title: Investigating cellular responses after inhibition of the thioredoxin system in lymphoma
Creators: name: Wang, Sicong
Publisher: name: Griffith University
Year: 2022
Subjects: subject: thioredoxin system
subject: Lymphoma
subject: haematological cancer

description: Lymphoma is a haematological cancer that develops in the lymphatic system. Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL) are the two main lymphoma subtypes. HL is commonly diagnosed in young people and in adults over the age of 55. NHL is a more aggressive subtype than HL, and it accounts for approximately 90% of all lymphoma cases. Despite the development of different chemotherapy regimens for lymphoma treatment, 40-50% of lymphoma patients fail to achieve long-term survival rates because some patients do not respond to chemotherapy or they become resistant to the treatment. Additionally, lymphoma patients also suffer from the side effects of chemotherapy. Therefore, improved chemotherapies are desired, including the identification and investigation of new targets. In this study, three lymphoma cell lines (KMH2, SUDHL2, and SUDHL4) representing three different subtypes were used. This thesis focused on evaluating the efficacy of inhibiting the thioredoxin system in lymphoma cells using [Au(d2pype)2]Cl, assessing the cellular response after inhibiting TrxR, and evaluating its potential role as part of a combination treatment strategy in lymphoma. Antioxidant systems, especially the thioredoxin (Trx) system, are known to provide cancer cells with a survival advantage, with thioredoxin reductase (TrxR) recently suggested as a potential anticancer target. In this project, overexpression of antioxidant system genes, including the Trx system, was observed in lymphoma patient samples. Furthermore, [Au(d2pype)2]Cl could significantly inhibit cell proliferation in three lymphoma cell lines by targeting TrxR as a specific TrxR inhibitor. Since the glutathione (GSH) system compensates for some functions of the Trx system, the GSH system was investigated in the lymphoma cell lines after the [Au(d2pype)2]Cl treatment. It was found that the activity of the Gpx selenoprotein was also inhibited by [Au(d2pype)2]Cl. In addition, the NHL cell lines were shown to be more sensitive to [Au(d2pype)]Cl than the HL cell line. Further investigation indicated that the KMH2 cell line (HL) had higher GSH levels than SUDHL2 and SUDHL4 cell lines (NHL), which may be a reason why KMH2 cells were not as sensitive to [Au(d2pype)2]Cl treatment. Although the gene expression of the Trx and GSH systems was altered after the [Au(d2pype)2]Cl treatment, it was still unknown whether other signaling pathways might be affected. Hence, RNA-seq was performed to investigate altered gene expression after the inhibition of TrxR using [Au(d2pype)2]Cl. Bioinformatic analysis suggested that the Nrf-2-mediated oxidative stress response was the most affected pathway after TrxR was inhibited using [Au(d2pype)2]Cl in lymphoma cells. Furthermore, cytokine signaling-related genes were strongly linked to the antioxidant genes, as assessed by STRING analysis. Follow-up experiments using RT-qPCR and western blotting analysis confirmed that the IL6/JAK/STAT3 pathway was downregulated after the [Au(d2pype)2]Cl treatment, leading to the downregulation of c-Myc expression in lymphoma cells. The B-cell receptor (BCR) signaling pathway is critical in lymphoma survival and development, and Bruton’s tyrosine kinase (BTK) is a key component of the BCR signaling pathway. In this project, the overexpression of BTK mRNA and protein was confirmed in lymphoma patients' samples from the TCGA and human protein atlas public databases. It was found that the mRNA expression levels of BTK and NF-κB (a downstream protein of the BCR pathway) were significantly correlated with the expression levels of TrxR in patient samples from the TCGA database. Further investigation indicated that TrxR inhibition using either specific siRNA or [Au(d2pype)2]Cl resulted in a decreased expression of both BTK mRNA and protein levels. In addition, the combination treatment performed by co-targeting TrxR and BTK using [Au(d2pype)2]Cl and ibrutinib showed a synergistic effect on inhibiting lymphoma cell proliferation. Meanwhile, cytoplasmic accumulation of p65 was observed after the combination treatment in DLBCL cells, indicating that the NF-κB pathway was inactive. The combination treatment also stimulated apoptosis in all three lymphoma cell lines. Moreover, the ferroptosis pathway was also activated after the combination treatment in the SUDHL4 cell line. Over several decades, metal-based compounds such as cisplatin have shown anticancer activity in the clinic. However, side effects or resistance to cisplatin still occur in treated patients. Therefore, alternative metal-based compounds need to be identified and studied to find potential drugs with fewer side effects, including drugs that will be effective in overcoming drug resistance. This study assessed 12 newly synthesized indole-metal-based compounds, including iron-based, cobalt-based, and gold-based. It was found that the indole-gold-based compounds showed the best anti-lymphoma activity via inhibiting selenoproteins TrxR and Gpx, compared to the iron-based and cobalt-based compounds. Further investigation revealed that two of the gold-based compounds, Inac-Au2, and Inac-Au3, decreased the GSH level in the DLBCL cell line and induced the expression of ferroptosis-related genes. In addition, lipid peroxidation was significantly increased, indicating the activation of ferroptosis in the DLBCL cell line after the Inac-Au2 and Inac-Au3 treatment. These results showed the potential of Inac-Au2 and Inac-Au3 as TrxR and Gpx inhibitors in lymphoma treatment, warranting further assessment in animal models. In conclusion, this thesis demonstrates the effectiveness of [Au(d2pype)2]Cl as a TrxR inhibitor in lymphoma and the validation of the Trx system as a potential target in malignant lymphoma treatment. Understanding the interaction between the Trx system and the BCR signaling pathway may provide a valid co-therapeutic strategy for lymphoma treatment.