Alteration of the Expression and Functional Activities of Myosin II Isoforms in Enlarged Hyperplastic Prostates.

INTRODUCTION: Benign prostatic hyperplasia (BPH) is a common pathologic process in aging men, and the contraction of the prostatic smooth muscles (SMs) in the stroma plays a vital role in this pathogenesis, leading to lower urinary tract symptoms (LUTSs). The isoforms of both the SM myosin (SMM) and non-muscle myosin (NMM) are associated with the contraction type of the prostatic SMs, but the mechanism has not been fully elucidated. METHODS: We collected prostate tissues from 30 BPH patients receiving surgical treatments, and normal human prostate samples were obtained from 12 brain-dead men. A testosterone-induced (T-induced) rat model was built, and the epithelial hyperplastic prostates were harvested. Competitive RT-PCR was used to detect the expression of SMM isoforms. We investigated the contractility of human prostate strips in vitro in an organ bath. RESULTS: The results regarding the comparisons of SMM isoforms varied between rat models and human samples. In comparison with T-induced rats and controls, competitive RT-PCR failed to show any statistically significant difference regarding the compositions of SMM isoforms. For human prostates samples, BPH patients expressed more SM-1 isoforms (66.8% vs. 60.0%, p < 0.001) and myosin light chain-17b (MLC17b) (35.9% vs. 28.5%, p < 0.05) when compared to young donors. There was a significant decrease in prostate myosin heavy chain (MHC) expression in BPH patients, with a 66.4% decrease in MHC at the mRNA level and a 51.2% decrease at the protein level. The upregulated expression of non-muscle myosin heavy chain-B (NMMHC-B) was 1.6-fold at the mRNA level and 2.1-fold at the protein level. The organ bath study showed that isolated prostate strips from BPH patients produced slower tonic contraction compared to normal humans. CONCLUSION: In this study, we claim that in the enlarged prostates of patients undergoing surgeries, MHC expression significantly decreased compared to normal tissues, with elevated levels of SM-1, MLC17b, and NMMHC-B isoforms. Modifications in SMM and NMM might play a role in the tonic contractile properties of prostatic SMs and the development of LUTS/BPH. Understanding this mechanism might provide insights into the origins of LUTS/BPH and facilitate the identification of novel therapeutic targets.

Dual Functionality of 6-Methylthioguanine: Synergistic Effects Enhancing the Photolability of DNA Nucleobases.

A small chemical modification of the nucleobase structure can significantly enhance the photoactivity of DNA, which may incur DNA damage, thus holding promising applications in photochemotherapy treatment of cancers or pathogens. However, single substitution confers only limited phototoxicity to DNA. Herein, we combine femtosecond and nanosecond time-resolved spectroscopy with high-level ab initio calculations to disentangle the excited-state dynamics of 6-methylthioguanine (me6-TG) under variable wavelength UVA excitation (310-330 nm). We find that double substitution of nucleobases (thionation and methylation) boosts the photoactivity by introducing more reactive channels. Intriguingly, 1nNπ*, rather than 1nSπ*, acts as the doorway state engendering the formation of the long-lived reactive triplet state in me6-TG. The 1nNπ* induces a low spin-orbit coupling of 8.3 cm-1, which increases the intersystem crossing (ISC) time (2.91 ± 0.14 ns). Despite the slowed ISC, the triplet quantum yield (ΦT) still accounts for a large fraction (0.6 ± 0.1), consistent with the potential energy surface that favors excited-state bifurcation to 1nNπ*min (3.36 ± 0.15 ps) rather than 1ππ*min (5.05 ± 0.26 ps), such that the subsequent ISC to triplet via 1nNπ*min constitutes the main relaxation pathway in me6-TG. Although this ΦT is inferior to its single-substituted predecessor 6-thioguanine (6-TG, 0.8 ± 0.2), the effect of thionation in synergy with methylation opens a unique C-S bond cleavage pathway through crossing to a repulsive 1πσ* state, generating thiyl radicals as highly reactive intermediates that may invoke biological damage. This photodissociation channel is extremely difficult for conventional nucleobases. These findings demonstrate the synergistic effects of double functionality substitution in modulating excited-state dynamics and enhancing the photolabile character of DNA nucleobases, providing inspirations for the rational design of advanced photodynamic and photochemotherapy approaches.

