Correlation of c-MET and CXCR4 proteins and microvessel density with liver metastasis in colorectal cancer / 肿瘤研究与临床

Objective:To explore the correlation of c-MET and CXCR4 proteins and microvessel density (MVD) with liver metastasis in colorectal cancer tissues.Methods:A total of 40 colorectal cancer tissue samples and 10 paracancerous (5 cm from the edge of the tumor) normal colorectal tissue samples were collected from March 2015 to December 2020 in Shanxi Traditional Chinese Medical Hospital. Among 40 patients with colorectal cancer, 15 patients had liver metastasis. Immunohistochemistry was used to detect c-MET protein, CXCR4 protein and CD34-labeled MVD in various tissues, and the relationships between them and liver metastasis and between the three were analyzed.Results:The positive rates of c-MET protein [72.5% (29/40) vs. 30.0% (3/10)], CXCR4 protein [47.5% (19/40) vs. 10.0% (1/10)] and MVD (20.1±5.2 vs. 11.5±4.3) in colorectal cancer tissues were higher than those in paracancerous tissues, and the differences were statistically significant (all P < 0.05). The positive rates of c-MET protein [86.7% (13/15) vs. 64.0% (16/25)] and CXCR4 protein [66.7% (10/15) vs. 36.0% (9/25)] in colorectal cancer liver metastasis group were significantly higher than those in non-liver metastasis group, and the differences were statistically significant (both P < 0.05). MVD in colorectal cancer liver metastasis group was significantly higher than that in non-liver metastasis group (21.5±5.3 vs. 12.4±5.7), and the difference was statistically significant ( P < 0.05). In colorectal cancer tissues, c-MET protein expression was positively correlated with CXCR4 protein expression ( r = 0.568, P < 0.05), and MVD in c-MET-positive patients or CXCR4-positive patients was higher than that in negative ones (both P < 0.05). Conclusions:The c-MET protein, CXCR4 protein and MVD may play important roles in the liver metastasis of colorectal cancer. The three indicators can provide a certain reference for the early diagnosis and prognosis prediction of liver metastasis of colorectal cancer.

[Expression and significance of chemokine CXCL12 and receptor CXCR4 in adenomyosis].

Objective: To observe the expression, correlation and significance of chemokine (C-X-C motif) ligand 12 (CXCL12) and chemokine (C-X-C motif) receptor 4 (CXCR4) in endometrium and myometrium of adenomyosis. Methods: Totally 38 patients were selected in this study, who underwent hysterectomy for adenomyosis at Beijing Obstetrics and Gynecology Hospital from October 2017 to December 2018 as the adenomyosis group, and, in the same period, selected 31 patients with cervical intraepithelial neoplasia Ⅲ or cervical cancer undergoing hysterectomy served as control group. The expression levels of mRNA and protein for CXCL12, CXCR4 in the endometrium and myometrium of the two groups were detected by immunohistochemistry and real-time PCR. Results: (1) The protein levels of CXCL12 and CXCR4 in endometrium in uterus with adenomyosis (0.229±0.025 and 0.226±0.016) were significantly higher than those in endometrium in uterus without adenomyosis (0.153±0.018 and 0.178±0.026); compared with each other, the differences were statistically significant (all P<0.05). And the expressions of CXCL12 and CXCR4 proteins in uterine myometrium of adenomyosis were 0.222±0.045 and 0.126±0.058, respectively, which were higher than those in the control group (0.091±0.029 and 0.099±0.020); compared with each other, the differences were statistically significant (all P<0.05). (2) The expression levels of CXCL12 and CXCR4 mRNA in endometrium of patients with adenomyosis were 6.31±0.12 and 8.49±0.21, respectively, which were higher than those in the control group (1.23±0.10 and 1.36±0.13); compared with each other, the differences were statistically significant (all P<0.05). Moreover, the expression levels of CXCL12 and CXCR4 mRNA in myometrium of patients with adenomyosis were 9.11±0.12 and 8.45±0.16, respectively, which were higher than those in the control group (1.18±0.08 and 1.46±0.13); compared with each other, the differences were statistically significant (all P<0.05). (3) In endometrium and myometrium of uterus with adenomyosis, CXCL12 and CXCR4 mRNA expression levels were positively associated (r=0.478, 0.542, all P<0.05). Conclusions: The levels of CXCL12 and CXCR4 in the endometrium and myometrium of adenomyosis are increased and positively correlated. The two chemokine may be involved in the development of adenomyosis.

Clinical Significance of C-X-C Motif Chemokine Receptor 4 and Integrin αvß6 Expression in Breast Cancer.

