Follistatin-like protein 1 attenuates doxorubicin-induced cardiomyopathy by inhibiting MsrB2-mediated mitophagy.

Doxorubicin (DOX) is a potent chemotherapeutic drug; however, its clinical use is limited due to its cardiotoxicity. Mitochondrial dysfunction plays a vital role in the pathogenesis of DOX-induced cardiomyopathy. Follistatin-like protein 1 (FSTL1) is a potent cardiokine that protects the heart from diverse cardiac diseases, such as myocardial infarction, cardiac ischemia/reperfusion injury, and heart failure. However, its role in DOX-induced cardiomyopathy is unclear. Therefore, the present study investigated whether administering recombinant FSTL1 could mitigate DOX-induced cardiomyopathy and clarified the underlying molecular mechanisms. FSTL1 treatment attenuated DOX-induced cardiac dysfunction, cardiac fibrosis, and cellular apoptosis by inhibiting excess mitochondrial matrix protein methionine sulfoxide reductase B2 (MsrB2)-mediated mitophagy. Furthermore, FSTL1 administration reduced the expression of apoptotic proteins, including MsrB2, Bax, caspase 3, mitochondrial Parkin, and LC3-II, increased myocardial ATP content, and decreased cardiac malondialdehyde levels, thus protecting mitochondrial function against DOX-induced cardiac injury. Furthermore, FSTL1 treatment protected the contractile properties of adult cardiomyocytes against DOX-induced injury in vitro. Furthermore, carbonyl cyanide m-chlorophenylhydrazone, a mitophagy inducer, impaired the protective effects of FSTL1 in DOX-treated H9c2 cardiomyocytes. In conclusion, these results show that FSTL1 is a novel therapeutic agent against DOX-induced cardiotoxicity that improves mitochondrial function and decreases mitophagy.

Protective role of forsythoside B in Kawasaki disease-induced cardiac injury: Inhibition of pyroptosis via the SIRT1-NF-κB-p65 signaling pathway.

Kawasaki disease (KD), an acute exanthematous febrile pediatric illness involving systemic non-specific inflammatory reactions in small- and medium-sized arteries, poses a significant risk of coronary artery and myocardial inflammatory injury. Developing new KD treatments with improved safety and fewer side-effects is highly desirable. Forsythoside B (FTS-B), extracted from the Forsythia suspensa plant, exerts anti-inflammatory activity by inhibiting NF-κB, which is regulated by SIRT1, the reduced expression of which is strongly associated with cardiovascular disease. However, it has yet to be established whether FTS-B influences KD-related inflammatory damage. In this study, we investigated the effects of FTS-B on inflammation in cellular and murine models of KD. Our findings revealed that KD is associated with cardiac dysfunction and inflammatory injury to myocardial and human coronary artery endothelial cells (HCAECs), resulting in a pyroptosis-feedback loop. Both cellular and KD models were characterized by reduced SIRT1 expression and increased NF-κB p65 expression. Contrastingly, the rates of pyroptosis in both murine model myocardial tissues and HCAECs were significantly alleviated in response to FTS-B treatment. Also in both models, we detected an increase of SIRT1 expression and a decrease in the expression of p65. Further examination of the protective mechanism of FTS-B using the SIRT1-specific inhibitor, EX 527, revealed that this inhibitor blocked the palliative effects of FTS-B on inflammatory injury-induced pyroptosis. These results highlight the potential utility of the SIRT1-NF-κB-p65 pathway as a therapeutic target for KD treatment and demonstrate that FTS-B can alleviate KD-induced cardiac and HCAEC inflammatory injury via inhibition of pyroptosis.

Comprehensive analysis of ALG3 in pan-cancer and validation of ALG3 as an onco-immunological biomarker in breast cancer.

ALG3 has significant modulatory function in the process of tumor development. Yet how ALG3 involves in the advancement of different malignancies isn't fully understood. We performed a pan-cancer assessment on ALG3 utilizing datasets from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) to examine its tumor-related roles across malignancies and its link to particular molecules and cells in the tumor microenvironment (TME). Furthermore, we focused on breast cancer to examine the influence of ALG3-mediated signaling pathways and intercellular interactions in the advancement of tumors. The biological effects of ALG3 were verified by breast cancer cells. Enhanced ALG3 expression was discovered to be substantially linked to patients' grim prognoses in a number of malignancies. Furthermore, the expression of ALG3 in the TME was linked to the infiltration of stromal and immune cells, and ALG3-related immune checkpoints, TMB, and MSI were also discovered. We also discovered that cancer patients having a high level of ALG3 exhibited a lower probability of benefiting from immunotherapy. Furthermore, our research found that KEGG enrichment, single-cell RNA and spatial sequencing analyses were effective in identifying key signaling pathways in ALG3-associated tumor growth. In vitro, knockdown of ALG3 could decrease the proliferation of breast cancer cells. In summary, our research offers a comprehensive insight into the advancement of tumors under the mediation of ALG3. ALG3 appears to be intimately associated with tumor development in the TME. ALG3 might be a viable treatment target for cancer therapy, particularly in the case of breast cancer.

