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ABSTRACT: This study aimed to examine the expression of immune suppression factors and the mechanisms of antitumor effects of cord blood dendritic cells (DCs) stimulated by soluble cluster of differentiation 40 ligand (sCD40L) and cytokines in vitro in ovarian cancer patients.
The expression levels of interleukin (IL)-10 and transforming growth factor (TGF)-β messenger RNA in peripheral blood were detected by reverse transcription polymerase chain reaction; expression levels of CD80 and CD86 in DCs stimulated by sCD40L were detected using flow cytometry and confocal laser scanning microscopy.
Expression levels of IL-10 and TGF-β genes in the peripheral blood of ovarian cancer patients were significantly increased compared with patients with benign ovarian tumors (P < 0.05). The expression levels of CD80 and CD86 in DCs cultured in the granulocyte-macrophage colony-stimulating factor + IL-4 + stem cell factor + Flt-3 ligand + sCD40L group were significantly increased compared with those in the control group, as assessed by flow cytometry and confocal laser scanning microscopy (P < 0.05).
A variety of cytokines in combination with sCD40L can promote the proliferation of cord blood-derived DCs and induce their maturation as well as stimulating a specific antitumor response.
OncoTargets and Therapy 01/2013; 6:503-15. · 1.26 Impact Factor
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ABSTRACT: Retinocytoma (RB) is a very common intraocular malignant tumor during infancy. Chemotherapy has gradually been used as the first-line treatment for intraocular RB in recent years. In this study, Livin and PTEN expressions were observed in the RB tissue, along with the growth-inhibiting and apoptosis-induced effects of topotecan (TPT) on RB HXO-Rb44 cell strain. This study aimed to investigate the antigrowth effects of TPT on RB cell strain HXO-Rb44.
Max-Vision(TM) rapid immunohistochemistry was adopted to detect Livin and PTEN expressions in the normal retina and in RB, and their relationship with RB clinicopathologic features was analyzed. Human RB cell strain HXO-Rb44 was cultivated and passaged. MTT method was used to measure the survival rates of HXO-Rb44 cell strains under various TPT concentrations. IC50 values were calculated. Flow cytometry was used to detect the effects of various TPT concentrations on HXO-Rb44 cell apoptosis. Western blotting was used to detect the differences of Livin and PTEN protein expressions during cell apoptosis.
The positive expressions of Livin and PTEN in the RB group were obviously different from those in the normal control group. In RB tissue, Livin expression was relevant to PTEN expression. TPT could significantly induce the occurrence of cell apoptosis and had a dependent relationship with drug concentration. Livin and PTEN expression levels varied with the extension of the effect time of TPT based on Western blotting analysis.
Livin and PTEN have high and low expression levels in the RB tissue, respectively. Both of them have key roles in RB occurrence and development. TPT could induce human RB cell strain HXO-Rb44 cell apoptosis, and its mechanism is associated with the inhibition of Livin and PTEN expressions.
Chinese medical journal 01/2013; 126(2):340-4. · 0.86 Impact Factor
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ABSTRACT: Cochinchina momordica seeds (CMS) have been widely used due to antitumor activity by Mongolian tribes of China. However, the details of the underlying mechanisms remain unknown. In the present study, we found that an EtOAc (ethyl ester) extract of CMS (CMSEE) induced differentiation and caused growth inhibition of melanoma B16 F1 cells. CMSEE at the concentration of 5-200 μg/ml exhibited strongest anti-proliferative effects on B16 F1 cells among other CMS fractions (water or petroleum ether). Moreover, CMSEE induced melanoma B16 F1 cell differentiation, characterized by dendrite-like outgrowth, increasing melanogenesis production, as well as enhancing tyrosinase activity. Western blot analysis showed that sustained phosphorylation of p38 MAP accompanied by decrease in ERK1/2 and JNK dephosphorylation were involved in CMSEE-induced B16 F1 cell differentiation. Notably, 6 compounds that were isolated and identified may be responsible for inducing differentiation of CMSEE. These results indicated that CMSEE contributes to the differentiation of B16 F1 cells through modulating MAPKs activity, which may throw some light on the development of potentially therapeutic strategies for melanoma treatment.