PURPOSE: C-X-C motif chemokine receptor 4 (CXCR4) and integrin αvß6 play important roles in the malignant progression of multiple cancers. However, it remains unclear whether the expression of one or both proteins in breast cancer (BC) is of clinical significance. In this study, we investigated the expression of CXCR4 and integrin αvß6 in BC tissues and their correlation with clinicopathological characteristics, including survival. METHODS: CXCR4 and αvß6 expression in 111 BC tissues was examined by immunocytochemistry. Correlations between the expression of the 2 proteins and patient clinicopathological characteristic were investigated using the Kaplan-Meier method and the Cox proportional hazards model. RESULTS: CXCR4 and αvß6 were overexpressed in BC tissue compared with normal breast tissue. Overexpression of both molecules was related to lymph node status (p = 0.013 and p = 0.022, respectively). αvß6 overexpression was also associated with tumor size (p = 0.044). A positive correlation was detected between the expression of CXCR4 and αvß6 (r = 0.649, p = 0.001), and co-overexpression of both molecules was associated with tumor size (p = 0.018) and lymph node metastasis (p = 0.015). Kaplan-Meier analysis revealed that overexpression of CXCR4, αvß6, or both molecules was associated with short overall survival (OS; p < 0.001, p < 0.001, and p = 0.009, respectively) and disease-free survival (DFS; p < 0.001, p = 0.005, and p = 0.019, respectively). Multivariate analysis indicated that lymph node metastasis was an independent prognostic factor for unfavorable OS and DFS (p = 0.002 and p = 0.005, respectively), whereas co-overexpression of CXCR4 and αvß6 was an independent prognostic factor only for OS (p = 0.043). CONCLUSION: CXCR4 and αvß6 may play synergistic roles in the progression of BC, and co-targeting of CXCR4 and αvß6 could be a potential strategy for the prevention and treatment of BC.

Expression and Functional Analysis of CXCL12 and Its Receptors in Human Term Trophoblast Cells.

Chemokine CXCL12 and its receptors CXCR4/CXCR7 play a pivotal role in many physiological and pathological situations, while the expression and function in human term trophoblast cells remain largely unknown. In the study, the expression and function of CXCL12 and its receptors CXCR4/CXCR7 in human term trophoblast cells were investigated. Immunocytochemistry and flow cytometry showed that the expression of CXCL12/CXCR4/CXCR7 could be detected in term trophoblast cells while expression level differed. The secretion of CXCL12 in human term trophoblast cells was confirmed by enzyme-linked immunosorbent assay (ELISA). In order to reveal the function of CXCL12, exogenetic recombinant human CXCL12 protein (rhCXCL12) was added to the cultured term trophoblast cells; results showed that cell proliferation ability was increased while cell apoptosis rate was decreased. Moreover, the effects of rhCXCL12 on term trophoblast cells could be diminished or attenuated by antibodies against CXCL12, CXCR4, or CXCR7, respectively. Therefore, these results revealed the important role of CXCL12 on human term trophoblast cells. Our study will provide new insights into understanding the role of CXCL12 on human term trophoblast cells.

The Effects of the CXCR4 Antagonist, AMD3465, on Human Retinal Vascular Endothelial Cells (hRVECs) in a High Glucose Model of Diabetic Retinopathy.

BACKGROUND High blood glucose levels in diabetes result in retinal angiogenesis, which is the key feature of diabetic retinopathy. This study aimed to investigate the effects of the CXCR4 antagonist, AMD3465, on human retinal vascular endothelial cells (hRVECs) [i]in vitro[/i]. MATERIAL AND METHODS Cell viability and the protein expression levels of CXCR4 and stromal cell-derived factor 1 (SDF-1) were evaluated in high glucose (HG)-treated human retinal vascular endothelial cells (hRVECs). The cell counting kit 8 (CCK-8) assay, the colony formation assay, immunofluorescence, and Western blot were used to investigate the effects of AMD3465 on hRVEC cell viability, colony formation, cell proliferation, and expression of CXCR4 and SDF-1. Cell apoptosis and angiogenesis were assessed by flow cytometry and Western blot. RESULTS Treatment with high glucose reduced the viability of hRVECs and increased the protein expression levels of CXCR4 and SDF-1. Following treatment with AMD3465, the colony formation capacity and cell proliferation in hRVECs increased, and there was a significant reduction in apoptosis rate compared with the untreated cells. AMD3465 significantly reduced the expression of angiogenesis-associated proteins, including ICAM1, VCAM1, VEGF, and AngII. AMD3465 significantly reduced the protein expression levels of TNF-α, IL-1ß, NF-κB, and p-p65. CONCLUSIONS The CXCR4 antagonist, AMD3465, reduced apoptosis of HG-treated hRVECs in an [i]in vitro[/i] model of diabetic retinopathy.

C-X-C Motif Chemokine Receptor 4 Blockade Promotes Tissue Repair After Myocardial Infarction by Enhancing Regulatory T Cell Mobilization and Immune-Regulatory Function.

