Synergistic antitumor activity by combining trastuzumab with retinoic acid in HER2 positive human breast cancer cells.

Breast cancer can be classified into molecular subtypes. Tumors overexpressing HER2 protein are more aggressive and metastatic; hence, patients have a poor prognosis. Anti-HER2 strategies, such as the monoclonal antibody Trastuzumab (Tz), have therefore been developed. Despite this progress, not all patients respond to the treatment. Retinoic acid (RA) has been proposed as an adjuvant treatment of breast carcinoma because of its ability to inhibit cell growth. We evaluated the effect of Tz in combination with RA on the viability, adhesion, migration, invasion and expression of migration-related proteins in SKBR3 and BT-474 human breast cancer cells. MTT, pharmacological interaction analysis, immunofluorescence, adhesion/migration/invasion and Western blot assays were performed. The coadministration of both drugs synergistically decreased cell survival. Tz+RA significantly decreased adhesion/migration/invasion in both cell types. Tz+RA strongly reduced FAK and HER2 expression and induced nuclear FAK translocation. In addition, a granular distribution of HER2 receptor was observed after the combined treatment. In conclusion, the coadministration of both drugs in patients with this type of cancer could contribute to the improvement of their prognosis and reduce the adverse effects of therapy because the applied Tz doses would be lower due to the adjuvant effect of RA.

Retinoic acid induces nuclear FAK translocation and reduces breast cancer cell adhesion through Moesin, FAK, and Paxillin.

Breast cancer is the most common malignancy in women, with metastases being the cause of death in 98%. In previous works we have demonstrated that retinoic acid (RA), the main retinoic acid receptor (RAR) ligand, is involved in the metastatic process by inhibiting migration through a reduced expression of the specific migration-related proteins Moesin, c-Src, and FAK. At present, our hypothesis is that RA also acts for short periods in a non-genomic action to cooperate with motility reduction and morphology of breast cancer cells. Here we identify that the administration of 10(-6) M RA (10-20 min) induces the activation of the migration-related proteins Moesin, FAK, and Paxillin in T-47D breast cancer cells. The phosphorylation exerted by the selective agonists for RARα and RARß, on Moesin, FAK, and Paxillin was comparable to the activation exerted by RA. The RARγ agonist only led to a weak activation, suggesting the involvement of RARα and RARß in this pathway. We then treated the cells with different inhibitors that are involved in cell signaling to regulate the mechanisms of cell motility. RA failed to activate Moesin, FAK, and Paxillin in cells treated with Src inhibitor (PP2) and PI3K inhibitor (WM), suggesting the participation of Src-PI3K in this pathway. Treatment with 10(-6) M RA for 20 min significantly decreased cell adhesion. However, when cells were treated with 10(-6) M RA and FAK inhibitor, the RA did not significantly inhibit adhesion, suggesting a role of FAK in the adhesion inhibited by RA. By immunofluorescence and immunoblotting analysis we demonstrated that RA induced nuclear FAK translocation leading to a reduced cellular adhesion. These findings provide new information on the actions of RA for short periods. RA participates in cell adhesion and subsequent migration, modulating the relocation and activation of proteins involved in cell migration.

Retinoic acid reduces migration of human breast cancer cells: role of retinoic acid receptor beta.

Breast cancer is the most common malignancy in women and the appearance of distant metastases produces the death in 98% of cases. The retinoic acid receptor ß (RARß) is not expressed in 50% of invasive breast carcinoma compared with normal tissue and it has been associated with lymph node metastasis. Our hypothesis is that RARß protein participates in the metastatic process. T47D and MCF7 breast cancer cell lines were used to perform viability assay, immunobloting, migration assays, RNA interference and immunofluorescence. Administration of retinoic acid (RA) in breast cancer cells induced RARß gene expression that was greatest after 72 hrs with a concentration 1 µM. High concentrations of RA increased the expression of RARß causing an inhibition of the 60% in cell migration and significantly decreased the expression of migration-related proteins [moesin, c-Src and focal adhesion kinase (FAK)]. The treatment with RARα and RARγ agonists did not affect the cell migration. On the contrary, the addition of the selective retinoid RARß-agonist (BMS453) significantly reduced cell migration comparable to RA inhibition. When RARß gene silencing was performed, the RA failed to significantly inhibit migration and resulted ineffective to reduce moesin, c-Src and FAK expressions. RARß is necessary to inhibit migration induced by RA in breast cancer cells modulating the expression of proteins involved in cell migration.