Complexed Photosensitizer of Hypericin with G-Quadruplex: Structure-Dependent Behavior.

Despite the increased interest of visible-light-absorbing compound Hypericin (Hyp) in photodiagnosis, photocatalysis, and photodynamic therapy (PDT) applications, a major obstacle still exists; i.e., the photoactivity is diminished due to the facile aggregation of Hyp in aqueous environment that induces excited-state quenching. Herein, we explore the excited-state property of Hyp bound to the DNA G-quadruplex by combining multiple steady-state and time-resolved spectroscopy. We find that the aggregation-induced quenching effect can be successfully prevented by appropriate G-quadruplex binders that disperse Hyp into monomer. The binding of Hyp/G-quadruplex is selective, however, exhibiting a preferential binding toward parallel G-quadruplexes (c-kit2, C14B1, STAT3, S50, and PS2.M), over antiparallel or hybrid G-quadruplex (Tel22, TBA). The excited-state property of Hyp is highly related to the binding behavior, showing a consistent trend that the better the Hyp/G-quadruplex binding, the longer the triplet 3Hyp* lifetime and the higher the efficiency to produce 1O2. For Hyp/c-kit2, the major binding mode is 5'-end stacking, which offers protection from collisional quenching reactions and ensures a stable photocycle of 3Hyp*-O2 energy transfer forming 1O2, leading to the highest 1O2 quantum yield (0.67) with superior photostability. These findings open possibilities of developing Hyp/G-quadruplex complex as a biocompatible photosensitizer for PDT applications, etc.

Can methylated purine bases act as photoionization hotspots?

The direct photoionization of DNA canonical bases under ultraviolet radiation is difficult due to the high ionization potentials. According to previous quantum chemical calculations, methylation can have great influence on the ionization potential. Are methylated nucleobases prone to photoionization and cause DNA damage? As an important epigenetic modification in transcription, expression, and regulation of genes, it is of great biological significance to explore the effect of methylation on base photoionization from the experimental perspective. Herein, we study the photoionization behavior of methylated purines 6 mA and 6mG at 266 nm using a nanosecond transient UV-Vis spectroscopy. The hydrated electron and methylated base radicals are observed, indicating the occurrence of photoionization for both 6mG and 6 mA. We measured one-photon ionization yields to be (5.0 ± 0.2) × 10-3 and (1.4 ± 0.2) × 10-3 for 6mG and 6 mA, respectively. These are higher than those of (dA)20 and (dA20 )·(dT20 ) previously reported, indicating that methylation significantly promotes base photoionization with a stronger effect than base stacking, consistent with calculations in literature. Given that the hydrated electrons and methylated base radicals from photoionization can trigger a cascade of deleterious reactions, the methylated purine bases may act as hotspots of DNA photoionization damage of living organisms.

Fluorescence Quenching Dynamics of 2-Amino-7-methyl-1,8-naphthyridine in Abasic-Site-Containing DNA Duplexes for Nucleobase Recognition.

Dramatic fluorescence quenching of small heterocyclic ligands trapped in the abasic site (AP) of DNA has been implemented as an unprecedented strategy recognizing single-base mutations in sequence analysis of cancer genes. However, the key mechanisms governing selective nucleobase recognition remain to be disentangled. Herein, we perform fluorescence quenching dynamics studies for 2-amino-7-methyl-1,8-naphthyridine (AMND) in well-designed AP-containing DNA single/double strands. The primary mechanism is discovered, showing that AMND only targets cytosine to form a pseudo-base pair, and therefore, fluorescence quenching of AMND arises through the DNA-mediated electron transfer (ET) between excited state AMND* and flanking nucleobases, most favorably with flanking guanines. Subtle dynamic conformational variations induced by different flanking nucleobases are revealed and found to modulate efficiencies of electron transfer and fluorescence quenching. These findings provide critical mechanistic insights for guiding the design of photoinduced electron transfer (PET)-based fluorescent ligands as sensitive single-base recognition reporters.

