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Sioux Falls, SD, United States

Dhuban K.B.,McGill University | D'Henneze E.,McGill University | Nashi E.,McGill University | Bar-Or A.,Montreal Neurological Institute | And 4 more authors.
Journal of Immunology | Year: 2015

Two distinct subsets of CD4+Foxp3+ regulatory T (Treg) cells have been described based on the differential expression of Helios, a transcription factor of the Ikaros family. Efforts to understand the origin and biological roles of these Treg populations in regulating immune responses have, however, been hindered by the lack of reliable surface markers to distinguish and isolate them for subsequent functional studies. Using a single-cell cloning strategy coupled with microarray analysis of different Treg functional subsets in humans, we identify the mRNA and protein expression of TIGIT and FCRL3 as a novel surface marker combination that distinguishes Helios+FOXP3+ from Helios-FOXP3+ memory cells. Unlike conventional markers that are modulated on conventional T cells upon activation, we show that the TIGIT/FCRL3 combination allows reliable identification of Helios+ Treg cells even in highly activated conditions in vitro as well as in PBMCs of autoimmune patients. We also demonstrate that the Helios-FOXP3+ Treg subpopulation harbors a larger proportion of nonsuppressive clones compared with the Helios+ FOXP3+ cell subset, which is highly enriched for suppressive clones. Moreover, we find that Helios- cells are exclusively responsible for the productions of the inflammatory cytokines IFN-γ, IL-2, and IL-17 in FOXP3+ cells ex vivo, highlighting important functional differences between Helios+ and Helios- Treg cells. Thus, we identify novel surface markers for the consistent identification and isolation of Helios+ and Helios- memory Treg cells in health and disease, and we further reveal functional differences between these two populations. These new markers should facilitate further elucidation of the functional roles of Heliosbased Treg heterogeneity. Copyright © 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00.

Zhuang Y.,Cancer Biology Research Center | Miskimins W.K.,Cancer Biology Research Center | Miskimins W.K.,University of South Dakota
Molecular Cancer Research | Year: 2011

There is substantial evidence that metformin, a drug used to treat type 2 diabetics, is potentially useful as a therapeutic agent for cancer. However, a better understanding of the molecular mechanisms through which metformin promotes cell-cycle arrest and cell death of cancer cells is necessary. It will also be important to understand how the response of tumor cells differs from normal cells and why some tumor cells are resistant to the effects of metformin. We have found that exposure to metformin induces cell death in all but one line, MDA-MB-231, in a panel of breast cancer cell lines.MCF10A nontransformed breast epithelial cells were resistant to the cytotoxic effects of metformin, even after extended exposure to the drug. In sensitive lines, cell death was mediated by both apoptosis and a caspase-independent mechanism. The caspase-independent pathway involves activation of poly(ADP-ribose) polymerase (PARP) and correlates with enhanced synthesis of PARP and nuclear translocation of apoptosis-inducing factor (AIF), which plays an important role in mediating cell death. Metformin-induced, PARP-dependent cell death is associated with a striking enlargement of mitochondria. Mitochondrial enlargement was observed in all sensitive breast cancer cell lines but not in nontransformed cells or resistant MDA-MB-231. Mitochondrial enlargement was prevented by inhibiting PARP activity or expression. A caspase inhibitor blocked metformin-induced apoptosis but did not affect PARP-dependent cell death or mitochondrial enlargement. Thus, metformin has cytotoxic effects on breast cancer cells through 2 independent pathways. These findings will be pertinent to efforts directed at using metformin or related compounds for cancer therapy. ©2011 AACR.

