Role of VEGF-C in lymphangiogenesis and lymphatic metastasis of breast cancer

AIM: To study the inhibitory effect of VEGF-C/Flt-4 system on lymphangiogenesis and lymphatic metastasis of breast cancer. METHODS: Lymphatic endothelial cells (LEC) were cultured in vitro, the effects of VEGF-C and anti-Flt-4 antibody on the proliferation of treated cells were observed. The antisense oligodeoxynucleotides (ASODN) targeting VEGF-C was designed and its effect on VEGF-C gene expression in vitro experiments was observed. The nude mice transplantation tumor model was made and the effects of VEGF-C ASODN on lymphangiogenesis and metastasis in the model were determined. RESULTS: The supernatant of cultured PC3 cells promoted LEC proliferation obviously while the cells treated with anti-Flt-4 antibody were obviously decreased whenever cell counting. The mRNA and protein expression of VEGF-C in MCF-7 cells treated with ASODN were significantly lower than that in control groups in vitro. In vivo ASODN also significantly reduced the VEGF-C mRNA expression detected by RT-PCR. The result of 5-Nase-ALPase enzyme -histochemistry showed that ASODN had obvious inhibitory effect on tumor lymphangiogenesis. Tumor growth velocity in ASODN group was much slower than that in control group. ASODN also inhibited tumor volume and lymphatic metastasis. CONCLUSION: The strong relationships between VEGF-C/Flt-4 system and lymphangiogenesis and lymphatic metastasis of breast cancer have been observed. If the expression of Flt-4 is blocked, the proliferation of LEC induced by tumor cells can be blocked in some degree. ASODN inhibits tumor lymphangiogenesis and lymphatic metastasis by down-regulating VEGF-C expressions.     

《中国蔬菜品种志》

本书由中国农业科学院蔬菜花卉研究所主编,已于2002年9月出版发行。全书分上、下卷,1～6章为上卷,包括根菜类、白菜类、芥菜类、甘蓝类、绿叶菜类及葱蒜类,计2263个品种,1347页;7～12章为下卷,包括瓜类、茄果类、豆类、薯芋类、水生蔬菜类和多年生蔬菜类,计2550个品种,1177页。入志的品种中,地方品种占     

Expression of CXC chemokine receptor 7 in atherosclerotic ApoE-deficient mice and therapeutic impact by atorvastatin

AIM: To evaluate the expression level of CXC chemokine receptor 7 (CXCR7) in atherosclerotic apolipoprotein E-deficient (ApoE^-/-) mice induced by high-fat diet (HFD) and the effects of atorvastatin on it. METHODS: ApoE^-/- male mice (8-week-old) were used and were randomly divided into 3 groups following 1-week normal rodent diet: normal diet control (NDC) group, HFD group and HFD+statins (HFD+Sat) group. HE staining and oil red O staining were used to observe the atherosclerotic lesion burdens in the aortas. The expression of CXCR7 on the aortas was detected by Western blot and immunohistochemistry. The expression of Akt and endothelial nitric oxide synthase (eNOS) in the aorta was determined by Western blot.RESULTS: Few lesions were found in the aortas in NDC group. Apparent atherosclerotic plaque burdens were seen in HFD group and HFD+Sat group, while the atherosclerotic plaque burdens in HFD+Sat group were notably reduced compared with HFD group. The protein levels of CXCR7, eNOS and Akt in aorta in HFD group and HFD+Sat group were significantly decreased compared with NDC group, while those in HFD+Sat group were increased compared with HFD group. The protein level of p-eNOS in the aorta and the concentration of NO in the plasma in HFD group were decreased compared with NDC group and HFD+Sat group. CONCLUSION: In ApoE^-/- mice, HFD increases the lipid level and promotes the development of atherosclerosis by downregulating the expression of CXCR7, Akt and eNOS. Atorvastatin reverses the above effect of hypercholesterolemia on the expression of CXCR7, Akt and eNOS, thus playing the role in treating atherosclerosis.     

