Paraquat (PQ) is a potential human neurotoxicant and is used in models of oxidative stress. We determined the toxicokinetics (TK) and toxicodynamics (TD) of PQ in adult mouse brain following repeated or prolonged PQ exposure. PQ accumulated in different brain regions and reached a plateau after ~18 i.p. (10 mg/kg) doses and resulted in modest morbidity and mortality unpredictably associated with dose interval and number. PQ had divergent effects on horizontal locomotor behavior depending on the number of doses. PQ decreased striatal dopamine levels after the 18th to 36th i.p. dose (10 mg/kg) and reduced the striatal level of tyrosine hydroxylase. Drinking water exposure to PQ (0.03– 0.05 mg/ml) did not result in any mortality and resulted in concentration and time dependent levels in the brain. The brain half-life of PQ varied with mouse strain. PQ accumulates and may saturate a site in mouse brain resulting in complex PQ level and duration-related consequences. These findings should alter our risk assessment of this compound and demonstrate a useful, but complex dynamic model for understanding the consequences of PQ in the brain.
Keywords: CDK, cyclin, paclitaxel, Smad3, triple negative breast cancer Abbreviations: BCSC, breast cancer stem cells; CDK, cyclin dependent kinase; CDKi, cyclin dependent kinase inhibitor; CK, cytokeratin; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; Pin1, peptidyL-prolyl cis-trans isomerase NIMA-interacting 1; PR, progesterone receptor; TNBC, triple negative breast cancerBreast cancer onset and disease progression have been linked to members of the TGFb superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFb in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant actions of Smad3. We hypothesized that CDK inhibition (CDKi) would restore Smad3 action and help promote cancer cell regression. Treatment of triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-436, Hs578T) with CDK2i or CDK4i resulted in increased Smad3 activity and decreased cell migration. Transfection with a 5M Smad3 construct containing inhibitory mutations in 5 CDK phosphorylation sites also resulted in decreased TNBC cell migration and invasion. MDA-MB-231 cells treated with CDK2i or CDK4i resulted in decreased Smad3 protein phosphorylation at the CDK phosphorylation T179 site, decreased MMP2 and c-myc expression, and increased p15 and p21 expression. Using a novel transfected cell array, we found that CDK2i treatment decreased activity of the epithelial-to-mesenchymal transition related transcription factors Snail and Twist. In vivo studies in an MDA-MB-231 tumor model showed that individual and combination treatment with paclitaxel and CDK2i resulted in decreased tumor volume and Ki67 staining. Collectively, these data support further investigation of targeted CDK inhibitors as a promising therapeutic strategy for TNBC, a breast cancer subtype with limited treatment options.
Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFβ superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.
A consensus about the functions of human wild-type or mutated α-synuclein (αSYN) is lacking. Both forms of αSYN are implicated in Parkinson’s disease, whereas the wild-type form is implicated in substance abuse. Interactions with other cellular proteins and organelles may meditate its functions. We developed a series of congenic mouse lines containing various allele doses or combinations of the human wild type αSYN (hwαSYN) or a doubly mutated (A30P*A53T) αSYN (hm2αSYN) in a C57Bl/6J line spontaneously deleted in mouse αSYN (C57BL/6JOla). Both transgenes had a functional role in the nigrostriatal system, demonstrated by significant elevations in striatal catecholamines, metabolites, and the enzyme tyrosine hydroxylase compared to null-mice without a transgene. Consequences occurred when the transgenes were expressed at a fraction of the endogenous level. Hemizygous congenic mice did not exhibit any change in the number or size of dopaminergic neurons in the ventral midbrain at nine months of age. Human αSYN was predominantly located in neuronal cell bodies, neurites, synapses, and in intraneuronal/intraneuritic aggregates. The hm2αSYN transgene resulted in more aggregates and dystrophic neurites than did the hw5 transgene. The hwαSYN transgene resulted in higher expression of two striatal proteins, synaptogamin 7 and UCHL1, compared to the levels of the hm2αSYN transgene. These observations suggest that mutations in αSYN may impair specific functional domains, leaving others intact. These lines may also be useful for exploring interactions between hαSYN and environmental or genetic risk factors in dopamine-related disorders using a mouse model.
