Role of ROS/RNS induced by anticancer treatments in the cancer resistance

Acquired drug resistance is a major cause of compromised chemotherapy outcomes, leading to cancer cell proliferation, tumor progression, and disease recurrence. Most chemotherapeutic agents induce the release of reactive oxygen species (ROS), nitric oxide (NO), and reactive nitrogen species (RNS) to exert a cytotoxic effect and stop tumor growth. However, activation of antioxidant systems is the strategy of cancer cells to lower ROS/RNS. Indeed, at low levels they are cytoprotective, acting as intracellular messengers that activate molecular signaling pathways involved in cell survival, proliferation, epithelial-to-mesenchymal transition (EMT), and metastasis. Photodynamic therapy (PDT) is a minimally invasive treatment in which the cancer cell is killed by oxidative stress. It can be used as a model to exclusively study the effect of ROS and NO/RNS on tumor progression. To this end, repeated suboptimal PDT treatments could mimic what happens during chronic chemotherapy in terms of ROS/RNS release. Previous data from my research group have shown that a human prostate cancer cell line with high metastatic potential (PC3 prostate cancer cells) develops a more aggressive population leading to tumor progression under such PDT conditions. These effect were attributed to low NO levels, which play a cytoprotective role as antioxidants. Furthermore, the data reported in this dissertation show that the release of low NO levels causes a bystander effect on neighboring PC3 tumor cells not directly treated with PDT. NO mainly works through the S-nitrosylation of protein Cys residues, resulting in different effects on protein function, depending on the specific primary function of each protein. To study this signal transduction, we focused on the expression of GSNOR, a denitrosylase that can revert the effect of S-nytrosilation, resulting responsible for the activation of survival-promoting genes such as NF-κB. The inhibition of NO release by the 1400W inhibitor of the NO inducible synthase (iNOS), by lowering protein S-nytrosilation rate, showed a decrease in the expression of GSNOR, suggesting a possible key role of GSNOR in tumor progression and resistance development. To investigate the role of ROS and NO/RNS induced by chronic drug treatment in the development of resistance, we repeatedly treated BxPC-3 pancreatic ductal adenocarcinoma (PDAC) cells with a low dose of gemcitabine (GEM), a first-line chemotherapeutic agent for PDAC therapy. We then characterized the BxPC-3/GEM Res cell line in terms of changes in cell morphology, aggressiveness and faster cell growth. In addition, we investigated the molecular signaling pathways and found progressive upregulation of pro-survival genes such as K-RAS, NF-κB, YY1, and Snail during resistance development. In order to re-sensitize BxPC-3/GEM Res cells to GEM therapy, a co-treatment with iNOS inhibitors (L-NAME and 1400W) or NO donor (DPTA) was performed. iNOS inhibition resulted in an increase in GEM cytotoxicity in BxPC-3/GEM Res cells, confirming the key role of NO in resistance development. The data obtained suggest that modulation of NO/RNS levels during therapy may help to prevent drug resistance. They also suggest that GSNOR is a potential target for improving cancer therapy.