Jiye Cai

Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca2+ concentration

Curcumin is a phytochemicals which is able to inhibit carcinogenesis in a variety of cell lines. However little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, we found that curcumin could inhibit the growth of human hepatocellular carcinoma cell line (HepG2), change the cell-surface morphology and trigger the pro-apoptotic factor to promote cell apoptosis. Cell counting kit results indicated that the cell viability had a dose-dependent relationship with the curcumin concentration in 24h. The 50% inhibiting concentration (IC50) was 17.5±3.2μM. It was clear that curcumin could lead to apoptosis, and the apoptosis increased as the reacting concentration goes up. Moreover, curcumin could also affect the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca(2+) concentration. All these alterations changed the cell morphology and cell-surface ultrastructure with atomic force microscopy (AFM) detecting at nanoscale level. AFM results indicated that cells in control group clearly revealed a typical long spindle-shaped morphology. Cell tails was wide and unrolled. The ultrastructure showed that cell membrane was made up of many nanoparticles. After being treated with curcumin, cell tail was narrowed. The size of membrane nanoparticles became small. These results can improve our understanding of curcumin which can be potentially developed as a new agent for treatment of hepatocellular carcinoma since it has been reported to have a low cytotoxic effect on healthy cell. AFM can be used as a powerful tool for detecting ultrastructures.

Tim-3-expressing CD4+ and CD8+ T cells in human tuberculosis (TB) exhibit polarized effector memory phenotypes and stronger anti-TB effector functions.

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4+ and CD8+ T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3+CD4+ and Tim-3+CD8+ T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3− counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3+CD4+ and Tim-3+CD8+ T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4+ and CD8+ T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.

Connection between biomechanics and cytoskeleton structure of lymphocyte and Jurkat cells: An AFM study

The mechanical properties of cells are important for many cellular processes. Here, atomic force microscopy (AFM) and laser scanning confocal microscopy (LSCM) were carried out to characterize lymphocyte and Jurkat cells. The average elastic modulus of lymphocyte is 1.24 +/- 0.09 kPa, which is almost twofold higher than that of Jurkat cell (0.51 +/- 0.06 kPa). LSCM images of sub-membrane cytoskeleton showed a significant difference in the organization of their F-actin structures. Lymphocyte cells had more and thicker actin bundles than that of Jurkat cells. Lymphocyte and Jurkat cells after adding the F-actin destabilizing agent Cytochalasin-B (Cyt-B) were also investigated by AFM. A decrease in the elastic modulus of lymphocyte from a value of 1.24 +/- 0.09 kPa down to 0.34 +/- 0.04 kPa for 24 h was observed, and that of Jurkat cell decreased from 0.51 +/- 0.06 kPa to 0.23 +/- 0.04 kPa. We really believe that this technology will be used for cancer detection and opens a door to study the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.

Detection of erythrocytes influenced by aging and type 2 diabetes using atomic force microscope.

The pathophysiological changes of erythrocytes are detected at the molecular scale, which is important to reveal the onset of diseases. Type 2 diabetes is an age-related metabolic disorder with high prevalence in elderly (or old) people. Up to now, there are no treatments to cure diabetes. Therefore, early detection and the ability to monitor the progression of type 2 diabetes are very important for developing effective therapies. Type 2 diabetes is associated with high blood glucose in the context of insulin resistance and relative insulin deficiency. These abnormalities may disturb the architecture and functions of erythrocytes at molecular scale. In this study, the aging- and diabetes-induced changes in morphological and biomechanical properties of erythrocytes are clearly characterized at nanometer scale using atomic force microscope (AFM). The structural information and mechanical properties of the cell surface membranes of erythrocytes are very important indicators for determining the healthy, diseased or aging status. So, AFM may potentially be developed into a powerful tool in diagnosing diseases.

BMP2 promotes migration and invasion of breast cancer cells via cytoskeletal reorganization and adhesion decrease: an AFM investigation

Bone morphogenetic protein 2 (BMP2) has been shown to modulate the proliferation and differentiation of breast cancer cells. However, the biochemical effects and mechanisms remain unknown. In this paper, the effects of recombinant human BMP2 on the migration of MCF-7 cells—one breast cancer cell line, using transwell and wound healing experiments, as well as on the cellular morphology, cytoskeleton, cell surface adhesion, and stiffness detected at subcellular level by an atomic force microscope, were investigated. After BMP2 treatment, the untreated round-shaped MCF-7 cells transformed to a spindle-like shape with lots of specialized structures, such as lamellipodia, filopodia, membrane protrusions, and others, which are essential for cellular migration or spreading. Moreover, flow cytometry quantitatively detected the BMP2-induced changes in the expression of adhesion molecules, a significant rise of CD44, and a remarkable drop of E-cadherin. The data indicated that BMP2 promoted the migration and invasion of MCF-7 cells by regulating the reorganization of cytoskeleton and the expression of adhesion molecules in/on the cells. Thus, it is very imperative to evaluate the oncogenicity of BMP2 when used in tissue engineering.

Highly sensitive detection of chromium (III) ions by resonance Rayleigh scattering enhanced by gold nanoparticles

Simple and sensitive determination of chromium (III) ions (Cr(3+)) has potential applications for detecting trace contamination in environment. Here, the assay is based on the enhancement of resonance Rayleigh scattering (RRS) by Cr(3+)-induced aggregation of citrate-capped gold nanoparticles (AuNPs). Transmission electron microscopy (TEM) and UV-vis absorption spectroscopy were employed to characterize the nanostructures and spectroscopic properties of the Cr(3+)-AuNP system. The experiment conditions, such as reaction time, pH value, salt concentration and interfering ions, were investigated. The combination of signal amplification of Cr(3+)-citrate chelation with high sensitivity of RRS technique allow a selective assay of Cr(3+) ions with a detection limit of up to 1.0 pM. The overall assay can be carried out at room temperature within only twenty minutes, making it suitable for high-throughput routine applications in environment and food samples.