Stem cell microenvironments decrease the invasiveness of cancer cells, and elucidating the mechanisms associated with disease regression could further the development of targeted therapies for aggressive cancer subtypes. To this end, we applied an emerging technology, TRanscriptional Activity CEll aRray (TRACER), to investigate the reprogramming of triple-negative breast cancer (TNBC) cells in conditions that promoted a less aggressive phenotype. The repressive environment was established through exposure to mouse embryonic stem cell conditioned media (mESC CM). Assessment of carcinogenic phenotypes indicated that mESC CM exposure decreased proliferation, invasion, migration, and stemness in TNBC cells. Protein expression analysis revealed that mESC CM exposure increased expression of the epithelial protein E-cadherin and decreased the mesenchymal protein MMP9. Gene expression analysis showed that mESC CM decreased epithelial to mesenchymal transition (EMT) markers fibronectin, vimentin, and Snail. Over a period of 6 d, TRACER quantified changes in activity of 11 transcription factors (TFs) associated with oncogenic progression. The EMT profile was decreased in association with the activity of 7 TFs (Smad3, NF-κΒ, MEF2, GATA, Hif1, Sp1, and RXR). Further examination of Smad3 and GATA expression and phosphorylation revealed that mESC CM exposure decreased noncanonical Smad3 phosphorylation and Smad3-mediated gene expression, increased GATA3 expression and phosphorylation, and resulted in a synergistic decrease in migration of GATA3 overexpressing MDA-MB-231 cells. Collectively, the application of TRACER to examine TF activity associated with the transition of cancer cells to a less aggressive phenotype, as directed by mESC CM, identified novel mechanistic events linking the embryonic microenvironment to both favorable changes and cellular plasticity in TNBC cell phenotypes.
Introduction: Several aspects of breast cancer onset and disease progression have been linked to members of the TGFβ superfamily and their associated downstream signaling components, the Smads. Alterations in Smad signaling have been directly implicated in the dichotomous role of TGFβ in malignancy, enacting both tumor suppressant and tumor promoting behaviors in breast carcinogenesis. Our previous work characterized Smad3 as a tumor suppressor and found that Smad3 action is inhibited upon phosphorylation by cyclin D/CDK4 in breast cancer cells. Others have shown that CDK phosphorylation of Smad3 can affect cell motility. Based on these findings, we hypothesized that activation of CDK4 leads to phosphorylation and inhibition of Smad3, thus releasing cell cycle arrest and promoting cell proliferation and metastasis. Methods: MCF7 parental and cyclin D overexpressing cells (MCF7 CD1) were treated with a CDK4 inhibitor alone or in combination with doxorubicin or paclitaxel and cell proliferation was determined using the MTS assay. MCF7 study cells were also transduced with lentiviral empty vector, wild-type (WT) Smad3, or Smad3 containing mutant CDK sites (T179V and 5M) resistant to inhibitory cyclin D/CDK phosphorylation. Stably transduced cells were plated in Matrigel 3-dimensional (3D) culture and treated with CDK4 inhibitor alone or with doxorubicin over a 12 day time course. Colony area was measured using MetaMorph software. Transwell migration and Matrigel invasion assays were used to determine the effect that transfection with Smad3 CDK phosphorylation site mutants or treatment with a CDK4 inhibitor had on highly metastatic cyclin D expressing MDA-MB-231 cells. Study wells were fixed/stained and the number of migrated or invaded cells was counted. Results: Treatment with the CDK4 inhibitor alone resulted in a growth inhibitory effect on proliferation of MCF7 parental and CD1 cells. Treatment with the CDK4 inhibitor in combination with chemotherapy resulted in the greatest inhibition of cell proliferation. In 3D culture, colonies formed by MCF7 study cells transduced with T179V or 5M Smad3 mutant constructs had smaller acinar size when compared with vector-transduced control cells. When compared to untreated cells, the MCF7 CD1 cells treated with CDK4 inhibitor or chemotherapy alone had smaller acinar size, with greatest decrease in size observed when the treatments were combined. Lastly, MDA-MB-231 cells transfected with the 5M Smad3 mutant construct, containing mutations in all 5 CDK phosphorylation sites, resulted in the greatest decrease in cell migration and invasion, when compared with cells transfected with both vector control or WT Smad3. Treatment of the MDA-MB-231 cells with the CDK4 inhibitor also resulted in a significant decrease in cell migration and invasion. Conclusions: Mutation of CDK phosphorylation sites in the Smad3 construct or direct inhibition of CDK4 resulted in a decrease in breast cancer cell proliferation, 3D colony formation and cell migration and invasion. Treatment with the CDK4 inhibitor in combination with chemotherapy further augmented these results. Inhibition of CDK4 and restoration of Smad3 activity may have a role in the treatment of breast cancers overexpressing cyclin D. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-06.
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