CD44 Protein Expression Research Articles

Effects of bio-strength electric field on the motility and CD9 expression of human epidermal cell line HaCaT and mouse epidermal cells

Objective: To investigate the regulatory effect of bio-strength electric field (EF) on the motility and CD9 expression of human epidermal cell line HaCaT and mouse epidermal cells. Methods: The experimental research method was used. Human immortal epidermal cell line HaCaT cells in logarithmic growth phase and primary epidermal cells isolated from 16 BALB/c mice (no matter male or female) aged 1-3 days were used for experiments. HaCaT cells were divided into EF group treated for 3 h at the EF intensity of 200 mV/mm and sham EF group with simulated treatment. The cell migration (direction, displacement velocity, and trajectory velocity, with 46 samples in EF group and 34 samples in sham EF group) and arrangement were observed in the living cell workstation, and the distribution and expression of CD9 protein were detected by immunofluorescence method. Both HaCaT cells and mouse epidermal cells were divided into sham EF group (simulated treatment) and EF groups treated respectively for 3 h at the corresponding EF intensity of 50, 100, 200, and 400 mV/mm. Both HaCaT cells and mouse epidermal cells were divided into blank control group without any treatment, and 1 h group, 3 h group, and 6 h group treated with EF at the intensity of 200 mV/mm for corresponding time respectively. The expression of CD9 protein was detected by Western blotting (n=3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test and least significant difference test. Results: Within 3 hours of treatment, HaCaT cells in EF group tended to move towards the negative electrode obviously, while HaCaT cells in sham EF group moved randomly around the origin; compared with those of sham EF group, the directivity of HaCaT cells in EF group was significantly enhanced, and the displacement velocity and trajectory velocity were significantly increased (Z=-3.975, -6.052, -6.299, P<0.01). After 3 hours of treatment, the long axis of HaCaT cells in EF group was perpendicular to the direction of EF, while HaCaT cells in sham EF group arranged randomly. After 3 hours of treatment, the expression of CD9 protein in HaCaT cells in EF group was significantly down-regulated compared with that of sham EF group (t=4.527, P<0.01), although both expressed on cytomembrane. After 3 hours of treatment, the expression of CD9 protein in HaCaT cells and mouse epidermal cells in sham EF group, 50 mV/mm group, 100 mV/mm group, 200 mV/mm group, and 400 mV/mm group were 0.332±0.021, 0.283±0.032, 0.254±0.020, 0.231±0.041, 0.212±0.031 and 0.565±0.021, 0.453±0.022, 0.389±0.020, 0.338±0.021, 0.233±0.011, respectively. For both types of cells, compared with that of sham EF group, the expression of CD9 protein in cells was significantly decreased in the four groups of EF treatment (P<0.01); compared with that of 50 mV/mm group, the expression of CD9 protein in cells was significantly decreased in the other three groups of EF treatment (P<0.01); compared with that of 100 mV/mm group, the expression of CD9 protein in cells was significantly decreased in 200 mV/mm group and 400 mV/mm group (P<0.01); compared with that of 200 mV/mm group, the expression of CD9 protein in cells was significantly decreased in 400 mV/mm group (P<0.01). The expression levels of CD9 protein in HaCaT cells and mouse epidermal cells in blank control group, 1 h group, 3 h group, and 6 h group were 0.962±0.031, 0.784±0.020, 0.531±0.021, 0.409±0.011 and 0.963±0.031, 0.872±0.031, 0.778±0.040, 0.591±0.041, respectively. For both types of cells, compared with that of blank control group, the expression of CD9 protein in cells was significantly decreased in 1 h group, 3 h group, and 6 h group (P<0.01); compared with that of 1 h group, the expression of CD9 protein in cells was significantly decreased in 3 h group and 6 h group (P<0.05 or P<0.01); compared with that of 3 h group, the expression of CD9 protein in cells was significantly decreased in 6 h group (P<0.01). Conclusions: The bio-strength intensity EF can induce the directional migration and arrangement of HaCaT cells and down-regulate the expression of CD9 in HaCaT cells and mouse epidermal cells in a time-dependent and intensity-dependent manner.

Calcium-Sensing Receptor Participates in High Glucose-Induced EndMT in Primary Human Aortic Endothelial Cells.

