Metformin reverses mesenchymal phenotype of primary breast cancer cells through STAT3/NF-κB pathways.

BACKGROUND: Breast cancer currently is the most frequently diagnosed neoplasm and the leading cause of death from cancer in women worldwide, which is mainly due to metastatic disease. Increasing our understanding of the molecular mechanisms leading to metastasis might thus improve the pharmacological management of the disease. Epithelial-mesenchymal transition (EMT) is a key factor that plays a major role in tumor metastasis. Some pro-inflammatory cytokines, like IL-6, have been shown to stimulate phenotypes consistent with EMT in transformed epithelial cells as well as in carcinoma cell lines. Since the EMT is one of the crucial steps for metastasis, we studied the effects of metformin (MTF) on EMT. METHODS: Cytotoxic effect of MTF was evaluated in eight primary breast cancer cell cultures by crystal violet assay. EMT markers and downstream signaling molecules were measured by Western blot. The effect of MTF on cell proliferation and cell migration were analyzed by MTT and Boyden chamber assays respectively. RESULTS: We observed that the response of cultured breast cancer primary cells to MTF varied; mesenchymal cells were resistant to 10 mM MTF and expressed Vimentin and SNAIL, which are associated with a mesenchymal phenotype, whereas epithelial cells were sensitive to this MTF dose, and expressed E-cadherin but not mesenchymal markers. Further, exposure of mesenchymal cells to MTF down-regulated both Vimentin and SNAIL as well as cell proliferation, but not cell migration. In an in vitro IL-6-induced EMT assay, primary breast cancer cells showing an epithelial phenotype underwent EMT upon exposure to IL-6, with concomitant activation of STAT3 and NF-κB; addition of MTF to IL-6-induced EMT reversed the expression of the mesenchymal markers Vimentin and SNAIL, decreased pSTAT3 Y705 and pNF-κB S536 and increased E-cadherin. In addition, downregulation of STAT3·activation was dependent on AMPK, but not NF-κB phosphorylation. Further, MTF inhibited cell proliferation and migration stimulated by IL-6. CONCLUSION: These results suggest that MTF inhibits IL-6-induced EMT, cell proliferation, and migration of primary breast cancer cells by preventing the activation of STAT3 and NF-κB. STAT3 inactivation occurs through AMPK, but not NF-κB.

Primary breast cancer cell culture yields intra-tumor heterogeneous subpopulations expressing exclusive patterns of receptor tyrosine kinases.

BACKGROUND: It has become evident that intra-tumor heterogeneity of breast cancer impact on several biological processes such as proliferation, migration, cell death and also might contribute to chemotherapy resistance. The expression of Receptor Tyrosine Kinases (RTKs) has not been analyzed in the context of intra-tumor heterogeneity in a primary breast cancer cell culture. Several subpopulations were isolated from the MBCDF (M serial-breast cancer ductal F line) primary breast cancer cells and were successfully maintained in culture and divided in two groups according to their morphology and RTKs expression pattern, and correlated with biological processes like proliferation, migration, anchorage-independent cell growth, and resistance to cytotoxic chemotherapy drugs and tyrosine kinase inhibitors (TKIs). METHODS: Subpopulations were isolated from MBCDF primary breast cancer cell culture by limiting dilution. RTKs and hormone receptors were examined by Western blot. Proliferation was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT assay). Cell viability was evaluated by Crystal Violet. Migration was assessed using Boyden chambers. Anchorage-independent cell growth was evaluated by colony formation in soft agar. RESULTS: Several subpopulations were isolated from the MBCDF breast cancer cells that were divided into two groups according to their morphology. Analysis of RTKs expression pattern showed that HER1, HER3, c-Met and VEGFR2 were expressed exclusively in cells from group 1, but not in cells from group 2. PDGFR was expressed only in cells from group 2, but not in cells from group 1. HER2, HER4, c-Kit, IGF1-R were expressed in all subpopulations. Biological processes correlated with the RTKs expression pattern. Group 2 subpopulations present the highest rate of cell proliferation, migration and anchorage-independent cell growth. Analysis of susceptibility to chemotherapy drugs and TKIs showed that only Paclitaxel and Imatinib behaved differently between groups. Group 1-cells were resistant to both Paclitaxel and Imatinib. CONCLUSIONS: We demonstrated that subpopulations from MBCDF primary cell culture could be divided into two groups according to their morphology and a RTKs excluding-expression pattern. The differences observed in RTKs expression correlate with the biological characteristics and chemoresistance of each group. These results suggest that intra-tumor heterogeneity contributes to generate groups of subpopulations with a more aggressive phenotype within the tumor.