BACKGROUND: Acute myocardial infarction (MI) elicits an inflammatory response that drives tissue repair and adverse cardiac remodeling. Inflammatory cell trafficking after MI is controlled by C-X-C motif chemokine ligand 12 (CXCL12) and its receptor, C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 antagonists mobilize inflammatory cells and promote infarct repair, but the cellular mechanisms are unclear. METHODS: We investigated the therapeutic potential and mode of action of the peptidic macrocycle CXCR4 antagonist POL5551 in mice with reperfused MI. We applied cell depletion and adoptive transfer strategies using lymphocyte-deficient Rag1 knockout mice; DEREG mice, which express a diphtheria toxin receptor-enhanced green fluorescent protein fusion protein under the control of the promoter/enhancer region of the regulatory T (Treg) cell-restricted Foxp3 transcription factor; and dendritic cell-depleted CD11c-Cre iDTR mice. Translational potential was explored in a porcine model of reperfused MI using serial contrast-enhanced magnetic resonance imaging. RESULTS: Intraperitoneal POL5551 injections in wild-type mice (8 mg/kg at 2, 4, 6, and 8 days) enhanced angiogenesis in the infarct border zone, reduced scar size, and attenuated left ventricular remodeling and contractile dysfunction at 28 days. Treatment effects were absent in splenectomized wild-type mice, Rag1 knockout mice, and Treg cell-depleted DEREG mice. Conversely, treatment effects could be transferred into infarcted splenectomized wild-type mice by transplanting splenic Treg cells from POL5551-treated infarcted DEREG mice. Instructive cues provided by infarct-primed dendritic cells were required for POL5551 treatment effects. POL5551 injections mobilized Treg cells into the peripheral blood, followed by enhanced Treg cell accumulation in the infarcted region. Neutrophils, monocytes, and lymphocytes displayed similar mobilization kinetics, but their cardiac recruitment was not affected. POL5551, however, attenuated inflammatory gene expression in monocytes and macrophages in the infarcted region via Treg cells. Intravenous infusion of the clinical-stage POL5551 analogue POL6326 (3 mg/kg at 4, 6, 8, and 10 days) decreased infarct volume and improved left ventricular ejection fraction in pigs. CONCLUSIONS: These data confirm CXCR4 blockade as a promising treatment strategy after MI. We identify dendritic cell-primed splenic Treg cells as the central arbiters of these therapeutic effects and thereby delineate a pharmacological strategy to promote infarct repair by augmenting Treg cell function in vivo.

Expression and clinical significance of SDF-1 and CXCR4 in peripheral blood of patients with hepatitis C cirrhosis / 中国医师杂志

Objective To detect the expression and clinical significance of stro-mal cell derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in peripheral blood of patients with hepatitis C cirrhosis.Methods From January 2015 to January 2018,120 patients with hepatitis C cirrhosis in our hospital were selected as the subjects,including 60 patients with compensated hepatitis C cirrhosis and 60 patients with decompensated hepatitis C cirrhosis,and 60 hepatitis C patients without cirrhosis in the same period were selected as the control group.Enzyme linked immunosorbent assay (ELISA) was used to detect the expression levels of SDF-1 and CXCR4 in peripheral blood;the correlation between SDF-1 and CXCR4 expressions in compensated cirrhosis and decompensated cirrhosis patients was analyzed.Results The expression levels of SDF-1 and CXCR4 in peripheral blood samples of patients with decompensated cirrhosis of hepatitis C were significantly higher than those of patients with compensated cirrhosis of hepatitis C (P ＜ 0.05);the expression levels of SDF-1 and CXCR4 in peripheral blood samples of patients with compensated and decompensated cirrhosis of hepatitis C were significantly higher than those of control group (P ＜ 0.05);the expressions of SDF-1 and CXCR4 in peripheral blood of patients with compensated and decompensated cirrhosis of hepatitis C were positively correlated (r =0.684,P ＜ 0.05,r =0.765,P ＜ 0.05).Albumin (AlB) level in peripheral blood of cirrhosis group was significantly lower than that of control group (P ＜ 0.05);alanine aminotransferase (ALT),aspartate aminotransferase (AST),total bilirubin (TBIL),while fasting insulin (FINs) and interleukin (IL)-6 in cirrhosis group were higher than those of control group (P ＜ 0.05);SDF-1 level and CXCR4 level were positively correlated with AlB,FINs and IL-6 (P ＜ 0.05);multiple regression analysis showed that FINs,IL-6,SDF-1 and CXCR4 were risk factors for hepatitis C cirrhosis.Conclusions The levels of SDF-1 and CXCR4 in peripheral blood of patients with hepatitis C cirrhosis increased,and the levels of SDF-1 and CXCR4 in peripheral blood were positively correlated,suggesting that they may be involved in the regulation of the occurrence and development of hepatitis C cirrhosis.