Asian Pacific journal of cancer prevention: APJCP 01/2012; 13(8):3795-802. · 0.66 Impact Factor
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ABSTRACT: To explore the effect of microRNA (miRNA)-mediated p65 gene knockdown on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells.
p65-targeted miRNA plasmid was constructed and transfected into MDA-MB-231 cells via lipofectamine(TM)2000. The expression of p65 gene in the transfected cells at the mRNA and protein levels were detected by RT-PCR and Western blotting, respectively. The cell proliferation and apoptosis were measured by MTT assay and flow cytometry (FCM), respectively. The expressions of apoptosis-related proteins were detected by Western blotting in the transfected cells.
Compared with the negative control group, the expressions of p65 mRNA and protein in p65miRNA-trasnfected cells were significantly down-regulated (P<0.05). MTT assay showed significantly lowered viability of MDA-MB-231 cells after the transfection (P<0.05). FCM showed an increased cell apoptosis rate in p65miRNA group compared with that in the negative control group (P<0.05). Caspase-3 and PARP were both cleaved into their active forms and the expression of these active forms was increased in p65miRNA group.
The miRNA targeting p65 gene can inhibit the proliferation and induce apoptosis of breast cancer cells, and p65 gene might become a new target in gene therapy for human breast cancer.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 10/2011; 31(10):1742-7.
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Zhi-Yu Wang,
Takanori Miki,
Yan Ding,
Shi-Jie Wang,
Yu-Huan Gao,
Xiao-Ling Wang,
Yu-Hua Wang,
Toshifumi Yokoyama,
Katsuhiko Warita,
Ken-ichi Ohta,
Shingo Suzuki,
Taira Ohnishi,
Takashi Obama,
Kuldip S Bedi,
Yoshiki Takeuchi, Bao-En Shan
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ABSTRACT: Apolipoprotein E (apoE) is one of the major transporters of cholesterol in the body and is essential for maintaining various neural functions in the brain. Given that hypercholesterolemia is a risk factor in Alzheimer's disease (AD), it has been suggested that altered cholesterol metabolism may be involved in the development of the pathogenesis, including neural degeneration, commonly observed in AD patients. Neurotrophic factors and their receptors, which are known to regulate various neural functions, are also known to be altered in various neurodegenerative diseases. We therefore hypothesized that cholesterol metabolism may itself influence the neurotrophin system within the brain. We decided to investigate this possibility by modulating the amount of dietary cholesterol given to apoE-knockout (apoE-KO) and wild-type (WT) mice, and examining the mRNA expression of various neurotrophin ligands and receptors in their hippocampal formations. Groups of eight-week-old apoE-KO and WT mice were fed a diet containing either "high" (HCD) or "normal" (ND) levels of cholesterol for a period of 12 weeks. We found that high dietary cholesterol intake elevated BDNF mRNA expression in both apoE-KO and WT mice and TrkB mRNA expression in apoE-KO animals. On the other hand, NGF and TrkA mRNA levels remained unchanged irrespective of both diet and mouse type. These findings indicate that altered cholesterol metabolism induced by HCD ingestion combined with apoE deficiency can elicit a differential response in the various neurotrophin ligand/receptor systems in the mouse hippocampus. Whether such changes can lead to neural degeneration, and the mechanisms that may be involved in this, awaits further research.
Metabolic Brain Disease 09/2011; 26(3):185-94. · 2.20 Impact Factor
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ABSTRACT: to observe the effect of ICA on the immunosuppressive and bone-marrow-suppressive mice after chemotherapy, and explore the machanism of hematopoietic and immunologic function in mice accentuated by ICA.
mice were injected Cy intraperitoneally except control group , then randomly divided into mode group(saline), positive (Shenqi, 1 mL/d)group, ICA groups(150, 80, 40 mg/kg.d). All mice were treated respectively for 10 successive days. The pathological changes of thymus were observed by HE staining. Killing activity of peritoneal macrophage were measured by LDH kits and its production of TNF-α and IL-12 was measured by ELISA kits. Population of white blood cells (WBC), red blood cell (RBC) and platelet (PLT) in the peripheral blood were detected with automated blood cell counter (ABCC). Hemogram of peripheral blood and bone marrow morphology were observed under the microscope.
