Edward Perkins

Mitochondrially Targeted Effects of Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium] on K1735-M2 Mouse Melanoma Cells: Comparison with Direct Effects on Isolated Mitochondrial Fractions

Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium] is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment.

Disruption of nucleocytoplasmic trafficking of cyclin D1 and topoisomerase II by sanguinarine

BackgroundThe quaternary isoquinoline alkaloid sanguinarine is receiving increasing attention as a potential chemotherapeutic agent in the treatment of cancer. Previous studies have shown that this DNA-binding phytochemical can arrest a number of different types of transformed cells in G0/G1, and upregulate the CKIs p21 and p27 while downregulating multiple cyclins and CDKs. To more closely examine the responses of some of these cell cycle regulatory molecules to sanguinarine, we used immunocytochemical methods to visualize cyclin D1 and topoisomerase II behavior in MCF-7 breast cancer cells.Results5 – 10 μM sanguinarine effectively inhibits MCF-7 proliferation after a single application of drug. This growth inhibition is accompanied by a striking relocalization of cyclin D1 and topoisomerase II from the nucleus to the cytoplasm, and this effect persists for at least three days after drug addition. DNA synthesis is transiently inhibited by sanguinarine, but cells recover their ability to synthesize DNA within 24 hours. Taking advantage of the fluorescence characteristics of sanguinarine to follow its uptake and distribution suggests that these effects arise from a window of activity of a few hours immediately after drug addition, when sanguinarine is concentrated in the nucleus. These effects occur in morphologically healthy-looking cells, and thus do not simply represent part of an apoptotic response.ConclusionIt appears that sub-apoptotic concentrations of sanguinarine can suppress breast cancer cell proliferation for extended lengths of time, and that this effect results from a relatively brief period of activity when the drug is concentrated in the nucleus. Sanguinarine transiently inhibits DNA synthesis, but a novel mechanism of action appears to involve disrupting the trafficking of a number of molecules involved in cell cycle regulation and progression. The ability of sub-apoptotic concentrations of sanguinarine to inhibit cell growth may be a useful feature for potential chemotherapeutic applications; however, a narrow effective range for these effects may exist.

Sanguinarine cytotoxicity on mouse melanoma K1735-M2 cells—Nuclear vs. mitochondrial effects

Sanguinarine (SANG) is an alkaloid recognized to have anti-proliferative activity against various human tumour cell lines. No data is available on the susceptibility of advanced malignant melanoma to SANG, although this disease has a very poor prognosis if not detected in time due to the resistance to conventional chemotherapy. The present work was designed to study the nuclear and mitochondrial involvement in the pro-apoptotic effect of SANG in an invasive mouse melanoma cell line. The results obtained show that SANG is primarily accumulated by the cell nuclei, causing inhibition of cell proliferation and inducing cell death, as confirmed by an increase in sub-G1 peaks. At low concentrations, SANG induces mitochondrial depolarization in a sub-population of melanoma cells, which also generally displayed strong nuclear labelling of phosphorylated histone H2AX. Western blotting revealed an increase in p53, but not Bax protein, in both whole-cell extracts and in mitochondrial fractions. Isolated hepatic mitochondrial fractions revealed that SANG affects the mitochondrial respiratory chain, and has dual effects on mitochondrial calcium loading capacity. We suggest that SANG is able to induce apoptosis in metastatic melanoma cells. The knowledge of mitochondrial vs. nuclear effects of SANG is important in the development of this promising compound for clinical use against aggressive melanoma.

Dimethylaminopyridine derivatives of lupane triterpenoids are potent disruptors of mitochondrial structure and function

Development of mitochondrially-targeted drugs is receiving increasing attention because of the central roles these organelles play in energy production, reactive oxygen generation, and regulation of cell death pathways. Previous studies have demonstrated that both natural and synthetic triterpenoids can disrupt mitochondrial structure and function. In this study, we tested the ability of a number of dimethylaminopyridine (DMAP) derivatives of lupane triterpenoids to target mitochochondria in two human melanoma cell lines and an untransformed normal fibroblast line. These compounds induced a striking fragmentation and depolarization of the mitochondrial network, along with an inhibition of cell proliferation. A range of potencies among these compounds was noted, which was correlated with the number, position, and orientation of the DMAP groups. Overall, the extent of proliferation inhibition mirrored the effectiveness of mitochondrial disruption. Thus, DMAP derivatives of lupane triterpenoids can be potent mitochondrial perturbants that appear to suppress cell growth primarily via their mitochondrial effects.