68Ga-NOTA-NFB PET/CT imaging in breast cancer: clinical study of a new targeted agent for chemokine receptor 4 / 中华核医学与分子影像杂志

Objective To investigate the clinical application of chemokine receptor 4 (CXCR4)-targeted PET/CT imaging in breast cancer using 68Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-TN14003 (NOTA-NFB) and the correlation between 68Ga-NOTA-NFB uptake and pathology.Methods From June 2014 to December 2014,11 female patients (age range:38-68 years) with non-specific invasive breast cancer were recruited in this study.All patients underwent neoadjuvant chemotherapy before surgery.68GaNOTA-NFB and 18F-fluorodeoxyglucose (FDG) PET/CT imaging were performed before the chemotherapy.Three patients also underwent 68Ga-NOTA-NFB PET/CT imaging after the fourth cycle of chemotherapy.The region of interest (ROI) method was used to measure the maximum standardized value (SUVmax) and tumor/non-tumor (T/NT) ratio was calculated.Paired t test and Spearman correlation analysis were used for statistical analysis.Results The SUVmax values of primary lesions were 3.78±2.03 and 8.11±5.14 (t=-3.01,P＜0.05) respectively in 68Ga-NOTA-NFB imaging and 18F-FDG imaging.The T/NT ratios for primary lesions were not significantly different between the two imaging methods (9.36±7.81 vs 15.62±14.51;t=-1.63,P＞0.05).In the metastatic lymph nodes,SUVmax values were not significantly different between 68Ga-NOTA-NFB imaging and 18F-FDG imaging (t=-2.02,P＞0.05),but T/NT ratios were significantly different (t=-2.43,P＜0.05).After neoadjuvant chemotherapy,T/NT ratios were decreased in the 3 patients.Correlation was not found between T/NT in 68Ga-NOTA-NFB imaging and Ki-67,but the P value was close to 0.05 (rs =0.600,P=0.051).Conclusion 68Ga-NOTA-NFB PET/CT can be used as a new CXCR4-targered imaging in diagnosis of breast cancer,and it may be beneficial to evaluate the effect of neoadjuvant chemotherapy.

The macrophage migration inhibitory factor pathway in human B cells is tightly controlled and dysregulated in multiple sclerosis.

In MS, B cells survive peripheral tolerance checkpoints to mediate local inflammation, but the underlying molecular mechanisms are relatively underexplored. In mice, the MIF pathway controls B-cell development and the induction of EAE. Here, we found that MIF and MIF receptor CD74 are downregulated, while MIF receptor CXCR4 is upregulated in B cells from early onset MS patients. B cells were identified as the main immune subset in blood expressing MIF. Blocking of MIF and CD74 signaling in B cells triggered CXCR4 expression, and vice versa, with separate effects on their proinflammatory activity, proliferation, and sensitivity to Fas-mediated apoptosis. This study reveals a new reciprocal negative regulation loop between CD74 and CXCR4 in human B cells. The disturbance of this loop during MS onset provides further insights into how pathogenic B cells survive peripheral tolerance checkpoints to mediate disease activity in MS.

The mechanism and biological function of chemokine receptor CXCR4 and its ligand CXCL12 in hepatocellular carcinoma / 天津医药

Objective To investigate the mechanism and function of chemokine receptor CXCR4 and its ligand CXCL12 (CXCL12 / CXCR4) in primary hepatocellular carcinoma (PHC). Methods Western blot assay, immunohistochemistry and Real-time PCR were used to detect the protein and mRNA expressions of CXCL12/CXCR4 in 60 PHC and corresponding paracancerous tissue samples. Four kinds of hepatoma cells (Huh7, MHCC97h, HepG2 and Hep3B) and normal hepatocytes (7702) were routinely cultured, and then real-time PCR was performed to detect the mRNA expressions of CXCL12/CXCR4 in these cells to screen suitable experimental cells. CXCR4 interference plasmid (sh-CXCR4) and corresponding empty vector (sh-control) were transfected into MHCC97h to construct stable transfected cell lines. The ability of invasion, migration, and proliferation of the 2 groups of cells were detected by Tanswell invasion experiment, cell scratch test and MTT test. The stably expressed sh-control and sh-CXCR4 MHCC97h cells were taken into the subcutaneous of six nude mice, and the growth of the tumor was observed. Western blot assay was used to detect the expressions of vascular endothelial growth factor-C (VEGF-C) in sh-control and sh-CXCR4 MHCC97h cell lines and corresponding xenografts in nude mice, as the same in MHCC97h, which was transfected with CXCR overexpressed plasmid. Results (1) The results of Western blot assay, immunohistochemistry and Real-time PCR showed that the expressions of CXCL12/CXCR4 protein and mRNA were significantly higher in liver cancer tissues than those of paracancerous tissues. (2) The expression levels of CXCL12/CXCR4 mRNA were higher in Huh7, MHCC97h, HepG2 and Hep3B cells than those of 7702 cells. MHCC97h was selected as the experimental cells. The ability of invasion, migration and proliferation of MHCC97h cells transfected with sh-CXCR4 were significantly lower than those of sh-control group. Meanwhile, the growth rate of nude mice transplanted with sh-CXCR4 MHCC97h cells was also significantly lower than that of sh-control group. (3) Both in vitro and in vivo experiments showed that the expression of VEGF-C was lower in sh-CXCR4 group than that in sh-control group, and the expression of VEGF-C was obviously up-regulated after overexpression of CXCR4. Conclusion High expressions of CXCL12/CXCR4 are found in primary cancer tissues and hepatoma cells. CXCL12/CXCR4 may inhibit the proliferation and invasion of hepatoma cells by regulating the expression of VEGF-C protein.

