Investigation into the level of Cellular Prion Protein (PrPc) in Glioblastoma Multiforme (GBM) Cells Treated with Alkylating Agents and Other Small Molecules

Abstract
Glioblastoma multiforme (GBM) is a malignant tumour of astrocytes in the brain which accounts for approximately one third of all tumours in the nervous system. Despite the availability of chemotherapy, the median survival rate for all patients is between 15 to 24 months. Temozolomide (TMZ) and carmustine (BCNU) are the most common chemotherapeutic agents used in combination with radiotherapy or surgery to treat GBM. The overexpression of PrPc in GBM has been reported and its involvement in the resistance to alkylating agents (TMZ and BCNU) was speculated. The exact role of PrPc in GBM, i.e. how it is affected by chemotherapeutic agents and small molecules, needs to be further investigated.
In this work, the effect of TMZ and BCNU on PrPc was evaluated using U87 glioblastoma cell line. The results showed that 24 h treatment with TMZ or BCNU followed by 48 h resting gave the highest level of G2/M cells (about 84%) when compared with untreated cells. The upregulations of p53 and p21 are believed to be the key cause for this increment. PRNP gene is also upregulated in TMZ or BCNU treated U87 cells for 24 h treatment followed by 48 h resting and this may contribute to the resistance these cells towards the treatments due to the cytoprotective effect of this protein despite less PrPc is detected at protein level. Some of these findings are in agreement while others are opposite to those previously reported.
The resistance to chemotherapy is considered as one of the main limitations for the effective treatment of cancer, especially for GBM. PRNP gene in U87 cell line was silenced to determine cell proliferation and chemoresistance. A decrease of 80% in PrPc level was obtained after 48 h of treatment with transfection reagents. Additionally, the level of the PrPc knockdown was retained 5 days post-knockdown. Silencing of the PRNP gene in glioblastoma cell line U87 reduces cell proliferation in a time-dependent manner. In addition, positive cells with proliferation marker ki67 were significantly decreased in knockdown cells after 5 days. This implies that PrPc contributes to the malignance of the cancer cells. It was also found that there was no significant reduction in cell proliferation in response to TMZ or BCNU in knockdown cells and p21 is upregulated in TMZ or BCNU knockdown cells. Therefore, silencing PRNP gene may impact the function of p21 which contributes to the resistance to alkylating agents TMZ and BCNU.
In an attempt to sensitise U87 cells to TMZ, the combination treatment of MG132 with TMZ did not improve the apoptosis rate and no accumulative effect was observed. High concentration of MG132 increased the level of PrPc in U87 cells after a short incubation time.
The level of PrPc was also evaluated in U87 cells treated with the compounds (FAPi) which inhibit the Fancony Anaemia (FA) pathway by reduction of FANCD2 using high content imaging by ImageXpress Micro. These compounds did not affect the level of PrPc in these cells with or without combination with TMZ or BCNU showing that these FAPi are target specific.
Several novel families of heteroaromatic compounds (30 compounds) previous shown to be cytotoxic to embryonic carcinoma cells (NT2) were screened on U87 cells and eight of them were found to be inhibit the proliferation of the cells. The compound 175 that belongs to the acridine family induced apoptosis for U87 cells. It also arrests U87 cells in G2/M and upregulate p53 and p21 proteins which are regulatory for cell cycle. That may open the door to examine more acridine compounds as anti-glioblastoma agents.