Recent advances in the development of activatable multifunctional probes for in vivo imaging of caspase-3

Caspases are a family of proteases that play critical roles in controlling inflammation and cell death.Apoptosis is a caspase-3 mainly controlled behavior to avoid inflammation and damage to surrounding cells,whereas anomalistic cell apoptosis may be associated with many diseases.The detection and imaging of caspase-3 will be of great significance in evaluating the early therapeutic effect of tumors.Developing smart fluorescent probes may be helpful for the visualization of the rapeutic effect compared with "always on" probes.Thus,more and more works toward activatable fluorescent probes for caspase-3 imaging have been reported.In addition,multifunctional probes have also been designed to further improve the imaging of caspase-3.Herein,this review systematically summarized the representative wo rk of caspase-3 from the perspective of molecular design that it will play a guiding role in the design of probes that respond to caspase-3.Also,challenges and perspectives toward the field for imaging of cell apoptosis(caspase-3) are also discussed.     

A Renal-Clearable Macromolecular Reporter for Near-Infrared Fluorescence Imaging of Bladder Cancer

Bladder cancer (BC) is a prevalent disease with high morbidity and mortality; however, in vivo optical imaging of BC remains challenging because of the lack of cancer-specific optical agents with high renal clearance. Herein, a macromolecular reporter (CyP1) was synthesized for real-time near-infrared fluorescence (NIRF) imaging and urinalysis of BC in living mice. Because of the high renal clearance (ca. 94 % of the injection dosage at 24 h post-injection) and its cancer biomarker (APN=aminopeptidase N) specificity, CyP1 can be efficiently transported to the bladder and specially turn on its NIRF signal to report the detection of BC in living mice. Moreover, CyP1 can be used for optical urinalysis, permitting the ex vivo tracking of tumor progression for therapeutic evaluation and easy translation of CyP2 as an in vitro diagnostic assay. This study not only provides new opportunities for non-invasive diagnosis of BC, but also reveals useful guidelines for the development of molecular reporters for the detection of bladder diseases.     

A Renal‐Clearable Macromolecular Reporter for Near‐Infrared Fluorescence Imaging of Bladder Cancer

Bladder cancer (BC) is a prevalent disease with high morbidity and mortality; however, in vivo optical imaging of BC remains challenging because of the lack of cancer‐specific optical agents with high renal clearance. Herein, a macromolecular reporter (CyP1) was synthesized for real‐time near‐infrared fluorescence (NIRF) imaging and urinalysis of BC in living mice. Because of the high renal clearance (ca. 94 % of the injection dosage at 24 h post‐injection) and its cancer biomarker (APN=aminopeptidase N) specificity, CyP1 can be efficiently transported to the bladder and specially turn on its NIRF signal to report the detection of BC in living mice. Moreover, CyP1 can be used for optical urinalysis, permitting the ex vivo tracking of tumor progression for therapeutic evaluation and easy translation of CyP2 as an in vitro diagnostic assay. This study not only provides new opportunities for non‐invasive diagnosis of BC, but also reveals useful guidelines for the development of molecular reporters for the detection of bladder diseases.     

Preparation of Catechin Nanoemulsion from Oolong Tea Leaf Waste and Its Inhibition of Prostate Cancer Cells DU-145 and Tumors in Mice

Anti-cancer activity of catechin nanoemulsions prepared from Oolong tea leaf waste was studied on prostate cancer cells DU-145 and DU-145-induced tumors in mice. Catechin nanoemulsions composed of lecithin, Tween-80 and water in an appropriate proportion was prepared with high stability, particle size of 11.3 nm, zeta potential of −67.2 mV and encapsulation efficiency of 83.4%. Catechin nanoemulsions were more effective than extracts in inhibiting DU-145 cell growth, with the IC₅₀ being 13.52 and 214.6 μg/mL, respectively, after 48 h incubation. Furthermore, both catechin nanoemulsions and extracts could raise caspase-8, caspase-9 and caspase-3 activities for DU-145 cell apoptosis, arresting the cell cycle at S and G2/M phases. Compared to control, catechin nanoemulsion at 20 μg/mL and paclitaxel at 10 μg/mL were the most effective in reducing tumor volume by 41.3% and 52.5% and tumor weight by 77.5% and 90.6% in mice, respectively, through a decrease in EGF and VEGF levels in serum.     

