ABSTRACT
Electrostimulation (ES) is an important therapeutic method for diseases caused by abnormal intracellular electrical activity. Also, it can induce apoptosis of cells, which is a potential tumor treatment method. At present, there are no relevant studies on changes in intracellular reactive oxygen species (ROS) levels produced in the process of ES, or on the effects of simultaneous implementation of conventional antioxidant inhibitor drugs and ES therapy. To reveal these, two organelle-targeting core-shell plasmonic probes were designed for measuring ROS produced during ES. The probes were delivered into target organelles (nucleus and mitochondrion) before the cells were electrically stimulated for different periods of time. Surface-enhanced Raman scattering (SERS) signals were detected in situ, and the sensing mechanism for the quantitative analysis of ROS is based on the signal reduction of SERS caused by the ROS-etching effect on the silver shell. The detection results revealed that ES could trigger ROS generation in cells, and the ROS levels localized around organelles were assessed by SERS. This study has great potential for exploring abnormal organelle microenvironments via organelle-targeting probes combined with SERS technology.
Subject(s)
Gold , Metal Nanoparticles , Organelles , Reactive Oxygen Species , Silver , Spectrum Analysis, Raman/methodsABSTRACT
Programmed cell death ligand 1 (PD-L1) is considered a major immune checkpoint protein that mediates antitumor immune suppression and response. Effectively regulating PD-L1 expression and dynamic monitoring has become a significant challenge in immunotherapy. Herein, we adopted smart surface-enhanced Raman scattering (SERS) nanoprobes to discriminate and monitor the dynamic expression of PD-L1 under external electrostimulation (ES). The PD-L1 expression levels in three cell lines (MCF-7 cells, HeLa cells, and H8 cells) were assessed before and after ES. The results reveal that ES could effectively and rapidly mediate a transformation in the PD-L1 content (or activity) on the cell membrane. Moreover, the molecular profiles of the cell membrane before and after ES were revealed by using the label-free SERS method with the help of immune plasmonic nanoparticles. The cell membrane protein information presented identifiable conformation changes after ES, showing a significant inhibitory effect on the bridge of PD-L1 and its antibody. This study indicates that ES is superior to chemical drugs due to lesser side effects because ES-based regulation does not depend on intracellular signalling pathways. This strategy is versatile and robust for discriminating and monitoring PD-L1 on cell membranes, thus providing potential clinical application value to PD-L1-mediated systems. This study also offers a practical way to assess the molecular profiles of cell membrane proteins in the presence of an external stimulus, which may be applicable to many membrane protein-related studies.
Subject(s)
B7-H1 Antigen , Nanoparticles , Cell Membrane , HeLa Cells , Humans , ImmunotherapyABSTRACT
A highly sensitive trypsin sensing system in serum was developed by using an anodic alumina oxide (AAO)-based, trypsin substrate-decorated hybrid ion permeation membrane. Owing to the trypsin-triggered peptide hydrolyzation reaction, the surface electrical feature of the peptide-decorated hybrid ion membrane changed. The electric double layer effect reduces the effective ion current diameter in the AAO nano unit, so that the ion current rectification ratio will be enhanced, realizing the quantitative detection of trypsin. The lowest detection concentration can be achieved as low as 0.1 pM. This method is no need for sample pre-preparation, easy to operate, highly sensitive, and also applicable to other enzyme evaluation systems by changing corresponding substrates. This study provides a new idea for selective measurements of proteases in complex biological samples.
Subject(s)
Aluminum Oxide/chemistry , Nanotechnology/instrumentation , Peptides/chemistry , Trypsin/analysis , Trypsin/blood , Electrochemical Techniques , Humans , Membranes, Artificial , Microscopy, Electron, ScanningABSTRACT
Caspase-3 is considered as one of the key proteases that can spontaneously regulate the life activities of cells, and its activation (usually is a slow process) will execute the apoptosis process of cells. Rapid activation of caspase-3 on demand in living-cells is therefore highly desired toward precise cancer therapy but it is still a key challenge. Herein, we applied electrostimulus (ES) to achieve fast activation of caspase-3 and trigger cell apoptosis, and developed a smart magnetic-plasmonic assembly nanoprobes (A-nanoprobes) to real-time trace cellular caspase-3 activation at the single cell level. The designer core-satellite A-nanoprobe, working specific to the activated caspase-3 via a disassembly tactic, provides strong "hot spots" to improve the sensitivity and therefore enables SERS sensing of cellular caspase-3 upon activated by ES. Single-cell analysis revealed that the ES can rapidly activate the apoptosis pathway of caspase-3 on demand to make the DNA fragmentation and ultimately induce the cell apoptosis. Such method and nanoplatform were further used to monitor ES-triggered caspase-3 activation in cell apoptosis process of different cell types, revealing that more caspase-3 will be activated for cancerous cells than normal cells during the ES to induce cells apoptosis. This strategy and platform are promising for detecting cellular caspase-3 and other enzymes in the process of cancer diagnosis and treatments.
