The use of 10-23 DNAzyme to selectively destroy the allele of mRNA with a unique purine-pyrimidine dinucleotide

10-23 DNAzyme is an oligodeoxyribonucleotide-based ribonuclease. It consists of a 15-nt catalytic domain flanked by two target-specific complementary arms. It has been shown to cleave target mRNA effectively at purine (R)-pyrimidine (Y) dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of a given allele at a unique RY dinucleotide while leaving the mRNA encoded from other alleles of the same gene intact. In this study, a p53-R249S (AGG-->AGT) mutant was tested. 10-23 DNAzyme was used to cut mutant mRNA at GT dinucleotide of codon 249. Both in vitro and in vivo studies showed that this DNAzyme could specifically cut the mutant p53 allele, leaving the wild-type unaffected. This proof-of-concept experiment provided a new way to knock down expression of a given allele with special single-base transversion.     

Suppression of platelet-type 12-lipoxygenase activity in human erythroleukemia cells by an RNA-cleaving DNAzyme.

Human platelet-type 12-lipoxygenase (12-LOX) and its metabolites play a crucial role in tumor angiogenesis. A "10-23" deoxyribozyme (DNAzyme) and its phosphorothioate-modified version were designed and synthesized against the 12-LOX mRNA. Both DNAzymes were able to cleave their substrate efficiently in a time- and concentration-dependent manner in vitro. Under a multiple turnover condition, both performed well at 37 degrees C, showing the k(cat) of 1 and 0.26 min(-1), respectively. The phosphorothioate modification of the DNAzyme significantly increased its stability in cells without a substantial loss of kinetic efficiency in vitro. In a cell culture system, transfection of the DNAzymes into HEL cells resulted in a significant down-regulation of the 12-LOX mRNA. Furthermore, the cell extracts from the DNAzyme-transfected cells exhibited a marked reduction in the 12-LOX enzyme activity. The present results indicated the potential use of DNAzyme technology for gene function study and cancer therapy.     

KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.     

Optimisation of the 10-23 DNAzyme-substrate pairing interactions enhanced RNA cleavage activity at purine-cytosine target sites

The 10–23 RNA cleaving DNAzyme has been shown to cleave any purine–pyrimidine (RY) junction under simulated physiological conditions. In this study, we systematically examine the DNAzymes relative activity against different RY combinations in order to determine the hierarchy of substrate core dinucleotide sequence susceptibility. The reactivity of each substrate dinucleotide compared in the same background sequence with the appropriately matched DNAzyme was found to follow the scheme AU = GU ≥≥ GC >> AC. The relatively poor activity of the DNAzyme against AC and GC containing substrates was found to be improved substantially by modifications to the binding domain which subtly weaken its interaction with the substrate core. The most effective modification resulting in rate enhancement of up to 200-fold, was achieved by substitution of deoxyguanine with deoxyinosine such that the base pair interaction with the RNA substrates core C is reduced from three hydrogen bonds to two. The increased cleavage activity generated by this modification could be important for application of the 10–23 DNAzyme particularly when the target site core is an AC dinucleotide.     

Loss of RhoA Exacerbates,Rather Than Dampens,Oncogenic K-Ras Induced Lung Adenoma Formation in Mice

Numerous cellular studies have indicated that RhoA signaling is required for oncogenic Ras-induced transformation, suggesting that RhoA is a useful target in Ras induced neoplasia. However, to date very limited data exist to genetically attribute RhoA function to Ras-mediated tumorigenesis in mammalian models. In order to assess whether RhoA is required for K-Ras-induced lung cancer initiation, we utilized the K-Ras^G12D Lox-Stop-Lox murine lung cancer model in combination with a conditional RhoA^flox/flox and RhoC^-/- knockout mouse models. Deletion of the floxed Rhoa gene and expression of K-Ras^G12D was achieved by either CCSP-Cre or adenoviral Cre, resulting in simultaneous expression of K-Ras^G12D and deletion of RhoA from the murine lung. We found that deletion of RhoA, RhoC or both did not adversely affect normal lung development. Moreover, we found that deletion of either RhoA or RhoC alone did not suppress K-Ras^G12D induced lung adenoma initiation. Rather, deletion of RhoA alone exacerbated lung adenoma formation, whereas dual deletion of RhoA and RhoC together significantly reduced K-Ras^G12D induced adenoma formation. Deletion of RhoA appears to induce a compensatory mechanism that exacerbates adenoma formation. The compensatory mechanism is at least partly mediated by RhoC. This study suggests that targeting of RhoA alone may allow for compensation and a paradoxical exacerbation of neoplasia, while simultaneous targeting of both RhoA and RhoC is likely to lead to more favorable outcomes.     

