Targeting Mitochondrial Cell Death Pathway to Overcome Drug Resistance with a Newly Developed Iron Chelate

Background: Multi drug resistance (MDR) or cross-resistance to multiple classes of chemotherapeutic agents is a major obstacle to successful application of chemotherapy and a basic problem in cancer biology. The multidrug resistance gene, MDR1, and its gene product P-glycoprotein (P-gp) are an important determinant of MDR. Therefore, there is an urgent need for development of novel compounds that are not substrates of P-glycoprotein and are effective against drug-resistant cancer. Methodology/Principal Findings: In this present study, we have synthesized a novel, redox active Fe (II) complex (chelate), iron N- (2-hydroxy acetophenone) glycinate (FeNG). The structure of the complex has been determined by spectroscopic means. To evaluate the cytotoxic effect of FeNG we used doxorubicin resistant and/or sensitive T lymphoblastic leukemia cells and show that FeNG kills both the cell types irrespective of their MDR phenotype. Moreover, FeNG induces apoptosis in doxorubicin resistance T lymphoblastic leukemia cell through mitochondrial pathway via generation reactive oxygen species (ROS). This is substantiated by the fact that the antioxidant N-acetyle-cysteine (NAC) could completely block ROS generation and, subsequently, abrogated FeNG induced apoptosis. Therefore, FeNG induces the doxorubicin resistant T lymphoblastic leukemia cells to undergo apoptosis and thus overcome MDR. Conclusion/Significance: Our study provides evidence that FeNG, a redox active metal chelate may be a promising ne

A Novel Copper Chelate Modulates Tumor Associated Macrophages to Promote Anti-Tumor Response of T Cells

At the early stages of carcinogenesis, the induction of tumor specific T cell mediated immunity seems to block the tumor growth and give protective anti-tumor immune response. However, tumor associated macrophages (TAMs) might play an immunosuppressive role and subvert this anti tumor immunity leading to tumor progression and metastasis.The Cu (II) complex, (chelate), copper N-(2-hydroxy acetophenone) glycinate (CuNG), synthesized by us, has previously been shown to have a potential usefulness in immunotherapy of multiple drug resistant cancers. The current study demonstrates that CuNG treatment of TAMs modulates their status from immunosuppressive to proimmunogenic nature. Interestingly, these activated TAMs produced high levels of IL-12 along with low levels of IL-10 that not only allowed strong Th1 response marked by generation of high levels of IFN-gamma but also reduced activation induced T cell death. Similarly, CuNG treatment of peripheral blood monocytes from chemotherapy and/or radiotherapy refractory cancer patients also modulated their cytokine status. Most intriguingly, CuNG treated TAMs could influence reprogramming of TGF-beta producing CD4(+)CD25(+) T cells toward IFN-gamma producing T cells.Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers through reprogramming of TAMs that in turn reprogram the T cells and reeducate the T helper function to elicit proper anti-tumorogenic Th1 response leading to effective reduction in tumor growth

A novel copper complex induces ROS generation in doxorubicin resistant Ehrlich ascitis carcinoma cells and increases activity of antioxidant enzymes in vital organs in vivo

BACKGROUND: In search of a suitable GSH-depleting agent, a novel copper complex viz., copper N-(2-hydroxyacetophenone) glycinate (CuNG) has been synthesized, which was initially found to be a potential resistance modifying agent and later found to be an immunomodulator in mice model in different doses. The objective of the present work was to decipher the effect of CuNG on reactive oxygen species (ROS) generation and antioxidant enzymes in normal and doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)-bearing Swiss albino mice. METHODS: The effect of CuNG has been studied on ROS generation, multidrug resistance-associated protein1 (MRP1) expression and on activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). RESULTS: CuNG increased ROS generation and reduced MRP1 expression in EAC/Dox cells while only temporarily depleted glutathione (GSH) within 2 h in heart, kidney, liver and lung of EAC/Dox bearing mice, which were restored within 24 h. The level of liver Cu was observed to be inversely proportional to the level of GSH. Moreover, CuNG modulated SOD, CAT and GPx in different organs and thereby reduced oxidative stress. Thus nontoxic dose of CuNG may be utilized to reduce MRP1 expression and thus sensitize EAC/Dox cells to standard chemotherapy. Moreover, CuNG modulated SOD, CAT and and GPx activities to reduce oxidative stress in some vital organs of EAC/Dox bearing mice. CuNG treatment also helped to recover liver and renal function in EAC/Dox bearing mice. CONCLUSION: Based on our studies, we conclude that CuNG may be a promising candidate to sensitize drug resistant cancers in the clinic

