Capsaicin attenuates Porphyromonas gingivalis-suppressed osteogenesis of periodontal ligament stem cells via regulating mitochondrial function and activating PI3K/AKT/mTOR pathway.

BACKGROUND AND OBJECTIVE: Prevention of periodontal bone resorption triggered by Porphyromonas gingivalis (P. gingivalis) is crucial for dental stability. Capsaicin, known as the pungent ingredient of chili peppers, can activate key signaling molecules involved in osteogenic process. However, the effect of capsaicin on osteogenesis of periodontal ligament stem cells (PDLSCs) under inflammation remains elusive. METHODS: P. gingivalis culture suspension was added to mimic the inflammatory status after capsaicin pretreatment. The effects of capsaicin on the osteogenesis of PDLSCs, as well as mitochondrial morphology, Ca2+ level, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and osteogenesis-regulated protein expression levels were analyzed. Furthermore, a mouse experimental periodontitis model was established to evaluate the effect of capsaicin on alveolar bone resorption and the expression of osteogenesis-related proteins. RESULTS: Under P. gingivalis stimulation, capsaicin increased osteogenesis of PDLSCs. Not surprisingly, capsaicin rescued the damage to mitochondrial morphology, decreased the concentration of intracellular Ca2+ and ROS, enhanced MMP and activated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. The in vivo results showed that capsaicin significantly attenuated alveolar bone loss and augmented the expression of bone associated proteins. CONCLUSION: Capsaicin increases osteogenesis of PDLSCs under inflammation and reduces alveolar bone resorption in mouse experimental periodontitis.

AID-induced CXCL12 upregulation enhances castration-resistant prostate cancer cell metastasis by stabilizing ß-catenin expression.

Prostate cancer (PCa) is one of the most common malignant diseases of urinary system and has poor prognosis after progression to castration-resistant prostate cancer (CRPC), and increased cytosine methylation heterogeneity is associated with the more aggressive phenotype of PCa cell line. Activation-induced cytidine deaminase (AID) is a multifunctional enzyme and contributes to antibody diversification. However, the dysregulation of AID participates in the progression of multiple diseases and related with certain oncogenes through demethylation. Nevertheless, the role of AID in PCa remains elusive. We observed a significant upregulation of AID expression in PCa samples, which exhibited a negative correlation with E-cadherin expression. Furthermore, AID expression is remarkably higher in CRPC cells than that in HSPC cells, and AID induced the demethylation of CXCL12, which is required to stabilize the Wnt signaling pathway executor ß-catenin and EMT procedure. Our study suggests that AID drives CRPC metastasis by demethylation and can be a potential therapeutic target for CRPC.

Extract of Jasminum grandiflorum L. alleviates CCl4-induced liver injury by decreasing inflammation, oxidative stress and hepatic CYP2E1 expression in mice.

Jasminum grandiflorum L. (JG) is a medicinal plant containing many bioactive ingredients. Herein, we analyzed the effects of four different extracts and two compounds of JG on acute liver injury caused by carbon tetrachloride (CCl4) and underlying molecular mechanisms. 7 weeks old C57BL/6 male mice were used to establish a liver injury model by injecting with 1% CCl4, 10 mL/kg ip. Four different extracts and two compounds of JG were given to mice by gavage for 3 days. Clinical and histological chemistry assays were performed to assess liver injury. Moreover, hepatic oxidative stress and inflammation related markers were determined by immunohistochemistry and western blotting. As a result, JG extracts and two functional components showed different degree of protect effects against CCl4-induced liver injury by the decrease of elevated serum transaminases and liver index, and the attenuation of histopathological changes in mice, among which JG extracted with petroleum ether (PET) had the most significant effect. In addition, PET remarkably alleviated hepatic oxidative stress and inflammation. Further studies revealed that PET significantly inhibited the TNF-α expression, signal pathway expression, NF-κB p65 and inflammatory factors IL-1ß and IL-6 expression in CCl4-induced liver injury mice. Nevertheless, hydroxytyrosol (HT) alleviated liver injury by reducing oxidative stress. Apart from PET extract, other extracts of JG can inhibit cytochrome CYP2E1 expression to protect liver tissue. These findings suggest that the extracts and its components of JG possesses the potential protective effects against CCl4-induced liver injury in mice by exerting antioxidative stress and anti-inflammation.

