Enhanced glycolysis by ATPIF1 gene inactivation increased the anti-bacterial activities of neutrophils through induction of ROS and lactic acid.

ATP synthase inhibitory factor 1 (ATPIF1) is a mitochondrial protein that regulates the activity of FoF1-ATP synthase. Mice lacking ATPIF1 throughout their bodies (Atpif1-/-) exhibit a reduction in the number of neutrophils. However, it remains unclear whether the inactivation of ATPIF1 impairs the antibacterial function of mice, this study aimed to evaluate it using a mouse peritonitis model. Mice were intraperitoneally injected with E. coli to induce peritonitis, and after 24 h, the colonies of E. coli were counted in agarose plates containing mice peritoneal lavage fluids (PLF) or extract from the liver. Neutrophils were analyzed for glucose metabolism in glycolysis following LPS stimulation. Reactive oxygen species (ROS) and lactic acid (LA) levels in neutrophils were measured using flow cytometry and Seahorse analysis, respectively. N-Acetylcysteine (NAC) and 2-Deoxy-d-glucose (2-DG) were employed to assess the role of ROS and LA in neutrophil bactericidal activity. RNA-seq analysis was conducted in neutrophils to investigate potential mechanisms. In ATPIF1-/- neutrophils, bactericidal activity was enhanced, accompanied by increased levels of ROS and LA compared to wildtype neutrophils. The augmented bactericidal activity of ATPIF1-/- neutrophils was reversed by pretreatment with NAC or 2-DG. RNA-seq analysis revealed downregulation of multiple genes involved in glutathione metabolism, pyruvate oxidation, and heme synthesis, along with increased expression of inflammatory and apoptotic genes. This study suggests that the inactivation of the Atpif1 gene enhances glucose metabolism in neutrophils, resulting in increased bactericidal activity mediated by elevated levels of ROS and LA. Inhibiting ATPIF1 may be a potential approach to enhance antibacterial immunity.

The protective role of microbiota in the prevention of MPTP/P-induced Parkinson's disease by resveratrol.

Parkinson's disease (PD) is a tricky neurodegenerative disease characterized with motor deficits and gastrointestinal (GI) dysfunction. Gut microbiota disturbance is reported to be involved in the clinical phenotypes of PD and its pathogenesis through the brain-gut-microbiota axis. Resveratrol is a natural polyphenol that possesses various biological activities in alleviating many diseases, including PD. The present study was aimed to investigate the role of gut microbiota in resveratrol-treated PD mice. A chronic mouse model of PD was generated via the injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid (MPTP/P) for 5 consecutive weeks. Resveratrol was orally administered once a day (30 mg kg-1 d-1) for a total of 8 weeks. From the 6th week to the 8th week, fecal microbiota transplantation (FMT) was performed from resveratrol-treated PD mice to PD mice to evaluate the contribution of resveratrol-shaped microbiota in the alleviation of PD. The results showed that FMT from resveratrol-shaped microbiota remarkably alleviated the mice phenotype from PD progression, including increased latency in the rotarod, shortened beam walking time, increased the number of tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta (SNpc) and enriched TH-positive fiber density in the striatum. Further experiments revealed that FMT could ameliorate the GI dysfunction by increasing the small intestinal transport rate and the colon length, decreasing the relative abundances of inflammatory cytokines (TNF-α, IL-6 and IL-1ß) in colon epithelial tissue. The 16S rDNA sequencing indicated that FMT attenuated the gut microbial dysbiosis in PD mice by increasing the abundances of Prevotellaceae, Rikenellaceae, Erysipelotrichaceae, Blautia and Alistipes, lowering the ratio of Fimicutes/Bacteroidetes, and decreasing the abundances of Lachnospiraceae and Akkermansia. Therefore, results in this study demonstrated that gut microbiota played a vital role in the prevention of PD progression, and the shaping of the gut microbiota was the pharmacological mechanism of resveratrol in alleviating the phenotype of Parkinson's disease in PD mice.

Sirtuin 6 maintains epithelial STAT6 activity to support intestinal tuft cell development and type 2 immunity.

