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Background N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a short peptide with an anti-silicosis effect. However, the short biological half-life and low plasma concentration of Ac-SDKP hamper discovery of specific targets in organisms and reduce the anti-silicosis effect. A novel peptide, Ac-SDK (biotin) proline, termed "Ac-B", with anti-fibrotic properties was synthesized. Methods Ac-B was detected quantitatively by high-performance liquid chromatography. Phagocytosis of Ac-B by the alveolar epithelial cell line A549 was investigated by confocal laser scanning microscopy and flow cytometry. To further elucidate the cellular-uptake mechanism of Ac-B, chemical inhibitors of specific uptake pathways were used. After stimulation with transforming growth factor-β1, the effects of Ac-B on expression of the myofibroblast marker vimentin and accumulation of collagen type I in A549 cells were analyzed by Western blotting. Sirius Red staining and immunohistochemical analyses of the effect of Ac-B on expression of α-smooth muscle actin (SMA) in a rat model of silicosis were undertaken. Results Ac-B had good traceability during the uptake, entry, and distribution in cells. Ac-B treatment prevented an increase in α-SMA expression in vivo and in vitro and was superior to that of Ac-SDKP. Caveolae-mediated uptake of Ac-B by A549 cells led to achieving anti-epithelial-mesenchymal transformation (EMT) effects. Conclusion Ac-B had an anti-fibrotic effect and could be a promising agent for the fibrosis observed in silicosis in the future.

Background Coronary artery disease (CAD) leads to the highest mortality worldwide, seriously threatening human health. Tanshinone IIA (Tan IIA), which could be extracted from Danshen, is applied in the treatment of cardiovascular and cerebrovascular diseases. MicroRNAs (miRNAs, miRs) play pivotal roles in cell proliferation and cell apoptosis of the cardiovascular system. The aim of the present study was to explore the role of Tan IIA in CAD in vitro and the underlying molecular mechanism. Methods Real-time polymerase chain reaction (RT-PCR) and Western blot were used for the detection of miRNA/mRNA and protein, respectively. Target genes of miR-133a-3p were searched in TargetScan, and the targeting relationship was verified by dual-luciferase reporter assay. Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8) and EdU labeling. Cell apoptosis was detected by flow cytometry and TUNEL staining. Results In the present study, lower miR-133a-3p level and higher epidermal growth factor receptor (EGFR; the target of miR-133a-3p) level were found in H2O2-induced H9c2 cells. In addition, Tan IIA upregulated miR-133a-3p and downregulated EGFR expression. Moreover, Tan IIA promoted cell proliferation and suppressed apoptosis and enhanced G0/G1, which was reversed by miR-133a-3p inhibitor, while siRNA-EGFR abolished the effects induced by miR-133a-3p in H2O2-induced H9c2 cells. Conclusion Tan IIA reversed H2O2-induced cell proliferation reduction, cell apoptosis induction, and G0/G1 arrest reduction in H9c2 cells by miR-133a-3p/EGFR axis. The findings suggested a potential molecular basis of Tan IIA in treating patients with CAD.

Objectives This study aims to evaluate the frequency and absolute number of circulating innate lymphoid cell (ILC) subsets and their associations with clinical and serological features in systemic lupus erythematosus (SLE). Patients and methods We recruited 28 SLE patients (6 males, 22 females; mean age 37.57 years; range, 18 to 56 years) and 13 healthy controls (4 males, 9 females; mean age 32.08 years; range, 19 to 48 years). Circulating ILC subsets were identified by flow cytometry. Associations between all detected cells and SLE disease activity, clinical manifestations, and serum autoantibodies were analyzed. Results In this study, significantly higher frequencies of ILC2s and ILC3s, lower frequencies of ILC1s, and higher ILC1/ILC3 and ILC1/ILC2 ratios were observed in SLE patients than in healthy controls. The frequencies and number of ILC3s were positively associated with SLE disease activity index 2000 score and anti-double stranded deoxyribonucleic acid titers in patients with SLE. Decreased ILC1 frequencies, increased ILC3 frequencies, and decreased ILC1/ILC3 and ILC2/ILC3 ratios were observed in patients with arthritis compared to those without arthritis. Conclusion Our results indicated biased altered distributions of circulating ILC subsets in SLE. ILC3s were associated with SLE disease activity, and ILC1s, ILC3s, and ILC1/ILC3 and ILC2/ILC3 ratios were associated with SLE accompanied with arthritis. Taken together, these results suggest that ILCs may serve as cellular biomarkers for disease activity and arthritis involvement in SLE.

Objective To evaluate 5-Aza-CdR's inhibited effects on migration, proliferation, and apoptosis in colon cancer cells and its potential mechanisms. Methods HCT-116, SW480, and SW620 were divided into HCT116 group, HCT116+5-Aza-CdR group, SW480 group, SW480+5-Aza-CdR group, SW620 group and SW620+5-Aza according to experimental needs. MTT test was chosen to investigate cell proliferation; Transwell test was used to evaluate cell migration; scratch assay was used to investigate cell invasion; flow cytometry was used to investigate apoptosis; immunofluorescence assay was used to investigate the protein level of DNMT1 and RASSF1A in cells; qRT-PCR was used to examine DNMT1, RASSF1A, RAS, Raf1, MEK, Grb2 and ERK transcription levels. Results Compared with HCT116 group, 5-Aza-CdR+HCT116 group inhibited cell proliferation, increased apoptosis rate, decreased invasive ability, decreased DNMT1 expression, increased expression of RASSF1A, decreased expression of RAS, Raf1, MEK, Grb2 and ERK. SW480 was compared with 5-Aza-CdR+SW480 group and SW620 group with 5-Aza-CdR+SW620 group. Their change trend of detection index was similar to that in HCT-116 group and HCT116+5-Aza-CdR group. Conclusion 5-Aza-CdR can obviously inhibit the proliferation, migration and invasion of three colon cancer cell lines. Its mechanism maybe relies on the inhibition of DNMT1 mRNA level and protein level and the enhancement of RASSF1A mRNA level and protein level.

Background/purpose Heavy alcohol drinking is associated with pancreatitis. Pancreatitis is initiated by the damage to the pancreatic acinar cells. The endoplasmic reticulum (ER) stress has been shown to play an important role in alcohol-induced pancreatic damage. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-inducible protein. The aim of the study was to determine whether MANF can ameliorate alcohol-induced ER stress and cellular damages to pancreatic acinar cells. Methods Alcohol-induced damage to mouse pancreatic 266-6 acinar cells was determined by MTT and flow cytometry. MANF expression was down-regulated by MANF siRNA using a Neon Transfection System. The over-expression of MANF was performed by the infection with the adenoviral vector carrying mouse MANF gene. The expression of ER stress markers was determined by immunoblotting and immunofluorescence. Results Alcohol caused ER stress, oxidative stress and induced apoptosis of 266-6 acinar cells. Recombinant human MANF alleviated alcohol-induced ER stress and cell death by inhibiting IRE1-caspase 12-caspase 3 apoptotic pathway. Overexpression of mouse MANF also protected cells against alcohol-induced apoptosis. In contrast, inhibiting MANF by siRNA exacerbated alcohol-induced cellular damage. Conclusions MANF was protective against alcohol-induced ER stress and cellular injury in pancreatic acinar cells. The findings suggest a potential therapeutic value of MANF for alcoholic pancreatitis.

