BackgroundResistance to chemotherapy is a major problem facing breast cancer patients, and identifying potential contributors to chemoresistance is a critical area of research. Bisphenol A (BPA) has long been suspected to promote carcinogenesis, but the high doses of BPA used in many studies generated conflicting results. In addition, the mechanism by which BPA exerts its biological actions is unclear. Although estrogen has been shown to antagonize anticancer drugs, the role of BPA in chemoresistance has not been examined.ObjectiveThe objective of our study was to determine whether BPA at low nanomolar concentrations opposes the action of doxorubicin, cisplatin, and vinblastine in the estrogen receptor-α (ERα)-positive T47D and the ERα-negative MDA-MB-468 breast cancer cells.MethodsWe determined the responsiveness of cells to anticancer drugs and BPA using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Specific ERα and ERβ inhibitors and real-time polymerase chain reaction were used to identify potential receptor(s) that mediate the actions of BPA. Expression of antiapoptotic proteins was assessed by Western blotting.ResultsBPA antagonizes the cytotoxicity of multiple chemotherapeutic agents in both ERα-positive and -negative breast cancer cells independent of the classical ERs. Both cell types express alternative ERs, including G-protein–coupled receptor 30 (GPR30) and members of the estrogen-related receptor family. Increased expression of antiapoptotic proteins is a potential mechanism by which BPA exerts its anticytotoxic effects.ConclusionsBPA at environmentally relevant doses reduces the efficacy of chemotherapeutic agents. These data provide considerable support to the accumulating evidence that BPA is hazardous to human health.
The liver exhibits an exquisitely controlled cell cycle, wherein hepatocytes are maintained in quiescence until stimulated to proliferate. The retinoblastoma tumor suppressor, pRB, plays a central role in proliferative control by inhibiting inappropriate cell cycle entry. In many cases, liver cancer arises due to aberrant cycles of proliferation, and correspondingly, pRB is functionally inactivated in the majority of hepatocellular carcinomas. Therefore, to determine how pRB loss may provide conditions permissive for deregulated hepatocyte proliferation, we investigated the consequence of somatic pRB inactivation in murine liver. We show that liver-specific pRB loss results in E2F target gene deregulation and elevated cell cycle progression during postnatal growth. However, in adult livers, E2F targets are repressed and hepatocytes become quiescent independent of pRB, suggesting that other factors may compensate for pRB loss. Therefore, to probe the consequences of acute pRB inactivation in livers of adult mice, we gave adenoviral-Cre by i.v. injection. We show that acute pRB loss is sufficient to elicit E2F target gene expression and cell cycle entry in adult liver, demonstrating a critical role for pRB in maintaining hepatocyte quiescence. Finally, we show that liver-specific pRB loss results in the development of nuclear pleomorphism associated with elevated ploidy that is evident in adult mice harboring both acute and chronic pRB loss. Together, these results show the crucial role played by pRB in maintaining hepatocyte quiescence and ploidy in adult liver in vivo and underscore the critical importance of delineating the consequences of acute pRB loss in adult animals. (Cancer Res 2005; 65(11): 4568-77)
Patients with advanced breast cancer often fail to respond to treatment, creating a need to develop novel biomarkers and effective therapeutics. Dopamine (DA) is a catecholamine which binds to five G-protein-coupled receptors. We discovered expression of DA type-1 receptors (D1R) in breast cancer, thereby identifying these receptors as novel therapeutic targets in this disease. Strong to moderate immunoreactive D1R expression was found in 30% of 751 primary breast carcinomas, and was associated with larger tumors, higher tumor grades, node metastasis, and shorter patient survival. DA and D1R agonists, signaling through the cGMP/protein kinase G (PKG) pathway, suppressed cell viability, inhibited invasion, and induced apoptosis in multiple breast cancer cell lines. Fenoldopam, a peripheral D1R agonist which does not penetrate the brain, dramatically suppressed tumor growth in two mouse models with D1R-expressing xenografts by increasing both necrosis and apoptosis. D1R-expressing primary tumors and metastases in mice were detected by fluorescence imaging. In conclusion, D1R overexpression is associated with advanced breast cancer and poor prognosis. Activation of the D1R/cGMP/PKG pathway induces apoptosis in vitro and causes tumor shrinkage in vivo. Fenoldopam, which is FDA-approved to treat renal hypertension, could be repurposed as a novel therapeutic agent for patients with D1R-expressing tumors.
