Mitochondrial Dynamics of Bcl-2 Family Proteins during 17-ß-Estradiol-Induced Apoptosis Correlate with the Malignancy of Endometrial Cancer Cells.

Excessive fat intake leads to an increase in cholesterol. Overexposure to estrogen derived from cholesterol is known to contribute to the malignancy of endometrial adenocarcinomas. However, it is not well understood the relationship between the exposure to estrogen and the malignancy of endometrial adenocarcinomas. We investigated how estrogen affected the malignancy of endometrial cancer cells, specifically HEC1 cells (a moderately differentiated adenocarcinoma) and HEC50B cells (a poorly differentiated adenocarcinoma). Cell viability was decreased by exogenous 17-ß-estradiol (E2) in a concentration-dependent manner. E2 disturbed the mitochondrial membrane potentials by changing the localization of the B-cell lymphoma 2 (Bcl-2) family protein; however, there were significant differences in the localization of Bcl-2 family proteins between HEC1 and HEC50B cells. In HEC1 cells, E2 increased the expression of B-cell lymphoma-extra large (Bcl-XL) and the Bcl-2-associated X protein (Bax) and decreased Bcl-2 and Bcl-2-associated death promoter (Bad) expression on the outer mitochondrial membrane. Conversely, E2 increased the expression of Bad and Bax, and it decreased Bcl-2 and Bcl-XL expressions on the outer mitochondrial membrane in HEC50B cells. The disturbance of the mitochondrial membrane potential led to the release of cytochrome c from the mitochondria to the cytosolic space followed by activating caspase-9. After that, caspase-3 was activated and induced apoptosis. These results suggested that the localization of the Bcl-2 family protein observed under E2-induced apoptosis is related to the malignancy of endometrial cancer cells. We hope that the dynamics of Bcl-2 family proteins such as Bcl-XL and Bad will be used to diagnose malignant endometrial adenocarcinomas.

Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry.

We report the first sequencing of morpholino antisense oligonucleotides (phosphorodiamidate morpholino oligomers, PMOs) using electron capture dissociation (ECD) mass spectrometry. In this research, we found dissociation of the backbone of 18- to 25-mer PMOs to produce d and z ions as the major ions, and 100% cleavage coverage (sequence coverage) was obtained with these ions. This is a critical contrast with beam-type collision-induced dissociation, which dominantly induces base loss, so it is difficult to obtain sequence information. The results showed that an electron beam energy (typically 15 eV) can be used universally for PMOs with different sequences, lengths, and charge states so that no detailed optimization is required for multiprecursor targeting liquid chromatography coupled with tandem mass spectrometry measurements. We also confirmed that the ECD reaction speed was compatible with the high-performance liquid chromatography time scale. Finally, we demonstrated a liquid chromatography electron capture dissociation tandem mass spectrometry workflow to survey the modification sites of the emulated PMO impurities.

Dynamic changes in the association between maternal mRNAs and endoplasmic reticulum during ascidian early embryogenesis.

Axis formation is one of the most important events occurring at the beginning of animal development. In the ascidian egg, the antero-posterior axis is established at this time owing to a dynamic cytoplasmic movement called cytoplasmic and cortical reorganisation. During this movement, mitochondria, endoplasmic reticulum (ER), and maternal mRNAs (postplasmic/PEM RNAs) are translocated to the future posterior side. Although accumulating evidence indicates the crucial roles played by the asymmetrical localisation of these organelles and the translational regulation of postplasmic/PEM RNAs, the organisation of ER has not been described in sufficient detail to date owing to technical difficulties. In this study, we developed three different multiple staining protocols for visualising the ER in combination with mitochondria, microtubules, or mRNAs in whole-mount specimens. We defined the internally expanded "dense ER" using these protocols and described cisterna-like structures of the dense ER using focused ion beam-scanning electron microscopy. Most importantly, we described the dynamic changes in the colocalisation of postplasmic/PEM mRNAs and dense ER; for example, macho-1 mRNA was detached and excluded from the dense ER during the second phase of ooplasmic movements. These detailed descriptions of the association between maternal mRNA and ER can provide clues for understanding the translational regulation mechanisms underlying axis determination during ascidian early embryogenesis.

Different reactivity of Sp and Rp isomers of phosphorothioate-modified oligonucleotides in a duplex structure.

