Aflatoxin B1 Up-Regulates Insulin Receptor Substrate 2 and Stimulates Hepatoma Cell Migration

<div><p>Aflatoxin B1 (AFB1) is a potent carcinogen that can induce hepatocellular carcinoma. AFB1-8,9-exo-epoxide, one of AFB1 metabolites, acts as a mutagen to react with DNA and induce gene mutations, including the tumor suppressor <em>p53</em>. In addition, AFB1 reportedly stimulates IGF receptor activation. Aberrant activation of IGF-I receptor (IGF-IR) signaling is tightly associated with various types of human tumors. In the current study, we investigated the effects of AFB1 on key elements in IGF-IR signaling pathway, and the effects of AFB1 on hepatoma cell migration. The results demonstrated that AFB1 induced IGF-IR, Akt, and Erk1/2 phosphorylation in hepatoma cell lines HepG2 and SMMC-7721, and an immortalized human liver cell line Chang liver. AFB1 also down-regulated insulin receptor substrate (IRS) 1 but paradoxically up-regulated IRS2 through preventing proteasomal degradation. Treatment of hepatoma cells and Chang liver cells with IGF-IR inhibitor abrogated AFB1-induced Akt and Erk1/2 phosphorylation. In addition, IRS2 knockdown suppressed AFB1-induced Akt and Erk1/2 phosphorylation. Finally, AFB1 stimulated hepatoma cell migration. IGF-IR inhibitor or IRS2 knockdown suppressed AFB1-induced hepatoma cell migration. These data demonstrate that AFB1 stimulates hepatoma cell migration through IGF-IR/IRS2 axis.</p> </div

AFB1 stimulates hepatoma cell migration through IGF-IR/IRS2 axis.

<p>(<b>A</b>) SMMC-7721 cells were seeded into 6-well plates. Upon confluency, scratches were made in cell cultures. To inhibit cell proliferation, the cells were treated with 2 µg/ml mitomycin C. Also, the cells were treated with or without 2.5 µM AFB1 and 10 µM IGF-IR inhibitor AG1024 for 4 days. <i>Bar</i>, 1000 µm. (<b>B</b>) SMMC-7721 cells were transfected with siCtrl or siIRS2. Twenty-four hours later scratches were made in cell cultures. The cells were treated with 2 µg/ml mitomycin C, and treated with or without 2.5 µM AFB1 for 4 days. <i>Bar</i>, 1000 µm. Cell lysates from siCtrl- or siIRS2-transfected cells were harvested and subjected to Western blot analysis of IRS2 expression.</p

AFB1 induces Akt and Erk1/2 phosphorylation.

<p>(<b>A</b>) HepG2 cells were treated with 2.5 µM AFB1 for 1, 3, or 5 days, or treated with 1, 2.5, and 5 µM AFB1 for 3 days, followed by western blot analysis of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2. (<b>B</b>) SMMC-7721 cells were treated with 2.5 µM AFB1 for 1, 3, or 5 days, or treated with 1, 2.5, and 5 µM AFB1 for 3 days, followed by western blot analysis of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2. (<b>C</b>) Chang liver cells were treated with 2.5 µM AFB1 for 3 days, followed by western blot analysis of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2. Immunoblots were subjected to densitometric analysis. The relative levels of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2 after normalization to actin were plotted. The relative levels of target proteins in cells treated without AFB1 were set as 1. A statistical analysis of densitometric quantification of immunoblots from individual experiments was shown. *, <i>p</i><0.05.</p

AFB1 induces IGF-IR phosphorylation, down-regulates IRS1 but up-regulates IRS2.