Objective: To establish a convenient and simple flow cytometry immunophenotyping panel to explore immune cellular alterations and potential cellular biomarkers in systemic lupus erythematosus. Materials and methods: This is a cross-sectional, case–control study including 60 patients with systemic lupus erythematosus and 20 sex- and age-matched healthy controls. A 14-color immunophenotyping panel was applied to detect proportions of circulating immune mononuclear cells, and comparisons between patients and healthy controls, and subgroups of patients, were performed. Correlations between cellular proportions and other parameters were investigated. Results: After multivariate analysis, significantly decreased proportions of CD4−CD8− T cells, natural killer cells and innate lymphoid cells were observed in patients compared with healthy controls. The proportions of basophils were decreased significantly in patients with lupus nephritis (LN) compared with those in patients without LN. Conclusion: In the present study, we found that basophil proportions may be a biomarker of LN., Lay abstract Systemic lupus erythematosus is a chronic, multisystem, autoimmune disorder that involves various abnormalities of immune cells and thus presents in a striking variety of ways. This study aimed to establish a biomarker panel that would enable the exploration of changes in immune cells and the relationships between immune cell subsets and clinical manifestations in patients with systemic lupus erythematosus. Our results showed that basophil cell proportions may be a biomarker of use in lupus nephritis.

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or "enteroids". Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2'-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

BackgroundAcute myeloid leukemia (AML) remains one of the most challenging hematological malignancies. Despite progress in therapeutics, majority of patients succumb to this neoplasm. CD33 is a proven therapeutic target, given its expression on most AML cells. Almost all anti-CD33 antibodies target the membrane distal immunoglobulin V (IgV) domain of the CD33 extracellular domain.MethodsIn this manuscript, we present data on three bispecific antibodies (BsAbs) against the CD33 IgV and membrane proximal immunoglobulin C (IgC) domains. We use in vitro binding and cytotoxicity assays to show the effect of these BsAbs on AML cell lines. We also use immunodeficient mice-bearing leukemias from cell lines and patient-derived xenografts to show the effect of these BsAbs in vivo.ResultsIn vitro, the IgV-targeting BsAb had higher binding to AML cell lines using flow cytometry and delivered more potent cytotoxicity in T-cell-dependent cytotoxicity assays; importantly, the IgC domain-targeting outperformed the IgV domain-targeting BsAb in medullary and extramedullary leukemia animal models.ConclusionsThese data support further clinical development of this BsAb for first-in-human phase I clinical trial.

Raddeanin A, one of the triterpenoid saponins extracted from Anemone raddeana rhizome of the Ranunculaceae family, has demonstrated the ability to inhibit the growth of human hepatic and gastric cancer cells. However, the effects of Raddeanin A on human colon cancer cells have not been investigated extensively. The present study aimed to examine the antiproliferative and apoptosis-inducing effects of Raddeanin A on the HCT-116 human colon cancer cell line in vitro, and evaluate the pharmacokinetic and biodistribution properties of Raddeanin A in mice following a single oral administration. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the in vitro cytotoxicity of Raddeanin A against HCT-116 cells. 4′,6-Diamidino-2-phenylindole, dihydrochloride staining and flow cytometry were performed to further examine the apoptosis-inducing capability of Raddeanin A. The concentrations of Raddeanin A in the plasma and tissues were analyzed using liquid chromatography-tandem mass spectrometry. Raddeanin A showed a dose-dependent antiproliferative effect towards the HCT-116 cells, with a half maximal inhibitory concentration of ~1.4 µM. Treatment with Raddeanin A resulted in a significant induction of apoptosis, observed as apparent morphological changes of the nuclei, with a total apoptotic ratio of 41.8% at a concentration of 3 µM. Low concentrations of Raddeanin A were detected in the heart, liver, spleen, lung, kidney and plasma of the mice following oral administration, however, the majority of the Raddeanin A was distributed in the intestinal tract, particularly in the colon and caecum. These present study confirmed the growth-inhibitory and apoptosis-inducing effects of Raddeanin A on HCT-116 cells and performed preliminary examinations of its pharmacokinetic properties, which provide a foundation for further investigating the inhibitory mechanism on the colon cancer cells in vivo.

Cynatratoside A (CyA) is a C21 Steroidal glycoside with pregnane skeleton isolated from the root of Cynanchum atratum Bunge (Asclepiadaceae). This study aimed to investigate the effects of CyA on concanavalin A (Con A)-induced autoimmune hepatitis (AIH) and the underlying mechanism. CyA was orally administered to mice at 10 and 40 mg/kg 8 h before and 1 h after Con A treatment. The effects of CyA on Con A-induced spleen and liver in mice were assessed via histopathological changes, T lymphocyte amounts and the expressions of IL-1β and ICAM-1. Con A-induced L-02 hepatocytes were used to evaluate whether CyA (0.1–10 μM) can directly protect hepatocytes from cytotoxicity and the possible mechanism. The results revealed that CyA treatment could significantly improve the histopathological changes of spleen and liver, reduce the proliferation of splenic T lymphocytes, and decrease the expressions of IL-1β and ICAM-1 in liver. The experiment in vitro showed that CyA inhibited Con A-induced hepatotoxicity in a concentration-dependent manner. CyA (10 μM) significantly increased/decreased the expression of Bcl-2/Bax and reduced the levels of cleaved caspases-9 and -3. Our study demonstrated for the first time that CyA has a significant protective effect on Con A-induced AIH by inhibiting the activation and adhesion of T lymphocytes and blocking hepatocyte apoptosis.

Pathogenesis of endometriosis is still unknown, and the relationship between NK cell activating receptors and endometriosis remains to be explored. We investigated the expression of NCRs and NKG2D in NK cells in peripheral blood (PB) and peritoneal fluid (PF) as well as expression of NKG2D ligands in endometrial cells, and illuminated their relationship with ovarian endometriosis. 20 patients with ovarian endometriosis and 13 subjects for control group were recruited. Flow cytometry was used for examining expressions of NCRs and NKG2D on NK cells. In PF with endometriosis, the expressions of NKp30 (P = 0. 006) and NKG2D (P = 0. 010) on CD56+NK cells were decreased, whereas the expression of NKp46 (P = 0. 040) on CD16+NK cells was higher than that of control. Real time PCR and Western blotting were used for detecting expression of NKG2D ligands. mRNA level of NKG2D ligands on endometrial cells showed no noticeable difference. As for protein expression, the ULBP-2 expression on eutopic endometrial cells with pelvic endometriosis was lower than that on ectopic endometrial cells and eutopic endometrial cells without endometriosis (P < 0.05), and the ULBP-3 expression on ectopic endometrial cells was lower than that on eutopic endometrial cells with or without endometriosis (P < 0.05). These findings indicate that change of NKp30, NKp46 and NKG2D on NK cells in PF and ULBP-2, 3 on endometrial cells may relate to the pathogenesis of pelvic endometriosis. Especially, change of NK cell activating receptors in PF implies that pelvic endometriosis is probably due to local immune changes.

With the rapid development of suspension array technology, microbeads-based barcodes as the core element with sufficient encoding capacity are urgently required for high-throughput multiplexed detection. Here, a novel structure-fluorescence combinational encoding strategy is proposed for the first time to establish a barcode library with ultrahigh encoding capacities. Based on the never revealed transformability of the structural parameters (e.g., porosity and matrix component) of mesoporous microbeads into scattering signals in flow cytometry, the enlargement of codes number has been successfully realized in combination with two other fluorescent elements of fluorescein isothiocyanate isomer I (FITC) and quantum dots (QDs). The barcodes are constructed with precise architectures including FITC encapsulated within mesopores and magnetic nanoparticles as well as QDs immobilized on the outer surface to achieve the ultrahigh encoding level of 300 accompanied with superparamagnetism. To the best of knowledge, it is the highest record of single excitation laser-based encoding capacity up to now. Moreover, a ten-plexed tumor markers bioassay based on the tailored-designed barcodes has been evaluated to confirm their feasibility and effectiveness, and the results indicate that the barcodes platform is a promising and robust tool for practical multiplexed biodetection.