The presence of a stereoisomeric center at the phosphorus atom in phosphorothioate-modified oligonucleotides (PS-ONs) has been recognized as an important feature since the early stages of their development. Therefore, several studies have been conducted on the chirality of PS-ONs. In this study, we evaluated the stereo-biased chemistry of PS-ON duplexes. Depending on their absolute configurations, PS-ON duplexes were found to have significantly different and stereospecific reactivities towards simple alkylating reagent.

Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro.

Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skipping in vitro using reverse transcription-polymerase chain reaction. We demonstrated that the number of LNAs in the SSO sequence and the melting temperature of the SSOs play important roles in inducing exon skipping and seem to be key factors for designing efficient LNA SSOs. LNA SSO length was an important determinant of activity: a 13-mer with six LNA modifications had the highest efficacy, and a 7-mer was the minimal length required to induce exon skipping. Evaluation of exon skipping activity using mismatched LNA/DNA mixmers revealed that 9-mer LNA SSO allowed a better mismatch discrimination. LNA SSOs also induced exon skipping of endogenous human dystrophin in primary human skeletal muscle cells. Taken together, our findings indicate that LNA SSOs are powerful tools for modulating pre-mRNA splicing.

Self-assembling polymer micelle/clay nanodisk/doxorubicin hybrid injectable gels for safe and efficient focal treatment of cancer.

The purpose of this study was to fabricate a safe and effective doxorubicin (DOX)-delivery system for focal cancer chemotherapy. A novel biodegradable injectable gel was developed through self-assembly of poly(D,L-lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide) (PLGA-PEG-PLGA) copolymer micelles, clay nanodisks (CNDs), and DOX. We discovered that DOX loaded in the hybrid gels acts as an anticancer drug and as a building block to organize new gel networks. Accordingly, long-term sustained release of DOX from hybrid injectable gels without initial burst release was achieved. Moreover, it was revealed that the DOX incorporated into gel networks controls its own release profile. This hybrid injectable gel is a self-controlled drug release system, which is a novel concept in controlled drug release. Importantly, a single injection of PLGA-PEG-PLGA/CND/DOX hybrid gel provides long-term sustained antitumor activity in vivo against human xenograft tumors in mice, suggesting the potential of hybrid gels as a valuable local DOX-delivery platform for cancer focal therapy.

Separation of the diastereomers of phosphorothioated siRNAs by anion-exchange chromatography under non-denaturing conditions.

In recent years, several small interfering RNA (siRNA) therapeutics have been approved, and most of them are phosphorothioate (PS)-modified for improving nuclease resistance. This chemical modification induces chirality in the phosphorus atom, leading to the formation of diastereomers. Recent studies have revealed that Sp and Rp configurations of PS modifications of siRNAs have different biological properties, such as nuclease resistance and RNA-induced silencing complex (RISC) loading. These results highlight the importance of determining diastereomeric distribution in quality control. Although various analytical approaches have been used to separate diastereomers (mainly single-stranded oligonucleotides), it becomes more difficult to separate all of them as the number of PS modifications increases. Despite siRNA exhibits efficacy in the double-stranded form, few reports have examined the separation of diastereomers in the double-stranded form. In this study, we investigated the applicability of non-denaturing anion-exchange chromatography (AEX) for the separation of PS-modified siRNA diastereomers. Separation of the four isomers of the two PS bonds tended to improve in the double-stranded form compared to the single-stranded form. In addition, the effects of the analytical conditions and PS-modified position on the separation were evaluated. Moreover, the elution order of the Sp and Rp configurations was confirmed, and the steric difference between them, i.e., the direction of the anionic sulfur atom, appeared to be important for the separation mechanism in non-denaturing AEX. Consequently, all 16 peak tops of the four PS modifications were detected in one sequence, and approximately 30 peak tops were detected out of 64 isomers of six PS bonds, indicating that non-denaturing AEX is a useful technique for the quality control of PS-modified siRNA therapeutics.

Specific recognition of cytosine by hypoxanthine in pyrrolidinyl peptide nucleic acid.

Hypoxanthine is an unnatural base that can potentially pair with all natural nucleobases. While hypoxanthine in DNA exhibits marginal preference for pairing with cytosine (C), little is known about its pairing behavior in other DNA analogues. In this study, we synthesized a hypoxanthine-containing monomer and incorporated it into pyrrolidinyl peptide nucleic acid with α/ß-peptide backbone derived from D-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). DNA binding studies clearly revealed that hypoxanthine in acpcPNA behaves like G-analogue because it can specifically form a stable base pair with dC in DNA. The ability to replace G by hypoxanthine without affecting the base pairing properties of acpcPNA can solve a number of problems associated with G-rich acpcPNA including difficult synthesis, formation of secondary structures and fluorescence quenching.

