Biološko vrednotenje himernih razgrajevalcev človeškega galektina-8 na celičnih linijah MDA-MB-231 in HUVEC

Durnik, Rebeka (author), Gobec, Martina (mentor), Smrdel, Lara (comentor)

Galektin-8 je protein, ki je močno izražen v rezistentnih vrstah raka. Njegova visoka izraženost korelira s slabšo prognozo bolezni ter procesoma angiogeneze ter zasevanja. Ena izmed najbolj rezistentnih vrst raka dojke je trojno negativni rak dojke, za katerega tarčno zdravljenje še ne obstaja. Himerni razgrajevalci proteinov (angl. Proteolysis Targeting Chimeras – PROTAC) so v zadnjih letih postali obetavna alternativa konvencionalnim zdravilom. Sestavljeni so iz dveh strukturnih ligandov. Eden izmed ligandov veže E3 ligazo, drugi pa tarčni protein, ki je v našem primeru galektin-8. To vodi v označevanje tarčnega proteina z ubikvitinom, kar je signal, da ga razgradi več-katalitični encim proteasom. Naša domneva je bila, da bodo molekule PROTAC, ki so jih sintetizirali na Katedri za farmacevtsko kemijo (UL FFA), razgradile galektin-8 na celičnih linijah MDA-MB-231 in HUVEC ter posredno vplivale na proces angiogeneze. V magistrski nalogi smo najprej izvedli test metabolične aktivnosti na obeh celičnih linijah in ugotovili, da nobena izmed spojin opazno ne zniža metabolne aktivnosti, s čimer smo posredno sklepali, da pri danih pogojih spojine niso citotoksične. Nato smo s sistemom Operetta preverili, če molekule preidejo v notranjost celice. Ugotovili smo, da v notranjost prehajajo le spojine, ki so bile v obliki estra, torej so kot ligand za galektin-8 imele spojino AEP11. Pri spojinah, ki so prešle v notranjost celice, smo s prenosom western preverili ali povzročijo razgradnjo galektina-8 in galektina-3. Razgradnjo obeh proteinov smo zaznali le pri spojini AEP70 in le na celični liniji MDA-MB-231. V nadaljevanju smo ovrednotili ali spojine vplivajo na izločanje VEGF in IL-8, ki ju povezujemo s procesom angiogeneze. Koncentracija VEGF se je pri vseh spojinah povišala, medtem ko se je koncentracija IL-8 znižala pri spojinah AEP65 in AEP100, a razlike niso bile statistično značilne. Za konec smo na celični liniji HUVEC pogledali še vpliv spojin na sam proces angiogeneze in ugotovili, da ga spojine ne zavrejo. Če povzamemo, smo pokazali, da le ena izmed spojin (AEP70) razgradi galektin-8, vendar pa ta spojina ne spremeni nivoja VEGF in IL-8 ter ne zavre procesa angiogeneze.

• Language: Slovenian
• Keywords: trojno negativni rak dojke, galektin-8, molekule PROTAC, proliferacija, angiogeneza, zasevanje
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2026
• PID: 20.500.12556/RUL-187504
• Publication date in RUL: 11.09.2026

Secondary language

• Language: English
• Title: Biological evaluation of proteolysis-targeting chimeras for human galectin-8 on MDA-MB-231 and HUVEC cell lines

Abstract

Galectin-8 is a protein highly expressed in resistant types of cancer. Its high expression correlates with poorer prognosis and enhanced processes of angiogenesis and metastasis. Triple-negative breast cancer is one of the most resistant forms of breast cancer, and no targeted therapy currently exists for it. In recent years, Proteolysis Targeting Chimeras (PROTACs) have emerged as a promising alternative to conventional drugs. They consist of two structural ligands: one binds to an E3 ligase, while the other binds to the target protein—in our case, galectin-8. This leads to the ubiquitination of the target protein, signaling its degradation by the multicatalytic proteasome enzyme. We hypothesized that PROTAC molecules synthesized at the Department of Pharmaceutical Chemistry (UL FFA) would degrade galectin-8 in MDA-MB-231 and HUVEC cell lines and indirectly influence the process of angiogenesis. In this master's thesis, we first performed a metabolic activity assay on both cell lines and found that none of the compounds significantly reduced metabolic activity, leading us to conclude that the compounds were not cytotoxic under the tested conditions. We then used the Operetta system to determine whether the molecules are cell permeable. We found that only compounds utilizing AEP11 as the ligand for galectin-8, which were in the form of an ester, were able to enter the cells. For the compounds that entered the cell, we used western blotting to determine whether they induce the degradation of galectin-8 and galectin-3. Degradation of both proteins was observed only with compound AEP70 and only in the MDA-MB-231 cell line. Next, we evaluated whether the compounds affect the secretion of VEGF and IL-8, which are associated with angiogenesis. VEGF levels increased with all compounds, whereas IL-8 levels decreased with compounds AEP65 and AEP100, however, the results were not statistically significant. Finally, we examined the effect of the compounds on the angiogenesis process using the HUVEC cell line and found that they do not inhibit it. To summarize, we demonstrated that only one of the compounds (AEP70) degrades galectin-8; however, this compound fails to significantly modulate VEGF and IL-8 levels and does not inhibit angiogenesis.

• Keywords: triple negative breast cancer, galectin-8, PROTAC molecules, proliferation, angiogenesis, metastasis