Dynamic regulation of intestinal epithelial cell (IEC) differentiation is crucial for both homeostasis and the response to helminth infection. SIRT6 belongs to the NAD+-dependent deacetylases and has established diverse roles in aging, metabolism and disease. Here, we report that IEC Sirt6 deletion leads to impaired tuft cell development and type 2 immunity in response to helminth infection, thereby resulting in compromised worm expulsion. Conversely, after helminth infection, IEC SIRT6 transgenic mice exhibit enhanced epithelial remodeling process and more efficient worm clearance. Mechanistically, Sirt6 ablation causes elevated Socs3 expression, and subsequently attenuated tyrosine 641 phosphorylation of STAT6 in IECs. Notably, intestinal epithelial overexpression of constitutively activated STAT6 (STAT6vt) in mice is sufficient to induce the expansion of tuft and goblet cell linage. Furthermore, epithelial STAT6vt overexpression remarkedly reverses the defects in intestinal epithelial remodeling caused by Sirt6 ablation. Our results reveal a novel function of SIRT6 in regulating intestinal epithelial remodeling and mucosal type 2 immunity in response to helminth infection.

scRNA-seq reveals ATPIF1 activity in control of T cell antitumor activity.

ATP synthase inhibitory factor 1 (ATPIF1) is a mitochondrial protein with an activity in inhibition of F1Fo-ATP synthase. ATPIF1 activity remains unknown in the control of immune activity of T cells. In this study, we identified ATPIF1 activity in the induction of CD8+ T cell function in tumor models through genetic approaches. ATPIF1 gene inactivation impaired the immune activities of CD8+ T cells leading to quick tumor growth (B16 melanoma and Lewis lung cancer) in ATPIF1-KO mice. The KO T cells exhibited a reduced activity in proliferation and IFN-γ secretion with metabolic reprogramming of increased glycolysis and decreased oxidative phosphorylation (OXPHOS) after activation. T cell exhaustion was increased in the tumor infiltrating leukocytes (TILs) of KO mice as revealed by the single-cell RNA sequencing (scRNA-seq) and confirmed by flow cytometry. In contrast, ATPIF1 overexpression in T cells increased expression of IFN-γ and Granzyme B, subset of central memory T cells in CAR-T cells, and survival rate of NALM-6 tumor-bearing mice. These data demonstrate that ATPIF1 deficiency led to tumor immune deficiency through induction of T cell exhaustion. ATPIF1 overexpression enhanced the T cell tumor immunity. Therefore, ATPIF1 is a potential molecular target in the modulation of antitumor immunity of CD8+ T cells in cancer immunotherapy. Induction of ATPIF1 activity may promote CAR-T activity in cancer therapy.

Discovery of novel ID2 antagonists from pharmacophore-based virtual screening as potential therapeutics for glioma.

Glioma, especially the most aggressive type glioblastoma multiforme, is a malignant cancer of the central nervous system with a poor prognosis. Traditional treatments are mainly surgery combined with radiotherapy and chemotherapy, which is still far from satisfactory. Therefore, it is of great clinical significance to find new therapeutic agents. Serving as an inhibitor of differentiation, protein ID2 (inhibitor of DNA binding 2) plays an important role in neurogenesis, neovascularization and malignant development of gliomas. It has been shown that ID2 affects the malignant progression of gliomas through different mechanisms. In this study, a pharmacophore-based virtual screening was carried out and 16 hit compounds were purchased for pharmacological evaluations on their ID2 inhibitory activities. Based on the cytotoxicity of these small-molecule compounds, two compounds were shown to effectively inhibit the viability of glioma cells in the micromolar range. Among them, AK-778-XXMU was chosen for further study due to its better solubility in water. A SPR (Surface Plasma Resonance) assay proved the high affinity between AK-778-XXMU and ID2 protein with the KD value as 129 nM. The plausible binding mode of ID2 was studied by molecular docking and it was found to match AGX51 very well in the same binding site. Subsequently, the cancer-suppressing potency of the compound was characterized both in vitro and in vivo. The data demonstrated that compound AK-778-XXMU is a potent ID2 antagonist which has the potential to be developed as a therapeutic agent against glioma.