Efficient silencing of gene expression by an ASON-bulge-DNAzyme complex

Background

DNAzymes are DNA molecules that can directly cleave cognate mRNA, and have been developed to silence gene expression for research and clinical purposes. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and exhibit good stability. The “10-23 DNA enzyme” is composed of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of approximately eight nucleotides in each direction, which provides the complementary sequence required for specific binding to RNA substrates. However, these eight nucleotides might not afford sufficient binding energy to hold the RNA substrate along with the DNAzyme, which would interfere with the efficiency of the DNAzyme or cause side effects, such as the cleavage of non-cognate mRNAs.

Methodology

In this study, we inserted a nonpairing bulge at the 5′ end of the “10–23 DNA enzyme” to enhance its efficiency and specificity. Different sizes of bulges were inserted at different positions in the 5′ end of the DNAzyme. The non-matching bulge will avoid strong binding between the DNAzyme and target mRNA, which may interfere with the efficiency of the DNAzyme.

Conclusions

Our novel DNAzyme constructs could efficiently silence the expression of target genes, proving a powerful tool for gene silencing. The results showed that the six oligo bulge was the most effective when the six oligo bulge was 12–15 bp away from the core catalytic domain.     

Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities

Two modified 2′-deoxynucleoside 5′-triphosphates have been used for the in vitro selection of a modified deoxyribozyme (DNAzyme) capable of the sequence-specific cleavage of a 12 nt RNA target in the absence of divalent metal ions. The modified nucleotides, a C5-imidazolyl-modified dUTP and 3-(aminopropynyl)-7-deaza-dATP were used in place of TTP and dATP during the selection and incorporate two extra protein-like functionalities, namely, imidazolyl (histidine analogue) and primary amino (lysine analogue) into the DNAzyme. The functional groups are analogous to the catalytic Lys and His residues employed during the metal-independent cleavage of RNA by the protein enzyme RNaseA. The DNAzyme requires no divalent metal ions or other cofactors for catalysis, remains active at physiological pH and ionic strength and can recognize and cleave a 12 nt RNA substrate with sequence specificity. This is the first example of a functionalized, metal-independent DNAzyme that recognizes and cleaves an all-RNA target in a sequence-specific manner. The selected DNAzyme is two orders of magnitude more efficient in its cleavage of RNA than an unmodified DNAzyme in the absence of metal ions and represents a rate enhancement of 10⁵ compared with the uncatalysed hydrolysis of RNA.     

A novel replicating circular DNAzyme

10–23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. However, the dependence on exogenous delivery limits its applications. The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector. By cloning the 10–23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz₇ and C-Dz₄₈₂, targeting the β-lactamase mRNA. These circular DNAzymes showed in vitro catalytic efficiencies (k_cat/K_M) of 7.82 × 10⁶ and 1.36 × 10⁷ M^–1·min^–1, respectively. Their dependence on divalent metal ions is similar to that found with linear 10–23 DNAzyme. Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting β-lactamase and bacterial growth. This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications.     

Lactate Formation in Primary and Metastatic Colon Cancer Cells at Hypoxia and Normoxia

High glucose consumption and lactate synthesis in aerobic glycolysis are a hallmark of cancer cells. They can form lactate also in glutaminolysis, but it is not clear how oxygen availability affects this process. We studied lactate synthesis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured with L‐Ser and/or L‐Asp. Glucose and lactate levels were determined colorimetrically, amino acids by HPLC, expression of AST1‐mRNA and AST2‐mRNA by RT‐PCR. In both lines glucose consumption and lactate synthesis were higher at 10% than at 1% oxygen, and lactate/glucose ratio was increased above 2.0 by L‐Asp. AST1‐mRNA expression was independent on oxygen and cell line, but AST2‐mRNA was lower at hypoxia in SW480. We conclude that, in both cell lines at 1% hypoxia, lactate is formed mainly from glucose but at 10% normoxia also from L‐Asp. At 10% normoxia, lactate synthesis is more pronounced in primary than metastatic colon cancer cells.     