Structural maintenance of chromosome 1 alpha (SMC1A) gene has been reported to be related to tumor development in some types of human cancers. However, the misregulation of SMC1A and its functions in castration-resistant prostate cancer (CRPC) have not been well understood. In the present study, we found that SMC1A was elevated in androgen-independent PCa cell lines PC-3 and DU-145 compared to androgen sensitive LNCap and 22RV1 cells by qPCR and western blot assay. Knockdown of SMC1A inhibited cell growth, colony formation and cell migration abilities of PC-3 and DU145 cells by MTT, colony formation and transwell assays, and affected cell cycle progression in PC-3 and DU145 cells by flow cytometry. Moreover, SMC1A knockdown significantly reduced tumor growth in vivo in a nude mouse model. Additionally, we also found that the expression of SMC1A gene was higher in prostate cancer tissues than in the adjacent normal tissues by immunohistochemical staining, and was positively correlated to tumor metastasis and recurrence by Oncomine database mining. Taken together, the present study indicates that SMC1A may play an important role in malignant transformation of PCa under conditions of androgen deprivation and act as a new target for PCa diagnosis and treatment.

Utilization of quercetin (QT) in clinics is limited by its instability and poor solubility. To overcome these disadvantages, we prepared QT as QT-loaded PLGA-TPGS nanoparticles (QPTN) and examined its properties and therapeutic efficacy for liver cancer. QT-loaded PLGA nanoparticles (QPN) and QT/coumarin-6-loaded PLGA-TPGS nanoparticles (QCPTN) with coumarin-6 as a fluorescent marker were also prepared to investigate the cellular uptake by HepG2 and HCa-F cells using a confocal laser scanning microscope (CLSM), and their effects on apoptosis of HepG2 cells were assessed with flow cytometry. The results measured using transmission electron microscopy, scanning electron microscopy and size analyses indicated that QPTN were stably dispersed sphere with diameter in the range of 100-200 nm. It indicated that the QT loading and encapsulation efficiency in QPTN reached 21.63% and 93.74%, respectively, and the accumulative drug release of QPTN was 85.8%, the QCPTN uptake in HCa-F and HepG2 cells were 50.87% and 61.09% using HPLC analysis, respectively. The results determined using an Annexin-PI flow cytometry indicated that QPTN could induce HepG2 cell apoptosis in a dose dependent manner. The results of histological examination and HPLC analysis confirmed that QPTN was targeted to liver cells. In vivo analysis using solid tumor-bearing mouse model indicated that QPTN could suppress tumor growth by 59.07%. Moreover, all the studied properties of QPTN were more desirable than those of QT-loaded PLGA nanoparticles (QPN). In conclusion, QPTN could be used as a potential intravenous dosage form for the treatment of liver cancer owing to the enhanced pharmacological effects of QT with increased liver targeting.

Aims To investigate the effect of 7-O-geranylquercetin (GQ), a derivative of quercetin (Q), on reversing drug resistance in breast cancer MCF-7/ADR cells and reveal the mechanisms related to P-glycoprotein (P-gp). Main methods Cell viability was determined by MTT assay. Accumulation of adriamycin (ADR) in cells was determined by confocal fluorescence microscope and microplate reader while that of rhodamine (Rh) was measured by flow cytometry. Expression levels of P-gp and MDR1 gene in cells were detected by western blot and Real-Time PCR, respectively. Molecular docking of GQ and Q with P-gp was conducted using AutoDock program. Xenograft model was established by inoculating MCF-7/ADR cells in BALB/c-nude mice. Tumor bearing mice were administered with ADR via tail vein injection and/or GQ (Q) by gavage. Expression levels of P-gp in tissues were detected by western blot and immunohistochemistry. Key findings GQ could reverse drug resistance of MCF-7/ADR cells to ADR. GQ inhibited the efflux of ADR by down-regulating the expression of P-gp protein and its encoding gene MDR1 in MCF-7/ADR cells. Molecular modeling showed that GQ matched with P-gp better than Q. GQ enhanced the antitumor effects of ADR and decreased the expression of P-gp in mice and its activities were higher than that of Q. GQ could reverse drug resistance of MCF-7/ADR cells by down-regulating the expression of P-gp in vitro and in vivo. Significances The reversal effect of GQ on P-gp-mediated drug resistance indicates its potential as a reversal agent for drug resistance in cancer chemotherapy.

BACKGROUND: Decidual γδ T cells are known to regulate the function of trophoblasts at the maternal–fetal interface; however, little is known about the molecular mechanisms of cross talk between trophoblast cells and decidual γδ T cells. METHODS: Expression of chemokine C-X-C motif ligand 6 (CXCL16) and its receptor CXCR6 was evaluated in first-trimester human villus and decidual tissues by immunohistochemistry. γδ T cells were isolated from first-trimester human deciduae and cocultured with JEG3 trophoblast cells. Cell proliferation and apoptosis-related molecules, together with cytotoxicity factor and cytokine production, were measured by flow cytometry analysis. RESULTS: Expression of CXCL16 and CXCR6 was reduced at the maternal–fetal interface in patients who experienced unexplained recurrent spontaneous abortion as compared to healthy pregnancy women. With the administration of pregnancy-related hormones or coculture with JEG3 cells, CXCR6 expression was upregulated on decidual γδ T cells. CXCL16 derived from JEG3 cells caused a decrease in granzyme B production of decidual γδ T cells. In addition, decidual γδ T cells educated by JEG3-derived CXCL16 upregulated the expression of Bcl-xL in JEG3 cells. CONCLUSION: This study suggested that the CXCL16/CXCR6 axis may contribute to maintaining normal pregnancy by reducing the secretion of cytotoxic factor granzyme B of decidual γδ T cells and promoting the expression of antiapoptotic marker Bcl-xL of trophoblasts.

The form of selenium appears to be important for preventing cancer in humans. Here, we evaluated selenium levels in the serum and bone tissue samples from osteosarcoma patients using atomic absorption spectrometry. The in vitro effects of Se-methylselenocysteine (Se-MSC) on growth, cell cycle status, and apoptosis of osteosarcoma cells were assessed using the WST-1 assay, Hoechst 33342/propidium iodide staining, and flow cytometry, respectively. In osteosarcoma cases, the mean serum selenium levels in osteosarcoma tissue and normal bone were 0.08 mg/kg and 0.03 mg/kg, respectively (P0.05). Serum selenium levels in osteosarcoma and non-osteosarcoma cases were 0.09 mg/L and 0.08 mg/L, respectively (P0.05). Se-MSC-treated MG63 cells showed altered cellular morphology, decreased viability in a time- and dose-dependent manner, and an increase in the sub-G1 cell population. Se-MSC also downregulated Bcl-2 expression and upregulated Bax. Se-MSC inhibited the proliferation of the drug-resistant osteosarcoma cell line Saos-2/MTX300 and enhanced the inhibitory effect of pirarubicin on MG63 cells. Our data demonstrate that selenium levels are significantly higher in osteosarcoma tissue than normal bone tissue in osteosarcoma patients. The results also support the anticancer effects of Se-MSC in osteosarcoma. Further development of Se-MSC as an ancillary chemotherapy agent in osteosarcoma is warranted.

Background: Rac3, a member of the Rac family of small guanosine triphosphatases (GTPases), regulates a variety of cell functions, including the organization of the cytoskeleton, cell migration, and invasion. Overexpression of Rac3 has been reported in several human cancers. However, the role of Rac3 in lung cancer (LC) has not been determined in detail. The purpose of this study was to investigate the effect of silencing of Rac3 expression in human LC cells and the consequences for cell survival. Materials and Methods: Lentivirus small hairpin RNA (shRNA) interference techniques were utilized to knock down the Rac3 gene. Gene and protein expression was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. LC cell apoptosis was examined by annexin V-APC /propidium iodide staining. Results: Efficient silencing of Rac3 strongly inhibited A549 cell proliferation and colony formation ability, and significantly decreased tumor growth. Moreover, flow cytometry analysis showed that knockdown of Rac3 led to G2/M phase cell cycle arrest as well as an excess accumulation of cells in the G1 and S phase. Conclusions: Thus, functional analysis using shRNAs revealed a critical role for Rac3 in the tumor growth of LC cells. shRNA silencing of Rac3 could provide an effective strategy to treat LC.