Effects of furin inhibitor on metastasis of human breast cancer MCF-7 cells

AIM：To investigate the mechanism underlying breast cancer metastasis and to provide theoretical data for studying the pathogenesis of breast cancer onset and development. METHODS：Human breast cancer MCF-7 cells were treated with different concentrations of furin inhibitor α1-PDX for 48 h. Wound healing assay and Transwell assay were applied to detect the migration and invasion abilities of the MCF-7 cells. The expression of cell migration-associated proteins, including membrane-type 1 matrix metalloproteinase (MT1-MMP), vascular endothelial growth factor (VEGF)-C and VEGF-D, was determined by Western blotting. The protein levels of MMP2 and MMP9 in the supernatant were measured by ELISA. RESULTS：Compared with control group, 200 nmol/L of furin inhibitor exerted significant inhibitory effects on the cell migration (P<0.05). The expression of cell migration-associated proteins MT1-MMP, VEGF-C and VEGF-D was significantly inhibited after treated with α1-PDX (P<0.05). Significant inhibitory effects of α1-PDX on the expression of MMP9 and MMP2 (P<0.05) in the supernatant were observed. CONCLUSION：Furin inhibitor suppresses the metastasis of MCF-7 cells via down-regulating the expression of MMPs and VEGFs.     

Expression of chemokine CXCL17 and its clinical relevance in gastric carcinoma

AIM: To investigate the expression and distribution of chemokine CXCL17 in gastric cancer and its clinical significance.METHODS: The CXCL17 expression was detected by real-time PCR and immunohistochemistry. The correlation between the CXCL17 expression and clinicopathological features was statistically analyzed and Kaplan-Meier survival analysis was used to evaluate the prognosis.RESULTS: A lower expression levels of CXCL17 were observed in the tumor tissues compared with the paired normal tissues (P < 0.05). Down-regulation of CXCL17 was associated with the degree of tumor differentiation and the size of tumor at primary site (P < 0.05). Kaplan-Meier survival analysis showed that increased CXCL17 in the tumor tissues was associated with longer survival time.CONCLUSION: The result might illustrate that CXCL17 acts as a key factor in the prognosis of gastric cancer and is closely associated with the progression of gastric cancer.     

Effects of axitinib on biological behavior of adrenocortical carcinoma cell line SW-13

AIM: To investigate the effects of axitinib on the biological behavior of adrenocortical carcinoma cell line SW-13. METHODS: CCK-8 assay was used to measured the viability of SW-13 cells treated with axitinib at different concentrations. The cell cycle distribution was analyzed by flow cytometry. The apoptotic rate was also analyzed by flow cytometry with Annexin V/PI double staining. Wound healing experiment and Transwell invasion assay were used to observe cell migration and invasion abilities,respectively. The protein levels of vascular endothelial growth factor receptor 2(VEGFR2), extracellular regulated protein kinases 1/2 (ERK1/2) and p-ERK1/2 were determined by Western blot. RESULTS: After treated with axitinib, the viability of SW-13 cells was significantly inhibited, the cell cycle was blocked in G₂/M phase, and the apoptosis rate was increased. The migration and invasion abilities of SW-13 cells were markedly inhibited by axitinib (P<0.01). The protein levels of VEGFR2 and p-ERK1/2 in the SW-13 cells were significantly decreased with axitinib treatment (P<0.01). CONCLUSION: Axitinib inhibits the viability, blocks the cell cycle, promotes cell apoptosis, and inhibits the migration and invasion abilities of SW-13 cells. The mechanism may be related to inhibition of VEGFR2 expression and reduction of ERK1/2 phosphorylation.     

Effects of phillyrin on VEGF and endostatin expression in Lewis lung carcinoma

AIM: To investigate the effects of phillyrin on vascular endothelial growth factor (VEGF) and endostatin expression in lung tumor tissues isolated from Lewis lung carcinoma. METHODS: The expression of VEGF and endostatin in control individuals and the patients with lung cancer was determined by immunohistochemistry. In the animal experiment, 5 groups of animals were examined: control, tumor model, and tumor model with 3 different concentrations of phillyrin treatments. For preparation of transplanted tumor model, Lewis cells were subcutaneously injected into the right limb armpit of the nude mice. After that, phillyrin was administered via oral gavage once daily for 20 d at dose of 5 or 10 g/kg, or twice daily at 10 g/kg. Lung tumor tissues isolated from each group were observed by hematoxylin-eosin staining. VEGF and endostatin expression were examined by immunohistochemistry. RESULTS: VEGF expression was increased in lung tumor tissues as compared with normal and pericarcinous tissues, while endostatin expression was decreased. Phillyrin significantly inhibited the tumor size and tumor tissue density dose-dependently, which was accompanied with a decrease in VEGF expression and an increase in endostatin expression. CONCLUSION: Phillyrin inhibits the development of lung tumor through reducing VEGF expression and increasing endostatin expression.     