Effect of RBP4 on neurocognitive function in diabetic nephropathy with silent cerebral infarction / 重庆医学

Objective To observe the impairment effect of retinol binding protein 4(RBP4) on neurocognitive function in diabetic nephropathy(DN) patients with silent cerebral infarction(SCI) and to explore its mechanism.Methods Sixty patients with newly diagnosed DN and 30 healthy volunteers were selected as the study subjects and the DN cases were divided into the complicating SCI group(SCI,n=30) and non-complicating SCI group(NSCI,n=30) according to the imaging results.The degrees of neurological function deficit and Montreal cognitive assessment(MoCA) were evaluated.Serum RBP4 level was determined by ELISA and expressions of Lp-PLA2 and C-X-C chemokine receptor type 4(CXCR4) were determined by Western blot.Results Compared with the NSCI group,the neurocognitive function in the SCI group was subsided,the expression levels of RBP4,Lp-PLA2 and CXCR4 were increased(P＜0.05).The RBP4 level was positively correlated with the neurocognitive function impairment in SCI patients,moreover,there existed a regression correlation between them.Conclusion Serum RBP4 may serve as the predictive factor of DN complicating SCI and is positively correlated with neurocognitive dysfunction.Lp-PLA2/CXCR4 pathway activation may be one of its pathogenesis.

Effect of atorvastatin on the expression of chemokine receptor-4 and migration ability of rat bone marrow mesenchymal stem cells cultivated in vitro / 中华眼底病杂志

Objective To investigate the effects of migration and expression from chemokine receptor 4 (chemokine receptor-4,CXCR4) of rat bone marrow mesenchymal stem cells (BMSCs) which were pretreated by atorvastatin (ATV) in vitro.Methods Isolated,cultivated,identified the BMSCs,pretreated P4-P6 of BMSCs with different concentrations of ATV for 12 hours.The experimental group was divided into control group,0.1 nM/L (group 0.1 nM),1 nM/L (1 nM group),10 nM/L (10 nM group),100 nM/L (100 nM group),1 000 nM/L (1 000 nM group).The mRNA and protein of CXCR4 were determined by real time-polymerase chain reaction and Western blot.Immunofluoreseence assay were used to detect the expression levels of CXCR4.The migration ability of BMSCs were measured by transwell chamber.Results Immunofluoreseence assay showed the protein level of CXCR4 of group 1 nM and 10 nM were significantly higher than the other group.RT-PCR and Western blot showed the protein and mRNA level of CXCR4 in 10 nM was higher than that in group 1 rM.The migration ability of group 10 nM was higher than 1 nM and control group.Conclusions ATV can be dose-dependent promote expression levels of CXCR4 of BMSCs cultivated in vitro.

Role of CXCR4 in dorsal root ganglia in incisional pain in rats / 中华麻醉学杂志

Objective To evaluate the role of C-X-C chemokine receptor type 4 ( CXCR4) in the dorsal root ganglia ( DRG) in incisional pain in rats. Methods Thirty-two male Sprague-Dawley rats, aged 7-10 weeks, weighing 250-300 g, in which intrathecal catheters were successfully implanted, were divided into 4 groups (n＝8 each) using a random number table method: sham operation group (group S), CXCR4 antagonist AMD3100 plus sham operation group (group A+S), incisional pain group (group I) and CXCR4 antagonist AMD3100 plus incisional pain group (group A+I). Rats were anesthetized with sevoflu-rane. AMD3100 20 μg (in 10 μl of normal saline) was intrathecally injected, and no incision was made 30 min later in group A+S. A 1-cm longitudinal incision was made through skin, fascia and muscle of the plantar aspect of the left hindpaw in group I. AMD3100 20 μg (in 10 μl of normal saline) was intrathecally injected, and 30 min later the model of incisional pain was established in group A+I. The mechanical paw withdrawal threshold ( MWT) and thermal paw withdrawal latency ( TWL) were measured at 24 h before surgery and 2, 4, 8, 16 and 24 h after surgery. The rats were sacrificed after the last measurement of pain threshold and the DRGs of the lumbar segment (L4-6) were removed for detecting the expression of CXCR4, phosphorylated extracellular regulated protein kinase ( p-ERK) and total ERK ( t-ERK) by Western blot. Results Compared with group S, MWT was significantly decreased and TWL was shortened at T1-5in group I and group A+I, and the expression of CXCR4 and p-ERK in DRGs was significantly up-regulated (P＜0. 05), and no significant change was found in the expression of t-ERK in group I, no significant change was found in the expression of CXCR4, p-ERK and t-ERK in group A+I, and no significant change was found in the parameters mentioned above in group A+S (P＞0. 05). Compared with group I, MWT was significantly increased and TWL was prolonged at T1-5, the expression of CXCR4 and p-ERK in DRGs was down-regulated (P＜0. 05), and no significant change was found in the expression of t-ERK in group A+I (P＞0. 05). Conclusion CXCR4 in DRGs is involved in incisional pain, and the mechanism may be re-lated to activating ERK1∕2 signaling pathway in rats.

