Bortezomib induces autophagy in human chondrosarcoma cells.

<p>A) Relative gene expression and B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC₅₀ values of bortezomib for 24 h. C) Western blot analysis for the expression of LC3BI-II. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II. D) Quantification of the relative LC3B protein expression.</p

Analysis of the mitochondria-caspase dependent pathway.

<p>A,B) Relative gene expression analysis of the pro- and anti-apoptotic markers Bax, Bak, Bcl-2, and Bcl-xl in Cal-78 and SW-1353 cells treated with the respective IC₅₀ values of bortezomib for 24 h (<i>n</i> = 10). C) The human apoptosis antibody protein array revealed the downregulation of the heat shock proteins HSP27, HSP60, and HSP70 as well as HTRA, Livin, p27, and p53 in both cell lines. D) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment.</p

Apoptotic induction of bortezomib.

<p>A,C) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3/7 activity than untreated cells. Untreated control cells served as reference value (ratio = 1). B,D) Cleavage of caspase-3 was detected after 24 h of bortezomib treatment by flow cytometry. The y-axis denotes cell counts and the x-axis represents fluorescence intensity of the APC antibody. The black filled histogram represents untreated control cells, the striated histogram represents 5 nM, and the checkered histogram shows 10 nM bortezomib treated cells.</p

Analysis of TRAIL/death receptor pathway.

<p>A,B) Relative gene expression analysis of IGF1R, Fas, and the death receptors TRAILR-1 and TRAILR-2 after bortezomib treatment with the respective IC₅₀ concentrations for 48 h. Untreated control cells served as reference value (ratio = 1). C) The human apoptosis antibody protein array supported the important role of the IGF binding proteins (IGFBP-1 to IGFBP-6), the TNF receptor family, and the TRAIL receptors in both cell lines. D) Western blot analysis showed the downregulation of Fas and TNF-R1.</p

Characterization of Dynamic Behaviour of MCF7 and MCF10A Cells in Ultrasonic Field Using Modal and Harmonic Analyses

<div><p>Treatment options specifically targeting tumour cells are urgently needed in order to reduce the side effects accompanied by chemo- or radiotherapy. Differences in subcellular structure between tumour and normal cells determine their specific elasticity. These structural differences can be utilised by low-frequency ultrasound in order to specifically induce cytotoxicity of tumour cells. For further evaluation, we combined <i>in silico</i> FEM (finite element method) analyses and <i>in vitro</i> assays to bolster the significance of low-frequency ultrasound for tumour treatment. FEM simulations were able to calculate the first resonance frequency of MCF7 breast tumour cells at 21 kHz in contrast to 34 kHz for the MCF10A normal breast cells, which was due to the higher elasticity and larger size of MCF7 cells. For experimental validation of the <i>in silico</i>-determined resonance frequencies, equipment for ultrasonic irradiation with distinct frequencies was constructed. Differences for both cell lines in their response to low-frequent ultrasonic treatment were corroborated in 2D and in 3D cell culture assays. Treatment with ~ 24.5 kHz induced the death of MCF7 cells and MDA-MB-231 metastases cells possessing a similar elasticity; frequencies of > 29 kHz resulted in cytotoxicity of MCF10A. Fractionated treatments by ultrasonic irradiation of suspension myeloid HL60 cells resulted in a significant decrease of viable cells, mostly significant after threefold irradiation in intervals of 3 h. Most importantly in regard to a clinical application, combined ultrasonic treatment and chemotherapy with paclitaxel showed a significantly increased killing of MCF7 cells compared to both monotherapies. In summary, we were able to determine for the first time for different tumour cell lines a specific frequency of low-intensity ultrasound for induction of cell ablation. The cytotoxic effect of ultrasonic irradiation could be increased by either fractionated treatment or in combination with chemotherapy. Thus, our results will open new perspectives in tumour treatment.</p></div

Proliferation analysis of ALDH1^high and ALDH1^low sarcoma cells.

<p>The immunohistochemical analysis using anti-Ki-67 proliferation marker revealed a decreased proliferation level of (A) SW-1353 ALDH1^low cells and compared to (B) SW-1353 ALDH1^high cells. (C–E) Dynamic proliferation curves for ALDH1^high and ALDH1^low cells seeded at 10,000 cells per well measured with the xCELLigence system.</p

Influence of (A) material properties (Young’s modulus for cytoplasm and nucleus are as first and second value in parenthesis), (B) cell dimensions, (C) thickness of the actin cortex in percent of the cell radius, and (D) cell embedding (Young’s modulus for agar in parenthesis) on natural frequencies of MCF10A cells (A) or MCF7 cells (A-D).

<p>Influence of (A) material properties (Young’s modulus for cytoplasm and nucleus are as first and second value in parenthesis), (B) cell dimensions, (C) thickness of the actin cortex in percent of the cell radius, and (D) cell embedding (Young’s modulus for agar in parenthesis) on natural frequencies of MCF10A cells (A) or MCF7 cells (A-D).</p

Primer Sequences used for real-time RT-PCR.

<p>Primer Sequences used for real-time RT-PCR.</p

Decreased survival of MCF7 cells after irradiation with an ultrasonic frequency of 24.5 kHz as determined by XCelligence (Roche).

<p>Decreased survival of MCF7 cells after irradiation with an ultrasonic frequency of 24.5 kHz as determined by XCelligence (Roche).</p

Increased death of MCF7 and MDA-MB-231 cells after irradiation with an ultrasonic frequency of 24.5 kHz.

<p>(A) Cells either cultivated in 2D culture or (B) growing in 3D culture on alginate beads (gems) were treated with 24.5 kHz and four different intensities for 4 min; 1 h later the proportion of dead cells (propidium iodide (PI) positive cells) was determined by FACS analysis. Results represent the means of data from eight (A) or three (B) independent experiments; the error bars represent the standard errors; p-values were calculated by the two-sided, paired Student’s t-test with * p<0.05, ** p<0.01.</p