ObjectivePrevious studies have shown that high glucose (HG) induces endothelial cell (EC) damage via endothelial-to-mesenchymal transition (EndMT). Although the underlying mechanisms are still unclear, recent studies have demonstrated the role of calcium-sensing receptor (CaSR) in mediating EC damage. Therefore, the aim of our study was to investigate whether CaSR mediates HG-induced EndMT and to determine the underlying mechanism.MethodsBioinformatics analysis of microarray profiles (GSE30780) and protein-protein interaction (PPI) analyses were performed to select the hub genes. As for in vitro research, the human aortic ECs (HAECs) were exposed to HG to induce EndMT. The expression of CaSR and β-catenin was determined, as well as their effects on EndMT (endothelial marker CD31, mesenchymal marker FSP1, and α-SMA).ResultsThe bioinformatics analysis indicated CaSR was significantly increased in HG-treated HAECs and was one of the hub genes. The in vitro results showed that HG significantly inhibited the expression of CD31 and increased FSP1 and α-SMA in a concentration- and time-dependent manner. Moreover, CaSR was increased in HAECs after HG treatment. The CaSR antagonist attenuated HG-induced expression of EndMT-related markers. Furthermore, HG treatment increased the nuclear translocation of β-catenin in HAECs. In contrast, blocking the nuclear translocation of β-catenin by DKK1 could attenuate HG-induced EndMT (increased the protein expression of CD31 by 30% and decreased the protein expression of FSP1 by 15% and α-SMA by 25%). CaSR siRNA further inhibited the HG-induced nuclear translocation of β-catenin in HAECs.ConclusionOur research demonstrated that HG-induced EndMT in HAECs might be mediated by CaSR and the downstream nuclear translocation of β-catenin.

CD47 is highly expressed in gliomas and targeting CD47 is a promising therapeutic strategy

Gliomas are very malignant brain tumors that are difficult to treat. CD47 is an antiphagocytic molecule that binds to SIPRα on phagocytes. It is overexpressed on the plasma membranes of multiple human tumor cell types and is an important diagnostic and prognostic biomarker in many types of cancer. However, the association between CD47 protein expression in glioma tissue and clinicopathological stage has not been investigated in detail. A total of 80 surgical glioma specimens were stained with anti-CD47 antibody to assess the relationship between CD47 protein expression and clinicopathological stage of the glioma. Wound healing assays were performed to analyze the influence of CD47 on the migration and invasion of glioma cells, and near-infrared fluorescence localization assays in a U-87 MG-bearing xenograft model were used to determine the distribution of anti-CD47 antibody in vivo. MTT assays and administration of anti-CD47 to a U251-bearing xenograft model were used to analyze the inhibitory effects of the antibody on gliomas. CD47 expression was higher in high-grade gliomas than in low-grade gliomas, and high CD47 expression was positively correlated with histology and tumor clinicopathological stage. CD47 over-expression promoted the growth and motility of two glioma cell lines (U-87 MG and U251) and a laboratory-developed anti-CD47 antibody accumulated at the glioma site. Proliferation of U251 and U-87 MG cells was not significantly inhibited by the anti-CD47 antibody in vitro, but the antibody significantly inhibited U251 growth in vivo. It also enhanced inhibition capacity by Taxol. Our results suggest that CD47 plays a critical role in the progression of gliomas from stage I to IV and may be a potential target for the treatment of gliomas. CD47 appears to play a critical role in the progression of gliomas from stage I to IV and an anti-CD47 antibody prepared in the laboratory may inhibit the growth of gliomas.

Estrogen modulates epithelial progenitor cells in rat vagina.

PurposeThe expression of epithelial progenitor cells (EPCs) in rat vagina was recently reported. The aims were to investigate the effects of estrogen on vaginal EPCs in the oophorectomized female rat model.Materials and MethodsFemale Sprague-Dawley rats (230–240 g, n=30) were divided into 3 groups: control (n=10), bilateral oophorectomy (OVX, n=10), and bilateral OVX followed by subcutaneous injections of 17β-estradiol (50 µg/kg/day, n=10). After 4 weeks, the expression of EPC-specific markers (CD44, estrogen receptor alpha [ERα], and progesterone receptor) were evaluated by immunohistochemistry and Western blot.ResultsThe CD44/ERα double-labeled cells were mainly expressed in basal cell layers and suprabasal layers as shown by confocal immunofluorescence. Confocal microscopy revealed that the number of CD44+/ERα+ cells decreased in the OVX group compared with the controls but was similar to control levels in rats receiving estrogen replacements. The protein expression of CD44 and ERα decreased after OVX and was restored to control levels after estrogen supplementation.ConclusionsMarkers of EPCs were expressed in the vagina, and the expression of resident EPCs was regulated by estrogen. These findings imply that resident EPCs may have an important role in the regeneration of vaginal mucosa by estrogen replacement.

Correlation between CD 34 and CD 68 expression in placental malaria with maternal anemia.

