RESUMEN
Human PC-3 prostate cancer cells were incubated in the presence of two cardenolides, i.e., ouabain and 19-hydroxy-2''-oxovoruscharin. Their effects were monitored by infrared spectroscopy of the cells after different exposure times to the cardenolides. Analysis of changes in absorbance intensities indicated that, for both compounds, the absorbance at one wavenumber with a minor contribution of a second wavenumber is sufficient to build a linear model accurate enough to assign more than 97% of the spectra to their correct time slot. Student t-tests and twodimensional correlation analysis (2D-COS) indicated that both drugs have very similar effects on PC-3 cells. However, asynchronous 2D maps revealed significant differences and allowed the sequence of the spectral changes to be determined: 1395 â 1695 cm(-1) for ouabain, and 1400 â 1655 â 1100 â 1250 â 1020 cm(-1) for 19-hydroxy-2''-oxovoruscharin. 2D correlation map subtraction allowed the identification of very specific differences in the impact of both compounds on PC-3 cells, in particular the ability of 19-hydroxy-2''-oxovoruscharin to affect nucleic acid of PC-3 cells.
Asunto(s)
Cardenólidos/química , Cardenólidos/farmacología , Neoplasias de la Próstata/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Línea Celular Tumoral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Histocitoquímica/métodos , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Neoplasias de la Próstata/patologíaRESUMEN
The number of anticancer agents that fail in the clinic far outweighs those considered effective, suggesting that the selection procedure for progression of drug molecules into the clinic requires improvement. Traditionally, new drugs are evaluated for their potential to kill cancer cell lines. This approach is obviously not sufficient, and molecules with new modes of action are required. We suggest here that the infrared spectrum of cells exposed to anticancer drugs could offer an opportunity to obtain a fingerprint of the metabolic changes induced by the drugs. Because the infrared spectrum of cells yields a precise image of all the chemical bonds present in the sample, different drug targets are likely to yield different infrared fingerprints characteristic of the 'mode of action' of the therapeutic agent under investigation. In turn, drug-induced metabolic disorders should be amenable to classification in the same way that bacteria gender, species, and strains can be classified. We examined here a human prostate cancer PC-3 cell line exposed to 7 well described antimitotics. In a first step the IC(50) values were determined. For FTIR imaging, PC-3 cells were exposed to the IC(50) concentration of each drug for 48 h. About one hundred images of 4096 IR spectra at 8 cm(-1) spectral resolution were acquired. We show with a Student t-test that the different molecules tested induced different infrared spectral modifications. Furthermore, drugs known to induce similar types of metabolic disturbances appear to cluster when spectrum shapes are analyzed. Finally, supervised statistical methods allowed the building of an efficient and discriminant model. When the discriminant model was applied to a full infrared image a good sorting was generally obtained and misclassified spectra generally belonged to a small number of specific cells. Taken all together these data suggest that FTIR could be used for the classification of drug action.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Antineoplásicos/clasificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Análisis por Conglomerados , Análisis Discriminante , Humanos , Concentración 50 Inhibidora , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factores de TiempoRESUMEN
Fourier transform infrared (FTIR) spectroscopy was used to investigate modifications of prostate cancer PC-3 cell lipidome after exposure to sub-lethal concentrations of ouabain. FTIR spectroscopy offered an overview of the lipid classes present in the whole sample. The method is simple, label free and some features can be detected on entire cells. We compared the achievements of FTIR spectroscopy with data obtained by mass spectrometry (MS) on the same samples. It appears that FTIR spectroscopy could identify content variations in some lipid classes, e.g., these containing choline head groups such as phosphatidylcholine and sphingomyelin. MS analysis could confirm this result as indicated by principal component analysis and 2D heterocorrelation maps. FTIR spectra were also able to report changes in ester/choline/phosphate ratios characterizing lipid changes induced by ouabain. Furthermore, quantization of major lipid classes (PC, PE, PG, SM) could be obtained by curve fitting of the FTIR spectra. Yet, FTIR failed to resolve lipid classes for which the polar heads do not display specific IR features such as phosphatidylglycerol and cardiolipin.
