Radiosensibilization of prostate cancer cells with targeted drugs

Uvod: Rak je bolezen, oziroma skupek bolezni, za katere je značilna nenadzorovana rast in delitev celic. Najpogostejši rak pri moških je rak prostate. Zdravljenje je lahko kirurško, obsevalno, hormonsko, kombinirano ali odloženo. Najpogostejše je obsevanje. Učinkovitost obsevanja se lahko izboljša z uporabo zdravil-radiosenzibilizatorjev. Namen: Namen diplomske naloge je bil ugotoviti, ali je zdravilo GnRHR-Gemicitabin (Gemcitabin vezan na peptid, ki se veže na receptor za gonadolinberin (GnRHR)) tarčno zdravilo, s katerim bi lahko zdravili rakave celice prostate in če lahko z njim radiosenzibiliziramo samo celice raka in ne ostalih celic. Metode dela: Izvedli smo test klonogenosti, ki se uporablja za določanje števila celic, ki so ohranile reproduktivno sposobnost. V poskusu smo uporabili dve celični liniji humanega raka prostate PC-3 ter DU145 in eno mišjo celično linijo raka debelega črevesa MC-38. Najprej smo z metodo kvantitativnega PCR v realnem času določili izražanja receptorja GnRHR v vseh treh celičnih linijah. Nato smo del vzorca vsake celične linije izpostavili različnim dozam sevanja, del pa kombinaciji Gemcitabina z obsevanjem ali GnRHR-Gemcitabina z obsevanjem. Po 14 dneh smo celice fiksirali, ter prešteli vidne kolonije. Rezultati: Pokazali smo, da so vse tri celične linije uporabljene v študiji občutljive na obsevanje, njihova radiosenzitivnost pa se poveča v kombinaciji z Gemcitabinom. Uporaba GnRHR-Gemcitabina pa je povečala radiosenzitivnost samo pri celičnih linijah PC-3 ter Du145, medtem ko je le ta pri celični liniji MC-38 ostala enaka samo obsevanim celicam. Pri mišji celični liniji MC-38 smo pokazali, da se GnRH receptor, na katerega se veže GnRHR-Gemcitabin, ne izraža, medtem ko se pri celičnih linijah PC-3 ter Du145 izraža, kar pojasni razlike v učinku GnRHR-Gemcitabina v kombinaciji z obsevanjem. Razprava in zaključek: Pri dodatku Gemcitabina se radiosenzibilitivnost vseh treh celičnih linij poveča, dodatek GnRHRGemcitabina pa poveča radiosenzibilnost celičnih linij PC-3 ter Du145, ne pa celične linije MC-38. S tem smo potrdili, da je GnRHR-Gemcitabin tarčno zdravilo ter radiosenzibilizator.Introduction: Cancer is a disease or a set of diseases characterized by uncontrolled growth and cell division. Prostate cancer is the most common cancer in men. Treatment can be surgical, irradiation, hormonal, combined or delayed. The most common is irradiation. The effectiveness of irradiation can be improved by the use of drugs called radiosensitizers. Purpose: The purpose of the diploma work was to determine whether GnRHRGemicitabin (Gemcitabine bound to a peptide binding to a gonadolinberin receptor (GnRHR)) is a targeted drug that can treat cancerous prostate cells and if it is possible to radiosensibilize only cancer cells and not healthy cells. Methods: We performed a clonogenic assay to determine the number of cells that maintained reproductive capacity. We used two cell lines of human prostate cancer PC-3 and DU145, and one murine cell line of colon cancer MC-38. We first determined the expression of GnRH receptor in all three cell lines with quantitative real time PCR. Then we either irradiated the cells lines or irradiate them in combination with Gemcitabine or GnRHR-Gemcitabine. After 14 days, the cells were fixed and stained and visible colonies counted. Results: We showed that all three cell lines were sensitive to irradiation, and their radiosensitivity increased in combination with Gemcitabine. The use of GnRHR-Gemcitabine increased radiosensitivity only in the PC-3 and Du145 cell lines, while it remained the same as for the irradiated cells in the MC-38 cell line. In the murine cell line MC-38 we showed that the GnRH receptor on which GnRHR-Gemcitabine is bound is not expressed, while in the cell lines PC-3 and Du145 it is expressed, which explains the differences in the effect of GnRHR-Gemcitabine in combination with irradiation. Discussion and conclusion: The addition of Gemcitabine increased the radiosensibility of all three cell lines, whereas the addition of GnRHR-Gemcitabine increased only the radiosensibility of PC-3 and Du145 cell lines, but not the MC-38 cell line. Thus, confirming that GnRHR-Gemcitabin is a targeted drug and a radiosenzitizor