Investigation of the cell death types on prion protein expression supressed multidrug resistant cell line, H69

Prion (PrP) is a highly conserved cell surface glycoprotein expressed in a wide range cell types and plays a critical role in many cellular events including cell signalling, differentiation and programmed cell death. Despite many significant works have been investigated the involvement of PrP in tumor biology, the mechanisms associated with biological activity of this protein in cancer remains largely unexplored. Recent findings suggested that PrP could promote metastasis and multidrug resistance (MDR). However, the exact mechanism underlying PrP-associated MDR in cancers have not been fully elucidated. In the present study, we focused two hypothetical questions: first, what is the possible effect of the knockdown of PrP by RNA interference to survival of drug-resistant cancer cells? The second point is the question of "which type of cell death progress can be seen after the siRNA transfection?" To clarify these questions, we therefore created an experimental design including knockdown of PrP by siRNA in a doxorubicin-resistant small cell lung cancer cell line, H69AR. The expression levels of PrP, CD44, Beclin-1 and Bax molecules in experimental groups, including the untreated group [Doxo (-); Group 1], the group were treated with doxorubicin at half maximal inhibitory concentration (IC50) [Doxo (+); Group 2], the group were transfected with siRNA targeting PrP [siRNA/Doxo (-); Group 3] and finally the group were transfected with siRNA plus treated with doxorubicin IC50 [siRNA/Doxo (+); Group 4], were evaluated by immunocytochemistry. Also, autophagy monitorized by monodansylcadaverine dye. Our immunocytochemical analyses indicated that PrP and Bax levels increased upon doxorubicin treatment, however the expression levels of Bax slightly decreased in the Groups 3 and 4 versus doxorubicin treated group (p> 0.05). When the CD44 expression levels were evaluated, non-critical differences between the groups was seen. This result suggest that CD44 expression in H69AR cells occurs PrP- and doxorubicin-independent manner. Interestingly, Beclin-1 expression levels were increased upon doxorubicin treatment (Group 2) (p< 0.05) and siRNA transfection (Group 3) as well as siRNA transfection + doxorubicin treatment (Group 4) (p< 0.05). Increased levels of autophagic vacuoles in these groups observed with monodansylcadaverine stainings could partially confirmed this result. Given this intriguing results we conclude that PrP has an important role in chemoresistance and knockdown of this protein induces a cell death mechanism, espacially autophagy rather than apoptosis in drug resistant H69AR cells. In conclusion, downregulation of PrP seems to be a new anti-cancer strategy when it combined with chemotherapeutics