Methazolamide Can Treat Atherosclerosis by Increasing Immunosuppressive Cells and Decreasing Expressions of Genes Related to Proinflammation, Calcification, and Tissue Remodeling.

It has been reported that carbonic anhydrase I (CA1) is a target for the diagnosis and therapy of atherosclerosis (AS) since CA1 can promote AS aortic calcification. We also found that methazolamide (MTZ), a drug for glaucoma treatment and an inhibitor of carbonic anhydrases, can treat AS by inhibiting calcification in aortic tissues. This study focused on the therapeutic mechanism of MTZ and the pathogenic mechanism of AS. In this study, a routine AS animal model was established in ApoE-/- mice, which were treated with MTZ. The aortic tissues were analyzed using single-cell sequencing. MTZ significantly increased the proportions of B-1/MZB B cells with high expressions of Nr4A1 and Ccr7, CD8+CD122+ Treg-like cells with high Nr4A1 expression, and smooth muscle cells with high Tpm2 expression. These cells or their marker genes were reported to exert immunosuppressive, anti-proinflammatory, and atheroprotective effects. MTZ also decreased the proportions of endothelial cells with high expressions of Retn, Apoc1, Lcn2, Mt1, Serpina3, Lpl, and Lgals3; nonclassical CD14+CD16++ monocytes with high expressions of Mt1, Tyrobp, Lgals3, and Cxcl2; and Spp1+ macrophages with high expressions of Mmp-12, Trem2, Mt1, Lgals3, Cxcl2, and Lpl. These cells or their marker genes have been reported to promote inflammation, calcification, tissue remodeling, and atherogenesis. A significant decrease in the proportion of CD8+CD183 (CXCR3)+ T cells, the counterpart of murine CD8+CD122+ T cells, was detected in the peripheral blood of newly diagnosed AS patients rather than in that of patients receiving anti-AS treatments. These results suggest that MTZ can treat AS by increasing immunosuppressive cells and decreasing expressions of genes related to inflammation, calcification, and tissue remodeling.

Overexpression of circulating CD38+ NK cells in colorectal cancer was associated with lymph node metastasis and poor prognosis.

Introduction: Lymph node metastasis (LNM) is a critical prognostic factor for colorectal cancer (CRC). Due to the potential influence of immune system on CRC progression, investigation into lymphocyte subsets as clinical markers has gained attention. The objective of this study was to assess the capability of lymphocyte subsets in evaluating the lymph node status and prognosis of CRC. Methods: Lymphocyte subsets, including T cells (CD3+), natural killer cells (NK, CD3- CD56+), natural killer-like T cells (NK-like T, CD3+ CD56+), CD38+ NK cells (CD3- CD56+ CD38+) and CD38+ NK-like T cells (CD3+ CD56+ CD38+), were detected by flow cytometry. Univariate and multivariate analyses were used to assess the risk factors of LNM. The prognostic role of parameters was evaluated by survival analysis. Results: The proportion of CD38+ NK cells within the NK cell population was significantly higher in LNM-positive patients (p <0.0001). However, no significant differences were observed in the proportions of other lymphocyte subsets. Poorer histologic grade (odds ratio [OR] =4.76, p =0.03), lymphovascular invasion (LVI) (OR =22.38, p <0.01), and CD38+ NK cells (high) (OR =4.54, p <0.01) were identified as independent risk factors for LNM. Furthermore, high proportion of CD38+ NK cells was associated with poor prognosis of CRC patients (HR=2.37, p =0.03). Conclusions: It was demonstrated that the proportion of CD38+ NK cells was a marker overexpressed in LNM-positive patients compared with LNM-negative patients. Moreover, an elevated proportion of CD38+ NK cells is a risk factor for LNM and poor prognosis in CRC.

Investigating the cell membrane localization of PADI4 in breast cancer cells and inhibition of anti-PADI4 monoclonal antibody.