We investigated the cellular uptake behavior and cell viability of semiconducting polymer dots (Pdots) on human gastric adenocarcinoma (SGC-7901) cells and human gastric mucosal (GES-1) cells. MTT studies indicate the Pdot treatment induces obvious cell proliferation in both types of cell lines. We performed further investigations such as reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) change, which indicate that the cell proliferation is in good agreement with the increase in the ROS and MMP levels. Moreover, expression of protein kinase B (Akt) decreased as the Pdot concentration increases, but the expression of protein dual-phosphorylated Erk (p-Erk) and phosphorylated c-Jun N-terminal kinases (p-JNK) were increased. These effects indicated that the Pdots could promote the growth of SGC-7901 cells and GES-1 cells by appropriately regulating the expressions of protein Akt, p-Erk, and p-JNK.

// Xiao-kun Wang 1 , Kenneth Kin Wah To 2 , Li-yan Huang 1 , Jing-hong Xu 3 , Ke Yang 1 , Fang Wang 1 , Zhen-cong Huang 1 , Sheng Ye 3 , Li-wu Fu 1 1 State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China 2 School of Pharmacy, The Chinese University of Hong Kong, Hong Kong, P.R. China 3 First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China Correspondence to: Li-wu Fu, e-mail: fulw@mail.sysu.edu.cn Keywords: Multidrug resistance, ABCG2, tyrosine kinase inhibitor, afatinib, combined chemotherapy Received: July 05, 2014 Accepted: October 27, 2014 Published: November 24, 2014 ABSTRACT Multidrug resistance (MDR) to chemotherapeutic drugs is a formidable barrier to the success of cancer chemotherapy. Expressions of ATP-binding cassette (ABC) transporters contribute to clinical MDR phenotype. In this study, we found that afatinib, a small molecule tyrosine kinase inhibitor (TKI) targeting EGFR, HER-2 and HER-4, reversed the chemoresistance mediated by ABCG2 in vitro , but had no effect on that mediated by multidrug resistance protein ABCB1 and ABCC1. In addition, afatinib, in combination with topotecan, significantly inhibited the growth of ABCG2- overexpressing cell xenograft tumors in vivo . Mechanistic investigations exhibited that afatinib significantly inhibited ATPase activity of ABCG2 and downregulated expression level of ABCG2, which resulted in the suppression of efflux activity of ABCG2 in parallel to the increase of intracellular accumulation of ABCG2 substrate anticancer agents. Taken together, our findings may provide a new and useful combinational therapeutic strategy of afatinib with chemotherapeutical drug for the patients with ABCG2 overexpressing cancer cells.

Excess fluoride intake could induce apoptosis in the cells. As an essential micronutrient and cytoprotectant, zinc is involved in many types of apoptosis. Here, we studied the effects of zinc and ZIP1 on fluoride-induced apoptosis in mouse MC3T3-E1 cells and examined the underlying molecular mechanisms. Our study found that fluoride not only inhibited cell proliferation and increased the intracellular reactive oxygen species (ROS) but also induced cell apoptosis. Whereas pretreatment with zinc significantly attenuated fluoride-induced ROS production and partly protected cells against fluoride-induced apoptosis through MAPK/ERK signaling pathway. Our study also found that fluoride upregulated the expression of ZIP1 in a time-dependent manner. Moreover, overexpression of ZIP1 also inhibited fluoride-induced apoptosis by activation of PI3K/Akt pathway. This cytoprotective effect of zinc and ZIP1 may be new factors that affect the physiological activity of fluoride and need study further.

Immunophenotype is critical for diagnosing common B-cell acute lymphoblastic leukemia (common ALL) and detecting minimal residual disease. We developed a protocol to explore the immunophenotypic profiles of common ALL based on the expression levels of the antigens associated with B lymphoid development, including IL-7Rα (CD127), cytoplasmic CD79a (cCD79a), CD19, VpreB (CD179a), and sIgm, which are successive and essential for progression of B cells along their developmental pathway. Analysis of the immunophenotypes of 48 common ALL cases showed that the immunophenotypic patterns were highly heterogeneous, with the leukemic cell population differing from case to case. Through the comprehensive analysis of immunophenotypic patterns, the profiles of patient-specific composite leukemia cell populations could provide detailed information helpful for the diagnosis, therapeutic monitoring, and individualized therapies for common ALL.

To investigate the biocompatibility of human gastric carcinoma cells (SGC-7901) with organic two-photon nanoparticles (NPs).Different concentrations of NPs were incubated with SGC-7901 cells for different times. The levels of cell apoptosis, reactive oxygen species (ROS), intracellular calcium, and mitochondrial membrane potential (MMP) were measured by staining the SGC-7901 cells with Annexin V-FITC/PI, 2',7'-dichlorofluorescin diacetate, Fluo-3 AM, and Rhodamine 123, followed by the flow cytometry assay. NPs at4 µg/ml, did not have any significant effect on apoptosis, necrosis, generation of ROS, increase of intracellular Ca(2+) concentration or decrease of MMP in SGC-7901 cells, but4 µg/ml had a major effects on all the above mentioned parameters.2,5,2',5'-Tetra(4-N,N-diphenylamine styryl) biphenyl NPs can be used at an appropriate concentration as a safe drug carrier or imaging marker and may serve as an effective tool for developing a photodynamic cancer therapy.

Algicidal microbes could effectively remove the harmful algae from the waters. In this study, we were concerned with the ecological influence of an algicide extracted from Streptomyces alboflavus RPS, which could completely lyse the Phaeocystis globosa cells within two days. In microcosms, 4 μg/mL of the microbial algicide could efficiently remove P. globosa cells without suppressing other aquatic organisms. Bioluminescent assays confirmed that the toxicity of microbial algicide at this concentration was negligible. Interestingly, the toxicity of P. globosa exudates was also significantly reduced after being treated with the algicide. Further experiments revealed that the microbial algicide could instantly increase the permeability of the plasma membrane and disturb the photosynthetic system, followed by the deformation of organelles, vacuolization and increasing oxidative stress. The pre-incubation of N-acetyl cysteine (NAC) verified that the rapid damages to the plasma membrane and photosynthetic system caused the algal death in the early phase and the increasing oxidative stress killed the rest. The late accumulation and possible release of CAT also explained the decreasing toxicity of the algal culture. These results indicated that this microbial algicide has great potential in controlling the growth of P. globosa on site.

We investigated the role of XPD in cell apoptosis of hepatoma and its relationship with p53 during the regulation of hepatoma bio-behavior. RT-PCR and Western blot were used to detect the expression levels of XPD, p53, c-myc, and cdk2. The cell apoptosis and cell cycle were analyzed with flow cytometry. Compared with the control cells, XPD-transfected cells displayed a lower viability and higher apoptosis rate. A decreased expression of p53 gene was detected in XPD-transfected cells. In contrast, both c-myc and cdk2 showed increased expressions of mRNAs and proteins in the transfected cells. Our results indicate that XPD may play an important role in cell apoptosis of hepatoma by inducing an over-expression of p53, but suppressing expressions of c-myc and cdk2.

Peritoneal metastasis is a distinct pathologic characteristic of advanced epithelial ovarian cancer (EOC), which is the most deadly disease of the female reproductive tract. The inflammatory environment of the peritoneum in EOC contains abundant macrophages, activated thrombin, and thrombin-associated receptors. However, little is known about the mechanism by which the thrombin-macrophages interaction contributes to tumor invasion and metastasis. We investigated the phenotype and cytokine/chemokine expression of thrombin-treated peripheral blood monocytes (MOs)/macrophages, it was found that the phenotype of MOs was altered toward a TAM-like macrophage CD163(high)IL-10(high)CCL18(high)IL-8(high) after thrombin stimulation. By Matrigel invasion assay, the conditioned medium of thrombin-stimulated MOs accelerated remarkable invasion of ES-2, SKOV3, and HO-8910, which was similar to invasive cell numbers of ascites stimuli (P0.05) and higher than MOs medium alone (P0.05). IL-8 was proposed as the major chemoattractant mediating EOC invasion based on MOs mRNA and protein expression profiling. It was observed that anti IL-8 monoclonal neutralizing antibody attenuated EOC cell invasion in a concentration-dependent manner. Increased transcriptional activation of NF-kappaB p50/p65 was identified in thrombin-treated MOs. This study provided insight the role of thrombin in the regulation of EOC peritoneal invasion via "educating" MOs.

