## 3 Results and Discussion
### RT-qPCR Results  
RT-qPCR was performed to analyze the expression levels of survivin, C-Myc, and Caspase 3 gene expression levels in U87 MG and T98G cells treated with etoposide, L-methioninease, and their combination. Survivin, also known as the baculoviral inhibitor of apoptosis repeat-containing 5 (BIRCS), is an anti-apoptotic protein that is highly expressed in cancer cells, including glioblastoma and contributes to their survival and resistance to cell death [36; 37]. It regulates cell cycle progression, prevents apoptosis, and causes chromosomal instability [38; 39]. Survivin plays an important role in apoptosis and cell division [40]. Survivin is an anti-apoptotic protein that is responsible for the inhibition of Caspase 3 and 7 [41; 42], and the downregulation of survivin expression is associated with cytotoxic conditions [43; 44; 45]. The overexpression of survivin in cancer cells is a suitable target for cancer treatment [46]. Because of its importance in the regulation of apoptosis, the alteration in mRNA expression was assessed in U87 MG and T98G cells subjected to a 24 h culture with L-methioninease and etoposide. The graph in Figure 23a clearly demonstrates a significant decrease in survivin's expression when treated with L-methioninease, etoposide, and the combined treatment. The combination of L-methioninease and etoposide almost completely inhibited survivin's expression. The decreased expression of Survivin mRNA following treatment suggests that these treatments successfully target the anti-apoptotic pathway, potentially leading to increased apoptosis and decreased cell survival. c-Myc is an oncogene that regulates cell proliferation and is frequently overexpressed in various human carcinomas, including glioblastoma [47; 48; 49]. The dysregulation of processes such as transcription, translation, or protein stability is often attributed to the overexpression of c-Myc in these cancers [50; 51]. A reduction in c-Myc expression can lead to apoptosis or the inhibition of cell growth [52]. In this study, it was observed that L-methioninease, etoposide, and its combined treatment resulted in the complete inhibition of c-Myc expression in U87 MG and T98G cells after 1 d of culture (Figure 23b). c-Myc is a key regulator of cell proliferation, and its downregulation suggests that this treatment affects the pathways involved in cell growth and division. The inhibition of c-Myc expression can lead to cell cycle arrest and reduced proliferation rates [53]. Based on these findings, the combined treatment of L-methioninease and etoposide appears to have a synergistic effect on decreasing the expression levels of c-Myc and survivin, potentially leading to decreased cell proliferation and enhanced apoptosis in U87 MG and T98G cells. This provides further evidence of the therapeutic potential of this combination in targeting the key molecular pathways involved in cancer cell survival and growth. These findings are consistent with the literature, which reported that L-methioninease could downregulate survivin expression. Similar effects have been observed with the use of methylselenocysteine in prostate cancer cells [54]. Caspase-3 is an important protein involved in the execution phase of apoptosis and activates and cleaves various cellular substrates, leading to characteristic apoptotic changes, such as DNA fragmentation, cytoskeletal reorganization, and the formation of apoptotic bodies [55; 56]. Evaluating the expression levels of Caspase-3 can provide insights into the apoptotic response and efficacy of apoptosis-inducing treatments. In this study, etoposide and L-methioninease induced the expression of Caspase-3. Furthermore, the combined treatment resulted in an increased expression compared to the individual treatments (Figure 23c). These findings are consistent with a recent study reporting that partially purified L-methioninease from Methylobacterium sp. is concurrent with tamoxifen, which increased Caspase-3 expression levels in breast cancer in athymic nude mice [57].