Role of the p53/p21 system in the response of human colon carcinoma cells to Doxorubicin

BACKGROUND: Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels. METHODS: The relationship between p53 and p21 protein levels and the cytotoxic effect of DOX was investigated, by MTT assay and western blot analysis, in HCT116 (p53-positive) and HT29 (p53-negative) colon cancer cells. We then assessed the effects of DOX in two isogenic cell lines derived from HCT116 by abrogating the expression and/or function of p53 and p21 (HCT116-E6 and HCT116 p21-/-, respectively). Finally, we evaluated the effect of pre-treatment with the piperidine nitroxide Tempol (TPL), an agent that was reported to induce p21 expression irrespective of p53 status, on the cytotoxicity of DOX in the four cell lines. Comparisons of IC50 values and apoptotic cell percentages were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations were performed by the combination Index method. RESULTS: Our results indicate that, in the colon carcinoma cell lines tested, sensitivity to DOX is associated with p21 upregulation upon drug exposure, and DOX cytotoxicity is potentiated by pre-treatment with TPL, but only in those cell lines in which p21 can be upregulated. CONCLUSIONS: p21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms

Proteomic analysis of dopamine and \u3b1-synuclein interplay in a cellular model of Parkinson's disease pathogenesis

Altered dopamine homeostasis is an accepted mechanism in the pathogenesis of Parkinson\u2019s disease. a-Synuclein overexpression and impaired disposal contribute to this mechanism. However, biochemical alterations associated with the interplay of cytosolic dopamine and increased a-synuclein are still unclear. Catecholaminergic SH-SY5Y human neuroblastoma cells are a suitable model for investigating dopamine toxicity. In the present study, we report the proteomic pattern of SH-SY5Y cells overexpressing a-synuclein (1.6-fold induction) after dopamine exposure. Dopamine itself is able to upregulate a-synuclein expression. However, the effect is not observed in cells that already overexpress a-synuclein as a consequence of transfection. The proteomic analysis highlights significant changes in 23 proteins linked to specific cellular processes, such as cytoskeleton structure and regulation, mitochondrial function, energetic metabolism, protein synthesis, and neuronal plasticity. A bioinformatic network enrichment procedure generates a significant model encompassing all proteins and allows us to enrich functional categories associated with the combination of factors analyzed in the present study (i.e. dopamine together with a-synuclein). In particular, the model suggests a potential involvement of the nuclear factor kappa B pathway that is experimentally confirmed. Indeed, a-synuclein significantly reduces nuclear factor kappa B activation, which is completely quenched by dopamine treatment.Altered dopamine homeostasis is an accepted mechanism in the pathogenesis of Parkinson's disease. \u3b1-Synuclein overexpression and impaired disposal contribute to this mechanism. However, biochemical alterations associated with the interplay of cytosolic dopamine and increased \u3b1-synuclein are still unclear. Catecholaminergic SH-SY5Y human neuroblastoma cells are a suitable model for investigating dopamine toxicity. In the present study, we report the proteomic pattern of SH-SY5Y cells overexpressing \u3b1-synuclein (1.6-fold induction) after dopamine exposure. Dopamine itself is able to upregulate \u3b1-synuclein expression. However, the effect is not observed in cells that already overexpress \u3b1-synuclein as a consequence of transfection. The proteomic analysis highlights significant changes in 23 proteins linked to specific cellular processes, such as cytoskeleton structure and regulation, mitochondrial function, energetic metabolism, protein synthesis, and neuronal plasticity. A bioinformatic network enrichment procedure generates a significant model encompassing all proteins and allows us to enrich functional categories associated with the combination of factors analyzed in the present study (i.e. dopamine together with \u3b1-synuclein). In particular, the model suggests a potential involvement of the nuclear factor kappa B pathway that is experimentally confirmed. Indeed, \u3b1-synuclein significantly reduces nuclear factor kappa B activation, which is completely quenched by dopamine treatment. \ua9 2010 The Authors Journal compilation \ua9 2010 FEBS

