Bladder dysfunction is associated with the overexpression of the intermediate filament (IF) proteins desmin and vimentin in obstructed bladder smooth muscle (BSM). However, the mechanisms by which ...these proteins contribute to BSM dysfunction are not known. Previous studies have shown that desmin and vimentin directly participate in signal transduction. In this study, we hypothesized that BSM dysfunction associated with overexpression of desmin or vimentin is mediated via c‐Jun N‐terminal kinase (JNK). We employed a model of murine BSM tissue in which increased expression of desmin or vimentin was induced by adenoviral transduction to examine the sufficiency of increased IF protein expression to reduce BSM contraction. Murine BSM strips overexpressing desmin or vimentin generated less force in response to KCl and carbachol relative to the levels in control murine BSM strips, an effect associated with increased JNK2 phosphorylation and reduced myosin light chain (MLC20) phosphorylation. Furthermore, desmin and vimentin overexpressions did not alter BSM contractility and MLC20 phosphorylation in strips isolated from JNK2 knockout mice. Pharmacological JNK2 inhibition produced results qualitatively similar to those caused by JNK2 knockout. These findings suggest that inhibition of JNK2 may improve diminished BSM contractility associated with obstructive bladder disease.
Abstract
Triple negative breast cancer (TNBC) is one of the most aggressive breast cancers and is characterized by poor prognosis and high possibilities of tumor relapse. TNBC lacks specific ...receptors that are targets for breast cancer treatment options. Plant flavonoids have been identified to inhibit tumor growth and metastasis in various cancers. Our study revealed that quercetin, one of the major flavonoids present in fruits, vegetables, green tea, and red wine inhibit TNBC migration and invasion. Our data indicate that quercetin is able to induce the expression of E-cadherin and also down regulate vimentin levels in TNBC cells. Further the cellular localization of β-catenin is also altered by quercetin treatment. Also, studies identified that quercetin is capable of inhibiting the chemotherapy induced TGF-β signaling in TNBC cells, and that it would decrease the development of chemotherapy resistance in TNBC. Quercetin inhibits the TGF-β induced phosphorylation status of smad-2 in drug resistant cells. Further, quercetin is able to inhibit the enhanced migration and mammosphere formation of chemoresistant TNBC cells. Taken together, our findings revealed that quercetin is a promising natural product that increases the anti-cancer effect of chemotherapy drug in TNBC. Future studies in TNBC animal models are needed to confirm the therapeutic efficacy of quercetin in TNBC.
Citation Format: Asha Srinivasan, Chellappagounder Thangavel, Yi Liu, Sunday Shoyele, Robert B. Den, Ponniah Selvakumar, Ashakumary Lakshmikuttyamma. Quercetin overcomes chemotherapy resistance in triple negative breast cancer. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5560. doi:10.1158/1538-7445.AM2015-5560
Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction ...in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease.
siRNAs have a high potential for silencing critical molecular pathways that are pathogenic. Nevertheless, their clinical application has been limited by a lack of effective and safe ...nanotechnology-based delivery system that allows a controlled and safe transfection to cytosol of targeted cells without the associated adverse effects. Our group recently reported a very effective and safe hybrid nanoparticle delivery system composing human IgG and poloxamer-188 for siRNA delivery to cancer cells. However, these nanoparticles need to be optimized in terms of particle size, loading capacity and encapsulation efficiency. In the present study, we explored the effects of certain production parameters on particle size, loading capacity and encapsulation efficiency. Further, to make these nanoparticles more specific in their delivery of siRNA, we conjugated anti-NTSR1-mAb to the surface of these nanoparticles to target NTSR1-overexpressing cancer cells. The mechanism of siRNA release from these antiNTSR1-mAb functionalized nanoparticles was also elucidated.
It was demonstrated that the concentration of human IgG in the starting nanoprecipitation medium and the rotation speed of the magnetic stirrer influenced the encapsulation efficiency, loading capacity and the size of the nanoparticles produced. We also successfully transformed these nanoparticles into actively targeted nanoparticles by functionalizing with anti-NTSR1-mAb to specifically target NTSR1-overexpressing cancer cells, hence able to avoid undesired accumulation in normal cells. The mechanism of siRNA release from these nanoparticles was elucidated to be by Fickian diffusion. Using flow cytometry and fluorescence microscopy, we were able to confirm the active involvement of NTSR1 in the uptake of these anti-NTSR1-mAb functionalized hybrid nanoparticles by lung adenocarcinoma cells.
This hybrid nanoparticle delivery system can be used as a platform technology for intracellular delivery of siRNAs to NTSR1-overexpressing tumor cells.
Abstract
Introduction: The retinoblastoma tumor suppressor protein (RB) is a critical mediator of cell proliferation via its regulation of cell cycle by binding to E2Fs. One of the mechanisms of RB ...activation is through CDK4/6 inhibition. Currently, multiple selective CDK4/6 inhibitors are being utilized in the clinical setting and are thought to drive cellular cytostasis. In this study, we demonstrate that RB activation not only results in cellular senescence, but for the first time, induces apoptosis by repressing IAPs (inhibitor of apoptosis protein).
Experimental Procedures: Isogenic pairs of RB knockdown and control cells were interrogated in vitro, and in xenograft tumors for CDK 4/6 inhibitor response. Cellular senescence and apoptosis were quantified. Molecular mechanism was determined using microarray, chromatin immunoprecipitation, and co-immunoprecipitation assays.
