HGF-Induced PKCζ Activation Increases Functional CXCR4 Expression in Human Breast Cancer Cells

The chemokine receptor CXCR4 and its ligand CXCL12 have been shown to mediate the metastasis of many malignant tumors including breast carcinoma. Interaction between hepatocyte growth factor (HGF) and the Met receptor tyrosine kinase mediates development and progression of cancers. HGF is able to induce CXCR4 expression and contributes to tumor cell invasiveness in breast carcinoma. However, the mechanism of the CXCR4 expression modulated by c-Met-HGF axis to enhance the metastatic behavior of breast cancer cells is still unclear. In this study, we found that HGF induced functional CXCR4 receptor expression in breast cancer cells. The effect of HGF was specifically mediated by PKCζ activity. After transfection with PKCζ-siRNA, the phosphorylation of PKCζ and CXCR4 was abrogated in breast cancer cells. Interference with the activation of Rac1, a downstream target of HGF, prevented the HGF-induced increase in PKCζ activity and CXCR4 levels. The HGF-induced, LY294002-sensitive translocation of PKCζ from cytosol to plasma membrane indicated that HGF was capable of activating PKCζ, probably via phosphoinositide (PI) 3-kinases. HGF treatment also increased MT1-MMP secretion. Inhibition of PKCζ, Rac-1 and phosphatidylinositol 3-kinase may attenuate MT1-MMP expression in cells exposed to HGF. Functional manifestation of the effects of HGF revealed an increased ability for migration, chemotaxis and metastasis in MDA-MB-436 cells in vitro and in vivo. Our findings thus provided evidence that the process of HGF-induced functional CXCR4 expression may involve PI 3-kinase and atypical PKCζ. Moreover, HGF may promote the invasiveness and metastasis of breast tumor xenografts in BALB/c-nu mice via the PKCζ-mediated pathway, while suppression of PKCζ by RNA interference may abrogate cancer cell spreading

Association between metabolic healthy obesity and female infertility: the national health and nutrition examination survey, 2013–2020

Abstract Background Obesity has been confirmed to be associated with infertility. However, the association between metabolically healthy obesity (MHO), a subset of obesity with no metabolic abnormalities, and female infertility has not yet been investigated. This study aimed to examine the association between MHO and the risk of female infertility among United States. Methods This study utilized a cross-sectional design and included 3542 women aged 20–45 years who were selected from the National Health and Nutrition Examination Survey (NHANES) 2013–2020 database. The association between MHO and the risk of infertility was evaluated using risk factor–adjusted logistic regression models. Results Higher BMI and WC were associated with increased infertility risk after adjusting for potential confounding factors (OR (95% CI): 1.04(1.02, 1.06), P = 0.001; OR (95% CI): 1.02 (1.01, 1.03), P < 0.001; respectively). After cross-classifying by metabolic health and obesity according to BMI and WC categories, individuals with MHO had a higher risk of infertility than those with MHN (OR (95% CI): 1.75(0.88, 3.50) for BMI criteria; OR (95% CI): 2.01(1.03, 3.95) for WC criteria). A positive linear relationship was observed between BMI/WC and infertility risk among metabolically healthy women (P non−linearity=0.306, 0.170; respectively). Conclusions MHO was associated with an increased risk of infertility among reproductive-aged women in the US. Obesity itself, regardless of metabolic health status, was associated with a higher infertility risk. Our results support implementing lifestyle changes aimed at achieving and maintaining a healthy body weight in all individuals, even those who are metabolically healthy

A case of Cardiobacterium valvarum endocarditis with cerebral hemorrhage after MVR, TVP and vegetation removal operation

Abstract Background Cardiobacterium is a fastidious Gram-negative bacillus, and is a rare human pathogen in clinical settings. Herein, we describe a case of Cardiobacterium valvarum (C. valvarum) endocarditis with a rare complication of cerebral hemorrhage after mitral valve replacement (MVR), tricuspid valve prosthesis (TVP) and vegetation removal operation. Case presentation A 41-year-old woman who had a history of gingivitis developed into infective endocarditis due to the infection of C. valvarum. Then, she was hospitalized to receive MVR, TVP and vegetation removal operation. The indicators of patient tended to be normal until the abrupt cerebral hemorrhage occurred on day 15 after operation. This is the first well-described case of C. valvarum infection in China, and the first report of C. valvarum endocarditis with cerebral hemorrhage after MVR, TVP and vegetation removal operation worldwide. Conclusions We reported the first case of C. valvarum infection in China clinically, with a rare complication of cerebral hemorrhage after MVR, TVP and vegetation removal operation

HGF enhances CXCR4 expression via PKCζ and promotes the invasion and metastasis of breast cancers in BALB/c-nu mice.