Role of the p53/p21 system in the response of human colon carcinoma cells to Doxorubicin

BACKGROUND: Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels. METHODS: The relationship between p53 and p21 protein levels and the cytotoxic effect of DOX was investigated, by MTT assay and western blot analysis, in HCT116 (p53-positive) and HT29 (p53-negative) colon cancer cells. We then assessed the effects of DOX in two isogenic cell lines derived from HCT116 by abrogating the expression and/or function of p53 and p21 (HCT116-E6 and HCT116 p21-/-, respectively). Finally, we evaluated the effect of pre-treatment with the piperidine nitroxide Tempol (TPL), an agent that was reported to induce p21 expression irrespective of p53 status, on the cytotoxicity of DOX in the four cell lines. Comparisons of IC50 values and apoptotic cell percentages were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations were performed by the combination Index method. RESULTS: Our results indicate that, in the colon carcinoma cell lines tested, sensitivity to DOX is associated with p21 upregulation upon drug exposure, and DOX cytotoxicity is potentiated by pre-treatment with TPL, but only in those cell lines in which p21 can be upregulated. CONCLUSIONS: p21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms

Biological evaluation of alginate-based hydrogels, with antimicrobial features by Ce(III) incorporation, as vehicles for a bone substitute

In this work three different hydrogels were developed to associate, as vehicles, with the synthetic bone substitute GR-HA. One based on an alginate matrix (Alg); a second on a mixture of alginate and chitosan (Alg/Ch); and a third on alginate and hyaluronate (Alg/HA), using Ca2+ ions as cross-linking agents. The hydrogels, as well as the respective injectable bone substitutes (IBSs), were fully characterized from the physical-chemical point of view. Weight change studies proved that all hydrogels were able to swell and degrade within 72 hours at pH 7.4 and 4.0, being Alg/HA the hydrogel with the highest degradation rate (80%). Rheology studies demonstrated that all hydrogels are non-Newtonian viscoelastic fluids, and injectability tests showed that IBSs presented low maximum extrusion forces, as well as quite stable average forces. In conclusion, the studied hydrogels present the necessary features to be successfully used as vehicles of GR-HA, particularly the hydrogel Alg/HA.The authors would like to acknowledge the financial support from FCT (Fundacao para a Ciencia e a Tecnologia) through the grant SFRH/BD/76237/2011 and project ENMED/0002/2010, from FEDER funds through the program COMPETE-Programa Operacional Factores de Competitividade-under the project PEst-C/EME/UI0285/2011, as well as to the project I&DT BIOMAT&CELL n. 1372

Stat3 Mediates Expression of Autotaxin in Breast Cancer

We determined that signal transducer and activator of transcription 3 (Stat3) is tyrosine phosphorylated in 37% of primary breast tumors and 63% of paired metastatic axillary lymph nodes. Examination of the distribution of tyrosine phosphorylated (pStat3) in primary tumors revealed heterogenous expression within the tumor with the highest levels found in cells on the edge of tumors with relatively lower levels in the central portion of tumors. In order to determine Stat3 target genes that may be involved in migration and metastasis, we identified those genes that were differentially expressed in primary breast cancer samples as a function of pStat3 levels. In addition to known Stat3 transcriptional targets (Twist, Snail, Tenascin-C and IL-8), we identified ENPP2 as a novel Stat3 regulated gene, which encodes autotaxin (ATX), a secreted lysophospholipase which mediates mammary tumorigenesis and cancer cell migration. A positive correlation between nuclear pStat3 and ATX was determined by immunohistochemical analysis of primary breast cancer samples and matched axillary lymph nodes and in several breast cancer derived cell lines. Inhibition of pStat3 or reducing Stat3 expression led to a decrease in ATX levels and cell migration. An association between Stat3 and the ATX promoter, which contains a number of putative Stat3 binding sites, was determined by chromatin immunoprecipitation. These observations suggest that activated Stat3 may regulate the migration of breast cancer cells through the regulation of ATX