Low curcumin concentration enhances the anticancer effect of 5-fluorouracil against colorectal cancer

BackgroundColon cancer treatments include surgery, radiotherapy, and chemotherapy. Chemotherapy using 5-fluorouracil (5-FU) has been widely applied to treat colorectal cancer (CRC). However, it is important to explore the use of chemotherapy drugs in combination with other agents to decrease severe adverse effects.PurposeThis study aimed to investigate the effects of curcumin in combination with 5-FU on the proliferation, migration, and apoptosis of CRC SW620 cell line both in vitro and in vivo.MethodsFlow cytometry was used to study the effect of curcumin on chemotherapy-induced apoptosis in CRC cells. The mechanism of curcumin's enhanced antitumor effect in vivo was investigated using gene knockdown, TUNEL, western blot, qRT-PCR and immunohistochemistry.ResultsThe results showed a synergistic effect of the two compounds on CRC cells. Considerable reduction in the proliferation and migration of SW620 cells was observed in the combination treatment group. Significantly increased apoptosis rate extended the survival of immunodeficient mice in the combination group as compared to that of the 5-FU group (p < 0.05). The results showed that curcumin significantly inhibited pERK signaling and downregulated L1 expression in SW620 cells.ConclusionsWe conclude that curcumin promotes chemosensitivity of CRC cells to 5-FU by downregulating L1 expression. Our findings provide experimental evidence for the synergism between curcumin and 5-FU, which can be utilized in clinical applications for reducing the toxicity and adverse effects of 5-FU.     

miR-129 promotes apoptosis and enhances chemosensitivity to 5-fluorouracil in colorectal cancer

Resistance to fluoropyrimidine-based chemotherapy is the major reason for the failure of advanced colorectal cancer (CRC) treatment. The lack of ability of tumor cells to undergo apoptosis after genotoxic stress is the key contributor to this intrinsic mechanism. Mounting evidence has demonstrated that non-coding microRNAs (miRNAs) are crucial regulators of gene expression, in particular, under acute genotoxic stress. However, there is still limited knowledge about the role of miRNAs in apoptosis. In this study, we discovered a novel mechanism mediated by microRNA-129 (miR-129) to trigger apoptosis by suppressing a key anti-apoptotic protein, B-cell lymphoma 2 (BCL2). Ectopic expression of miR-129 promoted apoptosis, inhibited cell proliferation and caused cell-cycle arrest in CRC cells. The intrinsic apoptotic pathway triggered by miR-129 was activated by cleavage of caspase-9 and caspase-3. The expression of miR-129 was significantly downregulated in CRC tissue specimens compared with the paired normal control samples. More importantly, we demonstrated that miR-129 enhanced the cytotoxic effect of 5-fluorouracil both in vitro and in vivo. These results suggest that miR-129 has a unique potential as a tumor suppressor and a novel candidate for developing miR-129-based therapeutic strategies in CRC.     

Suppression of TWIST1 enhances the sensitivity of colon cancer cells to 5-fluorouracil

The 5-fluorouracil (5FU)-based adjuvant chemotherapy improves the survival of patients with colorectal cancer, however the main obstacle affecting its effectiveness is a drug resistance. Our study aimed to investigate the impact of TWIST1 silencing on the sensitivity of cancer cells to 5FU. The suppression of TWIST1 expression in human colon cancer HT29 and HCT116 cell lines was achieved by transduction with lentiviral vector carrying the TWIST1 silencing sequence (pLL3.7-shTWIST1). The suppression of TWIST1 expression induced changes in the expression pattern of epithelial to mesenchymal transition markers, reduced the cells proliferation rate, increased their sensitivity to serum withdrawn, and increased the cytotoxic effect of 5FU. However, significantly higher 5FU cytotoxicity was observed in HT29 cell cultures. Cells with silenced TWIST1 displayed altered expression of enzymes metabolizing 5FU. The expression level of dihydropyrimidine dehydrogenase, and thymidylate synthase decreased significantly in HT29 shTWIST1 cells, but not in HCT116 shTWIST1 cells. On the other hand, significant increases in the expression levels of thymidine phosphorylase, and uridine phosphorylase 1 were seen in both cell lines with suppressed expression of TWIST1. The changes in enzymes expression were mirrored by enzymatic activities. In conclusion, our observations point to TWIST1 as a target protein to enhance the sensitivity of colorectal cancer cells to 5FU.     