Angiogenesis and expression of estrogen and progesterone receptors as predictive factors for recurrence of meningioma.

Meningiomas are benign tumors, with low rate of recurrence after surgery. The most important factor predicting recurrence is the extent of surgical resection; other factors have been studied with conflicting results. Angiogenesis, an important substratum for growth and spread of neoplasic cells, and the expression of estrogen and progesterone receptors (ER, PR), could play a role in the recurrence of meningioma. We evaluated 42 patients with meningioma diagnosis (confirmed by histopathology) treated exclusively by surgery between January 1995 and December 1999, and compared the recurring and non-recurring groups after a ten-year follow-up period. Recurrence was associated with several factors including vascular density (VD), cell proliferation index (CPI), ER, PR, and cyclin E (CE) tissue expression, as evaluated by immunohistochemistry. Complete surgical resection was achieved in 41% of patients. Recurrence of meningioma was found in 17 patients (40%). Median + or - standard deviation (SD) of recurrence time was 32 + or - 5 months. When recurrence versus no recurrence was compared, mean + or - SD of VD and CPI were 9 + or - 3.6 and 607.6 + or - 233 (40x/10 fields) respectively. Tissue expression was positive for ER, PR, and CE in 28, 62 and 91% of patients, respectively. The sole significant recurrence-associated factors were extent of resection (P = 0.003) and VD (P = 0.004). ER, PR, and CE-tissue expression were not statistically significant. The most important factor associated with meningioma relapse was vascular density, independently of hormonal status and extent of surgical resection. Patients with a high risk of recurrence could benefit from additional treatment.

[Differences in inflammatory markers in a non-smoking and smoking Mexican population]. / Diferencias de marcadores de inflamación entre fumadores y no fumadores en una población mexicana.

INTRODUCTION: Cigarette smoking is one of the main risk factors for the development of cancer, chronic obstructive pulmonary disease and cardiovascular disease, most of which exhibit an inflammatory component at some stage of their time-course. However, little is known about the early presence of proinflammatory markers in healthy smokers. MATERIAL AND METHODS: We conducted a 16-month, cross-sectional study to determine the presence of inflammatory markers in a group of smokers pronounced in good health after an exhaustive medical exam. Of an initial population of 1,806 smokers and non-smokers who underwent anthropometric, biochemical, radiographic and ultrasound studies plus exercise testing, 317 smokers and 297 non-smokers (the control group) found to have no alterations were ultimately selected and paired by age and gender. Their test data were then compared. RESULTS: In comparison with non-smokers, smokers showed higher levels of C-reactive protein, hemoglobin, hematocrit, platelets, lipid profile and cardiovascular risk. They also showed lower values of total proteins, albumin and lactic dehydrogenase. CONCLUSIONS: Even though laboratory value results were considered to be within normal range, smokers showed increased levels of prothrombotic and proinflammatory molecules. Therefore, tobacco smoking can be considered an inflammatory syndrome whose final outcome could be one of the many organic disorders that characterize and accompany this entity.

Doxorubicin induces atypical NF-κB activation through c-Abl kinase activity in breast cancer cells.

