ANALISIS LEVEL EKSPRESI PROTEIN ATM, ATR, DAN CASPASE-3 PADA OVARIAN CANCER STEM CELL CD133+ DARI LINI SEL SKOV3

• Main Author: Irsyad Ramadhan, Muhammad
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/86136
• Institution: Institut Teknologi Bandung

Epithelial ovarian cancer remains one of the leading causes of death among women globally. Approximately 60–80% of cases experience recurrence following initial treatment, leading to the development of chemoresistant tumors. Various studies have shown that a subpopulation of ovarian cancer stem cells (OCSC), which survive post-treatment, is involved in the development of these recurrent cancers. The isolation of OCSCs has so far relied on surface markers associated with stem cell properties, such as prominin-1 (CD133). However, the analysis of the expression of chemoresistance-related genes at the protein level in CD133+ OCSCs has not been extensively performed. It is known that chemoresistance mechanisms are commonly facilitated by DNA repair enhancement and self-renewal activities, thus, this study aimed to isolate CD133+ OCSCs and observe the expression of several proteins, including ATM, ATR, and Caspase-3 (CASP3). The research methodology began with the selection of CD133+ OCSCs from the SKOV3 cell line using Indirect MACS. Selection was carried out in two labeling stages followed by sorting through a magnetic column. This selection process yielded only 0.52-0.99% CD133+ OCSCs from the total cells that passed through. The proportion of OCSCs in the original population could not be precisely determined due to cell loss during the selection process. The ability of the original (heterogeneous) population and the two sorted SKOV3 subpopulations (CD133+ and CD133?) to initiate tumors was then observed through their ability to form spheroids. Initial culture condition showed that some cells in the CD133+ OCSC subpopulation were 3-4 times larger—giant cancer cells (GCC)—compared to the original population and non-OCSC CD133? cells. Asymmetric budding was likely occurred in these CD133+ GCCs. Furthermore, the diameter of CD133+ OCSC spheroids was significantly larger (p < 0.01) than that of non-OCSC CD133? spheroids from day 3 onward. The ability of the original population to form larger-diameter spheroids (p < 0.05) than CD133+ OCSC spheroids also indicated the necessity of interaction between OCSCs and non-OCSC subpopulations for optimal tumor initiation. Protein expression levels measured through Sandwich ELISA revealed that the CD133+ OCSC subpopulation expresses both ATM and CASP3 at significantly higher levels compared to the non-OCSC CD133? subpopulation (p < 0,01). The increased ATM expression is hypothesized to be triggered by sublethal activation of CASP3, which may induce spontaneous DNA double-strand breaks. The detection of DNA damage by ATM is further believed to prevent cell death during the cell cycle and to stimulate the secretion of pro-inflammatory proteins, suggesting that the elevated expression of ATM and CASP3 in CD133+ OCSCs may synergistically support tumor growth. Additionally, the expression level of ATR, which also plays a role in activating DNA repair checkpoints, did not differ significantly (p > 0.05) across all cell types, likely due to its crucial function in cell viability. Therefore, it can be concluded that the CD133+ OCSC subpopulation likely possesses higher tumorigenic and chemoresistant potential compared to the non-OCSC CD133? subpopulation, as indicated by their ability to form larger spheroids and the higher expression of ATM and CASP3 proteins.