Background:Malaria is the second most life-threatening infectious disease in Indonesia, causing approximately 1–3 million deaths annually. Histopathologic studies assessing CD 68 and CD 34 protein expression in placental malaria and its association with maternal anemia are essential to determine the prognosis of malaria in pregnancy.Materials and Methods:This cross-sectional study was carried out in 2017. Thirty biopsy samples of human placental tissue were obtained from Timika and Sumba, and ten normal biopsy samples were taken from the Pathological Anatomy Department of Cipto Mangunkusumo General Hospital as comparisons. CD 34 and CD 68 protein expressions were determined using immunohistochemistry, and the resulting data were analyzed using SPSS.Results:Average hemoglobin (Hb) level was 9.5 mg/dL, 11.5 mg/dL, and 9.9 mg/dL in acute infection, chronic infection, and latent infection, respectively. A positive correlation was found between CD 68 protein expression and maternal Hb level. No correlation was found between CD34 expression and maternal anemia.Conclusions:CD 68 expression in placental tissue biopsy from Timika and Sumba residents with placental malaria was shown to be positively correlated with maternal anemia. Immunohistochemical examination of CD 68 may play a role in the early diagnosis of malaria.

The cluster of differentiation-36 (CD-36) as a promising marker for many diseases

CD36 is a transmembrane protein involved in fatty acid translocation, scavenging for oxidized fatty acids acting as a receptor for adhesion molecules. It is expressed on macrophages, as well as other types of cells, such as endothelial and adipose cells. CD36 participates in muscle lipid uptake, adipose energy storage, and gut fat absorption. Recently, several preclinical and clinical studies demonstrated that the upregulation of CD36 is a prerequisite for tumor metastasis. Cancer metastasis-related research emerged much later and has been less investigated, though it is equally or even more important. CD36 protein expression can be modified by epigenetic changes and post-transcriptional interference from non-coding RNAs. Some data indicate modulation of CD36 expression in specific cell types by epigenetic changes via DNA methylation patterns or histone tails, or through miRNA interference, but this is largely unexplored. The few papers addressing this topic refer mostly to lipid metabolism-related pathologies, whereas in cancer research, data are even more scarce. The aim of this review was to summarize major epigenetic and post-transcriptional mechanisms that impact CD36 expression in relation to various pathologies while highlighting the areas in need of further exploration.

Desulfovibrio vulgaris, a potent acetic acid-producing bacterium, attenuates nonalcoholic fatty liver disease in mice

ABSTRACT The emerging evidence supports the use of prebiotics like herb-derived polysaccharides for treating nonalcoholic fatty liver disease (NAFLD) by modulating gut microbiome. The present study was initiated on the microbiota-dependent anti-NAFLD effect of Astragalus polysaccharides (APS) extracted from Astragalus mongholicus Bunge in high-fat diet (HFD)-fed mice. However, the exact mechanisms underlying the beneficial effects of APS on NAFLD formation remain poorly understood. Co-housing experiment was used to assess the microbiota dependent anti-NAFLD effect of APS. Then, targeted metabolomics and metagenomics were adopted for determining short-chain fatty acids (SCFAs) and bacteria that were specifically enriched by APS. Further in vitro experiment was carried out to test the capacity of SCFAs-producing of identified bacterium. Finally, the anti-NAFLD efficacy of identified bacterium was tested in HFD-fed mice. Our results first demonstrated the anti-NAFLD effect of APS in HFD-fed mice and the contribution of gut microbiota. Moreover, our results indicated that SCFAs, predominantly acetic acid were elevated in APS-supplemented mice and ex vivo experiment. Metagenomics revealed that D. vulgaris from Desulfovibrio genus was not only enriched by APS, but also a potent generator of acetic acid, which showed significant anti-NAFLD effects in HFD-fed mice. In addition, D. vulgaris modulated the hepatic gene expression pattern of lipids metabolism, particularly suppressed hepatic fatty acid synthase (FASN) and CD36 protein expression. Our results demonstrate that APS enriched D. vulgaris is effective on attenuating hepatic steatosis possibly through producing acetic acid, and modulation on hepatic lipids metabolism in mice. Further studies are warranted to explore the long-term impacts of D. vulgaris on host metabolism and the underlying mechanism.

