RESUMO
Chromosomes are intranuclear structures, their main function is to store and transmit genetic information during cell division. They are composed of tightly packed DNA in the form of chromatin, which is constantly exposed to various damaging factors. The resulting changes in DNA can have serious consequences (e.g. mutations) if they are not repaired or repaired incorrectly. In this article, we studied chromosomes isolated from human cervical cancer cells (HeLa) exposed to a genotoxic drug causing both single- and double-strand breaks. Specifically, we used bleomycin to induce DNA damage. We followed morphological and chemical changes in chromosomes upon damage induction. Atomic force microscopy was used to visualize the morphology of chromosomes, while Raman microspectroscopy enabled the detection of changes in the chemical structure of chromatin with the resolution close to the diffraction limit. Additionally, we extracted spectra corresponding to chromosome I or chromatin from hyperspectral Raman maps with convolutional neural networks (CNN), which were further analysed with the principal component analysis (PCA) algorithm to reveal molecular markers of DNA damage in chromosomes. The applied multimodal approach revealed simultaneous morphological and molecular changes, including chromosomal aberrations, alterations in DNA conformation, methylation pattern, and increased protein expression upon the bleomycin treatment at the level of the single chromosome.
Assuntos
Bleomicina , Cromossomos , Humanos , Bleomicina/farmacologia , Metáfase , Cromatina , DNARESUMO
This study presents a new approach to designing a lithocholic acid functionalized oligomer (OLithocholicAA-X) that can be used as a drug carrier with additional, beneficial activity. Namely, this novel oligomer can incorporate an anti-cancer drug due to the application of an effective backbone as its component (lithocholic acid) alone is known to have anticancer activity. The oligomer was synthesized and characterized in detail by nuclear magnetic resonance, attenuated total reflectance Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, thermal analysis, and mass spectrometry analysis. We selected lipid rafts as potential drug carrier-membrane binding sites. In this respect, we investigated the effects of OLithocholicAA-X on model lipid raft of normal and altered composition, containing an increased amount of cholesterol (Chol) or sphingomyelin (SM), using Langmuir monolayers and liposomes. The surface topography of the studied monolayers was additionally investigated by atomic force microscopy (AFM). The obtained results showed that the investigated oligomer has affinity for a system that mimics a normal lipid raft (SM:Chol 2:1). On the other hand, for systems with an excess of SM or Chol, thermodynamically unfavorable fluidization of the films occurs. Moreover, AFM topographies showed that the amount of SM determines the bioavailability of the oligomer, causing fragmentation of its lattice.
Assuntos
Lipossomos , Ácido Litocólico , Ácido Litocólico/análise , Ácido Litocólico/metabolismo , Lipossomos/química , Sistemas de Liberação de Medicamentos , Espectroscopia de Ressonância Magnética , Microdomínios da Membrana/química , Esfingomielinas/química , Colesterol/químicaRESUMO
The lack of specific and sensitive early diagnostic options for pancreatic cancer (PC) results in patients being largely diagnosed with late-stage disease, thus inoperable and burdened with high mortality. Molecular spectroscopic methodologies, such as Raman or infrared spectroscopies, show promise in becoming a leader in screening for early-stage cancer diseases, including PC. However, should such technology be introduced, the identification of differentiating spectral features between various cancer types is required. This would not be possible without the precise extraction of spectra without the contamination by necrosis, inflammation, desmoplasia, or extracellular fluids such as mucous that surround tumor cells. Moreover, an efficient methodology for their interpretation has not been well defined. In this study, we compared different methods of spectral analysis to find the best for investigating the biomolecular composition of PC cells cytoplasm and nuclei separately. Sixteen PC tissue samples of main PC subtypes (ductal adenocarcinoma, intraductal papillary mucinous carcinoma, and ampulla of Vater carcinoma) were collected with Raman hyperspectral mapping, resulting in 191,355 Raman spectra and analyzed with comparative methodologies, specifically, hierarchical cluster analysis, non-negative matrix factorization, T-distributed stochastic neighbor embedding, principal components analysis (PCA), and convolutional neural networks (CNN). As a result, we propose an innovative approach to spectra classification by CNN, combined with PCA for molecular characterization. The CNN-based spectra classification achieved over 98% successful validation rate. Subsequent analyses of spectral features revealed differences among PC subtypes and between the cytoplasm and nuclei of their cells. Our study establishes an optimal methodology for cancer tissue spectral data classification and interpretation that allows precise and cognitive studies of cancer cells and their subcellular components, without mixing the results with cancer-surrounding tissue. As a proof of concept, we describe findings that add to the spectroscopic understanding of PC.