ICA could protect the thymus and bone marrow from damage. The proliferation of lymphocyte and killing activity of macrophage cells in ICA treatment groups was all enhanced, moreover, the population of WBC, RBC and PLT were increased significantly.
ICA can improve the state of immunosuppressive and myelosuppressive mice caused by Cy thus could alleviate the side effect of chemotherapy effectively.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 10/2010; 26(10):976-9.
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ABSTRACT: Currently, no satisfactory biomarkers are available to screen for esophageal squamous cell carcinoma (ESCC). The goal of this study was to find biomarkers and establish a serum protein fingerprint model for early diagnosis of ESCC using the ClinProt protocol of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Serum samples were collected from 62 patients with ESCC, nine patients with esophageal adenocarcinoma (EA) and 38 healthy individuals. Proteomic spectra of mass to charge ratio (m/z) were generated following the application of plasma to weak cationic-exchanger magnetic beads (WCX-MB). The spectral data were analyzed using a support vector machine, and potential biomarkers were chosen for system training and used to construct diagnostic models.
Three differential patterns were established using MALDI-TOF MS. Pattern 1, consisting of 11 protein peaks, separated ESCC patients from the healthy individuals with a sensitivity of 90.0% and a specificity of 88.4%. Pattern 2, consisting of eight protein peaks, separated ESCC in stage I and stage II from stage III and stage IV with a sensitivity of 92.9% and a specificity of 82.3%. Pattern 3, consisting of seven protein peaks, separated ESCC from EA with a sensitivity of 91.3% and a specificity of 80.0%.
These results suggested that MALDI-TOF MS combined with MB separation yields significantly higher sensitivity and specificity for the detection of serum protein in patients with ESCC.
Clinical Chemistry and Laboratory Medicine 03/2010; 48(6):855-61. · 2.15 Impact Factor
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ABSTRACT: Background and Objective: Thrombospondin-1(TSP1) is an inhibitor of angiogenesis and its promoter hypermethylation has been found resulting in gene silencing in some primary human carcinomas. This study was to investigate the promoter methylation of TSP1 and its correlation with TGF-beta1 level and T cell immunity in gastric cardia adenocarcinoma (GCA). Methods: Methylation specific polymerase chain reaction (MSP) approach and immunohistochemistry method were used to examine the methylation status of the 5' CpG island and expression of TSP1 protein, respectively. The level of TGF-beta1 was measured by ELISA and T cell immunity of GCA by flow cytometry analysis. Results: TSP1 methylation frequency was significantly higher in tumor specimens than in corresponding normal tissues (35.4% vs. 3.1%, P<0.001) and significantly higher in Stages III and IV tumor tissues than in Stages I and II tumor tissues (P<0.05). TSP1 protein expression was significantly lower in the tumor tissues than in corresponding normal tissues (P<0.05) and statistically correlated with its methylation status (P<0.01). The total level of TGF-beta1 was significantly higher in the GCA patients than in healthy controls(P<0.05) and significantly higher in Stages III and IV GCA patients than in Stages I and II GCA patients (P<0.05). The level of active TGF-beta1 was significantly higher in the GCA patients with hypermethylation of TSP1 than in the GCA patients without methylation of TSP1(P<0.05), but there was no statistical difference(P>0.05). The function of T cell immunity was significantly different between the GCA patients with hypermethylation of TSP1 and those without methylation of TSP1 (P<0.05). Conclusion: Promoter hypermethylation of TSP1 may play an important role in the development of GCA and reflect the biological behaviours of GCA.
Ai zheng = Aizheng = Chinese journal of cancer 12/2009; 28(12):1298-303.
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ABSTRACT: Study the immunoregulatory function of rat bone marrow mesenchymal stem cells on spleen mononuclearcell (MNC) in vitro, and investigate the immunological regulation mechanism initially.