Myeloid derived suppressor cells (MDSCs) can induce the generation of Th17 response from naïve CD4+ T cells

IL-17 producing CD4+ T cells (Th17) are identified as a subset of proinflammatory T cells present at the tumor site of various murine and human cancer cases and plays a crucial role in shaping the neoplastic process through fostering tumor angiogenesis and metastasis. However, the development of Th17 response in the tumor microenvironment has not yet been fully elucidated. Herein, we make an attempt to disclose the involvement of tumor infiltrating antigen presenting cells (APCs), especially tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs) to polarize naïve CD4+ T cells toward IL-17+ T cells. We have found that MDSCs either isolated from the tumor site or generated in vitro are superior over TAMs to induce IL-17 production by naïve CD4+ T cells. Furthermore, we have shown that MDSCs mediated induction of IL-17+ T cell response is independent of MDSCs-T cell contact but crucially depends on the cytokines secreted by MDSCs. Our study will help to develop potential therapeutic strategies by harnessing the ability of MDSCs to induce IL-17 production by CD4+ T cells and thus restrict the generation of inflammatory Th17 population at the disease site

Interferon-gamma (IFN-γ)-mediated retinal ganglion cell death in human tyrosinase T cell receptor transgenic mouse.

We have recently demonstrated the characterization of human tyrosinase TCR bearing h3T-A2 transgenic mouse model, which exhibits spontaneous autoimmune vitiligo and retinal dysfunction. The purpose of current study was to determine the role of T cells and IFN-γ in retina dysfunction and retinal ganglion cell (RGC) death using this model. RGC function was measured by pattern electroretinograms (ERGs) in response to contrast reversal of patterned visual stimuli. RGCs were visualized by fluorogold retrograde-labeling. Expression of CD3, IFN-γ, GFAP, and caspases was measured by immunohistochemistry and Western blotting. All functional and structural changes were measured in 12-month-old h3T-A2 mice and compared with age-matched HLA-A2 wild-type mice. Both pattern-ERGs (42%, p = 0.03) and RGC numbers (37%, p = 0.0001) were reduced in h3T-A2 mice when compared with wild-type mice. The level of CD3 expression was increased in h3T-A2 mice (h3T-A2: 174 ± 27% vs. HLA-A2: 100%; p = 0.04). The levels of effector cytokine IFN-γ were also increased significantly in h3T-A2 mice (h3T-A2: 189 ± 11% vs. HLA-A2: 100%; p = 0.023). Both CD3 and IFN-γ immunostaining were increased in nerve fiber (NF) and RGC layers of h3T-A2 mice. In addition, we have seen a robust increase in GFAP staining in h3T-A2 mice (mainly localized to NF layer), which was substantially reduced in IFN-γ ((-/-)) knockout h3T-A2 mice. We also have seen an up-regulation of caspase-3 and -9 in h3T-A2 mice. Based on our data we conclude that h3T-A2 transgenic mice exhibit visual defects that are mostly associated with the inner retinal layers and RGC function. This novel h3T-A2 transgenic mouse model provides opportunity to understand RGC pathology and test neuroprotective strategies to rescue RGCs

Iron N-(2-hydroxy acetophenone) glycinate (FeNG), a non-toxic glutathione depletor circumvents doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo

Multidrug resistance-associated protein 1 (MRP1) reduces intracellular anticancer drug accumulation either by co transporting them with glutathione (GSH) or extruding drug-GSH conjugates outside of the cell. Thus, MRP1 confers multidrug resistance (MDR) and worsen successful chemotherapeutic treatment against cancer. Although the exact mechanism of MRP1 involved in MDR remains unknown, the elevated level of intracellular GSH is considered as a key factor responsible for MDR in cancer. Hence the quest for non-toxic molecules that are able to deplete intracellular GSH has profound importance to subdue MDR. The present preclinical study depicts the resistance reversal potentiality of an iron complex; viz. Ferrous N-(2-hydroxy acetophenone) glycinate (FeNG) developed by us in doxorubicin resistant Ehrlich ascites carcinoma (EAC/Dox) cells. FeNG potentiate cytotoxic effect of doxorubicin on EAC/Dox cells ex vivo and also increases the survivability EAC/Dox bearing Swiss albino mice in vivo as well. Moreover, in vivo administration of FeNG significantly depletes intracellular GSH with ensuant increase in doxorubicin concentration in EAC/Dox cells without alternation of MRP1 expression. In addition, intra-peritoneal (i.p.) application of FeNG in normal or EAC/Dox bearing mice does not cause any systemic toxicity in preliminary trials in mouse Ehrlich ascites carcinoma model. Therefore, the present report provides evidence that FeNG may be a promising new resistance modifying agent against drug resistant cancers

Reactive oxygen species (ROS) plays an important role in FeNG induced apoptosis.