Assuntos
Neoplasias Pancreáticas , Análise Espectral Raman , Humanos , Análise Espectral Raman/métodos , Pâncreas , Núcleo Celular , Neoplasias PancreáticasRESUMO
PURPOSE: Knowledge about pancreatic cancer (PC) biology has been growing rapidly in recent decades. Nevertheless, the survival of PC patients has not greatly improved. The development of a novel methodology suitable for deep investigation of the nature of PC tumors is of great importance. Molecular imaging techniques, such as Fourier transform infrared (FTIR) spectroscopy and Raman hyperspectral mapping (RHM) combined with advanced multivariate data analysis, were useful in studying the biochemical composition of PC tissue. METHODS: Here, we evaluated the potential of molecular imaging in differentiating three groups of PC tumors, which originate from different precursor lesions. Specifically, we comprehensively investigated adenocarcinomas (ACs): conventional ductal AC, intraductal papillary mucinous carcinoma, and ampulla of Vater AC. FTIR microspectroscopy and RHM maps of 24 PC tissue slides were obtained, and comprehensive advanced statistical analyses, such as hierarchical clustering and nonnegative matrix factorization, were performed on a total of 211,355 Raman spectra. Additionally, we employed deep learning technology for the same task of PC subtyping to enable automation. The so-called convolutional neural network (CNN) was trained to recognize spectra specific to each PC group and then employed to generate CNN-prediction-based tissue maps. To identify the DNA methylation spectral markers, we used differently methylated, isolated DNA and compared the observed spectral differences with the results obtained from cellular nuclei regions of PC tissues. RESULTS: The results showed significant differences among cancer tissues of the studied PC groups. The main findings are the varying content of ß-sheet-rich proteins within the PC cells and alterations in the relative DNA methylation level. Our CNN model efficiently differentiated PC groups with 94% accuracy. The usage of CNN in the classification task did not require Raman spectral data preprocessing and eliminated the need for extensive knowledge of statistical methodologies. CONCLUSIONS: Molecular spectroscopy combined with CNN technology is a powerful tool for PC detection and subtyping. The molecular fingerprint of DNA methylation and ß-sheet cytoplasmic proteins established by our results is different for the main PC groups and allowed the subtyping of pancreatic tumors, which can improve patient management and increase their survival. Our observations are of key importance in understanding the variability of PC and allow translation of the methodology into clinical practice by utilizing liquid biopsy testing.
Assuntos
Metilação de DNA , Neoplasias Pancreáticas , Humanos , Conformação Proteica em Folha beta , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/patologia , Análise Espectral , Neoplasias PancreáticasRESUMO
Pancreatic cancer (PC) is an aggressive and lethal neoplasm, ranking seventh in the world for cancer deaths, with an overall 5-year survival rate of below 10%. The knowledge about PC pathogenesis is rapidly expanding. New aspects of tumor biology, including its molecular and morphological heterogeneity, have been reported to explain the complicated "cross-talk" that occurs between the cancer cells and the tumor stroma or the nature of pancreatic ductal adenocarcinoma-associated neural remodeling. Nevertheless, currently, there are no specific and sensitive diagnosis options for PC. Vibrational spectroscopy (VS) shows a promising role in the development of early diagnosis technology. In this review, we summarize recent reports about improvements in spectroscopic methodologies, briefly explain and highlight the drawbacks of each of them, and discuss available solutions. The important aspects of spectroscopic data evaluation with multivariate analysis and a convolutional neural network methodology are depicted. We conclude by presenting a study design for systemic verification of the VS-based methods in the diagnosis of PC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/patologia , Diagnóstico Precoce , Análise Espectral , Biomarcadores Tumorais , Neoplasias PancreáticasRESUMO
DNA double-strand breaks (DSBs) are typical DNA lesions that can lead to cell death, translocations, and cancer-driving mutations. The repair process of DSBs is crucial to the maintenance of genomic integrity in all forms of life. However, the limitations of sensitivity and special resolution of analytical techniques make it difficult to investigate the local effects of chemotherapeutic drugs on DNA molecular structure. In this work, we exposed DNA to the anticancer antibiotic bleomycin (BLM), a damaging factor known to induce DSBs. We applied a multimodal approach combining (i) atomic force microscopy (AFM) for direct visualization of DSBs, (ii) surface-enhanced Raman spectroscopy (SERS) to monitor local conformational transitions induced by DSBs, and (iii) multivariate statistical analysis to correlate the AFM and SERS results. On the basis of SERS results, we identified that bands at 1050 cm-1 and 730 cm-1 associated with backbone and nucleobase vibrations shifted and changed their intensities, indicating conformational modifications and strand ruptures. Based on averaged SERS spectra, the PLS regressions for the number of DSBs caused by corresponding molar concentrations of bleomycin were calculated. The strong correlation (R2 = 0.92 for LV = 2) between the predicted and observed number of DSBs indicates, that the model can not only predict the number of DSBs from the spectra but also detect the spectroscopic markers of DNA damage and the associated conformational changes.