Zheng Y.,Cancer Biology Research Center | Zheng Y.,University of South Dakota | Zheng Y.,University of Houston | Miskimins W.K.,Cancer Biology Research Center
RNA Biology | Year: 2011

The cyclin dependent kinase inhibitor p27 Kip1 plays an important role in controlling the eukaryotic cell cycle. The 5′-untranslated region of the p27 mRNA harbors an internal ribosome entry site (IRES ) which may facilitate synthesis of p27 in certain conditions. In this study, the RNA-associated protein CU GBP1 was shown to interact with the human p27 5′-untranslated region. Overexpression of CU GBP1 inhibited endogenous p27 expression and reduced translation initiation through the p27 IRES . In contrast, repression of CU GBP1 by siRNA transfection enhanced p27 protein levels and stimulated p27 IRES activity. Addition of recombinant CU GBP1 repressed p27 IRES reporter mRNA translation in vitro. At last, our finding showed that cytosolic form of CU GBP1 binds efficiently to the p27 5′-untranslated region. © 2011 Landes Bioscience.

Chan D.K.,Cancer Biology Research Center | Miskimins W.K.,Cancer Biology Research Center
Journal of Ovarian Research | Year: 2012

Background: High mortality rates in ovarian cancer are largely a result of resistance to currently used chemotherapies. Expanding therapies with a variety of drugs has the potential to reduce this high mortality rate. Metformin and phenethyl isothiocyanate (PEITC) are both potentially useful in ovarian cancer, and they are particularly attractive because of their safety. Methods. Cell proliferation of each drug and drug combination was evaluated by hemacytometry with Trypan blue exclusion or Sytox green staining for cell death. Levels of total and cleaved PARP were measured by Western blot. General cellular and mitochondrial reactive oxygen species were measured by flow cytometry and live cell confocal microscopy with the fluorescent dyes dihydroethidine and MitoSOX. Results: Individually, metformin and PEITC each show inhibition of cell growth in multiple ovarian cancer cell lines. Alone, PEITC was also able to induce apoptosis, whereas metformin was primarily growth inhibitory. Both total cellular and mitochondrial reactive oxygen species were increased when treated with either metformin or PEITC. The growth inhibitory effects of metformin were reversed by methyl succinate supplementation, suggesting complex I plays a role in metformin's anti-cancer mechanism. PEITC's anti-cancer effect was reversed by N-acetyl-cysteine supplementation, suggesting PEITC relies on reactive oxygen species generation to induce apoptosis. Metformin and PEITC together showed a synergistic effect on ovarian cancer cell lines, including the cisplatin resistant A2780cis. Conclusions: Here we show that when used in combination, these drugs are effective in both slowing cancer cell growth and killing ovarian cancer cells in vitro. Furthermore, the combination of these drugs remains effective in cisplatin resistant cell lines. Novel combinations such as metformin and PEITC show promise in expanding ovarian cancer therapies and overcoming the high incidence of cisplatin resistant cancers. © 2012Chan and Miskimins;licensee BioMed Central Ltd.

Yallapu M.M.,Cancer Biology Research Center | Jaggi M.,Cancer Biology Research Center | Jaggi M.,University of South Dakota | Chauhan S.C.,Cancer Biology Research Center | Chauhan S.C.,University of South Dakota
Colloids and Surfaces B: Biointerfaces | Year: 2010

Curcumin, a hydrophobic polyphenolic compound derived from the rhizome of the herb Curcuma longa, possesses a wide range of biological applications including cancer therapy. However, its prominent application in cancer treatment is limited due to sub-optimal pharmacokinetics and poor bioavailability at the tumor site. In order to improve its hydrophilic and drug delivery characteristics, we have developed a β-cyclodextrin (CD) mediated curcumin drug delivery system via encapsulation technique. Curcumin encapsulation into the CD cavity was achieved by inclusion complex mechanism. Curcumin encapsulation efficiency was improved by increasing the ratio of curcumin to CD. The formations of CD-curcumin complexes were characterized by Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), thermo-gravimetric analysis (TGA), scanning electron microscope (SEM), and transmission electron microscope (TEM) analyses. An optimized CD-curcumin complex (CD30) was evaluated for intracellular uptake and anti-cancer activity. Cell proliferation and clonogenic assays demonstrated that β-cyclodextrin-curcumin self-assembly enhanced curcumin delivery and improved its therapeutic efficacy in prostate cancer cells compared to free curcumin. © 2010 Elsevier B.V.

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