Co-expression of CD24 and Hsp70 as a prognostic biomarker for lung cancer

Lung cancer is one of the most common malignant neoplasms worldwide. CD24 is a marker of tumor stem cells that plays an important role in tumorigenesis. Hsp70 is an important molecular chaperone. However, the co-expression and interaction of CD24 and Hsp70, as well as the significance for the prognosis of lung cancer are still unclear. The expression levels of CD24 and Hsp70 were detected by immunohistochemistry and their correlation was analyzed. The expression levels of CD24 mRNA and protein were examined using qRT-PCR and western blotting in SPCA1, A549, H1975, and H1650 cell lines. A CD24-overexpressing cell model was established. The interaction between CD24 and Hsp70 was verified by co-immunoprecipitation and western blotting. CD24 and Hsp70 expression were significantly higher in lung cancer tissues than in adjacent tissues (CD24: p=0.008; Hsp70: p<0.001). CD24 protein expression showed a positive correlation with lymph node metastasis, TNM stage, and vascular cancer thrombus. Hsp70 protein expression showed a positive correlation with differentiation, lymph node metastasis, and TNM stage. CD24 and Hsp70 high expression were also correlated with poor survival. The positive co-expression rate of CD24 and Hsp70 in lung cancer tissues was 52.7% (49/93). CD24 and Hsp70 expression in lung cancer were positively correlated (r=0.368, p<0.001), and co-immunoprecipitation was verified that both endogenous and exogenous CD24 co-precipitated with Hsp70 directly or indirectly. When Hsp70 inhibitor VER15508 was added to A549 cells, Hsp70 and CD24 protein expression were significantly decreased. The present study demonstrated that CD24 and Hsp70 were highly expressed in lung cancer tissues, and associated with invasion, metastasis, and poor survival. Hsp70 may regulate CD24 expression. Co-expression of CD24 and Hsp70 may be a prognostic biomarker for lung cancer.

Elevated serum and urine levels of progranulin (PGRN) as a predictor of microglia activation in the early phase of traumatic brain injury: a further link with the development of neurodegenerative diseases.

Traumatic brain injury (TBI) is afrequent finding during forensic autopsies and neuropathological examinations in medico-legal practices. Despite the unprecedented attention currently focused on TBI pathogenesis, there is aneed to improve its diagnostics through the use of novel biomarkers to facilitate detection, treatment, and prognosis. Recently, growth factor progranulin (PGRN) has attracted significant attention because of its neurotrophic and anti-inflammatory activities. The role of PGRN in TBI has not been widely discussed, although PGRN-related neuroinflammatory and neurodegenerative phenomena have been described. The aim of this study was to identify PGRN concentration levels in biofluids and examine PGRN and CD68 protein expression in brain tissue using immunohistochemical staining in individuals with fatal TBI in its early phase. The study was performed using cases (n = 30) of fatal head injury and control cases (n = 30) of sudden death. The serum and urine were collected within ~24 h after death and compared using the ELISA test, where brain specimens were stained with anti-PGRN and anti-CD68 antibodies. In our study, we observed elevated concentration levels of PGRN in the serum and urine of TBI individuals in the early phase of TBI. These changes were accompanied by increased expression of PGRN in the frontal cortex (1st-3rd layers), in which anti-CD68 immunostaining revealed disseminated cortical microglia activation. The possible implementation of performing such assays offers anovel and interesting tool for investigation and research regarding TBI diagnosis and pathogenesis. Furthermore, the above-mentioned surrogate biofluid assays may be useful in clinical prognosis and risk calculation of non-fatal cases of TBI, considering the development of neurodegenerative conditions of TBI individuals.

Interaction of CD147 and human epididymis protein 4 promotes invasion and metastasis of ovarian cancer.

Background: Ovarian cancer is one of the most common malignant tumors in female reproductive system. The expression of CD147 and human epididymis protein 4 (HE4) are both upregulated and associated with malignant progression in ovarian cancer. However, the important role of interaction between CD147 and HE4 in the invasion and metastasis of ovarian cancer remains unclear.Methods: The co-expression and co-localization of CD147 and HE4 in cells and tissues of ovarian cancer were detected by co-immunoprecipitation, immunohistochemistry and immunocytochemistry, double-labeling immunofluorescence method. The interaction between CD147 and annexin A2 (ANXA2) was also investigated. Furthermore, we detect the regulatory relationship between CD147 and HE4 by Western blot. Transwell assay and scratch test were conducted to explore the effect of CD147-HE4 interaction on migration and invasion of ovarian cancer.Results: The protein CD147 and HE4 were co-immunoprecipitated and co-located in the cytoplasm and membrane of ovarian cancer tissues and cells. Both of two proteins were highly expressed and positively associated with advanced FIGO stages, poor differentiation degree and poor prognosis of ovarian cancer, and HE4 was confirmed as an independent risk factor for CD147 in ovarian cancer tissues. What's more, AXNA2 was also identified as a CD147 interacting protein in ovarian cancer. It is further confirmed that interaction between CD147 and HE4 can affect the invasion and metastasis of ovarian cancer.Conclusion: This study demonstrated that HE4 and ANXA2 were both CD147 interacting proteins, the expression of CD147 and HE4 can affect each other, and HE4 could promote the invasion and metastasis of ovarian cancer by regulating the expression of CD147, which may provide novel thought for early diagnosis and therapeutic target of ovarian cancer.