Preclinical platforms to study therapeutic efficacy of human γδ T cells.

BACKGROUND: Gamma delta (γδ) T lymphocytes are promising candidate for adoptive T cell therapy, however, their treatment efficacy is not satisfactory. Vδ2 T cells are unique to primates and few suitable models are available to assay their anti-tumour function. METHODS: We tested human γδ T cell activation, tumour infiltration, and tumour-killing in four three-dimensional (3D) models, including unicellular, bicellular and multicellular melanoma spheroids, and patient-derived melanoma organoids. We studied the effects of checkpoint inhibitors on γδ T cells and performed a small molecule screen using these platforms. RESULTS: γδ T cells rapidly responded to melanoma cells and infiltrated melanoma spheroids better than αß T cells in PBMCs. Cancer-associated fibroblasts (CAFs) in bicellular spheroids, stroma cells in multicellular melanoma spheroids and inhibitory immune cells in organoids significantly inhibited immune cell infiltrates including γδ T cells and lessened their cytotoxicity to tumour cells. Tumour-infiltrating γδ T cells showed exhausted immunophenotypes with high checkpoints expression (CTLA-4, PD-1 and PD-L1). Immune checkpoint inhibitors increased γδ T cell infiltration of 3D models and killing of melanoma cells in all four 3D models. Our small molecule screen assay and subsequent mechanistic studies demonstrated that epigenetic modifiers enhanced the chemotaxis and cytotoxicity of γδ T cells through upregulating MICA/B, inhibiting HDAC6/7 pathway and downregulating the levels of PD-L1 and PD-L2 in CAFs and tumour cells. These compounds increased CXCR4 and CD107a expression, IFN-γ production and decreased PD-1 expression of γδ T cells. CONCLUSIONS: Tumour-infiltrating γδ T cells show exhausted immunophenotypes and limited anti-tumour capacity in melanoma 3D models. Checkpoint inhibitors and epigenetic modifiers enhance anti-tumour functions of γδ T cells. These four 3D models provided valuable preclinical platforms to test γδ T cell functions for immunotherapy.

TAS2R16 Activation Suppresses LPS-Induced Cytokine Expression in Human Gingival Fibroblasts.

Sustained and non-resolved inflammation is a characteristic of periodontitis. Upon acute inflammation, gingival fibroblasts release cytokines to recruit immune cells to counter environmental stimuli. The intricate regulation of pro-inflammatory signaling pathways, such as NF-κB, is necessary to maintain periodontal homeostasis. Nonetheless, how inflammation is resolved has not yet been elucidated. In this study, 22 subtypes of taste receptor family 2 (TAS2Rs), as well as the downstream machineries of Gα-gustducin and phospholipase C-ß2 (PLCß2), were identified in human gingival fibroblasts (HGFs). Various bitter agonists could induce an intensive cytosolic Ca2+ response in HGFs. More importantly, TAS2R16 was expressed at a relatively high level, and its agonist, salicin, showed robust Ca2+ evocative effects in HGFs. Activation of TAS2R16 signaling by salicin inhibited the release of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines, at least in part, by repressing LPS-induced intracellular cAMP elevation and NF-κB p65 nuclear translocation in HGFs. These findings indicate that TAS2Rs activation in HGFs may mediate endogenous pro-inflammation resolution by antagonizing NF-κB signaling, providing a novel paradigm and treatment target for the better management of periodontitis.

Comprehensive role of prostate-specific antigen identified with proteomic analysis in prostate cancer.