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Bleomicina/farmacologia , DNA/química , Dano ao DNARESUMO
Pancreatic cancer (PC) is one of the most aggressive and lethal malignant neoplasms, ranking in seventh place in the world in terms of the incidence of death, with overall 5-year survival rates still below 10%. The knowledge about PC pathomechanisms is rapidly expanding. Daily reports reveal new aspects of tumor biology, including its molecular and morphological heterogeneity, explain complicated "cross-talk" that happens between the cancer cells and tumor stroma, or the nature of the PC-associated neural remodeling (PANR). Staying up-to-date is hard and crucial at the same time. In this review, we are focusing on a comprehensive summary of PC aspects that are important in pathologic reporting, impact patients' outcomes, and bring meaningful information for clinicians. Finally, we show promising new trends in diagnostic technologies that might bring a difference in PC early diagnosis.
RESUMO
Even several thousands of DNA lesions are induced in one cell within one day. DNA damage may lead to mutations, formation of chromosomal aberrations, or cellular death. A particularly cytotoxic type of DNA damage is single- and double-strand breaks (SSBs and DSBs, respectively). In this work, we followed DNA conformational transitions induced by the disruption of DNA backbone. Conformational changes of chromatin in living cells were induced by a bleomycin (BLM), an anticancer drug, which generates SSBs and DSBs. Raman micro-spectroscopy enabled to observe chemical changes at the level of single cell and to collect hyperspectral images of molecular structure and composition with sub-micrometer resolution. We applied multivariate data analysis methods to extract key information from registered data, particularly to probe DNA conformational changes. Applied methodology enabled to track conformational transition from B-DNA to A-DNA upon cellular response to BLM treatment. Additionally, increased expression of proteins within the cell nucleus resulting from the activation of repair processes was demonstrated. The ongoing DNA repair process under the BLM action was also confirmed with confocal laser scanning fluorescent microscopy.
Assuntos
Bleomicina , Dano ao DNA , Bleomicina/farmacologia , Aberrações Cromossômicas , DNA , Reparo do DNA , HumanosRESUMO
Metabolic stress, such as lipotoxicity, affects the DNA methylation profile in pancreatic ß-cells and thus contributes to ß-cell failure and the progression of type 2 diabetes (T2D). Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that is involved in monounsaturated fatty acid synthesis, which protects pancreatic ß-cells against lipotoxicity. The present study found that SCD1 is also required for the establishment and maintenance of DNA methylation patterns in ß-cells. We showed that SCD1 inhibition/deficiency caused DNA hypomethylation and changed the methyl group distribution within chromosomes in ß-cells. Lower levels of DNA methylation in SCD1-deficient ß-cells were followed by lower levels of DNA methyltransferase 1 (DNMT1). We also found that the downregulation of SCD1 in pancreatic ß-cells led to the activation of adenosine monophosphate-activated protein kinase (AMPK) and an increase in the activity of the NAD-dependent deacetylase sirtuin-1 (SIRT1). Furthermore, the physical association between DNMT1 and SIRT1 stimulated the deacetylation of DNMT1 under conditions of SCD1 inhibition/downregulation, suggesting a mechanism by which SCD1 exerts control over DNMT1. We also found that SCD1-deficient ß-cells that were treated with compound c, an inhibitor of AMPK, were characterized by higher levels of both global DNA methylation and DNMT1 protein expression compared with untreated cells. Therefore, we found that activation of the AMPK/SIRT1 signaling pathway mediates the effect of SCD1 inhibition/deficiency on DNA methylation status in pancreatic ß-cells. Altogether, these findings suggest that SCD1 is a gatekeeper that protects ß-cells against the lipid-derived loss of DNA methylation and provide mechanistic insights into the mechanism by which SCD1 regulates DNA methylation patterns in ß-cells and T2D-relevant tissues.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Células Secretoras de Insulina/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Acetilação , Animais , Linhagem Celular , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo , Inativação Gênica , Histonas/metabolismo , Células Secretoras de Insulina/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirtuína 1/metabolismo , Análise Espectral Raman , Estearoil-CoA Dessaturase/antagonistas & inibidores , Estearoil-CoA Dessaturase/genética , Regulação para CimaRESUMO