<p>(<b>A</b>) HepG2 cells were treated with 2.5 µM AFB1 for 1, 3, or 5 days, or treated with 1, 2.5, and 5 µM AFB1 for 3 days, followed by western blot analysis of IGF-IR and phosphorylated IGF-IR, IRS1, and IRS2. (<b>B</b>) SMMC-7721 cells were treated with 2.5 µM AFB1 for 1, 3, or 5 days, or treated with 1, 2.5, and 5 µM AFB1 for 3 days, followed by western blot analysis of IGF-IR and phosphorylated IGF-IR, IRS1, and IRS2. (<b>C</b>) Chang liver cells were treated with 2.5 µM AFB1 for 3 days, followed by western blot analysis of IGF-IR and phosphorylated IGF-IR, IRS1, and IRS2. All blots were subjected to densitometric analysis. The relative levels of IGF-IR, phosphorylated IGF-IR, IRS1, and IRS2 after normalization to actin were plotted. The relative levels of target proteins in un-treated group were set as 1. A statistical analysis of densitometric quantification of immunoblots from individual experiments was shown. *, <i>p</i><0.05.</p

Inhibition of IGF-IR and IRS2 suppresses AFB1-induced Akt and Erk1/2 phosphorylation.

<p>(<b>A</b>) HepG2, SMMC-7721, and Chang liver cells were treated with or without 2.5 µM AFB1 and 10 µM IGF-IR inhibitor AG1024 for 3 days, followed by western blot analysis of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2, IGF-IR and phosphorylated IGF-IR. (<b>B</b>) HepG2, SMMC-7721, and Chang liver cells were transfected with control siRNA (siCtrl) or IGF-IR siRNA (siIGFIR). Twenty-four hours later, the cells were treated with or without 2.5 µM AFB1 for 3 days. Cell lysates were subjected to western blot analysis of Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2, IGF-IR and phosphorylated IGF-IR. (<b>C</b>) HepG2, SMMC-7721, and Chang liver cells were transfected with control siRNA (siCtrl) or IRS2 siRNA (siIRS2). Twenty-four hours later, the cells were treated with or without 2.5 µM AFB1 for 3 days. Cell lysates were subjected to western blot analysis of IRS2, Akt and phosphorylated Akt, Erk1/2 and phosphorylated Erk1/2.</p

AFB1 stimulates hepatoma cell growth and down-regulates IRS1 in a dose-dependent manner.

<p>(<b>A</b>) HepG2 and SMMC-7721 cells were plated into 96-well plates, and treated with or without AFB1 at indicated dose for 5 days. Cell growth was detected by CCK-8 reagent. The relative cell growth was plotted. <i>Bars</i>, SE. *, <i>p</i><0.05, compared with vehicle-treated cells. (<b>B</b>) HepG2 and SMMC-7721 cells were treated with or without AFB1 at indicated dose for 5 days. Cell lysates were subjected to western blot analysis of IRS1, IRS2 and phosphorylated IGF-IR.</p

Target-Responsive DNAzyme Cross-Linked Hydrogel for Visual Quantitative Detection of Lead

Because of the severe health risks associated with lead pollution, rapid, sensitive, and portable detection of low levels of Pb²⁺ in biological and environmental samples is of great importance. In this work, a Pb²⁺-responsive hydrogel was prepared using a DNAzyme and its substrate as cross-linker for rapid, sensitive, portable, and quantitative detection of Pb²⁺. Gold nanoparticles (AuNPs) were first encapsulated in the hydrogel as an indicator for colorimetric analysis. In the absence of lead, the DNAzyme is inactive, and the substrate cross-linker maintains the hydrogel in the gel form. In contrast, the presence of lead activates the DNAzyme to cleave the substrate, decreasing the cross-linking density of the hydrogel and resulting in dissolution of the hydrogel and release of AuNPs for visual detection. As low as 10 nM Pb²⁺ can be detected by the naked eye. Furthermore, to realize quantitative visual detection, a volumetric bar-chart chip (V-chip) was used for quantitative readout of the hydrogel system by replacing AuNPs with gold–platinum core–shell nanoparticles (Au@PtNPs). The Au@PtNPs released from the hydrogel upon target activation can efficiently catalyze the decomposition of H₂O₂ to generate a large volume of O₂. The gas pressure moves an ink bar in the V-chip for portable visual quantitative detection of lead with a detection limit less than 5 nM. The device was able to detect lead in digested blood with excellent accuracy. The method developed can be used for portable lead quantitation in many applications. Furthermore, the method can be further extended to portable visual quantitative detection of a variety of targets by replacing the lead-responsive DNAzyme with other DNAzymes