Vitamin E succinate exerts anti-tumour effects on human cervical cancer cells via the CD47-SIRPɑ pathway both in vivo and in vitro.

Vitamin E succinate (RRR-a-tocopheryl succinate, VES) acts as a potent agent for cancer therapy and has no toxic and side effects on normal tissue cells. However, the mechanism by which VES mediates the effects are not yet fully understood. Here, we hypothesised that VES mediates antitumour activity on human cervical cancer cells via the CD47-SIRPɑ pathway in vivo and in vitro. Results indicated that the human cervical cancer HeLa cells treated with VES were more efficiently engulfed by THP-1-derived macrophages. In response to VES, the protein expression of CD47 on cell membranes and the mRNA level of CD47 in different human cervical cancer cells significantly decreased. And the level of calreticulin (CRT) mRNA in the VES-treated cells increased. By contrast, CRT protein expression was not altered. miRNA-155, miRNA-133 and miRNA-326 were up-regulated in the VES-treated HeLa cells. Knocking down miRNA-155 and miRNA-133 by RNA interference increased CD47 protein expression in the VES-treated cells. In vivo efficacy was determined in BALB/C nude mice with HeLa xenografts. Results showed that VES reduced tumour growth, increased overall survival and inhibited CD47 in the tumour transcriptionally and translationally. Furthermore, inflammatory factors (TNF-α, IL-12, IFN-γ, IL-2 and IL-10) in the spleen were altered because of VES treatment. Our results suggest that VES-induced antitumour activity is coupled to the CD47-SIRPɑ pathway in human cervical HeLa cancer cells.

Benzidine promotes the stemness of bladder cancer stem cells via activation of the Sonic hedgehog pathway.

Substantial evidence suggests that cancer stem cells (CSCs) are the main cause of the initiation, progression and recurrence of tumors. Benzidine has been identified as a risk factor for bladder cancer. The aim of the present study was to investigate the effects of benzidine on bladder CSCs (BCSCs) and the possible mechanism underlying its action. The bladder cancer cell lines UM-UC-3 and EJ were maintained in serum-free medium and cells forming three-dimensional spheres were characterized as BCSCs. The sphere-forming cells were exposed to different concentrations of benzidine and vismodegib, and western blotting was performed to evaluate the expression of markers associated with CSCs and the Sonic hedgehog (SHH) signaling pathway. Flow cytometry was used to detect the distribution of cells in different phases of the cell cycle, and immunofluorescence staining was used to detect the protein expression of CD44. The results revealed that the levels of BCSC markers, namely CD133, CD44, aldehyde dehydrogenase 1-A1, Nanog and octamer-binding transcription factor-4, in the cell spheres were markedly elevated compared with those in cells cultured in serum-supplemented medium. Furthermore, benzidine increased the expression of BCSC markers and promoted the sphere-forming ability of the cells. In addition, it was observed that benzidine activated the SHH pathway, while inhibition of the Shh pathway using vismodegib diminished the promoting effects of benzidine on BCSCs. The findings of the present study indicate that benzidine promoted the stemness of BCSCs via activation of the SHH pathway, which may support further exploration of the molecular basis of the association between benzidine exposure and bladder oncogenesis.

Age, sex, and regional differences in scavenger receptor CD36 in the mouse brain: Potential relevance to cerebral amyloid angiopathy and Alzheimer's disease.

Scavenger receptor CD36 contributes significantly to lipid homeostasis, inflammation, and amyloid deposition, while CD36 deficiency is associated with restored cerebrovascular function in an Alzheimer's disease (AD) mouse model. Yet the distribution of CD36 has not been examined in the brain. Here, we characterized CD36 gene and protein expression in the brains of young, middle aged, aged, and elderly male and female C57BL/6J mice. Age-related increases in CD36 mRNA expression were observed in the male hippocampus and female midbrain. Additionally, male mice had greater CD36 mRNA expression than females in the striatum, hippocampus, and midbrain. CD36 protein was primarily expressed intravascularly, and this expression differed by region, age, and sex in the mouse brain. Although male mice brains demonstrated an increase in CD36 protein with age in several cortices, basal ganglia, hippocampus, and midbrain, a decrease with age was observed in female mice in the same regions. These data suggest that distinctive age, region, and sex expression of CD36 in the brain may contribute to Aβ deposition and neuroinflammation in AD.