A Renal‐Clearable Duplex Optical Reporter for Real‐Time Imaging of Contrast‐Induced Acute Kidney Injury

Despite its high morbidity and mortality, contrast‐induced acute kidney injury (CIAKI) remains a diagnostic dilemma because it relies on in vitro detection of insensitive late‐stage blood and urinary biomarkers. We report the synthesis of an activatable duplex reporter (ADR) for real‐time in vivo imaging of CIAKI. ADR is equipped with chemiluminescence and near‐infrared fluorescence (NIRF) signaling channels that can be activated by oxidative stress (superoxide anion, O₂^.?) and lysosomal damage (N‐acetyl‐β‐d ‐glucosaminidase, NAG), respectively. By virtue of its high renal clearance efficiency (80 % injected doses after 24 h injection), ADR detects sequential upregulation of O₂^.? and NAG in the kidneys of living mice prior to a significant decrease in glomerular filtration rate (GFR) and tissue damage in the course of CIAKI. ADR outperforms the typical clinical assays and detects CIAKI at least 8 h (NIRF) and up to 16 h (chemiluminescence) earlier.     

Antioxidative,Antiapoptotic, and Anti-Inflammatory Effects of Apamin in a Murine Model of Lipopolysaccharide-Induced Acute Kidney Injury

Sepsis is the major cause of acute kidney injury (AKI) in severely ill patients, but only limited therapeutic options are available. During sepsis, lipopolysaccharide (LPS), an endotoxin derived from bacteria, activates signaling cascades involved in inflammatory responses and tissue injury. Apamin is a component of bee venom and has been shown to exert antioxidative, antiapoptotic, and anti-inflammatory activities. However, the effect of apamin on LPS-induced AKI has not been elucidated. Here, we show that apamin treatment significantly ameliorated renal dysfunction and histological injury, especially tubular injury, in LPS-injected mice. Apamin also suppressed LPS-induced oxidative stress through modulating the expression of nicotinamide adenine dinucleotide phosphate oxidase 4 and heme oxygenase-1. Moreover, tubular cell apoptosis with caspase-3 activation in LPS-injected mice was significantly attenuated by apamin. Apamin also inhibited cytokine production and immune cell accumulation, suppressed toll-like receptor 4 pathway, and downregulated vascular adhesion molecules. Taken together, these results suggest that apamin ameliorates LPS-induced renal injury through inhibiting oxidative stress, apoptosis of tubular epithelial cells, and inflammation. Apamin might be a potential therapeutic option for septic AKI.     

An Activatable Polymeric Reporter for Near‐Infrared Fluorescent and Photoacoustic Imaging of Invasive Cancer

Discriminative detection of invasive and noninvasive breast cancers is crucial for their effective treatment and prognosis. However, activatable probes able to do so in vivo are rare. Herein, we report an activatable polymeric reporter (P‐Dex) that specifically turns on near‐infrared (NIR) fluorescent and photoacoustic (PA) signals in response to the urokinase‐type plasminogen activator (uPA) overexpressed in invasive breast cancer. P‐Dex has a renal‐clearable dextran backbone that is linked with a NIR dye caged with an uPA‐cleavable peptide substrate. Such a molecular design allows P‐Dex to passively target tumors, activate NIR fluorescence and PA signals to effectively distinguish invasive MDA‐MB‐231 breast cancer from noninvasive MCF‐7 breast cancer, and ultimately undergo renal clearance to minimize the toxicity potential. Thus, this polymeric reporter holds great promise for the early detection of malignant breast cancer.     