OBJECTIVE—Plasmacytoid precursor dendritic cell facilitating cells (p-preDC FCs) play a critical role in facilitation of syngeneic and allogeneic hematopoietic stem cell (HSC) engraftment. Here, we evaluated the phenotype and function of CD8+/TCR− FCs from NOD mice.RESEARCH DESIGN AND METHODS—The phenotype of CD8+/TCR− FCs was analyzed by flow cytometry using sorted FCs from NOD, NOR, or B6 mice. The function of NOD FCs was evaluated by colony-forming cell (CFC) assay in vitro and syngeneic or allogeneic HSC transplantation in vivo.RESULTS—We report for the first time that NOD FCs are functionally impaired. They fail to facilitate engraftment of syngeneic and allogeneic HSCs in vivo and do not enhance HSC clonogenicity in vitro. NOD FCs contain subpopulations similar to those previously described in B6 FCs, including p-preDC, CD19+, NK1.1+DX5+, and myeloid cells. However, the CD19+ and NK1.1+DX5+ subpopulations are significantly decreased in number in NOD FCs compared with disease-resistant controls. Removal of the CD19+ or NK1.1+DX5+ subpopulations from FCs did not significantly affect facilitation. Notably, Flt3 ligand (FL) treatment of NOD donors expanded FC total in peripheral blood and restored facilitating function in vivo.CONCLUSIONS—These data demonstrate that NOD FCs exhibit significantly impaired function that is reversible, since FL restored production of functional FCs in NOD mice and suggest that FL plays an important role in the regulation and development of FC function. FCs may therefore be linked to diabetes pathogenesis and prevention.

Background. Bone marrow (BM) chimerism has been shown to have a beneficial effect on allograft survival. We recently found that production of donor T-cells was highly correlated with induction of tolerance in minimally conditioned chimeras. In the present studies, we demonstrate that nonmyeloablative conditioning and BM cell infusion modulate innate and adaptive host immune responses. Methods. Chimeras were generated by bone marrow transplantation (B10.BR to B10). Recipients were preconditioned with T-cell depleting antibodies and total body irradiation with or without cyclophosphamide. Donor-specific tolerance was tested by skin grafting. Results. Transfer of tolerant splenocytes to immunocompetent secondary recipients did not transfer tolerance, nor did infusion of tolerant CD4+/CD25+ T-cells into chimeras without donor T-cell production, demonstrating that linked suppression is an unlikely mechanism in tolerance induction in the context of BM cell infusion. The addition of a single dose of cyclophosphamide to the conditioning enhanced engraftment and tolerance. This was associated with production of donor T-cells and effective clonal deletion, and a significant reduction in activated recipient plasmacytoid dendritic cells (pDC) and natural killer (NK) cells. Chimeras without donor T-cell production that eventually lost their chimerism did not generate an antidonor humoral response, whereas unconditioned controls infused with similar numbers of BM cells did, indicating that infusion of donor BM cells into conditioned recipients induced immune deviation for adaptive B-cell immunity, preventing sensitization to major histocompatibility complex (MHC) alloantigens. Conclusions. These results demonstrate that recipient T-cells, pDC, and NK cells contribute to the host barrier for establishing chimerism, implicate deletional tolerance as the mechanism for total body irradiation-based nonmyeloablative conditioning for BM transplantation, and show a beneficial effect of BM cells in preventing sensitization to MHC alloantigens.

Prohibitin (PHB), a potential tumor suppressor, has been shown to inhibit cell proliferation by repressing E2F-mediated transcription. But little is known about the role of PHB involved in tubulointerstitial fibrosis (TIF). Here, for the first time, we found PHB protein was positively expressed at normal renal tissues, strongly down-regulated in renal biopsy specimens, and negatively correlated with the expression of alpha-smooth-muscle actin (alpha-SMA) and with the degrees of tubulointerstitial lesions. Transforming growth factor-beta1 (TGF-beta1) is the most important profibrotic cytokine in the process of TIF and capable of inducing cell phenotypic change of interstitial fibroblasts characterized by the de novo expression of alpha-SMA. Confocal microscopy showed majority of PHB is located at cytoplasm as well as at nucleus in rat kidney fibroblasts cell (NRK-49F). As we found that PHB protein and mRNA expression were down-regulated in NRK-49F cells following TGF-beta1 stimulation. We used transient transfection to over-express PHB protein and found that cells with increased PHB levels had a significant reduction in the percentage entering cell cycle and abolished de novo expression of alpha-SMA following TGF-beta1 stimulation. Therefore, over-expression of PHB suppresses renal interstitial fibroblasts proliferation and cell phenotypic change induced by TGF-beta1, which indicates PHB as a potential therapeutic target to halt the progression of TIF.

Bone marrow transplantation offers great promise for treating a number of disease states. However, the widespread application of this approach is dependent upon the development of less toxic methods to establish chimerism and avoid graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FCs) have been shown to enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD. In the present studies, we have identified the main subpopulation of FCs as plasmacytoid precursor dendritic cells (p-preDCs). FCs and p-preDCs share many phenotypic, morphological, and functional features: both produce IFN-α and TNF-α, both are activated by toll-like receptor (TLR)-9 ligand (CpG ODN) stimulation, and both expand and mature after Flt3 ligand (FL) treatment. FL-mobilized FCs, most of which express a preDC phenotype, significantly enhance engraftment of HSCs and induce donor-specific tolerance to skin allografts. However, p-preDCs alone or p-preDCs from the FC population facilitate HSC engraftment less efficiently than total FCs. Moreover, FCs depleted of preDCs completely fail to facilitate HSC engraftment. These results are the first to define a direct functional role for p-preDCs in HSC engraftment, and also suggest that p-preDCs need to be in a certain state of maturation/activation to be fully functional.

An aquatic actinomycete capable of eliminating the brown tide causing marine alga Phaeocystis globosa was isolated from the surface sea water and the isolate named JS01 was characterized as Streptomyces on the basis of its 16S rRNA gene sequence. The supernatant of JS01 could lyse algal cells, implying that JS01 produced a latent alga-lytic compound. Considering this algicidal activity and the response of the algal cells, Chlorophyll a fluorescence decreased significantly in P. globosa in response to the JS01 supernatant when analyzed with flow cytometry. The algal cells experienced cell shrinkage and plasmolysis before disintegration after 72 h of treatment. The released algicide(s) were heat-tolerant, except above 121 °C, and fluctuation in pH variations; even so, algicidal activity was also over 60 %. The maximum toxicity of JS01 was on the seventh day of culture, and the relative luminosity was 0.49 at that time when detected by luminous bacteria Vibrio fischeri. These results indicated that the Streptomyces sp. JS01 could function as a potential controller of Phaeocystis globosa blooms.

Water-dispersed organic nanoparticles (NPs) were prepared from a conjugated molecule 2,5,2',5'-tetra(4'-N,N-diphenylaminostyryl)biphenyl (DPA-TSB). As a potential material for photodynamic therapy, we investigated cellular toxicity by using fluorescence confocal microscopy and Western blotting. Our results demonstrated that the cells treated with DPA-TSB NPs showed an increase in apoptosis accompanied by the increased expression of Bax protein, decreased expression of Bcl-2 protein and caspase-3 protein, the decreased mitochondrial membrane potential and the release of ROS. Using inhibitors of endocytosis can block nanoparticles uptake, indicating that DPA-TSB NPs entered the cells via an energy-dependent manner, mostly through clathrin-mediated and caveolae-mediated processes.