Subject(s)
Apoptosis , Caspase 3/metabolism , Gold/chemistry , Metal Nanoparticles/chemistry , Caspase 3/analysis , Cell Line, Tumor , Electric Stimulation Therapy , Humans , Spectrum Analysis, RamanABSTRACT
A kind of smart carbon nanodots (CNDs) with the pH response feature was prepared by the one-pot hydrothermal treatment of citric acid and dicyandiamide, which was used for the differentiation of cancer/normal cells and the selective photothermal therapy (PTT) of cancer cells. When the smart CNDs were cultured with cells, they were highly internalized in the lysosomes of cells. Since the small-sized CNDs (about 5â¯nm) tends to form aggregation (as large as about 20â¯nm or even larger) under an acid condition (pHâ¯=â¯4.7) due to the electrostatic attraction produced by the surface protonation, relatively severer aggregation of the CNDs were observed in liver cancer cells (HepG2 cells) relative to normal ones (LO2 cells) due to a relative lower pH in the lysosomes of HepG2 cells, which endows them a new strong absorption band at longer wavelengths (450-900â¯nm) and a higher photothermal conversion efficiency (42.13 %), benefiting to differentiated PTT. The flow cytometric data indicates strong photothermal ablation (8â¯min, 509.6â¯mW/cm2) for cancer cells with the assistance of these smart CNDs achieves 82 % death rate of cancer cells, while much less damage is observed on the normal cells (6.35 %). To the best of our knowledge, this is the first report about CNDs for selective PTT owing to their intrinsic property without the aid of any other targeting ligands. These smart CNDs are also available for other acid-responsive sensing systems, and this study inspires us in the synthesis of near-infrared featured carbon materials.
Subject(s)
Carbon/pharmacology , Nanoparticles/chemistry , Photosensitizing Agents/pharmacology , Phototherapy , Carbon/chemistry , Cell Survival/drug effects , Cells, Cultured , Citric Acid/chemistry , Guanidines/chemistry , Hep G2 Cells , Humans , Hydrogen-Ion Concentration , Molecular Structure , Particle Size , Photosensitizing Agents/chemistry , Surface PropertiesABSTRACT
Subcellular mitochondrion has become a target for improving the therapeutic efficiency and reducing side damage to normal cells via a combination of many therapeutic strategies. However, the underlying molecular mechanisms associated with cell death induced by subcellular dysfunction remain unknown or disputed. In this study, we investigated the dynamic molecular changes of living mitochondria upon phototherapy (photothermal therapy plus photodynamic therapy, PTT & PDT) by surface-enhanced Raman scattering spectroscopy (SERS) and intended to disclose the photo-induced cell death route in breast cancer cells (MCF-7) taking into account the mitochondrion. Indocyanine green (ICG), a Food and Drug Administration (FDA)-approved clinic blood-injection near-infrared angiographic contrast agent and a PTT & PDT drug, was used for the evaluation of the phototherapy effect. The results revealed that the content of phenylalanine (Phe) in mitochondria evidently increased during the phototherapy-induced cell death process. Moreover, the phototherapy-induced cell apoptosis was mainly regulated through the DNA structures. We expect that the understanding of mitochondrial molecular stress responses will be helpful for the diagnosis and therapy of cellular processes associated with mitochondria and provide valuable guidance for the further design and development of more effective therapeutic platforms and methods at the sub-cellular level.