The integrity of the chromatin structure is essential to every process occurring within eukaryotic nuclei. However, there are no reliable tools to decipher the molecular composition of metaphase chromosomes. Here, we have applied infrared nanospectroscopy (AFM-IR) to demonstrate molecular difference between eu- and heterochromatin and generate infrared maps of single metaphase chromosomes revealing detailed information on their molecular composition, with nanometric lateral spatial resolution. AFM-IR coupled with principal component analysis has confirmed that chromosome areas containing euchromatin and heterochromatin are distinguishable based on differences in the degree of methylation. AFM-IR distribution of eu- and heterochromatin was compared to standard fluorescent staining. We demonstrate the ability of our methodology to locate spatially the presence of anticancer drug sites in metaphase chromosomes and cellular nuclei. We show that the anticancer 'rule breaker' platinum compound [Pt[N(p-HC6F4)CH2]2py2] preferentially binds to heterochromatin, forming localized discrete foci due to condensation of DNA interacting with the drug. Given the importance of DNA methylation in the development of nearly all types of cancer, there is potential for infrared nanospectroscopy to be used to detect gene expression/suppression sites in the whole genome and to become an early screening tool for malignancy.
Assuntos
Cromossomos/ultraestrutura , DNA/ultraestrutura , Metáfase/genética , Espectrofotometria Infravermelho/métodos , Animais , Núcleo Celular/ultraestrutura , Eucromatina/ultraestrutura , Heterocromatina/ultraestrutura , Humanos , Interfase/genéticaRESUMO
Herein we report on the hydrolysis mechanism of [Pt{N(p-HC6F4)CH2}2(NC5H5)2(OH)2], a platinum(IV) complex that exhibits anti-cancer properties. Atomic telemetry, an in situ technique based on electron structure sensitive X-ray spectroscopy, revealed that hydrolysis preceded any reduction of the metal center. The obtained results are complemented with 19F NMR measurements and theoretical calculations and support the observation that this PtIV complex does not reduce spontaneously to PtII in HEPES buffer solution at pHâ¯7.4 and after 24â¯h incubation. These results are of importance for the design of novel Pt-based coordination complexes as well as understanding their behavior under physiological conditions.
Assuntos
Compostos Organoplatínicos/química , Platina/química , Espectroscopia de Ressonância Magnética , Espectroscopia por Absorção de Raios XRESUMO
Exposure to ionizing radiation can induce cellular defense mechanisms including cell activation and rapid proliferation prior to metastasis and in extreme cases can result in cell death. Herewith we apply infrared nano- and microspectroscopy combined with multidimensional data analysis to characterize the effect of ionizing radiation on single glioblastoma nuclei isolated from cells treated with 10 Gy of X-rays or 1 and 10 Gy of protons. We observed chromatin fragmentation related to the formation of apoptotic bodies following X-ray exposure. Following proton irradiation we detected evidence of a DNA conformational change (B-DNA to A-DNA transition) related to DNA repair and accompanied by an increase in protein content related to the synthesis of peptide enzymes involved in DNA repair. We also show that proton exposure can increase cholesterol and sterol ester synthesis, which are important lipids involved in the metastatic process changing the fluidity of the cellular membrane in preparation for rapid proliferation.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Nanotecnologia , Prótons , Neoplasias Encefálicas/radioterapia , Proliferação de Células/efeitos da radiação , Glioblastoma/radioterapia , Humanos , Espectrofotometria Infravermelho , Raios XRESUMO
The biological influence of radiation on living matter has been studied for years; however, several questions about the detailed mechanism of radiation damage formation remain largely unanswered. Among all biomolecules exposed to radiation, DNA plays an important role because any damage to its molecular structure can affect the whole cell and may lead to chromosomal rearrangements resulting in genomic instability or cell death. To identify and characterize damage induced in the DNA sugar-phosphate backbone, in this work we performed x-ray absorption spectroscopy at the P K-edge on DNA irradiated with either UVA light or protons. By combining the experimental results with theoretical calculations, we were able to establish the types and relative ratio of lesions produced by both UVA and protons around the phosphorus atoms in DNA.
