Targeting the Unique Methylation Pattern of Androgen Receptor (AR) Promoter in Prostate Stem/Progenitor Cells with 5-Aza-2′-deoxycytidine (5-AZA) Leads to Suppressed Prostate Tumorigenesis

Androgen receptor (AR) expression surveys found that normal prostate/prostate cancer (PCa) stem/progenitor cells, but not embryonic or mesenchymal stem cells, expressed little AR with high methylation in the AR promoter. Mechanism dissection revealed that the differential methylation pattern in the AR promoter could be due to differential expression of methyltransferases and binding of methylation binding protein to the AR promoter region. The low expression of AR in normal prostate/PCa stem/progenitor cells was reversed after adding 5-aza-2′-deoxycytidine, a demethylating agent, which could then lead to decreased stemness and drive cells into a more differentiated status, suggesting that the methylation in the AR promoter of prostate stem/progenitor cells is critical not only in maintaining the stemness but also critical in protection of cells from differentiation. Furthermore, induced AR expression, via alteration of its methylation pattern, led to suppression of the self-renewal/proliferation of prostate stem/progenitor cells and PCa tumorigenesis in both in vitro assays and in vivo orthotopic xenografted mouse studies. Taken together, these data prove the unique methylation pattern of AR promoter in normal prostate/PCa stem/progenitor cells and the influence of AR on their renewal/proliferation and differentiation. Targeting PCa stem/progenitor cells with alteration of methylated AR promoter status might provide a new potential therapeutic approach to battle PCa because the PCa stem/progenitor cells have high tumorigenicity.     

CAGE 基因mRNA水平表达与直肠癌关系的初步研究

目的:探讨直肠癌组织中癌相关基因(cancer-associated gene,CAGE)mRNA水平表达与直肠癌发生及临床分期、淋巴结转移的关系。方法:对我院2014年2月-2014年10月间肠镜室检查的标本,按病理检查结果分为3组:直肠癌组(80例;为直肠癌患者标本),大肠腺瘤组(38例;为大肠腺瘤患者标本),对照组(40例;为距肿瘤边缘10 cm以上标本)。采用RT-PCR法检测并比较各组CAGE在mRNA水平表达情况。甲基化特性PCR法(methylation-specific PCR,MSP)检测CAGE基因启动子的甲基化情况。将直肠癌患者分为CAGE基因甲基化患者组和CAGE基因去甲基化患者组,比较两组临床分期和淋巴结转移率。结果:与对照组相比,直肠癌组和大肠腺瘤组的CAGE mRNA水平表达明显升高(P0.01);直肠癌组高于大肠腺瘤组(P0.01)。3组间的基因启动子区的去甲基化阳性率差异有统计学意义(P0.01)。在直肠癌组中,CAGE去甲基化阳性患者的淋巴结转移率为73.8%(48/65),CAGE去甲基化阴性患者的淋巴结转移率为33.3%(5/15),两者相比差异有统计学意义(P0.01)。临床分期为I-Ⅱ期的CAGE基因启动子区的去甲基化直肠癌患者占24.6%(16/65),临床分期为Ⅲ-Ⅳ期的占75.4%(49/65);而甲基化的直肠癌患者中,临床分期为I-Ⅱ期的占66.7%(10/15),Ⅲ-Ⅳ期的占33.3%(5/15),两组间差异有统计学意义(P0.01)。结论:直肠癌组织和大肠腺瘤组织中,CAGE mRNA水平呈高表达。直肠癌的CAGE基因mRNA水平表达高于正常粘膜组织和大肠癌腺瘤组织。CAGE基因启动子区的去甲基化与淋巴结转移及临床分期密切相关。     

DNA-binding affinities and sequence specificities of protoberberine alkaloids and their demethylated derivatives: a comparative study

Berberrubine (1a), jatrorubine (2a), and palmatrubine (3a) have been chemically prepared by partial demethylation of berberine (1), jatrorrhizine (2), and palmatine (3), respectively. Their interactions with calf thymus (CT) DNA, poly(dA-dT)poly(dA-dT), poly(dG-dC)poly(dG-dC), and eight AT-rich 12-mer double-stranded DNAs have been investigated by means of competitive ethidium bromide (EB) displacement experiments. The results showed that DNA-binding affinities of these protoberberine alkaloids have been significantly improved by partial demethylation, and that all of these alkaloids have the preferable binding affinities with AT-rich DNA. Especially, the sequence specificities of DNA-binding of demethylated derivatives 1a, 2a, and 3a had changed to a certain extent when compared with the parent alkaloids 1, 2, and 3, respectively. The binding mode of these alkaloids was further confirmed by UV spectroscopic titration experiments. All the compounds bind to double-stranded DNA most probably via an intercalating mode.     