MSCs from bone marrow of rat were isolated and cultured in vitro. The morphological charactcrization of MSCs was observed after Wright-Giemsa's staining and the typical molecule on the cell surface were identified by flow cytometry(FCM). ConA was taken as stimuli origin. Effect of MSCs on proliferation of spleen MNC was determined by MTT method.Effect of MSCs on IL-2 and IL-10 secretion of spleen MNC was determined by ELISA.Analyze the effect of MSCs on MNC cell cycle distribution by FCM.Study the effect of MSCs on p27 and cyclin E expression of MNC by FCM. Investigate the effect of MSCs on spleen MNC killing colon26 and H22 by LDH release method.
Compared with positive control, proliferation of spleen MNC decreased obviously, when cultured with different number of MSCs (P<0.01), and the proliferation of MNC was inhibited markedly in a dose-dependent manner (P<0.01). After treated by MSCs, the level of IL-2 secreted by spleen MNC decreased, but the level of IL-10 increased markedly (P<0.01), and this function was also in a dose-dependent manner. MSCs could arrest spleen MNC to G(0);/G(1); phase (P<0.01), and inhibit spleen MNC entering S phase, when stimulated by ConA. MSCs could upregulate p27 expression and downregulate cyclin E expression of spleen MNC (P<0.05).After culturing with MSCs, ability of spleen MNC killing colon26 and H22 was impaired, compared with group cultured alone (P<0.05).
MSCs could inhibit spleen MNC proliferation in vitro, and this effect was related to that MSCs could alter cytokine secretion and regulate cell cycle distribution of MNC.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 12/2009; 25(12):1119-22.
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ABSTRACT: To investigate the immune mechanisms for Periplocin from Cortex Periplocae (CPP) in tumor-bearing mice.
H(22) tumor-bearing model BALB/c mice were applied to evaluated in vivo immunoregulatory effect of CPP. The influence of different dose CPP (0.25, 0.50 and 1.00 mg/kg) on immune organs in tumor-bearing mice were observed. T cell subsets of mice spleen were detected by flow cytometry. MTT assay was used to determine the influence of CPP on lymphocyte proliferation of mice spleen stimulated by ConA. The levels of TNF-alpha, IL-2 and IL-12 in serum from mice were detected by means of ELISA.
Thymus index and spleen index of H(22) tumor-bearing model control mice became less than that of normal mice (P<0.05). Compared to both model and normal control groups, thymus index and spleen index of H(22) tumor-bearing mice treated with CPP increased obviously (P<0.05). CPP had no influence on the number of CD8(+) T cells, but up-regulated markedly the number of CD3(+), CD4(+) T cells and the ratio of CD4(+)/CD8(+) in tumor-bearing mice. In CPP-treated mice, the percentage of CD3(+), CD4(+) T cells were not different from normal mice (P<0.05), the ratio of CD4(+)/CD8(+) was higher than that of normal mice (P<0.05). CPP enhanced obviously lymphocyte proliferation of mice spleen induced by ConA, the SI scores were even higher than that of normal mice. The levels of TNF-alpha, IL-2 and IL-12 in serum from CPP-treated mice, increased significantly compared to model control group (P<0.05) in a dose-dependent manner, were similar to or higher than that of normal mice.
CPP protected immune organs of tumor-bearing mice, increased obviously the percentage of CD4(+) and CD4(+)/CD8(+) among the T cell line, and enhanced lymphocyte proliferation of mice spleen significantly, stimulated the production of TNF-alpha, IL-2 and IL-12. The results suggested that CPP possessed potent immunoregulatory effect.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 10/2009; 25(10):887-90.
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ABSTRACT: To examine the in vivo anti-metastatic effect of enhanced expression of CD40L cDNA in murine ovarian cancer OVHM cells (CD40L-OVHM) injected into the spleen on liver metastasis in mice.