Subject(s)
Mitochondria/metabolism , Spectrum Analysis, Raman/methods , DNA Damage/drug effects , Gold/chemistry , Humans , Hyperthermia, Induced/methods , Indocyanine Green/pharmacology , Indocyanine Green/radiation effects , Indocyanine Green/toxicity , Infrared Rays , MCF-7 Cells , Nanotubes/chemistry , Phenylalanine/metabolism , Photochemotherapy/methodsABSTRACT
Cytochrome c (Cyt c) release and cellular pH change are two important mediators of apoptosis. Effective methods to regulate or monitor such two events are therefore highly desired for apoptosis research and cancer cell therapy. Herein, we exploited electrostimulation to regulate cellular Cyt c release and apoptosis process, and by designing and preparing a smart and efficient plasmonic nanorobot (with surface-modified Cyt c-specific aptamer and 4-mercaptobenzoic acid) that is capable of Cyt c capture and self-sensing, we achieved real-time SERS monitoring of dynamic Cyt c release and simultaneous cell acidification in apoptosis during electrostimulation. Distinctly different molecular stress responses in the two events for cancerous MCF-7 and HeLa cells and normal L929 cells were identified and revealed. The method and results are valuable and promising for apoptosis and Cyt c-mediated biology studies.
Subject(s)
Apoptosis , Cytochromes c/metabolism , Electric Stimulation , Nanotechnology/methods , Robotics , Animals , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/metabolism , Base Sequence , Benzoates/chemistry , Cell Line , Cytochromes c/chemistry , Humans , Hydrogen-Ion Concentration , Membrane Potential, Mitochondrial , Sulfhydryl Compounds/chemistryABSTRACT
Metallic plasmonic nanoparticles have been intensively exploited as theranostic nanoprobes for plasmonic photothermal therapy (PPT) and surface-enhanced Raman spectroscopy (SERS) applications. But the underlying molecular mechanisms associated with PPT-induced apoptosis between cancerous and normal cells have remained largely unknown or disputed. In this study, we designed an organelle-targeting theranostic plasmonic SERS nanoprobe (CDs-Ag/Au NS) composed of porous Ag/Au nanoshell (p-Ag/Au NSs) and carbon dots (CDs) for nucleus and mitochondria targeted PPT of cells. The differences in molecular stress response in the PPT-induced hyperthermia cell death between cancerous HeLa and normal L929 and H8 cells have been revealed by site-specific single-cell SERS detection. The contents of tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) in HeLa cells were found more evidently increased than L929 and H8 cells during the PPT-induced cell-death process. And from the mitochondria point of view, we found that the PPT-induced cell apoptosis for HeLa cells mainly stems from (or is regulated through) cellular thermal stress-responsive proteins, while for L929 and H8 cells it seems more related to DNA. Understanding molecular stress response difference of the PPT-induced cell apoptosis between cancerous and normal cells is helpful for diagnosis and treatment of cancer, and the method will open an avenue for single-cell studies.
Subject(s)
Cell Nucleus/metabolism , Mitochondria/metabolism , Nanoshells/chemistry , Quantum Dots/chemistry , Spectrum Analysis, Raman/methods , Theranostic Nanomedicine/methods , Apoptosis/drug effects , Carbon/chemistry , Carbon/radiation effects , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/metabolism , DNA/metabolism , DNA Fragmentation/drug effects , Gold/chemistry , Gold/radiation effects , HeLa Cells , Humans , Hyperthermia, Induced/methods , Infrared Rays , Nanoshells/radiation effects , Neoplasms/metabolism , Nuclear Localization Signals/chemistry , Nuclear Localization Signals/metabolism , Quantum Dots/radiation effects , Silver/chemistry , Silver/radiation effectsABSTRACT
A surface-enhanced Raman scattering (SERS) measurement of 3,3',4,4'-tetrachlorobiphenyl (PCB77) with aptamer capturing in a microfluidic device was demonstrated. To construct the microfluidic chip, an ordered Ag nanocrown array was fabricated over a patterned polydimethylsiloxane (PDMS) that was achieved by replicating an anodic aluminum oxide (AAO) template. The patterned PDMS sheet was covered with another PDMS sheet having two input channel grooves to form a close chip. The Ag nanocrown array was used for the SERS enhancement area and the detection zone. PCB 77 aptamers were injected into one channel and the other allows for analytes (PCBs). The mercapto aptamers captured the targets in the mixed zone and were immobilized to the SERS detection zone via S-Ag bonds so as to further improve both the SERS sensitivity and selectivity of PCB77. Such an aptamer-based microfluidic chip realized a rapid SERS detection. The lowest detectable concentration of 1.0 × 10(-8) M was achieved for PCB77. This work demonstrates that the aptamer-modified SERS microfluidic sensor can be utilized for selective detections of organic pollutants in the environment.
