Western blot method analysis of expression of proteins GAPDH, Akt and p53 in cells treated with N-sulfonylurea derivatives

Uvod : Tumorske bolesti, kao vodeći uzrok smrtnosti u svijetu, nameću potrebu za pronalaskom učinkovite antitumorske terapije. Većina antitumorskih lijekova nije ciljno specifična te napadaju i zdrave stanice. Novosintetizirani spojevi N-sulfoniluree koji su korišteni u ovom istraživanju pripadaju grupi antimetabolita koji postižu ciljnu specifičnost kroz interakciju s prirodnim metabolitima. Cilj istraživanja bio je odrediti moguće promjene ekspresije p53, Akt i GAPDH proteina u staničnim kulturama HeLa, CaCo-2, NCI-H358, K562 i Raji nakon tretmana spojevima D7-23 i R-191 N-sulfoniluree, ali i usporediti rezultate ekspresije proteina kod tretiranih i ne tretiranih stanica (kontrolna točka). Materijali i metode : Nakon 24-satnog tretmana, stanice su lizirane, a stanični proteini izolirani. Prisustvo analiziranih proteina p53, Akt i GAPDH određeno je Western blot metodom uz korištenje p53, Akt i GAPDH protutijela. Relativna kvantifikacija proteina na membrani napravljena je uz pomoć ImageJ programa. Rezultati : Western blot analize su pokazali promjene u ekspresiji p53, Akt i GAPDH proteina prije i nakon tretmana komponentama D7-23 i R-191 N-sulfonilurea. Značajna promjena u ekspresiji nakon tretmana s D7-23 i R-191 prisutna je kod proteina Akt u HeLa staničnoj liniji. Zaključak : Na temelju dobivenih rezultata možemo zaključiti da su testne komponente pokazale potencijalno antitumorsko djelovanje na Akt signalni put u HeLa staničnoj liniji.Introduction: Tumour diseases are a leading cause of death worldwide and there is a need to discover efficient form of tumour treatment. Most of anticancer drugs are not target-specific, thus affecting normal cells as well. Newly synthesized N-sulfonylureas derivatives that were used in this research belong to the group of antimetabolites that achieve specific effects through interaction with natural metabolites. Objectives: The aim of this study was to determine possible changes in the expression of the p53, Akt and GAPDH protein in HeLa, CaCo-2, NCI-H358, K562 and Raji cell lines after the treatment with test compounds D7-23 and R-191 N-sulfonylureas. The aim was also to compare expression of proteins in treated and nontreated cells (control point). Material and Methods: After 24 hours of treatment, cells were lysed and cellular proteins were isolated. Presence of p53, Akt and GAPDH proteins in total isolated cellular proteins was determined by Western blot method using p53, Akt and GAPDH antibodies. Relative quantification of the Akt protein obtained on the membrane was made with the ImageJ program. Results: Results of Western blot analysis showed change in expression level of p53, Akt and GAPDH proteins before and after the treatment with test compounds D7-23 and R-191 N-sulfonylureas. Significant change in expression level is presented in expression of Akt protein in HeLa cell line. Obtained results suggest that test compounds indicate potential antitumour activity. Key words : tumour diseases, N-sulfonylureas, compounds D7-23, R-191, p53, Akt, GAPDH, Western blot