Potent half-sandwich Ru(â ¡) N

Herein a new series of organometallic half-sandwich Ru(Ⅱ) complexes bearing aryl-BIAN chelating ligands with various electron-withdrawing and electron-donating substituents have been developed as theranostic agents. All the complexes display much higher anti-proliferative potency than the clinical chemotherapeutic drug cisplatin towards seven cancer cell lines. The anti-proliferative efficacy of these complexes is correlated to their electron-withdrawing ability. Interestingly, complex Ru1 also potently suppresses cancer cell migration in vitro and effectively inhibit tumor growth in vivo in a CT26 colon cancer mouse xenograft model. Mechanisms of action studies display that Ru1 can favorably accumulate in lysosome and exerts anti-cancer potency by inducing a series of events related to lysosomal dysfunction in CT26 cells. Interestingly, inhibition of lysosomal enzymes leads to suppression of cytotoxicity and apoptosis induced by Ru1. Our results elucidate that complex Ru1 can elicit cytotoxicity through lysosome-mediated apoptosis in vitro and suppress tumor growth in vivo.

Chitooligosaccharides Prevents the Development of Colitis-Associated Colorectal Cancer by Modulating the Intestinal Microbiota and Mycobiota.

Gut microbes play a crucial role in the development of colorectal cancer. Chitooligosaccharides (COS), are oligomer that are depolymerized from chitosan and possess a wide range of biological activities. In this study, the effects of COS on colorectal cancer (CRC) development were evaluated using azoxymethane and dextran sulfate sodium (AOM/DSS) induced mouse model of CRC (CACM). In the COS-treated CRC group (CMCOS), COS protected mice from CRC by decreasing the disease activity index, tumor incidences and multiplicity, and the mRNA levels of COX-2, IL-6, TNF-α, IL-1ß, IL-10, and IKK-ß mRNA in colonic epithelial cells. The results of a cage-exchanged experiment, in which mice from the CACMe and CMCOSe treatments exchanged cages every day to interact with microbes, showed that gut microbes play an important role in preventing CAC by COS. The abundances of fecal bacteria (total bacteria, Lactobacillus, Enterococcus, Fusobacterium nucleatum and butyrate-producing bacteria) were detected by qPCR on the 0th, 1st, 3rd, 6th, 9th, and 10th weekends. Furthermore, microbiota and mycobiota were analyzed by high-throughput sequencing on an Illumina MiSeq PE300 system. COS protected mice from CRC by reversing the imbalance of bacteria and fungi, especially by reducing the abundance of Escherichia-Shigella, Enterococcus, and Turicibacter, and increasing the levels of Akkermansia, butyrate-producing bacteria and Cladosporium.

Long Noncoding RNA HOXA-AS3 Integrates NF-κB Signaling To Regulate Endothelium Inflammation.

The long noncoding RNA HOXA-AS3 has recently been reported to act as a critical regulator in inflammation-linked lung adenocarcinoma. However, the roles of HOXA-AS3 in endothelium inflammation and related vascular disorders remain poorly defined. In the current study, we identified HOXA-AS3 to be a critical activator to promote NF-κB-mediated endothelium inflammation. HOXA-AS3, a chromatin-associated regulator which colocalizes with NF-κB at specific gene promoters, was found to interact with NF-κB and positively regulate its activity through control of the expression of the NF-κB inhibitor protein IκBα and the acetylation status at the K310 site of p65. More importantly, clinicopathological analysis showed that HOXA-AS3 expression has a significant positive correlation with atherosclerosis. Thus, we conclude that HOXA-AS3 may serve as a crucial biomarker for the clinical diagnosis of atherosclerosis, as well as a promising therapeutic target for the treatment of multiple inflammatory vascular diseases. In addition, this study suggests the functional importance of HOXA-AS3 in the regulation of inflammatory disorders.

LncRNA HOXA-AS2 represses endothelium inflammation by regulating the activity of NF-κB signaling.

BACKGROUND AND AIMS: Endothelium inflammation, which can lead to endothelial activation and dysfunction, is widely accepted as the major event in multiple vascular disorders. The lncRNA HOXA-AS2 was previously reported to be involved in multiple inflammation-linked cancers. However, the role of HOXA-AS2 in endothelium inflammation is poorly understood. This study aims to determine the regulatory role of HOXA-AS2 in endothelium inflammation and related vascular diseases. METHODS: High throughput mRNA sequencing was performed to establish expression profiles after HOXA-AS2 depletion. We extracted total RNAs of human peripheral blood mononuclear cells from normal control group and experimental group with carotid artery atherosclerosis, and performed qRT-PCR to assay the correlation between HOXA-AS2 expression and inflammatory vascular diseases. RESULTS: Inhibition of HOXA-AS2 can induce the activation of NF-κB signaling and subsequent inflammatory response. More importantly, HOXA-AS2 is inversely found to be inversely regulated by NF-κB in a negative feedback manner by helping recruit BRD4/P-TEFb complex to HOXA-AS2 promoter region, therefore facilitating release of the promoter-proximal paused RNA polymerase II and activating transcription elongation. CONCLUSIONS: We identify HOXA-AS2 as a critical repressor of endothelium inflammation. Moreover, this study offers us a new way to balance the NF-κB signaling-driven excessive endothelium inflammation by establishing a NF-κB/HOXA-AS2 negative feedback loop. Based on these findings, we conclude that HOXA-AS2 may serve as a crucial therapeutic target for various vascular disorders which are significantly associated with endothelium inflammation.

