Knockdown of Glutamate Cysteine Ligase Catalytic Subunit by siRNA Causes the Gold Nanoparticles-Induced Cytotoxicity in Lung Cancer Cells

<div><p>Gold nanoparticles (GNPs) have shown promising medical applications in cancer treatment involved in the regulation of intracellular redox balance. Previously, we have reported that GNPs can trigger apoptosis and necrosis in human lung cancer cells (A549) when L-buthionine-sulfoximine (BSO) was used to decrease the expression of intracellular glutathione (GSH). Herein, we investigated the cytotoxicity of GNPs toward lung cancer cells under the glutamate cysteine ligase catalytic subunit (GCLC) was silenced by siRNA. Our results showed that GNPs cause apoptosis and necrosis in cells transfected with GCLC siRNA by elevating intracellular reactive oxygen species (ROS). These findings demonstrated that the regulation of glutathione synthesis by GCLC siRNA in A549 cells can initiate the gold nanoparticles-induced cytotoxicity.</p></div

Flow cytometry analysis of mitochondrial membrane potential in GNPs-treated cells.

<p>After transfected with negative control siRNA or GCLC siRNA-1, cells were treated with GNPs (20μM) for additional 72 hours then collected and stained with JC-1 in darkness at 37°C, rinsed by PBS. The fluorescence shift (red to green) of samples was measured by flow cytometry. Each bar represents the mean (±SD n = 4) of triplicate determinations. *<i>p</i><0.05, compared with negative control group.</p

Knockdown of glutamate cysteine ligase catalytic subunit by siRNA.

<p>(A) GCLC mRNA levels in A549 cells following 24 hours transfected with negative control siRNA, GCLC siRNA-1, GCLC siRNA-2 and GCLC siRNA-3. GCLC siRNA significantly decreased the GCLC mRNA levels in A549 cells. (B) Representative Western blot gel documents for GCLC and summarized data showing that efficiency of gene silencing of GCLC by siRNA. Cytosolic proteins were isolated from transfected cells. GCLC protein levels in cell extracts were measured by Western blot analysis and were normalised to β-actin expression levels. *** P<0.001, compared to negative control.</p

Effect of GNPs on intracellular ROS levels in GCLC siRNA pretreated cells.

<p>Exponentially growing cells were transfected with negative control and GCLC siRNA-1 for 24 hours, following treated with GNPs (20μM), GNPs (20μM) and GSH (1mM), GNPs (20μM) and NAC (1mM). ROS levels were measured. Graphs indicate ROS (as determined by DCF) levels (%) compared with negative control cells. Each bar represents the mean (±SD n = 4) of triplicate determinations. **p<0.01</p

GNPs induce caspase activation in cells transfected with GCLC siRNA.

<p>Activation of caspase-3 was measured using specific antibodies by flow cytometry. Intracellular GSH was depleted by GCLC siRNA-1, after approximately 72 hours of GNPs treatment, the cells were collected, treated with 0.1% Triton X-100 and blocked with 1% BSA, then incubated with cleaved caspase-3 (Asp175) antibody (Alexa fluor 488 conjugate) for 30 minutes. The fluorescence intensity was measured by flow cytometry. Each bar represents the mean (±SD n = 4) of triplicate determinations. *<i>p</i><0.05, compared with negative control group.</p

Effect of GCLC siRNA on intracellular GSH levels.

<p>Cytosol was isolated from cells transfected with negative control siRNA, GCLC siRNA-1, GCLC siRNA-2 and GCLC siRNA-3. The intracellular GSH levels were determined at 412 nm absorbance with a multiwell plate reader. Data represent the mean percentage of negative control (n = 3) ±SD for 4 independent experiments. *p<0.05, ** P<0.01, compared to negative control.</p

GNPs induce apoptosis and necrosis in cells transfected with GCLC siRNA.

<p>Cells were transfected with either non-target control siRNA or GCLC-specific siRNA-1. One day later, cells were treated with GNPs (20μM), GNPs (20μM)﹢GSH (1mM) and GNPs (20μM)+NAC(1mM) for additional 72 hours. AnnexinV-FITC and PI cells were measured with flow cytometry. (A) The fluorescence pattern of AnnexinV-FITC and PI-stained A549 cells after treatment. (B) Percentages of Annexin V positive or PI positive cells for different treatments. Each bar represents the mean (±SD n = 3). **p<0.01, ***P<0.001, versus control.</p

Tumor-bearing mice display anemia and bone marrow erythropoiesis repression.

