Correlation of p16INK4A Expression and HPV Copy Number with Cellular FTIR Spectroscopic Signatures of Cervical Cancer Cells

Cervical cancer, a potentially preventable disease, has its main aetiology in infection by high risk human papillomavirus (HR-HPV). Approaches to improving cervical cancer screening and diagnostic methodologies include molecular biological analysis, targeting of biomarker proteins, but also exploration and implementation of new techniques such as vibrational spectroscopy. This study correlates the biomarker protein p16INK4A expression levels dependent on HPV copy number with the infrared absorption spectral signatures of the cervical cancer cell lines, HPV negative C33A, HPV-16 positive SiHa and CaSki and HPV-18 positive HeLa. Confocal fluorescence microscopy demonstrated that p16INK4A is expressed in all investigated cell lines in both nuclear and cytoplasmic regions, although predominantly in the cytoplasm. Flow cytometry was used to quantify the p16INK4A expression levels and demonstrated a correlation, albeit nonlinear, between the reported number of integrated HPV copies and p16INK4A expression levels. CaSki cells were found to have the highest level of expression, HeLa intermediate levels, and SiHa and C33A the lowest levels. FTIR spectra revealed differences in nucleic acid, lipid and protein signatures between the cell lines with varying HPV copy number. Peak intensities exhibited increasing tendency in nucleic acid levels and decreasing tendency in lipid levels with increasing HPV copy number, and although they were found to be nonlinearly correlated with the HPV copy number, their dependence on p16INK4A levels was found to be close to linear. Principal Component Analysis (PCA) of the Infrared absorption spectra revealed differences between nuclear and cytoplasmic spectroscopic signatures for all cell lines, and furthermore clearly differentiated the groups of spectra representing each cell line. Finally, Partial Least Squares (PLS) analysis was employed to construct a model which can predict the p16INK4A expression level based on a spectral fingerprint of a cell line, demonstrating the diagnostic potential of spectroscopic techniques

Understanding Hypoxia-Driven Tumorigenesis: The Interplay of HIF1A, DNA Methylation, and Prolyl Hydroxylases in Head and Neck Squamous Cell Carcinoma

Hypoxia-inducible factor 1-alpha (HIF1A) is a key transcription factor aiding tumor cells’ adaptation to hypoxia, regulated by the prolyl hydroxylase family (EGLN1-3) by directing toward degradation pathways. DNA methylation potentially influences EGLN and HIF1A levels, impacting cellular responses to hypoxia. We examined 96 HNSCC patients and three cell lines, analyzing gene expression of EGLN1-3, HIF1A, CA9, VEGF, and GLUT1 at the mRNA level and EGLN1 protein levels. Methylation levels of EGLNs and HIF1A were assessed through high-resolution melting analysis. Bioinformatics tools were employed to characterize associations between EGLN1-3 and HIF1A expression and methylation. We found significantly higher mRNA levels of EGLN3, HIF1A, GLUT1, VEGF, and CA9 (p = 0.021; p p p = 0.004, and p EGLN1 mRNA level in tumor tissues (p = 0.0013). In HNSCC patients with hypermethylation of HIF1A in normal tissue, we noted a reduction in HIF1A mRNA levels compared to tumor tissue (p = 0.04). In conclusion, the differential expression of EGLN and HIF1A genes in HNSCC tumors compared to normal tissues influences patients’ overall survival, highlighting their role in tumor development. Moreover, DNA methylation could be responsible for HIF1A suppression in the normal tissues of HNSCC patients

Metabolic syndrome is associated with similar long-term prognosis in non-obese and obese patients. An analysis of 45 615 patients from the nationwide LIPIDOGRAM 2004-2015 cohort studies

Aims We aimed to evaluate the association between metabolic syndrome (MetS) and long-term all-cause mortality. Methods The LIPIDOGRAM studies were carried out in the primary care in Poland in 2004, 2006 and 2015. MetS was diagnosed based on the National Cholesterol Education Program, Adult Treatment Panel III (NCEP/ATP III) and Joint Interim Statement (JIS) criteria. The cohort was divided into four groups: non-obese patients without MetS, obese patients without MetS, non-obese patients with MetS and obese patients with MetS. Differences in all-cause mortality was analyzed using Kaplan-Meier and Cox regression analyses. Results 45,615 participants were enrolled (mean age 56.3, standard deviation: 11.8 years; 61.7% female). MetS was diagnosed in 14,202 (31%) by NCEP/ATP III criteria, and 17,216 (37.7%) by JIS criteria. Follow-up was available for 44,620 (97.8%, median duration 15.3 years) patients. MetS was associated with increased mortality risk among the obese (hazard ratio, HR: 1.88 [95% CI, 1.79-1.99] and HR: 1.93 [95% CI 1.82-2.04], according to NCEP/ATP III and JIS criteria, respectively) and non-obese individuals (HR: 2.11 [95% CI 1.85-2.40] and 1.7 [95% CI, 1.56-1.85] according to NCEP/ATP III and JIS criteria respectively). Obese patients without MetS had a higher mortality risk than non-obese patients without MetS (HR: 1.16 [95% CI 1.10-1.23] and HR: 1.22 [95%CI 1.15-1.30], respectively in subgroups with NCEP/ATP III and JIS criteria applied). Conclusions MetS is associated with increased all-cause mortality risk in non-obese and obese patients. In patients without MetS obesity remains significantly associated with mortality. The concept of metabolically healthy obesity should be revised