<p>(A) CEM/ADR5000 cells were either kept untreated or treated with FeNG (10⁻⁴ M) and intra cellular ROS generation was measured [in terms of peroxide using dichlorofluorescein diacetate (DCF-DA)] as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011253#s4" target="_blank"><i>Materials and Methods</i></a> at different time points. Data are expressed as percent of control and are presented as mean±SD of 3 independent experiments. Differences between control and FeNG treated cells are significant *P<0.05, **P<0.01, ***P<0.001, by unpaired Student's <i>t</i> test. (B) NAC completely abrogated FeNG induced ROS generation in CEM/ADR5000 cells. Cells were either kept untreated or pretreated with NAC (5 mM) for 1 h. Then the cells were further cultured for 2 h, 4 h, 6 h and 8 h in the presence or absence of FeNG (10⁻⁴ M) and intra cellular ROS generation was measured. (C) Represents that NAC protects CEM/ADR5000 cells from FeNG induced cell death. CEM/ADR5000 cells were either left untreated or pretreated with different concentration of NAC for 1 h. The cells were then treated with FeNG (10⁻³ M or10⁻⁴ M) for 72 h and cell death was monitored by MTT assay. Value represents the mean ± SD of three independent experiments with four replicates in each. Significant difference at *P<0.05, ***P<0.001, respectively, from only FeNG treated cells. (D) FeNG depletes intra cellular glutathion (GSH) contents of CEM/ADR5000 cells. Cells were either kept untreated or treated with FeNG (10⁻⁴ M) for indicated time points and intra cellular GSH was measured as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011253#s4" target="_blank"><i>Materials and Methods</i></a>. Results are presented as mean±SD of 3 independent experiments. Differences between untreated control and FeNG treated cells are significant **P<0.01, ***P<0.001, by unpaired Student's <i>t</i> test.</p

Calculation of Resistance factor for FeNG.

<p>Anti-proliferative activity and resistance factor used to confirm multi-drug resistance phenotype and demonstrating whether iron complex (FeNG) was a substrate for P-glycoprotein. The resistance factor was calculated by division of the IC₅₀ for the drug resistance CEM-ADR 5000 cell line by the IC₅₀ for the drug sensitive CCRF-CEM cell line. Results presented are representative of three independent experiments.</p><p>*Reference<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011253#pone.0011253-Majumder2" target="_blank">[13]</a>.</p

FeNG induces apoptosis through mitochondrial cell death pathway.

<p>(A) CEM/ADR5000 cells of both untreated and FeNG treated for indicated time or rIFN γ treated were labeled with anti FasR antibody. Immunofluorescence analysis was performed by flow cytometry. Representative data of 3 independent experiments is presented. (B) CEM/ADR5000 cells were treated with or without FeNG for indicated time, and mitochondrial membrane potential was measured after JC1 staining. The ratio of red fluorescence (mitochondrial JC-1) to green fluorescence (cytoplasmic JC-1) was used as a surrogate for mitochondrial potential. Data represent mean ± SD of three independent experiments. Statistically significant difference from untreated control at *P<0.05, **P<0.01, ***P<0.001, respectively. (C) Effect of FeNG on the release of cytochrome c. Western blot analysis of cytosolic extracts from CEM/ADR5000 cells treated with FeNG (10⁻⁴ M) for indicated hours. Cytosolic fraction was prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011253#s4" target="_blank">Materials and Methods</a>. Membrane was probed with anticytochrome <i>c</i> antibody followed by incubation with peroxidase-conjugated secondary antibody. The protein was visualized by Lumi glow detection system. Membrane was blotted for β-actin (bottom panel) for loading correction. (D) Densitometric quantitation of cytochrome c levels in the cytoplasm. Immunoreactive bands were quantitated and expressed as the ratio of each band density to corresponding loading control (β actin) band density and values were represented after normalization to untreated control.</p

Morphological evidence of retina damage in h3T-A2 transgenic mice.

<p>Representative photomicrographs comparing morphologic changes in the 12-month-old wild-type HLA-A2 and h3T-A2 transgenic mice. Retina sections were stained with hematoxylin and eosin.</p