Cordycepin induces cell cycle arrest and apoptosis by inducing DNA damage and up-regulation of p53 in Leukemia cells

Cordycepin, an adenosine analog derived from Cordyceps militaris has been shown to exert anti-tumor activity in many ways. However, the mechanisms by which cordycepin contributes to the anti-tumor still obscure. Here our present work showed that cordycepin inhibits cell growth in NB-4 and U937 cells by inducing apoptosis. Further study showed that cordycepin increases the expression of p53 which promotes the release of cytochrome c from mitochondria to the cytosol. The released cytochrome c can then activate caspase-9 and trigger intrinsic apoptosis. Cordycepin also blocks MAPK pathway by inhibiting the phosphorylation of ERK1/2, and thus sensitizes the apoptosis. In addition, our results showed that cordycepin inhibits the expression of cyclin A2, cyclin E, and CDK2, which leads to the accumulation of cells in S-phase. Moreover, our study showed that cordycepin induces DNA damage and causes degradation of Cdc25A, suggesting that cordycepin-induced S-phase arrest involves activation of Chk2-Cdc25A pathway. In conclusion, cordycepin-induced DNA damage initiates cell cycle arrest and apoptosis which leads to the growth inhibition of NB-4 and U937 cells.     

蛹虫草菌生物合成虫草素的研究进展

蛹虫草Cordyceps militaris是我国传统的药用真菌,虫草素是蛹虫草的主要活性成分,具有抗癌、抗肿瘤、抗病毒等多种生理功能。蛹虫草菌液体发酵是最有希望实现高效生产虫草素的途径,但现阶段生产强度低,亟需应用发酵工程及代谢工程手段提高虫草素产量。文中对液体发酵体系中培养基组分(碳/氮源、前体物质、金属离子等)和培养条件(pH、溶氧量、光照等)对虫草素产量的影响进行了总结,并对虫草素的分离纯化、生物合成基因簇及合成代谢途径进行了阐述,最后探讨了实现虫草素高效生产的关键环节。     

mRNAs containing the histone 3′ stem–loop are degraded primarily by decapping mediated by oligouridylation of the 3′ end

Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem–loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 5′→3′ and 3′→5′ processes, but the relative contributions are not known. The 3′ end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 3′ UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of ∼40 min with the uncleavable cap compared to ∼8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 5′→3′ decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 5′ decay intermediates were oligouridylated. Preventing oligouridylation by 3′-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 3′→5′ degradation machinery stalls during initial degradation, whereupon reuridylation occurs.     

A new approach for improving cordycepin productivity in surface liquid culture of Cordyceps militaris using high‐energy ion beam irradiation

Aims: To obtain a higher cordycepin production using Cordyceps militaris mutant obtained by a new mutagenesis technique called ‘ion beam’. Methods and Results: Successful irradiation of C. militaris NBRC 9787 by a proton beam with high energy was performed, and 30 classes of 8‐azaadenine‐ and 28 classes of 8‐azaaguanine‐resistant mutants were obtained on mutant screening, of which seven classes were selected as promising preliminary mutants having an antibacterial ability as an index of cordycepin production. In a surface liquid culture technique, some of the 8‐azaadenine‐resistant mutants gave a better performance for the cordycepin productivity; in contrast, among the 8‐azaaguanine‐resistant mutants, it was shown that mutant no. G81‐3 was much better than the control in the metabolic rate of glucose and the cordycepin productivity. In primary optimization using the enriched medium, the cordycepin production was 3·1 and 1·8 g l^?1 on 21‐day culture for mutant no. G81‐3 and the control, respectively. The cordycepin production obtained by the mutant was 72% more than the control. Conclusions: The mutant obtained by proton beam irradiation had higher productivity of cordycepin than that of the control. Significance and Impact of the Study: The mutant obtained by irradiation had a superior production performance of cordycepin, and therefore, it could be used in the realm of applied industrial biotechnology for the large‐scale production of cordycepin.     