Target-Responsive DNA Hydrogel Mediated “Stop-Flow” Microfluidic Paper-Based Analytic Device for Rapid, Portable and Visual Detection of Multiple Targets

A versatile point-of-care assay platform was developed for simultaneous detection of multiple targets based on a microfluidic paper-based analytic device (μPAD) using a target-responsive hydrogel to mediate fluidic flow and signal readout. An aptamer-cross-linked hydrogel was used as a target-responsive flow regulator in the μPAD. In the absence of a target, the hydrogel is formed in the flow channel, stopping the flow in the μPAD and preventing the colored indicator from traveling to the final observation spot, thus yielding a “signal off” readout. In contrast, in the presence of a target, no hydrogel is formed because of the preferential interaction of target and aptamer. This allows free fluidic flow in the μPAD, carrying the indicator to the observation spot and producing a “signal on” readout. The device is inexpensive to fabricate, easy to use, and disposable after detection. Testing results can be obtained within 6 min by the naked eye via a simple loading operation without the need for any auxiliary equipment. Multiple targets, including cocaine, adenosine, and Pb²⁺, can be detected simultaneously, even in complex biological matrices such as urine. The reported method offers simple, low cost, rapid, user-friendly, point-of-care testing, which will be useful in many applications

A Synthetic Light-Driven Substrate Channeling System for Precise Regulation of Enzyme Cascade Activity Based on DNA Origami

Substrate channeling, in which a metabolic intermediate is directly passed from one enzyme to the next enzyme in an enzyme cascade, accelerates the processing of metabolites and improves substrate selectivity. Synthetic design and precise control of channeling outside the cellular environment are of significance in areas such as synthetic biology, synthetic chemistry, and biomedicine. In particular, the precise control of synthetic substrate channeling in response to light is highly important, but remains a major challenge. Herein, we develop a photoresponsive molecule-based synthetic substrate channeling system on DNA origami to regulate enzyme cascade activity. The photoresponsive azobenzene molecules introduced into DNA strands enable reversible switching of the position of substrate channeling to selectively activate or inhibit the enzyme cascade activity. Moreover, DNA origami allows precise control of interenzyme distance and swinging range of the swing arm to optimize the regulation efficiency. By combining the accurate and addressable assembly ability of DNA origami and the clean, rapid, and reversible regulation of photoresponsive molecules, this light-driven substrate channeling system is expected to find important applications in synthetic biology and biomedicine

Integrating Target-Responsive Hydrogel with Pressuremeter Readout Enables Simple, Sensitive, User-Friendly, Quantitative Point-of-Care Testing

Point-of-care testing (POCT) with the advantages of speed, simplicity, and low cost, as well as no need for instrumentation, is critical for the measurement of analytes in a variety of environments lacking access to laboratory infrastructure. In the present study, a hydrogel pressure-based assay for quantitative POCT was developed by integrating a target-responsive hydrogel with pressuremeter readout. The target-responsive hydrogels were constructed with DNA grafted linear polyacrylamide and the cross-linking DNA for selective target recognition. The hydrogel response to the target substance allows release of the preloaded Pt nanoparticles, which have good stability and excellent catalytic ability for decomposing H₂O₂ to O₂. Then, the generated O₂ in a sealed environment leads to significant pressure increase, which can be easily read out by a handheld pressuremeter. Using this target-responsive hydrogel pressure-based assay, portable and highly sensitive detection of cocaine, ochratoxin A, and lead ion were achieved with excellent accuracy and selectivity. With the advantages of portability, high sensitivity, and simple sample processing, the hydrogel pressure-based assay shows great potential for quantitative POCT of a broad range of targets in resource-limited settings