<p>(A) Micrographs of H&E-stained xenografts demonstrating the presence or absence of margin invasion by the MDA-MB-436 tumors untransduced (Un) or transduced with GFP-shRNA or PKCζ-shRNA (original magnification, 200×). (B) Representative micrographs of lung (upper) or liver (lower) tissue sections with H&E staining and PCNA immunohistochemical staining (original magnification, 200×). Each group from two independent experiments. (C) Mean ± SD wet lung weight in tumor-bearing mice. The number of mice in each group is indicated. * P<0.05 as compared to PBS-treated mice. (D) Expression of human HPRT mRNA relative to mouse 18S rRNA as determined by qRT-PCR in the lungs and livers of tumor-bearing mice. Data were normalized to PBS-treated mice. # P<0.01 as compared to PBS-treated mice. (E–F) Immunoblot of total PKCζ and p-PKCζ or total CXCR4 and p-CXCR4 protein, respectively, in breast tumor xenografts in female nude mice that were inoculated in the mammary fat pads with MDA-MB-436 cells treated as indicated. The experiment was repeated twice with similar results. A representative study is shown. (G) Representative microphotos of immunohistochemical results for p-PKCζ (middle) or p-CXCR4 (lower) expression in MDA-MB-436 tumors (original magnification, 400×).</p

Clinical characteristics, laboratory parameters, and clinical outcomes of the three groups.

<p>Note: Multiple groups of quantitative data were compared using analysis of variance, and the LSD test was performed for pairwise comparisons between any two of the three groups.</p><p>a The variance and LSD test were used to compare the three subgroups, and the values are presented as the mean ± standard deviation.</p><p>b A significant difference was detected among the three subgroups by analysis of variance (<i>P</i><0.05).</p><p>c Non-normally distributed data are presented as the median (range), as assessed by the Kruskal-Wallis test.</p><p>d Data were obtained using Pearson’s chi-square test.</p><p>^f Data were obtained using Fisher’s Exact Test.</p><p>* A significant difference was detected between the infected female and infected male groups.</p><p>^# A significant difference was detected between the infected female and infected couple groups.</p><p>^&A significant difference was detected between the infected male and infected couple groups.</p><p>Clinical characteristics, laboratory parameters, and clinical outcomes of the three groups.</p

Laboratory parameters and clinical outcomes of the syphilis and control groups.

<p>Note: The quality screening criteria for high-quality embryos included the presence of 7–8 blastomeres of equal size with an accumulation of <20% cell debris by day 3 (Embryology, 2011).</p><p>Normally fertilized oocytes were defined as those with two pronuclei (2PN) and 2 polar bodies at 16–20 hours after oocyte retrieval.</p><p>The normal fertilization rate was calculated as follows: number of normally fertilized oocytes / (number of oocytes with one pronucleus + number with two pronuclei + number with multiple pronuclei + number with late cleavage).</p><p>The implantation rate (IR) was defined as the number of gestational sacs per number of embryos transferred.</p><p>The live birth rate per embryos transferred was defined as the total number of births per the number of embryos transferred that resulted in clinical pregnancy.</p><p>The clinical pregnancy rate per embryo transferred was determined by the number of patients with a gestational sac in the uterus at 5 weeks after embryo transfer.</p><p>Early miscarriage was defined as the natural termination of pregnancy before 12 weeks, even in the presence of a positive serum hCG test or an ultrasound-detected intrauterine gestational sac after embryo transfer.</p><p>Biochemical pregnancy was defined as a pregnancy that did not clinically progress, accompanied by a β-hCG level of ≥25 U/L.</p><p>a The independent samples t-test was performed to compare the syphilis and control groups, and the values are presented as the mean ± standard deviation.</p><p>b A significant difference was detected between the syphilis and control groups using the independent samples t-test (<i>P</i><0.05).</p><p>c The Mann-Whitney test was conducted to compare the non-normally distributed data, and the results are shown as the median (range).</p><p>d Data were obtained using Pearson’s chi-square test.</p><p>e A significant difference was detected between the syphilis and control groups using Pearson’s chi-square test (<i>P</i><0.05).</p><p>^f Data were obtained using Fisher’ Exact Test.</p><p>Laboratory parameters and clinical outcomes of the syphilis and control groups.</p

Demographic and cycle characteristics of patients diagnosed with syphilis and their respective healthy controls.