BACKGROUND: Peptidyl arginine deiminase 4 (PADI4) is a post-translational modification enzymecan that converts arginine in protein into citrulline in the presence of calcium ions, which is called citrullination. PADI4 has been reported to be expressed in the cytoplasm and nucleus in a variety of malignant tumors. Based on the GeneCards database and our previous research, it is speculated that PADI4 may also be expressed on the cell membrane. This study aimed to confirm the membrane expression of PADI4 and the effect of anti-PADI4 antibodies on cell membrane PADI4. This may be another mechanism of action of anti-PADI4 monoclonal antibodies in the treatment of breast cancer. METHODS: The subcellular localizations of PADI4 in MDA-MB-231 and MCF-7 breast cancer cells were determined by immunofluorescence, immunoelectron microscopy, and Western blot analysis. The tumor cells were treated with PADI4 antibody, and cell proliferation, migration, colony formation, apoptosis, glycolysis, and epithelial-mesenchymal transition (EMT) were measured as well as the expression of some essential tumor genes. RESULTS: PADI4 was not only localized in the nucleus and cytoplasm of breast cancer cells but was also detected on the cell membrane. Following PADI4 antibody treatment, cell proliferation, migration, colony formation, EMT, and ATP production through glycolysis were decreased, and the mRNA expression of MYC proto-oncogene (MYC), FAT atypical cadherin 1 (FAT1), nuclear factor kappa B subunit 1 (NFκB), and tumor necrosis factor (TNF-α) in breast cancer cells was downregulated, while the mRNA expression of tumor protein p63 (TP63) was upregulated. CONCLUSIONS: PADI4 is expressed on the cell membrane in breast cancer cells. Anti-PADI4 antibodies can affect the biological functions of cell membrane PADI4, including proliferation, migration, apoptosis, and glycolysis, thereby inhibiting tumor progression.

Downregulation of Sirt6 by CD38 promotes cell senescence and aging.

Decreased nicotinamide adenine dinucleotide (NAD+) levels accompany aging. CD38 is the main cellular NADase. Cyanidin-3-O-glucoside (C3G), a natural inhibitor of CD38, is a well-known drug that extends the human lifespan. We investigated mechanisms of CD38 in cell senescence and C3G in antiaging. Myocardial H9c2 cells were induced to senescence with D-gal. CD38 siRNA, C3G and UBCS039 (a chemical activator of Sirt6) inhibited D-gal-induced senescence by reducing reactive oxygen species, hexokinase 2 and SA-ß-galactosidase levels. These activators also stimulated cell proliferation and telomerase reverse transcriptase levels, while OSS-128167 (a chemical inhibitor of Sirt6) and Sirt6 siRNA exacerbated the senescent process. H9c2 cells that underwent D-gal-induced cell senescence increased CD38 expression and decreased Sirt6 expression; CD38 siRNA and C3G decreased CD38 expression and increased Sirt6 expression, respectively; and Sirt6 siRNA stimulated cell senescence in the presence of C3G and CD38 siRNA. In D-gal-induced acute aging mice, CD38 and Sirt6 exhibited increased and decreased expression, respectively, in myocardial tissues, and C3G treatment decreased CD38 expression and increased Sirt6 expression in the tissues. C3G also reduced IL-1ß, IL-6, IL-17A, TNF-α levels and restored NAD+ and NK cell levels in the animals. We suggest that CD38 downregulates Sirt6 expression to promote cell senescence and C3G exerts an antiaging effect through CD38-Sirt6 signaling.

Anti-PADI4 antibody suppresses breast cancer by repressing the citrullinated fibronectin in the tumor microenvironment.

BACKGROUND: PADI4, an enzyme catalyzing arginine residues to citrulline residues, is highly expressed in malignant tumors. This study prepared a monoclonal anti-human PADI4 antibody and investigated the therapeutic effect of the antibody on breast tumors and the functional mechanism. METHODS: After treatment with PADI4 antibody, the changes in tumor-bearing mice were examined by PET-CT, pathological assays, biochemical tests, routine blood tests, cytokine assays and metabolic assays. We used PADI4 recombinant protein to catalyze fibronectin (Fn) and then used citrullinated fibronectin (Cit-Fn) to culture MDA-MB-231 cells. We also treated Cit-Fn cultured cells with PADI4 antibody. The cultured cells were examined using cell proliferation, apoptosis, colony formation, migration and glycolic ATP production. Citrullination in the tumor tissues and peripheral blood was measured using Western blotting and ELISA, respectively. RESULTS: Following PADI4 antibody treatment, tumor growth was significantly suppressed, and the number of apoptotic cells in tumor tissues was increased. The citrullination level in peripheral blood and tumor tissues was decreased, EMT-related gene expression in tumors was also decreased, and the spontaneous movement of tumor-bearing mice was increased following treatment. Following antibody treatment, the serum concentrations of IL-10, IL-12p70, IL-23, ALT and AST were significantly decreased. MDA-MB-231 cells treated with Cit-Fn showed increased cell proliferation, cell migration, colony formation and glycolytic ATP production and decreased apoptosis. The growth and migration of MDA-MB-231 cells were reduced following PADI4 antibody treatment, and PADI4 antibody inhibited the citrullination of fibronectin in vitro. CONCLUSIONS: The PADI4 antibody had a therapeutic effect on breast tumors by inhibiting the citrullination of fibronectin to change the tumor tissue microenvironment. PADI4 antibody is a potential means for tumor treatment.

