Abstract C3: Bcl-2 inhibition in Chondrosarcoma leads to increased apoptosis and is a promising therapeutic target
Authors: van Oosterwijk Jolieke G. , Meijer Danielle , Gelderblom Hans J. , van de Water Bob , Bovee Judith VMG 
Published: June 2012

Abstract
Chondrosarcomas are malignant cartilage-forming tumors of the bone and are notorious for their resistance to chemo- and radiotherapy. Chondrosarcomas consist of slowly dividing cells and show overexpression of BCL2. A correlation between BCL2 expression and malignant transformation of osteochondromas to peripheral chondrosarcomas has been found as well as BCL2 positivity in 30–67% of central chondrosarcomas, increasing with grade. As BCL2 is an anti-apoptotic protein, inhibition could lead to resensitization of tumor cells to conventional therapy. This study aims at inducing apoptosis in chondrosarcomas insensitive to conventional treatment measures. ABT737 is a BH3 mimetic which inhibits anti-apoptotic proteins. BCL2 inhibition has been proven to work in leukemia cells.
Expression of pro- and anti-apoptotic genes was studied using Illumina human expression bead chip v3 microarrays on 23 solitary chondrosarcomas and 6 normal cartilage tissues for comparison using R software. ABT737 was tested for its ability to induce apoptosis in 4 chondrosarcoma cell lines and 4 primary cultures (SW1353, OUMS27, CH2879, JJ012, and L835, L869, L1081, L1250, respectively). HL60, an ALL cell line, was used as a positive control. ABT737 was also tested in combination with Doxorubicin and Cisplatin. Cell viability was assessed using WST assay measuring mitochondrial activity; cell count was performed with the Casey® cell counter. Induction of apoptosis was determined using Life Cell Imaging where apoptotic cells were labeled with Cy5-labelled AnnexinV. During Life Cell Imaging ABT737 was also combined with Roscovitine, an MCL1 inhibitor. FACS analysis on cell cycle was performed with DAPI staining.
After comparing solitary chondrosarcomas to normal cartilage tissue, the anti-apoptotic MCL1 (logFc 0.6, p=0.02) and BAD (logFc=0.5, p=0.007) were overexpressed in chondrosarcoma. P-values were False Discovery Rate adjusted. A loss in cell number (IC50: 2 M) and viability (IC50: 20 M) after 72 hours incubation with ABT737 was achieved. 25µMABT737 in combination with 10µM Doxorubicin led to 85% reduction in cell viability in L869 and 65% reduction in other cultures. Life Cell Imaging showed a strong induction of apoptosis in cells treated with ABT737 alone (650%) over 72 hours of treatment. For Doxorubicin 10 M 560% increase in apoptotic cells was observed and for Roscovitine 10 M the total increase in apoptotic cells was 400%. Combining ABT737 with Roscovitine or Doxorubicin showed a total increase in apoptotic cell number of 430%. Combining ABT737 with doxorubicin showed no additional onset of apoptosis during the last 24 hours. By adding the BH3 mimic ABT737 to Doxorubicin treatment in chondrosarcoma cultures, an additional 20% decrease in cell viability can be obtained. However, by counting apoptotic cells, no such decrease could be observed, suggesting that the additional 20% is a cytostatic effect rather than a cytotoxic. Accordingly, preliminary data obtained with FACS analysis on cell cycle show a 10% increase of cells in G1 phase after ABT737 and Doxorubicin treatment, compared to ABT737 treatment alone.
In conclusion, BCL2 overexpression seems to play an important part in chemoresistance in chondrosarcoma, and co-treatment of ABT737 with Doxorubicin causes the vast majority of chondrosarcoma cells to enter apoptosis while the remaining cells enter cell cycle arrest.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C3.