Isovitexin alleviates hepatic fibrosis by regulating miR-21-mediated PI3K/Akt signaling and glutathione metabolic pathway: based on transcriptomics and metabolomics.

BACKGROUND: Effective drugs for the treatment of hepatic fibrosis have not yet been identified. Isovitexin (IVT) is a promising hepatoprotective agent owing to its efficacy against acute liver injury. However, the role of IVT in liver fibrosis has not been reported. PURPOSE: To explore the effect of IVT on liver fibrosis both in vitro and in vivo. STUDY DESIGN AND METHODS: A mouse model of liver fibrosis induced by carbon tetrachloride (CCl4) and two types of hepatic stellate cell models induced by platelet-derived growth factor-BB (PDGF-BB) were established to evaluate the effect of IVT on hepatic fibrosis. Transcriptomics and metabolomics were used to predict the underlying targets of IVT and were validated by a combination of in vitro and in vivo experiments. Exploration of miRNA and N6-methyladenosine (m6A) modifications was also carried out to detect the key upstream targets of the above targets. RESULTS: IVT reduced collagen deposition and hepatic stellate cell activation to alleviate liver fibrosis. The transcriptomics and metabolomics analyses showed that phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling and the glutathione (GSH) metabolic pathway may be the main regulatory processes of IVT in hepatic fibrosis. Both the in vitro and in vivo experiments confirmed the inhibitory effect of IVT on the PTEN-PI3K-Akt-mTOR axis and activation of the GSH metabolic pathway. A miR-21 mimic inhibited the effects of IVT on these two pathways, suggesting that miR-21 is the hub for IVT regulation of PI3K-Akt signaling and the GSH metabolic pathway. IVT also increased pri-miR-21 level and reduced the m6A enrichment of pri-miR-21, demonstrating that IVT may regulate pri-miR-21 through m6A modification, thereby affecting the maturation of miR-21. CONCLUSION: This study is the first to propose a protective effect of IVT against liver fibrosis. The mechanism of IVT against hepatic fibrosis is based on the regulation of miR-21, targeting PTEN-Akt signaling and the GSH metabolic pathway, which is also a novel discovery.

Asiatic acid ameliorates rifampicin- and isoniazid-induced liver injury in vivo by regulating sphingolipid metabolism and mitogen-activated protein kinase signalling pathways.

In this study, we aimed to determine whether asiatic acid (AA) exerts any therapeutic effects on rifampicin (RFP)- and isoniazid (INH)-induced liver injury and elucidate the underlying mechanisms. Briefly, liver injury in mice was induced via RFP and INH administration. We investigated the effects and potential action mechanisms of AA on liver injury using transcriptomics, metabolomics and various examinations. We found that AA significantly ameliorated the pathological changes in liver tissues and decreased the transaminase activity, inflammation and oxidative stress damage. Transcriptomics revealed 147 differentially expressed genes (DEGs) between the AA and model groups that were enriched in metabolic and mitogen-activated protein kinase (MAPK) signalling pathways. Metabolomics revealed 778 differentially expressed metabolites between the AA and model groups. Furthermore, integrated transcriptomics and metabolomics analyses revealed strong correlations between DEGs and differentially expressed metabolites and indicated that AA regulates the sphingolipid metabolism by inhibiting the expression of delta 4-desaturase, sphingolipid 1. Experimental results confirmed that AA inhibited the MAPK signalling pathway. In summary, AA inhibits inflammation and oxidative stress damage by regulating the sphingolipid metabolism pathway and blocking the MAPK signalling pathway, thereby relieving the RFP/INH-induced liver injury.

Sequence-specific binding behavior of coralyne toward triplex DNA: An ultrafast time-resolved fluorescence spectroscopy study.