Novel half-sandwich iridium OˆC (carbene)-Complexes: In vitro and in vivo tumor growth suppression and pro-apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes.

Herein we present half-sandwich IrIII complexes [(η5-Cpxbiph)Ir(OˆC)Cl] containing OˆC(NHC)-chelating ligand as anticancer and antimetastasis agents. All the complexes displayed high potency in vitro against a wide range of cancer cells. In addition, Ir2 significantly curb tumor growth in a colon cancer mouse xenograft model in vivo. Further mechanism of action studies indicate that Ir2-initiated apoptosis occurs through ROS-mediated cross-talk between mitochondria and lysosomes.

LncRNA HOXA-AS2 positively regulates osteogenesis of mesenchymal stem cells through inactivating NF-κB signalling.

As is previously reported, mesenchymal stem cells have potential ability to differentiate into osteocytes. However, the underlying mechanism during this biological process is poorly understood. In the present study, we identify a novel long non-coding RNA named HOXA-AS2 as a critical regulator during the formation of osteogenesis. Attenuation of HOXA-AS2 can reduce the calcium deposition and repress the alkaline phosphatase activity. Moreover, the expressions of osteogenic marker genes are markedly downregulated after HOXA-AS2 depletion. Mechanistically, we found HOXA-AS2 can regulate the transcriptional activity of NF-κB, a critical inhibitor of osteogenesis. More importantly, HOXA-AS2 knockdown could result in the transcriptional repression of the osteogenic master transcription factor SP7 by a NF-κB/HDAC2-coordinated H3K27 deacetylation mechanism. Based on these studies, we conclude that HOXA-AS2 may serve as a promising therapeutic target for bone tissue repair and regeneration in the near future.

An arginine-rich cell penetrating peptide contained anti-gelatinase scFv-LDM fusion protein shows potent antitumor efficacy in pancreatic cancer.

Pancreatic cancer (PC) is one of the most dangerous cancers with less than 5% survival rate in 5 years. This study was to evaluate the antitumor activities of dFv-LDP-AE and dFv-R-LDP-AE, two energized fusion protein targeting gelatinases, on pancreatic cancer. The fusion protein dFv-LDP-AE consists of two tandem anti-gelatianses scFv and an enediyne antibiotic lidamycin (LDM) for receptor binding and cell killing. To improve the penetration capability, the fusion protein dFv-LDP-AE was integrated with an arginine-rich cell penetrating peptide (Arg)9 and then generated the fusion protein dFv-R-LDP-AE. The current study demonstrated that dFv-LDP and dFv-R-LDP had high affinity with the antigen gelatinases and PC cells, the integration of (Arg)9 could increase the penetration rate of fusion protein in SW-1990 and PANC-1 cells. After enediyne-energized with chromophore of lidamycin, the energized fusion protein dFv-LDP-AE and dFv-R-LDP-AE showed potent cytotoxicity to PC cells and could induced the robust cell apoptosis and necrosis in vitro. Western blot showed that dFv-R-LDP-AE could increase PARP cleavage, and inhibited the expression of VEGF, Cyclin D1, Cox-2 and Bcl-2 in SW-1990 and PANC-1 cells. In vivo, at a tolerated dosage, dFv-LDP, dFv-LDP-AE and dFv-R-LDP-AE inhibited tumor growth by 20.42%, 56.31% (P < 0.01, compared to that of control) and 74.2% (P < 0.05, compared to that of dFv-LDP-AE) in pancreatic cancer SW-1990 xenografted mice, respectively. Moreover, the results of in vivo optical imaging showed that fusion protein dFv-R-LDP displayed prominent accumulation in the tumor in SW-1990 xenografted mice and Capan-2 orthotopic transplanted mice. These results showed that dFv-R-LDP-AE possessed potent antitumor efficacy on PC, which indicating it could be a promising candidate for targeting therapy of PC.

