GM2/GM3 controls the organizational status of CD82/Met microdomains: further studies in GM2/GM3 complexation.

At cell surface gangliosides might associate with signal transducers proteins, grown factor receptors, integrins, small G-proteins and tetraspanins establishing microdomains, which play important role in cell adhesion, cell activation, motility, and growth. Previously, we reported that GM2 and GM3 form a heterodimer that interacts with the tetraspanin CD82, controlling epithelial cell mobility by inhibiting integrin-hepatocyte growth factor-induced cMet tyrosine kinase signaling. By using molecular dynamics simulations to study the molecular basis of GM2/GM3 interaction we demonstrate, here, that intracellular levels of Ca2+ mediate GM2/GM3 complexation via electrostatic interaction with their carboxyl groups, while hydrogen bonds between the ceramide groups likely aid stabilizing the complex. The presence of GM2/GM3 complex alters localization of CD82 on cell surface and therefore downstream signalization. These data contribute for the knowledge of how glycosylation may control signal transduction and phenotypic changes.

Expression levels of survivin, Bcl-2, and KAI1 proteins in cervical cancer and their correlation with metastasis.

Cervical cancer is associated with abnormal expression of multiple genes. Survivin and Bcl-2 proteins are apoptosis inhibitors. The tumor suppressor gene CD82, which encodes the protein KAI1, is downregulated in cervical cancer, and is associated with differentiation degree. We investigated the expression levels of three proteins and their correlation with metastasis in cervical cancer by comparing them in different cervical lesions. Immunohistochemistry was used to detect their three protein expression levels in the normal cervix, chronic cervicitis, cervical intraepithelial neoplasia (CIN) lesions, and cervical cancer. The relationships between the protein expression levels and tumor type, clinical stage, tissue differentiation, invasion, and metastasis were analyzed. Survivin and Bcl-2 expression levels in cervical cancer were significantly higher than in the normal cervix, chronic cervicitis, or CIN (P < 0.05). KAI1 expression was markedly lower in cervical cancer than in the normal cervix, chronic cervicitis, or CIN (P < 0.05). There was no statistical difference between the expression levels of the three proteins in CIN and chronic cervicitis, but there were differences in expression between CIN and normal cervical tissues (P < 0.05). Bcl-2 and survivin levels were positively correlated while KAI1 expression was negatively correlated with clinical stage. Survivin and KAI1 expression levels were associated with lymph node metastasis (P < 0.05), and KAI1 expression was positively related with differentiation degree (P < 0.05). Survivin, Bcl-2, and KAI1 are metastasis-related factors in cervical cancer. Overexpression of survivin and Bcl-2, and low expression of KAI1 promotes cervical cancer progress and metastasis.

Hypoxia and Serum deprivation protected MiaPaCa-2 cells from KAI1-induced proliferation inhibition through autophagy pathway activation in solid tumors.

PURPOSE: KAI1 closely correlates with pancreatic cancer metastasis. There might be some factors that protect the cells from a proliferation inhibition by KAI1 in the solid tumors' microenvironment. Hypoxia and ischemia are the main characteristics of the microenvironment within solid tumors. Whether they affect the KAI1 inhibitory effects on cell proliferation is still unclear. METHODS: MiaPaCa-2 human pancreatic cancer cells do not express KAI1 protein. However, after being infected with Ad5-KAI1, they expressed KAI1 protein. We cultured them under hypoxic and serum-free conditions to simulate the solid tumor hypoxic-ischemic microenvironment. The cells were divided into the control, hypoxic, serum-free, and hypoxic with serum-free groups. The proliferation and apoptosis were observed by CCK8 and Annexin V-FITC/PI, respectively. The green fluorescent protein-labeled light chain 3 association with autophagosome membranes was detected by confocal microscopy. The ratio of LC3-II-LC3-I expression level was detected by western blot. Pretreatment of 3-MA was used to inhibit the autophagy. We, then observed whether the hypoxic and serum-free conditions could change the effect of KAI1 on cell survival and whether the pretreatment of 3-MA could inhibit the effect of hypoxic and serum-free conditions on KAI1 function. RESULTS: Hypoxia and serum-free media effectively reduced the apoptosis and proliferation inhibition caused by KAI1 and was beneficial to the cell survival. 3-MA pretreatment effectively blocked the protective effect of hypoxia and serum-free media on the cells by autophagy block. CONCLUSIONS: Serum-free media and hypoxia protected the MiaPaCa-2 cells from a KAI1-induced apoptosis and proliferation inhibition via autophagy induction.

Effect of a probiotic fermented milk on the thymus in Balb/c mice under non-severe protein-energy malnutrition.