T-Cell Immune Imbalance in Rheumatoid Arthritis Is Associated with Alterations in NK Cells and NK-Like T Cells Expressing CD38.

BACKGROUND: CD38+ NK (CD3- CD16+ CD38+ CD56+) cells were increased in rheumatoid arthritis (RA), which suppressed Treg cell differentiation. This study explored how CD38+ NK cells regulated CD4+ T-cell differentiation into Treg cells in RA. METHODS: Proportions of CD38+ NK cells and their counterpart CD38+ NK-like T (CD3+ CD16+ CD38+ CD56+) cells were measured in RA and rats with collagen-induced arthritis (CIA). CD38+ NK cells and CD38+ NK-like T cells were cocultured with CD4+ T cells, respectively. RESULTS: A significantly increased proportion of CD38+ NK cells and a decreased proportion of CD38+ NK-like T cells were detected in RA and CIA blood and synovial fluids. When CD4+ T cells were cocultured with CD38+ NK cells, mammalian target of rapamycin (mTOR) signaling was activated, and Th1/Th2 and Th17/Treg ratios were increased. When CD38+ NK cells were pretreated with anti-CD38 antibody, Treg cell proportion was increased, and Th1/Th2 and Th17/Treg ratios were decreased. CD38+ NK-like T cells showed the opposite results. CD38+ NK cells and CD38+ NK-like-T cells activated differential gene expressions and pathways in CD4+ T cells and initiated Th1 and Th2 cell differentiation by differential gene nodes. CONCLUSIONS: This study suggest that the high CD38+ NK cell proportion and low CD38+ NK-like T cell proportion in RA suppress Treg cell differentiation by stimulating mTOR signaling in CD4+ T cells, which consequentially disturbs the immune tolerance.

ARHGEF10L Promotes Cervical Tumorigenesis via RhoA-Mediated Signaling.

BACKGROUND: Rho guanine nucleotide exchange factor 10-like protein (ARHGEF10L) is a member of the guanine nucleotide exchange factor family, which regulates Rho GTPase activities, thus contributing to tumorigenesis. Our previous study demonstrated a strong association between the ARHGEF10L gene and the risk of cervical carcinoma. This study investigated the pathogenic role and mechanism of ARHGEF10L in cervical tumors. METHODS: The HeLa cell line, which was derived from cervical carcinoma, was transfected with ARHGEF10L-overexpressing plasmids or anti-ARHGEF10L siRNA. Cell counting kit-8 assays, wound-healing assays, and cell apoptosis assays were performed to investigate the effects of ARHGEF10L on cell activities. A Rho pull-down assay and RNA-sequencing analysis were performed to investigate the pathogenic pathway of ARHGEF10L involvement in cervical tumors. RESULTS: ARHGEF10L overexpression promoted cell proliferation and migration, reduced cell apoptosis, and induced epithelial-to-mesenchymal transition (EMT) via downregulation of E-cadherin and upregulation of N-cadherin and Slug in transfected HeLa cells. The overexpression of ARHGEF10L also upregulated GTP-RhoA, ROCK1, and phospho-ezrin/radixin/moesin (ERM) expression in HeLa cells. RNA-sequencing analysis detected altered transcription of 31 genes in HeLa cells with ARHGEF10L overexpression. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) pathway analyses identified significant differences in cyclin-dependent protein serine/threonine kinase activity, cell responses to vitamin A, and Toll-like receptor signaling pathways. Both real-time PCR and Western blotting verified the increased expression of heat shock 70 kDa protein 6 (HSPA6) in ARHGEF10L-overexpressing HeLa cells. Since we reported that ARHGEF10L played a role through RhoA-ROCK1-ERM signaling, an important pathway in tumorigenesis, and stimulated EMT and HSPA6 expression in liver tumors and gastric tumor cells, we suggest that ARHGEF10L is a novel oncogene in many tumors.

RCC2 Expression Stimulates ER-Positive Breast Tumorigenesis.