Asunto(s)
Cardiotónicos/farmacología , Lípidos/análisis , Ouabaína/farmacología , Neoplasias de la Próstata/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectroscopía Infrarroja por Transformada de Fourier , Línea Celular Tumoral , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológicoRESUMEN
Recently, the possibility of using IR spectroscopy to fingerprint the mode of action of potent antitumor drugs on cancer cells at sub-lethal concentrations has been demonstrated by comparing spectra recorded from untreated or drug-treated cells. The present study investigates the potential interference of the cell culture confluence rate on cell FTIR signature. Significant spectral differences were observed on cells harvested at different confluence rates. Yet, these differences were weak when compared to those induced by sub-lethal ouabain concentrations, used as a model of cardenolide drug. Furthermore, principal component analysis reveals that the impact of the confluence rate, above 50% coverage, on the FTIR spectra is essentially unique and orthogonal to the one induced by the drug model.
Asunto(s)
Línea Celular Tumoral , Neoplasias de la Próstata/patología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Ouabaína/farmacología , Análisis de Componente Principal , Neoplasias de la Próstata/tratamiento farmacológicoRESUMEN
Platinum complexes remain widely used to combat various types of cancers. Three platinum complexes, cisplatin, carboplatin and oxaliplatin, are marketed for various oncological purposes. Additionally, nedaplatin, lobaplatin and heptaplatin have gained regionally limited approval for oncology purposes. Furthermore, various platinum derivatives are currently under clinical trials. More than 40 years after their discovery, however, the precise mechanism of action of platinum antitumor complexes remains elusive, partly because these compounds display numerous intracellular targets. Structure-activity-relationship analyses are therefore difficult to conduct to optimize the synthesis of novel platinum derivatives. The aim of the present study is to illustrate the potential of using Fourier Transform Infrared (FTIR) analyses to monitor the cellular modifications induced by the new platinum derivatives that we have synthesized. We show in the present study the advantages of combining an in vitro assay to determine the IC50 growth inhibition concentrations of a series of compounds belonging to a given chemical series and FTIR analyses carried out at the IC50 concentrations for each compound to identify potential hits within this series of compounds. The original pharmacological approach proposed here could, therefore, avoid large-scale pharmacological experiments to find hits within a given chemical series.
Asunto(s)
Neoplasias/tratamiento farmacológico , Compuestos Organoplatinos/farmacología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Animales , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Concentración 50 Inhibidora , RatonesRESUMEN
We show in the present work that the infrared (IR) spectrum of human PC-3 prostate cancer cells exposed to anticancer drugs could offer a unique opportunity to get a fingerprint of all the major biochemical components (DNA, RNA, proteins, lipids, etc.) present in the cells and to identify with high sensitivity the signature of the metabolic changes induced by anticancer drugs. We investigated here the FTIR-related signatures of the effect of 4 structurally-related cardiotonic steroids (CS), i.e. ouabain, 19-hydroxy-2â³-oxovoruscharin, hellebrin and 19-hydroxy-hellebrin on PC-3 cancer cells incubated between 0 and 36 h in the absence (control) or the presence of the CS. For each molecule a single spectral signature described the largest part of the time dependent modifications with a possible very minor second component. The spectral signatures characterizing the effects of each of the four CS were unique but very similar when compared to the signature of the effect of an intercalating anticancer drug, i.e. doxorubicin, selected as a positive reference compound in our study, suggesting a fully distinct set of cellular perturbations. The current study thus illustrates that Fourier Transform Infrared (FTIR) analyses can be used to identify, among the perturbations induced on a given cancer cell line, the features common to a group of anticancer compounds as well as features specific to every single drug.