Summary: CDK4/6 inhibition diminished cell growth in an RB dependent manner both in in vitro and in vivo models. Immunohistochemistry from the xenografts demonstrated increased levels of apoptosis in the RB proficient isogenic lines. This was confirmed via western blot analysis for Caspase-3. Mechanistically, microarray analysis demonstrated that RB activation downregulated several key anti-apoptotic proteins (e.g. FOXM1, Survivin). ChIP assay and in silico modeling showed that FOXM1 and Survivin are repressed by RB/E2F binding. In the RB deficient setting, immunoprecipitation demonstrates that FOXM1 and Survivin interact directly with Caspase-3 resulting in decreased apoptosis. This was reversed using a competitive assay with overexpression of the SMAC (second mitochondrial) protein.
Conclusion: Data from multiple isogenic NSCLC models demonstrate that RB activation via CDK4/6 inhibitors results in apoptosis in an RB dependent manner. Mechanistically, RB represses transcription of multiple IAPs (including FOXM1 and Survivin), which allows pro-apoptotic factors such as SMAC to activate Caspase-3. These findings suggest that CDK4/6 inhibitors should be viewed as cytotoxic, as opposed to purely cytostatic, agents and warrant further clinical investigation.
Citation Format: Chellappagounder Thangavel, Ettickan Boopathi, Yi Liu, Alex Haber, Maryna Perepelyuk, Sankar Addya, Sunday Shoyele, Adam P. Dicker, Karen E. Knudsen, Robert B. Den. Retinoblastoma protein orchestrates cellular apoptosis in non-small cell lung cancer in response to CDK4/6 inhibition: novel targets and key mechanisms. abstract. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B08.
In the present study, we describe the effects of medium composition in primary cultures of rat hepatocytes on the expression of two major constituent female-dependent CYP isoforms, CYP2C12 and ...CYP2A1. When female rat hepatocytes were cultured with the serum-free medium HepatoZYME, currently used to attain long-term maintenance of hepatocyte phenotypic expression, CYP2C12 mRNA and protein levels were markedly suppressed, despite the constant presence of growth hormone, the essential regulator of liver CYP2C12. Conversely, rat hepatocytes cultured in the serum-free medium Dulbecco's modified Eagle's medium-F12K, also supplemented with growth hormone, sustained near normal expression levels of CYP2C12 mRNA and protein for the 7 days of observations. Although media composition had no significant effect on mRNA expression of CYP2A1, protein content decreased dramatically in hepatocytes cultured with HepatoZYME medium. We were able to demonstrate the plasticity of the cells by restoring/suppressing the expression of CYP2C12 and CYP2A1 mRNA by reverting the culture conditions. Addition of the mitogen epidermal growth factor present in the HepatoZYME formulation to the Dulbecco's modified Eagle's medium-F12K culture media appreciably decreased expression of both CYP2C12 and CYP2A1 in female hepatocytes, while briefly sustaining levels of the cyclin inhibitor p21. Lastly, reduced CYP protein content observed in hepatocytes cultured with epidermal growth factor was not the result of an absence or reduction in the CCAAT/enhancer-binding protein alpha, a requisite transcription factor for CYP2C12 expression.
The RB pathway plays a critical role in proliferation control that is commonly subverted in tumor development. However, restoration of RB pathway function can be elicited in many tumor cells by the ...inhibition of CDK4/6 activity that leads to dephosphorylation of RB and subsequent repression of E2F-mediated transcription. In this context, active RB/E2F complexes inhibit the expression of a critical program of coding genes that promote cell cycle progression. However, the non-coding RNA target genes downstream from RB that could be relevant for tumor biology remain obscure. Here, miRNA gene expression profiling identified the miR106b cluster as being efficiently repressed with CDK4/6 inhibition in an E2F and RB-dependent manner. Importantly, the miR106B-cluster is intragenic of MCM7, and through a series of functional studies, the basis of MCM7 regulation and concordant expression of the miRNA species within the 106b cluster was determined. Importantly, RB-mediated repression of the 106b cluster enhances the transcript levels of p21Cip1 and PTEN. These data provide a mechanistic basis for cross-talk between the RB pathway and p21 and PTEN through the regulation of the MCM7/miR106b locus.
Second generation antiandrogens have improved overall survival for men with metastatic castrate resistant prostate cancer; however, the antiandrogens result in suppression of androgen receptor (AR) ...activity in all tissues resulting in dose limiting toxicity. We sought to overcome this limitation through encapsulation in a prostate specific membrane antigen (PSMA)–conjugated nanoparticle. We designed and characterized a novel nanoparticle containing an antiandrogen, enzalutamide. Selectivity and enhanced efficacy was achieved through coating the particle with PSMA. The PSMA-conjugated nanoparticle was internalized selectively in AR expressing prostate cancer cells. It did not elicit an inflammatory effect. The efficacy of enzalutamide was not compromised through insertion into the nanoparticle; in fact, lower systemic drug concentrations of enzalutamide resulted in comparable clinical activity. Normal muscle cells were not impacted by the PSMA-conjugated containing antiandrogen. This approach represents a novel strategy to increase the specificity and effectiveness of antiandrogen treatment for men with castrate resistant prostate cancer. The ability to deliver higher drug concentrations in prostate cancer cells may translate into improved clinical end points including overall survival.
PDF