Assuntos
Dano ao DNA , DNA/efeitos da radiação , Espectroscopia por Absorção de Raios X/métodos , Animais , Bovinos , Prótons , Raios UltravioletaRESUMO
Platinum-based drugs are commonly used in cancer treatment. The biological activity of a metallodrug is obviously closely related to its chemical and stereochemical characteristics. An overlooked aspect is the effect of the ligand to the electronic structure of the metal atom (coordinated atom). We report herein a Resonant X-ray Emission Spectroscopy (RXES) study on the chemical speciation of chiral platinum complexes in which diastereomers are distinguished on the basis of their metal electronic configuration. This demonstrates RXES high chemical speciation capabilities, a necessary property to further investigate the reactivity of the Pt atom towards nucleophiles or bionucleophiles, and an important complement the previously reported RXES abilities, namely that it can be employed for in situ studies at physiological concentrations.
Assuntos
Antineoplásicos/química , Compostos Organoplatínicos/química , Espectrometria por Raios X , Flúor/química , Ligantes , EstereoisomerismoRESUMO
This review presents a new application of Resonant X-ray Emission Spectroscopy (RXES) to study the mechanism of action of metal containing anticancer derivatives and in particular platinum in situ and in vivo. The technique is an example of a photon-in photon-out X-ray spectroscopic approach, which enables chemical speciation of drugs to be determined and therefore to derive action mechanisms, and to determine drug binding rates under physiological conditions and therapeutic concentrations. This is made feasible due to the atomic specificity and high penetration depth of RXES. The review presents examples of the three main types of information that can be obtained by RXES and establishes an experimental protocol to perfect the measurements within cells.
Assuntos
Complexos de Coordenação/química , Nanopartículas Metálicas/química , Neoplasias/diagnóstico por imagem , Espectrometria por Raios X/métodos , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Complexos de Coordenação/uso terapêutico , Humanos , Nanopartículas Metálicas/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , RadiografiaRESUMO
The structure-activity relationships of chiral 1,2-diaminophenylalkane platinum(II) anticancer derivatives are studied, including interactions with telomeric- and genomic-like DNA sequences, the pKa of their diaqua species, structural properties obtained from DFT calculations and resonant X-ray emission spectroscopy. The binding modes of the compounds to telomeric sequences were elucidated, showing no major differences with conventional cis-platinum(II) complexes like cisplatin, supporting that the cis-square planar geometry governs the binding of small Pt(II) complexes to G4 structures. Double-stranded DNA platination kinetics and acid-base constants of the diaqua species of the compounds were measured and compared, highlighting a strong steric dependence of the DNA-binding kinetics, but independent to stereoisomerism. Structural features of the compounds are discussed on the basis of dispersion-corrected DFT, showing that the most active series presents conformers for which the platinum atom is well devoid of steric hindrance. If reactivity indices derived from conceptual DFT do not show evidences for different reactivity between the compounds, RXES experiments provide new insight into the availability of platinum orbitals for binding to nucleophiles.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , DNA de Neoplasias/efeitos dos fármacos , Hidrocarbonetos Clorados/farmacologia , Compostos Organoplatínicos/química , Compostos Organoplatínicos/farmacologia , DNA de Neoplasias/genética , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Quadruplex G/efeitos dos fármacos , Humanos , Hidrocarbonetos Clorados/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Estrutura Molecular , Teoria Quântica , Relação Estrutura-AtividadeRESUMO
SR-FTIR in combination with Principal Component Analysis (PCA) was applied to investigate macromolecular changes in a population of melanocytes and their extracted nuclei induced by environmentally relevant fluxes of UVR (Ultraviolet Radiation). Living cells and isolated cellular nuclei were investigated post-irradiation for three different irradiation dosages (130, 1505, 15,052 Jm(-2) UVR, weighted) after either 24 or 48 hours of incubation. DNA conformational changes were observed in cells exposed to an artificial UVR solar-simulator source as evidenced by a shift in the DNA asymmetric phosphodiester vibration from 1236 cm(-1) to 1242 cm(-1) in the case of the exposed cells and from 1225 cm(-1) to 1242 cm(-1) for irradiated nuclei. PCA Scores plots revealed distinct clustering of spectra from irradiated cells and nuclei from non-irradiated controls in response to the range of applied UVR radiation doses. 3D Raman confocal imaging in combination with k-means cluster analysis was applied to study the effect of the UVR radiation exposure on cellular nuclei. Chemical changes associated with apoptosis were detected and included intra-nuclear lipid deposition along with chromatin condensation. The results reported here demonstrate the utility of SR-FTIR and Raman spectroscopy to probe in situ DNA damage in cell nuclei resulting from UVR exposure. These results are in agreement with the increasing body of evidence that lipid accumulation is a characteristic of aggressive cancer cells, and are involved in the production of membranes for rapid cell proliferation.
