Comparing biochemical and biophysical methodologies for the characterization of induced cell death

Abstract

Background: In specific circumstances, the induction of cell death can be an appropriate treatment of disease. Cell death can occur because of internal programming or external stress caused by infection, radiation or chemotherapies. There are many ways to investigate cell death and this work utilized vibrational spectroscopy to distinguish viable from dead cells. Cell death was achieved in this study by the addition of chemical stressors. Methodology: Cervical adenocarcinoma (HeLa) cells and African green monkey kidney (Vero) cells were used. Two organic diphosphino gold complexes designated AE 76 and AE125, auranofin, an extract of Plectranthus ciliatus, actinomycin D and methanol were used as external cell death inducers. Biochemical methods used included Sodium 3’-[phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) colorimetric assays, Real Time Cell Electronic Sensing (RT-CES), flow cytometry, Transmission Electron Microscopy (TEM) and caspases 3 / 7 assays. The biophysical experiments were Fourier Transform Infrared (FTIR) spectroscopy and single cell Raman microspectroscopy. Multivariate analysis (Principal Component Analysis (PCA)) and one way Analysis Of Variance (ANOVA) was used to determine significantly altered vibrational bands. Results and discussion: Using XTT, it was found that the cell death inducers were cytotoxic at low concentrations. RT-CES analysis detected that the treatments induced concentration dependent cellular responses (nontoxic or cytostatic to cytotoxic in both cell lines). Early apoptosis was detected after treatment using flow cytometry, with the exception of methanol treated cells being necrotic. Caspase dependent apoptosis was detected in HeLa and Vero cells. Thirteen FTIR spectral bands associated with cytotoxicity were significantly (p <0.05) altered when HeLa cells were treated with naturally derived products. These bands were related to nucleic acids, proteins and lipids. Two of the bands associated with amide I were also indicative of early stress responses. FTIR microspectroscopy confirmed cytostatic cells were viable and could still recover. Based on significant FTIR changes in both cell lines, flow cytometrically sorted populations (viable, apoptotic and necrotic) could be distinguished. Glycogen and high wavenumber region alterations were distinctly different between viable and necrotic cells while apoptotic cells were mostly altered in the regions of nuclear material. Flow cytometrically sorted cells of different populations (viable and dead) were confirmed using TEM based on morphological characteristics. Raman spectroscopy was utilized in investigating metallodrug induced apoptosis. Vibrational peaks assigned to phosphatidylethaolamine (762 cm-1) and ester bonds significantly increased in intensity which could be a molecular signature of induced apoptosis. Treated cells also had higher intensities for glucose and glycogen, which could be a survival mechanism of cancer cells. Raman spectroscopy detected cell death biomarkers in sorted cells, where dead cells had increased intensities at 762 cm-1 and 1578 cm-1. Conclusion: Investigations of cell death using vibrational spectroscopy initially started around 2000 with limited numbers of articles available on the subject matter. Vibrational spectroscopy proved to be a powerful tool for detecting spectral markers of early cell stress, cytostatic cellular responses, survival mechanisms of cancer cells and cell death. The approach presented here may find application in the (in vitro) evaluation of diseases (e.g. HIV / AIDS, TB, malaria and cancer) where induced cell death is part of the pathology. Data produced in this investigation substantially supplemented vibrational spectroscopic knowledge into understanding cellular stress, cell death and potential cell death markers in vitro