5-Aminolaevulinic acid enhances ultrasound-induced mitochondrial damage in K562 cells

Background

Ultrasound therapy is a new modality in the control of malignant cancers. The aim of the present study was to investigate the effect of 5-aminolaevulinic acid on the ultrasonic killing action in the cancer cells.

Materials/methods

The K562 cells as a cancer cell model were subjected to investigate the effect of 5-aminolaevulinic acid (5-ALA) on the ultrasonic killing action, in which the 5-ALA concentration was 2 mM and the ultrasound exposure was 15 s at the intensity of 0.46 W/cm² and the frequency of 1.7 MHz. Cytotoxicity was investigated 24 h after ultrasound exposure using the trypan blue exclusion test. Ultrastructural cell morphology and mitochondrial changes were observed using transmission electron microscopy (TEM). Mitochondrial membrane potential (ΔΨm) was evaluated using Rhodamine 123 assay.

Results

The death rates of the K562 cells in the controls including sham radiation and 5-ALA treatment alone were 1.81 ± 0.13%, 1.27 ± 0.20%, respectively. Those in ultrasound radiation alone and 5-ALA-ultrasound treatment were 12.61 ± 2.63%, 46.87 ± 4.09%, respectively. There were significant differences between 5-ALA-ultrasound treatment, ultrasound radiation alone and the controls (P < 0.05). TEM showed that the mitochondria expanding and some vacuoles were found in the ultrasound-treated cells. After the treatment of ultrasound and 5-ALA together some cells presented typical characteristics of apoptotic cells, such as nuclear condensation and crescent formation. Mitochondria of the cells were damaged more seriously than those treated by ultrasound alone, there were obvious swollen mitochondria and mitochondria in which cristae were almost perfectly disappeared, and more vacuolar mitochondria were founded. Mitochondrial membrane potential (ΔΨm) was more significantly collapsed when the K562 cells were exposed to 2 mM 5-ALA for 4 h and then 0.46 W/cm² irradiation of ultrasound than ultrasound radiation alone.

Conclusion

5-ALA pretreatment significantly enhanced the cytotoxicity of ultrasound radiation in K562 cells. The damage of mitochondria structure and function might be an important cause of cell death in K562 cells induced by the treatment of ultrasound radiation and 5-ALA together.     

Volume quantification by fuzzy logic modelling in freehand ultrasound imaging

Introduction

Many algorithms exist for 3D reconstruction of data from freehand 2D ultrasound slices. These methods are based on interpolation techniques to fill the voxels from the pixels. For quantification purposes, segmentation is involved to delineate the structure of interest. However, speckle and partial volume effect errors can affect quantification.

Objective

This study aimed to assess the effect of the combination of a fuzzy model and 3D reconstruction algorithms of freehand ultrasound images on these errors.

Methods

We introduced a fuzzification step to correct the initial segmentation, by weighting the pixels by a distribution function, taking into account the local gray levels, the orientation of the local gradient, and the local contrast-to-noise ratio. We then used two of the most wide-spread reconstruction algorithms (pixel nearest neighbour (PNN) and voxel nearest neighbour (VNN)) to interpolate and create the volume of the structure. Finally, defuzzification was used to estimate the optimal volume.

Validation

B-scans were acquired using 5 MHz and 8 MHz ultrasound probes on ultrasound tissue-mimicking phantoms. Quantitative evaluation of the reconstructed structures was done by comparing the method output to the real volumes. Comparison was also done with classical PNN and VNN algorithms.

Results

With the fuzzy model quantification errors were less than 4.3%, whereas with classical algorithms, errors were larger (10.3% using PNN, 17.2% using VNN). Furthermore, for very small structures (0.5 cm³), errors reached 24.3% using the classical VNN algorithm, while they were about 9.6% with the fuzzy VNN model.

Conclusion

These experiments prove that the fuzzy model allows volumes to be determined with better accuracy and reproducibility, especially for small structures (<3 cm³).     

Simultaneous grayscale and subharmonic ultrasound imaging on a modified commercial scanner

Objective

To demonstrate the feasibility of simultaneous dual fundamental grayscale and subharmonic imaging on a modified commercial scanner.

Motivation

The ability to generate signals at half the insonation frequency is exclusive to ultrasound contrast agents (UCA). Thus, subharmonic imaging (SHI; transmitting at f₀ and receiving at f₀/2) provides improved visualization of UCA within the vasculature via suppression of the surrounding tissue echoes. While this capability has proven useful in a variety of clinical applications, the SHI suppression of surrounding tissue landmarks (which are needed for sonographic navigation) also limits it use as a primary imaging modality. In this paper we present results using a commercial ultrasound scanner modified to allow imaging in both grayscale (f₀ = 4.0 MHz) and SHI (f₀ = 2.5 MHz, f₀/2 = 1.25 MHz) modes in real time.