Effect of Silencing CXCR4 Gene by siRNA on Biological Behavior of Gastric Cancer / 胃肠病学

Background:CXCR4 is widely expressed in tumor cells and participates in tumor invasion and metastasis. RNA interference technology can effectively reduce or shut down the expression of genes and block tumor invasion and metastasis at different levels. Aims:To investigate the mRNA and protein expressions of CXCR4 and their relationship with clinicopathological features of gastric cancer,and to explore the effect of silencing CXCR4 by siRNA on biological behavior of gastric cancer. Methods:A total of 86 gastric cancer tissues and their adjacent normal mucosa were collected. mRNA and protein expressions of CXCR4 were detected by RT-PCR and Western blotting,respectively,and their relationship with clinicopathological features was analyzed. The CXCR4 RNA interference plasmid vector was constructed,and were transfected into gastric cancer cell line MKN45. Cell proliferation,migration and invasion,apoptosis were detected by MTT assay,Transwell assay and flow cytometry,respectively. Results:mRNA and protein expressions of CXCR4 in gastric cancer tissues were significantly higher than those in adjacent normal tissues (P ＜0.05),and CXCR4 expression was related to TNM staging,tumor differentiation,lymph node metastasis (P＜0.05). Compared with negative control cells, cell proliferation in siRNA transfection group at 24,48,and 72 hours were significantly decreased (P ＜0.05 ),cell migration rate and cell invasion rate were significantly decreased (P ＜0.05),and cell apoptosis rate was significantly increased (P＜0.05). Conclusions:CXCR4 plays an important role in the development of gastric cancer. The silencing CXCR4 gene by siRNA can significantly inhibit the proliferation,migration and invasion of MKN45 cells,and increase apoptosis,thereby providing a new strategy for the treatment of gastric cancer.

[Evaluation of clinical characteristics, MYD88(L265P) mutation, CXCR4(WHIM) mutation and prognosis in Waldenström macroglobulinemia: A single center retrospective study of 93 patients].

Objective: To evaluate the clinical characteristics, MYD88(L265P) mutation, CXCR4(W)HIM mutation and prognosis in patients with Waldenström macroglobulinemia (WM). Methods: The clinical characteristics, International Prognostic Scoring System for symptomatic WM (WPSS) , and overall survival (OS) were retrospectively assayed in 93 patients with newly diagnosed WM at Peking Union Medical College Hospital during January 2000 to August 2016. The MYD88(L265P) mutation and CXCR4(W)HIM mutation were tested among 34 patients. Results: The median age of the 93 patients was 64 years (range, 33-85 years) with a male-to-female ratio of 2.44. According to WPSS, we included 16 (17.2%) low-risk, 44 (47.3%) intermediate-risk and 33 (35.5%) high-risk patients. Eight patients had secondary amyloidosis. With a median follow-up of 44 (1-201) months, the median OS was 84 months. Cox regression multifactor analysis showed WPSS risk group (HR=2.342, 95% CI 1.111-4.950, P=0.025) , whether patients had secondary amyloidosis (HR=5.538, 95% CI 1.958-15.662, P=0.001) and whether patients received new drugs (HR=3.392, 95% CI 1.531-7.513, P=0.003) were independent factors associated with OS. We have investigated the presence of the MYD88(L265P) and CXCR4(WHIM) mutation in 34 patients and found that MYD88(L265P) mutation was occurred in 32 patients (94.1%) and CXCR4(WHIM) mutation was occurred in 8 patients (23.5%). Seven of 8 patients who harbored CXCR4(WHIM)-mutated also exhibited the MYD88(L)265P mutation. Patients with MYD88(L265P)CXCR4(WHIM) vs MYD88(L265P)CXCR4(WT) presented with more severe anemia, lower platelet level, higher M protein level and more hyper-viscosity syndrome. Conclusion: WPSS risk group, whether patients had secondary amyloidosis or received new drugs are independent factors for OS in WM. MYD88(L265P) and CXCR4(WHIM) mutation, the most common somatic variants in WM, often occur together and impact the clinical presentation.

Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity: Evidence From Mouse and Human Studies.

BACKGROUND: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. METHODS: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2- or TaglnCre-driven)-specific deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/ß-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. RESULTS: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=12-15) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/ß-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. CONCLUSIONS: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.

[Ibrutinib inhibits mesenchymal stem cells-mediated drug resistance in diffuse large B-cell lymphoma].