北虫草栽培残基中主要活性物质含量分析及评价

对北虫草栽培残基中蛋白质、虫草素等主要营养及活性物质含量分析,为其资源化合理利用研究提供参考。采用反相高效液相色谱法测定氨基酸含量,半微量凯氏定氮法测定粗蛋白,稀酸碱处理法测定粗纤维含量,旋光度法测量淀粉含量,电感耦合等离子体原子发射光谱法测定矿质元素钙及磷含量,苯酚硫酸法测定多糖的含量,高效液相色谱法测定虫草素。结果显示,北虫草栽培残基中主要营养成分氨基酸、粗蛋白、粗纤维、淀粉、含量检测分别为8. 17%、18. 4%、4. 9%、43. 1%;矿质元素钙及磷含量分别为0. 09%、0. 46%;主要活性成分虫草多糖含量可达4. 8%,与子座含量(5. 1%)相当;虫草素含量0. 318%,显著高于子座(0. 166%)中虫草素含量。结果表明,北虫草栽培残基中除富含蛋白质、氨基酸、淀粉等营养物质外,还含有虫草素、虫草多糖等北虫草特有的活性物质,具有较大营养价值。北虫草栽培残基综合利用资源化研究应用前景广阔。     

Effects of ammonium feeding on the production of bioactive metabolites (cordycepin and exopolysaccharides) in mycelial culture of a Cordyceps sinensis fungus

AIMS: To examine the effects of ammonium feeding on the production of cordycepin (3'-deoxyadenosine, a nucleoside analogue) and exopolysaccharides (EPS) in mycelial culture of a new Cordyceps sinensis fungus Cs-HK1. METHODS AND RESULTS: Cs-HK1 fungus was cultivated in a liquid medium containing glucose, yeast extract, peptone and a few major inorganic salts. NH(4)Cl was fed to the mycelial culture at various concentrations from 5 to 40 mmol l(-1) on day 3 (during exponential phase). NH(4)Cl, fed at 10 mmol l(-1), stimulated the cordycepin production most significantly, with nearly fourfold increase in the cordycepin content of mycelia (from 28.5 to 117 microg g(-1)), and also increased the EPS production by 40% (from 2.6 to 3.7 g l(-1)). The ammonium feeding had a slightly positive effect at 5-10 mmol l(-1), but a negative effect at higher concentrations on the mycelium growth. Ammonium feeding also caused a sharp drop of the medium pH, owing perhaps to the uptake of NH(3) and the release of H(+) by the fungal cells. CONCLUSIONS: Ammonium feeding to the mycelial culture of Cs-HK1 fungus enhanced the intracellular cordycepin accumulation and the EPS production. The enhanced cordycepin production may be attributed to the uptake of ammonia for nucleoside synthesis, and the enhanced EPS to the increased uptake of glucose for EPS biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: It is useful for the production of bioactive metabolites and for understanding ammonium metabolism and its relationship to the biosynthesis of nucleosides in a precious medicinal fungus.     

蛹虫草磷酸甲羟戊酸激酶和焦磷酸甲羟戊酸脱羧酶基因的功能分析

【目的】确定蛹虫草甲羟戊酸途径中的2个关键酶——磷酸甲羟戊酸激酶(CmErg8)和焦磷酸甲羟戊酸脱羧酶(CmErg19)的功能及其对麦角甾醇和虫草素含量的影响。【方法】通过生物信息学分析鉴定蛹虫草中CmErg8和CmErg19,并采用酵母互补确定其功能是否保守;以蛹虫草尿嘧啶营养缺陷型CmΔpyrG为背景菌株,利用农杆菌介导的转化方法对CmErg8和CmErg19进行过表达,观察其对麦角甾醇和虫草素含量的影响。【结果】CmErg8和CmErg19不能互补酵母erg8和erg19突变体的温度敏感表型;CmErg8和CmErg19过表达菌株中麦角甾醇和虫草素含量均有所增加,特别是CmErg19基因过表达可以使虫草素含量提升5倍左右。【结论】本研究揭示了蛹虫草CmErg8和CmErg19的功能,并且发现蛹虫草麦角甾醇合成通路基因可能会影响虫草素含量。     