Nanostructure and nanomechanics analysis of lymphocyte using AFM: from resting, activated to apoptosis.

The ultrastructural and mechanical properties of single resting, activated and apoptosis lymphocyte have been investigated by atomic force microscopy (AFM). Using topographic imaging, we showed that the surface of the resting lymphocyte is smooth, while lymphocyte activation and apoptosis are often accompanied by changes in cell morphology. The apoptosis lymphocyte is rougher than those of the two other morphotypes, and coated with many big particles. Using spatially resolved force-distance curves, we found that the valve of the activated lymphocyte is about two to three times stiffer (Youngs modulus of approximately 20 kPa) than those of the two other morphotypes (5-11 kPa). These results can improve our understanding of the mechanical properties of cells during growth and differentiation.

Pathway of cytotoxicity induced by folic acid modified selenium nanoparticles in MCF-7 cells.

Selenium nanoparticles (Se NPs) have been recognized as promising materials for biomedical applications. To prepare Se NPs which contained cancer targeting methods and to clarify the cellular localization and cytotoxicity mechanisms of these Se NPs against cancer cells, folic acid protected/modified selenium nanoparticles (FA–Se NPs) were first prepared by a one-step method. Some morphologic and spectroscopic methods were obtained to prove the successfully formation of FA–Se NPs while free folate competitive inhibition assay, microscope, and several biological methods were used to determine the in vitro uptake, subcellular localization, and cytotoxicity mechanism of FA–Se NPs in MCF-7 cells. The results indicated that the 70-nm FA–Se NPs were internalized by MCF-7 cells through folate receptor-mediated endocytosis and targeted to mitochondria located regions through endocytic vesicles transporting. Then, the FA–Se NPs entered into mitochondria; triggered the mitochondria-dependent apoptosis of MCF-7 cells which involved oxidative stress, Ca2+ stress changes, and mitochondrial dysfunction; and finally caused the damage of mitochondria. FA–Se NPs released from broken mitochondria were transported into nucleus and further into nucleolus which then induced MCF-7 cell cycle arrest. In addition, FA–Se NPs could induce cytoskeleton disorganization and induce MCF-7 cell membrane morphology alterations. These results collectively suggested that FA–Se NPs could be served as potential therapeutic agents and organelle-targeted drug carriers in cancer therapy.

Artesunate induced morphological and mechanical changes of Jurkat cell studied by AFM.

In this study, we have used atomic force microscopy (AFM) to study the morphology and mechanical property changes of Jurkat cells exposed to different concentrations of Artesunate (ART) for 24 h at single cellular level. Cell viability and proliferation assays were performed by using the Cell Counting Kit-8. The concentration of ART, which resulted in the inhibition rate >50% was selected. The AFM images revealed that the cell membrane changed and the ultrastructure also became complex. Mechanical properties of individual cell were tracked with AFM-based force spectroscopy. The force curves revealed that when a cell was exposed to the ART, the mechanical properties changed obviously. Treated cells had a lower adhesion force of 416.8+/-37.9 pN, whereas control group had a higher adhesion force of 1064.2+/-97.0 pN. The Youngs modulus decreased to nearly one-third, from control group of 0.648+/-0.037 kPa to treated group of 0.254+/-0.035 kPa and the stiffness increased to nearly 1.5 times, from control group of 1.231+/-0.084 mN/m to treated group of 1.917+/-0.137 mN/m. These results suggest that ART can inhibit the proliferation of Jurkat and induce changes in the morphological structure and mechanical properties of Jurkat cells. The high resolution and high sensitivity of AFM can be used to detect morphological and mechanical properties of cells exposed to ART. The AFM may be developed to be a useful tool for detecting the cell death and evaluating the anti-carcinogen efficacy against tumor cell. SCANNING 31: 83-89, 2009. (c) 2009 Wiley Periodicals, Inc.

Time-dependent surface adhesive force and morphology of RBC measured by AFM

Atomic force microscopy (AFM) is a rapidly developing tool recently introduced into the evaluation of the age of bloodstains, potentially providing legal medical experts useful information for forensic investigation. In this study, the time-dependent, morphological changes of red blood cells (RBC) under three different conditions (including controlled, room-temperature condition, uncontrolled, outdoor-environmental condition, and controlled, low-temperature condition) were observed by AFM, as well as the cellular viscoelasticity via force-vs-distance curve measurements. Firstly, the data indicate that substrate types have different effects on cellular morphology of RBC. RBC presented the typical biconcave shape on mica, whereas either the biconcave shape or flattened shape was evident on glass. The mean volume of RBCs on mica was significantly larger than that of cells on glass. Surprisingly, the adhesive property of RBC membrane surfaces was substrate type-independent (the adhesive forces were statistically similar on glass and mica). With time lapse, the changes in cell volume and adhesive force of RBC under the controlled room-temperature condition were similar to those under the uncontrolled outdoor-environmental condition. Under the controlled low-temperature condition, however, the changes in cell volume occurred mainly due to the collapse of RBCs, and the curves of adhesive force showed the dramatic alternations in viscoelasticity of RBC. Taken together, the AFM detections on the time-dependent, substrate type-dependent, environment (temperature/humidity)-dependent changes in morphology and surface viscoelasticity of RBC imply a potential application of AFM in forensic medicine or investigations, e.g., estimating age of bloodstain or death time.