A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine-Pyrimidine Junction

Several RNA-cleaving deoxyribozymes (DNAzymes) have been reported for efficient cleavage of purine-containing junctions, but none is able to efficiently cleave pyrimidine-pyrimidine (Pyr-Pyr) junctions. We hypothesize that a stronger Pyr-Pyr cleavage activity requires larger DNAzymes with complex structures that are difficult to isolate directly from a DNA library; one possible way to obtain such DNAzymes is to optimize DNA sequences with weak activities. To test this, we carried out an in vitro selection study to derive DNAzymes capable of cleaving an rC-T junction in a chimeric DNA/RNA substrate from DNA libraries constructed through chemical mutagenesis of five previous DNAzymes with a k_obs of ∼ 0.001 min^− 1 for the rC-T junction. After several rounds of selective amplification, DNAzyme descendants with a k_obs of ∼ 0.1 min^− 1 were obtained from a DNAzyme pool. The most efficient motif, denoted “CT10-3.29,” was found to have a catalytic core of ∼ 50 nt, larger than other known RNA-cleaving DNAzymes, and its secondary structure contains five short duplexes confined by a four-way junction. Several variants of CT10-3.29 exhibit a k_obs of 0.3-1.4 min^− 1 against the rC-T junction. CT10-3.29 also shows strong activity (k_obs  > 0.1 min^− 1) for rU-A and rU-T junctions, medium activity (> 0.01 min^− 1) for rC-A and rA-T junctions, and weak activity (> 0.001 min^− 1) for rA-A, rG-T, and rG-A junctions. Interestingly, a single-point mutation within the catalytic core of CT10-3.29 altered the pattern of junction specificity with a significantly decreased ability to cleave rC-T and rC-A junctions and a substantially increased ability to cleave rA-A, rA-T, rG-A, rG-T, rU-A, and rU-T junctions. This observation illustrates the intricacy and plasticity of this RNA-cleaving DNAzyme in dinucleotide junction selectivity. The current study shows that it is feasible to derive efficient DNAzymes for a difficult chemical task and reveals that DNAzymes require more complex structural solutions for such a task.     

Liriodenine induces G1/S cell cycle arrest in human colon cancer cells via nitric oxide- and p53-mediated pathway

Liriodenine is an aporphine alkaloid compound extracted from the leaves of Michelia compressa var. lanyuensis. It had been reported to have an anti-colon cancer effect, but the mechanism remains unclear. In the present study, the antiproliferative mechanisms of liriodenine were investigated in the human colon cancer SW480 cells. Flow cytometry analysis indicated that liriodenine notably induced the G1/S phase arrest. The G1/S phase cycle-related proteins analysis illustrated that the expressions of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6, and of cyclin D1 and A, as well as the phosphorylation of retinoblastoma tumor suppressor protein (ppRB) were found to be markedly reduced by liriodenine, whereas the protein levels of the CDK inhibitors (CKIs), p21 and p27 were increased. Moreover, the intracellular nitric oxide (NO) production, protein levels of inducible NO synthase (iNOS) and, p53 were increased. The p53 overexpression was a downstream event of NO production in liriodenine-induced G1/S-arrested SW480 cells, and the up-regulation of p21 and p27 was found to be mediated by a p53-dependent pathway. The inhibition of p53 by pifithrin-α (PFT-α), down-regulation of p21 and p27 by siRNA, or NO reduction by S-ethylisothiourea (ETU) entirely abolished the liriodenine-induced G1/S phase arrest. We concluded that liriodenine potently inhibited the cell cycle of SW480 cancer cells via NO- and p53-dependent G1/S phase arrest pathway. These results suggest that liriodenine might be a powerful agent against colon cancer.     

Interplay between Menin and K-Ras in Regulating Lung Adenocarcinoma

MEN1, which encodes the nuclear protein menin, acts as a tumor suppressor in lung cancer and is often inactivated in human primary lung adenocarcinoma. Here, we show that the inactivation of MEN1 is associated with increased DNA methylation at the MEN1 promoter by K-Ras. On one hand, the activated K-Ras up-regulates the expression of DNA methyltransferases and enhances the binding of DNA methyltransferase 1 to the MEN1 promoter, leading to increased DNA methylation at the MEN1 gene in lung cancer cells; on the other hand, menin reduces the level of active Ras-GTP at least partly by preventing GRB2 and SOS1 from binding to Ras, without affecting the expression of GRB2 and SOS1. In human lung adenocarcinoma samples, we further demonstrate that reduced menin expression is associated with the enhanced expression of Ras (p < 0.05). Finally, excision of the Men1 gene markedly accelerates the K-Ras^G12D-induced tumor formation in the Men1^f/f;K-Ras^G12D/+;Cre ER mouse model. Together, these findings uncover a previously unknown link between activated K-Ras and menin, an important interplay governing tumor activation and suppression in the development of lung cancer.     

