Proučevanje antiproliferativnega delovanja zaviralcev proteina toplotnega šoka 90 na celičnih modelih raka dojk in Ewingovega sarkoma

Trunkelj, Natalija (avtor), Tomašič, Tihomir (mentor), Urbančič, Dunja (komentor)

V razvitem svetu rak predstavlja drugi najpogostejši vzrok smrti. Zaradi zgodnjega odkrivanja in številnih terapevtskih možnosti se umrljivost za rakom v zadnjih letih zmanjšuje, a še vedno ostaja del malignih obolenj neozdravljiv ali razvije rezistenco na terapijo. Rakave celice povečano proizvajajo proteine toplotnega šoka (Hsp), ki zato predstavljajo dobro tarčo pri zdravljenju raka. V sklopu magistrske naloge smo proučevali vpliv zaviralcev Hsp90 na celice raka dojke in Ewingovega sarkoma. Na celičnem modelu raka dojk MCF-7 smo testirali dva zaviralca C-končne domene Hsp90 (1 in 2) ter selektivni zaviralec N-končne domene Hsp90β, spojino 3. Na celicah Ewingovega sarkoma SK-N-MC smo vrednotili in vitro tri različne zaviralce Hsp90 (1, 2 in 4), od katerih se vsi vežejo na C-končno domeno Hsp90, eden izmed njih (spojina 4) pa zavira še N-končno domeno topoizomeraze IIⱺ. Celice smo izpostavili spojinam različnih koncentracij, ki smo jih določili glede na rezultate predhodnih presejalnih testov proliferacije. Na celicah MCF-7 smo testirali spojine v naslednjih koncentracijah: spojino 1 v koncentracijah 0,7 µM; 1,5 µM in 7,0 µM, spojino 2 v koncentraciji 7,0 µM in spojino 3 v koncentracijah 7,0 µM in 37,5 µM. Na celičnih modelih SK-N-MC smo testirali spojine v naslednjih koncentracijah: spojino 1 v koncentracijah 0,7 µM; 1,5 µM; 7,0 µM; 37,5 µM in 70 µM, spojino 2 v koncentraciji 7,0 µM, 37,5 µM in 70 µM in spojino 4 v koncentracijah 0,7 µM; 1,5 µM; 7,0 µM; 37,5 µM. Izvedli smo primerjavo učinkovitosti posameznih spojin na različne tipe rakavih celic. S kombinacijo fluorescenčno označenega aneksina V in barvila SytoxBlue smo s pretočno citometrijo merili obseg apoptoze pri izbranih celicah, z uporabo propidijevega jodida pa preverili zastoj celic v celičnem ciklu. Pri vrednotenju spojin smo uporabili tri časovne točke: 24-urno, 48-urno in 72-urno. Rezultati so pokazali, da vsi proučevani zaviralci Hsp90 učinkovito izzovejo apoptozo tako pri celicah MCF-7 kot tudi pri SK-N-MC. Spojine 1, 2 in 3 po 24-urnem tretmaju povzročijo zastoj celic MCF-7 v fazi G2/M celičnega cikla. Na celični liniji SK-N-MC pa tudi opazimo zastoj v fazi G2/M po 24 urah po tretiranju s spojino 2. Spojina, ki deluje kombinirano tako na C-končno domeno Hsp90 kot na N-končno domeno topoizomeraze IIⱺ (spojina 4), v večji meri povzroči apoptozo celic v primerjavi s selektivnimi zaviralci C-končne domene na celicah SK-N-MC. Spojina 1 pa je najučinkovitejša zoper celice raka dojke. V prihodnosti bi bilo zato smiselno testirati in vitro izbrane zaviralce Hsp90 v kombinaciji z drugimi zaviralci signalnih poti pri raku in vrednotiti sinergistične učinke. Na ta način bi lahko uporabili nižje koncentracije zaviralcev Hsp90 in s tem povzročili manj neželenih učinkov.

• Jezik: Slovenski jezik
• Ključne besede: Hsp90, rak dojke, Ewingov sarkom, apoptoza, celični cikel, zaviralci Hsp90
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-158898
• Datum objave v RUL: 22.06.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Evaluation of antiproliferative activity of heat shock protein 90 inhibitors in breast cancer and Ewing sarcoma cell lines

Izvleček

Cancer ranks as the second leading cause of death in developed countries. Due to advances in early detection and therapeutic options, the cancer mortality rate has been decreasing in recent years, but some malignant diseases remain incurable or develop resistance. Cancer cells overproduce heat shock proteins (Hsps), which makes them a promising target for cancer treatment. As part of a Master's thesis, we investigated the effect of Hsp90 inhibitors on breast cancer cells and Ewing's sarcoma cells. We tested two different inhibitors of the C-terminal domain of Hsp90 (1, 2) and a selective inhibitor of the Hsp90β N-terminal domain (compound 3), on a breast cancer cell model MCF-7. We also tested three different Hsp90 inhibitors (1, 2, and 4) on Ewing's sarcoma cells – SK-N-MC. They all bind to the C-terminal domain of Hsp90, with one of them (compound 4) also inhibiting the N-terminal domain of topoisomerase IIⱺ. Cells were exposed to compounds at different concentrations, which were determined based on the results of previous proliferation screening assays. Compounds were tested on MCF-7 cells at the following concentrations: compound 1 at 0.7 µM; 1.5 µM and 7.0 µM, compound 2 at 7.0 µM, and compound 3 at 7.0 µM and 37.5 µM. On SK-N-MC cells, we tested compounds at the following concentrations: compound 1 at 0.7 µM; 1.5 µM; 7.0 µM; 37.5 µM; and 70 µM, compound 2 at 7.0 µM; 37.5 µM; and 70 µM, and compound 4 at 0.7 µM; 1.5 µM; 7.0 µM; and 37.5 µM. We compared the efficacy of each compound on specific cells. Apoptosis was measured in selected cells by flow cytometry using a combination of fluorescently labelled annexin V and SytoxBlue dye. Cell cycle arrest was measured using propidium iodide. The results were obtained at three time points: 24 hours, 48 hours, and 72 hours. Results showed that all investigated Hsp90 inhibitors were effective in induction of apoptosis in both MCF-7 and SK-N-MC cells. The compounds 1, 2 and 3 triggered cell cycle arrest in the G2/M phase after 24 hours of treatment. In the cell line SK-N-MC, we also observed cell cycle arrest in the G2/M phase after 24 hours of treatment with compound 2. A compound acting on both the C-terminal domain of Hsp90 and the N-terminal domain of topoisomerase IIⱺ (compound 4) induced apoptosis on SK-N-MC cells to a greater extent than selective inhibitors of the C-terminal domain. Compound 1 proved to be the most effective against breast cancer cells. Based on our results, future studies could address the in vitro evaluation of selected Hsp90 inhibitors in combination with other inhibitors of different signaling pathways in cancer to investigate synergistic effects. This approach would allow lower concentrations of Hsp90 inhibitors to be used, potentially reducing the associated side effects.

• Ključne besede: Hsp90, breast cancer, Ewing sarcoma, apoptosis, cell cycle, Hsp90 inhibitors