New derivatives of lupane triterpenoids disturb breast cancer mitochondria and induce cell death

Novel cationic dimethylaminopyridine derivatives of pentacyclic triterpenes were previously described to promote mitochondrial depolarization and cell death in breast and melanoma cell lines. The objective of this work was to further investigate in detail the mechanism of mitochondrial perturbations, correlating those effects with breast cancer cell responses to those same agents. Initially, a panel of tumor and non-tumor cell lines was grown in high-glucose or glucose-free glutamine-containing media, the later forcing cells to synthesize ATP by oxidative phosphorylation only. Cell proliferation, cell cycle, cell death and mitochondrial membrane polarization were evaluated. Inhibition of cell proliferation was observed, accompanied by an arrest in the G1-cell cycle phase, and importantly, by loss of mitochondrial membrane potential. On a later time-point, caspase-9 and 3 activation were observed, resulting in cell death. For the majority of test compounds, we determined that cell toxicity was augmented in the galactose media. To investigate direct evidences on mitochondria isolated rat liver mitochondria were used. The results showed that the compounds were strong inducers of the permeability transition pore. Confirming our previous results, this work shows that the novel DMAP derivatives strongly interact with mitochondria, resulting in pro-apoptotic signaling and cell death.

Differentiation of pluripotent stem cells on multiwalled carbon nanotubes

This paper studies the adhesion, growth, and differentiation of stem cells on carbon nanotube matrices. Glass coverslips were coated with multiwalled carbon nanotube (MWNT) thin films using layer-by-layer self-assembling techniques. Pluripotent P19 mouse embryonal carcinoma stem cells were seeded onto uncoated or MWNT-coated coverslips, and either maintained in an undifferentiated state or induced to differentiate by the addition of retinoic acid. We found that cell adhesion was increased on the MWNT-coated surfaces, and that the expression patterns of some differentiation markers were altered in cells grown on MWNTs. The results suggest that MWNTs will be useful in directing pluripotent stem cell differentiation for tissue engineering purposes.

Vital imaging of multicellular spheroids.

Cell behavior is significantly different in two-dimensional and three-dimensional culture conditions, and a number of methods have been developed to establish and study three-dimensional cellular arrays in vitro. When grown under nonadherent conditions, many types of cells form structures called multicellular spheroids (MCSs), which have been popular models to study cell behavior in a three-dimensional environment. The histoarchitecture of MCSs derived from malignant cells resembles that of tumors, and there is rapidly increasing interest in using these structures to more accurately understand the dynamics of cancer cells in situ, including their responses to chemotherapeutics. Confocal microscopy is an extremely useful method to investigate cell behavior in MCSs due to its ability to more clearly image fluorescent probes at some depth in three-dimensional structures. This chapter describes some basic approaches toward visualizing a variety of fluorescent probes in MCSs.

Downstream bioengineering of ACE chromosomes for incorporation of site-specific recombination cassettes.

Advances in mammalian artificial chromosome technology have made chromosome-based vector technology amenable to a variety of biotechnology applications including cellular protein production, genomics, and animal transgenesis. A pivotal aspect of this technology is the ability to generate artificial chromosomes de novo, transfer them to a variety of cells, and perform downstream engineering of artificial chromosomes in a tractable and rational manner. Previously, we have described an alternative artificial chromosome technology termed the ACE chromosome system, where the ACE platform chromosome contains a multitude of site-specific, recombination sites incorporated during the creation of the ACE platform chromosome. In this chapter we review a variant of the ACE chromosome technology whereby site-specific, recombination sites can be integrated into the ACE chromosome following its de novo synthesis. This variation allows insertion of user-defined, site-specific, recombination systems into an existing ACE platform chromosome. These bioengineered ACE platform chromosomes, containing user-defined recombination sites, represent an ideal circuit board to which an array of genetic factors can be plugged-in and expressed for various research and therapeutic applications.