Attenuation of Perfluorooctane Sulfonate-Induced Steatohepatitis by Grape Seed Proanthocyanidin Extract in Mice

Perfluorooctane sulfonate (PFOS), an environmentally persistent pollutant, has been revealed to elicit hepatic toxicity. In the current study, we investigated the protective role of grape seed proanthocyanidin extract (GSPE) against PFOS-caused steatohepatitis in mice. Animals were exposed intragastrically to PFOS (10 mg/kg/day), GSPE (150 mg/kg/day), or their combination. After 21 days of treatment, mice exposed to PFOS exhibited steatosis, oxidative stress, and inflammation in the liver. Nevertheless, simultaneous administration of GSPE resumed the declined serum hepatic enzyme activities and histological abnormalities in PFOS-exposed mice. Furthermore, GSPE supplementation reduced the contents of triglyceride (TG) and total cholesterol (TC) and expression of lipid metabolism-associated genes CD36 and fatty acid-binding protein 4 (FABP4) in the liver of mice treated with PFOS. Moreover, GSPE suppressed the generation of lipid peroxidative product malondialdehyde and restored the activity of superoxide dismutase in the liver of PFOS-exposed mice. In addition, GSPE repressed the PFOS-induced hepatic overproduction of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Our results demonstrate that GSPE attenuates PFOS-caused steatohepatitis in mice by regulating lipid metabolism, oxidative stress, and inflammatory response.

Exosome EpCAM promotes the metastasis of glioma by targeting the CD44 signaling molecule on the surface of glioma cells.

Glioma, the most common primary tumor in the central nervous system, originates from glial cells and has a poor prognosis. This experimental laboratory study was designed to explore the role of epithelial cell adhesion molecule (EpCAM) in the metastasis of glioma. Serum samples were collected from patients with non-metastatic or metastatic glioma (n = 20 per group), and healthy volunteers (n = 8). Exosomes were isolated from the serum and the morphological characteristics were observed under a scanning electron microscope (SEM). The expression of CD81 and CD63 was measured to identify exosomes. Glioma tissue and the adjacent normal tissue samples were obtained from patients with non-metastatic or metastatic glioma (n = 12 per group). Meanwhile, 4 normal brain tissue samples were collected. The expression of CD44, hyaluronan-mediated motility receptor (HMMR), and matrix metalloproteinase-9 (MMP-9) was determined in each group using immunohistochemistry. The protein expression of CD44, HMMR, matrix metalloproteinase-2 (MMP-2), MMP-9, and selectin E (SELE) was measured with western blotting. Exosomes were present in the serum, and the proteins CD81 and CD63 were expressed in all 3 groups. CD44 was highly expressed in the non-metastasis and metastasis groups. The expression of HMMR and MMP-9 in the Adj-metastasis and Adj-non-metastasis groups was high, while in the other groups, the levels were low. The expression of CD44 in the metastasis and non-metastasis groups was significantly higher than that of the negative control (NC) group, and the expression in the metastasis group was higher than that of the non-metastasis group. The MMP-2 and MMP-9 were not found in either the metastasis or non-metastasis group. The protein expression of HMMR and SELE was high in all groups. Exosome EpCAM promoted the metastasis of glioma by targeting CD44.

Key Role of CD151-integrin Complex in Lung Cancer Metastasis and Mechanisms Involved.

Tetraspanin CD151 was found to be upregulated in malignant cell types and has been identified as a tumor metastasis promoter. In this study, we aimed to examine the role of the CD151-integrin complex in lung cancer metastasis and the underlying mechanisms. CD151 QRD194-196 →AAA194-196 mutant was generated and used to transfect A549 human lung adenocarcinoma cells. We found that there was no significant difference in CD151 protein expression between CD151 and CD151-AAA mutant groups. In vitro, CD151-AAA mutant delivery abrogated the migration and invasion of A549 cells, which was promoted by CD151 gene transfer. Furthermore, CD151-AAA delivery failed to activate FAK and p130Cas signaling pathways. Western blot and immunohistochemical staining showed strong CD151 expression in lung cancerous tissues but not in adjacent normal tissues. Increased level of CD151 protein was observed in 20 of the patients and the positive rate of CD151 protein in specimens was 62.5% (20/32). In addition, CD151 was co-localized with α3 integrin at the cell-cell contact site in carcinoma tissues. These results suggested that the disruption of the CD151-α3 integrin complex may impair the metastasis-promoting effects and signaling events induced by CD151 in lung cancer. Our findings identified a key role for CD151-α3 integrin complex as a promoter in the lung cancer.

