Abstracts Division 2

40. Extracellular cathepsin B inhibition in hepatocellular carcinoma leads to decreased tumor growth

H. van Mourik1,2, L. Stoffels1,2, A. Westheim1,2, D. Meesters1, M. Li1, M. Kramer3, T. Gevers3, J. Theys2, R. Shiri-Sverdlov1

1 Department of Genetics and Cell Biology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University, Maastricht, the Netherlands 
2 Department of Precision Medicine, School for Oncology & Reproduction (GROW), Maastricht University Medical Centre, Maastricht, the Netherlands 
3 Department of Gastroenterology and Hepatology, Maastricht University Medical Centre, Maastricht, the Netherlands 

Background
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Ample data points towards the involvement of cathepsins, lysosomal proteases, in many types of cancers. Cathepsins have various physiological roles, however, upon secretion into the extracellular space, they often take on pathological roles. Previously we determined that plasma levels of cathepsin (CTS)D and CTSS were significantly increased in HCC patients compared to healthy controls. In addition, we determined that in vitro inhibition of extracellular CTSB led to a decrease in liver cancer cell viability. However, data regarding the mechanism of action of CTSB in HCC are lacking. In the current study, we investigate the effect of CTSB inhibition in an in vivo HCC model.

Methods

HepG2 cells were injected into the chorioallantoic membrane (CAM) of fertilized White Leghorn eggs (chicken) on embryonic day (E)9. Eggs were divided into four groups: 1) vehicle (n=23), 2) paclitaxel (n=22), 3) extracellular CTSB inhibitor (n=24), 4) extracellular CTSB inhibitor combined with paclitaxel (combination therapy) (n=23). At E18 the embryos were sacrificed and tissues were collected. Tumors were weighed and angiogenesis was determined by counting the number of blood vessels in the tumor. Furthermore, immunohistochemical analyses were performed to study cell death and proliferation.

Results

Embryos treated with the extracellular CTSB inhibitor alone or with the combination therapy did not have decreased survival compared to the vehicle. In addition, embryos treated with the combination therapy showed decreased angiogenesis, tumor growth and proliferation in comparison to the vehicle. Both the extracellular CTSB inhibitor and the combination study demonstrated a reduced Ki67/TUNEL ratio compared to the vehicle, which indicates that there was more cell death than proliferation.

Conclusion

Extracellular cathepsin B inhibition in combination with paclitaxel leads to a decrease in angiogenesis, which then leads to increased cell death and reduced proliferation and thus reduced tumor growth.

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