Selective and Sensitive Detection of Heavy Metal Ions in 100% Aqueous Solution and Cells with a Fluorescence Chemosensor Based on Peptide Using Aggregation-Induced Emission

A fluorescent peptidyl chemosensor for the detection of heavy metal ions in aqueous solution as well as in cells was synthesized on the basis of the peptide receptor for the metal ions using an aggregation-induced emission fluorophore. The peptidyl chemosensor (<b>1</b>) bearing tetraphenylethylene fluorophore showed an exclusively selective turn-on response to Hg²⁺ among 16 metal ions in aqueous buffered solution containing NaCl. The peptidyl chemosensor complexed Hg²⁺ ions and then aggregated in aqueous buffered solution, resulting in the significant enhancement (OFF-On) of emissions at around 470 nm. The fluorescent sensor showed a highly sensitive response to Hg²⁺, and about 1.0 equiv of Hg²⁺ was enough for the saturation of the emission intensity change. The detection limit (5.3 nM, <i>R</i>² = 0.99) of <b>1</b> for Hg²⁺ ions was lower than the maximum allowable level of Hg²⁺ in drinking water by EPA. Moreover, the peptidyl chemosensor penetrated live cells and detected intracellular Hg²⁺ ions by the turn-on response

β-lap in combination with IR induces apoptotic cell death via nuclear translocation of AIF.

<p>(A) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. The data represent a typical experiment conducted three times with similar results. (B) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 12 h. Cytosolic fractions from NQO1⁺-MDA-MB-231 cells were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. The data represent a typical experiment conducted three times with similar results. (C) NQO1⁺-MDA-MB-231 cells were treated with combination of IR and β-lap for 12 h in the presence or absence of z-VAD-fmk. The data represent a typical experiment conducted three times with similar results. (D) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of z-VAD-fmk (30 µM). After 24 h, the percentage of the cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (E) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h. Nuclear fractions from NQO1⁺-MDA-MB-231 cells were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. (F) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of siRNA targeting AIF. After 24 h, the percentage of the cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (G) NQO1⁺-MDA-MB-231 cells were treated with combination of IR and β-lap for 24 h in the presence or absence of NAC, PD98059, Sal or SP600125. Nuclear fractions were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times.</p

β-lap in combination with IR rapidly activates ERK and JNK.

<p>(A) NQO1⁻ or NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. The data represent a typical experiment conducted three times with similar results. (B) and (C) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of PD98059 (30 µM), SP600125 (30 µM), SB203580 (30 µM), or siRNA targeting ERK1/2 or JNK2. The percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs.</p

β-lap in combination with IR induces positive feedback regulation between ERK and ROS.

<p>(A) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 30 min in the presence or absence of NAC. The data represent a typical experiment conducted three times with similar results. (B) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 3 h in the presence or absence of siRNA targeting ERK1/2 or JNK2. After 3 h, the cells were incubated with 10 µM H2DCF-DA for 30 min and then analyzed by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (C) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 30 min in the presence or absence of PD98059 or SP600125. The data represent a typical experiment conducted three times with similar results.</p

β-lap induces radiosensitization in an NQO1-dependent manner.

<p>(A) NQO1⁻ or NQO1⁺-MDA-MB-231 cells were treated with various concentrations of β-lap. Cells were allowed to grow for 10 to 14 days and were stained with 0.5% crystal violet and scored for colony formation. Results from three independent experiments are expressed as means ± SEM. *Significant difference between NQO1⁻- and NQO1⁺-MDA-MB-231 cells after β-lap treatment at p<0.05. (B) Cells were treated with 2 µM β-lap and then exposed to increasing doses of IR 30 min after treatment with β-lap. After 14 days, cells were scored for colony formation. Results from three independent experiments are expressed as means ± SEMs. (C) Cells were treated with IR alone, β-lap alone or the combination of IR and β-lap for the indicated times. The percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs.</p

β-lap in combination with IR enhances ROS generation and leads to apoptotic cell death.

<p>(A) and (B) Cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. After 3 h, the cells were incubated with 10 µM H2DCF-DA and 4 µM DHE, respectively, for 30 min and then analyzed by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (C) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of NAC (10 mM). After 24 h, the percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (D) NQO1⁺-MDA-MB-231 cells were treated with combination of IR (2 Gy) and β-lap (2 µM) in the presence or absence of NAC (10 mM). Cells were allowed to grow for 10 to 14 days and were stained with 0.5% crystal violet and scored for colony formation. Results from three independent experiments are expressed as means ± SEM. *Significant difference between cells in the presence or absence of NAC after combined treatment with IR and β-lap, at p<0.05.</p

β-lap in combination with IR causes Bax cleavage and translocation of cleaved Bax to mitochondria.