Determination of cytokeratins 1, 13 and 14 in oral lichen planus.

UNLABELLED: INTRODUCCION: Cytokeratins (CK) are molecules of the cytoskeleton that contribute to the cellular differenciation. We studied the expression of CK1, CK13 and CK14 in thirty-three patients with OLP. The biopsied lesions were located in the dorsal surface of the tongue, the palatal keratinized mucosa and the nonkeratinized buccal mucosa. OBJECTIVES: This study aimed to determine the expression of CK1, CK13 and CK14 in oral lichen planus (OLP) and its relations with: clinical patterns, prognosis, drugs and tobacco intake and histopathological features. STUDY DESIGN: Immunohistochemical analysis, retrospective, descriptive, observational and no randomized study. RESULTS: No significant difference was observed in the expression of CK1 in patients with or without drug treatment. No association was found with the amount of drugs intake or smoking nor with the histopathological features examined. Samples immunostained with CK13 were all positive in the suprabasal layers, and 13 of them in the basal layer. In these last ones, statistical analysis showed significance in the grade of vacuolization of the basal layer (p=0.023) and in the degree of exocytosis (p=0.0025), this, making the degree of affection higher for both parameters. Thirty-two tissue sections were immunostained with CK14. CK14 was expressed in the basal layer in 97% of samples and in the suprabasal layer in 94% of samples. CONCLUSIONS: The three CK were altered in OLP. CK1 does not have a direct connection with the presence of orthokeratosis. The finding of the CK13 in the basal layer is related to the agression of the lymphocytic infiltration in the epithelium, due to the basal stratum vacuolization and the increase in lymphocytic exocitosis. The presence of CK14 in the suprabasal stratums is not a parameter to predict malignancy. The CK in OLP do not follow the normal pattern of keratinized or non-keratinized mucosa.

Impaired mammary gland T cell population during early lactation in hypoprolactinemic lactation-deficient rats.

Mammary stroma is composed of various cell types, including migratory leukocytes. Although mammary antibody-secreting cells have been extensively studied, reports focusing on mammary T cells are scarce. It is thought that the recruitment mechanism of leukocytes to the mammary gland (MG) is controlled by pregnancy- and lactation-specific stimuli. But whether prolactin (PRL) modulates the T-cell population in MG is still unknown. Our aim was to study the relationship between PRL levels and T and B cells during early lactation (L2, day 2 post partum) and mid-lactation (L12, day 12 of lactation). In order to investigate whether PRL is associated with homing events to MG, female Sprague Dawley (SD) and SD-derived desmoglein 4(-/-) hairless (phenotype with lactation deficit, OFA hr/hr) rats were killed during estrus, pregnancy, and post partum, and blood, MG, and corpora lutea were obtained to perform fluorescent-activated cell sorting (FACS), real-time PCR, and histological and RIA studies. Serum PRL levels were lower in OFA hr/hr rats than in SD rats during early lactation. MG of OFA hr/hr rats showed less secretory material compared with SD rats. FACS analysis showed lower percentage of MG CD3+ cells in OFA hr/hr rats compared with SD rats on L2 and L12. OFA hr/hr rats showed higher absolute numbers of circulating CD3+ cells compared with SD rats on L2 but not on L12. These results show that T-cell population in MG is affected in early lactating OFA hr/hr rats and strongly suggest that serum PRL levels may be involved in the homing events to MG, probably helping antibody-secreting cells and protecting the gland during lactation development.

DNA damage induced by paclitaxel and DNA repair capability of peripheral blood lymphocytes as evaluated by the alkaline comet assay.

This study was designed to assess whether the chemotherapeutic drug paclitaxel can induce DNA damage in peripheral blood lymphocytes of human healthy donors, and to evaluate if such damage could be repaired. Venous blood was collected by routine venipuncture, the lymphocytes were isolated and cultured and then treated with 100nM, 500nM, 10microM, and 30microM of taxol for 4h. The alkaline comet assay technique was used to quantify the level of DNA damage and the DNA repair in lymphocytes. A significant increase in DNA damage was achieved when the cells were incubated with paclitaxel concentrations of 10microM or above. To test the DNA repair capability, the lymphocytes were allowed to recover for 2, 4, 6, and 24h. The DNA damage was almost completely repaired after 24h of incubation demonstrating a time-dependent repair capability. In conclusion, we demonstrate that paclitaxel induces DNA damage in peripheral blood lymphocytes and that this damage can be repaired.