Methods

A Logiq 9 ultrasound scanner (GE Healthcare, Milwaukee, WI) with a 4C curvilinear probe was modified to provide this capability. Four commercially available UCA (Definity, Lantheus Medical Imaging, North Billerica, MA; Optison, GE Healthcare, Princeton, NJ; SonoVue, Bracco Imaging, Milan, Italy; and Sonazoid, GE Healthcare, Oslo, Norway) were all investigated in vitro over an acoustic output range of 3.34 MPa. In vivo the subharmonic response of Sonazoid was investigated in the portal veins of four canines (open abdominal cavity) and four patients with suspected portal hypertension.

Results

In vitro, the four UCA showed an average maximum subharmonic amplitude of 44.1 ± 5.4 dB above the noise floor with a maximum subharmonic amplitude of 48.6 ± 1.6 dB provided by Sonazoid. The average in vivo maximum signal above the noise floor from Sonazoid was 20.8 ± 2.3 dB in canines and 33.9 ± 5.2 dB in humans. Subharmonic amplitude as a function of acoustic output in both groups matched the S-curve behavior of the agent observed in vitro. The dual grayscale imaging provided easier sonographic navigation, while the degree of tissue suppression in SHI mode varied greatly on a case by case basis.

Conclusions

These results demonstrate the feasibility of dual grayscale and SHI on a modified commercial scanner. The ability to simultaneously visualize both imaging modes in real time should improve the applicability of SHI as a future primary clinical imaging modality.     

Ultrasound induces cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin

Objectives

Curcumin, a natural pigment from the traditional Chinese herb, has shown promise as an efficient enhancer of ultrasound. The present study aims to investigate ultrasound-induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin in vitro.

Methods

Nasopharyngeal carcinoma cell line CNE2 cells were incubated by 10 μm curcumin and then were treated by ultrasound for 8 s at the intensity of 0.46 W/cm². Cytotoxicity was evaluated using MTT assay and light microscopy. Mitochondrial damage was analyzed using a confocal laser scanning microcopy with Rhodamine 123 and ultrastructural changes were observed using a transmission electron microscopy (TEM).

Results

MTT assay showed that cytotoxicity induced by ultrasound treatment alone and curcumin treatment alone was 18.16 ± 2.37% and 24.93 ± 8.30%, respectively. The cytotoxicity induced by the combined treatment of ultrasound and curcumin significantly increased up to 86.67 ± 7.78%. TEM showed that microvillin disappearance, membrane blebbing, chromatin condensation, swollen mitochondria, and mitochondrial myelin-like body were observed in the cells treated by ultrasound and curcumin together. The significant collapse of mitochondrial membrane potential (MMP) was markedly observed in the CNE2 cells after the combined treatment of curcumin and ultrasound.

Conclusions

Our findings demonstrated that ultrasound sonication in the presence of curcumin significantly killed the CNE2 cells and induced ultrastructural damage and the dysfunction of mitochondria, suggesting that ultrasound treatment remarkably induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin.     

Hyperfine structure investigations of some odd levels of Co I by level crossing and optical methods

Using the level crossing technique the ratios and absolute values of the hyperfine structure (hfs) constants of the levelsz ⁴F_9/2 andz ⁴F_7/2 of the configuration 3d ⁷4s4p of Co I were measured:z ⁴ F _9/2: ¦A¦=(811±12)MHz; ¦B¦=(48±93) MHz;B/A=?0.06±0.11 A>0; B<0z ⁴ F _7/2: ¦A¦ = (659 ±11)MHz; ¦B¦=(33±84)MHz;B/A=?0.05±0.13 A>0; B<0. In addition the hfs constants of three other excited levels of Co I could be determined by optical methods:z ⁴ F _9/2:A=525±26 MHz;B=200 MHzy ⁴ F _9/2:A=300±30 MHz;B=?500 MHzy ⁴ G _11/2:A=315±20 MHz;B=400 MHz. The experimental results are compared with known experimental and also with theoretical values which where calculated using the parametric potential method.     

Directed neuronal differentiation of human embryonic stem cells

Background

We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons.HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4⁺/SSEA-4^- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII).

Results

A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7–10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons.

Conclusion

This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4⁺ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.

