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

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

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

Influence of sesquiterpene lactones on cell cycle distribution.

<p>A) Untreated TE-671 cells were measured as control. B) Treatment of TE-671 cells with the IC₅₀ of costunolide led to a very small change in the distribution, whereas, C) the IC₅₀ of dehydrocostus lactone caused a significant decrease in the number of cells in G1 phase after 48 h which was accompanied by an increase of the number of S and G2/M phase cells. Values are expressed as percentage of the cell population in the G1, S, and G2/M phase of cell cycle. Graphical representations of the cell cycle distribution of D) TE-671, E) SW-872, and F) SW-982 cells.</p

Relative mRNA expression of MMP-1, -2 and -9 measured by real-time RT-PCR.

<p>A) Costunolide increased MMP-2 and -9 expression levels significantly in SW-982 and TE-671 cell lines. B) Dehydrocostus lactone increased MMP-1 expression level significantly in SW-872 and TE-671 cells. Furthermore, the levels of MMP-2 and -9 were significantly reduced in TE-671, but increased in SW-982 cells.</p

Western blot analysis of G2/M arrest regulator proteins.

<p>Total protein analysis revealed a significant downregulation of cdc2 and cyclin B1 in dehydrocostus lactone treated STS cells, whereas, costunolide treatment did not affect the protein levels of these G2/M checkpoint regulators. β-actin was used as loading control. Data shown are representative from at least three independent experiments.</p

Primer Sequences used for RT-PCR.

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

Relative mRNA expression and Western blot analysis of CDK2.

<p>A) Dehydrocostus lactone decreased the CKD2 mRNA expression levels significantly in SW-982 (*p = 0.041) and TE-671 (*p = 0.007) cells. B) This downregulation was also found on the protein level. No significant alteration could be observed after costunolide treatment. β-actin was used as loading control. Data shown are representative from at least three independent experiments.</p

Sesquiterpene lactones inhibited the invasion potential.

<p>Costunolide and dehydrocostus lactone pre-treated DiI stained STS cells were seeded in the upper compartment of matrigel coated 8 µm pore size HTS FluoroBlok™ cell culture inserts in serum free medium. Invasion towards the lower compartment containing medium supplemented with 5% FBS was monitored by fluorescent microscope and quantified after overnight incubation at 37°C. The invasion potential was significantly reduced in all three STS cell lines.</p