Abstract B88: Local delivery of breast cancer chemopreventives using mesenchymal stem cells

The overall goal of this study is to evaluate the ability of bone marrow mesenchymal stem cells (MSCs) to act as vehicles for local delivery of chemopreventives in mice that develop mammary cancer. Recently, efforts to use adult‐derived stem cells to deliver therapeutic agents to tumor sites have shown great promise and we currently are extending this concept to include the delivery of gene therapy in the form of chemopreventives. Our goals in this initial project were to (1) isolate and study the behavior of transplanted MSCs in mammary glands of mice and (2) develop a gene expression system which robustly produces gene therapeutics. For gene therapy we aimed to target epidermal growth factor (EGFR/ErbB1/HER1) because activation of this growth factor pathway is a frequent component driving breast cancer proliferation and survival and recent evidence demonstrated that the EGFR pathway is more active in hyperplastic enlarged lobules compared with normal terminal ductal lobular units (AJP 2007 171, 253–262), indicating that EGFR activation is a very early event that precedes cancer development and therefore inhibiting EGFR may abrogate cancer development. To carry out this goal we developed an expression system that produces the natural inhibitory ligand of EGFR, decorin. Decorin is a small leucine rich proteoglycan with known effects in extracellular matrix assembly as well as growth inhibition via reduction in levels and activity of EGFR along with other signaling molecules. Thus far we have demonstrated the ability to isolate, culture and maintain murine MSCs from the FVB/N strain in an undifferentiated state. In addition, these MSCS do not form tumors upon transplantation into mammary glands of syngeneic mice. Furthermore, we have produced secreted decorin in Chinese hamster ovarian cells and MSCs. Finally, we demonstrate the ability of conditioned media containing decorin to inhibit mammary tumor cell proliferation in vitro. Our long-term goal is to test the efficacy of decorin‐engineered MSCs as a breast cancer prevention modality in mouse models. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B88.

Abstract A197: Development of an immunocompetent, triple negative, breast cancer model for stem cell based delivery of therapeutics

Breast cancer represents a broad‐spectrum cancer with diverse morphological and immunohistochemical features and varied clinical outcomes. Affected breast cancer patients fall into one of three classifications: 1) hormone (estrogen receptor (ER), progesterone receptor (PR))‐positive tumors, 2) tumors with demonstrated ERBB2 (HER2 + ) amplification and, 3) tumors that are ER − , PR − and ERBB2 lo . For hormone receptor‐positive (ER) tumors, a variety of ER‐targeted therapy regimens with and without chemotherapy are available. Patients whose tumors are classified as ER − , PR − and ERBB2 lo (triple negative breast cancer; TNBC) are largely limited to chemotherapy as the only therapeutic modality. We have developed an autologous, immunocompetent triple negative breast cancer (TNBC) model whose features may overcome deficiencies of xenograft models for development of TNBC therapeutics. The model consists of: FVB/N immunocompetent mice; bone derived mesenchymal stem cells (MSCs) from FVB/N mice; and a triple negative cell line (UMD‐227), which is derived from a mammary tumor that developed in an NRL‐TGFα FVB/N mouse. Our hypothesis is that expression of antitumor factors (interferon beta (IFNβ), TNF‐related apoptosis‐inducing ligand (TRAIL), and/or the epidermal growth factor receptor (EGFR) inhibitor decorin (DCN)) from MSCs, either singly or in combination, will efficiently kill triple negative breast cancers. To test this hypothesis in vitro , we have applied each therapeutic individually, as conditioned medium produced from MSCs, on our novel, triple negative, mouse mammary cancer line, UMD‐227, and measured cell proliferation by a sulforhodamine B assay. In addition, we have tested for an additive effect with IFNβ and TRAIL using MSC/IFNβ conditioned media and commercial TRAIL. The addition of MSC/IFN conditioned medium to UMD‐227 cells significantly inhibits their growth in a proliferation assay similar to that shown with the TNBC line MDA‐MB‐231. The addition of commercial TRAIL 24 hours after placing UMD‐227 cells in MSC/IFNβ conditioned medium, produces a significant decrease in cell proliferation as compared to MSC/IFN conditioned medium alone or TRAIL alone. This is consistent with the knowledge that IFNβ upregulates TRAIL receptors, making the cell more susceptible to the addition of TRAIL at the 24 hour timepoint. These results suggest that MSCs engineered to express combinations of anticancer molecules may be a more efficacious therapeutic modality for TNBC. The next step in the development of our autologous immunocompetent triple breast cancer model is the insertion of multiple factors into the MSCs. We will then test the efficacy of the combinatorial therapy delivered from MSCs on the proliferation and motility of UMD‐227s in vitro. Our final goal is to move the entire system into the autologous mouse model (NRL‐TGFα) and test the efficacy of the combinatorial therapy in vivo. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A197.