Isobaric Tags for Relative and Absolute Quantitation-Based Quantitative Proteomic Analysis of X-Linked Inhibitor of Apoptosis and H2AX in Etoposide-Induced Renal Cell Carcinoma Apoptosis
Renal cell carcinoma (RCC) is one of the most common malignant urological tumors, accounting for approximately 2% of all cancer cases worldwide. The 5-year survival rate of patients with metastatic RCC is less than 10%, largely due to the resistance of this malignancy to chemotherapy and radiotherapy. RCC exhibits a high apoptosis threshold, which is influenced by various factors, including the overexpression of X-linked inhibitor of apoptosis (XIAP). XIAP is a critical anti-apoptotic protein that inhibits caspases, thereby preventing cell death. In RCC, XIAP expression is significantly higher than in autologous normal kidneys, and its overexpression is predictive of poor prognosis. This study aimed to investigate the regulatory mechanism of XIAP in etoposide-induced apoptosis in RCC cells, focusing on its interaction with the histone variant H2AX.
The study utilized two Caki-1 cell lines with high or low XIAP expression, established through RNA interference (RNAi) technology. The differentially expressed proteins in these cell lines were globally analyzed using an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics approach. This method identified 255, 375, 362, and 5 differentially expressed proteins after 0, 0.5, 3, and 12 hours of etoposide stimulation, respectively. These proteins were involved in numerous biological processes, with significant alterations observed in the expression of histone proteins, including H1.4, H2AX, H3.1, H3.2, and H3.3. Notably, XIAP silencing was accompanied by the marked downregulation of H2AX. Protein-protein interactions were further confirmed through immunofluorescence and Western blot analyses.
The results suggested that XIAP plays a crucial role as a cell signal regulator, influencing the expression of DNA repair-related proteins such as H2AX and thereby affecting the DNA repair process. This regulatory function of XIAP may be decisive in determining the sensitivity of RCC cells to apoptosis induction in response to chemotherapeutic agents. The findings provide new insights into the mechanisms underlying the resistance of RCC to apoptosis and highlight the potential of XIAP as a therapeutic target.
Introduction
RCC is highly resistant to chemotherapy due to its high apoptosis threshold, which is influenced by the overexpression of XIAP. XIAP is a member of the inhibitor of apoptosis (IAP) family and is characterized by its ability to inhibit caspases, thereby preventing cell death. In RCC, XIAP expression is significantly higher than in autologous normal kidneys, and its overexpression is associated with poor prognosis. Previous research has shown that RCC cells with high XIAP expression are resistant to apoptosis stimulation, while those with low XIAP expression are sensitive to apoptotic signals. XIAP contains three Baculovirus IAP Repeats (BIR) domains and a RING domain, which confer E3 ubiquitin ligase activity, promoting the degradation of target proteins through ubiquitination.
Recent advancements in technology have expanded our understanding of XIAP’s functions beyond caspase inhibition. XIAP is involved in various signal transduction pathways, including the TGF-Beta Activated Kinase 1 Binding Protein 1 (TAB1)-TGF-Beta-Activated Kinase 1 (TAK1)-Jun Proto-Oncogene 1 (JNK1) pathway, and may also participate in mammary development and T-cell maturation. Upon drug-induced apoptosis, XIAP translocates from the cytosol to the nucleus, although its role in the nucleus remains unclear. Given that RCC cells are resistant to radiation-induced apoptosis, which causes DNA damage in the nucleus, this study focused on the effect of nuclear XIAP on apoptosis induced by DNA damage.
The histone variant H2AX plays a critical role in the DNA damage response. Upon DNA damage, H2AX is phosphorylated on Ser139 by PI3K family members, including ataxia telangiectasia-mutated gene (ATM), ATM and Rad-3 related (ATR), and DNA-dependent protein kinase (DNA-PK), generating γ-H2AX. This phosphorylation facilitates the recruitment of signaling and repair proteins to sites of DNA double-strand breaks. The phosphorylation of tyrosine 142 in H2AX prevents the recruitment of the repair complex and promotes the binding of pre-apoptotic factors, such as JNK1, thereby directly promoting cell apoptosis. Although XIAP and H2AX are both implicated in apoptosis, no study has explored the interaction between these two proteins.