Inflammation plays an essential role in the pathophysiology of atherosclerosis. Our study was aimed to investigate whether salvianolate, a novel water-soluble phenolic compound of Danshen, alleviates atherosclerosis via regulating the inflammation in rats. High fat diet feeding plus vitamin D3 injection was used to induce atherosclerosis in rats. Salvianolate (60, 120 or 240 mg/kg) or placebo was given to atherosclerotic rats. The plasma lipids, interleukin 6 (IL-6) and C reactive protein (CRP) were measured by ELISA. CD4+CD25+Foxp3+ cells were determined by flow cytometry. Histological changes were examined by hematoxylin and eosin staining. The results showed that the levels of plasma IL-6 and CRP were elevated in the rats fed on high fat diet, and the histological analysis demonstrated the successful establishment of atherosclerosis models. Treatment with salvianolate alleviated the atherosclerotic process and decreased the levels of plasma IL-6 and CRP. Also the number of CD4+CD25+Foxp3+ cells was increased in salvianolate-treated rats. It was concluded that salvianolate could treat atherosclerosis via modulating the inflammation at cytokine and cell levels.

This study was purposed to compare the effect of 3 different cell components for expanding CD4(+) CD25(+) Treg in vitro, and identify their immunosuppressive function. CD4(+) T cells, CD4(+) CD25(-)T cells and CD4(+) CD25(+)T cells were isolated from mouse splenocytes by MACS and then expanded in vitro. Phenotype of the T cell lines and expression of the FOXP3 was determined by flow cytometry. The inhibitory effect of expanded CD4(+) CD25(+) T cells on CD4(+) CD25(-)T cells was tested by MLR method. The results showed that the Treg cells from all the three groups were expanded significantly after culture for 2 weeks. In the CD4(+) T cells group, the proliferation rate was (77.8 ± 5.32) folds with a percentage of Treg cells increasing from (6.61 ± 1.00)% to (15.33 ± 1.31)%. The proliferation rate in the CD4(+) CD25(-) T cells group was (95.20 ± 7.67) folds, with the percentage of CD4(+) CD25(+) T cells raising from (0.37 ± 0.13)% to (9.84 ± 0.98)%. The proliferation rate in the CD4(+) CD25(+) T cells group was (41.20 ± 6.92) folds, the proportion of Treg cells decreased from (86.75 ± 1.25)% to (85.32 ± 1.62)%, and the expression of Foxp3 decreased from (76.92 ± 1.72)% to (75.33 ± 2.11)% during the culture, there were not significant differences in the cell purity and the expression of Foxp3, compared with pre-amplification. The inhibitory test showed that the expanded CD4(+) CD25(+) T cells could inhibit the proliferation of CD4(+) CD25(-) T cells in vitro in a cell dose-dependent manner. It is concluded that the amplification of CD4(+) CD25(+) Treg cells is successful in vitro, especially in the CD4(+) CD25(+) T cells group, the cell purity and Foxp3 gene is not obviously changes after amplification.

Purpose: To investigate whether modified acidic fibroblast growth factor (MaFGF) can protect NRK52E cell against apoptotic death induced by actinomycin D (Act D) and the effect of MaFGF on PI3K/Akt signaling pathway.Methods: NRK52E cell apoptotic death was measured by several methods including cell morphologic observation, Hoechst 33342 staining and flow cytometry. In addition, the levels of phosphorylated-Akt protein were analyzed by Western blotting method.Results: The results showed that 0.75 mg/L Act D-treated NRK52E cell for 20 h was the optimal conditions for establishing NRK52E cell apoptotic model. Different doses of MaFGF (0.01, 0.03, 0.1, 0.3 and 1.0 mg/L) decreased apoptotic rate but enhanced the expression of phosphorylated Akt protein. However, MaFGF’s protection against Act D-induced apoptosis was significantly (p < 0.05) prevented when NRK52E cells were exposed to wortmannin.Conclusion: These results reveal that MaFGF can reduce the level of ActD-induced apoptotic cell death in 20 h, and the protective mechanism of MaFGF may be associated with the activation of PI3K/Akt signaling pathway by up-regulation of expression of phosphorylated Akt protein.Keywords: Modified acidic fibroblast growth factor (MaFGF), Renal injury, Apoptotic death, Actinomycin D (Act D)

Aquaporin 5 (AQP5) promotes the progression and invasion of several cancers, but its role in the tumorigenesis of human gastric carcinoma (GC) has not been clearly defined. Here, we investigated the potential functions of AQP5 in the proliferation and migration of human GC. RT-PCR and western blotting were used to detect the expression of AQP5 in human GC cell lines. Immunohistochemistry was applied to evaluate the expression of AQP5 in human GC tissues and corresponding normal tissues. Following ectopic overexpression of AQP5 or inhibition of AQP5 by its inhibitor, acetazolamide (AZA), cell proliferation and migration of AGS cells were analyzed by MTT assay, colony formation assay, and wound healing assay. Heterogeneous expression of AQP5 mRNA and protein was observed in human GC cell lines MKN45, MKN28, AGS, and SGC7901. AQP5 was up-regulated in GC tissues in comparison to corresponding normal tissues. AQP5 protein was mainly localized in the cell membrane. Overexpression of AQP5 was correlated with enhanced lymph node metastasis. In vitro, overexpression of AQP5 notably enhanced, while inhibition of AQP5 by AZA significantly attenuated the proliferation and migration of AGS cells. Our data indicate that AQP5 may play an important role in the tumorigenesis and progression of human GC and suggest that AQP5 is a potential therapeutic target against GC.

Serum response factor (SRF) is a transcription factor of the MADS box family. To date, DNA binding sites for SRF [serum response elements (SREs)] have been found in the promoters of approximately 50 different genes known to be involved in the regulation cell proliferation, differentiation, and apoptosis. Recent studies have indicated that SRF plays a role in the development of some tumors, including hepatocellular, thyroid, esophageal, and lung carcinomas. However, expression of SRF and its roles in gastric carcinoma are unclear. We found SRF to be highly expressed in human gastric carcinoma as well as ectopic or reduced expression for E-cadherin and β-catenin. Blockage of SRF expression was found to inhibit proliferation, invasion, and migration. We also found that an inhibitor (Y-27632) of Rho-associated coiled kinase (ROCK1), a regulator of actin cytoskeleton that regulates cell adhesion, migration, and motility, suppressed SRF expression as well. These results demonstrate that SRF is involved in the aggressive behavior of gastric carcinoma cells. We also found that the inhibition of ROCK1 by Y-27632 can inhibit the invasion and migration of gastric cells done at least, in part, by attenuating SRF expression.

Supplemental Digital Content is available in the text, B cell acute lymphoblastic leukemia (B-ALL) exhibits phenotypes reminiscent of normal stages of B-cell development. As demonstrated by flow cytometry, the immunophenotypes are able to determine the stages of B cell development. Multicolor flow cytometry (MFC) is more accurate at identifying cell populations. In this study, 9-color panels, including CD10, CD19, CD20, CD22, CD34, CD79a, CD179a, and IgM, which are sequentially expressed during B cell development, were designed to detect the leukemia cell subpopulations in adult B-ALL patients. In 23 patients at diagnosis, 192 heterogeneous subpopulations of leukemia cells were detected. Compared with their counterparts at diagnosis and after the 1st course of induction therapy, the responses of the subpopulations were also heterogeneous. In the CD10+ population, the residual B cell subpopulations in the BCR/ABL+ patients were obviously reduced compared to those in the BCR/ABL− patients. New subpopulations were detected in 22 of 23 patients and were primarily located in the CD34+CD10− populations. Subpopulations of clonal evolution were heterogeneous after induction therapy. Our results suggest that the subpopulations in B-ALL patients should be dynamically monitored by development-associated immunophenotyping before, during, and after induction therapy and to predict the prognosis of the disease.