Electroacupuncture Pretreatment Mitigates Myocardial Ischemia/Reperfusion Injury via XBP1/GRP78/Akt Pathway.

Myocardial ischemia/reperfusion injury is a common clinical problem and can result in severe cardiac dysfunction. Previous studies have demonstrated the protection of electroacupuncture against myocardial ischemia/reperfusion injury. However, the role of X-box binding protein I (XBP1) signaling pathway in the protection of electroacupuncture was still elusive. Thus, we designed this study and demonstrated that electroacupuncture significantly improved cardiac function during myocardial ischemia/reperfusion injury and reduced cardiac infarct size. Electroacupuncture treatment further inhibited cardiac injury manifested by the decrease of the activities of serum lactate dehydrogenase and creatine kinase-MB. The results also revealed that electroacupuncture elevated the expressions of XBP1, glucose-regulated protein 78 (GRP78), Akt, and Bcl-2 and decreased the Bax and cleaved Caspase 3 expressions. By using the inhibitor of XBP1 in vitro, the results revealed that suppression of XBP1 expression could markedly increase the activities of lactate dehydrogenase and creatine kinase-MB and cell apoptosis, thus exacerbating stimulated ischemia/reperfusion-induced H9c2 cell injury. Compared with stimulated ischemia/reperfusion group, inhibition of XBP1 inhibited the downstream GRP78 and Akt expressions during stimulated ischemia/reperfusion injury. Collectively, our data demonstrated that electroacupuncture treatment activated XBP1/GRP78/Akt signaling to protect hearts from myocardial ischemia/reperfusion injury. These findings revealed the underlying mechanisms of electroacupuncture protection against myocardial ischemia/reperfusion injury and may provide novel therapeutic targets for the clinical treatment of myocardial ischemia/reperfusion injury.

A novel lymphatic pattern promotes metastasis of cervical cancer in a hypoxic tumour-associated macrophage-dependent manner.

Lymphatic remodelling in the hypoxic tumour microenvironment (TME) is critically involved in the metastasis of cervical squamous cell carcinoma (CSCC); however, its underlying mechanisms remain unclear. Here, we uncovered a novel lymphatic pattern in the hypoxic TME, wherein lymphatic vessels (LVs) are encapsulated by tumour-associated macrophages (TAMs) to form an interconnected network. We describe these aggregates as LVEM (LVs encapsulated by TAMs) considering their advantageous metastatic capacity and active involvement in early lymph node metastasis (LNM). Mechanistic investigations revealed that interleukin-10 (IL-10) derived from hypoxic TAMs adjacent to LVs was a prerequisite for lymphangiogenesis and LVEM formation through its induction of Sp1 upregulation in lymphatic endothelial cells (LECs). Interestingly, Sp1high LECs promoted the transactivation of C-C motif chemokine ligand 1 (CCL1) to facilitate TAM and tumour cell recruitment, thereby forming a positive feedback loop to strengthen the LVEM formation. Knockdown of Sp1 or blockage of CCL1 abrogated LVEM and consequently attenuated LNM. Notably, CSCCnon-LNM is largely devoid of hypoxic TAMs and the resultant LVEM, which might explain its metastatic delay. These findings identify a novel and efficient metastasis-promoting lymphatic pattern in the hypoxic TME, which might provide new targets for anti-metastasis therapy and prognostic assessment.

CD40 Accelerates the Antigen-Specific Stem-Like Memory CD8+ T Cells Formation and Human Papilloma Virus (HPV)-Positive Tumor Eradication.