虫草素生理功效的研究进展

综述了虫草素生理功效的研究进展。虫草素的分子结构为3′-脱氧腺苷,它具有抗菌、抗病毒、抗肿瘤、调节免疫等作用,在医药、食品等领域具有广阔的应用前景。     

蛹虫草菌丝产虫草素液体培养条件的研究

通过对蛹虫草菌丝产虫草素液体培养条件的研究,明确蛹虫草菌丝产虫草素的适宜碳源及浓度,适宜氮源及浓度,最适pH值,最适培养温度,最适转速以及最适培养时间,以便应用于虫草素的工厂化生产。结果表明,蛹虫草菌丝产虫草素的条件：适宜碳源为D-果糖,最适浓度为10g/L;适宜氮源为蛋白胨,最适浓度为15g/L;最适初始pH为7,最适培养温度为24℃,最适转速为180r/min,最适培养时间为9d,其培养液虫草素含量可达到0.537g/L。     

COVID-19 disease and malignant cancers: The impact for the furin gene expression in susceptibility to SARS-CoV-2

Furin is a proprotein convertase that activates different kinds of regulatory proteins, including SARS-CoV-2 spike protein which contains an additional furin-specific cleavage site. It is essential in predicting cancer patients'' susceptibility to SARS-CoV-2 and the disease outcomes due to varying furin expressions in tumor tissues. In this study, we analyzed furin''s expression, methylation, mutation rate, functional enrichment, survival rate and COVID-19 outcomes in normal and cancer tissues using online databases, and our IHC. As a result, furin presented with biased expression profiles in normal tissues, showing 12.25-fold higher than ACE2 in the lungs. The furin expression in tumors were significantly increased in ESCA and TGCT, and decreased in DLBC and THYM, indicating furin may play critical mechanistic functions in COVID-19 viral entry into cells in these cancer patients. Line with furin over/downexpression, furin promoter hypo-/hyper-methylation may be the regulatory cause of disease and lead to pathogenesis of ESCA and THYM. Furthermore, presence of FURIN-201 isoform with functional domains (P_proprotein, Peptidase_S8 and S8_pro-domain) is highest in all cancer types in comparison to other isoforms, demonstrating its use in tumorigenesis and SARS-Cov-2 entry into tumor tissues. Furin mutation frequency was highest in UCES, and its mutation might elevate ACE2 expression in LUAD and UCEC, reduce ACE2 expression in COAD, elevate HSPA5 expression in PAAD, and elevate TMPRSS2 expression in BRCA. These results showed that furin mutations mostly increased expression of ACE2, HSPA5, and TMPRSS2 in certain cancers, indicating furin mutations might facilitate COVID-19 cell entry in cancer patients. In addition, high expression of furin was significantly inversely correlated with long overall survival (OS) in LGG and correlated with long OS in COAD and KIRC, indicating that it could be used as a favorable prognostic marker for cancer patients'' survival. GO and KEGG demonstrated that furin was mostly enriched in genes for metabolic and biosynthetic processes, retinal dehydrogenase activity, tRNA methyltransferase activity, and genes involving COVID-19, further supporting its role in COVID-19 and cancer metabolism. Moreover, Cordycepin (CD) inhibited furin expression in a dosage dependent manner. Altogether, furin''s high expression might not only implies increased susceptibility to SARS-CoV-2 and higher severity of COVID-19 symptoms in cancer patients, but also it highlights the need for cancer treatment and therapy during the COVID-19 pandemic. CD might have a potential to develop an anti-SARS-CoV-2 drug through inhibiting furin expression.