Objective: To explore the mechanism of ibrutinib on drug resistance diffuse large B-cell lymphoma (DLBCL) cells. Methods: DLBCL cell line was cultured with mesenchymal stem cells (MSC) , and DLBCL cells which migrated and adhered to MSC under microscope was counted. The secretion of CXCL12 by MSC were measured by ELISA. The expression of CXCR4 on DLBCL cells were measured by flow cytometry, HBL-1 cells were transfected with a CXCR4-lentivector. An Annexin Ⅴ-binding assay was used to detect the induction of apoptosis. Clonogenic growth of DLBCL cells was evaluated on MethoCult media. Ibrutinib was injected into NOD/SCID mice, tumor growth was assessed via caliper measurements every 3 days. Results: MSC promoted migration and adhesion of DLBCL cells to MSC. Ibrutinib inhibited migration and adhesion of DLBCL cells to MSC in a dose-dependent manner (P<0.05) . CXCL12 secreted by MSC and CXCR4 expressed on DLBCL cells could induce each other, which upgraded the levels of secretion and expression. Ibrutinib could inhibit the secretion of CXCL12 (SUDHL10: 660 pg/ml vs 1 400 pg/ml, P=0.004; HBL-1: 720 pg/ml vs 1 490 pg/ml, P=0.018; DLBCL:850 pg/ml vs 1 450 pg/ml, P=0.004) and expression of CXCR4 (P<0.05) . When co-cultured with MSC, the ratio of HBL-1 cells apoptosis in the group of control, mitoxantrone, ibrutinib, mitoxantrone+ibrutinib were respectively 15.1%, 17.5%, 23.5%, 58.7%. After transfected with a CXCR4-lentivector and overexpressed CXCR4, the ratios of HBL-1 cells apoptosis were 14.2%, 16.1%, 22.5%, 38.3% respectively. The ratio of DLBCL cells apoptosis induced by mitoxantrone was lower when co-cultured with MSC (P<0.05) . But with the addition of ibrutinib, the ratio of apoptosis was increaed and it was similar to cultivation without MSC, which suggested ibrutinib could inhibit drug-resistance induced by MSC. But after transfected with a CXCR4-lentivector, the overexpression of CXCR4 was detected and the ratio of apoptosis was significantly lower when co-cultured with MSC which demonstrated that ibrutinib inhibited drug-resistance by inhibiting the expression of CXCR4. MSC enhanced lymphoma clonogenicity in vitro and lymphoma cell growth in vivo. The number of colonies of control, MSC, Ibrutinib, MSC+Ibrutinib were 113±5, 205±4, 62±9, 123±3 (2.5×10(3)/well, x±s) , respectively. The tumor volume of NOD/SCID mice were respectively 6 500, 17 000, 4 000, 10 000 mm(3). Ibrutinib inhibited lymphoma clonogenicity in vitro and lymphoma cell growth in vitro. Conclusion: Ibrutinib targeted the CXCL12/CXCR4 axis, inhibited the expression of CXCR4 and inhibited MSC-mediated drug resistance. Ibrutinib also inhibited lymphoma clonogenicity in vitro and lymphoma cell growth in vivo. These results provided a scientific rationality for relapsed/refractory DLBCL treatment with ibrutinib.

Ibrutinib inhibits mesenchymal stem cells-mediated drug resistance in diffuse large B-cell lymphoma / 中华血液学杂志

Objective@#To explore the mechanism of ibrutinib on drug resistance diffuse large B-cell lymphoma (DLBCL) cells.@*Methods@#DLBCL cell line was cultured with mesenchymal stem cells (MSC) , and DLBCL cells which migrated and adhered to MSC under microscope was counted. The secretion of CXCL12 by MSC were measured by ELISA. The expression of CXCR4 on DLBCL cells were measured by flow cytometry, HBL-1 cells were transfected with a CXCR4-lentivector. An Annexin Ⅴ-binding assay was used to detect the induction of apoptosis. Clonogenic growth of DLBCL cells was evaluated on MethoCult media. Ibrutinib was injected into NOD/SCID mice, tumor growth was assessed via caliper measurements every 3 days.@*Results@#MSC promoted migration and adhesion of DLBCL cells to MSC. Ibrutinib inhibited migration and adhesion of DLBCL cells to MSC in a dose-dependent manner (P<0.05) . CXCL12 secreted by MSC and CXCR4 expressed on DLBCL cells could induce each other, which upgraded the levels of secretion and expression. Ibrutinib could inhibit the secretion of CXCL12 (SUDHL10: 660 pg/ml vs 1 400 pg/ml, P=0.004; HBL-1: 720 pg/ml vs 1 490 pg/ml, P=0.018; DLBCL:850 pg/ml vs 1 450 pg/ml, P=0.004) and expression of CXCR4 (P<0.05) . When co-cultured with MSC, the ratio of HBL-1 cells apoptosis in the group of control, mitoxantrone, ibrutinib, mitoxantrone+ibrutinib were respectively 15.1%, 17.5%, 23.5%, 58.7%. After transfected with a CXCR4-lentivector and overexpressed CXCR4, the ratios of HBL-1 cells apoptosis were 14.2%, 16.1%, 22.5%, 38.3% respectively. The ratio of DLBCL cells apoptosis induced by mitoxantrone was lower when co-cultured with MSC (P<0.05) . But with the addition of ibrutinib, the ratio of apoptosis was increaed and it was similar to cultivation without MSC, which suggested ibrutinib could inhibit drug-resistance induced by MSC. But after transfected with a CXCR4-lentivector, the overexpression of CXCR4 was detected and the ratio of apoptosis was significantly lower when co-cultured with MSC which demonstrated that ibrutinib inhibited drug-resistance by inhibiting the expression of CXCR4. MSC enhanced lymphoma clonogenicity in vitro and lymphoma cell growth in vivo. The number of colonies of control, MSC, Ibrutinib, MSC+Ibrutinib were 113±5, 205±4, 62±9, 123±3 (2.5×103/well, ±s) , respectively. The tumor volume of NOD/SCID mice were respectively 6 500, 17 000, 4 000, 10 000 mm3. Ibrutinib inhibited lymphoma clonogenicity in vitro and lymphoma cell growth in vitro.@*Conclusion@#Ibrutinib targeted the CXCL12/CXCR4 axis, inhibited the expression of CXCR4 and inhibited MSC-mediated drug resistance. Ibrutinib also inhibited lymphoma clonogenicity in vitro and lymphoma cell growth in vivo. These results provided a scientific rationality for relapsed/refractory DLBCL treatment with ibrutinib.