Recent studies indicate that serum response factor (SRF) is highly expressed in tumors such as hepatocellular, thyroid, esophageal and lung carcinoma. However, the expression and roles of SRF in esophageal squamous cell carcinoma (ESCC) are unclear. In this study, immunohistochemistry was used to compare the expression of SRF in ESCC cases (n=73) and normal controls (n=30). The RNA interference (RNAi) technique was used to knock down the expression of SRF in Eca-109 cells. Cell proliferation, cell cycle stage and invasion were measured with cell counting kit (CCK)-8, flow cytometry and Transwell assays, respectively. Western blotting was used to measure SRF, E-cadherin, β-catenin and cyclin D1 expression in Eca-109 cells treated with siRNA. The study demonstrated that human ESCC has increased expression of SRF. In addition, blocking SRF expression inhibited tumor proliferation and invasion. In conclusion, SRF has the potential to be a new marker for ESCC diagnosis and therapy.

The aim of this study was to separate and amplify CD4(+)CD25(+)Treg cells from splenocytes of sensitized mice. The percentage of CD4(+)CD25(+)Treg cells was detected by flow cytometry in sensitized and normal control mice. CD4(+)T, CD4(+)CD25(+)Treg and CD4(+)CD25(-) T cells were isolated from mouse splenocytes by MACS. CD4(+)CD25(+)Treg cells were expanded in vitro cultures in addition of CD3/CD28 MACSiBead and IL-2. The activity of cells was detected with 0.4 trypan blue staining. The purity of cells after sorting, the main surface marker and the level of Foxp3 were detected by flow cytometry. The results showed that CD4(+)CD25(+)Treg cell proportion was higher in sensitized mice than normal control mice (P0.05). The average purity of CD4(+)CD25(+)Treg cells was 87. The activity of these cells was more than 97, and the expression of Foxp3 in these cells was high. The amplification multiples achieved 42 times after 2 weeks in vitro. The percentage of CD4(+)CD25(+) regulatory T cells was 85.32, and the expression of Foxp3 decreased from (76.92 ± 1.72) to (75.33 ± 2.11) (P0.05). It is concluded that the sorting of CD4(+)CD25(+)Treg cells is isolated successfully by MACS without affecting the vitality of target cells. The amplification of CD4(+)CD25(+)Treg cells is successful in vitro. Expression of surface markers and Foxp3 gene does not obviously change after amplification, so that to establish a practical method to recover and enlarge the amount of CD4(+)CD25(+)Treg cells in good condition.

Docetaxel (DOC) therapy is well tolerated and shows high response rates in patients with hormone refractory prostate cancer (HRPC). There are many reports on the effect of rapamycin (RPM) on the treatment of carcinogenesis. The goal of this study was to test whether RPM could enhance the susceptibility of both androgen-dependent and -independent prostate carcinoma cells to DOC.Prostate cancer (PC) cell lines (LNCap, PC3 and AILNCap) were cultured and treated with RPM and DOC alone or in combination. The effects of therapeutic agents on cells were determined by the WST-1 assay. Apoptosis induction was confirmed by flow cytometric analysis. The apopcyto caspase colorimetric assay kit was applied to measure the activities of caspases 3 and 9. The antitumor effects of RPM and DOC against PC cells were also assessed in nude mice using four randomized groups: control, RPM, DOC and combination drug therapy by measuring tumor size. All the animals tolerated both RPM and DOC without significant weight loss.RPM and DOC caused dosage-dependent growth suppression of PC cells. RPM could increase the susceptibility of PC cells to DOC significantly, and combined treatment with RPM and DOC caused synergistic growth suppression in all examined PC cell lines by isobolographic analysis. Both RPM and DOC significantly induced apoptosis in a dosage-dependent manner. RPM (10 nmol/L), DOC (1 nmol/L), and combined treatment induced apoptosis rate were 8%, 17% and 38%, respectively (the control was 2%). RPM could promote the apoptosis induced by DOC in PC cell lines. Both RPM and DOC significantly increased the caspase activity in a dosage-dependent manner. The relative activities of caspase 9 in control, RPM, DOC and RPM + DOC groups were 0.22 +/- 0.02, 0.36 +/- 0.06, 0.47 +/- 0.05 and 0.84 +/- 0.08, respectively. The relative activities of caspase 3 were 0.21 +/- 0.02, 0.24 +/- 0.05, 0.42 +/- 0.06 and 0.81 +/- 0.09, respectively. Either RPM or DOC alone significantly inhibited the growth of PC cells in nude mice compared to the control. The combination of RPM and DOC produced a significant reduction in tumor volume when compared to RPM or DOC alone. After 5-week treatment, the tumor sizes of LNCap in control, RPM, DOC and RPM + DOC groups were (570 +/- 56) mm(3), (412 +/- 41) mm(3), (425 +/- 46) mm(3) and (221 +/- 26) mm(3), respectively.RPM could significantly increase the susceptibility of both androgen-dependent and -independent PC cells to DOC; the synergy of RPM and DOC was demonstrated. RPM enhanced the DOC-induced upregulation of caspase activity, resulting in an increasing number of cells in sub-G1 phases. The synergy of the combined treatment might be observed in both androgen-dependent and -independent PC cell lines.

The low rate of engraftment in children with beta-thalassemia has seriously restricted the popularity of the hematopoietic stem cell transplantation (HSCT). Panel reactive antibody (PRA) has been regarded as one of the important factors for the success of kidney transplantation. Poly-transfusion before transplantation is associated with the production of PRA. Also PRA is produced in the children with beta-thalassemia major who need poly-transfusion for life. PRA might be one of factors inducing the low rate of engraftment in children with beta-thalassemia. This study focused on observing the effect of PRA on the proliferation, differentiation, apoptosis and necrosis of cord blood CD34(+) cells in vitro by incubating the cord blood CD34(+) cells with serum containing PRA.Seven samples of cord blood were collected and the HLA typing for every sample was done. Seven sera positive for PRA and seven negative sera were selected respectively. Mononuclear cells (MNCs) were obtained by Ficoll-Hypaque density gradient centrifugation. CD34(+) cells were isolated from MNCs by positive selection using an immunomagnetic separation (CD34(+) progenitor cell isolation kit and auto-MACS). The CD34(+) cells of umbilical cord blood were incubated with the serum and complement in the following groups: A (absence of serum), B (presence of PRA positive serum), C (presence of PRA positive serum and complement), D (presence of complement), and E (presence of PRA negative serum). After incubation the samples were centrifuged and the supernatant was collected for LDH detection. At the same time the CD34(+) cells were harvested for assessing the expression of Annexin V and CD95 of the CD34(+) cells by flow cytometry and also for the detection of the DNA synthesis by (3)H-TaR incorporation. Meanwhile the cells were inoculated into the methylcellulose cultural system. The proliferation and hematopoietic potential of the CD34(+) cell of cord blood by the colony formation assay were detected on the day 10.The concentration of LDH in group A was (20.71 +/- 2.81) U/L, which was significantly lower than that in group B (64.28 +/- 5.12) U/L and group C (84.29 +/- 4.99) U/L. The concentration of LDH in group B was significantly lower than that in group C, while there were no significant differences in the concentration of LDH among groups A, D and E (P0.05). The cpm in group A was (22 629 +/- 3288), which was significantly higher than that in group B (4598 +/- 2178) and group C (1626 +/- 1192). And the cpm in group B was significantly higher than that in group C. There were no significant differences in the cpm among groups A, D and E (P0.05). On day 10 of culture, the total colonies, granulocyte-macrophage colony forming unit (CFU-GM), mixed colony forming unit (CFU-GEMM) and erythroid burst colony forming unit (BFU-E) in group A were significantly higher than that in group B and C. The total colonies, CFU-GM and CFU-GEMM in group B were significantly higher than those in group C. No significant differences were found in the total colonies, CFU-GM, CFU-GEMM and BFU-E among groups A, D and E (P0.05). There were no statistically significant differences in the CD95 and Annexin V expression among all the groups (P0.05).PRA could impair the membrane, decrease the DNA synthesis, and inhibit the colony formation of CD34(+) cord blood cells, which could be strengthened by the presence of the complement at the given concentration in our study. PRA had no significant influence on the apoptosis of CD34(+) cells in vitro.