Subject(s)
Aptamers, Nucleotide/chemistry , Microfluidic Analytical Techniques , Polychlorinated Biphenyls/analysis , Spectrum Analysis, Raman , Aluminum Oxide/chemistry , Metal Nanoparticles/chemistry , Microfluidic Analytical Techniques/instrumentation , Silver/chemistry , Spectrum Analysis, Raman/instrumentation , Surface PropertiesABSTRACT
The focus of this work was to study the photothermal effect of a silica-gold-silica nanocomposite in the near-infrared (NIR) region. The NIR region is considered a biological window because living cells and tissues have low light scattering and adsorption in this region. Both a laser source and a tungsten lamp source were used in this study. The critical parameters for photothermal efficiency, including nancomposite concentration and irradiation time, were evaluated. The penetration of the nanocomposites into mammalian cells was also investigated. With laser irradiation, the nanocomposite showed a significant photothermal effect in the NIR region. The maximal temperature that the nanocomposites could reach was 51.9 °C. Vybrant assays showed that 5 min of laser irradiation along with the nanocomposite caused target cell death through both apoptosis (59%) and necrosis (31%), while controls showed minimal effects. The nanocomposite may be a potential light-absorbing agent for NIR fluorescence-guided photothermal therapy.
Subject(s)
Gold/chemistry , Nanocomposites/chemistry , Phototherapy/instrumentation , Silicon Dioxide/chemistry , Animals , Apoptosis/radiation effects , Cell Line , Infrared Rays , Macrophages/cytology , Macrophages/radiation effects , MiceABSTRACT
Alzheimer's disease (AD) is associated with damage to hippocampal neurons and declines in cognitive functions. The accumulation of amyloid peptides is regarded as a crucial event in the initiation of AD. The neurotoxicity induced by Aß25-35 peptides was used to screen for cytoprotective factors in vitro, and the cognitive deficits induced by the injection of Aß25-35 into the hippocampus were used to evaluate effect on learning and memory. Our previous study revealed that hydrolysate of polygalasaponins (HPS) clearly improve the cognitive deficits induced by the injection of Aß25-35 in mice, but the potential active constituent of HPS remains unclear. The purposes of this study were to separate and purify the secondary saponins of HPS, screen for neuroprotective effects of the constituents in vitro, and to evaluate the effect of cognition in vivo. Various chromatographic methods were used to separate and purify the HPS. The neuroprotective effects were examined in Aß25-35-damage-induced PC12 cells. The protective effect of tenuifolin on the cognitive impairments induced by Aß25-35 injection was assessed using the Morris water maze and step-through passive avoidance tests. Tenuifolin and fallaxsaponin A were isolated from the HPS. Tenuifolin possessed neuroprotective effects against Aß25-35-induced apoptosis in PC12 cells and significantly improved the cognitive deficits induced by the intrahippocampal injection of Aß25-35 in mice. Thus, tenuifolin is one of the active constituents of HPS against the neurotoxicity induced by Aß25-35 peptides in vitro and in vivo.
