Role of nitric oxide in the response to photooxidative stress in prostate cancer cells
Graphical abstract
Introduction
Reactive oxygen species and reactive nitrogen species (ROS/RNS) are highly reactive molecules generated by exogenous [1] and endogenous [2] sources that can induce carcinogenesis and tumorigenesis [3], [4], [5], [6], [7]. They are produced in different cellular compartments, such as the mitochondria [8], endoplasmic reticulum [9], [10], [11] and peroxisomes [12]. Other sources of ROS/RNS are hypoxia conditions, oncogenes [13], [14] and many antitumor drugs employed in the clinic such as 5-FU, paclitaxel, cyclophosphamide, methotrexate, cisplatinum, epirubicin, doxorubicin and vinorelbine. In addition to generating ROS/RNS, these drugs cause chemoresistance and tumor recurrence [15], [16], [17], [18], [19], [20]. ROS/RNS can interact with each other and generate new reactive species. For example, the superoxide anion radical •O2– reacts with nitric oxide (•NO) to yield peroxynitrite (ONOO–) [21], [22].
•NO is generated by three different isoforms of nitric oxide synthase (NOS): endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). It plays a key role in cancer because it is implicated in angiogenesis, cell cycle, invasion and metastasis [23], [24], [25]. However, the exact role of •NO in cancer is highly controversial because of the contrasting data reported in the literature. High •NO levels induce DNA damage and apoptosis, while low •NO levels promote tumor survival, metastasis and drug resistance [26], [27], [28], [29], [30]. Recent data have suggested that iNOS, which catalyzes the production of •NO from L-arginine, is involved in the carcinogenesis induced by chronic inflammation [31], [32]. Furthermore, evidence that RNS favor the development of cancer stem cells (CSCs) [14] that are responsible for tumor resistance and recurrence has been provided [33]. The dual behavior of •NO depends on several factors, including its local concentration, redox state of the cell, the cell compartment where it is generated [34], the presence of antioxidants and the relative concentrations of L-arginine (the substrate of NOS enzymes) [35]. Due to its complex behavior, we hypothesized that •NO could be a mediator of the signaling pathways activated in response to oxidative stress associated with chemotherapy. Although the oxidative stress induced by chemotherapeutic drugs is known to cause side effects in patients [16], it remains unclear whether it induces drug resistance and recurrence.
To replicate the oxidative stress produced by anticancer drugs in tumor cells and investigate its sole effects, i.e. by excluding the anticancer effect of the drugs itself, we used a photosensitizer that can generate ROS/RNS upon irradiation in a very controlled way [36]. In addition to ROS, the photooxidative insult stimulates •NO production by NOS enzymes [36], [37], [38], [39], [40], [41]. To investigate how tumor cells respond to different levels of oxidative stress, we set up two photooxidative treatments, each inducing a specific response regarding activated molecular pathways. In the first treatment, we used repeated low-dose Pba/light treatments to generate a low chronic level of ROS/RNS in cancer cells. In the second treatment, we used a single dose Pba/light treatment to generate either low or high ROS/RNS levels in the cells. Previous work from our laboratory showed that the response of tumor cells to photooxidative stress is mediated by the NF-κB/YY1/Snail/RKIP loop [42], which is influenced by •NO [41], [43]. The behavior of this loop is complex and involves anti-apoptotic and pro-survival Snail and Yin Yang 1 (YY1) [44], [45], which are also modulators of the epithelial–mesenchymal transition (EMT) and metastatic disease [46], [47], [48], [49], as well as pro-apoptotic and metastatic suppressor Raf kinase inhibitor protein (RKIP) [50], [51], [52], [53], [54]. When the NF-κB/YY1/Snail/RKIP loop is dysregulated, tumor cells become resistant to apoptosis [55], [56], [57].
In addition to the NF-κB/YY1/Snail/RKIP loop, other redox-sensitive signaling pathways are involved in the tumor response to oxidative stress [58]. Of particular interest are those involving BTB and CNC homology (BACH-1), glycogen synthase kinase 3β (GSK-3β) and the nuclear factor erythroid 2-related factor 2 (Nrf2). BACH-1 is a basic leucine-zipper transcription factor that regulates oxidative stress [59], [60] and inhibits the metastasis suppressor RKIP in breast cancer cells, promoting invasion and metastasis [61], [62]. GSK-3β is also involved in signal transduction under oxidative stress [63], [64]. Indeed, by downregulating Nrf2, GSK-3β affects the antioxidant response of the cell [65]. Nrf2 is considered the master regulator of the antioxidant response [66], [67], [68], [69]. Mounting evidence indicates that the Nrf2 pathway is a driver of cancer progression [70], [71], metastasis [72] and chemoresistance [73].
In this study, we have compared the response of cancer cells exposed to chronic low levels of ROS/RNS, as occurs with chemotherapeutic treatments, to an acute dose of ROS/RNS. In particular, we have analyzed the following: (i) how iNOS/•NO acts as a “redox switch” regulating the molecular pathways stimulated in response to oxidative stress; (ii) the behavior of Nrf2 in regulating the expression of cytoprotective genes; (iii) how NF-κB and Nrf2 communicate with each other in response to oxidative stress.
Our study will be useful to identify new therapeutic targets to reduce tumor resistance and recurrence and strengthen the efficacy of chemotherapy.
Section snippets
Cell culture and reagents
Human prostate cancer PC3 cells (obtained from CNR, ISOF, Bologna, Italy) were cultured in RPMI medium containing 10% fetal bovine serum, antibiotics (100 U/ml of penicillin and 100 μg/ml of streptomycin) and 2 mM glutamine (Euroclone, Pero (MI), Italy). The cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C. All the experiments were performed using cells in the exponential growth phase. The cells were used between the third and fifteenth passages of culture. Pheophorbide a
Chronic photooxidative exposure of PC3 prostate cancer cells leads an aggressive and resistant population
Anticancer therapies are normally administered in repeated cycles in order to destroy most proliferating tumor cells and reduce the side effects, allowing normal cells to recover easily [77], [78], [79]. Most anticancer drugs generate moderate levels of ROS/RNS that are likely to induce chemoresistance and tumor recurrence [80]. To mimic the chronic oxidative stress to which cancer cells are exposed during the chemotherapeutic treatments, we used repeated photooxidative insults. First, we
Discussion
Many drugs used in cancer therapy release ROS/RNS and induce tumor resistance [15], [16], [17], [18], [19], [20]. In this study we have investigated how the production of ROS/RNS associated to chemotherapeutic drugs can induce tumor progression. To this aim we have explored the role of •NO/RNS as mediators of the molecular pathways implicated in the tumor response to oxidative damage. Unlike many other signaling molecules, •NO is freely diffusible, reacting with several intracellular targets
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported by AIRC “Associazione Italiana per la Ricerca sul Cancro”, IG 2017, Project Code 19898, The National FFABR, VQ3 DAME. The authors wish to thank Dr. Daniela Cesselli (DAME) for the FACS sorting experiments. We also sincerely thank Prof. Benjamin Bonavida (Department of Microbiology, Immunology & Molecular Genetics and David Geffen School of Medicine, UCLA, Los Angeles, CA, USA) for critical reading the manuscript.
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These authors contributed equally to this work.