Antigen-specific stem-like memory CD8+ T cells (Tscm) have a series of stem cell characteristics, including long-term survival, self-renewal, anti-apoptosis and persistent differentiation into cytotoxic T cells. The effective induction of tumor-specific CD8+ Tscm could persistently eradicate tumor in pro-tumor hostile microenvironment. This study was to investigate the role of CD40 in HPV16-specific CD8+ Tscm induction and its anti-tumor function. We found that CD40 activation accelerated vaccine-induced HPV16 E7-specific CD8+ Tscm formation. Comparing to other HPV-specific CD8+ T cells, CD8+ Tscm were found to be stronger and long-term anti-tumor function, in vivo and in vitro, even in the adoptive cellular transferring model. Furthermore, high frequencies of Tscm might prevent the HPV infection to move on to the development of cancer. And the CD40 effect on Tscm involved Wnt/ß-catenin activation. Our study suggest that CD40 activation supports the generation of tumor-specific CD8+ Tscm, thus providing new insight into cancer immunotherapy.

The over-expression of aquaporin-1 alters erythroid gene expression in human erythroleukemia K562 cells.

Aquaporin genes are differentially expressed in primitive versus definitive erythropoiesis. Our previous research results showed that over-expression of aquaporin-1 (AQP1) gene greatly promotes the erythroid differentiation of erythroleukemia K562 cells, using benzidine staining and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis for representative erythroid-related genes, including γ-globin. But the molecular mechanisms underlying erythroid-specific gene regulation remain unknown. In this study, we demonstrated that AQP1 induced hemoglobins expression and altered erythroid gene expression by microarray analysis in K562 cells. The retroviral expression vector of AQP1 (pBABE-puro-AQP1) was constructed and infected K562 cells to establish a stable AQP1 over-expression cell line (K562-AQP1). AQP1 over-expression effectively inhibited cell proliferation and induced cell growth arrest in G1 phase of K562 cells. Then microarray profile was applied to analyze the differentially expressed genes which involved the mechanism of AQP1 in erythroid differentiation induction. The DAVID functional annotation clustering tool was used to identify biological functions enriched with the differentially expressed genes (n = 466 genes) and to group genes into clusters based on their functional similarity. Significant enrichment of genes involved in "oxygen transporter activity" (p = 3.8E-7) including hemoglobins (HBD, HBG, HBB, HBE1, and HBQ1), HEMGN, and EBP42 were validated by qRT-PCR. Moreover, silencing of HEMGN by RNA interference in K562-AQP1 cells resulted in down-regulation of these genes. These data provide a better understanding of the role of AQP1 in erythroid differentiation, by promoting HEMGN induction and other potential signaling pathways associated with hemoglobin induction.

CHD5 a tumour suppressor is epigenetically silenced in hepatocellular carcinoma.

BACKGROUND: Chromodomain helicase DNA binding protein 5 (CHD5) has recently been identified as a potent tumour suppressor by acting as a master regulator of a tumour-suppressive network. Its inactivation resulted from aberrant methylation in the promoter occurs in several types of human malignancy and is associated with malignant tumour behaviour. In human hepatocellular carcinoma (HCC), CHD5 gene expression, methylation status and tumour-suppressive function have not been elucidated. AIMS: In this study, we focused on the epigenetic modification and tumour-suppressive mechanism of CHD5 gene in HCC. METHODS: CHD5 expression in nine HCC cell lines and 30 pairs of HCC specimens and adjacent non-cancerous tissues were analysed by quantitative reverse transcription PCR and Western blotting. Methylation-specific sequencing and methylation-specific PCR were performed to examine DNA methylation status of the CHD5 promoter in HCC cell lines and samples. The effect of CHD5 restoration on proliferation, colony formation, senescence, apoptosis and tumourigenicity were examined. RESULTS: CHD5 expression was sinificantly down-regulated in HCC cell lines and tissues examined, and the -841 to -470 region of CHD5 promoter was hypermethylated in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2-deoxycytidine resulted in a striking regional demethylation of the -841 to -470 region of CHD5 promoter and an increase in CHD5 expression. The restoration of CHD5 expression inhibited tumour cell proliferation, colony formation and tumourigenicity and caused cellular senescence. CONCLUSIONS: Our findings demonstrate that CHD5 is a potential tumour suppressor gene epigenetically silenced in HCC.

Silencing of CHD5 gene by promoter methylation in leukemia.

