OBJECTIVES: This study aims to investigate the effects of tumor-stromal fibroblasts (TSFs) on the proliferation, invasion, and migration of salivary gland pleomorphic adenoma (SPA) cells in vitro. METHODS: Salivary gland pleomorphic adenoma cells (SPACs), TSFs, and peri-tumorous normal fibroblasts (NFs) were obtained by tissue primary culture and identified by immunocytochemical staining. The conditioned medium was obtained from TSF and NF in logarithmic phase. SPACs were cultured by conditioned medium and treated by TSF (group TSF-SPAC) and NF (group NF-SPAC). SPACs were used as the control group. The proliferation, invasion, and migration of the three groups of cells were detected by MTT, transwell, and scratch assays, respectively. The expression of vascular endothelial growth factor (VEGF) in the three groups was tested by enzyme linked immunosorbent assay (ELISA). RESULTS: Immunocytochemical staining showed positive vimentin expression in NF and TSF. Results also indicated the weak positive expression of α-smooth muscle actin (SMA) and fibroblast activation protein (FAP) in TSFs and the negative expression of α-SMA and FAP in NFs. MTT assay showed that cell proliferation in the TSF-SPAC group was significantly different from that in the NF-SPAC and SPAC groups (P<0.05). Cell proliferation was not different between the NF-SPAC and SPAC groups (P>0.05). Transwell and scratch assays showed no difference in cell invasion and migration among the groups (P>0.05). ELISA showed that no significant difference in VEGF expression among the three groups (P>0.05). CONCLUSIONS TSFs may be involved in SPA biological behavior by promoting the proliferation of SPACs but has no effect on the invasion and migration of SPACs in vitro. Hence, TSF may be a new therapeutic target in SPA treatment.
Humans ; Adenoma, Pleomorphic/metabolism* ; Vascular Endothelial Growth Factor A ; Culture Media, Conditioned/metabolism* ; Fibroblasts/metabolism* ; Salivary Glands/metabolism*

OBJECTIVETo observe the effects of supernatants of normal skin keratinocytes(NK) and scar keratinocytes(SK) on proliferation and collagen synthesis of hypertrophic scar fibroblasts(HSFB).

METHODSThe supernatant, collected from cultured NK and SK, was added to the cultivated HSFB. The MTT-method, 3H-proline incorporation and radioimmunoassay were employed to measure the cell proliferation, collagen synthesis and secretion.

RESULTSNK supernatant could inhibit HSFB proliferation and increase the collagen synthesis, but inhibit collagen secretion, as compared with the control group. On the contrary, SK supernatant could increase collagen synthesis and secretion, which had little effects on HSFB proliferation.

CONCLUSIONKeratinocytes derived from normal skin and hypertrophic scar show different effects on hypertrophic scar fibroblasts.

Cell Division ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Collagen ; biosynthesis ; Culture Media, Conditioned ; Fibroblasts ; physiology ; Humans ; Keratinocytes ; physiology

The purpose of this study is to provide a culture for mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages (PM) and to characterize their molecular and cellular biology. The cell number and purity from the primary culture were assessed by cell counter and flow cytometry, respectively. Morphological features were evaluated by inverted microscope. Phagocytosis by macrophages was detected by the neutral red dye uptake assay. Phenotypic markers were analyzed by real-time fluorescent quantitative PCR. Our results show that the cell number was much higher from culture of BMDM than PM, while there was no significant difference regarding the percentage of F4/80+CD11b+ cells (98.30%±0.53% vs. 94.83%±1.42%; P>0.05). The proliferation rate of BMDM was significantly higher than PM in the presence of L929 cell conditioned medium, by using CCK-8 assay. However, PM appeared to adhere to the flask wall and extend earlier than BMDM. The phagocytosis capability of un-stimulated BMDM was significantly higher than PM, as well as lipopolysaccharide (LPS)-stimulated BMDM, except the BMDM stimulated by low dose LPS (0.1 μg/mL). Furthermore, Tnfα expression was significantly higher in un-stimulated BMDM than PM, while Arg1 and Ym1 mRNA expression were significantly lower than PM. The expression difference was persistent if stimulated by LPS+IFN-γ or IL-4. Our data indicate that bone marrow can get larger amounts of macrophages than peritoneal cavity. However, it should be aware that the molecular and cellular characteristics were different between these two culture systems.
Animals ; Bone Marrow Cells ; physiology ; Cells, Cultured ; Culture Media, Conditioned ; Lipopolysaccharides ; metabolism ; Macrophages ; classification ; physiology ; Mice ; Phagocytosis