Subject(s)
Amyloid beta-Peptides , Diterpenes, Kaurane , Neuroprotective Agents , Neurotoxins , Peptide Fragments , Animals , Humans , Mice , Rats , Alzheimer Disease/drug therapy , Alzheimer Disease/etiology , Alzheimer Disease/psychology , Amyloid beta-Peptides/physiology , Diterpenes, Kaurane/isolation & purification , Diterpenes, Kaurane/pharmacology , Hippocampus/drug effects , Hippocampus/physiopathology , Hydrolysis , In Vitro Techniques , Learning/drug effects , Memory/drug effects , Models, Animal , Neuroprotective Agents/isolation & purification , Neuroprotective Agents/pharmacology , Neurotoxins/toxicity , PC12 Cells , Peptide Fragments/physiology , Phytotherapy , Saponins/chemistry , Saponins/pharmacologyABSTRACT
This study uses the powerful fingerprint features of Raman spectroscopy to distinguish different types of breast tissues including normal breast tissues (NB), fibroadenoma (FD), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC). Thin frozen tissue sections of fresh breast tissues were measured by Raman spectroscopy. Due to the inherent low sensitivity of Raman spectra, Au@SiO2 shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) technique was utilized to provide supplementary and more informative spectral features. A total of 619 Raman spectra were acquired and compared to 654 SHINERS spectra. The maximum enhancement effect of distinct and specific bands was characterized for different tissue types. When applying the new criteria, excellent separation of FD, DCIS, and IDC was obtained for all tissue types. Most importantly, we were able to distinguish ADH from DCIS. Although only a preliminary distinction was characterized between ADH and NB, the results provided a good foundation of criteria to further discriminate ADH from NB and shed more light toward a better understanding of the mechanism of ADH formation. This is the first report to detect the premalignant (ADH and DCIS) breast tissue frozen sections and also the first report exploiting SHINERS to detect and distinguish breast tissues. The results presented in this study show that SHINERS can be applied to accurately and efficiently identify breast lesions. Further, the spectra can be acquired in a minimally invasive procedure and analyzed rapidly facilitating early and accurate diagnosis in vivo/in situ.
Subject(s)
Breast Neoplasms/diagnosis , Breast Neoplasms/pathology , Gold/chemistry , Metal Nanoparticles/chemistry , Nanotechnology/methods , Silicon Dioxide/chemistry , Spectrum Analysis, Raman/methods , Adult , Aged , Breast/pathology , Carcinoma, Ductal, Breast/diagnosis , Carcinoma, Ductal, Breast/pathology , Carcinoma, Intraductal, Noninfiltrating/diagnosis , Carcinoma, Intraductal, Noninfiltrating/pathology , DNA/chemistry , Female , Fibroadenoma/diagnosis , Fibroadenoma/pathology , Humans , Hyperplasia/diagnosis , Hyperplasia/pathology , Middle Aged , Young Adult , beta Carotene/chemistryABSTRACT
Chemotherapy is the most common way to treat malignancies, but myelosuppression, one of its common side-effects, is a formidable problem. The present study described the protective role of dammarane sapogenins (DS), an active fraction from oriental ginseng, on myelosuppression induced by cyclophosphamide (CP) in mice. DS was orally administered at different dosages (37.5, 75, and 150 mg/kg) for 10 d after CP administration (200 mg/kg intraperitoneally). The results showed that DS increased the number of white blood cells (WBC) on day 3 and day 7 (P < 0.05), such that WBC levels were increased by 105.7 ± 29.5% at 75 mg/kg of DS on day 3 (P < 0.05, compared with the CP group). Similar results were observed in red blood cells and platelets in DS-treated groups. The colony-forming assay demonstrated that the depressed numbers of CFU-GM (colony-forming unit-granulocyte and macrophage), CFU-E (colony-forming unit-erythroid), BFU-E (burst-forming unit-erythroid), CFU-Meg (colony-forming unit-megakaryocyte) and CFU-GEMM (colony-forming unit-granulocyte, -erythrocyte, -monocyte and -megakaryocyte) induced by CP were significantly reversed after DS treatment. Moreover, the ameliorative effect of DS on myelosuppression was also observed in the femur by hematoxylin/eosin staining. In DS-treated groups, ConA-induced splenocyte proliferation was enhanced significantly at all the doses (37.5, 75, 150 mg/kg) on day 3 at the rate of 50.3 ± 8.0%, 77.6 ± 8.5% and 44.5 ± 8.4%, respectively, while lipopolysaccharide-induced proliferation was increased mainly on day 7 (P < 0.01), with an increased rate of 39.8 ± 5.6%, 34.9 ± 6.6% and 38.3 ± 7.3%, respectively. The thymus index was also markedly increased by 70.4% and 36.6% at 75 mg/kg on days 3 and 7, respectively, as compared with the CP group. In summary, DS has a protective function against CP-induced myelosuppression. Its mechanism might be related to stimulating hematopoiesis recovery, as well as enhancing the immunological function.