Actin Filament in the First Cell Cycle Contributes to the Determination of the Anteroposterior Axis in Ascidian Development.

In many animal species, the body axis is determined by the relocalization of maternal determinants, organelles, or unique cell populations in a cytoskeleton-dependent manner. In the ascidian first cell cycle, the myoplasm, including mitochondria, endoplasmic reticulum (ER), and maternal mRNAs, move to the future posterior side concomitantly (called ooplasmic segregation or cytoplasmic and cortical reorganization). This translocation consists of first and second phases depending on the actin and microtubule, respectively. However, the transition from first to second phase, that is, translocation of myoplasmic components from microfilaments to microtubules, has been poorly investigated. In this study, we analyzed the relationship between these cytoskeletons and myoplasmic components during the first cell cycle and their role in morphogenesis by inhibitor experiments. Owing to our improved visualization techniques, there was unexpected F-actin accumulation at the vegetal pole during this transition period. When this F-actin was depolymerized, the microtubule structure was strongly affected, the myoplasmic components, including maternal mRNA, were mislocalized, and the anteroposterior axis formation was disordered. These results suggested the importance of F-actin during the first cell cycle and the existence of interactions between microfilaments and microtubules, implying the enigmatic mechanism of ooplasmic segregation. Solving this mystery leads us to an improved understanding of ascidian early development.

An improved synthesis of 2'-O,4'-C-ethylene nucleic acid (ENA) and thermodynamic studies of duplex formation containing the guanosine ENA unit.

Oligonucleotides containing 2'-O,4'-C-ethylene nucleic acids (ENA) have been proven highly effective for antisense therapeutics. 2'-O,4'-C-Ethyleneguanosine and its phosphoramidite were previously obtained from 3,5-di-O-benzy1-4-C-(p-tolulenesulfonyloxyethyl)-1,2-di-O-acetyl-α-D-erythropentofuranose by glycosylation, but with limited efficiency. Using 3,5-di-O-benzy1-4-C-(2-t-butyldiphenylsilyloxyethyl)-1,2-di-O-acetyl-α-D-erythropentofuranose as an alternative substrate, we developed several methods to obtain 2'-O,4'-C-ethyleneguanosine derivatives with much higher yields than previously reported. These methods were also applicable for the synthesis of 2'-O,4'-C-ethyleneadenosine and 2'-O,4'-C-ethylene-5-methyluridine derivatives. Moreover, we investigated the thermodynamic benefit of DNA strands containing 2'-O,4'-C-ethyleneguanosines during duplex formation with complementary RNA. Only a single modification by the nucleoside resulted in a 10-fold greater binding constant of the DNA/RNA duplex.

Enhancement of exon skipping activity by reduction in the secondary structure content of LNA-based splice-switching oligonucleotides.

PAGE and UV melting analysis revealed that longer LNA-based splice-switching oligonucleotides (SSOs) formed secondary structures by themselves, reducing their effective concentration. To avoid such secondary structure formation, we introduced 7-deaza-2'-deoxyguanosine or 2'-deoxyinosine into the SSOs. These modified SSOs, with fewer secondary structures, showed higher exon skipping activities.

Recognition of a flipped base in a hairpinloop DNA by a small peptide.

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258-259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 x 10(6) variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use pi-pi stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 x 10(6) M(-1) at 25 degrees C and the resulting binding free energy change at 25 degrees C (DeltaG degrees (25)) was -8.2 kcal mol(-1). The binding of the peptide to AP2 was also analyzed and the resulting binding constant and DeltaG degrees (25) were about 4.2 x 10(4) M(-1) and -6.3 kcal mol(-1), respectively. The difference in the binding free energy changes (DeltaDeltaG degrees (25)) of 1.9 kcal mol(-1) was comparable to the values reported in other systems and was considered a consequence of the loss of pi-pi stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.

Quantitative relationship between chemical properties and bioactivities of anti-microRNA oligonucleotides targeted to tumor-associated microRNA-21.