Triplex DNA structure has potential therapeutic application in inhibiting the expression of genes involved in cancer and other diseases. As a DNA-targeting antitumor and antibiotic drug, coralyne shows a remarkable binding propensity to triplex over canonical duplex and thus can modulate the stability of triplex structure, providing a prospective gene targeting strategy. Much less is known, however, about coralyne-binding interactions with triplex. By combining multiple steady-state spectroscopy with ultrafast fluorescence spectroscopy, we have investigated the binding behaviors of coralyne with typical triplexes. Upon binding with a G-containing triplex, the fluorescence of coralyne is markedly quenched owing to the photoinduced electron transfer (PET) of coralyne with the G base. Systematic studies show that the PET rates are sensitive to the binding configuration and local microenvironment, from which the coexisting binding modes of monomeric (full and partial) intercalation and aggregate stacking along the sugar-phosphate backbone are distinguished and their respective contributions are determined. It shows that coralyne has preferences for monomeric intercalation within CGG triplex and pure TAT triplex, whereas CGC+ triplex adopts mainly backbone binding of coralyne aggregates due to charge repulsion, revealing the sequence-specific binding selectivity. The triplex-DNA-induced aggregation of coralyne could be used as a probe for recognizing the water content in local DNA structures. The strong π-π stacking of intercalated coralyne monomer with base-triplets plays an important role in stabilizing the triplex structure. These results provide mechanistic insights for understanding the remarkable propensity of coralyne in selective binding to triplex DNA and shed light on the prospective applications of coralyne-triplex targeted anti-gene therapeutics.

TiO2 as a Nanozyme Mimicking Photolyase to Repair DNA Damage.

Cyclobutane pyrimidine dimer (CPD) is the most abundant DNA photolesion, and it can be repaired by photolyases based on electron-transfer mechanisms. However, photolyase is absent in the human body and lacks stability for applications. Can one develop natural enzyme mimetics utilizing nanoparticles (termed nanozymes) to mimic photolyase in repairing DNA damage? Herein, we observe the successful reversal of thymine dimer T<>T to normal T base by TiO2 under UVA irradiation. Time-resolved spectroscopy provides direct evidence that the photogenerated electron of TiO2 transfers to T<>T, causing structural instability and initiating the repair process. T-T- would then undergo bond cleavage to form T and T-, and T- returns an electron to TiO2, finishing the photocatalytic cycle. For the first time, TiO2 is discovered to exhibit photocatalytic properties similar to those of natural enzymes, pointing to its extraordinary application potential as a nanozyme to mimic photolyase in repairing DNA damage.

A nano-preparation approach to enable the delivery of daphnoretin to potentiate the therapeutical efficacy in hepatocellular cancer.

Daphnoretin (DAP), isolated from a traditional Chinese medicine Wikstroemia indica (Linn. C. A. Meyer), could induce apoptosis of hepatocellular cancer (HCC) and inhibit tumor growth. However, the application of DAP in cancer therapies was hampered because to its poor solubility. Herein, this study aimed to design an approach of double-targeted nano-preparation to enable the delivery of DAP to potentiate the therapeutical efficacy in liver cancer via glycyrrhetinic acid-polyethylene glycol-block-poly (D,L-lactic acid)/polyethylene glycol-block-poly (D,L-lactic acid)-DAP (GPP/PP-DAP). In particular, the purity of separated DAP was up to 98.12% for preparation research. GPP/PP-DAP was successfully prepared by the thin-film hydration method. Subsequently, the GPP/PP-DAP was optimized by univariate analysis and the response surface methodology, producing a stable and systemically injectable nano-preparation. Impressively, on the one hand, cytotoxicity studies showed that the IC50 of the GPP/PP-DAP was lower than that of free DAP. On the other hand, the GPP/PP-DAP was more likely to be endocytosed by HepG2 cells and targeted to the liver with orthotopic tumors, potentiating the therapeutical efficacy in HCC. Collectively, both in vitro and in vivo results indicated the excellent tumor inhibition and liver targeting of GPP/PP-DAP, suggesting the nano-preparation could serve as a potential drug delivery system for natural ingredients with anti-hepatoma activity to lay the theoretical foundation for clinical application.

