Liling Tang

Effect of actin cytoskeleton disruption on electric pulse-induced apoptosis and electroporation in tumour cells

Electric pulses are known to affect the outer membrane and intracellular structures of tumour cells. By applying electrical pulses of 450 ns duration with electric field intensity of 8 kV/cm to HepG2 cells for 30 s, electric pulse‐induced changes in the integrity of the plasma membrane, apoptosis, viability and mitochondrial transmembrane potential were investigated. Results demonstrated that electric pulses induced cell apoptosis and necrosis accompanied with the decrease of mitochondrial transmembrane potential and the formation of pores in the membrane. The role of cytoskeleton in cellular response to electric pulses was investigated. We found that the apoptotic and necrosis percentages of cells in response to electric pulses decreased after cytoskeletal disruption. The electroporation of cell was not affected by cytoskeletal disruption. The results suggest that the disruption of actin skeleton is positive in protecting cells from killing by electric pulses, and the skeleton is not involved in the electroporation directly.

Apoptosis induction with electric pulses - a new approach to cancer therapy with drug free.

Electrical pulses have been widely used in biomedical fields, whose applications depend on the parameters such as durations and electric intensity. Conventional electroporation (0.1-1kV/cm, 100micros) has been used in cell fusion, transfection and electrochemotherapy. Recent studies with high-intensity (MV/cm) electric field applications with durations of several tens of nanoseconds can affect intracellular signal transduction and intracellular structures with plasma intact, resulting in an application of intracellular manipulation. The most recent development is the finding that parameters between those two ranges could be used to induce apoptosis of cancer cells. Proposal of apoptosis induction and tumor inhibition has advantages to pursue the treatment of cancer free of cytotoxic drugs.

Irreversible electroporation and apoptosis in human liver cancer cells induced by nanosecond electric pulses

The goal of this study was to assess the effect of nanosecond electric pulses on HepG2 human liver cancer cells. Electric pulses with a high strength of 10 kV/cm, duration of 500 ns and frequency of 1 Hz were applied to the cells. After delivery of electric pulses, apoptosis, intracellular calcium ion concentrations, transmembrane mitochondrial potentials, electropermeabilization and recovery from electropermeabilization in cells were investigated. The results showed that electric pulse treatment for 20 s and more could trigger apoptosis in cells. Real-time observation indicated an immediate increase in intracellular calcium ion concentration and a dramatic decrease in mitochondrial membrane potential in cells responding to electric pulses. In subsequent experiments, propidium iodide uptake in cells emerged after exposure to electric pulses, indicating electropermeabilization of the cell membrane. Furthermore, recovery from electropermeabilization was not observed even 4 h after the stimulation, demonstrating that irreversible electropermeabilization was induced by electric pulses. In conclusion, electric pulses with a high strength and nanosecond duration can damage cancer cells, accompanied by a series of intracellular changes, providing strong evidence for the application of electric pulses in cancer treatment.

Induction of apoptosis of liver cancer cells by nanosecond pulsed electric fields (nsPEFs)

The application of nanosecond pulsed electric fields (nsPEFs) is a novel method to induce the death of cancer cells. NsPEFs could directly function on the cell membrane and activate the apoptosis pathways, then induce apoptosis in various cell lines. However, the nsPEFs-inducing-apoptosis action sites and the exact pathways are not clear now. In this study, nsPEFs were applied to the human liver cancer cells HepG2 with different parameters. By apoptosis assay, morphological observation, detecting the mitochondrial membrane potential (ΔΨm), intracellular calcium ion concentration ([Ca2+]i) and the expressions of key apoptosis factors, we demonstrated that nsPEFs could induce the morphology of cell apoptosis, the change in ΔΨm, [Ca2+]i and the upregulation of some key apoptosis factors, which revealed the responses of liver cancer cells and indicated that cells may undergo apoptosis through the mitochondria-dependent pathway after nsPEFs were applied.

A ns-μs duration, millitesla, exponential decay pulsed magnetic fields generator for tumor treatment

In order to research a new tumor treatment method using exponential decay pulsed magnetic fields (EDPMF), we developed a EDPMF generator by means of the integration of pulsed power technology and modern power electronics technology. After charging a capacitor with a high voltage dc power supply, EDPMF was generated inside the Helmholtz coils by means of discharging the capacitor through a switch to the discharge resistor. After a brief introduction of basic principle of EDPMF generator, steep pulse formation circuit and Helmholtz coils, two main parts of EDPMF generator, were discussed in detail, especially on design techniques. A height-adjustable platform was designed inside the Helmholtz coils. Cell solution and little animals could be treated on this platform. The peak value, width and repeat rate of the output EDPMF waveform could be adjusted independently in the range of 0 - 1 mT, 160 ns-55 μs and 1 Hz-1 kHz, respectively. The rise time of the output EDPMF waveform was shortened to 70-220 ns by means of choosing quickly operating switch and reducing circuit stray inductance. Magnetic field distribution uniformity was improved via designing better geometrical structure of Helmholtz coils. This was proved by finite element simulation and measurement. Cell experiment in vitro was performed by exposing human liver cancer cells (Hep-G2) to pulsed magnetic field (0.8 mT, 55 μs, 15 Hz) using this generator and the result showed that proliferation of cancer cells in treated group was significantly (P<;0.01) inhibited. This suggests possible clinical application using this kind of EDPMF for local tumor treatment.