Essential oil of Cephalotaxus griffithii needle inhibits proliferation and migration of human cervical cancer cells: involvement of mitochondria-initiated and death receptor-mediated apoptosis pathways

This study was conducted to determine the effect of Cephalotaxus griffithii needle essential oil (CGNO) on proliferation and migration of human cervical cancer (HCC) cells. CGNO treatment decreased the viability of all the tested HCC (HeLa, ME-180 and SiHa) cells. Morphological and DNA fragmentation analysis of CGNO-treated HeLa cells indicated the involvement of apoptosis in inducing HCC cell death. CGNO increased mitochondrial membrane depolarisation and upregulated the expression of caspase-9, caspase-8, caspase-3 and cleaved-PARP. The activity of caspase-8 and caspase-9 was also significantly increased. Wound healing and transwell migration assay demonstrated that CGNO significantly inhibited the migration of HeLa cells to close a scratched wound and also inhibited their migration through filter towards a chemotactic stimulus. Taken together, these results indicated that CGNO inhibited the proliferation and migration of HCC cells. Of note, CGNO induced HeLa cell death through mitochondria-initiated and death receptor-mediated apoptosis pathway.     

Single domain antibody–quantum dot conjugates for ricin detection by both fluoroimmunoassay and surface plasmon resonance

The combination of stable biorecognition elements and robust quantum dots (QDs) has the potential to yield highly effective reporters for bioanalyses. Llama-derived single domain antibodies (sdAb) provide small thermostable recognition elements that can be easily manipulated using standard DNA methods. The sdAb was self-assembled on dihydrolipoic acid (DHLA) ligand-capped CdSe–ZnS core–shell QDs made in our laboratory through the polyhistidine tail of the protein, which coordinated to zinc ions on the QD surface. The sdAb–QD bioconjugates were then applied in both fluorometric and surface plasmon resonance (SPR) immunoassays for the detection of ricin, a potential biothreat agent. The sdAb–QD conjugates functioned in fluoroimmunoassays for the detection of ricin, providing equivalent limits of detection when compared to the same anti-ricin sdAb labeled with a conventional fluorophore. In addition, the DHLA-QD–sdAb conjugates were very effective reporter elements in SPR sandwich assays, providing more sensitive detection with a signal enhancement of ∼10-fold over sdAb reporters and 2–4 fold over full sized antibody reporters. Commercially prepared streptavidin-modified polymer-coated QDs also amplified the SPR signal for the detection of ricin when applied to locations where biotinylated anti-ricin sdAb was bound to target; however, we observed a 4-fold greater amplification when using the DHLA-QD–sdAb conjugates in this format.     

Ultraviolet A radiation induces rapid apoptosis of human leukemia cells by Fas ligand-independent activation of the Fas death pathways

Endogenous cellular chromophores absorb ultraviolet A radiation (UVA, 290-320 nm), the major UV component of terrestrial solar radiation, leading to the formation of reactive oxidizing species that initiate apoptosis, gene expression and mutagenesis. UVA-induced apoptosis of T helper cells is believed to underlie the UVA phototherapy for atopic dermatitis and other T cell-mediated inflammatory skin diseases. We have evaluated the involvement of the Fas-Fas ligand (FasL) pathway in rapid UVA-induced apoptosis in human leukemia HL-60 cells. UVA-induced apoptosis was not inhibited by pretreatment with a neutralizing anti-Fas antibody, although the same UVA treatment initiated cleavage of caspase-8 and subsequent processing of Bid and caspase-3-like proteases. Inhibition of caspase-8 by Lle-Glu (OMe)-Thr-Asp(OMe)-fluoromethyl ketone completely blocked caspase-3 cleavage and apoptosis in UVA-treated cells, suggesting that apoptosis was initiated by the Fas pathway. This inference was supported by demonstrating that immunoprecipitates obtained from UVA-treated cells using anti-Fas antibody contained caspase-8 and Fas-associating protein with death domain (FADD). In addition, Fas clustering in response to UVA treatment was observed by immunofluorescence microscopy. These data support a mechanism for rapid, UVA-induced apoptosis in HL-60 cells involving initial formation of the Fas-FADD-caspase-8 death complex in an FasL-independent manner.     