Etoposide, a topoisomerase II inhibitor, is widely used to treat various human cancers. It induces DNA strand breaks and promotes tumor apoptosis in a dose-dependent manner. This study combined RNAi technology with iTRAQ-based quantitative proteomics to globally profile the function of XIAP in etoposide-induced apoptosis of RCC cells. The study aimed to identify the differentially expressed proteins in XIAP knockdown cells compared to the original Caki-1 cells following etoposide treatment, with a focus on the role of XIAP in regulating DNA repair mechanisms through H2AX.
Methods
Caki-1 cells were cultured and treated with etoposide to induce apoptosis. XIAP expression was knocked down using RNAi technology, and the efficiency of XIAP knockdown was confirmed through Western blot analysis. The proteomes of XIAP knockdown cells and original Caki-1 cells were compared using iTRAQ-based quantitative proteomics. Protein samples were labeled with iTRAQ isobaric tags and analyzed through liquid chromatography tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins were identified and quantified using the Mascot search engine. Protein-protein interactions were assessed through immunofluorescence and Western blot analyses.
Results
The study successfully established XIAP knockdown in Caki-1 cells, as confirmed by Western blot analysis. The apoptosis rates of XIAP knockdown cells and original Caki-1 cells were assessed using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining and flow cytometry. The results showed that the total apoptosis rate of XIAP knockdown cells was 4.2-fold higher than that of the original Caki-1 cells after 24 hours of etoposide treatment.
iTRAQ-based proteomic analysis identified 1783 non-redundant differentially expressed proteins in the two cell lines. Applying a fold-change cut-off of ≥1.3 or ≤0.77, the study identified 255, 375, 362, and 5 significantly altered proteins at 0, 0.5, 3, and 12 hours of etoposide treatment, respectively. Gene ontology (GO) enrichment analysis revealed that these differentially expressed proteins were involved in various biological processes, including DNA repair and damage response.
Among the differentially expressed proteins, the study focused on histone variants, given their role in DNA damage and repair. The expression levels of five histone proteins (H1.4, H2AX, H3.1, H3.2, and H3.3) were significantly altered by etoposide treatment. Notably, H2AX was downregulated in Caki-1 cells but not in XIAP knockdown cells. Western blot analysis confirmed the downregulation of H2AX in Caki-1 cells, consistent with the iTRAQ results.
Immunofluorescence analysis revealed that XIAP and H2AX did not colocalize in the nucleus or cytoplasm of Caki-1 cells, suggesting that XIAP does not directly interact with H2AX via E3 ubiquitin ligase activity. Instead, XIAP may indirectly regulate H2AX through other signaling pathways.
Discussion
The study demonstrated that XIAP plays a crucial role in regulating apoptosis in RCC cells through multiple pathways, including DNA damage and repair mechanisms. XIAP knockdown sensitized Caki-1 cells to etoposide-induced apoptosis, as evidenced by the increased apoptosis rate in XIAP knockdown cells compared to the original Caki-1 cells. The iTRAQ-based proteomic analysis revealed that XIAP influences the expression of numerous proteins involved in various biological processes, with a particular focus on histone variants.
The downregulation of H2AX in Caki-1 cells, but not in XIAP knockdown cells, suggests that XIAP may regulate H2AX expression. Although XIAP and H2AX did not colocalize, indicating no direct interaction, XIAP may indirectly influence H2AX through other signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway. The study’s findings provide new insights into the mechanisms underlying the resistance of RCC to apoptosis and highlight the potential of XIAP as a therapeutic target.
Conclusions
The study concluded that XIAP mediates apoptosis in RCC cells through multiple pathways, including the regulation of DNA damage and repair mechanisms via H2AX. XIAP knockdown sensitized RCC cells to etoposide-induced apoptosis, suggesting that high XIAP expression contributes to the resistance of RCC cells to apoptosis. The findings provide a new direction for studying the radiation resistance of RCC and the potential function of XIAP in regulating DNA repair mechanisms. Further research is needed to elucidate the precise mechanisms by which XIAP influences H2AX and other DNA repair-related proteins.
doi.org/10.1097/CM9.0000000000000553
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