To construct the TRAIL gene eukaryotic expression vector modulated by the human telomerase reverse transcription gene core promoter and to evaluate the expression of the TRAIL gene in ovarian cancer cell line SKOV3 in vitro.The amplified TRAIL gene fragment was subsequently cloned into hTERT promoter-pIRES2-EGFP vector and CMVpromoter-pIRES2-EGFP vector. The hTERT promoter-pIRES2-EGFP-TRAIL and CMVpromoter-pIRES2-EGFP-TRAIL eukaryotic expression vectors were obtained, respectively. The plasmids were transfected into human ovarian carcinoma SKOV3 cell line by lipofeclin mediation and the positive clones were screened by G418. The expression of TRAIL gene was examined by RT-PCR, Western blot, immunocytochemistry and flow cytometry.All the constructed vectors were verified by enzyme digestion. The TRAIL gene of transfected cells was increased significantly (P0.01).An eukaryotic expression vector of the TRAIL gene modulated by the human telomerase reverse transcription gene core promoter has been constructed successfully and its steady expression in human ovarian cancer cell line SKOV3, which will be beneficial to further research into its role in regulating the biological behavior of ovarian cancer cells.

To investigate whether the protective effects of puerarine (Pur) against cerebral ischemia is associated with depressing the extracellular levels of amino acid transmitters in brain of rats.Male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) for 60 min followed by 24 h reperfusion. Pur (50, 100 mg/kg, i.p.) was administered at the onset of MCAO. The infarct rate and edema rate were detected on TTC (2,3,5-triphenyltetrazolium chloride)-stained coronal sections. The extracellular levels of amino acid transmitters were monitored in striatum of rats with ischemic/reperfusion injury using in vivo microdialysis technique. Furthermore, the protective effects of Pur against glutamate-induced neurotoxicity were detected. Glutamate-induced apoptotic and necrotic cells in hippocampus were estimated by flow cytometric analysis of Annexin-V and PI labeling cells.Pur (100 mg/kg) significantly decreased infarct size by 31.6% (P0.05), reduced edema volume (P0.05), and improved neurological functions (P0.05) following MCAO. In these rats, the ischemia-induced extracellular levels of aspartate (Asp), glutamate (Glu), y-aminobutyric acid (GABA), and taurine (Tau) were significantly reduced in striatum of vehicle-treated animals by 54.7%, 56.7%, 75.8%, and 68.1% (P0.01 and P0.05). Pur reduced the peak values of Glu and Asp more obviously than those of GABA and Tau, and the rate of Glu/GABA during MCAO markedly decreased in Pur-treated MCAO rats, compared with the vehicle-treated MCAO rats. Meanwhile, apoptosis and necrosis induced by Glu in cultured hippocampal neurons were significantly reduced after Pur treatment.Acute treatment with Pur at the onset of occlusion significantly depresses ischemia-induced efflux of amino acids, especially, excitotoxicity in the striatum, a mechanism underlying the neuroprotective effect on cellular survival.

Although the BRCA1 tumor suppressor has been implicated in a number of cellular processes, it plays an especially important role in the DNA damage response as a regulator of cell cycle checkpoints and DNA repair pathways. In vivo, BRCA1 exists as a heterodimer with the BARD1 protein, and many of its biological functions are mediated by the BRCA1-BARD1 complex. Here, we show that BARD1 is phosphorylated in a cell cycle-dependent manner and that the hyperphosphorylated forms of BARD1 predominate during M phase. By mobility shift analysis and mass spectrometry, we have identified seven sites of mitotic phosphorylation within BARD1. All sites exist within either an SP or TP sequence, and two sites resemble the consensus motif recognized by cyclin-dependent kinases. To examine the functional consequences of BARD1 phosphorylation, we used a gene targeting knock-in approach to generate isogenic cell lines that express either wild-type or mutant forms of the BARD1 polypeptide. Analysis of these lines in clonogenic survival assays revealed that cells bearing phosphorylation site mutations are hypersensitive to mitomycin C, a genotoxic agent that induces interstrand DNA cross-links. These results implicate BARD1 phosphorylation in the cellular response to DNA damage.

The aim of this experiment was to develop a cytotoxic cancer vaccine (EC109-DC) prepared by fusions of esophageal carcinoma cells with dendritic cells derived from cord blood and to study the biological characteristics and resultant induction of antitumor immunity. CD34+ hematopoietic stem cells were isolated from cord blood using a CD34+ Progenitor Cell Isolation Kit by magnetic cell sorting system (MACS). CD34+ cells were incubated with rhGM-CSF, rhTNF-alpha and rhSCF for 2 weeks as DC (dendritic cells), and then by PEG-3600 to fuse with an esophageal carcinoma cell line. Selection with MACS marked with HLA-DR MicroBeads generated EC109-DC. Phenotypes and proliferation were analyzed by flow cytometry and cell culture in vitro. The lymphocyte proliferation reaction and CTL cytotoxicity were examined by MTT assay. The EC109-DC cells could proliferate slowly in vitro and highly expressed CD80, CD83 and CD86. The lymphocyte proliferation reaction and specific cytotoxicity against EC109 induced by EC109-DC cells were significantly higher than in control groups (p0.05). EC109-DC cells obtained by PEG fusion acquired the immuno-stimulating phenotype and could significantly stimulate the lymphocyte proliferation reaction and CTL activity. The results of this research provide the basis for materials to develop the DC-based vaccine against esophageal carcinoma.

CD45 congenic mice have been used to study stem cell engraftment in the absence of alloreactivity. Recently, impaired engraftment was reported in this model and attributed to weak immune reactivity. We have confirmed that there is indeed low-level reactivity mediated by CD8(+) cells and alpha beta-TCR(+) T cells. B6 (CD45.2) recipients were conditioned with total body irradiation (TBI) and transplanted with increasing doses of B6 (CD45.1) bone marrow cells (BMCs). Although chimerism was present at 1 month in all recipients, durable engraftment did not occur with

Interactions between cytotoxic lymphocytes and their targets require the T cell antigen receptor (TCR) and the integrin lymphocyte function-associated molecule-1 (LFA-1, CD11a/CD18). LFA-1 is not constitutively avid for its counter-receptors, intercellular adhesion molecules (ICAMs)-1 and -2. Cross-linking of the TCR transiently converts LFA-1 to a high avidity state and thus provides a mechanism for regulating cellular adhesion and de-adhesion in an antigen-specific manner. Truncation of the cytoplasmic domain of the beta, but not the alpha, subunit of LFA-1 eliminated binding to ICAM-1 and sensitivity to phorbol esters. Thus, LFA-1 binding to ICAM-1 was found to be regulated by the cytoplasmic domain of the beta subunit of LFA-1.

Histone acetylation is emerging as a major regulatory mechanism thought to modulate gene expression by altering the accessibility of transcription factors to DNA. In this study, treatment of human tumor cells with the histone deacetylase inhibitor, trapoxin (TPX), resulted in selective changes in genes that control the cell cycle. TPX activated p21(waf1) transcription that led to elevated p21(waf1) protein levels in three human tumor cell lines without altering the protein levels of cdk2, cdk4, or cyclin B. In addition, TPX increased cyclin E transcription without increasing the levels of Rb, E2F, dihydrofolate reductase, or glyceraldehyde-3-phosphate dehydrogenase. The elevated levels of p21(waf1) protein led to decreased Rb phosphorylation and cdk2 activity. These effects resulted in G(1) and G(2) cell cycle arrest in H1299 human lung and MDA-MB-435 breast carcinoma cells and apoptosis in A549 lung carcinoma cells. Chromatin immunoprecipitation assays revealed that TPX increased the level of chromatin acetylation associated with histone H3 in the trapoxin-responsive region of the p21(waf1) promoter. This study demonstrates that inhibition of HDAC by TPX increases acetylation of H3-associated chromatin and alters gene expression with marked selectivity.