Expression of STING and PD-L1 in colorectal cancer and their correlation with clinical prognosis.

OBJECTIVE: To investigate the expression of PD-L1 and STING in colorectal cancer and analyze their correlation with disease prognosis. METHODS: The colorectal tissue chip was taken as the research object. Immunohistochemical methods were used to detect expression of PD-L1 and STING of 87 cases of colorectal cancer tissue and corresponding adjacent tissue in the tissue chip. Chi-square test or Fisher's test were used to analyze the relationship of expression of PD-L1 and STING and with the clinical pathological features. Kaplan-Meier curves were used to analyze the relationship between expression of PD-L1 and STING and the prognosis of colorectal cancer. RESULTS: The positive expression rate of PD-L1 in 87 cases of colorectal cancer tissue and corresponding adjacent tissue was 79.3% and 42.5% respectively, and the difference was significant (P < 0.05). The positive expression rate of PD-L1 in colorectal carcinoma tissues was not related with age, gender, tumor size, differentiation, TNM clinical stage, or depth of invasion (P > 0.05). The positive expression rate of STING in 87 cases colorectal cancer tissues and corresponding adjacent tissue was 9.2% and 40.2% respectively and the difference was significant (P < 0.05). The positive expression rate of STING in colorectal carcinoma tissue was correlated with gender, age, and differentiation (P < 0.05), but not associated with tumor size, depth of invasion, and TNM clinical stage (P > 0.05). The expression rate of PD-L1 was related to the prognosis of colorectal cancer (P=0.018). The expression rate of STING was not correlated with prognosis of colorectal cancer (P=0.784). In four independent groups (PD-L1+STING+ group, PD-L1-STING- group, PD-L1-STING+ group, PD-L1+STING- group), there were significant differences in 5-year survival (P=0.047 < 0.05), the 5-year survival rate of the PD-L1-STING+ group was significantly higher than that of the other three groups. In the STING positive group, the PD-L1+STING+ group had a worse prognosis than the PD-L1-STING+ group. In the STING negative group, the PD-L1-STING-group had a higher survival rate than the PD-L1+STING- group. CONCLUSION: PD-L1 might promote the occurrence of disease and STING might play an important role in anti-tumor immunity. PD-L1 was related with the prognosis of patients with colorectal cancer but the expression of STING was not obviously associated with the prognosis. Survival rates of patients with colorectal cancer were higher in patients with PD-L1 negative expression or STING positive expression. When PD-L1 expression was negative and STING expression was positive, the 5-year survival rate of patients with colorectal cancer was highest.

A bispecific enediyne-energized fusion protein targeting both epidermal growth factor receptor and insulin-like growth factor 1 receptor showing enhanced antitumor efficacy against non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) both overexpressed on non-small cell lung cancer (NSCLC) and are known cooperatively to promote tumor progression and drug resistance. This study was to construct a novel bispecific fusion protein EGF-IGF-LDP-AE consisting of EGFR and IGF-IR specific ligands (EGF and IGF-1) and lidamycin, an enediyne antibiotic with potent antitumor activity, and investigate its antitumor efficacy against NSCLC. Binding and internalization assays showed that EGF-IGF-LDP protein could bind to NSCLC cells with high affinity and then internalized into cells with higher efficiency than that of monospecific proteins. In vitro, the enediyne-energized analogue of bispecific fusion protein (EGF-IGF-LDP-AE) displayed extremely potent cytotoxicity to NSCLC cell lines with IC50<10-11 mol/L. Moreover, the bispecific protein EGF-IGF-LDP-AE was more cytotoxic than monospecific proteins (EGF-LDP-AE and LDP-IGF-AE) and lidamycin. In vivo, EGF-IGF-LDP-AE markedly inhibited the growth of A549 xenografts, and the efficacy was more potent than that of lidamycin and monospecific counterparts. EGF-IGF-LDP-AE caused significant cell cycle arrest and it also induced cell apoptosis in a dosage-dependent manner. Pretreatment with EGF-IGF-LDP-AE inhibited EGF-, IGF-stimulated EGFR and IGF-1R phosphorylation, and blocked two main downstream signaling molecules AKT and ERK activation. These data suggested that EGF-LDP-IGF-AE protein would be a promising targeted agent for NSCLC patients with EGFR and/or IGF-1R overexpression.