PURPOSE: NF-κB transcription factor has been associated with cancer development and chemoresistance. We studied the signaling pathway activated by doxorubicin (DOX) leading to NF-κB activation in breast cancer cells. METHODS: NF-κB activity was evaluated by electrophoretic mobility shift in T47D, ZR75.30 and primary culture (MBCDF) from a ductal infiltrating carcinoma. Cell viability was measured by crystal violet. Western blotting was performed to check the expression and phosphorylation of IκBα Ser-32/36. c-Abl was inhibited with Imatinib or by overexpressing a dominant negative form of c-Abl (K290R). RESULTS: We found a correlation between sensitivity to DOX and amplitude of NF-κB activation. In cells least sensitive to DOX, NF-κB remained activated for longer time (T47D and MBCDF). The opposite effect was observed in cells sensitive to DOX (ZR75.30). DOX did not induce IκBα degradation or Ser-32/36 phosphorylation. Instead, there were modifications in the levels of IκBα tyrosine phosphorylation, suggesting an atypical NF-κB activation. In DOX-resistant cells, Imatinib treatment reduced IκBα tyrosine phosphorylation and NF-κB activity. The Imatinib-DOX combination significantly enhanced cell death of T47D and MBCDF breast cancer cells. Overexpression of c-Abl K290R in T47D and MBCDF cells reduced basal and DOX-induced NF-κB activation as well as IκBα tyrosine phosphorylation. In c-Abl K290R cells, DOX treatment did not mimic the combination Imatinib-DOX-induced cell death. CONCLUSIONS: Inhibition of c-Abl inactivated IκBα/NF-κB pathway is associated with IκBα tyrosine phosphorylation in breast cancer cells. These results also raise the potential use of a combined therapy with Imatinib and DOX for breast cancer patients.

Efecto del daño de vías dopaminérgicas mesencefálicas en la conducta adictiva al tabaco: Revisión generadora de una hipótesis / Mesencephalic dopaminergic tract lesions in tobacco addiction behavior: A review of mechanisms of disease and an hypothesis

El tabaquismo es la enfermedad prevenible que se relaciona con más problemas de salud y causas de muerte en el mundo. Se ha estimado una prevalencia de 1,100 millones de personas en el mundo con adicción al tabaco. La adicción a la nicotina depende de múltiples factores; está documentado que el sistema nervioso central desempeña un importante papel en su desarrollo a través de la estimulación de receptores neuronales dopaminérgicos por la nicotina. La dopamina tiene su principal síntesis en las neuronas de la sustancia nigra y área tegmental ventral del mesencéfalo, las cuales se proyectan hacia los núcleos básales y núcleo accumbens. Los estudios en animales sugieren que el sistema dopaminérgico está involucrado de manera importante en la adicción a la nicotina. Las lesiones neurotóxicas del sistema mesolfmbico o la administración sistémica de un bloqueador del receptor de nicotina reduce la administración de nicotina en roedores; sin embargo, estos hallazgos se han realizado en modelos animales, siendo difícil corroborarlos en humanos porque no se puede experimentar en ellos y causarles daño tóxico o vascular de las vías dopaminérgicas mesencefálicas. Una estrategia circunstancial apropiada podría llevarse a cabo analizando la conducta en términos de adicción de sujetos con lesiones del mesencéfalo, secundarias a un evento vascular cerebral.

Tobacco use, a well known cause of death, represents a preventable disease related to many health problems. Epidemiológical studies estimate a prevalence of 1,100 million tobacco dependent people. Nicotine addiction depends on multiple factors; studies have reported that the central nervous system plays an important role through the stimulation of neuronal dopaminergic nicotine receptors. The main dopamine synthesis sites are located in the neurons of the substantia nigra and the ventral tegmental area of the mesencephalus, which are projected towards the basal ganglia and accumbens nuclei. In vivo studies suggest that the dopaminergic cistern is highly implicated in nicotine dependence, showing that the neurotoxic lesions of the mesolimbic system or the systemic administration of nicotine receptor blockers reduce the quantity of nicotine administered. Nevertheless, these studies have been carried out in animal models; thus, such findings can not be confirmed in humans due to methodological limitations (i.e. it is unethical to produce toxic or vascular damage of the mesencephalic dopaminergic tracts). It could be possible to carry out such a study in patients with mesencephalic injuries secondary to stroke.