OVHM cells were inoculated into the spleen of 6 to 8 week-old female B6C3F1 (C57BL/6N x C3H/He) mice. The established liver metastasis was identified by histopathology (HE staining). OVHM cells, DNA-pMKITneo-OVHM cells or CD40L-OVHM cells were inoculated into the spleen of female B6C3F1 mice and the expressions of CD11c in splenic cells were detected by flow cytometry. The specific cytotoxicity of splenic cells was detected by MTT assay, and the serum cytokines of IFN-gamma, TNF-alpha, IL-12, IL-4 and IL-10 of the mice were measured by enzyme linked immunoabsorbent assay. The liver metastases and the survival time of the mice were also recorded.
The mouse models with liver metastasis by injecting tumor cells into the spleen of mice were established. The expression of CD11c and the specific killing rate in CD40L-OVHM cells group was significantly higher than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group. The expressions of IFN-gamma, TNF-alpha and IL-12 in the CD40L-OVHM cells group were much more increased than OVHM cells group and DNA-pMKITneo-OVHM cells group, but the expressions of IL-4 and IL-10 in the CD40L-OVHM cells group were decreased significantly (p < 0.05). The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lower than those of DNA-pMKITneo-OVHM cells group and OVHM cells group. The survival time of mice in CD40L-OVHM cells group was also significantly longer than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group (P < 0.05).
The data directly demonstrate that the expression of CD40L in ovarian cancer cells (CD40L-OVHM) can enhance the proliferation and differentiation of dendritic cells in the spleen and induce specific cytotoxic effect of T cells in the spleen, and may regulate the immune function of peripheral blood cells and the immune balance between Th1 cells and Th2 cells, which maybe the possible mechanism induced by CD40L in mice inhibiting the development of liver metastasis.
Zhonghua zhong liu za zhi [Chinese journal of oncology] 07/2009; 31(7):505-9.
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ABSTRACT: The Wnt/beta-catenin signaling pathway plays an important role in the development and progression of human cancers, especially in colorectal carcinomas. This study was to analyze the inhibition effect of periplocin extracted from cortex periplocae (CPP) on proliferation of human colon carcinoma cell line SW480 and the underlying mechanism.
Cell proliferation of SW480 cells was measured by MTT assay. Cell apoptosis and cell cycle were analyzed by flow cytometry. Protein expression of beta-catenin in total cell lysates, cytosolic extracts, and nuclear extracts were detected by Western blot. Binding activity of the T cell factor (TCF) complex in nucleus to its specific DNA binding site was measured by electrophoretic mobility shift assay (EMSA). Expressions of beta-catenin, survivin, c-myc and cyclin D1 mRNA in cells after the treatment with CPP were detected by semi-quantitative RT-PCR.
CPP significantly inhibited the proliferation of SW480 cells in a time-and dose-dependent manner (P<0.01). CPP (0.5 microg/mL) also caused G0/G1 cell cycle arrest of SW480 cells and induced cell apoptosis (P<0.05). Compared to untreated control cells, after the treatment with CPP, the protein levels of beta-catenin in total cell lysates, cytosolic extracts, and nuclear extracts were reduced (P<0.01); the binding activity of the TCF complex in nucleus to its specific DNA binding site was suppressed; mRNAs of the downstream target genes survivin, c-myc and cyclin D1 were decreased (P<0.01) while beta-catenin mRNA remained unchanged.
CPP could significantly inhibit the proliferation of SW480 cells, which may be through down-regulating the Wnt/beta-catenin signaling pathway.
Ai zheng = Aizheng = Chinese journal of cancer 06/2009; 28(5):456-60.
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ABSTRACT: Celecoxib can inhibit cell proliferation, regulate cell cycle and induce apoptosis, but the underlying mechanisms are still unclear. This study was to investigate the association between the NF-kappaB (kappaB) pathway and the apoptosis of breast cancer cell line MDA-MB-231 induced by celecoxib.
The expression of cyclo-oxygenase-2 (COX-2) mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Cell proliferation and cell cycle were detected by MTT and flow cytometry (FCM), respectively. The protein expressions of caspase-3 and p65 in MDA-MB-231 cells were detected by western blot.
