Cytotoxic Effects of 5-Azacytidine on Primary Tumour Cells and Cancer Stem Cells from Oral Squamous Cell Carcinoma: An In Vitro FTIRM Analysis

Abstract

In the present study, the cytotoxic effects of 5-azacytidine on primary Oral Squamous Cell Carcinoma cells (OSCCs) from human biopsies, and on Cancer Stem Cells (CSCs) from the same samples, were investigated by an in vitro Fourier Transform InfraRed Microscospectroscopy (FTIRM) approach coupled with multivariate analysis. OSCC is an aggressive tumoral lesion of the epithelium, accounting for ~90% of all oral cancers. It is usually diagnosed in advanced stages, and this causes a poor prognosis with low success rates of surgical, as well as radiation and chemotherapy treatments. OSCC is frequently characterised by recurrence after chemotherapy and by the development of a refractoriness to some employed drugs, which is probably ascribable to the presence of CSCs niches, responsible for cancer growth, chemoresistance and metastasis. The spectral information from FTIRM was correlated with the outcomes of cytotoxicity tests and image-based cytometry, and specific spectral signatures attributable to 5-azacytidine treatment were identified, allowing us to hypothesise the demethylation of DNA and, hence, an increase in the transcriptional activity, together with a conformational transition of DNA, and a triggering of cell death by an apoptosis mechanism. Moreover, a different mechanism of action between OSSC and CSC cells was highlighted, probably due to possible differences between OSCCs and CSCs response.

Evaluation of Controlled Ovarian Stimulation Protocols in Patients with Normal and Low Ovarian Reserve: Analyses of miRNAs and Selected Target Genes Involved in the Proliferation of Human Cumulus Cells and Oocyte Quality

“…The low level of RNA in these two latter groups seemed to be caused by two different mechanisms: first, the LR patients treated with rFSH displayed the highest values of the 1240/1220 band area ratio, which indicates high amounts of the unordered A-form DNA, which is less efficiently transcribed [32,38,39]; second, in NR patients treated with hMG, the lower transcriptional levels may be associated with the highest levels of the 924/970 band area ratio, representative of the Z-DNA amount. Z-DNA is a left-handed conformation of DNA, often found in specialized sequences as a result of DNA methylation processes, which strongly influences the transcriptional activity via the exclusion of transcription factors [33,40,41,45]. Hence, the different stimulation treatment in ART patients with normal and low ovarian reserve seems to determine a change in the conformational state of the CCs DNA; in particular, the different order (A-DNA and B-DNA) and the methylation state (Z-DNA) [33,37,46], representing the real novelty of this study.…”

“…FTIRM is a vibrational technique, widely applied to investigate the biomolecular building and composition of cells and tissues, allowing one to obtain, in a label-free way, the unique molecular fingerprint of the most relevant biological molecules [29,30]. The spectral profiles obtained by the FTIRM analysis can be correlated with biological processes, such as metabolism, stress status, and apoptosis [31][32][33]. A previous study from our laboratory evidenced the re-lationship between the metabolic profile of granulosa cells and the fate of the corresponding oocyte, as assessed by gene expression and Fourier transform infrared microspectroscopy (FTIRM).…”

“…Recently, FTIRM was also exploited to characterize both human oocytes [34] and granulosa cells [22,35], and to assess the effects on human cumulus cells of three controlled ovarian stimulation protocols, based on urinary FSH, recombinant FSH, or hMG [36]. In particular, FTIRM is able to elucidate possible conformational changes in DNA conformation, by evaluating the relative amounts of B-form, A-form, and Z-form DNAs [33]. For example, the B-to A-form DNA transition has been reported to occur, in a reversible way, upon dehydration, possibly as a mechanism to reduce DNA damages [37].…”

Vibrational Imaging Techniques for the Characterization of Hard Dental Tissues: From Bench-Top to Chair-Side

“…In recent years, vibrational spectroscopic techniques, such as Raman Microspectroscopy (RMS) and Fourier Transform Infrared (FTIR) spectroscopy, have been employed as emerging tools for studies in the biomedical field, including those on the structural and macromolecular composition of tissues and cells [1][2][3]. Both of these non-destructive and label-free analytical techniques, based on the interaction between matter and electromagnetic radiation, provide relevant information on the chemical bonds, and hence the functional groups of the macromolecules within the analyzed sample [4,5].…”

“…For example, IR spectra are not affected by fluorescence, which often influences Raman measurements [14]. On the other hand, RMS is more suitable for the analysis of samples in aqueous solution; in fact, Raman spectra are not altered by water, which conversely massively influences IR spectra and requires complex subtraction procedures [2,14].…”