Expression of IL-1α and TNF-α modified by Toll-like receptor 4 genetic polymorphism is associated with colorectal carcinoma

AIM: To investigate the association of D299G, T399I and A896G polymorphisms of Toll-like receptor 4 (TLR4) and colorectal carcinoma (CRC). METHODS:
The genotypes of these 3 loci among 268 patients with CRC and 268 healthy controls were determined by polymerase chain reaction-restriction fragment lengthy polymorphism (PCR-RFLP). The protein levels of IL-1α, IL-8, TGF-β and TNF-α in the homogenate of CRC biopsies were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: No significant difference of the genotype frequencies of TLR4 A896G and D299G between the cases and the controls was observed. CT combined TT genotype of T399I was significantly associated with increased CRC risk. The individuals with the T allele of T399I showed a 1.843-fold increase in CRC risk as compared with the C allele. The concentrations of IL-1α and TNF-α in CRC biopsies were significantly elevated in the individuals with the genotype of T399I CT combined with TT as compared with the genotype of CC. CONCLUSION: TLR4 T399I promotes the development of CRC by modifying the expression of IL-1α and TNF-α in CRC tissues.     

Protein expression of EGFR and c-Kit is associated with primary tumor progression in nasopharyngeal carcinoma

AIM: To investigate the protein expression of 2 receptor tyrosine kinases,epidermal growth factor receptor(EGFR) and c-Kit, in non-keratinizing nasopharyngeal carcinoma (NPC) and to analyze the relationship of that with the clinicopathological parameters. METHODS: Ninety-five samples of stage II and III non-keratinizing NPC biopsies were collected from the Department of Pathology, Sun Yat-sen University Cancer Center. The 10% formalin-fixed paraffin-embedded biopsy blocks were re-sectioned. Besides HE routine staining, immunohistochemistry was performed for detecting the expression of EGFR, c-Kit, latent membrane protein 1(LMP1) and Ki-67. RESULTS: The protein expression rates of EGFR and c-Kit were 70.53% (67/95) and 63.16% (60/95), respectively. The expression score of EGFR was positively correlated with that of c-Kit protein (P<0.05). They were both correlated with T staging (EGFR,P<0.05; c-Kit, P<0.01). Furthermore, the staining intensities of EGFR and c-Kit proteins were also correlated with T staging(EGFR, P<0.05; c-Kit,P<0.01). CONCLUSION: The proteins of EGFR and c-Kit are usually expressed in non-keratinizing NPC cells. The immunoreactive scores of these 2 receptor tyrosine kinases are positively correlated with each other. Either the expression rate or the immunoreactive intensity of EGFR and c-Kit proteins is correlated with primary tumor progression. Immunohistochemical staining of EGFR or c-Kit protein in NPC biopsies could be recognized as an insight into further gene analysis for target therapy.     

Expression and significance of stanniocalcin 2 in diabetic retinopathy

AIM To investigate the expression level of stanniocalcin 2 (STC2) in vitreous tissues of rats with diabetic retinopathy (DR), and to explore the relationship between STC2 and vascular endothelial growth factor (VEGF). METHODS Wistar rats were randomly divided into control group and DR group. The DR model was constructed by injection of streptozotocin. RT-qPCR, Western blot and ELISA were used to detect the expression levels of STC2 and VEGF in rat vitreous tissues. Rat retinal ganglion cells were treated with VEGFA, and the expression of STC2 was detected by RT-qPCR, Western blot and ELISA. The retinal ganglion cells were also treated with STC2 protein, and then the expression of VEGF was detected by RT-qPCR, Western blot and ELISA. Co-immunoprecipitation was used to detect the interaction between VEGF and STC2. RESULTS Compared with control group, the mRNA and protein levels of STC2 were significantly increased in vitreous tissues of the rats with DR, and the expression level of VEGF was also significantly increased in DR group (P<0.01). The expression level of STC2 was positively correlated with VEGF expression. VEGF induced the expression of STC2 in rat retinal ganglion cells and promoted its secretion. STC2 protein induced the expression and secretion of VEGF in rat retinal ganglion cells, and VEGF had certain interaction with STC2. CONCLUSION STC2 expression is significantly increased in vitreous tissues of the rats with DR, and is closely related to VEGF.     