Cerivastatin enhances the cytotoxicity of 5-fluorouracil on chemosensitive and resistant colorectal cancer cell lines

Cerivastatin is one of the synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used for the treatment and prevention of hypercholesterolaemia. The observation that patients receiving this drug had a lower incidence at cancer led to our interest in using it as a putative anticancer agent. In this study, we tested the cytotoxicity of cerivastatin on a panel of 5-fluorouracil (5FU) sensitive and resistant cell lines in vitro. Cerivastatin was cytotoxic to both 5FU sensitive and resistant cells. Cerivastatin significantly augmented the cytotoxic effect of 5FU on drug sensitive (6-22-fold) and resistant (229-310-fold) cell lines. Cerivastatin and 5FU acted synergistically. Cerivastatin inhibited nuclear factor kappaB DNA binding activity. The enhancing effect of cerivastatin on 5FU was partially mevalonate pathway independent. Cerivastatin may allow successful 5FU therapy in chemoresistant patients.     

The calculation of plant 5S rRNAs secondary structure

Using commercially available computer software package for ribonucleic acid (RNA) secondary structure analysis we calculated the free energy (delta G) of all higher plant 5S rRNA species. To gain insight into the relation between structure (nucleotide sequence) and free energy we generated point mutants of plant 5S rRNA and calculated their secondary structure. This analysis permitted to identify single sites which affect the stability and conformation of RNA molecule. Furthermore, the calculated data were compared with the electrophoretic mobility of 5S rRNA on polyacrylamide gels.     

Differentiation of oocyte- and somatic-type 5S rRNAs in animals

In some amphibians and bony fishes, oocyte- and somatic-type 5S rRNA genes are expressed differently in oocytes and somatic cells. In order to determine at what stage of animal evolution this differential expression system appeared and how it is regulated, the sequences of oocyte and somatic 5S rRNAs from three invertebrates (sea urchin, sea hare, and silkworm) and two vertebrates (lamprey and chick) were analyzed. It was found that the oocyte 5S rRNA from lamprey consists of two components, while its somatic 5S rRNA consists of only one. In other animals, such differential expression of 5S rRNA in oocytes and somatic cells was not seen. A phylogenetic tree of 63 animal 5S rRNAs was constructed by means of the parsimony method, and the evolution of oocyte and somatic-type 5S rRNAs was discussed.     

Nucleotide sequences of 5S rRNAs from four jellyfishes.

The nucleotide sequences of 5S rRNAs from four jellyfishes, Spirocodon saltatrix, Nemopsis dofleini, Aurelia aurita and Chrysaora quinquecirrha have been determined. The sequences are highly similar to each other. A fairly high similarity was also found between these jellyfishes and a sea anemone, Anthopleura japonica.     

Secondary structure and phylogeny of Staphylococcus and Micrococcus 5S rRNAs

Nucleotide sequences of 5S rRNAs from four bacteria, Staphylococcus aureus Smith (diffuse), Staphylococcus epidermidis ATCC 14990, Micrococcus luteus ATCC 9341 and Micrococcus luteus ATCC 4698, were determined. The secondary structural models of S. aureus and S. epidermidis sequences showed characteristics of the gram-positive bacterial 5S rRNA (116-N type [H. Hori and S. Osawa, Proc. Natl. Acad. Sci. U.S.A. 76:381-385, 1979]). Those of M. luteus ATCC 9341 and M. luteus ATCC 4698 together with that of Streptomyces griseus (A. Simoncsits, Nucleic Acids Res. 8:4111-4124, 1980) showed intermediary characteristics between the gram-positive and gram-negative (120-N type [H. Hori and S. Osawa, 1979]) 5S rRNAs. This and previous studies revealed that there exist at least three major groups of eubacteria having distinct 5S rRNA and belonging to different stems in the 5S rRNA phylogenic tree.     

Regulated alpha-globin mRNA decay is a cytoplasmic event proceeding through 3'-to-5' exosome-dependent decapping

The alpha-globin mRNA contains a C-rich stability element (CRE) in its 3' untranslated region (3' UTR) which is critical for the stability of this long-lived mRNA. A protein complex, termed the alpha-complex, forms on the CRE and has been shown to contribute to stabilization of the mRNA by at least two mechanisms, first by interacting with the poly(A)-binding protein (PABP) to prevent deadenylation, and second by protecting the mRNA from attack by an erythroid endoribonuclease. In this report, we demonstrate that the alpha-globin 3' UTR can confer stability on a heterologous mRNA in cells, and this stability is dependent on the alpha-complex. Moreover, the stability was exclusively detected with cytoplasmic mRNA, suggesting that the regulation of alpha-globin mRNA stability is a cytoplasmic event. An additional mechanism by which the alpha-complex can confer stability on an RNA in vitro was also identified and shown to involve inhibition of 3' to 5' exonucleolytic degradation. Furthermore, using an in vitro mRNA decay system, we were able to follow the demise of the alpha-globin RNA and demonstrate that the decay was initiated by deadenylation followed by 3'-to-5' decay carried out by the exosome and ultimately hydrolysis of the residual cap structure.     