��-Tocopheryl succinate potentiates the paclitaxel-induced apoptosis through enforced caspase 8 activation in human H460 lung cancer cells

Paclitaxel is one of the chemotheraputic drugs widely used for the treatment of nonsmall cell lung cancer (NSCLC) patients. Here, we tested the ability of α-tocopheryl succinate (TOS), another promising anticancer agent, to enhance the paclitaxel response in NSCLC cells. We found that sub-apoptotic doses of TOS greatly enhanced paclitaxel-induced growth suppression and apoptosis in the human H460 NSCLC cell lines. Our data revealed that this was accounted for primarily by an augmented cleavage of poly(ADP-ribose) polymerase (PARP) and enhanced activation of caspase-8. Pretreatment with z-VAD-FMK (a pan-caspase inhibitor) or z-IETD-FMK (a caspase-8 inhibitor) blocked TOS/paclitaxel cotreatment-induced PARP cleavage and apoptosis, suggesting that TOS potentiates the paclitaxel-induced apoptosis through enforced caspase 8 activation in H460 cells. Furthermore, the growth suppression effect of TOS/paclitaxel combination on human H460, A549 and H358 NSCLC cell lines were synergistic. Our observations indicate that combination of paclitaxel and TOS may offer a novel therapeutic strategy for improving paclitaxel drug efficacy in NSCLC patient therapy as well as for potentially lowering the toxic side effects of paclitaxel through reduced drug dosage.     

An Activatable Polymeric Reporter for Near-Infrared Fluorescent and Photoacoustic Imaging of Invasive Cancer

Discriminative detection of invasive and noninvasive breast cancers is crucial for their effective treatment and prognosis. However, activatable probes able to do so in vivo are rare. Herein, we report an activatable polymeric reporter (P-Dex) that specifically turns on near-infrared (NIR) fluorescent and photoacoustic (PA) signals in response to the urokinase-type plasminogen activator (uPA) overexpressed in invasive breast cancer. P-Dex has a renal-clearable dextran backbone that is linked with a NIR dye caged with an uPA-cleavable peptide substrate. Such a molecular design allows P-Dex to passively target tumors, activate NIR fluorescence and PA signals to effectively distinguish invasive MDA-MB-231 breast cancer from noninvasive MCF-7 breast cancer, and ultimately undergo renal clearance to minimize the toxicity potential. Thus, this polymeric reporter holds great promise for the early detection of malignant breast cancer.     

Apoptogenic effects of β-sitosterol glucoside from Castanopsis indica leaves

β-Sitosterol glucoside (BSSG) is a natural biologically active substance isolated from the Castanopsis indica leaves. This study explored the apoptogenic mechanistic studies of BSSG against Ehrlich's ascites carcinoma (EAC) treated mice through morphological study, comet assay, flow cytometry (FACS) and Western blotting assay method. AO/EB staining and FACS analysis showed that BSSG possessed apoptosis induction activities on EAC cells. Dose dependent induction of DNA damage was observed after BSSG treatment. Increase the expression of apoptotic protein p53 and p21 in EAC, multiple downstream factors contributing to apoptosis pathway. The increase of caspase-9 and caspase-3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by BSSG, and up-regulation of Bax and down-regulation of anti-apoptotic protein Bcl-2 resulted in the decrease of Bcl-2/Bax ratio. Owing to the combination of significant antitumour activity by inducing apoptosis, BSSG holds the promise of being an interesting chemo-preventive agent active in cancer therapy.     

