Testing the inhibition of modified liver type 6-phosphofructo-1-kinaze in metastatic cancer cells with selected compounds.

Posttranslacijska modifikacija 6-fosfofrukto-1-kinaze (PFK-1) pri rakastih celicah je ključna za nastanek dereguliranega metabolnega pretoka preko glikolize, zaradi katerega prihaja do viška laktata v medceličnem prostoru tumorjev. V humanih celicah so izražene tri različne izooblike encima, in sicer mišična (PFK-M), trombocitna (PFK-P) in jetrna (PFK-L) oblika. V predhodnih raziskavah na Kemijskem inštitutu so s pomočjo super računalnika odkrili šestnajstih nizko molekularnih spojin (inhibitorji druge generacije), ki naj bi se vezale na ATP-vezavno mesto encima PFK-L. Te spojine smo testirali na treh različnih metastaznih tumorigenih celičnih linijah, Caco-2, Colo-829 in MDA-MB-231, da bi zmanjšali tvorbo laktata. Največjo inhibicijo pri tvorbi laktata smo zaznali pri inhibitorjih številka 23, 29, 30, 31 in 32, ki smo jih nato uporabili še v nadaljnjih raziskavah. Ugotovili smo, da je njihova učinkovitost odvisna od dodane koncentracije. Izbrane inhibitorje smo nato dodajali vsakih 24 ur v nizkih koncentracijah (10–30 μM). Zaporedno dodajanje je bilo zelo učinkovito, saj nam je že pri najnižji koncentraciji dodanega inhibitorja uspelo znižati nadaljnjo tvorbo laktata. Zaradi tkivno specifičnih pojavljanj izooblik smo testirali tudi kombinacije inhibitorjev prve (vežejo se na izoobliki PFK-M in PFK-P) in druge generacije. Nižjo tvorbo laktata v primerjavi s kontrolo smo opazili že ob dodanih nizkih koncentracijah inhibitorja (20 μM). Inhibitorji tudi ne kažejo zaviralnega vpliva na rast celic (citostatičen učinka) in negativnega vpliva na preživetje celic (citotoksični učinek). Na podlagi rezultatov sklepamo, da smo odkrili obetavne molekule, ki bi jih lahko v povezavi z že znanimi terapijami v prihodnosti uporabili za preprečevanje širjenja raka.Post-translational modification of the 6-phosphofructo-1-kinase (PFK-1) in cancers results in the formation of highly-active enzyme that might be the pivotal factor of deregulated glycolytic flux and consequently lactate generation and excretion. Human cells express three different PFK1 isozymes: muscle type (PFK-M), platelet type (PFK-P), and liver type (PFK-L). At the National Institute of Chemistry in Ljubljana, sixteen low-molecular compounds were virtually selected by a supercomputer that might bind to the to the ATP-binding of the 3-D crystal structure of human PFK-L enzyme. My goal was to test the selected compounds for preventing lactate generation by inhibiting PFK-L activity in three selected tumorigenic cell lines. Preliminary tests revealed the strongest inhibition of lactate formation by inhibitors designated 23, 29, 30, 31, and 32. By more detailed dose dependent experiments, the efficiencies of individual inhibitors in specific tumorigenic cell lines were estimated. The obtained results showed different reduction of lactate formation by selected inhibitors at individual cell lines. Because the reductions in the strengths of inhibitions were occasionally observed during the prolonged 90 hours incubation, sequential addition of the inhibitors has been tested. By repeated addition of inhibitors at low (10-30 µM) concentrations at 24 hours intervals, lactate generation was largely prevented in all tested tumorigenic cell lines. Due to tissue-specific occurrence of isozymes, we also tested a combination of first- and second-generation inhibitors. Lower concentrations of the lactate were recorded already at the lowest concentrations of inhibitors (20 µM). Based on these results, it can be concluded that discovered small-molecule-inhibitors can decrease the deregulated glycolytic flux the cancer cells and effectively prevent lactate generation. The selected inhibitors might in combination with other therapeutic methods help to prevent the growth of different tumors