Lidocaine inhibits glioma cell proliferation, migration and invasion by modulating the circEZH2/miR-181b-5p pathway.

Lidocaine is well known as a local anesthetic that has been reported to play an antitumor role in numerous cancers, including glioma. Circular RNAs (circRNAs) play multiple biological roles in cancers. The aim of this study was to determine the effects of lidocaine in glioma in vitro and in vivo and explore functional mechanisms. The effects of lidocaine on glioma progression were investigated by cell proliferation, migration and invasion using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, colony formation assay and transwell assay. The expression of CD133 and glial fibrillary acidic protein (GFAP) was quantified by western blot to assess cell differentiation. The expression of circEZH2 and miR-181b-5p was detected by a quantitative real-time PCR (qRT-PCR). The target relationship between circEZH2 and miR-181b-5p was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The effect of lidocaine on tumor growth in vivo was investigated by establishing Xenograft models. Lidocaine inhibited proliferation, migration, invasion and induced differentiation of glioma cells in vitro. Lidocaine suppressed the expression of circEZH2, and circEZH2 was highly expressed in glioma tissues and cells. CircEZH2 overexpression partly inhibited the function of lidocaine. CircEZH2 was a sponge of miR-181b-5p, and miR-181b-5p was downregulated in glioma tissues and cells. Besides, miR-181b-5p restoration reversed the effects of circEZH2 overexpression to repress the malignant behaviors of glioma cells. In addition, lidocaine mediated the circEZH2/miR-181b-5p axis to inhibit tumor growth in vivo. Lidocaine suppressed glioma progression by modulating the circEZH2/miR-181b-5p pathway.

USP10 deletion inhibits macrophage-derived foam cell formation and cellular-oxidized low density lipoprotein uptake by promoting the degradation of CD36.

Foam cell formation process is involved in the pathogenesis of atherosclerosis (AS). Activation of this biological process depends on lipid uptake by scavenger receptors, such as CD36, SR-A and SR-B1. Among these receptors, CD36 is the principal one because it dominates roughly 50% lipid uptake in monocytes. In this study, our western blotting and RT-qPCR assays revealed that USP10 inhibition promotes the degradation of CD36 protein but does not change its mRNA level. In addition, Co-IP results showed that USP10 interacts with CD36 and stabilizes CD36 protein by cleaving poly-ubiquitin on CD36. Significantly, USP10 promotes foam cell formation. Immunofluorescence and Oil red O staining assays show that inhibition or knockdown of USP10 suppresses lipid uptake and foam cell formation by macrophages. In conclusion, USP10 promotes the development and progression of atherosclerosis through stabilizing CD36 protein expression. The regulation of USP10-CD36 may provide a significant therapeutic scheme in atherosclerosis.

Relationship between CD45 Expression and Outcomes in B Lymphoblastic Leukemia/Lymphoma

Introduction : B lymphoblastic leukemia/lymphoma (B-ALL) constitutes the most common type of lymphoblastic leukemia, with about two thirds of the cases being of B-cell lineage. It represents a significant cause of cancer related morbidity and mortality in both pediatric and adult populations. B-ALL immunophenotype has been extensively studied and used for diagnosis. Recent data underscores the importance of the CD45 transmembrane phosphatase in the modulation of signal transduction pathways in leukemic cells. In fact, the gene encoding CD45, PTPRC, has been identified as a tumor suppressor that is deleted in 10% of cases of T-ALL. Based on the impact of CD45 on leukemic cell biology, we hypothesized a relationship between CD45 protein expression and clinical parameters. Methods : This is a retrospective, single center analysis of patients diagnosed with B-ALL at Indiana University Simon Comprehensive Cancer Center from December 2003 until December 2018. Patients were followed until July 2020. Variables included patient demographics, and clinical and treatment characteristics. B-ALL diagnosis, molecular profiles, and clinical outcomes were abstracted by chart review. Descriptive statistics were used to summarize baseline characteristics and outcomes. Flow cytometry analysis was performed using 4- and 10-color leukemia screening panel. Median and geometric mean of CD45 antigen intensity was analyzed using FCS Express V6 software. Statistical analysis was performed using SPSS statistical package. Results: A total of 148 BALL patients were included in the analysis. 19 (12.8%) were adults (&amp;gt;18 years old) and 13 (8.7%) were Philadelphia chromosome positive. Average Median CD45 expression and CD45 Geometric Mean were 22.9 and 21.9 respectively for the entire cohort. Relevant clinical outcomes collected included complete remission (CR) at days 14 and 29, presence of minimal residual disease (MRD) at days 7 and 29, relapse of disease and death. Mann-Whitney Test was used to establish association between CD45 levels and clinical outcomes. A significant association was found between achieving CR at 29 days and lower levels of CD45 expression (as defined by median and geometric mean, p=0.03 for both). The association was was maintained when excluding Philadelphia positive cases (p=0.05 for both) and excluding patients over 30 years old (p=0.05 for both). There were no significant correlations between CD45 expression and other clinical variables. Conclusions: Lower CD45 expression as determined by flow cytometry is associated with higher rates of CR at 29 days. This finding is significant across different age groups and Philadelphia negative cohorts. This finding needs to be explored in further studies. CD45 protein expression levels could be an important biomarker for adjusting induction chemotherapy to achieve complete remission. Disclosures No relevant conflicts of interest to declare.