Evaluation of clinical characteristics, MYD88L265P mutation, CXCR4WHIM mutation and prognosis in Waldenström macroglobulinemia: A single center retrospective study of 93 patients / 中华血液学杂志

Objective@#To evaluate the clinical characteristics, MYD88L265P mutation, CXCR4WHIM mutation and prognosis in patients with Waldenström macroglobulinemia (WM).@*Methods@#The clinical characteristics, International Prognostic Scoring System for symptomatic WM (WPSS) , and overall survival (OS) were retrospectively assayed in 93 patients with newly diagnosed WM at Peking Union Medical College Hospital during January 2000 to August 2016. The MYD88L265P mutation and CXCR4WHIM mutation were tested among 34 patients.@*Results@#The median age of the 93 patients was 64 years (range, 33-85 years) with a male-to-female ratio of 2.44. According to WPSS, we included 16 (17.2%) low-risk, 44 (47.3%) intermediate-risk and 33 (35.5%) high-risk patients. Eight patients had secondary amyloidosis. With a median follow-up of 44 (1-201) months, the median OS was 84 months. Cox regression multifactor analysis showed WPSS risk group (HR=2.342, 95% CI 1.111-4.950, P=0.025) , whether patients had secondary amyloidosis (HR=5.538, 95% CI 1.958-15.662, P=0.001) and whether patients received new drugs (HR=3.392, 95% CI 1.531-7.513, P=0.003) were independent factors associated with OS. We have investigated the presence of the MYD88L265P and CXCR4WHIM mutation in 34 patients and found that MYD88L265P mutation was occurred in 32 patients (94.1%) and CXCR4WHIM mutation was occurred in 8 patients (23.5%). Seven of 8 patients who harbored CXCR4WHIM-mutated also exhibited the MYD88L265P mutation. Patients with MYD88L265PCXCR4WHIM vs MYD88L265PCXCR4WT presented with more severe anemia, lower platelet level, higher M protein level and more hyper-viscosity syndrome.@*Conclusion@#WPSS risk group, whether patients had secondary amyloidosis or received new drugs are independent factors for OS in WM. MYD88L265P and CXCR4WHIM mutation, the most common somatic variants in WM, often occur together and impact the clinical presentation.

Studies of the mechanism of endothelial dysfunction in rats under intermittent hypoxia / 天津医药

Objective To explore the mechanism of vessel endothelial dysfunction in rats under intermittent hypoxia (IH). Methods The respiratory simulation system was used to simulate IH. Sixty C57BL/6J rats (male) were randomized into control group and IH group. The rats of IH group were exposed to IH 8 hours per day for 6 weeks. The serum levels of hypoxia inducible factor (HIF)-1a and stromal cell derived factor (SDF)-1a were assessed by ELISA. The serum levels of reactive oxygen species (ROS) were detected in two groups. The serum expression of miR-199a-5p was detected by real-time fluorescent quantitative PCR in two groups. The dual luciferase report system and point mutation test were used to verify target gene for HIF-1a. Results The serum levels of HIF-1a and SDF-1a were significantly higher in IH group than those of control group (μg/L:1.60±0.02 vs. 1.19±0.02, 1 823.00±8.97 vs. 1 444.00±17.90, P<0.01). The serum level of ROS was significantly higher in IH group than that of control group (U/mL:487.66±35.73 vs. 211.57±23.82, P<0.01). The serum level of miR-199a-5p expression was significantly lower in IH group compared to that of control group (1.31±0.07 vs. 3.47± 0.17, P<0.01). The result of dual luciferase reporter gene detection confirmed that target gene of miR-199a-5p was HIF-1a. Conclusion The serum level of miR-199a-5p is decreased first due to IH, and then its target gene (HIF-1a) is increased. HIF-1a can induce the increased level of SDF-1a, and its receptor (CXCR-4 ) is also increased. Finally, HIF-1a can increase the serum level of ROS, resulting in the endothelial dysfunction.