N-Demethylation of nocathiacin I via photo-oxidation

In order to improve aqueous solubility of nocathiacin I (1), a potent antibacterial agent, N-demethylation of the amino-sugar moiety was sought. Irradiation of 1 in DMF/CH₂Cl₂ with UV light of 380 nm led to a cyclic product 2, which was hydrolyzed to yield the desired nocathiacin VI (3). Treatment of 1 with shorter UV light caused trans–cis isomerization of a c–c double bond.     

Catalyzation of cocaine N-demethylation by cytochromes P4502B,P4503A,and P4502D in fish liver

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver-Burk plot, two K(m) values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 micro M concentrations of these inhibitors. At 100 micro M final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 micro M final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 micro M. IC(50) values were calculated to be 20 micro M for ketoconazole, 48 micro M for cimetidine (both specific P4503A inhibitors), 164 micro M for quinidine (P4502D inhibitor), and 59 micro M for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes.     

Relationship of demethylation processes to veratric acid concentration and cell density in cultures of Rhodococcus erythropolis

The aim of this study was to investigate the correlation between veratrate degradation, veratric acid concentration and cell density in Rhodococcus erythropolis cultures. The optimum culture conditions for veratrate demethylation proved to be a cell density of A(660)=1 and a concentration of 0.02% veratrate. All the products of demethylation (i.e. vanillic and protocatechuic acids) were found to be present and correlated with the appearance of high levels of free radicals and formaldehyde after contact of the cells with veratrate. Demethylation was accompanied by oscillatory changes in the levels of endogenous oxygen uptake and phenolic products. Changes in veratrate concentration and cell density caused a disturbance in the demethylation process and also in the efficiency of phenolics, formaldehyde and reactive oxygen species.     

The lysine-specific demethylase 1 is a novel substrate of protein kinase CK2

Protein kinase CK2 is a pleiotropic serine/threonine kinase responsible for the generation of a substantial proportion of the human phosphoproteome. CK2 is generally found as a tetramer with two catalytic, α and α′ and two non catalytic β subunits. CK2α C-terminal tail phosphorylation is regulated during the mitotic events and the absence of these phosphosites in α′ suggests an isoform specialization. We used a proteomic approach to identify proteins specifically phosphorylated by a CK2α phosphomimetic mutant, CK2αT344ET360ES362ES370E (CK2α4E), in human neuroblastoma SKNBE cellular extract. One of these proteins is lysine-specific demethylase 1 (LSD1 or KDM1A), an important player of the epigenetic machinery. LSD1 is a FAD-dependent amine oxidase and promotes demethylation of lysine 4 and lysine 9 of mono- and di-methylated histone H3. We found that LSD1 is a new substrate and an interacting partner of protein kinase CK2. Three CK2 phosphosites, (Ser131, Ser137 and Ser166) in the N-terminal region of LSD1 have been identified. This domain is found in all chordates but not in more ancient organisms and it is not essential for LSD1 catalytic event while it could modulate the interaction with CK2 and with other partners in gene repressing and activating complexes. Our data support the view that the phosphorylation of the N-terminal domain by CK2 may represent a mechanism for regulating histone methylation, disclosing a new role for protein kinase CK2 in epigenetics.     

Synthesis of novel lignan-like compounds and their antimicrobial activity

Herein we report the preparation of 3,4-dibenzylfurans and some oxidized derivatives with lignan backbone. The compounds were prepared using the Friedel-Crafts reaction with BF₃ etherate as catalyst, demethylation with iodocyclohexane, acetylation and oxidation reactions. The antimicrobial activity was evaluated through their capacity to inhibit the growth of Gram positive and Gram negative bacteria, and of the yeast Candida albicans. Among ten products assayed four furans displayed a good antimicrobial activity against Staphylococcus aureus, S. epidermidis and C. albicans; on the contrary, none of the compounds were active against Pseudomonas aeruginosa. One of them inhibited the growth of S. aureus, S. epidermidis (biofilm producer strain) and C. albicans at 16 μg/mL, showing a bactericidal activity already after one hour of treatment. In summary, the results suggest a possible use of these derivatives for general disinfection practices or antimicrobial agents in cosmesis skin-care.