Expression of KDM5B gene in breast cancer and its clinical significance

AIM:To investigate the expression of KDM5B gene in breast cancer tissues and its relationship with clinical data and prognosis of the patients. METHODS:Data sets of breast cancer were collected from The Cancer Genome Atlas (TCGA) database, and KDM5B mRNA expression profiles were downloaded. The mRNA expression of KDM5B in breast cancer tissues and adjacent tissues was detected by real-time PCR. The cases were divided into high expression group and low expression group according to the median expression of KDM5B, and the relationship with clinical data and case characteristics were analyzed. The relationship between KDM5B and prognosis of breast cancer patients was analyzed by Kaplan-Meier plotter. RESULTS:The expression of KDM5B in breast cancer tissues was significantly higher than that in normal breast tissues (P<0.01). In TCGA breast cancer data, the expression of KDM5B was significantly correlated with human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), age, histopathological type and lymph node metastasis (P<0.01), but not with progesterone receptor (PR), menopause and distant metastasis. The expression of KDM5B was significantly correlated with HER2, age and lymph node metastasis, but not with ER, PR, menopause, pathological type and distant metastasis. The higher the expression of KDM5B, the shorter the total survival time and the disease-free survival time of breast cancer patients. CONCLUSION:KDM5B is over-expressed in breast cancer tissues and correlated with prognosis of the patients. KDM5B expression is significantly correlated with HER2, age and lymph node metastasis. KDM5B may play an important role in the development of breast cancer.     

Expression of P-gp,HER-2 and microRNA-296 is associated with radiation resistance in esophageal carcinoma

AIM: To investigate whether the expression of P-glycoprotein (P-gp),human epidermal growth factor receptor 2(HER-2)and microRNA-296 is associated with the radiation resistance in esophageal cancer. METHODS: The human esophageal squamous carcinoma cell line Eca109 was divided into control group and treatment group. The cells in treatment group were irradiated by X-ray repetitiously (cumulative radiation dose 60 Gy). The difference of the cell proliferation inhibition between the 2 groups was determined by MTT assay. The expression of P-gp and HER-2 in the cells was detected by immunocytochemical method. The differential expression of microRNA-296 in the cells of the 2 groups were identified by Northern blotting. RESULTS: Compared with control group, a clear radiation resistance and lower growth inhibition were observed in treatment group. The expression of P-gp and HER-2 in treatment group increased significantly than that in control group. No significant difference of microRNA-296 expression between the 2 groups was observed. CONCLUSION: P-gp and HER-2 are relevant with radiation resistance in esophageal cancer. No significant association between microRNA-296 and radiation resistance in Eca109 cells is showed.     

Expression of VEGF and bFGF in rat model of pressure ulcer

AIM:To study the effects of vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) on the molecular pathogenesis of pressure ulcer.METHODS:SD rats were randomly divided into control group and experiment group. The pressure ulcer model was established by magnetic disk circulating compression method. HE staining was used to observe the pathological changes of the skin in the rats. The expression of VEGF and bFGF in the tissues was detected by immunohistochemical method. RESULTS:The expression of VEGF and bFGF in the tissues of rat Ⅲ-degree pressure ulcer was lower than that in the surrounding tissues and normal skin(P<0.01). The changes of VEGF and bFGF were consistent(κ=0.58). CONCLUSION:The expression levels of VEGF and bFGF are decreased in the tissues of rat pressure ulcer, suggesting that they may be the potential key factors in the difficult healing of pressure ulcer.     

Change of serum vascular endothelial growth factor level in patients with bladder transitional cell carcinoma

AIM:To investigate serum vascular endothelial growth factor (VEGF) level in patients with bladder transitional cell carcinoma(BTCC) and its clinical significance.METHODS:ELISA method was used to examine the serum VEGF level in 42 cases of bladder transitional cell carcinoma and in 10 cases of normal people as control. The change of VEGF in blood of the pre-operation and post-operation patients with BTCC was also compared.RESULTS:The VEGF level in blood of the patients was higher than that of the normal people, in spite of pre-operation, post-chemotherapy, and post-operation, but VEGF level decreased obviously after chemotherapy or operation. In addition, the plasma VEGF level was related to the grade and invasion of tumor.CONCLUSION:Detecting serum VEGF level can help us to assess the change of tumor and therapeutic effect.     

4-Hydroxytamoxifen-stimulated activation of ezrin is mediated via G-protein-coupled receptor 30 and promotes human breast cancer MCF-7 cell migration

AIM：To investigate the effect of 4-hydroxytamoxifen (OHT) on the migration of human breast cancer cell line MCF-7. METHODS：The cell migration ability was assayed by scratch healing experiment. The protein expression of Src, p-Src, ezrin and p-ezrin were examined by Western blotting. RESULTS：The results of scratch healing experiment confirmed that both OHT and estradiol (E2) promoted MCF-7 cell migration and the E2-enhanced the cells migration was not inhibited by OHT. The most effective concentration of OHT that enhanced cell migration was 5 μmol/L. Significant promotion of the cell migration was observed at 6 h after OHT treatment. Increased p-ezrin and p-Src expression was observed after treatment with OHT and G-protein-coupled receptor 30 (GPR30) agonists G1. The expression of p-ezrin after OHT treatment was inhibited by G15 (GPR30 blocker) or PP2（Src inhibitor）. CONCLUSION：4-Hydroxytamoxifen promotes MCF-7 cell migration by activation of ezrin via GPR30 and c-Src.     

