Can ethanol affect the cell structure? A dynamic molecular and Raman spectroscopy study.

The role that tobacco consumption plays in the etiology of oral cancer carcinogenesis, and of alcohol consumption acting as a co-factor, have been well established. However, in recent years, the contribution of alcohol consumption alone to oral cancer has been proposed. In fact, a high percentage of patients who develop oral cancer have both habits (tobacco and alcohol consumption), and other small patient groups only consume alcohol or do not have any other identifiable bad habits. In the present study we demonstrate, using a combination of dynamic molecular modelling and Raman spectroscopy, that ethanol has a significant effect on oral cells in vitro, mainly interacting with the lipids of the cell membrane, changing their conformation. Thus, it is possible to conclude that ethanol can affect the cell permeability, and by consequence serve as a possible trigger in oral carcinogenesis.

Raman spectroscopic analysis of oral cells in the high wavenumber region.

Raman spectroscopy can provide a molecular-level signature of the biochemical composition and structure of cells with submicrometer spatial resolution and could be useful to monitor changes in composition for early stage and non-invasive cancer diagnosis, both ex-vivo and in vivo. In particular, the fingerprint spectral region (400-1800cm-1) has been shown to be very promising for optical biopsy purposes. However, limitations for discrimination of dysplastic and inflammatory processes based on the fingerprint region have been demonstrated. In addition, the Raman spectral signal of dysplastic cells is one important source of misdiagnosis of normal versus pathological tissues. The high wavenumber region (2800-3600cm-1) provides more specific information based on NH, OH and CH vibrations and can be used to identify the subtle changes which could be important for discrimination of samples. In this study, we demonstrate the potential of the high-wavenumber spectral region in this context by collecting Raman spectra of nucleolus, nucleus and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (DOK) cell lines and from normal oral epithelial primary cells, in vitro, in water immersion, which were then analyzed by principal components analysis as a method to discriminate the spectra. Analysis was performed before and after digital subtraction of the bulk water signal. In the normal cell line, the three subcellular regions are well differentiated before water subtraction, although the discrimination of the two nuclear regions is less well defined after water subtraction. Comparing the respective subcellular regions of the three cell lines, before water subtraction, the cell lines can be discriminated using sequential PCA and Feature Discriminant Analysis with up to ~100% sensitivity and 97% specificity for the cytoplasm, which is improved to 100% sensitivity and 99% specificity for the nucleus. The results are discussed in terms of discrimination comparing the CH vibrational modes of nucleic acids, proteins and lipids. The potential role of the OH vibrations, considering free water and confined water, in the discrimination of cell cultures and pathological processes are also discussed.

Induction of apoptosis in oral squamous carcinoma cells by pyrrolo-1,5-benzoxazepines.

Oral cancer (OC) is a largely asymptomatic disease, resulting in one of the highest mortality rates of any cancer. OC is currently ranked as the sixth most common cancer in the world, according to a recent World Health Organization analysis, and its prevalence is increasing, both in western and developing regions. Depending on the stage of OC, treatment strategies include surgery, radiation therapy and chemotherapy, or a combination thereof. As with numerous other types of cancer, resistance to conventional chemotherapeutic drugs is increasing in oral squamous cell carcinoma (OSCC). The present study aimed to investigate the use of a novel group of compounds, the pyrrolo­1,5­benzoxazepines (PBOXs), as a therapeutic alternative for the treatment of OC. PBOXs are microtubule­targeting agents that are able to induce apoptosis in numerous cancer cell types, thereby preventing tumour cell proliferation. Ca9.22 gingival and TR146 buccal cell lines were used as models for OSSC. Cell viability and proliferation in the presence of two PBOXs: PBOX­6 and PBOX­15, was monitored using an AlamarBlueTM assay. Flow cytometric analysis of propidium iodide­stained cells was used to determine the DNA content, and therefore the percentage of cells in each phase of the cell cycle. Microtubule disruption was determined by indirect immunofluorescence staining. Changes in protein expression and degradation were determined by western blotting. The results of the present study indicated that both PBOX­6 and ­15 were able to induce apoptotic cell death by disrupting the microtubule network in both cell lines. The EC50 values were subsequently calculated for both PBOX­6 and ­15, and PBOX­15 was shown to possess a higher potency. Both compounds displayed anti­proliferative effects mediated through sustained G2/M arrest accompanied by tubulin disruption, and a decrease in DNA repair protein poly (ADP ribose) polymerase expression. These findings suggest that PBOXs may prove useful, either alone or in combination with other agents, in the treatment of chemotherapeutic resistant OSCC.

Raman micro-spectroscopy for rapid screening of oral squamous cell carcinoma.

Raman spectroscopy can provide a molecular-level fingerprint of the biochemical composition and structure of cells with excellent spatial resolution and could be useful to monitor changes in composition for dysplasia and early, non-invasive cancer diagnosis (carcinoma in situ), both ex-vivo and in vivo. In this study, we demonstrate this potential by collecting Raman spectra of the nucleoli, nuclei and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (pre-cancerous, DOK) cell lines and from normal oral epithelial primary cell cultures, in vitro, which were then analysed by principal component analysis (PCA) as a multivariate statistical method to discriminate the spectra. Results show significant discrimination between cancer and normal cell lines. Furthermore, the dysplastic and cancer cell lines could be discriminated based on the spectral profiles of the cytoplasmic regions. The principal component loading plot, which elucidates the biochemical features responsible for the discrimination, showed significant contributions of nucleic acid and proteins for nucleolar and nuclear sites and variation in features of lipids for the cytoplasmic area. This technique may provide a rapid screening method and have potential use in the diagnosis of dysplasia and early, non-invasive oral cancer, the treatment of which involves much less extensive and complex surgery and a reduction in associated co-morbidity for the patient.