Ultraviolet (UV) radiation-induced loss of dermal extracellular matrix is associated with skin photoaging. Recent studies demonstrated that keratinocyte-releasable stratifin (SFN) plays a critical role in skin collagen metabolism by inducing matrix metalloproteinase 1 (MMP1) expression in target fibroblasts. In the present study, we examined whether SFN released from UVB-irradiated epidermal keratinocytes increases MMP1 release from dermal fibroblasts, and whether these events are affected by p-coumaric acid (p-CA), a natural phenolic compound with UVB-shielding and antioxidant properties. HaCaT cells were exposed to UVB in the absence and presence of p-CA, and the conditioned medium was used to stimulate fibroblasts in medium transfer experiments. The cells and media were analyzed to determine the expressions/releases of SFN and MMP1. UVB exposure increased SFN release from keratinocytes into the medium. The conditioned medium of UVB-irradiated keratinocytes increased MMP1 release from fibroblasts. The depletion of SFN using a siRNA rendered the conditioned medium of UVB-irradiated keratinocytes ineffective at stimulating fibroblasts to release MMP1. p-CA mitigated UVB-induced SFN expression in keratinocytes, and attenuated the MMP1 release by fibroblasts in medium transfer experiments. In conclusion, the present study demonstrated that the use of UV absorbers such as p-CA would reduce UV-induced SFN-centered signaling events involved in skin photoaging.
Collagen ; Culture Media, Conditioned ; Extracellular Matrix ; Fibroblasts* ; Keratinocytes* ; Matrix Metalloproteinase 1* ; Metabolism ; Phenol ; RNA, Small Interfering ; Skin

OBJECTIVE: To investigate the effect of interference of P2X4 receptor expression in tumor-associated macrophages (TAMs) on invasion and migration of glioma cells. METHODS: C57BL/6 mouse models bearing gliomas in the caudate nucleus were examined for glioma pathology with HE staining and expressions of Iba-1 and P2X4 receptor with immunofluorescence assay. RAW264.7 cells were induced into TAMs using conditioned medium from GL261 cells, and the changes in mRNA expressions of macrophage polarization-related markers and the mRNA and protein expressions of P2X4 receptor were detected with RT-qPCR and Western blotting. The effect of siRNA-mediated P2X4 interference on IL-1β and IL-18 mRNA and protein expressions in the TAMs was detected with RT-qPCR and Western blotting. GL261 cells were cultured in the conditioned medium from the transfected TAMs, and the invasion and migration abilities of the cells were assessed with Transwell invasion and migration experiment. RESULTS: The glioma tissues from the tumor-bearing mice showed a significantly greater number of Iba-1-positive cells, where an obviously increased P2X4 receptor expression was detected (P=0.001), than the brain tissues of the control mice (P < 0.001). The M2 macrophage markers (Arg-1 and IL-10) and M1 macrophage markers (iNOS and TNF-α) were both significantly up-regulated in the TAMs derived from RAW264.7 cells (all P < 0.01), but the up-regulation of the M2 macrophage markers was more prominent; the expression levels of P2X4 receptor protein and mRNA were both increased in the TAMs (P < 0.05). Interference of P2X4 receptor expression significantly lowered the mRNA(P < 0.01)and protein (P < 0.01, P < 0.05)expression levels of IL-1β and IL-18 in the TAMs and obviously inhibited the ability of the TAMs to promote invasion and migration of the glioma cells (P < 0.05). CONCLUSION Interference of P2X4 receptor in the TAMs suppresses the migration and invasion of glioma cells possibly by lowering the expressions of IL-1β and IL-18.
Animals ; Culture Media, Conditioned ; Glioma ; Interleukin-18 ; Mice ; Mice, Inbred C57BL ; RNA, Messenger ; Receptors, Purinergic P2X4/metabolism* ; Tumor-Associated Macrophages

OBJECTIVETo explore the differences of metabolic footprint in the conditioned culture medium of placental explants between early-onset and late-onset severe preeclampsia.

METHODSIn 13 cases of early-onset severe preeclampsia and 14 cases of late-onset severe preeclampsia, the placentas were sampled at the surface of the maternal placenta. High performance liquid chromatography-mass spectrometry (HPLC-MS) was used to determine the differences in the metabolites in the conditioned culture medium of the placental villous explants cultured in 6% atmospheric O(2) for 96 h. Standard samples were used to establish the tryptophan and kynurenine chromatography library by HPLC-MS to analyze the concentration of tryptophan and kynurenine in the conditioned culture medium.