D4-GDI is cleaved by caspase-3 during daunorubicin-induced apoptosis in HL-60 cells

Daunorubicin, an anti-cancer drug, is known to induce apoptosis in HL-60 cells in a dose-dependent manner through the activation of caspase-3 (CPP32). Caspase-3 selective inhibitor, Ac-DEVD-CHO, prevented both the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). D4-GDI is a GDP dissociation inhibitor for the Ras-related Rho family GTPase in hematopoietic cells. Here we report that D4-GDI is a substrate for the caspase-3. D4-GDI was cleaved to a 23 kDa fragment by daunorubicin treatment in HL-60 cells with kinetics that parallel the onset of apoptosis. D4-GDI cleavage as well as DNA fragmentation was inhibited by treatment with Ac-DEVD-CHO but not with Ac-YVAD-CHO, a caspase-1 inhibitor. These data suggest that D4-GDI of Rho family GTPase may be regulated during apoptosis through the caspase-3 mediated cleavage of the GDI protein.     

Long‐Wavelength Fluorescent Reporters for Monitoring Protein Kinase Activity

In vivo optical imaging must contend with the limitations imposed by the optical window of tissue (600–1000 nm). Although a wide array of fluorophores are available that are visualized in the red and near‐IR region of the spectrum, with the exception of proteases, there are few long wavelength probes for enzymes. This situation poses a particular challenge for studying the intracellular biochemistry of erythrocytes, the high hemoglobin content of which optically obscures subcellular monitoring at wavelengths less than 600 nm. To address this, tunable fluorescent reporters for protein kinase activity were developed. The probing wavelength is preprogrammed by using readily available fluorophores, thereby enabling detection within the optical window of tissue, specifically in the far‐red and near‐IR region. These agents were used to monitor endogenous cAMP‐dependent protein kinase activity in erythrocyte lysates and in intact erythrocytes when using a light‐activatable reporter.     

Ultrasonic-Assisted Enzymolysis Extraction and Protective Effect on Injured Cardiomyocytes in Mice of Flavonoids from Prunus mume Blossom

Prunus mume blossom is an edible flower that has been used in traditional Chinese medicine for thousands of years. Flavonoids are one of the most active substances in Prunus mume blossoms. The optimal ultrasonic-assisted enzymatic extraction of flavonoids from Prunus mume blossom (FPMB), the components of FPMB, and its protective effect on injured cardiomyocytes were investigated in this study. According to our results, the optimal extraction process for FPMB is as follows: cellulase at 2.0%, ultrasonic power at 300 W, ultrasonic enzymolysis for 30 min, and an enzymolysis temperature of 40 °C. FPMB significantly promoted the survival rate of cardiomyocytes and reduced the concentration of reactive oxygen species (ROS). FPMB also improved the activities of proteases caspase-3, caspase-8, and caspase-9 in cardiomyocytes. The cardiomyocyte apoptosis rate in mice was significantly reduced by exposure to FPMB. These results suggest that the extraction rate of FPMB may be improved by an ultrasonic-assisted enzymatic method. FPMB has a protective effect on the injured cardiomyocytes.     

Pectin-Lyase-Modified Ginseng Extract and Ginsenoside Rd Inhibits High Glucose-Induced ROS Production in Mesangial Cells and Prevents Renal Dysfunction in db/db Mice

Diabetes increases the incidence rate of chronic renal disease. Pectin-lyase-modified ginseng (GS-E3D), with enhanced ginsenoside Rd content, has been newly developed. In this study, renal protective roles of GS-E3D in type-2 diabetic db/db mice were investigated. The generation of reactive oxygen species (ROS) induced by high glucose (25 mM) was reduced by ES-E3D (75%) and ginsenoside Rd (60%). Diabetic db/db mice received 100 or 250 mg/kg/day of GS-E3D daily via oral gavage for 6 weeks. Albuminuria and urinary 8-hydroxy-2′-deoxyguanosine (8-OhdG, an oxidative stress marker) levels were increased in db/db mice and the levels recovered after GS-E3D treatment. In renal tissues, TUNEL-positive cells were decreased after GS-E3D treatment, and the increased apoptosis-related protein expressions were restored after GS-E3D treatment. Therefore, GS-E3D has a potent protective role in diabetes-induced renal dysfunction through antioxidative and antiapoptotic activities. These results may help patients to select a dietary supplement for diabetes when experiencing renal dysfunction.     