Effects of electric pulses on apoptosis induction and mitochondrial transmembrane potential of cancer cells

Application of electric pulses is becoming a promising strategy to killing cancer. In this study, the effects of different pulses on the apoptosis and mitochondrial transmembrane potential of human cancer cells SMMC7721 were studied. In our experiments, the apoptosis in cells was determined using Annexin-V-FITC. Results showed that 1.8µs, 200V/cm and 250 V/cm electric fields induced cells apoptosis while 120ns, 600V/cm pulses didn’t have significant apoptotic effect on cells. Using confocal microscopy, changes of mitochondrial transmembrane potential (Δφm) were investigated when cells exposed to electric fields in real-time way. Results showed that the electric stimulation decreased mitochondrial transmembrane potential. Interestingly, the mitochondrial transmembrane potential decreased more significantly in cells that were exposed to the 120ns, 600V/cm electric fields (67.09%) compared to the cells in 1.8µs, 200V/cm and 250V/cm (23.71%,28.44%). In conclusion, electric pulses can induce cell apoptosis and decrease the mitochondrial transmembrane potential, depending on the duration and electric intensity of electric pulses. Moreover, shorter pulses have more effect on intracellular structure.

hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway

&NA; hnRNP A1 acts as a critical splicing factor in regulating many alternative splicing events in various physiological and pathophysiological progressions. hnRNP A1 is capable of regulating UVB‐induced hdm2 gene alternative splicing according to our previous study. However, the biological function and underlying molecular mechanism of hnRNP A1 in cell survival and cell cycle in response to UVB irradiation are still unclear. In this study, silencing hnRNP A1 expression by siRNA transfection led to decreased cell survival after UVB treatment, while promoting hnRNP A1 by lentiviruse vector resulted in increased cell survival. hnRNP A1 remarkably enhanced PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of Akt, mTOR and P70S6 protein. Inhibition of PI3K/Akt signaling by LY294002 suppressed the expression of hnRNP A1. While mTOR signaling inhibitors, rapamycin and AZD8055, did not influence hnRNP A1 expression in HaCaT cells, suggesting that hnRNP A1 may be an upstream mediator of mTOR signaling. Furthermore, hnRNP A1 could alleviate UVB‐provoked cell cycle arrest at G0/G1 phase and promoted cell cycle progression at G2/M phase. Our results indicate that hnRNP A1 promotes cell survival and cell cycle progression following UVB radiation.

The structure-function relationships of insulin-like growth factor 1 Ec in C2C12 cells

ABSTRACT Insulin-like growth factor 1 (IGF1) is a crucial growth factor, that regulates skeletal muscles development during cell growth and repair. Recently, its alternative splicing variant, named IGF1Ec, also named mechano-growth factor (MGF), has gained attentions as a new damage repair factor. However, the structure-function relationships of IGF1Ec have not been fully clarified due to contradictory reports. In this study, we systematically investigated physiologic responses of C2C12 muscle cells to IGF1Ec, IGF1 and MGF E peptide. Our data indicate that while the N-terminal sequence of IGF1Ec, which is homolog in part with IGF1, promotes proliferation; the C-terminal sequence of IGF1Ec, which is identical to MGF E, promotes differentiation and migration of C2C12 cells. Our results suggest that MGF E cannot completely replace all the functions of IGF1Ec on muscle repair and regeneration, and elucidate the relationships between structure and function of IGF1Ec.

SPARC acts as a mediator of TGF-β1 in promoting epithelial-to-mesenchymal transition in A549 and H1299 lung cancer cells: SPARC acts as a mediator of TGF-β1

Migration and metastasis of tumor cells greatly contributes to the failure of cancer treatment. Recently, the extracellular protein secreted protein acidic and rich in cysteine (SPARC) has been reported closely related to tumorigenesis. Some articles have suggested that SPARC promoted metastasis in several highly metastatic tumors. However, there are also some studies shown that SPARC acted as an antitumor factor. SPARC-induced epithelial-to-mesenchymal transition (EMT) in melanoma cells and promoted EMT in hepatocellular carcinoma. Therefore, the role of SPARC in tumorigenesis and its relationship with EMT is still unclear. In this study, we investigated the expression change of SPARC in A549 and H1299 lung cancer cells undergoing EMT process. Our study indicated that SPARC was upregulated in A549 and H1299 cells EMT process. We further investigated the function of SPARC on proliferation, migration, and EMT process of A549 and H1299 cells. Overexpression of SPARC promoted the migration and EMT of A549 and H1299 cells. Knockdown SPARC inhibited the EMT of A549 cells. Overexpression of SPARC induced the increased expression of p-Akt and P-ERK. Furthermore, exogenous SPARC peptide promoted transforming growth factor (TGF)-β1-induced EMT of A549 and H1299 cells. SPARC knockdown partially eliminated TGF-β1 function in inducing EMT of A549 cells. SPARC follistatin-like functional domain reduced the expression of E-cadherin, but had no effect on the expression of p-Akt and p-ERK. In conclusion, we elucidated that SPARC contributes to tumorigenesis by promoting migration and EMT of A549 and H1299 lung cancer cells. These results will provide some new suggestion for lung cancer treatment.

Comparative Study of the Biological Responses to Conventional Pulse and High-Frequency Monopolar Pulse Bursts

Given its nonthermal property and other advantages, irreversible electroporation (IRE) has quickly translated into clinical applications. An increasing number of clinical cases using IRE have also revealed crucial issues. The uneven distribution of the electric field caused by the heterogeneity of biological tissues has not been completely and effectively addressed to date. The use of high-frequency monopolar pulse bursts (HFMPBs) is expected to solve this problem. The high-frequency content of the HFMPB is used to ensure the uniform distribution of the electric field in the tissue. A temperature increase similar to that of the conventional pulse method does not cause thermal damage and results in a killing effect similar to that of the conventional technique. Meanwhile, the subpulse has a pulse duration of 1–