In order to control the emergence and progression of cancer, survival mechanisms, especially cancer stem cell factors, and inflammation, become very important [5, 8, 10, 12]. In contrast to traditional cytotoxic chemotherapies, targeted cancer therapy involves treating cancer cells with greater sensitivity and potentially fewer side effects and mechanisms of carcinogenesis . In this study, we analyzed the effects of low doses of sorafenib treatment on DEN-induced lung carcinogenesis in rats and determined that DEN effectively induced lung carcinogenesis when applied alone and sorafenib was significantly effective in decreasing the inflammatory parameters in the tumor microenvironment and level of SOX2 protein, which mediates the self-renewal of cancer stem cells.
Diethylnitrosamine (DEN) is a well-known and commonly used chemical compound to induce cancers of the esophagus, liver, and lung in experimental animals . Some literature data demonstrated that DEN may be a potent lung carcinogen in strains with a higher incidence of lung tumors than other cancers [19, 20]. We selected a dose of 150 mg/kg DEN based on previous studies, which demonstrated that the application of DEN at this dose remarkably reflects the pathological and biochemical findings in lung cancer in humans [17, 21]. In parallel with the previous literature, we determined that DEN administration significantly increased histopathological scores and inflammation in the present study. Lung tissues displayed extensive pathological changes in the DEN group. Widespread inflammatory cell infiltration, thickened alveolar wall, proliferation, degeneration in the bronchiole epithelium, hyperemia in the vessels were observed in the DEN group. In contrast to the DEN group, the histopathological scores decreased and partial histopathological improvement was observed in the group treated with SOR.
Chronic inflammation is often a hallmark of tumor initiation and progression . JNK takes an important role in these inflammation-related events. Many studies demonstrated that JNK is a positive regulator of tumor growth in the lung tissue of experimental models [6, 22]. In line with such roles of JNK, it has been documented that JNK maintain the tumor-initiating capacity of lung stem cells and another role has been added to it as "one of the pre-tumor roles of JNK" . Besides this, JNK has activated the inflammatory lung environment to support tumor development by regulating cytokines including IL-1β and TNF-α . IL-1β, a proinflammatory cytokine, is associated with tumor progression in lung cancer patients in multiple studies [24, 25]. An examination of the Cancer Genome Atlas database shows that for lung cancer, IL-1 and TNF reveals a tendency as an oncogene, as patients with higher IL-1β and TNF-α mRNA levels are linked to shorter overall survival, although this correlation does not reach statistical significance . In another study, it is well documented that TNF-α and IL-1β signaling support COX-2 promoter activity as well as COX-2 and mRNA expressions. Moreover, recent studies have demonstrated that COX-2 expression also requires the activity of JNK intracellular signaling [28, 29].
In our study, JNK and COX-2 mRNA in lung tissue and TNF-α and IL-1β protein levels in serum increased in animals as a result of DEN administration. Studies on lung tissue and serum demonstrate that DEN treatment increased COX-2, TNF-α, and IL-1β levels [30, 31]. To the best of our knowledge, the results of the present study were first demonstrating that DEN leads to high level of JNK expression in lung tissue. With the application of SOR, it was observed that JNK and COX-2 mRNA in lung tissue decrease with the amelioration of tissue damage and inflammatory response. In parallel to this, serum TNF-α and IL-1β levels were decreased with the administration of SOR. There were no studies evaluating the effect of SOR on lung JNK and serum TNF-α and IL-1β levels after DEN administration in rats previously. However, it has been reported that sorafenib reduces JNK expression in lung cancer cell line in vitro , and it has therapeutic properties by reducing TNF-α and IL-1β levels in liver cancer . It has also been reported that, treatment of low concentrations of SOR significantly inhibits the proliferation of A549 tumor cells in vitro and suppressed tumor growth in vivo .
Inflammation also leads to cancer cells dedifferentiating into CSCs through several signaling pathways such as COX-2 and JNK signaling [6, 9]. SOX2 has demonstrated the potential to be a clinically useful biomarker to maintain lung CSCs . There are also studies in the literature reporting that SOX2 is overexpressed and acts as an oncogene in lung cancer . Although the role of SOX2 in lung cancer has been determined as a result of clinical and preclinical studies [11, 35] the effect of DEN administration on SOX2 expression in experimental animals remains unclear. In our study, we observed that DEN increased SOX2 levels as compared to the control group. In the literature, we could not find any study demonstrating SOX2 level in the DEN-induced lung cancer model. However, in line with our result, a recent study declared that stem cell gene SOX2 increased significantly in liver tissue with DEN application . In this study, SOR treatment significantly reduced SOX2 level in comparison to the DEN group. Consistent with this result, SOR was reported to decrease SOX2 level in an in vitro study . In the present study, we show for the first time that DEN exposure in rats promotes SOX2 levels through JNK/COX-2 signaling, endowing lung cells with malignant properties. These effects of DEN were reversed by the administration of sorafenib. It was also shown for the first time in this study that SOR reduces inflammation-associated JNK and COX-2 signaling and TNF and IL-1β protein levels in the tumor microenvironment and decreases the level of SOX2, which is a marker related to CSCs. These findings are noteworthy because of the close proximity to clinical trials investigating sorafenib for the treatment of lung cancer.