<p>(A) Hematological parameters in 4T1 tumor-bearing mice. BALB/c mice were inoculated 4T1 cells and tumors were grown for 28 days. Blood was collected from the mice, and RBC counts, reticulocytes, hemoglobin (Hb) concentrations and hematocrit were measured on the automated blood cell analyzer. (B) Serum levels of proinflammatory cytokines and serum iron parameters in 4T1 tumor-bearing mice. Serum was harvested at the time points indicated following injection of 4T1 cells. Serum concentrations of IFN-γ and IL-6 were analyzed by ELISA; serum iron and transferring saturation were measured using an Iron/TIBC reagent set. (C) (D) RBC lifespan of 4T1 tumor-bearing mice. Sulfo-NHS-biotin was injected into mice intravenously on day 7 after tumor cells injection. A drop of blood was collected from the tail on days 8, 15, 22, 29. The percent of biotinylated RBCs was analyzed by flow cytometry. (C) The decay curves are shown for 4 mice in each group. (D) Representative histograms of data from one mouse in each group are shown on day 22 after tumor cells implantation. (E) Serum levels of G-CSF in control and 4T1 tumor-bearing mice. (F) Photograph of femurs dissected from control and 4T1 tumor-bearing mice on day 21 after tumor cells implantation. (G) Total cells of bone marrow (BM) in one femur were counted at indicated times after tumor induction. (H) The percentage of Ter119 and CD71 positive cells of bone marrow in control and 4T1 tumor-bearing mice at indicated times after tumor growth. (I) Representative example of CD71 and Ter119 profiles from the bone marrow of control and 4T1 tumor-bearing mice on day 21 after tumor cells implantation. (J) Graphs show the total number of cell subsets expressing CD71 or Ter119 staining in bone marrow. (K)The numbers of BFU-E and CFU-E derived colonies from the bone marrow of control and 4T1 tumor-bearing mice. All data are expressed as the mean ± SEM; n = 4–6 mice per group for one out of three independent experiments. <i>*p</i> < 0.05, <i>**p</i> < 0.01, <i>***p</i> < 0.001 compared with control group.</p

The essential role of spleen in tumor-stress erythropoiesis.

<p>(A) (B) (C) Splenomegaly in tumor-bearing mice. (A) Spleens were collected and photographed at the indicated times after tumor cells transplantation. (B) Graph shows the mean spleen weight ± SEM of control and tumor-bearing mice from six to eight mice per time points and is representative of three separate experiments. (C) Splenocytes were counted at indicated times after tumor induction. Each bar represents the mean (±SEM n = 4–6) of triplicate determinations. <i>*p</i> < 0.05, <i>***p</i> < 0.001, compared with control group. (D) The percentage of Ter119 and CD71 positive cells of spleen in control and 4T1 tumor-bearing mice at indicated times after tumor growth. Each bar represents the mean (±SEM n = 4) of triplicate determinations. <i>**p</i> < 0.01, <i>***p</i> < 0.001, compared with control group. (E) Representative examples of CD71 and Ter119 profiles from the spleen of control and 4T1 tumor-bearing mice on day 21 after tumor cells transplantation. (F) Numbers of erythroid populations in spleen of control and 4T1 tumor-bearing mice. Each bar represents the mean (±SEM n = 4) of triplicate determinations. <i>**p</i> < 0.01, <i>***p</i> < 0.001, compared with control group. (G) The numbers of BFU-E and CFU-E derived colonies from the spleen of control and 4T1 tumor bearing mice. Each bar represents the mean (±SEM n = 4) of triplicate determinations. <i>**p</i> < 0.01, <i>***p</i> < 0.001, compared with control group. (H) BALB/c mice were splenectomized and allowed to recover for at least 2 weeks. Mice with or without spleen were implanted 4T1 cells. Blood was collected at indicated times after tumor cells transplantation. Hematological parameters were analyzed. All data are expressed as the mean ± SEM; n = 6 mice per group for one out of three independent experiments. <i>*p</i> < 0.05, ** p < 0.01, ***p < 0.001, compared with sham-operated control group.</p

Tumor growth alters serum Epo levels and splenic microenvironment of mice.

<p>(A)Serum levels of Epo in control and 4T1 tumor-bearing mice at indicated times after tumor induction. (B) Quantification of spleen BMP4 mRNA by quantitative reverse-transcribed polymerase chain reaction, normalized to S14 mRNA. (C) Analysis of spleen BMP4 by immunochemistry. Spleens were collected at the indicated times after 4T1 tumor cells implantation. Representative images of BMP4 staining from spleens of control and 4T1 tumor-bearing mice were photographed by microscope. (D) Confocal analysis of F4/80 and BMP4 colocalization in spleen macrophages. Spleens were collected on day 21 after tumor cells transplantation. Representative images of F4/80, BMP4, and DAPI staining sections from spleens of control and 4T1 tumor-bearing mice were photographed by confocal microscope. Data are means ± SEM (n = 4–6) calculated from three independent experiments. **p<0.01, ***p<0.001 compared with control group.</p