Current treatments including androgen deprivation fail to prevent prostate cancer (PrCa) from progressing to castration-resistant PrCa (CRPC). Accumulating evidence highlights the relevance of prostate-specific antigen (PSA) in the development and progression of PrCa. The underlying mechanism whereby PSA functions in PrCa, however, has yet been elucidated. We demonstrated that PSA knockdown attenuated tumorigenesis and metastasis of PrCa C4-2 cells in vitro and in vivo, whereas promoted the apoptosis in vitro. To illuminate the comprehensive role of PSA in PrCa, we performed an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis to explore the proteomic change induced by PSA knockdown. Among 121 differentially expressed proteins, 67 proteins were up-regulated, while 54 proteins down-regulated. Bioinformatics analysis was used to explore the mechanism through which PSA exerts influence on PrCa. Protein-protein interaction analysis showed that PSA may mediate POTEF, EPHA3, RAD51C, HPGD and MCM4 to promote the initiation and progression of PrCa. We confirmed that PSA knockdown induced the up-regulation of MCM4 and RAD51C, while it down-regulated POTEF and EPHA3; meanwhile, MCM4 was higher in PrCa para-cancerous tissue than in cancerous tissue, suggesting that PSA may facilitate the tumorigenesis by mediating MCM4. Our findings suggest that PSA plays a comprehensive role in the development and progression of PrCa.

Activation-Induced Cytidine Deaminase Expression Facilitates the Malignant Phenotype and Epithelial-to-Mesenchymal Transition in Clear Cell Renal Cell Carcinoma.

Although advances have been made in the development of antiangiogenesis targeted therapy and surgery, metastatic clear cell renal cell carcinoma (ccRCC) is still incurable. Activation-induced cytidine deaminase (AID) is mainly expressed in a variety of germ and somatic cells, and induces somatic hypermutation and class-switch recombination, playing a vital role in antibody diversification. We confirmed that AID was expressed at a higher level in ccRCC tissues than in the corresponding nontumor renal tissues. We explored the impact of AID on ccRCC proliferation, invasion, and migration. In 769-p and 786-0 cells, expression of an AID-specific short hairpin RNA significantly reduced AID expression, which markedly inhibited tumor cell invasion, proliferation, and migration. Previous studies showed that AID is associated with Wnt ligand secretion mediator (WLS/GPR177), cyclin-dependent kinase 4 (CDK4), and stromal cell-derived factor-1 (SDF-1/CXCL12) regulation, which was further confirmed in human ccRCC tissues. Therefore, we studied the relationship between AID and these three molecules, and the impact of AID on epithelial-to-mesenchymal transition in ccRCC. WLS/GPR177, SDF-1/CXCL12, and CDK4 were sensitive to 5-azacytidine (a DNA demethylation agent), which reverted the inhibition of carcinogenesis caused by AID repression. In summary, AID is an oncogene that might induce tumorigenesis through DNA demethylation. Targeting AID may represent a novel therapeutic approach to treat metastatic ccRCC.

AID modulates carcinogenesis network via DNA demethylation in bladder urothelial cell carcinoma.

Bladder cancer is one of the most common malignant diseases in the urinary system, with poor survival after metastasis. Activation-induced cytidine deaminase (AID), a versatile enzyme involved in antibody diversification, is an oncogenic gene that induces somatic hypermutation and class-switch recombination (CSR). However, the contribution of AID-mediated DNA demethylation to bladder urothelial cell carcinoma (BUCC) remains unclear. Herein, we evaluated the impact on BUCC caused by AID and explored the gene network downstream of AID by using a proteomic approach. Lentiviral vector containing AID-specific shRNA significantly reduced AID expression in T24 and 5637 cells. Silencing AID expression remarkably inhibited tumour malignancies, including cell proliferation, invasion and migration. We used Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics analysis technology to study the underpinning mechanism in monoclonal T24 cells, with or without AID knockdown. Among the 6452 proteins identified, 99 and 142 proteins in shAICDA-T24 cells were significantly up- or downregulated, respectively (1.2-fold change) compared with the NC-T24 control. After a pipeline of bioinformatics analyses, we identified three tumour-associated factors, namely, matrix metallopeptidase 14 (MMP14), C-X-C motif chemokine ligand 12 and wntless Wnt ligand secretion mediator, which were further confirmed in human BUCC tissues. Nonetheless, only MMP14 was sensitive to the DNA demethylation molecule 5-aza-2'-deoxycytidine (5-azadC; 5 µM), which reversed the inhibition of carcinogenesis by AID silence in T24 and 5637 cells. Overall, AID is an oncogene that mediates tumourigenesis via DNA demethylation. Our findings provide novel insights into the clinical treatment for BUCC.