OBJECTIVE: Regulator of chromosome condensation 2 (RCC2) has been reported to be involved in the regulation of cell cleavage. This study investigated the effect of RCC2 expression on breast tumorigenesis. METHODS: MCF-7 cells originating from estrogen receptor-positive (ER+) breast cancer were transfected with anti-RCC2 siRNA or RCC2-expressing plasmids. Cell proliferation, apoptosis, migration, and cytokine production in the transfected cells were examined using the CCK-8 assay, wound healing assay, and flow cytometry, respectively. PCR array was used to investigate the tumorigenic pathway of RCC2 in MCF-7 cells transfected with the anti-RCC2 siRNA. MCF-7 cells were also transfected with lentivirus-containing anti-RCC2 short hairpin RNA and were injected into BALB/c nude mice to generate tumor-bearing mice. Tumor growth in the mouse model was examined using magnetic resonance imaging by diffusion-weighted imaging analysis. RESULTS: Western blotting and immunohistochemistry detected significantly increased expression of RCC2 in ER + breast tumor tissues compared with breast fibroadenoma samples. Inhibiting RCC2 expression decreased cell migration and stimulated apoptosis in MCF-7 cells, while overexpressing RCC2 stimulated cell migration and inhibited apoptosis. The inhibition of RCC2 expression significantly decreased breast tumor growth and IL-6 levels in the tumor-bearing mice. PCR array demonstrated that inhibiting RCC2 expression significantly decreased the expression of IGF1 and TWIST1, two well-known tumor-enhancing genes, in MCF-7 cells; conversely, overexpressing RCC2 increased the expression levels of these two genes in the transfected cells. This result was verified in the mouse model following inhibition of RCC2 expression in MCF-7 cells. Additionally, estradiol-17ß suppressed MCF-7 cell apoptosis, stimulated cell proliferation and cell migration, and increased RCC2, IGF1, and TWIST1 expression. The siRNA-mediated inhibition of RCC2 expression alleviated the inhibitory effects of estrogen on apoptosis in MCF-7 cells, while overexpressing RCC2 enhanced the estrogen-driven inhibition of apoptosis. Modifying RCC2 expression had no impact on MCF-7 cell proliferation in the presence or absence of estradiol-17ß. CONCLUSIONS: Our results suggest that estrogen-induced RCC2 expression prompts IGF1, TWIST1, and IL-6 expression, stimulates cell migration, and inhibits apoptosis to contribute to ER + breast tumorigenesis.

Investigating the pathogenic role of PADI4 in oesophageal cancer.

PADI4 post-translationally converts peptidylarginine to citrulline. PADI4 can disrupt the apoptotic process via the citrullination of histone H3 in the promoter of p53-target genes. The current study focused on PADI4 expression in various subtypes of oesophageal carcinoma (EC) by immunohistochemistry, western blotting and real time PCR. The study also investigated the effect of bile acid deoxycholate (DCA) on PADI4 expression in Eca-109 cells that originated from EC. Apoptosis and DCA-induced toxicity were analyzed by TUNEL, MTT assay and flow cytometry. Additionally, the present study investigated the correlation between single nucleotide polymorphism (SNP) in PADI4 gene and EC risk in Chinese population using Illumina GoldenGate assay. Compared with paraneoplastic tissues, the transcriptional and translational levels of PADI4 were significantly elevated in oesophageal squamous cell carcinoma (ESCC, n=9) and oesophageal adenocarcinoma (EAC, n=5) tissues. Immunolabeling detected expression of PADI4 in ESCC tissues (98.56%, n=139), EAC samples (87.5%, n=16) and oesophageal small cell undifferentiated carcinoma (91.7%, n=12) but not in normal tissues (0%, n=16). Furthermore, PADI4 levels is positively correlated with the pathological classification of ESCC (p=0.009). PADI4 expression levels were consistent with the number of apoptotic cells in the induced Eca-109 cells. rs10437048 [OR= 0.012831; 95% CI, 0.001746~0.094278; p=1.556×10(-12)] were significantly associated with decreased risk of EC, whereas rs41265997 [OR=12.7; 95% CI, 0.857077~33.207214; p=3.896×10(-8)] were significantly associated with increased risk of EC. rs41265997 in exon 3 of PADI4 gene is non-synonymous and converts ACG to ATG resulting in a threonine /methionine conversion at position 274 of the protein. Haplotypes GC that carries the variant alleles for rs2501796 and rs2477134 was significantly associated with increased risk of EC (frequency=0.085, p=0.0256, OR=2.7). The results suggest that PADI4 expression is related to the tumorigenic process of EC and the DCA-induced apoptosis. The PADI4 gene may be a valid EC susceptibility gene.

PADI4 and tumourigenesis.

PADI4 post-translationally converts peptidylarginine to citrulline, a process called citrullination. Studies have demonstrated the high expression of PADI4 in various malignant tumour tissues. PADI4 is also expressed at high levels in the blood of patients with some malignant tumours. Thus far, citrullination of histone, cytokeratin, antithrombin and fibronectin have been confirmed to be involved in abnormal apoptosis, high coagulation, and disordered cell proliferation and differentiation, all of which are main features of malignant tumours. PADI4 is expressed in CD34+ stem cells in normal tissues, and many more CD34+ cells expressing PADI4 are present in tumour tissues. These findings suggest that PADI4 may play an important role in tumourigenesis.