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

Structure and Mass spectral study of Iron Complex.

<p>(A)Chemical Structure of iron complex, iron (II) N-(2-hydroxyacetophenone) glycinate (FeNG). (B) Mass fragments of FeNG.</p

Activation of caspase 3 in CEM/ADR5000 cells after FeNG treatment.

<p>(A) Effect of FeNG and caspase inhibitors on the activity of caspase-3 of CEM/ADR5000 cells. Cells were treated with either vehicle (medium) control or FeNG (10⁻⁴ M) for 12 h, 24 h, 48 h, 72 h or caspases inhibitors; Ac-DEVD-cho (caspase 3 specific inhibitor) and z-VAD-fmk (pan caspase inhibitor) alone (50 µM) or in combination with FeNG for 72 h. After completion of these treatments, cells were harvested and cell lysates were prepared. The enzymatic activity of cell lysates towards tetrapeptide chromogenic substrates Ac-DEVD-AMC was determined. Caspase activities are expressed as fold change of control and presented as mean±SD of three independent experiments. Differences between untreated control and FeNG treated cells are significant ***P<0.001, by unpaired Student's t test. (B) Effects of caspase inhibitor on FeNG induced cell death of CEM/ADR5000 cells. Cells were either left untreated or treated with FeNG (10⁻³ M or10⁻⁴ M) or Ac-DEVD-cho (50 µM) and z-VAD-fmk (50 µM) alone or in combination with FeNG 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<0.01, respectively, from only FeNG treated cells. (C) Effect of FeNG on the cleavage patterns of PARP in CEM/ADR5000 cells. Cells were grown at standard culture conditions as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011253#s4" target="_blank">Materials and methods</a>, and treated with FeNG (10⁻⁴ M) for 24 h to 72 h, and cell lysates were prepared. Immunoblot analysis was performed to identify the full (116 kDa) and cleaved (89 kDa) PARP using specific primary antibodies. Loading was checked by immunoblotting of β -actin. Bands were visualized by Lumi glow detection system. Data shown are representative of three independent experiments. (D) Densitometric quantitation of cleaved (89 kDa) frgments of PARP 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

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

Comparison of the cytotoxic effect of iron complex on different cell types.

<p>Dose response curves for iron complex (FeNG) using (A) CEM/ADR5000 (B) CCRF-CEM, and (C) Human PBMC cells, as assessed by MTT assay. Cells were seeded into 96-well plates (4×10⁴ cells/well) and allowed to overnight incubation at 37°C in 5% CO₂ incubator. Next day, cells were treated with increasing concentrations of FeNG for 24 h, 48 h, and 72 h incubation. Results are expressed as percentage viability of solvent-treated control cells. Value represents the mean ± SD of three independent experiments with four replicates in each.</p

Changes in nuclear morphology of CEM/ADR5000 cells after FeNG treatment.

<p>(A) Morphological changes of CEM/ADR5000 cells treated with 0.75×10⁻³ M FeNG alone or in combination with 5 mM NAC (one hour prior to FeNG treatment). CEM/ADR5000 cells after treatments with drugs were fixed with 1% paraformaldehyde and stained with Hoechst 33258. The cells were observed under a fluorescence microscope. Apoptotic cells showed condensed or fragmented chromatin in the nucleus (arrowhead). (B) Represents the temporal kinetics of apoptotic percentage of CEM/ADR5000 cells. Cells were treated with FeNG alone or in combination with 5 mM NAC for the indicated times. After treatment, cells were harvested and stained with Hoechst 33258. Apoptotic cells were examined by counting the cells with condensed and fragmented nuclei. Each point represents an average of three independent experiments, and standard deviation bars are shown.</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

Combination of high IL-12 and low IL-10 can skew induction of Th1 response.