Cyclin D1 is a proto-oncogene that functions by inactivation of the retinoblastoma tumor suppressor protein, RB. A common polymorphism in the cyclin D1 gene is associated with the production of an alternate transcript of cyclin D1, termed cyclin D1b. Both the polymorphism and the variant transcript are associated with increased risk for multiple cancers and the severity of a given cancer; however, the underlying activities of cyclin D1b have not been elucidated relative to the canonical cyclin D1a. Because cyclin D1b does not possess the threonine 286 phosphorylation site required for nuclear export and regulated degradation, it has been hypothesized to encode a stable nuclear protein that would constitutively inactivate the RB pathway. Surprisingly, we find that cyclin D1b protein does not inappropriately accumulate in cells and exhibits stability comparable to cyclin D1a. As expected, the cyclin D1b protein was constitutively localized in the nucleus, whereas cyclin D1a was exported to the cytoplasm in S-phase. Despite enhanced nuclear localization, we find that cyclin D1b is a poor catalyst of RB phosphorylation/inactivation. However, cyclin D1b potently induced cellular transformation in contrast to cyclin D1a. In summary, we demonstrate that cyclin D1b specifically disrupts contact inhibition in a manner distinct from cyclin D1a. These data reveal novel roles for D-type cyclins in tumorigenesis.Cyclin D1 is an essential regulator of cell cycle progression, and aberrant induction of cyclin D1 activity is well established in human tumorigenesis (1-7). Initially, cyclin D1 was identified as the PRAD1 oncogene by mapping the sites of amplification in parathyroid adenomas to 11q13 (1,8). Subsequently, deregulation of cyclin D1 has been observed to occur in a variety of cancer types (9). For example, the BCL-1 translocation in centrocytic lymphoma deregulates the expression of cyclin D1 (8). In animal models, cyclin D1 has also been shown to exhibit oncogenic activity when overexpressed in specific tissues (10). Lastly, cyclin D1 activity is critical for tumor formation induced by other oncogenes (e.g. Ras), as mice deficient in cyclin D1 are resistant to tumorigenesis (11,12). Because of the intimate connections between cyclin D1 and oncogenesis, extensive analyses have focused on the mechanisms through which cyclin D1 contributes to human cancer.Cyclin D1 exerts its effects on cellular proliferation by integrating external signals (e.g. mitogens) with the cell cycle machinery (3-7, 13-15). Given this critical role, cyclin D1 action is highly regulated. Cyclin D1 transcription is stimulated as a delayed early response to mitogenic signaling cascades (4, 16). Additionally, protein stability is regulated through the glycogen synthase kinase 3 signal transduction pathway to coordinately enhance accumulation of cyclin D1 protein (17, 18). Once synthesized, cyclin D1 interacts with and activates the G 1 cyclin-dependent kinases (CDK), 1 CDK4 and CDK6 (19,20). This interaction is assisted through the action of both m...