RESULTSThirty-six metabolites showed statistically significant differences between early-onset and late-onset severe preeclampsia (P<0.05). The concentration of kynurenine was significantly higher in early-onset severe preeclampsia than in late-onset severe preeclampsia (P<0.05).

CONCLUSIONEarly-onset and late-onset severe preeclampsia may have different pathogeneses. By detecting the concentration of metabolites, metabolomic strategies provide a new means for predicting the onset time of severe preeclampsia.

Chorionic Villi ; metabolism ; Culture Media, Conditioned ; chemistry ; Female ; Humans ; In Vitro Techniques ; Kynurenine ; metabolism ; Ornithine ; metabolism ; Placenta ; metabolism ; Pre-Eclampsia ; metabolism ; Pregnancy ; Tryptophan ; metabolism

OBJECTIVETo determine the expression of TREM-1 (triggering receptor expressed on myeloid cells-1) in macrophages after coxsackievirus B3 (CVB3) infection and the cardiomyocytes viability after culturing with supernatant of macrophages in the absence and presence of TREM-1 inhibitor LP-17 to explore if TREM-1 is involved in the pathogenesis of CVB3 infection induced inflammation and cardiomyocytes injury.

METHODSTREM-1 mRNA and TREM-1 and DAP-12 protein expression in macrophages were detected by Real-time PCR at 0, 1, 4, 8 and 12 h and by Western blot at 0, 16, 24 and 48 h post CVB3 infection. TNF-α secretion of macrophages was measure by ELISA, vitality and the apoptosis degree of cardiomyocytes was assessed by CCK8 and Annexin V-FITC after the cardiomyocytes were cultured with the supernatant of macrophages in normal control group, CVB3 infection group and LP-17 pretreated CVB3 infection group.

RESULTSTREM-1 mRNA expression was significantly upregulated at 4, 8, and 12 h (peaked at 8 h) and TREM-1 protein expression was significantly upregulated at 16 and 24 h and returned to baseline level at 48 h after CVB3 infection. The protein expression of DAP-12, a direct downstream signaling molecule of TREM-1, also significantly increased at 24 and 48 h post CVB3 infection (P < 0.01). Level of macrophages secreted TNF-α post CVB3 infection was significantly reduced in LP-17 pretreated cells (P < 0.01), LP-17 pretreatment also significantly improved viability and significantly reduced apoptosis of cardiomyocytes cultured with supernatant of CVB3 infected macrophages (P < 0.01).

CONCLUSIONTREM-1 might be an important mediator post CVB3 infection and a major player on inducing excess macrophages-related inflammation and resulting in an indirect injury to cardiomyocytes.

Animals ; Coxsackievirus Infections ; metabolism ; Culture Media, Conditioned ; Macrophages ; metabolism ; Male ; Myocarditis ; metabolism ; virology ; Myocytes, Cardiac ; cytology ; virology ; Rats ; Rats, Sprague-Dawley ; Receptors, Immunologic ; metabolism

BACKGROUNDAlthough the introduction of serum prostate-specific antigen (PSA) measurements into clinical practice has revolutionized the care of patients with prostate cancer, there are well-recognized limitations of PSA, and there is a critical need to identify additional prostate cancer biomarkers to assist in early detection and prognosis. In this regard, high resolution proteomic technology has the unexceptionable superiority to find those high abundance biomarkers. The purpose of this study was to search new tumor markers by proteomic technology.

METHODSThe proteins in conditioned medium (CM) of BPH-1 and LNCaP cells were profiled by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). The corresponding mRNA levels of some identified proteins were analyzed by RT-PCR.

RESULTSTotally 11 differentially expressed proteins (6 up-regulated including creatine kinase, brain (CKB), triosephosphate isomerase 1 (TPI1), isocitrate dehydrogenase 2 (IDH2) and 5 down-regulated including glutathione S-transferase pi (GST-pi)) in the CM were identified using MALDI-TOF-MS and database search. The expression pattern between mRNA and CM protein levels of CKB, IDH2, TPI1 and GST-pi in BPH-1 and LNCaP was similar.

CONCLUSIONWe proved a feasible and effective way to search new tumor markers by a proteomics-based strategy and identified 11 potentially useful proteins in CM of BPH-1 and LNCaP cells to distinguish prostate cancer from benign prostatic hypertrophy.