<p>(A) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. Cell lysates were subjected to Western blot analysis. The data represent a typical experiment conducted three times with similar results. (B) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 12 h. Mitochondrial fractions of NQO1⁺-MDA-MB-231 cells were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. (C) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 12 h. Whole cell lysates and mitochondrial fractions of NQO1⁺-MDA-MB-231 cells were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. (D) NQO1⁺-MDA-MB-231 cells were treated with combination of IR and β-lap for 24 h in the presence or absence of MG132. The data are representative a typical experiment conducted three times. (E) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. After 12 h, the concentration of retained DiOC₆(3) in cells was measured by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (F) NQO1⁺-MDA-MB-231 cells were treated with combination of IR and β-lap for 12 h in the presence or absence of NAC, PD98059, Sal or SP600125. Mitochondrial fractions were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. (G) NQO1⁺-MDA-MB-231 cells were treated with combination of IR and β-lap for 12 h in the presence or absence of NAC, PD98059, Sal or SP600125. After 12 h, the concentration of retained DiOC₆(3) in cells was measured by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (H) Schematic model of combined treatment (IR+β-lap)-induced apoptotic cell death. Combined treatment with IR and β-lap increases mitochondrial apoptotic cell death in an NQO1 dependent manner. As described in detail in the text, positive feedback regulation between ERK and ROS induced by combined treatment plays a critical role in the induction of ER stress. This enhanced ER stress is required for JNK activation, which leads to subsequent mitochondrial apoptotic cell death. Moreover, JNK activation induces cleavage of Bax and mitochondrial translocation of cleaved Bax, which causes loss of mitochondrial transmembrane potential and consequent release of AIF.</p

The induction of ER stress by combined treatment with IR and β-lap is required for JNK activation.

<p>(A) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. Cell lysates were subjected to Western blot analysis. The data represent a typical experiment conducted three times with similar results. (B) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of Sal (10 µM). After 24 h, the percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (C) NQO1⁺- MDA-MB-231 cells were treated with IR alone β-lap alone or combination of IR and β-lap for 3 h in the presence or absence of Sal (10 µM). After 3 h, the cells were incubated with 10 µM H2DCF-DA for 30 min and then analyzed by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (D) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 30 min in the presence or absence of NAC. The data represent a typical experiment conducted three times with similar results. (E) and (F) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 30 min in the presence or absence of Sal, SP600125 or PD98059. The data represent a typical experiment conducted three times with similar results.</p

Percent of CSCs in MCF-7 human breast cancer cells treated with hyperthermia and metformin.

<p>(A) Cells were incubated with 0–5 mM metformin for 48 h at 37°C, dispersed to single cells and analyzed for CD44^high/CD24^low cells (CSCs) with FACS. The effect of heating alone was studied by heating the cells at 42°C for 1 h followed by 47 h incubation at 37°C. The combined effect of heating and metformin was studied by heating the cells for 1 h at 42°C with metformin and then incubating at 37°C for 47 h. (B) Experiments described in A were repeated four times and the average of % of CSCs was obtained. Averages of 5 experiments ±1 S.E. are shown. The decreases in CSCs by 1 mM and 5 mM alone were statistically significant. The decrease in CSCs by heating at 42°C for 1 h was also statistically significant. The combinations of heating and metformin were statistically more effective than metformin alone. (C) MIA PaCa-2 cells were incubated with 0–10 mM metformin for 48 h at 37°C, dispersed to single cells and analyzed for CD44^high/CD24^high cells (CSCs) with FACS. The effect of heating alone was studied by heating the cells at 42.5°C for 1 h followed by 47 h incubation at 37°C. The combined effect of heating and metformin was studied by heating the cells for 1 h at 42.5°C with metformin and then incubating at 37°C for 47 h. (D, E) MCF-7 cells were plated in ultralow attachment plate (1,000 cells/plate) in sphere media. Metformin was added to the media (0.5–5 mM), then heating at 42°C for 1 h. Thereafter, the cells were incubated for 8 days under the standard culture conditions. The numbers of spheres with diameter >50 µm were counted under a microscope. The combinations of heating and metformin were statistically more effective than metformin alone.</p

Western blotting of AMPK/mTOR pathway in MCF-7 and MDA-MB-231 cells treated with hyperthermia and metformin.

<p>(A, C) Cells were heated at 42°C for 1 h and then incubated at 37°C for 47 h (total 48 h treatment). (B, D) The effects of metformin alone were studied by incubating cells with 5 mM metformin for 48 h at 37°C. The combined effects of metformin and heating were studied by heating the cells at 42°C for 1 h with 5 mM metformin and then incubating at 37°C for 47 h. (E) The combined effects of metformin and heating were studied by heating the cells at 39.5–41°C for 1 h with 5 mM metformin and then incubating at 37°C for 47 h. (F) The combined effects of metformin and heating were studied by heating the cells at 39.5°C for 6 h with 30 µM metformin and then incubating at 37°C for 47 h. Experiments were repeated 4–5 times and the representative results are shown.</p