Synthetic anti-microRNA oligonucleotides (AMOs) are promising drug candidates to inactivate disease-related microRNAs because of their sequence-specific binding to their targets and the variety of chemical modifications available. Over the last decade, the qualitative relationships between the chemical properties of AMOs and bioactivity (inactivation of their target miRNAs) have been studied to enhance their bioactivity. On the other hand, in real-world drug development, drugs must be designed case-by-case, taking many factors into account. Thus, in order to design AMOs that target specific miRNA, understanding the quantitative relationship between the chemical properties of AMOs and inactivation of their target miRNA is necessary. Here, we aimed to find the specific quantitative relationship of AMOs targeted to tumor-associated miR-21 through direct comparison of their inactivation efficacies with systematically varied chemical properties, including sequence-specific binding affinity, nuclease resistance, and RNase H activation. As a result, we newly found the quantitative relationships; (1) sequence-specific binding affinity of AMOs against miR-21 is the main determining factor for inactivation efficacy, (2) nuclease resistance of AMOs impacts their miR-21 inactivation efficacy acting cooperatively with the binding affinity, although nuclease resistance alone does not affect the miRNA inactivation efficacy, and (3) RNase H activation is unnecessary. This study also demonstrates the utility of the obtained relationship for the design of AMO-based drugs targeted to miR-21, through cell-based analyses. Thus, the obtained quantitative relationship would make it possible to predict the miR-21 inactivation efficacy of AMOs which are newly designed.

A novel stable RNA pentaloop that interacts specifically with a motif peptide of lambda-N protein.

To achieve a novel specific peptide-nucleic acid binding model, we designed an in vitro selection procedure to decrease the energetic contribution of the electrostatic interaction in the total binding energy and to increase the contribution of hydrogen bonding and pi-pi stacking. After the selection of hairpin-loop RNAs that specifically bound to a model peptide of lambda N protein (N peptide), a new thermostable pentaloop RNA motif (N binding thermostable RNA hairpin: NTS RNA) was revealed. The obtained NTS RNA was able to bind to the N peptide with superior specificity to the boxB RNA, which is the naturally occurring partner of the lambda N protein.

Discovery of a new function of curcumin which enhances its anticancer therapeutic potency.

Curcumin has received immense attention over the past decades because of its diverse biological activities and recognized as a promising drug candidate in a large number of diseases. However, its clinical application has been hindered due to extremely low aqueous solubility, chemical stability, and cellular uptake. In this study, we discovered quite a new function of curcumin, i.e. pH-responsive endosomal disrupting activity, derived from curcumin's self-assembly. We selected anticancer activity as an example of biological activities of curcumin, and investigated the contribution of pH-responsive property to its anticancer activity. As a result, we demonstrated that the pH-responsive property significantly enhances the anticancer activity of curcumin. Furthermore, we demonstrated a utility of the pH-responsive property of curcumin as delivery nanocarriers for doxorubicin toward combination cancer therapy. These results clearly indicate that the smart curcumin assemblies act as promising nanoplatform for development of curcumin-based therapeutics.

Thermodynamic analysis of duplex formation of the heterochiral DNA with L-deoxyadenosine.

An L-DNA, the mirror-image isomer of natural DNA, has extraordinary nuclease resistance, and thus the molecules should be promising reagents for many applications, such as antisense technology. However, little is known about the structural and thermodynamic properties of DNAs with this modified nucleotide. In this study, we prepared the L-nucleotide (L-dA) and introduced it into oligodeoxyribonucleotides to assess the ability of the L-nucleotide as a functional molecule for many applications based on the DNA hybridization. Two decamers with an L-dA at the center were synthesized and duplexes with the complementary DNA strand were applied to structural and thermodynamic analyses. The structural study by CD spectra showed that the structures of both modified "L/D-D" duplexes were the typical B-form. This result suggests that the global structure of DNA was not collapsed by the introduction of an L-DNA. Thermodynamic parameters (deltaH degrees, deltaS degrees, and deltaG degrees 37) of the duplex formation, determined by UV melting experiments, indicated that the both duplexes were destabilized at about 2.5 to 3.0 kcal mol(-1) by the introduced L-dA, mainly due to an unfavorable enthalpic effect. In conjunction with information by other researchers, these results suggest that the L-DNA affect on the duplex structure and the stability vary locally; thus, the thermodynamic stability of modified L/D-D duplexes should be predictable by the nearest-neighbor thermodynamic parameters.