Asunto(s)
Antineoplásicos/farmacología , Glicósidos Cardíacos/farmacología , Proliferación Celular/efectos de los fármacos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Antineoplásicos/química , Bufanólidos/química , Bufanólidos/farmacología , Cardenólidos/química , Cardenólidos/farmacología , Glicósidos Cardíacos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Humanos , Concentración 50 Inhibidora , Masculino , Estructura Molecular , Ouabaína/química , Ouabaína/farmacología , Análisis de Componente Principal , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Tiazoles/química , Tiazoles/farmacología , Factores de TiempoRESUMEN
The paper presents a simple and robust method to determine protein secondary structure from circular dichroism, transmission and attenuated total reflection (ATR) Fourier transform infrared spectra. It is found that the different spectroscopic methods bring valuable but roughly identical information on the secondary structure of proteins. ATR and transmission FTIR spectra display distinct differences, yet the secondary structure can be predicted from their spectra with roughly the same success. It is also found that one wavenumber or wavelength includes the large majority of the information correlated with secondary structure content and no more than 3 significant independent wavenumbers/wavelengths could be found for any of the spectroscopic data. This finding indicates that more complex linear combinations of the absorbance or ellipticities will not further improve secondary structure predictions. Furthermore, the information content in CD, transmission and ATR FTIR spectra is largely redundant. If combining CD and FTIR results in some improvement of structure prediction quality, the improvement is too modest to prompt spectroscopists to collect different spectroscopic data for structure prediction purposes. On the other hand, the data collected show that the quality of the FTIR spectrometers is such that biosensors or imaging methods sampling from 10(-9) to 10(-15) g yield spectra of sufficient quality to analyze protein secondary structure. These new techniques open the way to a new area of research, both in protein conformational response to ligand and imaging at sub-cellular scales.
Asunto(s)
Dicroismo Circular/métodos , Estructura Secundaria de Proteína , Proteínas/análisis , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Biopsia , Técnicas Biosensibles , Neoplasias de la Mama/química , Línea Celular Tumoral , Femenino , Humanos , Masculino , Neoplasias de la Próstata/químicaRESUMEN
There is a growing interest for screening antitumor drugs for their mechanism of action on cancer cells. Yet, screening for "modes of action" presents a technical challenge that is beyond the capability of conventional methods used in cellular or molecular biology. Several studies have highlighted the advantages of using infrared spectroscopy for diagnostic purposes at the clinical level for identifying cell types. In the present work, we suggest that the Fourier Transform Infrared (FTIR) spectrum of cells exposed to anti-cancer drugs could offer a unique opportunity to obtain a fingerprint of all molecules present in the cells and to observe, with a high sensitivity, the metabolic changes induced by potential anti-cancer drugs. Ouabain is one of the most potent cardenolides, which acts by inhibiting sodium pump activity. Cardenolides represent a class of compounds that are intended to soon enter clinical trials in oncology. In order to evaluate the potential of infrared spectroscopy to yield a signature for ouabain action on cancer cells, human prostate cancer PC-3 cells were treated with 36 nM ouabain, a sub-lethal concentration. Using ouabain as a model, we have thus demonstrated the possibility of using IR spectroscopy in the assessment of the global effects of an investigational compound on the cell constituents, thus contributing to setting up a new method for screening for novel anti-cancer agents in general, and potential anti-cancer cardenolides in particular. The most spectacular data obtained strongly suggest a modification in the nature of the cell lipids.
Asunto(s)
Antineoplásicos/química , Ouabaína/química , Ouabaína/toxicidad , Espectroscopía Infrarroja por Transformada de Fourier/métodos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Masculino , Modelos Moleculares , Neoplasias de la Próstata/patologíaRESUMEN
Multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette superfamily. Using the energy provided by ATP hydrolysis, it transports a broad spectrum of substrates across the plasma membrane, including hormones, leukotriene C(4), bile salts, and anti-cancer drugs. Recent works have suggested that P-glycoprotein is associated to cholesterol and sphingolipid-rich membrane microdomains and that cholesterol upregulates its ATPase and drug transport activities. Confocal microscopy experiments and Triton X-100 extraction of detergent-resistant membranes provide evidence that MRP1 is not located in raft-like structures and that its activity is downregulated by cholesterol. The data are discussed in terms of cholesterol-protein interaction and topology.