Cell Line ; Cell Line, Tumor ; Culture Media, Conditioned ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Male ; Prostate-Specific Antigen ; metabolism ; Prostatic Neoplasms ; metabolism ; Proteome ; analysis

BACKGROUNDTrichophyton rubrum is superficial fungi characteristically confined to dead keratinized tissues. These observations suggest that the soluble components released by the fungus could influence the host immune response in a cell in contact-free manner. Therefore, this research aimed to analyze whether the culture supernatant derived from T. rubrum grown in the nail medium could elicit the immune response of keratinocyte effectively.

METHODSThe culture supernatants of two strains (T1a, T XHB ) were compared for the β-glucan concentrations and their capacity to impact the innate immunity of keratinocytes. The β-glucan concentrations in the supernatants were determined with the fungal G-test kit and protein concentrations with bicinchoninic acid protein quantitative method, then HaCaT was stimulated with different concentrations of culture supernatants by adopting morphological method to select a suitable dosage. Expressions of host defense genes were assessed by quantitative polymerase chain reaction after the HaCaT was stimulated with the culture supernatants. Data were analyzed with one-way analysis of variance, followed by the least significant difference test.

RESULTSThe T. rubrum strains (T1a and T XHB ) released β-glucan of 87.530 ± 37.581 pg/ml and 15.747 ± 6.453 pg/ml, respectively into the media. The messenger RNA (mRNA) expressions of toll-like receptor-2 (TLR2), TLR4, and CARD9 were moderately up-regulated in HaCaT within 6-h applications of both supernatants. HaCaT cells were more responsive to T1a than T XHB . The slight increase of dendritic cells-specific intercellular adhesion molecule 3-grabbing nonintegrin expression was faster and stronger, induced by T1a supernatant than T XHB . The moderate decreases of RNase 7, the slight up-regulations of Dectin-1 and interleukin-8 at the mRNA level were detected only in response to T1a rather than T XHB . After a long-time contact, all the elevated defense genes decreased after 24 h.

CONCLUSIONThe culture supernatant of T. rubrum could directly and transiently activate the innate immune response of keratinocytes.

Cell Line, Tumor ; Culture Media, Conditioned ; pharmacology ; Humans ; Immunity, Innate ; drug effects ; Keratinocytes ; drug effects ; metabolism ; Trichophyton ; metabolism ; beta-Glucans ; metabolism

BACKGROUND: Progressive lipid loss of adipose tissue is a major feature of cancer-associated cachexia. In addition to systemic immune/inflammatory effects in response to tumor progression, tumor-secreted cachectic ligands also play essential roles in tumor-induced lipid loss. However, the mechanisms of tumor-adipose tissue interaction in lipid homeostasis are not fully understood. METHODS: The yki -gut tumors were induced in fruit flies. Lipid metabolic assays were performed to investigate the lipolysis level of different types of insulin-like growth factor binding protein-3 (IGFBP-3) treated cells. Immunoblotting was used to display phenotypes of tumor cells and adipocytes. Quantitative polymerase chain reaction (qPCR) analysis was carried out to examine the gene expression levels such as Acc1 , Acly , and Fasn et al . RESULTS: In this study, it was revealed that tumor-derived IGFBP-3 was an important ligand directly causing lipid loss in matured adipocytes. IGFBP-3, which is highly expressed in cachectic tumor cells, antagonized insulin/IGF-like signaling (IIS) and impaired the balance between lipolysis and lipogenesis in 3T3-L1 adipocytes. Conditioned medium from cachectic tumor cells, such as Capan-1 and C26 cells, contained excessive IGFBP-3 that potently induced lipolysis in adipocytes. Notably, neutralization of IGFBP-3 by neutralizing antibody in the conditioned medium of cachectic tumor cells significantly alleviated the lipolytic effect and restored lipid storage in adipocytes. Furthermore, cachectic tumor cells were resistant to IGFBP-3 inhibition of IIS, ensuring their escape from IGFBP-3-associated growth suppression. Finally, cachectic tumor-derived ImpL2, the IGFBP-3 homolog, also impaired lipid homeostasis of host cells in an established cancer-cachexia model in Drosophila . Most importantly, IGFBP-3 was highly expressed in cancer tissues in pancreatic and colorectal cancer patients, especially higher in the sera of cachectic cancer patients than non-cachexia cancer patients. CONCLUSION Our study demonstrates that tumor-derived IGFBP-3 plays a critical role in cachexia-associated lipid loss and could be a biomarker for diagnosis of cachexia in cancer patients.
Humans ; Insulin-Like Growth Factor Binding Protein 3/metabolism* ; Culture Media, Conditioned/pharmacology* ; Cachexia/pathology* ; Gastrointestinal Neoplasms ; Somatomedins/metabolism* ; Insulins/metabolism* ; Lipids