The role of a new CD44st in increasing the invasion capability of the human breast cancer cell line MCF-7

<p>Abstract</p> <p>Background</p> <p>CD44, a hyaluronan (HA) receptor, is a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines and growth factors to the corresponding receptors, and docking of proteases at the cell membrane, as well as in signaling for cell survival. The CD44 gene contains 20 exons that are alternatively spliced, giving rise to many CD44 isoforms, perhaps including tumor-specific sequences.</p> <p>Methods</p> <p>Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to detect CD44st mRNA and CD44 protein in sensitive MCF-7, Lovo, K562 and HL-60 cell lines as well as their parental counterparts, respectively. The full length cDNA encoding CD44st was obtained from the total RNA isolated from MCF-7/Adr cells by RT-PCR, and subcloned into the pMD19-T vector. The CD44st gene sequence and open reading frame were confirmed by restriction enzyme analysis and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44st was transfected into MCF-7 cells using Lipofectamine. After transfection, the positive clones were obtained by G418 screening. The changes of the MMP-2 and MMP-9 genes and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells penetrating through the artificial matrix membrane in each group (MCF-7, MCF-7+HA, MCF-7/neo, MCF-7/neo+HA, MCF-7/CD44st, MCF-7/CD44st+HA and MCF-7/CD44st+Anti-CD44+HA) was counted to compare the change of the invasion capability regulated by the CD44st. Erk and P-Erk were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by the CD44st.</p> <p>Results</p> <p>Sensitive MCF-7, Lovo, K562 and HL-60 cells did not contain CD44st mRNA and CD44 protein. In contrast, the multidrug resistance MCF-7/Adr, Lovo/Adr, K562/Adr and HL-60/Adr cells expressed CD44st mRNA and CD44 protein. The CD44st mRNA gene sequence was successfully cloned into the recombinant vector pcDNA3.1 and identified by the two restriction enzymes. It was confirmed that the reconstructed plasmid contained the gene sequence of CD44st that was composed of exons 1 to 4, 16 to 17, and 1 to 205 bases of exons 18. The new gene sequence was sent to NCBI for publication, and obtained the registration number FJ216964. The up-regulated level of the mRNA of the CD44 gene and the CD44 protein were detected, respectively, by RT-PCR and flow cytometry in MCF-7 cells transfected with pcDNA3.1-CD44st. The invasiveness of the cells and the activity of MMP-2 and MMP-9 were clearly activated by HA treatment, and blocked by CD44 neutralizing antibody. MCF-7/CD44st cells pretreated with the neutralizing antibody against CD44, and the inhibitor of MAPKs signaling pathway, could strongly block the expression of P-Erk.</p> <p>Conclusions</p> <p>A new CD44st was expressed in multidrug resistant MCF-7/Adr, Lovo/Adr, K562/Adr and HL-60/Adr cells. The expression vector pcDNA3.1-CD44st was cloned and constructed successfully, and stably transfected into MCF-7 cells. HA could interact with the new CD44st and regulate the expression of MMP-2 and MMP-9, which could increase the invasion capability of MCF-7 cells through the Ras/MAPK signaling pathway.</p

Influencia de la infección subclínica por agentes de la fiebre por garrapatas en vacas lecheras

ABSTRACTObjective. The objective of this study was to evaluate the effect of subclinical infection by agents of tick fever in dairy cattle on milk parameters, such as production, composition, and quality. Materials and methods. The study was conducted in a private farm with 75 free-stall-housed dairy cows, from which 37 were evaluated. Monthly, individual milk samples were collected for compositional (fat, lactose, protein, and total solids) and quality (somatic cell counts (SCC)) analyses. In addition, blood samples were collected in order to identify cows that were tick fever-negative and positive by PCR for one or more of the following etiological agents: Babesia bovis, Babesia bigemina and Anaplasma marginale. Results. The results showed increased SCC in positive animals for at least one of the agents when compared to non-infected cows (p&lt;0.05). Milk production was significantly lower in A. marginale positive animals (p&lt;0.05). An increase of about 40% in milk solids content was found in B. bovis positive cows. Also, an increment of approximately 23% in lactose was found on cows positives for B. bigemina. Conclusions. We may conclude that the presence of at least one of these parasites in dairy cattle affects composition or quality of their milk.RESUMENObjetivo. El objetivo de este estudio fue evaluar el efecto de la infección subclínica por agentes de la fiebre por garrapatas en el ganado lechero en producción de leche, la composición y calidad. Materiales y métodos. El estudio se realizó en una finca privada con 75 vacas lecheras alojadas-libre puesto, y de estas se evaluaron 37. Se recogieron muestras de leche individuales mensuales para determinar la composición (grasa, lactosa, proteína y sólidos totales) y la calidad (recuento de células somáticas (SCC)). Además, se recogieron muestras de sangre para identificar vacas que fueron negativas a fiebre de garrapatas y positivos por PCR para uno o más de los siguientes agentes etiológicos: Babesia bovis, Babesia bigemina y Anaplasma marginale. Resultados. Los resultados mostraron un aumento de SCC en los animales positivos, al menos para uno de los agentes cuando se comparó con vacas no infectadas (p&lt;0.05). La producción de leche fue significativamente menor en A. marginale animales positivos (p&lt;0.05). Un aumento de aproximadamente el 40% en el contenido de sólidos de la leche fue encontrado en vacas positivas a B. bovis. También, un incremento de aproximadamente el 23% de la lactosa se encontró en vacas positivas para B. bigemina. Conclusiones. Se puede concluir que la presencia de al menos uno de estos parásitos en el ganado lechero afecta composición o calidad de su leche