Subject(s)
Cyclophosphamide/adverse effects , Drug-Related Side Effects and Adverse Reactions , Myeloablative Agonists/antagonists & inhibitors , Myeloablative Agonists/toxicity , Protective Agents/administration & dosage , Sapogenins/administration & dosage , Triterpenes/administration & dosage , Administration, Oral , Animals , Colony-Forming Units Assay/methods , Cyclophosphamide/administration & dosage , Femur/pathology , Histocytochemistry , Leukocyte Count , Mice , Panax/chemistry , Protective Agents/isolation & purification , Sapogenins/isolation & purification , Stem Cells , Triterpenes/isolation & purification , DammaranesABSTRACT
OBJECTIVE: To study the inhibitive effects of an effective section of a prescription of traditional Chinese medicine (TCM-ES) on influenza virus A FM1 strain in vitro. METHODS: The experiments were performed by microcytopathic-inhibiting-assay, Neutral Red stain and inhibiting plaque-forming units (PFU) test on MDCK cell strain. By means of observing the cytopathic effects (CPE), measuring the absorbance [D(lambda)] and counting the PFU, according to Reed-Muench assay, the TCM-ES's effective dosage of 50 percentage (EC50) and treatment index (TI) to FM1 were calculated. The inhibiting dose of 50 percentage of PFU (IC50) was also figured up. RESULTS: By CPE assay, TCM-ES'S EC50, MTC and TI to 100TCID50 FM1 strain infection were (300 +/- 18.3) mg/L, (75 +/- 6.8) mg/L and (7.1 +/- 0.7), respectively; Whereas, ribavirin's EC50, MTC and TI was (52.3 +/- 10.1) mg/L, (25 +/- 4.1) mg/L and (20.8 +/- 5.1), respectively. By Neutral Red stain assay,TCM-ES's IC50 and TI was (285.0 +/- 19.2) mg/L and (7.2 +/- 0.6), respectively; whereas ribavirin's IC50 and TI was (45.3 +/- 4. 9) mg/L and (21.2 +/- 3.1), respectively. By reducing PFU assay, the IC50 of TCM-ES and ribavirin was 300 mg/L and 50 mg/L, respectively. All the results above were almost consistent with each other (P > 0.05). CONCLUSIONS: TCM-ES assumes antiviral action on IFV-FM1 strain in a certain degree in vitro and can rebel intracellular virus. But it is worse than the positive control medicine of ribavirin and is worthy of further study.
Subject(s)
Antiviral Agents/pharmacology , Drugs, Chinese Herbal/pharmacology , Influenza A virus/drug effects , Plants, Medicinal/chemistry , Animals , Antiviral Agents/isolation & purification , Cells, Cultured , Dogs , Drug Combinations , Drugs, Chinese Herbal/isolation & purification , Influenza A virus/physiology , Inhibitory Concentration 50 , RNA, Viral/drug effects , Ribavirin/pharmacology , Virus Replication/drug effectsABSTRACT
Paeonol, a phenolic component from the root bark of Paeonia moutan, is traditionally used as a Chinese herbal medicine to activate the blood flow and remove blood stasis. Evidence shows that paeonol have anti-tumor, anti-inflammatory, and analgesic effects; however, the underlying mechanisms remain unknown. In this study, we investigated the molecular mechanisms by which paeonol exerts the anti-tumor effects by using a murine model of hepatoma established by in vivo injection of mouse HepA-hepatoma cells. Treatment of mice with 100, 200, or 400 mg/kg/day of paeonol significantly inhibited the growth of the HepA tumor in mice, induced HepA cell apoptosis as demonstrated by light microscopy and electron microscopy analyses, decreased the expression of Bcl-2 and increased the expression of Bax in HepA tumor tissues in a dose-related manner. Administration of paeonol in vivo also elevated serum levels of IL-2 and TNF-alpha in tumor-bearing mice. Moreover, splenocytes and macrophages isolated from paeonol-treated HepA tumor-bearing mice produced higher levels of IL-2 and TNF-alpha in response to concanavalin A and lipopolysaccharide stimulation, respectively, compared to these isolated from non-treated HepA tumor-bearing mice. In vitro treatment with paeonol was able to directly stimulate IL-2 and TNF-alpha production in splenocytes and macrophages from tumor-bearing mice, respectively. In conclusion, paeonol has the anti-tumor effect against hepatoma cells, which are likely mediated via induction of tumor cell apoptosis and stimulation of IL-2 and TNF-alpha production. Paeonol could be a promising drug to treat hepatocellular carcinoma.
