## 4 Discussion
A time longer than 15 years since smoking cessation significantly increased the number of \(ABCB1\) transcripts. An age-associated upregulation of the investigated gene was another characteristic of NSCLC patients, regardless of the histological cancer subtype. It is possible to hypothesize that the progressive restoration of \(ABCB1\) expression in cancer cells with age may be due to environmental factors, including longer exposure to air pollution or increases in drug consumption. Conversely, older people often have decreased P-gp levels in the blood-brain barrier that are connected, i.a., with concomitant neurodegenerative disease [40]. The results of animal studies indicate that the directions of P-gp expression/function changes are likely to be tissue-specific [41]. It is known that the amounts of P-gp are also different between men and women. For example, hepatic P-gp expression is lower in females than in males [42]. In our analysis, the \(ABCB1\) expression appeared to be upregulated in males but only in adenocarcinoma cells. In summary, differences in the P-gp level may result from tissue diversity, hormonal influences, ethnicity, and interindividual conditions. Due to the limited number of studies addressing the influences of smoking, age, gender, and ethnicity on the \(ABCB1\) transcript levels in lung cancer patients, these observations have not been confirmed elsewhere. The present analysis demonstrated that \(ABCB1\) gene expression negatively correlates with NSCLC staging. Surprisingly, in the most advanced clinical stage of cancer (IV), the number of transcripts increased, which could suggest an association between \(ABCB1\) amplifications and patient survival. Patients diagnosed with a primary tumor in stage IV could be exposed to the strong effects of harmful environmental compounds or prolonged drug use for reasons other than cancer. Unfortunately, according to our best knowledge, there have been no studies that thoroughly assessed the correlation between the expression of the investigated gene and the lung cancer staging adjusted for data on the degree of environmental influence. Moreover, a high level of \(ABCB1\) mRNA was associated with a longer recurrence-free survival/overall survival for patients with lung adenocarcinoma. Conversely, [PERSON] et al. showed a trend toward worse survival in tumors with P-gp expression in both LUAD and LUSC; however, the difference for lung squamous-cell carcinoma was not statistically significant [43]. Thus, all these results suggest that \(ABCB1\) expression is useful for predicting prognoses in LUAD patients. However, the level of gene transcripts should be regarded as only one of many factors determining the expression of a protein that requires the interaction of multiple signaling pathways. These, in turn, may undergo interindividual and cancer-specific modifications, e.g., depending on the \(EGFR\) mutation status. On the other hand, many mechanisms have been identified that may alter the \(ABCB1\) expression in tumors, including genomic instability in regions of chromosome 7, the activity of transcription factors/miRNAs, genetic mutations/polymorphisms, and epigenetic regulation. Among the epigenetic modifications, DNA methylation, histone modifications, and the effect of non-coding RNAs are distinguished, but the methylation of the \(ABCB1\) promoter region is analyzed the most often. The organization of the gene encoding P-gp was first described by [PERSON] et al. [44]. According to the aforementioned study, there are two regions in \(ABCB1\) where the transcription of this gene can be initiated. The downstream/proximal promoter acts as the major promoter. A distal promoter is located upstream and usually remains inactive, but under certain conditions it could produce a total transcript in some drug-resistant cell lines. In addition, the upstream promoter is located in a region poor in CpG islands/dinucleotides, and its complementary DNA strand serves as a template for the transcription of the \(RUNDC3B\) gene.