Protein­energy malnutrition (PEM) causes a significant impairment of the immune system, the thymus being one of the most affected organs. It has been demonstrated that the administration of probiotic fermented milk (PFM) recovered the intestinal barrier, histological alterations and mucosal and systemic immune functions in a non-severe malnutrition model using BALB/c mice. The aim of the present study was to evaluate, in the same model of malnutrition, the effect of a PFM added to a re-nutrition diet on the recovery of the thymus, analysing histological and functional alterations caused by malnutrition. Mice were undernourished and divided into three groups according to the dietary supplement received during re-nutrition: milk, PFM or its bacterial-free supernatant (BFS). They were compared with well-nourished and malnourished mice. PFM was the most effective re-nutrition supplement to improve the histology of the thymus, decreasing cellular apoptosis in this organ and recovering the percentage of CD4þ/CD82 single-positive thymocytes. Immature doublepositive thymocytes were increased in the malnourished control (MC). The production of different cytokines in the thymus was increased in mice given PFM, compared with the mice that received other dietary supplements and MC. Mice given the BFS presented an improvement in the thymus similar to those that received milk. We demonstrated the importance of the whole PFM supplementation on the histological and functional recovery of the thymus in a non-severe PEM model.

P504S expressing circulating prostate cells as a marker for prostate cancer.

Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer deaths. The serum prostate specific antigen (PSA) is the only biomarker routinely used in screening. The aim of this study was to develop a system to test the presence of circulating prostate cells in men without a diagnosis of prostate cancer in relation with age, serum PSA levels and prostate biopsy by determining the co-expression of several markers such as CD82, HER-2 and matrix metalloproteinase 2 (MMP-2). For this purpose mononuclear cells were separated from blood using differential centrifugation and then prostate cells were identified by using standard immunocytochemical method. Results indicated that among 409 men screened for prostate cancer 16.6% were positive for circulating prostate cells. Cells were positive for MMP-2 and HER-2 in 100 and 14.3% of cases, respectively, without an association with age or PSA levels. However, CD82 protein expression was associated with older age and low grade tumors. It can be concluded that the study of circulating prostate cells with various markers could be a useful complementary screening test for prostate cancer in men with increased PSA level.

Downregulation of CD9 protein expression is associated with aggressive behavior of oral squamous cell carcinoma.

Squamous cell carcinoma of the oral cavity (OSCC) is a malignancy characterized by a high degree of local aggression and metastasis to cervical lymph nodes. Tetraspanins are proteins with functional roles in a wide array of cellular processes and are reported to be associated with tumor progression. The present study investigated the expression of the CD9, CD37, CD63, CD81 and CD82 tetraspanins in OSCC using immunohistochemistry (IHC) and quantitative Real Time-PCR (qRT-PCR). Tissue microarray (TMA) analysis of samples from 179 cases of OSCC and 10 normal samples oral mucosa were evaluated immunomorphologically. We analyzed CD9 and CD82 expression by qRT-PCR in 66 OSCC cases and 4 normal samples of oral mucosa. Expression of CD63, CD37 and CD81 was not detected in the samples studied. CD82 was downregulated or negative in 127 of 179 (80%) specimens; no correlation was observed between CD82 expression, clinicopathological parameters, disease-free survival and 5-year overall survival. CD9 expression was downregulated or negative in 75 of 129 (42%) OSCC samples. Loss of CD9 expression in OSCC samples correlated with the incidence of lymph node metastasis (p=0.017). Disease-free survival and the 5-year overall survival of patients with downregulated or negative CD9 expression were significantly lower than in patients with positive CD9 expression (p=0.010 and p=0.071, respectively). No correlation was found between CD9 or CD82 expression and clinicopathological parameters by qRT-PCR. Our results suggest that the downregulation or lack of expression of the CD9 protein might indicate a more aggressive of OSCC.

CD63 tetraspanin slows down cell migration and translocates to the endosomal-lysosomal-MIICs route after extracellular stimuli in human immature dendritic cells.

We analyzed herein whether members of the tetraspanin superfamily are involved in human immature dendritic cell (DC) functions such as foreign antigen internalization, phagocytosis, and cell migration. We show that CD63, CD9, CD81, CD82, and CD151 are present in immature DCs. Whereas CD9 and CD81 are mostly expressed at the cell surface, CD63 and CD82 are also located in intracellular organelles. Complexes of monoclonal antibody (Mab) FC-5.01-CD63 or Fab-5.01-CD63 were rapidly translocated "outside-in" and followed the endocytic pathway through early endosomes and lysosomes, reaching major histocompatibility complex (MHC) class II-enriched compartments (MIICs) in less than one hour. Internalization of CD63 was also observed during Saccharomyces cerevisiae phagocytosis. Moreover, an association of CD63 with the beta-glycan receptor dectin-1 was observed. Mabs against CD9, CD63, CD81, and CD82 enhanced by 50% the migration induced by the chemokines macrophage inflammatory protein-5 (MIP-5) and MIP-1alpha. Concomitantly, Mabs against CD63 and CD82 diminished the surface expression of CD29, CD11b, CD18, and alpha5 integrins. By immunoprecipitation experiments we found that CD63 associated with integrins CD11b and CD18. These results suggest that CD9, CD63, CD81, and CD82 could play a role in modulating the interactions between immature DCs and their environment, slowing their migratory ability. However, only CD63 would intervene in the internalization of complex antigens.