After incubation with different concentrations of celecoxib for 48 h, COX-2 mRNA expression in MDA-MB-231 cells was decreased in a dose-dependent manner compared with untreated cells (P<0.05). Proliferation of MDA-MB-231 cells was reduced drastically in a dose-and time-dependent manner after celecoxib treatment (P<0.05). Combination of prostaglandin E2 (PGE2) and celecoxib exerted similar inhibition effect to celecoxib alone on cell growth (P>0.05). High-dose celecoxib induced an increase in the percentage of G0/G1 phase cells accompanied by the change in DNA ploidy. The cellular caspase-3 level was enhanced whereas the p65 level was decreased in celecoxib-treated MDA-MB-231 cells after 24 h in comparison to those in the control cells.
Celecoxib could inhibit MDA-MB-231 cell proliferation and promote cell apoptosis by down-regulating the NF-kappaB signaling pathway.
Ai zheng = Aizheng = Chinese journal of cancer 06/2009; 28(6):569-74.
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ABSTRACT: The Wnt signaling pathway plays a pivotal role in cellular developmental processes and human carcinogenesis. The aim of this study was to investigate the effects of quercetin on the growth of the colon carcinoma cell line and the regulation effect of quercetin on the Wnt/beta-catenin signaling pathway.
MTT assay was used to determine the reduction of cell viability of quercetin on SW480 cells and clone 26 cells. The apoptotic rate and cell-cycle analysis after treatment with quercetin was determined by flow cytometry. Effects of quercetin on mRNA expression of cyclin D(1) and survivin were detected by semiquantitative RT-PCR. After treatment with quercetin, the protein expression of cyclin D(1) and survivin in SW480 cells was analyzed by Western blot analysis. We built a Wnt/beta-catenin signaling pathway reporter gene model. The regulation effect of quercetin on the Wnt/beta-catenin signaling transcription was investigated by using this reporter gene model.
Quercetin reduced cell viability in a dose- and time-dependent manner in SW480 and clone 26 cells. The percentages of SW480 cells and clone 26 cells at G(2)/M phase were increased significantly after treatment with 40 approximately 80 micromol/L quercetin for 48 hours. Quercetin induced the apoptosis of SW480 cells in a dose-dependent manner at the concentration of 20, 40, 60, anf 80 micromol/L. However, quercetin only induced the apoptosis of clone 26 cells at the concentration of 80 micromol/L. Quercetin downregulated transcriptional activity of beta-catenin/Tcf in SW480 cells transiently transfected with the TCF-4 reporter gene. Within 24 hours of treatment, a 160-mumol/L concentration of quercetin reduced beta-catenin/Tcf transcriptional activity by about 18-fold. Cyclin D(1) and the survivin gene were downregulated markedly by quercetin in a dose-dependent manner at both the transcription and protein expression levels.
The results indicate that the molecular mechanism underlying the antitumor effect of quercetin in SW480 colon cancer cells is related to the inhibition of expression of cyclin D(1) and survivin as well as the Wnt/beta-catenin signaling pathway. Therefore, the Wnt/beta-catenin signaling pathway could be qualified as one of the promising targets for innovative treatment strategies of colorectal cancer.
Cancer Investigation 06/2009; 27(6):604-12. · 1.85 Impact Factor
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ABSTRACT: Although non-steroidal anti-inflammatory drugs (NSAIDs) have been demonstrated to have cancer-preventive effects and induce apoptosis of cancer cells, the mechanism of their effects is not clearly known. We studied the mechanism in human esophageal cancer cell line TE13. The esophageal squamous cell carcinoma cell line TE-13 was cultured with NS-398 at different concentrations or for different times. Proliferation and apoptosis were measured by MTT reduction and flow cytometry. Prostaglandin F(1alpha) was determined with radioimmunoassay. Expression of COX-2 mRNA was measured by RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic IkappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. NS-398 significantly inhibited cell proliferation and induced apoptosis at concentrations of 0.001, 0.01, 1, and 100 micromol/L. NS-398 dose-dependently decreased the levels of COX-2 mRNA, COX-2 protein, nuclear NF-kappaB protein and production of PGF(1alpha) and increased the cytoplasmic IkappaB protein. In conclusion, NS-398 inhibits the proliferation of, and induced apoptosis in, the cultured TE-13 SCC cell line. These changes correlate with a reduction in COX-2 mRNA and protein expression, prostaglandin synthesis, an inhibition of NF-kappaB nuclear translocation, and an increase in cytoplasmic IkappaB.