<p>A) RT-PCR. B) Densitometric analysis. C, D, E & F) Flow cytometry. CD4⁺ population from TALs (obtained from untreated EAC/Dox bearing mice) was purified and challenged either with single or combine dose of recombinant IL-12 and IL-10 and cultured for 96 h. Purified CD4⁺ population from TALs derived from untreated EAC/Dox bearing mice, cultured without any treatment was taken as untreated control. Equivalent amount of mRNA (2 µg) from each experimental group was used for semi-quantitative RT-PCR analysis and in all cases GAPDH was used as housekeeping gene control (A). Densitometry analysis of mRNA expression of each gene transcript was expressed as a ratio of cytokine mRNA to GAPDH mRNA (B). Intracellular cytokines specific for Th1 (IFN-γ) or Th2 (IL-4) or suppressive (TGF-β) production profile in the above mentioned experimental groups were also analyzed by flow cytometry and a representative data is shown (C). CD4+ TALs were co-cultured with untreated TAMs or CuNG treated TAMs either unfixed or fixed with paraformaldehyde or CuNG treated fixed TAMs along with high rIL-12 and low rIL-10. In some cases CD4+ TALs and CuNG treated unfixed TAMs were separated by transwell insert (0.45 µ Meter pore) in culture. After 96 h of culture intracellular IFN-γ and TGF-β production pattern were studied by flow cytometry (D). Mean fluorescence intensity for IFN-γ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007048#pone-0007048-g005" target="_blank">Fig. 5E</a>) and TGF-β (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007048#pone-0007048-g005" target="_blank">Fig. 5F</a>) production by these experimental groups were also analyzed from the flow cytometric statistical data and represented graphically. Representative data from three independent experiments is presented.</p

Both in vitro and in vivo CuNG treatment caused alteration of cytokines profile of TAMs.

<p>A) ELISA. B) Flow cytometry. C) Fluorometric analysis. D, E & F) ELISA. In vitro CuNG treatment (2.5 µg/ml) did not change IFN-γ production from CD4⁺ T cells of TALs of untreated EAC/Dox bearing mice (A). TAMs were purified from peritoneal ascitic fluid of both untreated and 15 days of CuNG treated EAC/Dox bearing mice and labeled with anti F4/80 antibodies and with either intracellular IL-10 or IL-12 or TGF-β or with specific isotype control Abs. Immunofluorescence analysis were performed by flow cytometry. Representative data of 3 independent experiments is presented (B). Purified TAMs were either kept untreated or treated with CuNG in vitro and ROS was measured [in terms of peroxide using dichlorofluorescein diacetate (DCF-DA)] at different time points. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007048#s2" target="_blank">Results</a> are presented as mean±SD of 3 independent experiments (C). Purified TAMs from untreated EAC/Dox bearing mice were plated (2×10⁶ cells/500 µl). Cells were either kept untreated or pretreated with tocopherol (50 µM) for 1 h. Then the cells were further cultured for 12 h, 24 h and 48 h in the presence or absence of CuNG (2.5 µg/ml). The culture supernatants were collected and analyzed for cytokines IL-10 (D), IL-12 (E) and TGF-β (F) by ELISA and results are presented as mean±SE of 3 independent experiments, each experiment having every measurement in triplicate.</p

In vitro CuNG treatment caused reprogramming of Treg.

<p>A, B & C) Flow cytometry. CD4⁺CD25⁺ Treg populations were purified from TALs of untreated EAC/Dox bearing mice. (A) Treg cells were labeled with CFSE and then cultured for 96 h with cell free supernatant of TAMs (isolated from untreated EAC/Dox bearing mice) either kept untreated or treated in vitro with CuNG for 48 h. Intracellular IFN-γ and TGF-β production was analyzed with respect to specific isotype control by flow cytometry. Representative data of three independent experiments is shown. (B) Treg cells were cultured for 96 h with supernatant of 48 h culture of untreated or in vitro CuNG treated TAMs and fresh medium (1∶1). Intracellular IFN-γ and TGF-β production versus FoxP3 expression was analyzed with respect to specific isotype control by flow cytometry. Representative data of four independent experiments is shown. (C) Tregs (CD4⁺CD25⁺ cells) isolated from ascitic fluid of untreated EAC/Dox bearing mice were cultured for 96 h in presence of cell-free supernatants from 48 h cultures of untreated or CuNG treated TAMs (culture supernatant: fresh medium being 1∶1). Now, these cells were washed and cultured with CFSE loaded CD4⁺ T cells isolated from inguinal and axillary lymph nodes of normal mice (Treg and CD4⁺ T cells were taken in a proportion of 1∶5) for 96 h. Fluorescence levels of CFSE were measured by flow cytometry. Proliferation of normal CD4⁺ T cells either in the presence or absence of Treg cells were also analyzed by CFSE fluorescence level. Representative data of 3 independent experiments is presented here.</p