Switch (SWI)/sucrose nonfermentable (SNF)is an evolutionarily conserved complex with ATPase function, capable of regulating nucleosome position to alter transcriptional programs within the cell. It is known that the SWI/SNF complex is responsible for regulation of many genes involved in cell cycle control and proliferation, and it has recently been implicated in cancer development. The ATPase action of SWI/SNF is conferred through either the brahma-related gene 1 (Brg1) or brahma (Brm) subunit of the complex, and it is of central importance to the modification of nucleosome position. In this study, the role of the Brg1 and Brm subunits were examined as they relate to chromatin structure and organization. Deletion of the Brg1 ATPase results in dissolution of pericentromeric heterochromatin domains and a redistribution of histone modifications associated with these structures. This effect was highly specific to Brg1 and is not reproduced by the loss of Brm or SNF5/BAF47/INI1. Brg1 deficiency is associated with the appearance of micronuclei and aberrant mitoses that are a by-product of dissociated chromatin structure. Thus, Brg1 plays a critical role in maintaining chromatin structural integrity. INTRODUCTIONThe switch/sucrose nonfermentable (SWI/SNF) complex uses the energy of ATP hydrolysis to modulate nucleosome position and density across promoters and thus plays an important role in regulating gene expression. The SWI/SNF ATPase function is necessary for its chromatin remodeling activities (Laurent et al., 1993), and this function is manifested through one of two mutually exclusive subunits, brahma-related gene 1 (Brg1) or brahma (Brm). Although both Brg1 and Brm can function as the central ATPase in the SWI/SNF complex, each defines a discrete complex with unique biochemical activity. In addition, specific accessory factors interact with the ATPase subunits and play critical roles in chromatin remodeling activity. For example, SNF5/ BAF47/INI1 is present in both Brg1-and Brm-associated complexes and is required for maximal chromatin remodeling activity, both in vitro and in yeast. Surprisingly, although Brg1 and Brm have Ͼ75% sequence homology, the impact of each individual subunit on chromatin biology and underlying cell biology is poorly understood (Phelan et al., 1999).Due to its central function in chromatin remodeling, SWI/ SNF function is required in many facets of gene regulation. Correspondingly, it is estimated that approximately 5% of the yeast genome is transcriptionally regulated by the SWI/ SNF complex (Holstege et al., 1998;Sudarsanam et al., 2000). In mammalian cells, SWI/SNF plays a critical role in the activation of a diverse set of genes and has been shown to be recruited directly to transcriptional activation domains (De- Hassan et al., 2001). In contrast, SWI/ SNF is also required for transcriptional repression. For example, transcriptional repression of cell cycle genes mediated by the retinoblastoma tumor suppressor is dependent on SWI/SNF, implicating the involvement of SWI/SNF in cell...
Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease with high mortality. Treatments, which can result in significant morbidity, have not substantially changed in three decades. The second messenger cyclic GMP (cGMP), which targets protein kinase G (PKG), is generated by guanylate cyclases (GCs), and is rapidly hydrolyzed by phosphodiesterases (PDEs). Activation of the cGMP/PKG pathway is antineoplastic in several cancer types, but its impact on HNSCC has not been fully exploited. We found differential expression of critical components of this pathway in four HNSCC cell lines. Several activators of soluble GC (sGC), as well as inhibitors of PDE5, increased intracellular cGMP, reduced cell viability, and induced apoptosis in HNSCC cells. The apoptotic effects of the sGC activator BAY 41-2272 and the PDE5 inhibitor Tadalafil (Cialis) were mediated by PKG. Furthermore, Tadalafil substantially reduced the growth of CAL27-derived tumors in athymic mice. Several drugs which either activate sGC or inhibit PDE5 are approved for treatment of nonmalignant conditions. These drugs could be repurposed as novel and effective therapeutics in patients with head and neck cancer.
The 117-nucleotide (nt) RNA, called the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor, has been shown to be an efficient vector for the construction of RNA nanoparticles for the delivery of small interfering RNA (siRNA) into specific cancer or viral-infected cells. Currently, chemical synthesis of 117-nt RNA is not feasible commercially. In addition, labeling at specific locations on pRNA requires the understanding of its modular organization. Here, we report multiple approaches for the construction of a functional 117-base pRNA using two synthetic RNA fragments with variable modifications. The resulting bipartite pRNA was fully competent in associating with other interacting pRNAs to form dimers, as demonstrated by the packaging of DNA via the nanomotor and the assembly of phi29 viruses in vitro. The pRNA subunit assembled from bipartite fragments harboring siRNA or receptor-binding ligands were equally competent in assembling into dimers. The subunits carrying different functionalities were able to bind cancer cells specifically, enter the cell, and silence specific genes of interest. The pRNA nanoparticles were subsequently processed by Dicer to release the siRNA embedded within the nanoparticles. The results will pave the way toward the treatment of diseases using synthetic pRNA/siRNA chimeric nanoparticles.
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