Low Energy Photoionization of Phosphorothioate DNA-Oligomers and Ensuing Hole Transfer.

Phosphorothioate (PS) modified oligonucleotides (S-DNA) naturally exist in bacteria and archaea genome and are widely used as an antisense strategy in gene therapy. However, the introduction of PS as a redox active site may trigger distinct UV photoreactions. Herein, by time-resolved spectroscopy, we observe that 266 nm excitation of S-DNA d(Aps)20 and d(ApsA)10 leads to direct photoionization on the PS moiety to form hemi-bonded -P-S∴S-P- radicals, in addition to A base ionization to produce A+•/A(-H)•. Fluorescence spectroscopy and global analysis indicate that an unusual charge transfer state (CT) between the A and PS moiety might populate in competition with the common CT state among bases as key intermediate states responsible for S-DNA photoionization. Significantly, the photoionization bifurcating to PS and A moieties of S-DNA is discovered, suggesting that the PS moiety could capture the oxidized site and protect the remaining base against ionization lesion, shedding light on the understanding of its existence in living organisms.

Reminiscence therapy-based care program serves as an optional nursing modality in alleviating anxiety and depression, improving quality of life in surgical prostate cancer patients.

PURPOSE: Reminiscence therapy is reported to attenuate the psychological disorders in cancer patients, such as colorectal and lung cancer patients. However, relevant report on surgical prostate cancer patients is scarce. This study put forward a reminiscence therapy-based care program (RTCP + UC) combing reminiscence therapy with usual care (UC), and aimed to evaluate the impact of RTCP + UC on anxiety, depression, quality of life and survival in surgical prostate cancer patients. METHODS: Totally, 108 prostate cancer patients receiving surgical resection were enrolled, who were subsequently randomized and allocated to the RTCP + UC group (N = 55) and UC group (N = 53) at a 1:1 ratio. Hospital Anxiety and Depression Scale (HADS) and QLQ-C30 were assessed at month M0, M3, M6, M9 and M12 during the intervention period. After intervention, patients were followed up for another 24 months to calculate disease-free survival (DFS) and overall survival (OS). RESULTS: RTCP + UC decreased HADS-anxiety score at M9 and M12, declined HADS-depression score at M6, M9 and M12, reduced depression rate and the severity level of depression at M12, while did not affect these issues at other time points. Meanwhile, RTCP + UC enhanced the QLQ-C30 global health status score at M3, M6, M9 and M12, but did not influence the QLQ-C30 function score and QLQ-C30 symptom score at any time points. Meanwhile, RTCP + UC had no effect on the accumulating DFS and OS of surgical prostate cancer patients. CONCLUSION: RTCP + UC serves as an optional nursing modality in alleviating anxiety and depression, improving quality of life in surgical prostate cancer patients.

Association of a Novel DOCK2 Mutation-Related Gene Signature With Immune in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality worldwide. Many studies have shown that dedicator of cytokinesis 2 (DOCK2) has a crucial role as a prognostic factor in various cancers. However, the potentiality of DOCK2 in the diagnosis of HCC has not been fully elucidated. In this work, we aimed to investigate the prognostic role of DOCK2 mutation in HCC. The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) cohorts were utilized to identify the mutation frequency of DOCK2. Then, univariate Cox proportional hazard regression analysis, random forest (RF), and multivariate Cox regression analysis were performed to develop the risk score that was significantly related to DOCK2 mutation. Moreover, Gene Set Enrichment Analysis (GSEA), Gene Set Variation Analysis (GSVA), and immune correlation analysis were conducted for an in-depth study of the biological process of DOCK2 mutation involved in HCC. The results revealed that the mutation frequency of DOCK2 was relatively higher than that in non-cancer control subjects, and patients with DOCK2 mutations had a low survival rate and a poor prognosis compared with the DOCK2-wild group. In addition, the secretin receptor (SCTR), tetratricopeptide repeat, ankyrin repeat and coiled-coil domain-containing 1 (TANC1), Alkb homolog 7 (ALKBH7), FRAS1-related extracellular matrix 2 (FREM2), and G protein subunit gamma 4 (GNG4) were found to be the most relevant prognostic genes of DOCK2 mutation, and the risk score based on the five genes played an excellent role in predicting the status of survival, tumor mutation burden (TMB), and microsatellite instability (MSI) in DOCK2 mutant patients. In addition, DOCK2 mutation and the risk score were closely related to immune responses. In conclusion, the present study identifies a novel prognostic signature in light of DOCK2 mutation-related genes that shows great prognostic value in HCC patients; and this gene mutation might promote tumor progression by influencing immune responses. These data may provide valuable insights for future investigations into personalized forecasting methods and also shed light on stratified precision oncology treatment.