Decytabine enhances cytotoxicity induced by oxaliplatin and 5-fluorouracil in the colorectal cancer cell line Colo-205

Background  

DNA methylation is an epigenetic phenomenon known to play an important role in the development of cancers, including colorectal cancer (CRC). Aberrant methylation of promoter regions of genes is potentially reversible, and if methylation is important for cancer survival, demethylation should do the opposite. To test this we have addressed the hypothesis that DNA methyltransferase inhibitors (DNMTi), decytabine and zebularine, potentiate inhibitory effects of classical anti-CRC cytostatics, oxaliplatin and 5-fluorouracil (5-FU), on survival of CRC cells in vitro.     

Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules

5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator.     

Structural analysis of prokaryotic and eukaryotic 5S rRNAs by RNase H

The availabilities of single-stranded 5S rRNA regions c, d and d' for base pairing interactions were analyzed by using synthetic DNA oligomers. Hybrid formation was detected by the endonucleolytical mode of the RNA-DNA specific action of RNase H. Provided that the hybrid interaction involved 6 successive base pairs, 5S rRNA loop c nucleotides 42-47 displayed accessibility in Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus 5S rRNAs as well as in eukaryotic 5S rRNAs from Saccharomyces carlsbergensis, Rattus rattus and Equisetum arvense. Investigating eubacterial 5S rRNA regions d and d' (nucleotides 71-76 and 99-105, respectively), susceptibility was observed in E. coli 5S rRNA which, however, decreases in B. stearothermophilus and even more so in T. thermophilus 5S rRNA. For additional evaluation of the data obtained by RNase H cleavage, association constants of the hexanucleotides were determined by equilibrium dialysis at 4 degrees C for B. stearothermophilus 5S rRNA. The results obtained reveal that nucleotides 36-41 of B. stearothermophilus 5S rRNA are inaccessible for Watson-Crick interaction, which suggests that this part of loop c is in a structurally constrained configuration, or buried in the tertiary structure or involved in tertiary interactions.     

Secondary structure and domain architecture of the 23S and 5S rRNAs

We present a de novo re-determination of the secondary (2°) structure and domain architecture of the 23S and 5S rRNAs, using 3D structures, determined by X-ray diffraction, as input. In the traditional 2° structure, the center of the 23S rRNA is an extended single strand, which in 3D is seen to be compact and double helical. Accurately assigning nucleotides to helices compels a revision of the 23S rRNA 2° structure. Unlike the traditional 2° structure, the revised 2° structure of the 23S rRNA shows architectural similarity with the 16S rRNA. The revised 2° structure also reveals a clear relationship with the 3D structure and is generalizable to rRNAs of other species from all three domains of life. The 2° structure revision required us to reconsider the domain architecture. We partitioned the 23S rRNA into domains through analysis of molecular interactions, calculations of 2D folding propensities and compactness. The best domain model for the 23S rRNA contains seven domains, not six as previously ascribed. Domain 0 forms the core of the 23S rRNA, to which the other six domains are rooted. Editable 2° structures mapped with various data are provided (http://apollo.chemistry.gatech.edu/RibosomeGallery).     

The nucleotide sequences of 5S rRNAs from three ciliated protozoa.

The nucleotide sequences of 5S rRNAs from three ciliated protozoa, Paramecium tetraurelia, Tetrahymena thermophila and Blepharisma japonicum have been determined. All of them are 120 nucleotides long and the sequence of probable tRNA binding site of position 41-44 is GAAC which is characteristic of the plant 5S rRNAs. The sequence similarity percents are 87% (Paramecium/Tetrahymena), 86% (Paramecium/Blepharisma) and 79% (Tetrahymena/Blepharisma), suggesting a close relationship of these three ciliates.     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

Reduction of the antineoplastic fluoropoyrimidine, 5-fluorouracil,to 5,6-dihydro-5-fluorouracil inEscherichia coli

A two-step culture ofE. coli, in presence of 5-fluorouracil (FU), allowed to obtain 5,6-dihydro-5-fluorouracil in the external medium. The first step led to large quantities of cells in which FU catabolic pathway was induced (complex culture medium, presence of thymidine, and growth up to the stationary phase); the second step was carried out in conditions in which FU catabolic pathway was favoured (minimal medium).     

An analysis of G-U base pair occurrence in eukaryotic 5S rRNAs

The structure-function relationship in RNA molecules is a key to understanding of the expression of genetic information. Various types of RNA play crucial roles at almost every step of protein biosynthesis. In recent years, it has been shown that one of the most important structural elements in RNA is a wobble pair G-U. In this paper, we present for the first time an analysis of the distribution of G-U pairs in eukaryotic 5S ribosomal RNAs. Interestingly, the G-U pair in 5S rRNA species is predominantly found in two intrahelical regions of the stems I and V and at the junction of helix IV and loop A. The distribution of G-U pairs and the nature of adjacent bases suggests their possible role as a recognition site in interactions with other components of protein biosynthesis machinery.