Subject(s)
Acetophenones/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Interleukin-2/metabolism , Liver Neoplasms, Experimental/drug therapy , Tumor Necrosis Factor-alpha/metabolism , Acetophenones/therapeutic use , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Line, Tumor , Cells, Cultured , Dose-Response Relationship, Drug , Interleukin-2/blood , Liver Neoplasms, Experimental/immunology , Liver Neoplasms, Experimental/pathology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Mice , Proto-Oncogene Proteins c-bcl-2/metabolism , Spleen/drug effects , Spleen/immunology , Tumor Necrosis Factor-alpha/blood , Up-Regulation , bcl-2-Associated X Protein/metabolismABSTRACT
A rice gene, OsPIPK 1, encoding a 792-aa putative phosphatidylinositol 4-phosphate 5-kinase (PIPK), was identified and characterized. Comparison between the cDNA and genomic sequences revealed the presence of 10 exons (39-1050 bp) and 9 introns (88-745 bp) in OsPIPK 1 gene. The deduced amino acid sequence of OsPIPK1 contains a lipid kinase domain that is highly homologous to those of previously isolated PIPKs, and structural analysis revealed the intriguing presence of multiple MORN motifs at the N-terminus. The MORN motifs have also been detected in PIPKs from Arabidopsis thaliana and Oryza sativa, but not in the well-characterized PIPKs from animal and yeast cells. RT-PCR analysis indicated that OsPIPK 1 was expressed almost constitutively in roots, shoots, stems, leaves and flowers, and up-regulated following treatment with plant hormones or application of various stresses. An antisense transgenic strategy was used to suppress the expression of OsPIPK 1, and homozygous transgenic plants showed earlier heading (7-14 days earlier) than control plants, suggesting that OsPIPK 1 negatively regulates floral initiation. This was further confirmed by morphologic observation showing earlier floral development in antisense plants, as well as leaf emergence measurement indicating delayed leaf development under OsPIPK 1 deficiency, a common phenotype observed with earlier flowering. RT-PCR analysis and cDNA chip technology were used to examine transcripts of various genes in the transgenic plants and the results showed altered transcriptions of several flowering-time or -identity related genes, suggesting that OsPIPK 1 is involved in rice heading through regulation of floral induction genes, signaling and metabolic pathways.
Subject(s)
Flowers/genetics , Oryza/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Amino Acid Motifs/genetics , Amino Acid Sequence , Animals , Calcium/pharmacology , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Flowers/growth & development , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Plant/drug effects , Molecular Sequence Data , Mutation , Oryza/enzymology , Oryza/growth & development , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Plant Growth Regulators/pharmacology , Plant Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Salicylic Acid/pharmacology , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Time Factors , Yeasts/enzymology , Yeasts/geneticsABSTRACT
This paper reports a novel immunoassay based on surface-enhanced Raman scattering (SERS) and immunogold labelling with silver staining enhancement. Immunoreactions between immunogold colloids modified by a Raman-active probe molecule (e.g., 4-mercaptobenzoic acid) and antigens, which were captured by antibody-assembled chips such as silicon or quartz, were detected via SERS signals of Raman-active probe molecule. All the self-assembled steps were subjected to the measurements of ultraviolet-visible (UV-vis) spectra to monitor the formation of a sandwich structure onto a substrate. The immunoassay was performed by a sandwich structure consisting of three layers. The first layer was composed of immobilized antibody molecules of mouse polyclonal antibody against Hepatitis B virus surface antigen (PAb) on a silicon or quartz substrate. The second layer was the complementary Hepatitis B virus surface antigen (Antigen) molecules captured by PAb on the substrate. The third layer was composed of the probe-labelling immunogold nanoparticles, which were modified by mouse monoclonal antibody against Hepatitis B virus surface antigen (MAb) and 4-mercaptobenzoic acid (MBA) as the Raman-active probe on the surface of gold colloids. After silver staining enhancement, the antigen is identified by a SERS spectrum of MBA. A working curve of the intensity of a SERS signal at 1585 cm(-1) due to the [small nu](8a) aromatic ring vibration of MBA versus the concentration of analyte (Antigen) was obtained and the non-optimized detection limit for the Hepatitis B virus surface antigen was found to be as low as 0.5 [micro sign]g mL(-1).