The Integrated Analysis of Transcriptomics and Metabolomics Unveils the Therapeutical Effect of Asiatic Acid on Alcoholic Hepatitis in Rats.

The present study was to investigate the therapeutical effects and mechanisms of Asiatic acid from Potentilla chinensis against alcoholic hepatitis. Rats were intragastrically fed with alcohol for 12 weeks to induce alcoholic hepatitis and then treated with various drugs for further 12 weeks. The results showed that Asiatic acid significantly alleviated liver injury caused by alcohol in rats, as evidenced by the improved histological changes and the lower levels of AST, ALT, and TBIL. Besides, Asiatic acid significantly enhanced the activity of ADH and ALDH, promoting alcohol metabolism. Asiatic acid suppressed CYP2E1 activity and NADP+/NADPH ratio, resulting in low ROS production. Further study revealed that Asiatic acid markedly reduced hepatocyte apoptosis by regulating the expression levels of apoptosis-related protein. Moreover, Asiatic acid could regulate the Nrf2 and NF-κB signaling pathway, attenuating oxidative stress and inflammation as a result. Interestingly, the comprehensive analysis of transcriptomics and metabolomics indicated that Asiatic acid inhibited the gene expression of Gpat3 and thereby affected the biosynthesis of the metabolites (1-acyl-Sn-glycerol-3-phosphocholine, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine), regulating the glycerophospholipid metabolism pathway and ultimately ameliorating hepatocyte damage. In conclusion, this study demonstrates that Asiatic acid can ameliorate alcoholic hepatitis by modulating the NF-κB and Nrf2 signaling pathways and the glycerophospholipid metabolism pathway, which may be developed as a potential medicine for the treatment of alcoholic hepatitis.

Isovitexin protects against acute liver injury by targeting PTEN, PI3K and BiP via modification of m6A.

Isovitexin (IVT) has been shown to have a potential therapeutic effect on acute liver injury (ALI), but its underlying mechanisms especially the targets remain unclear, which was investigated in the present study. Briefly, the targets of IVT were predicted by bioinformatics and then were verified by multiple examinations using molecular docking, cellular thermal shift assay (CETSA), and Lipopolysaccharide/D-Galactosamine (LPS/D-GalN)-induced ALI animal model. The bioinformatic analysis predicted that the target genes of IVT against ALI were enriched into the PI3K/Akt and ERS-related pathways, in which, molecular docking and CETSA examination verified that the binding sites of IVT likely were PTEN, PI3K and BiP. Furthermore, the possible targets were also verified by animal experiments. The results revealed that IVT significantly ameliorated the hepatic injury, as evidenced by the attenuation of histopathological changes and the reduction in serum aminotransferase and total bilirubin activities. In addition, IVT treatment led to the reduction of PTEN, BiP and ERS-related targets expressions, as well as the elevation of PI3K, Akt and mTOR expressions. Notably, IVT significantly decreased total hepatic m6A level and m6A enrichment of PTEN and BiP, suggesting IVT regulated PTEN and BiP by modulating m6A modification. To sum up, the results indicate that IVT significantly ameliorates ALI, which is attributed to its ability to regulate the PI3K/Akt pathway and ERS by targeting PTEN, PI3K and BiP via modification of m6A. Our finding demonstrates that IVT may be a promising natural medicine for the treatment of ALI.