Electron Transport Chain Inhibition Suppresses CD40 Expression and Sensitizes Chronic Lymphocytic Leukaemia Cells to Venetoclax

Alterations in expression of specifically BCL-XL and MCL-1 dictate sensitivity of CLL cells to the Bcl-2 specific inhibitor venetoclax (VEN). We and others have shown upregulation of these anti-apoptotic proteins by interaction of CLL cells with CD4+ T helper cells within their lymph node microenvironment (LN-ME) mediated by CD40 signalling. We also reported significant metabolic changes of LN-ME activated CLL cells but whether metabolic alterations can be linked to VEN resistance remains unclear. As VEN is increasingly used in early stages of CLL, better understanding and tools to circumvent VEN resistance are highly needed. We aim to reveal the metabolic adaption of CLL to CD40 signalling in connection with VEN resistance. After in vitro CD40 signalling stimulation of peripheral blood (PB) CLL cells, mitochondrial mass and glucose uptake were measured by flow cytometry, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured on Seahorse XF Analyser. The result demonstrated that CD40 stimulation enhances both oxidative phosphorylation (OXPHOS) and glycolysis. This was also confirmed by microarray and metabolomics analyses, as genes and metabolites involved in these two metabolic pathways are significantly upregulated by CD40 stimulation. To find out whether these pathways are linked to VEN resistance, PB CLL cells were treated with OXPHOS or glycolysis inhibitors during CD40 stimulation. Remarkably, OXPHOS inhibition by electron transport chain (ETC) inhibitors (rotenone, antimycin A and oligomycin) counteracted strongly for VEN resistance, while glycolysis inhibition by 2-Deoxy-D-glucose (2DG) did not. The three ETC inhibitors also attenuated CLL activation, ATP production and NAD levels. Interestingly, complex II inhibition of the ETC (TTFA and DMM) did not affect VEN resistance. Regarding BCL-2 family members induced by CD40 ligation, both MCL-1 and BCL-XL were downregulated by these ETC inhibitors. In addition, OXPHOS inhibition strongly elevates glycolysis, and vice versa, which illustrates a strong metabolic plasticity of CLL cells. To further investigate the cross-talk between CD40 signalling, VEN resistance and mitochondrial metabolism, the three main fuels of the TCA cycle were inhibited: pyruvate (by UK5099), glutamine (by DON) and fatty acids (by etomoxir). Even though the OCR and ECAR were slightly decreased by (combinations of) these fuel inhibitors, neither CD40 signalling nor VEN sensitivity was affected. Next, we inhibited PI3K by idelalisib, BTK by ibrutinib and mTOR by rapamycin, which are three downstream targets of CD40 signalling. The results showed that only rapamycin inhibited CD40 activation and metabolic activities, and none of the three inhibitors counteracts VEN resistance. Lastly, we investigated CD40 splicing and overall expression. Interestingly, CD40 stimulation has a huge impact on CD40 expression itself, and these changes were blocked by ETC inhibition. These data indicate that ETC inhibition affects CD40 signals to counteract VEN resistance, by directly affecting the expression of CD40 protein on the cell membrane. In conclusion, after CD40 stimulation, CLL cells become metabolically activated and highly flexible in the use of mitochondrial fuels. The enhanced OXPHOS but not glycolysis contributes to VEN resistance, while ETC inhibition reverses CLL VEN resistance by directly suppressing CD40 expression on CLL. These findings link CLL metabolism directly to CD40 transcription and signalling, which may contribute to clinical VEN resistance. Disclosures van der Windt: genmab: Current Employment. Kater:Abbvie: Research Funding; Roche: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Genentech: Research Funding. Eldering:Genentech: Research Funding; Celgene: Research Funding; Janssen: Research Funding.