Curcumisome nanovesicles generated by self-assembly of curcumin amphiphiles toward cancer theranostics.

Curcumin (CCM) is an important molecule for achieving cancer theranostics because CCM is a naturally-occurring biocompatible material that exhibits both anticancer activity and strong fluorescence property that can be used for bio-imaging. However, CCM has never been utilized in clinical trials due to its extremely low water solubility, its rapid hydrolysis in aqueous conditions at neutral pH, and its low cellular uptake into cancer cells. Taking advantage of the strong hydrophobicity, π-conjugated frameworks, and ketone and enol groups that generate hydrogen bonds in CCM, we herein fabricated novel CCM-based biodegradable nanovesicles, which we termed as "curcumisome", through the self-assembly of amphiphilic CCM-poly(ethylene glycol) conjugates in aqueous media to develop multifunctional nanobiomaterials for use in cancer theranostics. A high CCM loading content in the curcumisomes was achieved, and the curcumisomes showed high water dispersibility with improved hydrolysis resistance. Importantly, the curcumisomes were effectively internalized into cancer cells and exhibited strong fluorescence for a long period, which is favorable for cancer cell imaging, although only a small amount of the curcumisomes penetrated into normal cells and showed very weak fluorescence. Moreover, curcumisomes effectively induced apoptosis of cancer cells. Thus, curcumisomes may act as multifunctional nanobiomaterials for the development of CCM-based cancer theranostics.

Triplet Analysis That Identifies Unpaired Regions of Functional RNAs.

We developed a novel method for analyzing RNA sequences, deemed triplet analysis, and applied the method in an in vitro RNA selection experiment in which HIV-1 Tat was the target. Aptamers are nucleic acids that bind a desired target (bait), and to date, many aptamers have been identified by in vitro selection from enough concentrated libraries in which many RNAs had an obvious consensus primary sequence after sufficient cycles of the selection. Therefore, the higher-order structural features of the aptamers that are indispensable for interaction with the bait must be determined by additional investigation of the aptamers. In contrast, our triplet analysis enabled us to extract important information on functional primary and secondary structure from minimally concentrated RNA libraries. As a result, by using our method, an important unpaired region that is similar to the bulge of TAR was readily predicted from a partially concentrated library in which no consensus sequence was revealed by a conventional sequence analysis. Moreover, our analysis method may be used to assess a variety of structural motifs with desired function.

Pyrrolidinyl peptide nucleic acid with α/ß-peptide backbone: A conformationally constrained PNA with unusual hybridization properties.

We describe herein a new conformationally constrained analog of PNA carrying an alternating α/ß amino acid backbone consisting of (2'R,4'R)-nucleobase-subtituted proline and (1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). The acpcPNA has been synthesized and evaluated for DNA, RNA and self-pairing properties by thermal denaturation experiments. It can form antiparallel hybrids with complementary DNA with high affinity and sequence specificity. Unlike other PNA systems, the thermal stability of acpcPNA·DNA hybrid is largely independent of G+C contents, and is generally higher than that of acpcPNA·RNA hybrid with the same sequence. Thermodynamic parameters analysis suggest that the A·T base pairs in the acpcPNA·DNA hybrids are enthalpically stabilized over G·C pairs. The acpcPNA also shows a hitherto unreported behavior, namely the inability to form self-pairing hybrids. These unusual properties should make the new acpcPNA a potentially useful candidate for various applications including microarray probes and antigene agents.

Accurate curve fitting procedure for UV melting analysis of highly thermostable RNA hairpins.

Thermostable RNA hairpins with specific loop sequences are critically important structural elements for proper folding of functional RNAs. Thermodynamic parameters of the structural elements are indispensable for secondary structure prediction. However when RNA folds into highly thermostable hairpin-loop structures, it is difficult to determine the melting temperature and other thermodynamic parameters from the normal UV melting analysis. In this study, we demonstrate a procedure to solve the problem using our software, Jfit. UV absorbance in the higher temperature region above 95 degrees C and the upper baseline were estimated using this analytical procedure. The ideal lines of -RlnK vs. 1/T were generated from the predicted thermodynamic parameters and the solution candidates were compared with the van't Hoff plot generated from the measured melting curve to judge their self-consistency. As a result, our analysis was able to reduce the number of the possible solution and to specify the best set of thermodynamic parameters for the stable hairpin formation.