Chromodomain helicase DNA binding protein 5 (CHD5) was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network. CHD5 is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of CHD5 down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that CHD5 was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the CHD5 promoter was hypermethylated in human leukemia cells and samples. Thus, CHD5 expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) activates CHD5 expression in human leukemia cell lines. In vitro luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2) as a potential transcription factor involved in CHD5 expression and indicated that treatment with DAC increases the recruitment of AP2 to the CHD5 promoter. In vitro transcription-factor activity studies showed that AP2 over-expression was able to activate CHD5 promoter activity. Our findings indicate that repression of CHD5 gene expression in human leukemia is mediated in part by DNA methylation of its promoter.

Knockdown of CD44 enhances chemosensitivity of acute myeloid leukemia cells to ADM and Ara-C.

It is known that chemoresistance is a major cause of treatment failure in acute myeloid leukemia (AML). Substantial data indicate that the CD44 adhesion molecule is strongly expressed on AML blasts and that it can also inhibit apoptosis. Our study shows that drug resistance of the AML cell line HL60/ADM is due to overexpression of CD44. In an in vitro study, we knocked down CD44 in the HL60/ADM cell line using small interfering RNA (siRNA). Cell proliferation and the 50% inhibitory concentrations (IC50) were determined by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis and intracellular ADM accumulation were detected by flow cytometry. Expression of CD44, Bcl-2, c-Myc were assayed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The results indicate that the expression of CD44 in HL60/ADM cell line was much higher than in HL60 cell, and siRNA targeted CD44 (siRNA/CD44) could silence its expression in both mRNA and protein levels effectively. siRNA/CD44 substantially induces cell apoptosis, inhibits cell proliferation, enhances susceptibility to ADM and Ara-C, and at the same time increases intracellular ADM accumulation even reverses chemoresistance to ADM and Ara-C. Furthermore, by qRT-PCR and Western blot, we found that siRNA/CD44 decreases Bcl-2 and c-Myc synthesis. Our study provides a novel clue that CD44 plays a significant role in the chemoresistance of AML cells to Ara-C and ADM. Moreover, this provides a new direction to the approaches that combination therapy including targeting CD44 may overcome drug resistance and improve treatment effects.

[Role of aquaporin-1 gene in erythroid differentiation of erythroleukemia K562 cells induced by retinoic acid].

OBJECTIVE: To explore the role of aquaporin-1 (AQP1) gene in erythroid differentiation of erythroleukemia K562 cells induced by retinoic acid (RA). METHODS: K562 cells were cultured in the presence of 1 µmol/L RA for varying lengths of time, and γ-globin mRNA expression and hemoglobin content in the cells were detected by real-time PCR (RT-PCR) and ultraviolet spectrophotometry, respectively, to evaluate the erythroid differentiation of K562 cells. RT-PCR and Western blotting were used to examine AQP1 expression in the cells following RA treatment. A retroviral expression vector of AQP1 small interfering RNA (pSUPER-retro-puro-shAQP1) was constructed and transfected into K562 cells to establish a K562 cell line with stable AQP1 down-regulation (K562-shAQP1), in which the changes in γ-globin and hemoglobin expressions after RA treatment were detected. RESULTS: RA treatment significantly increased γ-globin and hemoglobin expressions in K562 cells (P<0.01), causing also significantly enhanced AQP1 mRNA and protein expressions over time (P<0.01). Transfection with the recombinant plasmids pSuper-retro-puro-shAQP1 resulted in stable AQP1 suppression in K562 cells (P<0.01), which showed markedly reduced γ-globin and hemoglobin expressions after RA induction as compared to the control K562 cells (P<0.01). CONCLUSION: K562 cells show a significant increase of AQP1 expression after RA-induced erythroid differentiation, and suppression of AQP1 expression can partially block the effect of RA, suggesting the important role of AQP1 in RA-induced erythroid differentiation of K562 cells.

[Survey of the evolutionary characteristics of influenza H1N1 hemagglutinin gene HA1 in 2000-2009].

OBJECTIVE: To study the global evolutionary characteristics of hemagglutinin gene HA1 of influenza H1N1 infecting different species during 2000-2009. METHODS: The target sequences were downloaded from NCBI and analyzed using bioinformatic software to construct the phylogenetic tree. RESULTS: The HA1 amino acid sequences of influenza H1N1 contained four mutated antigenic sites and receptor-binding sites, and the novel influenza virus shared most of the mutated amino acid sites with swine H1N1 influenza virus. CONCLUSION: The HA1 gene of novel influenza virus might originate from the early swine H1N1 influenza virus from North America, and in the evolutionary process, a number of important sites of HA1 gene mutated to result in the outbreak of influenza.