Luminescent kinase activity biosensors based on a versatile bimolecular switch

Real-time tracking of kinase activity in living systems has revealed new modes of encoding signaling information into spatiotemporal activity patterns and opened new avenues for screening kinase modulators. However, the sensitivity of kinase activity detection, which is commonly coupled to a fluorescence resonance energy transfer (FRET)-based readout, has often been a limiting factor. Here we show that a kinase-inducible bimolecular switch consisting of a substrate for the kinase of interest and a phosphoamino acid binding domain can be designed to sense different kinase activities and coupled to various readouts, thereby allowing for examination of dynamic kinase activity with increased sensitivity and versatility. Specifically, we demonstrate that bimolecular switches designed to sense protein kinase A (PKA) or protein kinase C (PKC) activities can turn on FRET as well as bioluminescence signals. Notably, the FRET-based sensors gain larger dynamic ranges in comparison with their unimolecular counterparts; the novel bioluminescence-based reporters for PKA and PKC show high sensitivity and a unique capability to detect basal kinase activities and should enable new applications in in vivo imaging of kinase activity and high-throughput compound screening. Thus, this generalizable design advances the molecular toolkit of kinase activity detection and provides a means for versatile and sensitive detection of kinase activity in various biological systems.     

Hispolon Induces Apoptosis,Suppresses Migration and Invasion of Glioblastoma Cells and Inhibits GBM Xenograft Tumor Growth In Vivo

Hispolon, a polyphenol compound isolated from Phellinus linteus, has been reported to exhibit antioxidant, antiproliferative, and antitumor activities. This study aimed to explore the antitumor effects of hispolon on glioblastoma multiforme (GBM) cells in vitro and in vivo. The results revealed that hispolon significantly inhibited GBM cell proliferation and induced apoptosis through caspase-9 and caspase-3 activation and PARP cleavage. Hispolon also induced cell cycle G2/M phase arrest in GBM cells, as supported by flow cytometry analysis and confirmed by a decrease in cyclin B1, cdc2, and cdc25c protein expressions in a dose- and time-dependent manner. Furthermore, hispolon suppressed the migration and invasion of GBM cells by modulating epithelial–mesenchymal transition (EMT) markers via wound healing, transwell assays, and real-time PCR. Moreover, hispolon significantly reduced tumor growth in DBTRG xenograft mice and activated caspase-3 in hispolon-treated tumors. Thus, our findings revealed that hispolon is a potential candidate for the treatment of GBM.     

Lipid rafts mediate ultraviolet light-induced Fas aggregation in M624 melanoma cells

Ultraviolet light (UV) induces aggregation of Fas-receptor through a Fas-ligand-independent pathway. However, the mechanism of ultraviolet light-induced Fas-receptor aggregation is not known. In this report, we show that lipid rafts mediate ultraviolet light-induced aggregation of Fas. Our data show that UV induces a redistribution of Fas-receptor in a 25-5% Optiprep continuous gradient. The amount of Fas-receptorS is significantly increased in a gradient fraction that contain lipid rafts and is associated with an increase of FADD and caspase-8. Our data also show that the active dimeric form of caspase-8 (p44/p41) is increased in the lipid raft fraction. In addition, our data show that cholesterol, a major component of lipid rafts, is significantly reduced in only the lipid raft fractions after UV-irradiation. However, ceramide, another major lipid raft component, is increased evenly in all gradient fractions after UV-irradiation. These results suggest that UV alters the composition of major lipid raft components, which leads to the recruitment of Fas-receptor and FADD, with subsequent activation of caspase-8. Based on our results, we propose a novel mechanism by which UV induces apoptosis through a membrane lipid raft-mediated signaling pathway.