     

Hypocrellin B-mediated sonodynamic action induces apoptosis of hepatocellular carcinoma cells

Objective

The present study aims to investigate apoptosis of hepatocellular carcinoma cells induced by hypocrellin B-mediated sonodynamic action.

Methods

The hypocrellin B concentration was kept constant at 2.5 μM and cells from the hepatocellular carcinoma HepG2 cell line were exposed to ultrasound with an intensity of 0.46 W/cm² for 8 s. Cell cytotoxicity was quantified using an MTT assay 24 h after sonodynamic therapy (SDT) of hypocrellin B. Apoptosis was investigated using a flow cytometry with Annexin V-FITC and propidium iodine staining. Intracellular reactive oxygen species (ROS) levels were detected using a flow cytometry with 2,7-dichlorodihydrofluorecein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of hypocrellin B-mediated sonodynamic action on HepG2 cells was significantly higher than those of other treatments including ultrasound alone, hypocrellin B alone and sham treatment. Flow cytometry showed that hypocrellin B-induced sonodynamic action markedly enhanced the apoptotic rate of HepG2 cells. Increased ROS was observed in HepG2 cells after being treated with hypocrellin B-mediated sonodynamic action.

Conclusions

Our data demonstrated that hypocrellin B-mediated sonodynamic action remarkably induced apoptosis of HepG2 cells, suggesting that apoptosis is an important mechanism of cell death induced by hypocrellin B-mediated SDT.     

High-frequency EPR of Cr<Superscript>2+</Superscript> ions in CdGa<Subscript>2</Subscript>S<Subscript>4</Subscript>

High-frequency broad-band (65–240 GHz) EPR is used to study impurity centers of bivalent chromium in a CdGa₂S₄ crystal. It is found that the EPR spectra correspond to tetragonal symmetry. The spin Hamiltonian H = βB · g · S + B ₂ ⁰ O ₂ ⁰ + B ₄ ⁰ O ₄ ⁰ + B ₄ ⁴ O ₄ ⁴ with the parameters B ₂ ⁰ =23659±2 MHz, B ₄ ⁰ =1.9±1 MHz, |B ₄ ⁴ |=54.2±2 MHz, g_‖=1.93±0.02, and g_⊥=1.99±0.02 is used to describe the observed spectra. It is concluded that chromium ions occupy one of the tetrahedrally coordinated cation positions.     

Bestimmung der Kernmomente des Ho165 aus der Hyperfeinstruktur des Grundzustandes im Ho I-Spektrum

In an atomic beam magnetic resonance experiment, the hyperfine interaction constantsA andB of the⁴ I _2/15-groundstate of Ho¹⁶⁵ were found to beA=800,58389 (50) MHz,B=?1667,997 (50) MHz. Using an effective value for 〈r ^?3〉, the magnetic moment of the Ho¹⁶⁵ nucleus was calculated to beμ=4·1(4)μ _n . The quadrupolement was determined by use of the 〈r ^?3〉 given byWatson andFreeman. The result isQ=2·4·10^?24 cm².     

Apoptosis of ovarian cancer cells induced by methylene blue-mediated sonodynamic action

Objective

The present study aims to investigate apoptosis of ovarian cancer cells induced by methylene blue (MB)-mediated sonodynamic therapy (SDT).

Methods

The MB concentration was kept constant at 100 μM and ovarian cancer HO-8910 cells were exposed to ultrasound therapy for 5 s with an intensity of 0.46 W/cm². The cytotoxicity was investigated 24 h after MB-mediated sonodynamic action. Apoptosis was analyzed using a flow cytometer with Annexin V-FITC and propidium iodine (PI) staining as well as fluorescence microscopy with Hoechst 33258 staining. Intracellular reactive oxygen species (ROS) level was measured by flow cytometer with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of MB-mediated SDT on HO-8910 cells after MB-mediated SDT was significantly higher than those of other treatments including ultrasound alone, MB alone and sham treatment. Flow cytometric analysis showed a significant increase in the early and late apoptotic cell populations by MB-mediated SDT of HO-8910 cells. Nuclear condensation and increased ROS levels were also found in HO-8910 cells treated by MB-mediated SDT.

Conclusions

Our findings demonstrated that MB-mediated sonodynamic action significantly induced apoptosis of HO-8910 cells and an increase in intracellular ROS level. This indicates that apoptosis is an important mechanism of cell death induced by MB-mediated SDT. Thus, MB-mediated SDT might be a potential therapeutic strategy for combating ovarian cancer.