Jarid2 is essential for the maintenance of tumor initiating cells in bladder cancer.

Bladder cancer is the most common urologic malignancy in China, with an increase of the incidence and mortality rates over past decades. Recent studies suggest that bladder tumors are maintained by a rare fraction of cells with stem cell proprieties. Targeting these bladder tumor initiating cell (TICs) population can overcome the drug-resistance of bladder cancer. However, the molecular and genetic mechanisms regulating TICs in bladder cancer remain poorly defined. Jarid2 is implicated in signaling pathways regulating cancer cell epithelial-mesenchymal transition, and stem cell maintenance. The goal of our study was to examine whether Jarid2 plays a role in the regulation of TICs in bladder cancer. We found that knockdown of Jarid2 was able to inhibit the invasive ability and sphere-forming capacity in bladder cancer cells. Moreover, knockdown of Jarid2 reduced the proportion of TICs and impaired the tumorigenicity of bladder cancer TICs in vivo. Conversely, ectopic overexpression of Jarid2 promoted the invasive ability and sphere-forming capacity in bladder cancer cells. Mechanistically, reduced Jarid2 expression led to the upregulation of p16 and H3K27me3 level at p16 promoter region. Collectively, we provided evidence that Jarid2 via modulation of p16 is a putative novel therapeutic target for treating malignant bladder cancer.

Isoliquiritigenin decreases the incidence of colitis-associated colorectal cancer by modulating the intestinal microbiota.

Imbalances in intestinal bacteria correlate with colitis-associated colorectal cancer (CAC). Traditional Chinese medicines have been used to adjust the gut microbiota, and isoliquiritigenin (ISL), a flavonoid extracted from licorice, has shown antitumor efficacy. In this study, the effects of ISL on CAC development and the gut microbiota were evaluated using an azoxymethane and dextran sulphate sodium (AOM/DSS)-induced mouse model of CAC (CACM). Histopathological analysis suggested that ISL reduced tumor incidence in vivo. Moreover, high-throughput sequencing and terminal restriction fragment length polymorphism (T-RFLP) studies of the bacterial 16S rRNA gene revealed that the structure of the gut microbial community shifted significantly following AOM/DSS treatment, and that effect was alleviated by treatment with high-dose ISL (150 mg/kg). Compared to the microbiota in the control mice (CK), the levels of Bacteroidetes decreased and the levels of Firmicutes increased during CAC development. ISL reversed the imbalance at the phylum level and altered the familial constituents of the gut microbiota. Specifically, the abundance of Helicobacteraceae increased after treatment with high-dose ISL, while the abundance of Lachnospiraceae and Rikenellaceae decreased. At the genus level, ISL reduced the abundance of opportunistic pathogens (Escherichia and Enterococcus), and increased the levels of probiotics, particularly butyrate-producing bacteria (Butyricicoccus, Clostridium, and Ruminococcus). Thus, ISL protects mice from AOM/DSS-induced CAC, and ISL and the gut microbiota may have synergistic anti-cancer effects.

Long non-coding RNA HoxA-AS3 interacts with EZH2 to regulate lineage commitment of mesenchymal stem cells.

Long non-coding RNAs (lncRNAs) play an important role in gene regulation and are involving in diverse cellular processes. However, their roles in reprogramming of gene expression profiles during lineage commitment and maturation of mesenchymal stem cells (MSCs) remain poorly understood. In the current study, we characterize the expression of a lncRNA, HoxA-AS3, during the differentiation of MSCs. We showed that HoxA-AS3 is increased upon adipogenic induction of MSCs, while HoxA-AS3 remains unaltered during osteogenic induction. Silencing of HoxA-AS3 in MSCs resulted in decreased adipogenesis and expression of adipogenic markers, PPARG, CEBPA, FABP4 and ADIPOQ. Conversely, knockdown of HoxA-AS3 expression in MSCs exhibited an enhanced osteogenesis and osteogenic markers expression, including RUNX2, SP7, COL1A1, IBSP, BGLAP and SPP1. Mechanistically, HoxA-AS3 interacts with Enhancer Of Zeste 2 (EZH2) and is required for H3 lysine-27 trimethylation (H3K27me3) of key osteogenic transcription factor Runx2. Our data reveal that HoxA-AS3 acts as an epigenetic switch that determines the lineage specification of MSC.