The antitumor activity of PNA modified vinblastine cationic liposomes on Lewis lung tumor cells: In vitro and in vivo evaluation.

Non-small cell lung cancer (NSCLC) is one of the frequently-occurring disease in the world, and the treatment effects are usually unsatisfactory. Vinblastine is an anti-microtubule drug in clinic. In this study, a nanostructured liposome was designed and prepared for treating NSCLC. In the liposomes, peanut agglutinin (PNA) was modified on the liposomal surface, 3-(N-(N',N'-dimethylaminoethane)carbamoyl) cholesterol was used as cationic materials, and vinblastine was encapsulated in the aqueous core of liposomes, respectively. The PNA modified vinblastine cationic liposomes were approximately 100 nm in size with a positive potential. In vitro results showed that the targeting liposomes could significantly enhance cellular uptake, selectively accumulate in LLT cells, and dramatically initiate apoptosis via activating pro-apoptotic proteins and apoptotic enzymes, thus leading to the strongest antitumor efficacy to LLT cells. In vivo results demonstrated that the targeting liposomes could display a prolonged circulation time in the blood, accumulate more drug in tumor location, and induce most of tumor cells apoptosis. As a result, a robust overall antitumor efficacy in tumor-bearing mice was observed subsequently. In conclusion, the chemotherapy using the PNA modified vinblastine cationic liposomes could provide a potential strategy for treating non-small cell lung cancer.

PEGylated VRB plus quinacrine cationic liposomes for treating non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common form of lung cancer, and the treatment effects are usually unsatisfactory. Vinorelbine (VRB) is extensively used in cancer treatment, but it has some disadvantages when used alone. PEGylated liposomes have been extensively used as a delivery carrier for antitumor drugs via prolonging the circulation time in the blood. PURPOSE: The nanostructured liposomes were designed and prepared for treating NSCLC. METHODS: In the liposomes, PEG was modified on the liposomal surface, DC-Chol was used as cationic materials, and VRB plus quinacrine were encapsulated in an aqueous core of the liposomes as an antitumor drug and an apoptosis-inducing agent, respectively. Evaluations were performed on A549 cells, tubular network formations and xenografts of the A549 cells. RESULTS: The PEGylated drugs-loaded cationic liposomes could significantly enhance cellular uptake and selectively accumulate in A549 cells, thus leading to show strongest antitumor efficacy to tumor cells and to tumor-bearing mice. Action mechanisms showed that the enhanced efficacy in treating NSCLC was related to activate caspase 9 and caspase 3, to activate Bax and P53, and to suppress Bcl-2 and Mcl-1. CONCLUSION: The PEGylated VRB plus quinacrine cationic liposomes showed a potential strategy for treating NSCLC.

Overexpression of immunoglobulin G prompts cell proliferation and inhibits cell apoptosis in human urothelial carcinoma.

Only B lymphocytes can express immunoglobulins according to the traditional immunological theories, and the expression of immunoglobulin G (IgG) messenger RNA (mRNA) and protein was found in certain human cancer cells recently. However, the expression pattern of IgG and its possible role in human urothelial carcinoma are still elusive. In this study, we investigated the expression of IgG in two human urothelial carcinoma cell lines, T24 and BIU-87, and in 56 cases of clinical urothelial carcinoma tissues. The mRNA of IgG was positively detected by in situ hybridization and reverse transcription PCR; furthermore, IgG protein was also positively detected by immunohistochemistry and Western blot. Moreover, blockade of tumor-derived IgG by either antihuman IgG antibody or antisense oligonucleotides increased cell apoptosis and inhibited cell growth in bladder cancer cell lines in vitro, and antihuman IgG antibody could suppress the growth of xenotransplant tumor in vivo. In addition, either antihuman IgG antibody or antisense oligonucleotides enhanced the sensitivity to mitomycin C in bladder cancer cell line T24. Furthermore, blockade of IgG in bladder cancer cell T24 resulted in upregulation of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Our results indicated that bladder cancer cells were capable of expressing IgG, and blockade of IgG expression induced cell apoptosis through activation of caspase-dependent pathway. A novel potential targeted therapy for bladder cancer will be possibly developed based on these data.