Assuntos
Núcleo Celular/efeitos da radiação , Conformação de Ácido Nucleico/efeitos da radiação , Pele/citologia , Pele/efeitos da radiação , Linhagem Celular Tumoral , Núcleo Celular/química , DNA/química , Humanos , Análise de Célula Única , Pele/química , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Raios UltravioletaRESUMO
The binding of the antitumor drug cisplatin with DNA was determined by means of in situ resonant inelastic X-ray scattering (RIXS) spectroscopy. Because of the penetrating properties of hard X-rays, we could determine, under physiological conditions, the identity and number of platinum complexes present. In situ RIXS revealed that under physiological conditions, water molecules replace chloride ligands owing to drug hydration. The subsequent interaction with DNA, led to the bonding of the aqua complexes into the DNA structure with simultaneous loss of the coordinating water and chloride ion. The data analysis reveals that Pt is coordinated by two adjacent guanines giving cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] upon losing its coordinating water or chloride ligands.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , DNA/química , Compostos de Platina/química , Compostos de Platina/farmacologia , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido NucleicoRESUMO
The effectiveness of two alkylphosphocholines (APCs), hexadecylphosphocholine (miltefosine) and erucylphosphocholine to combat prostate cancer has been studied in vitro with artificial cancerous membrane, modelled with the Langmuir monolayer technique, and on cell line (Du-145). Studies performed with the Langmuir method indicate that both the investigated drugs have the affinity to the monolayer mimicking prostate cancer membrane (composed of cholesterol:POPC = 0.428) and the drug-membrane interactions are stronger for erucylphosphocholine as compared to hexadecylphosphocholine. Moreover, both studied drugs were found to fluidize the model membrane, which may lead to apoptosis. Indeed, biological studies confirmed that in Du-145 cell line both investigated alkylphosphocholines cause cell death primarily by apoptosis while necrotic cells constitute only a small percentage of APC-treated cells.
Assuntos
Membrana Celular/metabolismo , Fosforilcolina/metabolismo , Neoplasias da Próstata/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Humanos , Masculino , Modelos Biológicos , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/farmacologiaRESUMO
The aim of this research was to find out whether the passage number effect may influence on the PC-3 cells (the human prostate cancer line derived from bone metastases) response to proton radiation. 2 MeV horizontally focused proton microbeam was used as a radiation source. The cells were treated with a counted number of H(+) ions (50-8000) corresponding to doses of 1.3-209 Gy/cell. For comparison, cell death was also induced by UVC radiation. All cells were stained with Hoechst 33342 and propidium iodide and visualized under a fluorescence microscope. Necrosis was observed at: a) 8000 protons per cell (corresponding to â¼209 Gy/cell) after 2-4 passages, b) 3200 protons per cell (corresponding to â¼84 Gy/cell) for cells after 11-14 passages and c) only 800 protons per cell (corresponding to â¼2 Gy/cell ) after 47-50 passages. Apoptosis was efficiently induced, by protons, only in cells after 50 passages. The results showed that the laboratory conditions affected cellular response of PC-3 cell line to the proton irradiation. The cellular response to the radiation treatment strongly depends on number of passages.