Identification of uPAR-positive Chemoresistant Cells in Small Cell Lung Cancer

BACKGROUND: The urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are major regulators of extracellular matrix degradation and are involved in cell migration and invasion under physiological and pathological conditions. The uPA/uPAR system has been of great interest in cancer research because it is involved in the development of most invasive cancer phenotypes and is a strong predictor of poor patient survival. However, little is known about the role of uPA/uPAR in small cell lung cancer (SCLC), the most aggressive type of lung cancer. We therefore determined whether uPA and uPAR are involved in generation of drug resistant SCLC cell phenotype. METHODS AND FINDINGS: We screened six human SCLC cell lines for surface markers for putative stem and cancer cells. We used fluorescence-activated cell sorting (FACS), fluorescence microscopy and clonogenic assays to demonstrate uPAR expression in a subpopulation of cells derived from primary and metastatic SCLC cell lines. Cytotoxic assays were used to determine the sensitivity of uPAR-positive and uPAR-negative cells to chemotherapeutic agents. The uPAR-positive cells in all SCLC lines demonstrated multi-drug resistance, high clonogenic activity and co-expression of CD44 and MDR1, putative cancer stem cell markers. CONCLUSIONS: These data suggest that uPAR-positive cells may define a functionally important population of cancer cells in SCLC, which are resistant to traditional chemotherapies, and could serve as critical targets for more effective therapeutic interventions in SCLC

Podoplanin Associates with CD44 to Promote Directional Cell Migration

Podoplanin, a cancer-associated glycoprotein, interacts with CD44. Both glycoproteins are coordinately upregulated during tumor progression. Podoplanin–CD44 interaction in the cell membrane occurs mainly in migrating cells, and it seems to be required for podoplanin-mediated cell migration and directionality

Breast cancer stem cells: implications for therapy of breast cancer

The concept of cancer stem cells responsible for tumour origin, maintenance, and resistance to treatment has gained prominence in the field of breast cancer research. The therapeutic targeting of these cells has the potential to eliminate residual disease and may become an important component of a multimodality treatment. Recent improvements in immunotherapy targeting of tumour-associated antigens have advanced the prospect of targeting breast cancer stem cells, an approach that might lead to more meaningful clinical remissions. Here, we review the role of stem cells in the healthy breast, the role of breast cancer stem cells in disease, and the potential to target these cells

Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer

Background: Hyaluronan (HA) an important component of the extracellular matrix, has been linked to tumor progression and drug resistance in several malignancies. However, limited data is available for ovarian cancer. This study investigated the role of hyaluronan (HA) and a potential link between the HA-CD44 pathway and membrane ATP binding cassette (ABC) transporter proteins in ovarian cancer chemoresistance. Methods: We investigated the ability of HA to block the cytotoxic effects of the chemotherapy drug carboplatin, and to regulate the expression of ABC transporters in ovarian cancer cells. We also examined HA serum levels in ovarian cancer patients prior to and following chemotherapy and assessed its prognostic relevance. Results: HA increased the survival of carboplatin treated ovarian cancer cells expressing the HA receptor, CD44 (OVCAR-5 and OV-90). Carboplatin significantly increased expression of HAS2, HAS3 and ABCC2 and HA secretion in ovarian cancer cell conditioned media. Serum HA levels were significantly increased in patients following platinum based chemotherapy and at both 1st and 2nd recurrence when compared with HA levels prior to treatment. High serum HA levels (>50 μg/ml) prior to chemotherapy treatment were associated with significantly reduced progression-free (P = 0.014) and overall survival (P = 0.036). HA production in ovarian cancer cells was increased in cancer tissues collected following chemotherapy treatment and at recurrence. Furthermore HA treatment significantly increased the expression of ABC drug transporters (ABCB3, ABCC1, ABCC2, and ABCC3), but only in ovarian cancer cells expressing CD44. The effects of HA and carboplatin on ABC transporter expression in ovarian cancer cells could be abrogated by HA oligomer treatment. Importantly, HA oligomers increased the sensitivity of chemoresistant SKOV3 cells to carboplatin. Conclusions: Our findings indicate that carboplatin chemotherapy induces HA production which can contribute to chemoresistance by regulating ABC transporter expression. The HA-CD44 signaling pathway is therefore a promising target in platinum resistant ovarian cancer.Carmela Ricciardelli, Miranda P Ween, Noor A Lokman, Izza A Tan, Carmen E Pyragius, and Martin K Oehle

Outpatient vascular clinic management in COVID-19 pandemic

INTRODUCTION: The recent Sars-CoV2 pandemic has dramatically slowed patients' access to our clinic for vascular pathology when the contagion curve peaked. The need to restore the assistance activity has led us to adopt new individual prophylaxis and hygiene measures. METHODS: Doctors and staff must wear dedicated clothes. Mask and gloves are mandatory for patients. A visit is scheduled every 60 minutes to allow the sanitation of the rooms. The day before the visit patients are contacted by telephone for the Covid-19 risk triage. In the presence of symptoms the visit is postponed. In the presence of other risk factors a IgG/IgM Rapid Test for Covid-19 is performed on admission to the clinic. In the presence of fever, if an extraordinary rapid test cannot be performed, the visit must be postponed. Rapid test positive patients cannot be visited: they are placed in solitary confinement at their home waiting for a nasopharyngeal swab for Covid-19. When the rapid test is positive, immediate room sanitation also occurs. The rooms dedicated to the outpatient clinic as well as medical and not medical instruments are disinfected. CONCLUSION: The one adopted can be a useful management model for any type of care activity in order to guarantee the safety of patients and all the staff. KEY WORDS: COVID-19, Management, vascular, Outpatient clinic