a 120-min mitotic delay by about 80 min and shifted the X-ray transition point about 10 min farther away from mitosis. Further studies showed that synchronous cells, treated continuously with 15 μg/ml of cordycepin starting at mid-to-late S phase, proceeded into mitosis approx. 40 min ahead of controls. This acceleration was associated with a 30-min lengthening of S phase and a reduction in the length of G2 from 80 to about 10 min. Furthermore, cordycepin reduced the 70-min mitotic delay observed for cells irradiated in S phase by 20 min. In contrast to the results for treatment at mid-S phase, continuous treatment during G2 of unirradiated synchronous cells with 15 μg/ml of cordycepin had little effect on accelerating cells into mitosis, yet did reduce by about 60 min the 170-min mitotic delay observed for cells irradiated in G2. Unirradiated synchronous cells treated with cordycepin starting before mid-S did not reach mitosis. Thus, there are the following transition points or intervals for cordycepin: for treatment prior to mid-S phase, cell cycle progression through S is blocked; for treatment between mid-S and late S, progression through S continues but progression through G2 is accelerated; and for treatment during G2, the rate of progression in accelerated only if the cells have been irradiated. These results are discussed in relation to the synthesis during late S and G2 of critical protein molecules essential for mitosis.     

Effects of cordycepin,hydroxyurea and cycloheximide on histone mRNA synthesis in synchronized HeLa cells

The effects of cordycepin, hydroxyurea and cycloheximide on the synthesis of histone mRNA in synchronized HeLa cells were studied by quantitating the RNA, using translation in a rabbit reticulocyte cell-free system. It was found that biologically active histone mRNA is synthesized in the presence of cordycepin. This result strengthens the findings by others that histone mRNA does not contain poly(A). Hydroxyurea and cycloheximide, when used at concentrations that inhibit cellular DNA synthesis, had different effects on histone messenger activity. While polyribosomal messenger activity rapidly declined after addition of hydroxyurea it was not impaired by cycloheximide. These results might help to elucidate regulatory mechanisms involved in the coupled synthesis of DNA and histones in HeLa cells.     

Cordycepin inhibits albumin-induced epithelial-mesenchymal transition of renal tubular epithelial cells by reducing reactive oxygen species production

Albumin induced epithelial-mesenchymal transition (EMT) of renal tubular cells through reactive oxygen species (ROS) pathway plays an important role in tubulointerstitial fibrosis. Cordycepin (3 -deoxyadenosine), a potential antioxidant, was demonstrated to have various pharmacological effects and could inhibit EMT of some cells. However, the role of cordycepin on albumin-induced EMT in renal tubular cells (HK2) is unclear. In this study, we investigated the effect of cordycepin on albumin-induced EMT of HK2 cells and its mechanisms. HK-2 cells were exposed to bovine serum albumin with or without pretreatment with cordycepin. Results showed that albumin significantly induced EMT formation of HK-2 which associated with NADPH oxidase activation and intracellular ROS overproduction through increased Rac1 activity and expression of NOX4, p22phox and p47phox, while these effects were abolished in that pretreated with cordycepin. In conclusion, cordycepin could ameliorate albumin-induced EMT of HK2 cells by decreasing NADPH oxidase activity and inhibiting ROS production.     

Synchronization of MEL cell commitment with cordycepin.

The response of differentiating MEL cells to the nucleotide analogue cordycepin reveals a previously unrecognized aspect of the molecular events which cause commitment of these cells to terminal erythroid differentiation. Cordycepin rapidly inhibits commitment of DMSO-treated MEL cells in a dose range which does not cause cytotoxicity. Reversal of cordycepin treatment in the presence of inducer leads to a rapid and synchronous commitment of a significant proportion of cells in the culture. These results suggest that MEL cells can be blocked just prior to the point of commitment by cordycepin treatment.     

An early response to gibberellic Acid not requiring protein synthesis

Cell-free extracts from gibberellic acid-treated barley (Hordeum vulgare L. cv. Himalaya) aleurone layers show phosphorylcholine glyceride transferase activity greater than that from control layers. The increase in activity is not prevented by a mixture of amino acid analogs nor by cordycepin under conditions in which it is demonstrated that the analogs and the cordycepin are entering the cells in effective concentrations. We conclude therefore that the GA₃-dependent increase in phosphorylcholine glyceride transferase activity (which occurs within the first 4 hours of GA₃ treatment) does not require RNA synthesis or protein synthesis.     

Cordycepin disrupts the microtubule networks and arrests Nil 8 hamster fibroblasts at the onset of mitosis

The nucleoside analogue 3'-deoxyadenosine (cordycepin) arrests dividing cells at the onset of mitosis in prometaphase. The microtubules in the arrested prometaphase cells depolymerize to two small asters. A minimum of 80 micrograms/ml cordycepin or 20 micrograms/ml cordycepin in combination with 2 micrograms/ml of the deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA) to inhibit its degradation is required to see these effects. Analysis of cell extracts by high-pressure liquid chromatography indicates that cordycepin enters the cells rapidly and is phosphorylated to 3'-dATP. The intracellular concentration rises almost linearly from 0.7 mM after 15 min to 7 mM by 210 min. Concomitantly the ATP concentration shows a rapid drop from the 4 mM present in controls. However, the direct reduction of ATP levels does not mimic the same rapid effects of cordycepin on the microtubules. In addition, similar effects are not produced by a variety of other adenosine analogues with alterations in the 2' and 3' ribose positions. Although other pharmacological reagents arrest cells at the onset of mitosis, cordycepin is unusual because of the collapse of the microtubule networks to two small asters that radiate from the microtubule-organizing center. 3'-dATP can replace the requirement for ATP or GTP in the vitro polymerization of microtubules from microtubule protein: however, at limiting concentrations of nucleotide it requires approximately two times the concentration of 3'-dATP as ATP to support an equivalent level of microtubule polymerization. This suggests that the effects of cordycepin in vivo may be the result of the depletion of cellular ATP pools and the altered ability of 3'dATP to substitute for ATP-dependent reactions. Current experiments are testing this hypothesis.