Free-Radical-Mediated Photoinduced Electron Transfer between 6-Thioguanine and Tryptophan Leading to DNA-Protein-Like Cross-Link.

The nucleobase analog 6-thioguanine (6-TG) has emerged as important immunosuppressant, anti-inflammatory, and anticancer drug in the past few decades, but its unique photosensitivity of absorbing strongly ultraviolet UVA light elicits photochemical hazards in many ways. The particularly intriguing yet unresolved question is whether the direct photoreaction of 6-TG can promote DNA-protein cross-links (DPCs) formation, which are large DNA adducts blocking DNA replication and physically impede DNA-related processes. Herein, by real-time observation of radical intermediates using time-resolved UV-vis absorption spectroscopy in conjunction with product analysis by HPLC-MS, we discover that UVA excitation of 6-TG triggers direct covalent cross-linking with tryptophan (TrpH) via an exquisite radical mechanism of electron transfer. The photoexcitation prepares the redox-active triplet 36-TG*, which initiates electron transfer with TrpH, creating TrpH•+ and 6-TG•- in the first step. The deprotonated Trp• undergoes radical-recombination with its geminate partner 6-TG•- and eliminates a H2S, leading to the cross-linking product 6-TG-Trp. The photoadduct structures (two chiral isomers and one constitutional isomer) are identified unambiguously, validating further the mechanism. These findings pinpoint the exact amino acid that is vulnerable to photo-cross-linking with 6-TG and establish a mechanistic framework for understanding mutagenic DPCs formation and developing photoprobes based on this new type of photo-cross-linking.

Reactivity and DNA Damage by Independently Generated 2'-Deoxycytidin-N4-yl Radical.

Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2'-deoxycytidin-N4-yl radical (dC·) plays a role in DNA damage mediated by one electron oxidants, such as HOCl and ionizing radiation. However, the reactivity of dC· is not well understood. To reduce this knowledge gap, we photochemically generated dC· from a nitrophenyl oxime nucleoside and within chemically synthesized oligonucleotides from the same precursor. dC· formation is confirmed by transient UV-absorption spectroscopy in laser flash photolysis (LFP) experiments. LFP and duplex DNA cleavage experiments indicate that dC· oxidizes dG. Transient formation of the dG radical cation (dG+•) is observed in LFP experiments. Oxidation of the opposing dG in DNA results in hole transfer when the opposing dG is part of a dGGG sequence. The sequence dependence is attributed to a competition between rapid proton transfer from dG+• to the opposing dC anion formed and hole transfer. Enhanced hole transfer when less acidic O6-methyl-2'-deoxyguanosine is opposite dC· supports this proposal. dC· produces tandem lesions in sequences containing thymidine at the 5'-position by abstracting a hydrogen atom from the thymine methyl group. The corresponding thymidine peroxyl radical completes tandem lesion formation by reacting with the 5'-adjacent nucleotide. As dC· is reduced to dC, its role in the process is traceless and is only detectable because of the ability to independently generate it from a stable precursor. These experiments reveal that dC· oxidizes neighboring nucleotides, resulting in deleterious tandem lesions and hole transfer in appropriate sequences.

The cell-impermeable Ru(II) polypyridyl complex as a potent intracellular photosensitizer under visible light irradiation via ion-pairing with suitable lipophilic counter-anions.