Spectral karyotyping of the human colon cancer cell lines SW480 and SW620

The cell lines SW480 and SW620, derived from different stages of colon carcinoma in the same patient, have been used for a number of biochemical, immunological, and genetic studies on colon cancer. A comparative analysis of their karyotypes may identify chromosomal aberrations that might represent markers for metastatic spread. In the present study spectral karyotyping (SKY) was applied to these two colon cancer cell lines. Compared to previously reported G-banded karyotypes, 9 (SW480) and 7 (SW620) markers were identical, 3 (SW480) and 3 (SW620) markers could be redefined, 5 (SW480) and 8 (SW620) markers were newly identified, and 4 (SW480) and 5 (SW620) of the previous described markers could not be confirmed. The redefined aberrations include very complex rearrangements, such as a der(16) t(3;16;1;16;8;16; 1;16;10) and a der(18)t(18;15;17)(q12; p11p13;??) in SW620 and a der(19)t(19;8;19;5) in SW480, that have not been identified by conventional banding techniques. The resulting chromosome gains (5q11-->5q15, 7pter-->q22, 11, 13q14-->qter, 20pter-->p12, X) and losses (8pter-->p2, 18q12-->qter, Y) found in both SW480 and SW620 were in good agreement with those frequently described in colorectal tumors as primary changes in the stem cell. Abnormalities found exclusively in SW620 cells only (gains of 5pter-->5q11, 12q12-->q23, 15p13-->p11, and 16q21-->q24 and losses of 2pter-->2p24, 4q28-->qter, and 6q25-->qter) can be viewed as changes that occurred in a putative metastatic founder cell.     

CCDC12 promotes tumor development and invasion through the Snail pathway in colon adenocarcinoma

Integrative expression Quantitative Trait Loci (eQTL) analysis found that rs8180040 was significantly associated with Coiled-coil domain containing 12 (CCDC12) in colon adenocarcinoma (COAD) patients. Immunohistochemical staining and western blotting confirmed CCDC12 was highly expressed in COAD tissues, which was consistent with RNA-Seq data from the TCGA database. Knockdown of CCDC12 could significantly reduce proliferation, migration, invasion, and tumorigenicity of colon cancer cells, while exogenous overexpression of CCDC12 had the opposite effect. Four plex Isobaric Tags for Relative and Absolute Quantitation assays were performed to determine its function and potential regulatory mechanism and demonstrated that overexpression of CCDC12 would change proteins on the adherens junction pathway. Overexpressed Snail and knocked down CCDC12 subsequently in SW480 cells, and we found that overexpression of Snail did not significantly change CCDC12 levels in SW480 cells, while knockdown of CCDC12 reduced that of Snail. CCDC12 plays a significant role in tumorigenesis, development, and invasion of COAD and may affect the epithelial to mesenchymal transformation process of colon cancer cells by regulating the Snail pathway.Subject terms: Diagnostic markers, Colon cancer     

Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.     

10-23型DNA酶作为鉴定mRNA靶点有效性的新工具

10-23DNA酶是能主动切割mRNA的一类反义寡核苷酸.利用10-23DNA酶的直接切割作用验证mRNA结构靶点的有效性.对筛选的绿色荧光蛋白(GFP)基因mRNA的4个靶点平行设计了4条反义寡核苷酸和4条10-23DNA酶,对照组反义寡核苷酸将最佳靶点——靶点2的反义寡核苷酸突变2个碱基,对照组10-23DNA酶将靶点2的10-23DNA酶结合臂中央突变2个碱基.体外4条10-23DNA酶切割mRNA的结果和相应的4条反义寡核苷酸依赖的RNaseH降解结果完全相似,细胞内4条10-23DNA酶对绿色荧光蛋白的表达抑制作用与相应的4条反义寡核苷酸相似,表明10-23DNA酶显示的最佳作用靶点同样是最佳作用效果的反义寡核苷酸结合靶.10-23DNA酶可以作为评价mRNA结构靶点有效性的新工具.     

2′-Functional group of adenosine in 10–23 DNAzyme promotes catalytic activity

10–23 DNAzyme is an artificially selected catalytic DNA molecule. Its great potential as genetic therapeutics promoted chemical modifications for more efficient DNAzymes. Here, 10–23 DNAzyme was modified on its six deoxyadenosine residues (A5, A9, A11, A12, A15 in the catalytic domain and A0 of the recognition arm next to the cleavage site) with compound 1, an adenosine analogue with 2′-O-[N-(aminoethyl)carbamoyl]methyl group. A positive effect of compound 1 at A15 was observed (HJDS-05, k_obs = 0.0111 min⁻¹). Compared to the effect of 2′-H and 2′-OMe at A15, this result provided an approach for more efficient DNAzyme by combining 2′-substituted amino group of adenosine with A15 as the lead structure.