Cancer Investigation 02/2009; 27(1):17-23. · 1.85 Impact Factor
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ABSTRACT: To study the antitumor effect of IL-23 in mouse mammary cancer and the change of its immune function.
IL-23/MA-891, LXSN/MA-891 and the parental MA-891 cells were inoculated in the subcutaneous tissues of mice, respectively. The mouse models were used to observe the tumorigenic activity of IL-23/MA-891 cells. On the thirtieth day, the spleens and tumors of the mice in three groups were extracted and then IFN-gamma, TNF-alpha, IL-12 and IL-4 were detected by ELISA. The expression of MHC-I, MHC-II, CD80 and CD86 in tumor cells, the ratio of positive cells of CD11c, and the selection of CD4 and CD8 in the spleens were detected by flow cytometry, respectively. CD4 and CD8 in three groups were immuno-stained and then they were examined by microscope.
The tumor in the mouse inoculated with IL-23/MA891 cells developed much more slowly than that in the other two groups. Meanwhile, the spleen cells of this group secreted higher IFN-gamma, TNF-alpha and IL-12 than the other two groups (P<0.01), but the secretion of IL-4 in the three groups had no difference (P>0.05). In IL-23/MA891 group, CD4+, CD8+ lymphocytes, CD11c+ cells in spleens and infiltration of CD4+, CD8+ lymphocytes in tumor tissues were also markedly higher than those in LXSN/MA-891 and MA-891 groups (P<0.01). The expression of MHC-I, MHC-II, CD80 and CD86 in IL-23/MA-891 groups was also higher than that in the other two groups (P<0.01).
In the mice the antitumor effect of the mammary cancer cells transferred by IL-23 gene is obvious, which can enhance the cellular immune function and play an important role in inhibiting the growth of tumor.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 10/2008; 24(9):853-6.
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ABSTRACT: To investigate the effects of IL-18 gene transfection on proliferation, apoptosis and tumorigenesis of mouse ovarian cancer cell line OVHM.
OVHM were transfected with retrovirus encoding IL-18 gene (OVHM/IL-18). The wild OVHM and OVHM transfected with vector without IL-18 gene (OVHM/LXSN) were enrolled as the controls. The proliferation, cell cycle and apoptosis were measured by MTT and FCM respectively. The nude mice were subcutaneously inoculated with the three different cells respectively. The observation of transplanted tumor's growth and calculation of tumorigenesis ratio was made. The expression of IL-18 mRNA in tumor tissues was detected by RT-PCR, and the apoptosis of tumor cell in tissue was analyzed by electron microscope and FCM.
The three cells cultured in vitro showed no apoptotic peaks, and no differences in proliferation and cell cycle (All P>0.05). Following inoculation, the ratios of tumorigenesis were similar in all the three groups. But in OVHM/IL-18 group, the latent period of tumorigenesis was longer with slower tumor growth rate and positive expression of IL-18 mRNA in tumor tissue. It was also observed that in the tumor cells from nude mice inoculated by OVHM/IL-18, the cells in phase of G0/G1 increased with typical morphology of apoptosis, and those in S phase decreased with decreased proliferation index and increased apoptosis index (All P<0h01).
Although IL-18 gene has no cytotoxic effects in vitro, its inhibiting effects on tumorigenesis of OVHM in vivo were exerted by interdicting cell cycle and accelerating apoptosis of tumor cells.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 07/2008; 24(6):577-80.
