Doxorubicin enhances protein ubiquitination and exerts synergic cytotoxic effect in combination with Bortezomib

Neuroblastoma (NB) is a neoplasm that originates in the neural crest and is the most common extracranial solid tumor in children. Advanced NB is still frequently fatal despite aggressive management. Doxorubicin, an anti-tumour agent of the anthracycline family widely used in the treatment of solid tumors, is commonly considered to exert its anti-tumor activity leading to the apoptosis of cancer cells through the damage of DNA but our recent results evidenced that Doxorubicin also induces Heat Shock Proteins (HSP) expression, normally triggered by protein damage, and that HSP inhibition or their silencing enhance Doxorubicin apoptotic effect in Neuroblastoma (NB) cells. A combination of Doxorubicin and Bortezomib, a proteasome inhibitor, has been proposed in the treatment of a variety of tumour. In different NB cell lines it has been demonstrated a cooperation of the two drugs. Those data suggest that Doxorubicin may exert its anti-tumor effect also through a mechanism triggered by protein damage. AIMS Aim of the present work, is to explore the effect of Doxorubicin on protein modification. RESULTS The neuroblastoma cell line SJNKP was treated with different concentration of Doxorubicin and Bortezomib at different times. We observed that after Doxorubicin treatment HSP27 decreased its solubility possibly due to its binding to denatured proteins. We also observed a Doxorubicin dose dependent protein over-ubiquitination. Following the identification of the ubiquitinated proteins and their ubiquitination sites by mass spectrometry, we noticed a remarkable similarity between the ubiquitinated protein patterns induced by Doxorubicin or Bortezomib treatments. We did not detect any inhibitory effect of Doxorubicin on the proteasome activity indicating that Doxorubicin induces protein over-ubiquitination because of protein damage. We also confirm a synergic effect of Doxorubicin and Bortezomib on cell death. CONCLUSIONS Our results suggest that Doxorubicin may exert its cytotoxic effect through cellular protein damage and their accumulation. This finding may help the comprehension of the cooperative effects of Doxorubicin and Bortezomib suggesting a rationale for the design of novel therapeutic associations