<p>Note: BMI = body mass index; FSH = follicle-stimulating hormone; LH = luteinizing hormone.</p><p>a The independent samples t-test was conducted to compare the syphilis and control groups, and the values are presented as the mean ± standard deviation.</p><p>b A significant difference was detected between the syphilis and control groups using the independent samples t-test (<i>P</i><0.05).</p><p>Demographic and cycle characteristics of patients diagnosed with syphilis and their respective healthy controls.</p

Overexpressed CXCR4 in HGF-stimulated MDA-MB-436 cells is functional.

<p>(A–B). Confluent cells were grown in 0.5% FBS medium for 24 hours and were then wounded with a tip. The cells were washed, and the medium was replaced with or without addition of HGF, PSζ peptides or AMD3100. Representative micrographs of the wounds are shown together with the results of the migration quantification. Results are presented as the mean ± SD of 3 independent experiments. # P<0.01 as compared to PBS. Original magnification, 200×. (C).Dose and time-dependent response of HGF-induced MDA-MB-436 cell migration. (D).Dose-dependent response of HGF-induced MDA-MB-436 cell chemotaxis. (E).PKC and CXCR4 regulate HGF-mediated chemotaxis. MDA-MB-436 cells were incubated for 30 minutes with 10 µM chelerythrine chloride, an inhibitor of all PKC, or for 1 hour with 10 µM of PS-α/β, PS-ε, or PSζ peptide. (F). Boyden chamber assays were performed using the SDF-1 ligand for CXCR4 as a chemotactic attractive agent in the lower chamber. AMD3100, PSζ, PSα/β, PSε and NSC23766 (upper) or PKCζ-siRNA (lower) was added to the cell culture for the blocking assay. Data are shown as the mean ± SD of three experiments. A representative study is shown. (G). qRT-PCR analysis of the MT1-MMP mRNA extracted from MDA-MB-436 cells cultured for 24 hours with the indicated agents. Results are presented as the mean ± SD of three independent experiments. # P<0.01 as compared to PBS. (H). Western blot analysis of the total protein expression levels of MT1-MMP in MDA-MB-436 cells cultured for 24 hours with the indicated agents. The experiment was repeated three with similar results. A representative study is shown.</p

HGF upregulates CXCR4 expression and membrane presentation in human breast cancer cells.

<p>(A). qRT-PCR analysis of the total CXCR4 mRNA extracted from HGF- or SDF-1-treated MDA-MB-436 and MCF-7 cells. Results are presented as the mean ± SD of three independent experiments. # P<0.01 as compared to PBS. (B). Western blot analysis of the total protein expression and phosphorylation levels of Met or CXCR4 in MDA-MB-436 and MCF-7 cells cultured for 24 hours with and without the presence of 50 ng/ml HGF or 20 ng/ml SDF-1. The experiment was repeated twice with similar results. A representative study is shown. (C). Time course of the relative extracted CXCR4 mRNA expression in MDA-MB-436 and MCF-7 cells following stimulation with 50 ng/ml HGF. Results are presented as the mean ± SD of three independent experiments. # P<0.01,* P<0.05 as compared to PBS. (D). Western blot analyses of CXCR4 protein expression in HGF-treated MDA-MB-436 and MCF-7 cells. The experiment was repeated three times with similar results. A representative study is shown. (E). Flow cytometric analysis of MDA-MB-436 cells stained for the expression of CXCR4 using a CXCR4 monoclonal antibody and isotype controls. The experiment was repeated three times with similar results. A representative study is shown. (F). Increased membrane and intracellular CXCR4 labeling in MDA-MB-436 cells incubated for 24 hours in the presence of 50 ng/ml HGF compared with untreated cells (PBS) taken as 1. Flow cytometry analysis data (mean±SD of three independent experiments) are shown. # P<0.01,* P<0.05 as compared to PBS. (G). Decreased internalization rate of anti-CXCR4-PE mAb in MDA-MB-436 cells treated for 24 hours with HGF compared with PBS. Results are presented as the mean ± SD of three independent experiments. * P<0.05 as compared to PBS at each time point.</p