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ABSTRACT: Our previous study showed that aspirin induced apoptosis of esophageal cancer cells in vitro by inhibiting the pathway of NF-kappaB downstream regulation of cyclooxygenase-2. The purpose of this study was to determine if similar changes occurred in vivo in the tumors of patients with SCC of the esophagus who were given a preferential COX-2 inhibitor, meloxicam. Fifty-three patients who had an esophagectomy for SCC were allocated randomly to either a Treatment group (n = 25) or a control group (n = 28). Patients in the Treatment group were given 7.5 mg/day of meloxicam, for between 10 and 14 days before surgery. Patients in the control group did not take any type of NSAID during this time interval. Samples of the tumor taken from the resected specimens were collected. Proliferation and apoptosis were measured by flow cytometry. The concentration of 6-keto-prostaglandin F(1)alpha in cancer tissue was determined by radio-immuno-assay. Expression of COX-2 mRNA was measured with RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic I kappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. There were significantly more apoptotic cells in the tumors of patients who were using meloxicam. It also decreased the levels of COX-2 mRNA, COX-2 protein and nuclear NF-kappaB protein and increased the cytoplasmic I kappaB protein in the cancer. We conclude that meloxicam induces apoptosis in SCC of the esophagus in vivo by inhibiting the pathway of NF-kappaB downstream regulation of COX-2.
International Journal of Cancer 05/2008; 122(7):1639-44. · 5.44 Impact Factor
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ABSTRACT: To examine whether the enhanced expression of CD40L cDNA on murine ovarian cancer (OVHM) cells could induce the secretion of Th1 cytokines from dendritic cells (DC).
OVHM cells were transfected with the full-length mouse CD40L cDNA by lipofectamine 2000 and then G418 resistant cells as positive cells were selected. They were examined for their expression of CD40L with flow cytometry. Bone marrow cells were firstly depleted of erythrocytes, macrophages, T and B cells with PE-conjugated magnetic beads, and then cultured in 10% FCS RPMI 1640 medium supplemented with recombinant mouse GM-CSF and IL-4 for 10 days. PKH67-labeled tumor cells were cultured with DC, and then the stained cells were analyzed for the expression of MHC-I, MHC-II, CD80, CD86, CCR7 in DC with flow cytometry. The expression of p40, p19, p35, p28, EBI3 subunits, IL-18, IFN-gamma, Mig gene in cocultured DC-tumor cells were detected by RT-PCR.
The CD40L cDNA was successfully transfected into OVHM cells. Bone marrow-derived DCs, when cultured with CD40L/OVHM, formed clusters with the tumors and showed an upregulated expression of MHC- I, MHC-II, CD80, CD86, CCR7. Expression of the IL-12, IL-23, IL-27, IL-18, interferon-gamma (IFN-gamma) and Mig (monokine induced by IFN-gamma) genes was induced in the DCs that were cultured with CD40L/OVHM but not with OVHM cells.
These data directly showed that the expression of CD40L on ovarian cancer cells facilitates the interaction between DCs and tumors, enhances the maturation of DCs, induces secretion of Th1 cytokines, especially for IL-12, IL-23 and IL-27, which maybe one of the possible antitumor mechanism for CD40L-transfected ovarian cancer cell line.
Zhonghua zhong liu za zhi [Chinese journal of oncology] 04/2008; 30(3):174-8.
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ABSTRACT: To construct the eukaryotic expression vector of human spleen tyrosine kinase (Syk) and study the effect of Syk on MHC-I expression of human breast cancer cells.
A CDS fragment of human syk was obtained by RT-PCR from human breast cancer cells MDA-MB-468 and then the fragment was cloned into the expression vector pcDNA3.1D/V5-His-TOPO. After restriction endonuclease digestion, PCR and DNA sequencing confirmation, the recombinant hsyk expression plasmid was transfected into human breast cancer cells MDA-MB-231 by using lipofectamine protocols. After G418 selection, the cells stably expressed Syk. The expression of Syk, MHC-I and ICAM-I in the transfected cells was detected by Western blot, RT-PCR and flow cytometry (FCM).
The eukaryotic expression plamid pcDNA3.1D/V5-His-TOPO/hsyk was constructed and it was expressed in the human breast cancer cells MDA-MB-231. The MDA-MB-231/Syk cells highly expressed MHC-I and ICAM-I.
The successful construction of eukaryotic expression plamid pcDNA3.1D/V5-His-TOPO/hsyk and its expression in eukaryotic cells will be useful for further study of tumor immunity.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 02/2008; 24(1):3-5.