Effects of propofol on expression of CXCR4/CXCR7 and migration ability of human breast cancer MCF-7 cells in vitro

AIM:To investigate the effects of propofol on the expression of CXC-chemokine receptor (CXCR)4/CXCR7 and migration ability of human breast cancer MCF-7 cells in vitro. METHODS:MCF-7 cells were randomly divided into 4 groups,control group, lipid emulsion group, 3 mg/L and 8 mg/L propofol group. The cell viability was measured by MTT assay. The migration ability was detected by wound-healing assay and Transwell assay. The mRNA level of CXCR4/CXCR7 was detected by RT-qPCR. The protein expression leve of CXCR4/CXCR7 was determined by Western blot. RESULTS:Compared with control group, the scratching healing rates in 3 and 8 mg/L propofol group were decreased (P<0.05), and the chemotactic index was also decreased (P<0.05). The protein expression level of CXCR4/CXCR7 was decreased in 3 and 8 mg/L propofol group(P<0.05). However, both the mRNA level of CXCR4/CXCR7 and the viability of the MCF-7 cells kept no change. CONCLUSION:Propofol down-regulates the protein expression of CXCR4/CXCR7 and inhibits the migration ability of breast cancer MCF-7 cells in vitro.     

TLR7 agonist enhances anti-tumor activity of PBMCs from patient of renal cell carcinoma

AIM:To investigate the effect of Toll-like receptor 7(TLR7) agonist on the anti-tumor activity of peripheral blood mononuclear cells (PBMCs) in the patient with renal cell carcinoma.METHODS:Primary renal cancer cells from the postoperative specimens of the patient were co-cultured with peripheral blood mononuclear cells from the same patient stimulated by TLR7 agonist. The cytokine levels in culture medium were measured by ELISA, and the cell cycle distribution of the renal cell carcinoma cells was analyzed by flow cytometry. The anti-tumor activity of PBMCs was evaluated by[⁵¹Cr] release trial. The protein levels of Skp2 and its downstream pathway molecules were determined by Western blot. RESULTS:TLR7 agonist increased the expression of interferon-γ(IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) in the co-cultured medium, which significantly inhibited the proliferation index of the renal cell carcinoma cells. The cytotoxicity of PBMCs to renal cell carcinoma cells was markedly increased (P<0.05). The protein level of Skp2 in renal cell carcinoma cells was decreased significantly after stimulation, which was consistent with the change of cell proliferation index of renal cell carcinoma cells(P<0.05). The protein level of p27 was increased significantly (P<0.05), which was opposite to the change of Skp2. However, no significant difference in the expression of p21 and p53 was observed. CONCLUSION:TLR7 agonist effectively enhances the anti-tumor activity of PBMCs and results in the growth of renal cell carcinoma cells inhibiting. The mechanism may be relate to the cell cycle arrest by inhibiting the Skp2/p27 pathway.     

Association of tumor budding with tumor-infiltrating lymphocytes and prognosis in breast cancer patients

AIM: To investigate the relationship of tumor budding with clinicopathologic parameters, tumor-infiltrating lymphocytes (TILs) of tumor microenvironment and the prognosis in breast cancer patients.METHODS: A total of 178 HE section samples were collected from the breast cancer patients treated with surgery in the First Affilated Hospital of Jinan University during Jan. 2012 to Dec. 2016. The tumor budding and stromal tumor-infiltrating lymphocytes were observed under light microscope. The correlation of tumor budding with the clinicopathologic status and TILs were analyzed by χ² test. Kaplan-Meier survival analysis and Log-rank test were used to estimate the disease-free survival (DFS) and overall survival (OS).RESULTS: High tumor budding level was associated with more positive lymph nodes, higher grade, and more lymphovascular invasion. In addition, the patients with higher tumor budding level showed fewer TILs, while the patients with lower tumor budding level had more TILs. Furthermore, the patients with higher tumor budding level had a worse disease-free survival and overall survival than those with lower tumor budding level.CONCLUSION: Tumor budding is significantly associated with adverse clinicopathological characteristics of breast cancer and negatively correlated with TILs. Therefore, tumor budding may serve as a potential biomarker to predict the prognosis of breast cancer.