Transarterial oily chemoembolization with lidamycin shows potent therapeutic efficacy in VX2 rabbit liver tumor.

Transarterial oily chemoembolization (TOCE) is one of the most effective approaches for the treatment of patients with hepatocellular carcinoma (HCC), who are not suitable for surgical therapy. Lidamycin (LDM), a potent antitumor antibiotic, demonstrates good antitumor efficacy in various tumor types, both in vitro and in vivo. In this study, the antitumor efficacy of LDM combined with TOCE against the rabbit VX2 tumor was assessed. A toxicity assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) demonstrated that a combination of LDM with lipiodol did not impair the cytotoxicity of LDM against HepG2 cells in vitro. Using TOCE in rabbit VX2 tumor models, LDM showed a more powerful inhibitory effect against the tumor and lowered the expression levels of proliferating cell nuclear antigen (PCNA), cluster of differentiation 31 (CD31), and vascular endothelial growth factor (VEGF) compared to Adriamycin (ADM); moreover, this improvement was not accompanied by an increase of hepatotoxicity as shown by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. These results suggested that LDM combined with TOCE may be a feasible strategy in HCC therapy in the future.

PTEN plays an important role in thrombin-mediated lung cancer cell functions.

Thrombin and its membrane receptor, protease-activated receptor 1 (PAR1), have been reported to promote the development of lung cancer in vitro and in vivo. However, the intracellular molecular mechanism or signaling pathway that mediates the cytological effects after the thrombin-receptor interaction is poorly understood. Our previous study observed that the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was downregulated in thrombin-stimulated lung cancer. In this study, the role of PTEN in thrombin-mediated cell function and the corresponding cell signaling pathway were studied in lung cancer cell Glc-82. The results indicated that thrombin downregulates the PTEN expression level and that PTEN plays an important role in thrombin-mediated Glc-82 functions, including cell cycle progression, cell apoptosis, and cell migration. The PI3K/AKT signaling pathway and its related proteins, including p27 and S phase kinase associated protein 2 (Skp2), are involved in the effects induced by PTEN downregulation. PAR1 plays a role in thrombin-mediated reduction of PTEN expression. This study suggested that the PTEN/PI3K/AKT signaling pathway plays an important role in thrombin/PAR1-mediated lung cancer cell growth and migration.

Small antibody fusion proteins with complementarity-determining regions and lidamycin for tumor targeting therapy.

Gelatinases are overexpressed in several types of maligancies and tumor stromal cells. Lidamycin is an enediyne antitumor antibiotic, which is composed of an apoprotein (LDP) and an active chromophore (AE). It is known that the heavy-chain complementarity-determining region-3 (CDR3) domain of scFv is important in antibody affinity. The aim of this study was to prepare the enediyne-energized fusion proteins with a heavy-chain CDR3 domain of anti-gelatinases scFv and lidamycin, and to evaluate their antitumor efficiency. Fusion proteins comprising the CDR3 domain and the lidamycin apoprotein were generated, and ELISA, immunofluorescence and FACS were used to analyze the binding of the fusion protein with antigen gelatinases. The purified fusion proteins were assembled with the lidamycin chromophore, and the antitumor effects were evaluated in vitro and in vivo. It was found that the CDR3-LDP and CDR3-LDP-CDR3 fusion proteins demonstrated high affinity towards antigen gelatinases. Following stimulation of CDR3-LDP with enediyne, the results of MTT showed potent cytotoxicity towards tumor cells; the IC50 values of CDR3-LDP-AE to HepG2 and Bel-7402 tumor cells were 1.05×10-11 and 6.6×10-14 M, respectively. In addition, CDR3-LDP-AE displayed a potent antitumor effect in H22 cell xenografts in mice; the combination of CDR3-LDP (10 mg/kg) and CDR3-LDP-AE (0.25 and 0.5 mg/kg) revealed that the tumor inhibitory rates were 85.2 and 92.7%, respectively (P<0.05 compared with CDR3-LDP-AE). In conclusion, these results suggest that the CDR3-LDP fusion protein and its analog CDR3-LDP-AE may both be promising candidates for tumor targeting therapy.