Developing the cell-impermeable Ru(II) polypyridyl cationic complexes as effective photosensitizers (PS) which have high cellular uptake and photo-toxicity, but low dark toxicity, is quite challenging. Here we found that the highly reactive singlet oxygen (1O2) can be generated by the irradiation of a typical Ru(II) polypyridyl complex Ru(II)tris(tetramethylphenanthroline) ([Ru(TMP)3]2+) under visible light irradiation by ESR with TEMPO (2,2,6,6-tetramethyl-4-piperidone-N-oxyl) as 1O2 probe. Effective cellular and nuclear delivery of cationic [Ru(TMP)3]2+ was achieved through our recently developed ion-pairing method, and 2,3,4,5-tetrachlorophenol (2,3,4,5-TeCP) was found to be the most effective among all chlorophenols tested. The accelerated cellular, especially nuclear uptake of [Ru(TMP)3]2+ results in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and DNA strand breaks, caspase 3/7 activation and cell apoptosis in HeLa cells upon light irradiation. More importantly, compared with other traditional photosensitizers, [Ru(TMP)3]2+ showed significant photo-toxicity but low dark toxicity. Similar effects were observed when 2,3,4,5-TeCP was substituted by the currently clinically used anti-inflammatory drug flufenamic acid. This represents the first report that the cell-impermeable Ru(II) polypyridyl complex ion-paired with suitable lipophilic counter-anions functions as potent intracellular photosensitizer under visible light irradiation mainly via a 1O2-mediated mechanism. These findings should provide new perspectives for future investigations on other metal complexes with similar characteristics as promising photosensitizers for potential photodynamic therapy.

Ultrafast excited state dynamics and light-switching of [Ru(phen)2(dppz)]2+ in G-quadruplex DNA.

The triplet metal to ligand charge transfer (3MLCT) luminescence of ruthenium (II) polypyridyl complexes offers attractive imaging properties, specifically towards the development of sensitive and structure-specific DNA probes. However, rapidly-deactivating dark state formation may compete with 3MLCT luminescence depending on different DNA structures. In this work, by combining femtosecond and nanosecond pump-probe spectroscopy, the 3MLCT relaxation dynamics of [Ru(phen)2(dppz)]2+ (phen = 1,10-phenanthroline, dppz = dipyridophenazine) in two iconic G-quadruplexes has been scrutinized. The binding modes of stacking of dppz ligand on the terminal G-quartet fully and partially are clearly identified based on the biexponential decay dynamics of the 3MLCT luminescence at 620 nm. Interestingly, the inhibited dark state channel in ds-DNA is open in G-quadruplex, featuring an ultrafast picosecond depopulation process from 3MLCT to a dark state. The dark state formation rates are found to be sensitive to the content of water molecules in local G-quadruplex structures, indicating different patterns of bound water. The unique excited state dynamics of [Ru(phen)2(dppz)]2+ in G-quadruplex is deciphered, providing mechanistic basis for the rational design of photoactive ruthenium metal complexes in biological applications.

Mechanism of transmembrane and coiled-coil domain 1 in the regulation of proliferation and migration of A549 cells.

Bioinformatics analyses have shown that transmembrane and coiled-coil domain 1 (TMCO1) may be associated with lung adenocarcinoma. However, to the best of our knowledge, no current research has determined whether TMCO1 is involved in the development of lung adenocarcinoma. The present study aimed to identify the association between TMCO1 and lung adenocarcinoma. The present study demonstrated that the positive immunohistochemical staining of TMCO1 in lung adenocarcinoma tissues was significantly higher compared with paracarcinoma tissues. Additionally, knockdown of TMCO1 was demonstrated to downregulate B-cell lymphoma-2 protein expression levels and upregulate cysteinyl aspartate specific proteinase (caspase)-3 and caspase-9 protein expression levels in A549 cells. These changes resulted in decreased apoptosis of A549 cells uponTMCO1 downregulation. In addition, knockdown of TMCO1 decreased matrix metalloproteinase (MMP)-2 and MMP-9 expression levels. The expression of N-cadherin and vimentin also decreased. By contrast, the expression levels of E-cadherin protein increased. Knockdown of TMCO1 resulted in the inhibition of A549 cell migration. The results of the present study demonstrated that TMCO1 was associated with lung adenocarcinoma and that inhibition of TMCO1 expression levels negatively regulated the apoptosis and migration of lung adenocarcinoma cells. Therefore, the present study suggests the potential for TMCO1 to be used in the clinical treatment of lung adenocarcinoma.